Biochemistry

Chemical Basis of Life Page |1
Chemical Basis of Life
 Biochemistry can be defined as the chemical basis of life.
 It can also be described as the study of the chemical constituents of living cells and the chemical
reactions and processes occurring in the cells.
Cellular foundations
 Cells are the structural
and functional units

of all living organisms.
 All living organisms fall
into one of three large

groups (domains) that
define three branches
of the evolutionary
tree of life originating
from a common
progenitor; bacteria,
archea and eukarya.
Two large groups of
single-celled micro-
organisms can be distinguished on genetic and
biochemical grounds: Bacteria and Archaea. Animals,
plants and fungi belong to the eukaryotes.
 A eukaryotic cell consists of cytoplasm and a nucleus.
It is enclosed by a plasma membrane. The cytoplasm

contains a complex system of inner membranes that
form cellular structures (organelles). The cytoplasm
is composed of an aqueous solution, the cytosol, and
a variety of suspended particles with specific
functions.
 The main organelles are the mitochondria, the
endoplasmic reticulum, the Golgi apparatus, peroxisomes and lysosomes. Centrioles, small
cylindrical particles made up of microtubules, play an essential role in cell division. Ribosomes are not
classified as organelle since they are not enclosed by a membrane and serve as the sites of protein
synthesis. Many cells present granules or droplets in the cytoplasm which contains stored nutrients
such as starch and fat.
 Plant cells also contain vacuoles (which store large quantities of organic acids) and chloroplasts (in
which sunlight drives the synthesis of ATP in the process of photosynthesis).

 The cytosol (the cytoplasm minus its membrane-bounded organelles) is organized by the
cytoskeleton, an extensive array of filaments that also gives the cell its shape and the ability to move.
 All cells have, for at least some part of their life, either a nucleoid or a nucleus. The nucleoid, in
bacteria and archaea, is not separated from the cytoplasm by a membrane; the nucleus, in
eukaryotes, is enclosed within a double membrane, the nuclear envelope. The nucleoid contains a
single, circular molecule of DNA, and the cytoplasm (like that of most bacteria) contains one or more
smaller circular segments of DNA called plasmids. In nature, some plasmids confer resistance to
toxins and antibiotics in the environment.
 Typical eukaryotic cells are much larger than bacteria—commonly 5 to 100 μm in diameter, with cell
volumes a thousand to a million times larger than those of bacteria. The distinguishing characteristics
of eukaryotes are the nucleus and a variety of membrane-enclosed organelles with specific functions.
Nucleus
 All eukaryotic cells except mature erythrocytes (red blood
cells) contain a nucleus (nuclei) where the cell’s genomic

DNA resides. Every normal human cell contains 23 pairs of
chromosomes within the nucleus of every cell.
 The outermost structure of the nucleus is the nuclear
envelope which is a double-layered phospholipid

membrane with nuclear pores to permit transfer of
materials between the nucleus and the cytosol.
 The interior of the nucleus contains the nucleoplasm, the fluid in which the chromosomes are found.
 A prominent structure within the nucleus is a suborganelle called the nucleolus which is rich in RNA,
particularly rRNA. It is also rich in enzymes in DNA and RNA synthesis.
Mitochondrion
 Mitochondria are regarded as the power house of the cell.
Mitochondria, like gram-negative bacteria, have two membranes.

 The inner mitochondrial membrane forms folded structures called
cristae that protrude into the mitochondrial lumen (space) known

as the mitochondrial matrix. The components of electron
transport chain and oxidative phosphorylation are embedded in
the inner mitochondrial membrane.
 The mitochondrial matrix contains the pyruvate dehydrogenase complex and the enzymes of the
citric acid cycle, the fatty acid β-oxidation pathway, and the pathways of amino acid oxidation—all
the pathways of fuel oxidation except glycolysis. Part of urea cycle, heme synthesis and
gluconeogenesis take place in mitochondrial matrix.
 Mitochondria also contain DNA (mtDNA) and ribosomes for the production of RNA and some
mitochondrial proteins. mtDNA is approximately 1% of total cellular DNA and exists in a circular
arrangement within the mitochondrial matrix.
 Mitochondria self replicate or divide by fission, as do bacteria. Mitochondria are actually believed to
have arisen from bacteria that were engulfed by ancestral eukaryotic cells.
 Mitochondrial proteins such as cytochrome c involve in apoptosis process. Thus, mitochondrial
involvement is important to ensure cell survival when it is appropriate and also to facilitate
programmed cell death when necessary. Cytochrome c in the cytosol stimulates a cascade of
biochemical events resulting in apoptotic death of the cell.
Endoplasmic reticulum
 Appearing like a series of interconnected, flattened tubes, the ER
is often observed to surround the nucleus. The outer layer of the

nuclear envelope is actually contiguous with the ER. In muscle
cells, this organelle is known as the sarcoplasmic reticulum.
 Regions of ER where ribosomes are bound to the outer
membrane are called rough endoplasmic reticulum (rough ER or

RER). RER is involved in the production and modification of
membrane bound and export proteins.
 Smooth endoplasmic reticulum (SER) refers to the regions of ER without attached ribosomes. SER is
site of synthesis of steroid hormone, phospholipids and metabolism of foreign compounds

(xenobiotics).
 Both RER and SER function in the glycosylation (addition of carbohydrate) of proteins and in the
synthesis of lipids.
 During the process of cell fractionation, rough ER is disrupted to form small vesicles known as
microsomes (microsomal fraction). It may be noted that microsomes as such do not occur in the cell.
Golgi complex
 Extending outward from the nucleus and the ER, the next organelle is the Golgi complex. This
organelle appears as flat, stacked, membranous sacs.

 It is responsible for protein modifications, such as glycosylations, phosphorylations to form mature,
functional proteins. It is also involved with packaging of proteins to form membrane enclosed
vesicles which are secreted from the cell after the appropriate signal.
Lysosome
 Lysosomes are membrane-enclosed organelles of various sizes that have an acidic internal pH (pH 5).
 They are actively involved in digestion of cellular substances. Lysosomes contain potent enzymes
known collectively as acid hydrolases. They function within the acidic environment of lysosomes to
hydrolyze or break down macromolecules (proteins, nucleic acids, carbohydrates, and lipids).
 In lysosomal storage diseases, nonfunctional macromolecules build up to toxic levels since they are
not degraded within lysosomes and properly recycled for reuse within the cell due to defective
lysosomal acid hydrolases.
Peroxisomes
 Peroxisomes resemble lysosomes in size and in structure. They have single membranes enclosing
them and contain hydrolytic enzymes.

 Within peroxisomes, fatty acids and purines (AMP and GMP) are broken down. Hydrogen peroxide,
a toxic by-product of many metabolic reactions, is detoxified in peroxisomes by catalase.
Cytoskeleton and cytosol

 Several types of protein filaments crisscrossing the eukaryotic cell, forming an interlocking three-
dimensional meshwork are known as the cytoskeleton. There are three general types of cytoplasmic
filaments—actin filaments, microtubules, and intermediate filaments. All types provide structure
and organization to the cytoplasm and shape to the cell. Actin filaments and microtubules also help
to produce the motion of organelles or of the whole cell.
 Cytosol is the site for many metabolic processes such as glycolysis, pentose phosphate pathway,
nucleotide synthesis, fatty acid synthesis and part of gluconeogenesis, urea cycle and heme
synthesis.
Chemical foundations
 Biochemistry aims
to explain biological
form and function
in chemical terms.
 Fewer than 30 of
the more than 90
naturally occurring
chemical elements
are essential to
organisms. Most of the elements in living matter have relatively low atomic numbers.
 The four most abundant elements in living organisms, in terms of percentage of total number of
atoms, are hydrogen, oxygen, nitrogen, and carbon, which together make up more than 99% of the
mass of most cells. The atoms of these elements are linked together by covalent bonds to form
C h e m ic a l B as i s o f L i f e P a g e |5
molecules. Two different molecules can be held together by non-covalent bonds, which are much
weaker than covalent bonds.
 The chemistry of living organisms is organized around carbon. Carbon can form single bonds with
hydrogen atoms, and both single and double bonds with oxygen and nitrogen atoms.
 Most biomolecules can be regarded as derivatives of hydrocarbons, with hydrogen atoms replaced
by a variety of functional groups that confer specific chemical properties on the molecule, forming
various families of organic compounds. Typical of these are alcohols, which have one or more
hydroxyl groups; amines, with amino groups; aldehydes and ketones, with carbonyl groups; and
carboxylic acids, with carboxyl groups.
 A second biologically important group of
elements, which together represent only about
0.5% of the body mass, are present almost
exclusively in the form of inorganic ions. This
group includes the alkali metals sodium (Na) and potassium (K), and the alkaline earth metals
magnesium (Mg) and calcium (Ca). The halogen chlorine (Cl) is also always ionized in the cell. All
other elements important for life are present in such small quantities that they are referred to as
trace elements. These include transition metals such as iron (Fe), zinc (Zn), copper (Cu), cobalt (Co),
manganese (Mn) and a few non-metals such as selenium (Se), iodine (I).
 The small organic molecules of the cell are carbon-based compounds that contain up to 30 or so
carbon atoms. Some are used as monomer subunits to construct giant polymeric macromolecules—
proteins, nucleic acids and large polysaccharides. Others act as energy sources and are broken down
and transformed into other small molecules in a network of intracellular metabolic pathways.
Broadly speaking, cells contain four major families of small organic molecules: the sugars, the fatty
acids, the nucleotides, and the amino acid.
 Macromolecules are the major constituents of cells. The chemistry of cells is dominated by
macromolecules with remarkable properties. Macromolecules are the most abundant carbon-
containing molecules in a
living cell. Macromolecules
themselves may be further
assembled into supra-
molecular complexes, forming functional units such as ribosomes. The macromolecules in cells are
polymers that are constructed by covalently linking small organic molecules (called monomers)
into long chains. There are three major families of macromolecules in the body; proteins, nucleic
acids and polysaccharides.
Molecular composition of the human body

 Water
 Carbohydrate
 Lipids
 Proteins
 Nucleic acids
 Inorganic salts and minerals
Water
 Water is the solvent in which most biochemical reactions take
place, and its properties are essential to the formation of
macromolecular structures and the progress of chemical reactions.
 Water (H2O) is a hydride of oxygen in which the highly
electronegative oxygen atom attracts the bonding electrons from
two hydrogen atoms. This leads to polar H — O bonds in which the
hydrogen atoms have a slightly positive charge (δ⁺) and the oxygen atom has a slightly negative
charge (δ⁻). Therefore the water molecule forms an electrical dipole.
 Unlike charges attract each other. Therefore the hydrogen atoms of a water molecule are attracted
by the oxygen atoms of other water molecules, forming hydrogen bonds. The hydrogen bonds
determine the physical properties of water, including its boiling point.
 Networks of hydrogen bonds hold the water molecules together and account for the cohesion of
liquid water. The polar nature of water is responsible for its high dielectric constant of water.
 Molecules in aqueous solution interact with water molecules through the formation of hydrogen
bonds and through ionic interactions. These interactions make water a versatile solvent, able to
readily dissolve many species, especially polar and charged compounds that can participate in these
interactions.
 Hydrophilic molecules
o Water is an excellent solvent for both ionic compounds (e.g., NaCl) and low molecular weight
nonionic polar compounds (e.g., sugars and alcohols).
o Substances that dissolve readily in water are termed hydrophilic. They are composed of ions or
polar molecules that attract water molecules through electrical charge effects.
o Ionic substances such as sodium chloride dissolve because water molecules are attracted to the
positive (Na+) or negative (Cl-) charge of each ion. Non-ionic polar substances such as urea
dissolve because their molecules form hydrogen bonds with the surrounding water molecules.
 Hydrophobic molecules
o Molecules that contain a preponderance of nonpolar bonds are usually insoluble in water and are
termed hydrophobic. Hydrocarbons, which contain many C–H bonds are not attracted to water
molecules and so cannot participate in hydrogen bonding or ionic interactions with water.
 Water is both the solvent in which metabolic reactions occur and a reactant in many biochemical
processes, including hydrolysis, condensation, and oxidation-reduction reactions.
 Water has slight but important tendency to dissociate. It can act either as an acid or as a base. Water
has a pH of 7.
 Functions of water
o Transport of molecules (gas, nutrients, waste products, etc.)
o Thermoregulation
o Solvent
o Reaction medium
o Reactant in many biochemical reactions such as hydrolysis
Carbohydrate
 Carbohydrates are polyhydroxy aldehydes or ketones, or substances that yield such compounds on
hydrolysis.
 Smaller carbohydrates such as milk sugar and table sugar are soluble in aqueous solution, while
polymers such as starch or cellulose form colloidal dispersions or are insoluble.
 Carbohydrate is major source of energy and is stored in the body in the form of glycogen.
 They bound to proteins or lipids and contribute important structural and regulatory functions.
 Carbohydrates are classified as follows: monosaccharides, disaccharides, oligosaccharides and
polysaccharides.
Monosaccharides
 Monosaccharides, or simple sugars, consist of a
single polyhydroxy aldehyde or ketone unit.
The most abundant monosaccharide in nature
is the six-carbon sugar. They cannot be
hydrolyzed into simpler carbohydrates. The
smallest monosaccharides, composed of three carbon atoms, are dihydroxyacetone and D - and L -
glyceraldehyde.
 They may be classified depending upon the number of carbon atoms, e.g., trioses, tetroses,
pentoses, hexoses, or heptoses and depending on the functional group (an aldehyde or ketone
group) e.g., aldoses or ketoses.
 In addition to aldehydes and ketones, the polyhydric alcohols (sugar alcohols or polyols), in which
the aldehyde or ketone group has been reduced to an alcohol group, also occur naturally in foods.
They are synthesized by reduction of monosaccharides for use in the manufacture of foods for
weight reduction and for diabetics. They are poorly absorbed, and have about half the energy yield of
sugars.
 Numbering of the carbon begins from the end containing the aldehyde group or for ketoses, the
terminal carbon closest to the keto carbon.
 In aqueous solution, aldotetroses and all monosaccharides with five or more carbon atoms in the
backbone occur predominantly as cyclic (ring) structures in which the carbonyl group has formed a
covalent bond with the oxygen of a hydroxyl group along the chain.
 Carbohydrates can exist in a wide variety of isomeric forms
o Glucose, with four asymmetric carbon atoms, can form 16 isomers.
o Constitutional isomers
 They have identical molecular formulas but differ in how the atoms are ordered.
 E.g., fructose has the same molecular formula as glucose but differs in that there is a
potential keto group in position 2, the anomeric carbon of fructose, whereas in glucose there
is a potential aldehyde group in position 1, the anomeric carbon.
o Epimers
 Monosaccharides that differ in the orientation of
substituents around one of their asymmetrical
carbons are called epimers e.g., D –mannose is a
C-2 epimer of glucose, and D -galactose is a C-4
epimer of glucose. Epimers are diastereomers,
not enantiomers. This means that they have different physical and chemical properties.
o Stereoisomers
 They are also called L and D isomerism that differ in spatial
arrangement 0f the constituents around an asymmetric
carbon. Stereoisomers are designated as having either D or L
configuration. These molecules are a type of stereoisomer
called enantiomers, which are mirror images of each other.
 According to convention, the D and L isomers are determined
by the configuration of the asymmetric carbon atom farthest
from the aldehyde or keto group. When the —OH group on this carbon is on the right, the
sugar is the D isomer; when it is on the left, it is the L isomer. Most of the naturally occurring
monosaccharides are D sugars, and the enzymes responsible for their metabolism are
specific for this configuration.
 Dihydroxyacetone is the only monosaccharide without at least one asymmetric carbon atom.
 The presence of asymmetric carbon atoms also confers optical activity on the compound. In
solution, glucose is dextrorotatory, and glucose solutions are sometimes known as dextrose.
C h e m ic a l B as i s o f L i f e P a g e |9
o Pyranose and furanose ring structures

 Predominant forms of sugars in solution inside the cell are
cyclic or ring structures rather than linear chain.
 The chemical basis for ring formation is the reaction
between alcohols and aldehydes or ketones to form
derivatives called hemiacetals or hemiketals.
 The ring structures of monosaccharides are similar to the
ring structures of either pyran (a six-membered ring) or
furan (a five-membered ring).
 More than 99% of total glucose in solution is pyranose ring
structure. Fructopyranose is the primary structure of
fructose in aqueous solution but the furanose form predominates in many fructose
derivatives.
o Alpha and beta anomers
 Isomeric forms of monosaccharides that differ only in their configuration about the
hemiacetal or hemiketal carbon atom are called anomers, and the carbonyl carbon atom is
called the anomeric carbon. In cyclic structures, asymmetric center at C-1 in glucose and C-2 in
fructose are known as anomeric carbon.
 The furanose-ring form of fructose also has anomeric forms, in which α and β refer to the
hydroxyl groups attached to C-2, the anomeric carbon atom.
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Biochemical significance of monosaccharides

 Glucose, galactose, fructose, and mannose are physiologically the most important hexoses. Glucose
is the major fuel for energy production in the body.
 Pentoses are important constituent in nucleotides, nucleic acids, and several coenzymes.
 Mannose, glucose and galactose are found in glycolipids and glycoproteins.
Derivatives of simple sugars

 When certain hydroxy groups are replaced by other groups or a carbon atom is oxidized to a carboxyl
group, sugar derivatives are formed. These occur especially in polysaccharides.
 Acid sugars
o Oxidation of the carbonyl (aldehyde)
carbon of glucose to the carboxyl
level produces gluconic acid. Other
aldoses yield other aldonic acids.
o Oxidation of the carbon at the other end of the carbon chain—C-6 of
glucose, galactose, or mannose—forms the corresponding uronic acid:
glucuronic, galacturonic, or mannuronic acid.
o The sialic acids are a family of sugars with the same nine-carbon backbone.
One of them, N-acetyl neuraminic acid (often referred as “sialic acid”) is a
component in many glycoproteins and glycolipids on animal cell surfaces,
providing sites of recognition by other cells or extracellular carbohydrate-
binding proteins.
 Sugars esters
o Condensation of phosphoric acid with one of the hydroxyl groups of a sugar
forms a phosphate ester, as in glucose 6-phosphate. In the synthesis and
metabolism of carbohydrates, many metabolic intermediates occur as
phosphate esters of sugars. One effect of sugar phosphorylation within cells is
to trap the sugar inside the cell.
o Several important phosphorylated derivatives of sugars e.g., ribose 5-
phosphate are components of nucleotides.
 Sugar alcohols
o If the aldehyde of a sugar is reduced, all of the carbon atoms
contain alcohol (hydroxyl) groups, and the sugar is a polyol, e.g.,
sorbitol, mannitol, xylitol.
o Glycerol and myo-inositol are components of membrane
phospholipids.
o Xylitol is widely used to sweeten sugarless gums and mints.

 Deoxysugars
o They are monosaccharides with one or more
hydroxyl groups replaced by hydrogen.
o 2-deoxy-ribose is a constituent of DNA in
organisms. Deoxysugars also occur frequently in glycoprotein and polysaccharides.
o The substitution of hydroxyl group with hydrogen at C-6 of L–galactose or L-mannose produces
L-fucose or L-rhamnose, respectively. L-Fucose is found in the complex oligosaccharide
components of glycoproteins and glycolipids. L -rhamnose is found in plant polysaccharides.
 Aminosugars
o Amino sugars such as galactosamine and glucosamine
contain an amino group instead of a hydroxyl group on one
of the carbon atoms, usually carbon 2.
o Glucosamine is seen in hyaluronic acid, heparin and blood group
substances.
o Galactosamine is present in chondroitin of cartilage, bone and tendons.
o Amino group in the sugar may be further acetylated to form an N-
acetylated sugar such as N-acetyl-glucosamine (GluNAc), N-acetyl
galactosamine (GalNAc). In complex molecules termed proteoglycans,
many of the N-acetylated sugars also contain negatively charged sulfate
groups attached to a hydroxyl group on the sugar. They are important constituents of
glycoproteins, mucopolysaccharides and cell membrane antigens.
Glycosides
 Glycosides are compounds containing a
carbohydrate and another carbohydrate or a
non-carbohydrate residue in the same molecule.
 The non-carbohydrate residue present in the
glycoside is called aglycone. Aglycones may be
methy alchohol, glycerol, phenol, sterols.
 Glycosides are found in many drugs and spices.
 Cardiac glycoside, e.g., digoxin is important in
clinical medicine and used for cardiac failure.
 Ouabain inhibits active transport of Na+ in
cardiac muscle (sodium pump inhibitor).
 Streptomycin is an aminoglycoside which is used as antibiotics.
Formation of glycosidic bond

Glycosidic bonds form when the hydroxyl group on the anomeric carbon of a monosaccharide
reacts with an –OH or –NH group of another compound.
α-glycosides or β-glycosides are produced depending on the position of atom attached to the
anomeric carbon of the sugar.
O-glycosidic bond is formed when a hydroxyl group of one sugar molecule reacts with the anomeric
carbon of the other to form disaccharides, oligosaccharides and polysaccharides.
N-glycosidic bonds join the anomeric carbon of a sugar to a nitrogen atom in glycoproteins and
nucleotides, e.g., between adenine and ribose.
Reducing sugars
Fehling and Benedict assays
 If the oxygen atom on the anomeric carbon of a sugar is  Use alkaline cupric salt solutions.
not attached to any other structure (free anomeric  With heating, the glucose decomposes
carbon), that sugar can act as a reducing agent and is oxidatively, yielding a complex mixture
of organic acids and aldehydes.
termed as reducing sugar.
 Oxidation of the sugar reduces cupric
 All monosaccharides react with cupric (Cu2+) ion reducing
ion (blue-green color) to cuprous ion
1+
it to cuprous ion (Cu ), while itself being oxidized. The (orange-red color) in solution. The
carbonyl carbon is oxidized to a carboxyl group. color yield produced is directly

proportional to the glucose
 Disaccharides are also reducing sugar (one free anomeric
content of the sample.
carbon on one end) with the exception of sucrose and
 They do not distinguish glucose
trehelose (anomeric carbons of both sugars involve in from other reducing sugars, such
glycosidic bond formation). as fructose or galactose.
 Solutions of cupric ion (known as Fehling’s solution) provide a simple test for the presence of sugars
such as glucose.
 Benedict’s reagent can be used to determine if a reducing sugar is present in urine or feces. A
positive result is indicative of an underlying pathology because sugars are not normally present in
urine or feces.
Disaccharides and oligosaccharides

 When the anomeric hydroxyl group of one monosaccharide is linked with one of the OH groups of
another monomeric sugar by a glycosidic bond, a disaccharide is formed, e.g., lactose, maltose,
isomaltose, sucrose and trehalose.
 Common diassacharides in our diet are lactose (milk sugar), sucrose (table sugar), maltose and
isomaltose (from digestion of starch).
 In disaccharides or polysaccharides, the end of a chain with a free anomeric carbon (one not
involved in a glycosidic bond) is commonly called the reducing end.
 Lactose
o Principal sugar in milk
o It contains galactose and glucose linked by β (1 – 4) glycosidic bond.
o It has a free anomeric carbon and is a reducing sugar.
 Sucrose
o Sucrose (table sugar) is a disaccharide of glucose and fructose
linked by α (1 – 2) glycosidic bond. It is formed by plants but not by
animals.
o Sucrose contains no free anomeric carbon atom; the anomeric
carbons of both monosaccharide units are involved in the glycosidic
bond. Sucrose is therefore a non-reducing sugar and its stability
toward oxidation makes it a suitable molecule for the storage and
transport of energy in plants.
 Maltose
o Maltose contains two glucose molecules joined by α (1 – 4) linkage.
It retains a free anomeric carbon, maltose is a reducing sugar.
 Isomaltose
o Isomaltose consists of two glucose units linked by α (1 – 6) glycosidic
bond. It is derived from hydrolysis of branched chain
polysaccharides, e.g., dextran, glycogen.
 Trehalose
o Trehalose is a disaccharide of glucose joined by α (1 – 1) linkage. It is
a non-reducing sugar like sucrose.
o It is a major constituent of the circulating fluid (hemolymph) of
insects, serving as an energy-storage compound.
 Oligosaccharides consist of short chains of monosaccharide residues (3 – 10) joined by glycosidic

bonds.
o They are naturally occurring substances or as hydrolysis products of natural materials.
Oligosaccharides also occur widely as components of antibiotics e.g., erythromycin,
streptomycin.
o Biomedical importance of oligosaccharides
 Oligosaccharides units are covalently attached to the extracellular side of intergral
membrane proteins.
 Many secreted proteins such as antibodies and coagulation factors also contain
oligosaccharide units.
Polysaccharides
 They are condensation products of more than ten monosaccharide units, e.g., starches and dextrins,
which may be linear or branched polymers.
 Classification
o Homopolysaccharides (homoglycans) –
polymer of same monosaccharide units,
e.g., starch, glycogen, inulin, cellulose,
dextrins, dextrans.
o Heteropolysaccharides (heteroglycans) – polymer of different monosaccharide units or their
derivatives, e.g., mucopolysaccharides (glycosaminoglycans or GAGs).
 Polysaccharides serve as energy storage, structure and protective functions.
 Starch and glycogen provide energy reserves for the cells.
 There are a wide variety of other polysaccharides that are collectively known as non-starch
polysaccharides which cannot be digested by human
enzymes. They are the major component of dietary
fiber, e.g., cellulose from plant cell walls (glucose
polymer) and inulin, the storage carbohydrate in some
plants (a fructose polymer).
 Starch
o Homopolymer of glucose forming α-glucosidic
chain, called glucan
o Most important dietary carbohydrate in cereals,
potatoes, legumes, and other vegetables
o The two main constituents are amylose (13 – 20%),
which has a non-branching helical structure, and
amylopectin (80 – 87%), which consists of branched chains consists of 24 to 30 glucose residues
with α 1 → 4 linkages in the chains and by α 1 → 6 linkages at the branch
points.
o The glycemic index of a starchy food is a measure of its digestibility, based
on the extent to which it raises the blood concentration of glucose
compared with an equivalent amount of glucose or a reference food.
 Glycogen
o Storage polysaccharide in animals
o More highly branched structure than amylopectin, with chains of 12 to 15 glucose residues (in α 1
→ 4 glucosidic linkage) with branching by means of α 1 → 6 glucosidic bonds
o Storage form of carbohydrate in the body (stored in liver and muscles)
 Inulin
o Polymer of fructose found in tubers, bulbs of onion and garlic
o It is readily soluble in water and is used to determine the glomerular filtration rate.
o It is not hydrolyzed by intestinal enzymes, so it has no nutritional value.
 Dextrins – intermediates in the hydrolysis of starch
 Dextrans
o Bacterial and yeast polysaccharides consisting of polymer of D-glucose with α (1 – 6) linkages
with α (1 – 3), α (1 – 2) and α (1 – 4) branches.
o Dental plaque, formed by bacteria growing on the surface of teeth, is rich in dextrans.
o Synthetic dextrans are used in several commercial products e.g., Sephadex in chromatography,
infusion fluid for plasma volume expander.
 Cellulose
o It is the chief constituent of plant cell walls. It is insoluble and consists of polymer of β-D-glucose
linked by β 1 → 4 bonds to form long, straight chains strengthened by cross-linking hydrogen
bonds.
o Mammals lack lack enzymes to hydrolyze β 1 → 4 bonds and so cannot digest cellulose.
 Chitin
o Structural polysaccharide in
the exoskeleton of
crustaceans and insects,
and also in mushrooms
o It consists of N-acetyl-d-
glucosamine units joined
by β 1 → 4 glycosidic bonds.
 Pectin
o It occurs in fruits consisting of a polymer of galacturonic acid linked by α-1→ 4 linkages, with
some galactose and/or arabinose branches and is partially methylated.
Mucopolysaccharides or glycosaminoglycans (GAGs)

 GAG is the most abundant polysaccharides in the body. They are a family of linear polymers
composed of repeating disaccharide units containing amino sugar and acid sugar. Amino sugar is
always either N-acetylglucosamine or N-acetylgalactosamine and acid sugar is a uronic acid in most
cases, usually D-glucuronic or L-iduronic acid. Some glycosaminoglycans contain esterified sulfate
groups.
 They may be attached to a protein to form a proteoglycan.
 GAGs are located primarily on cell surface or in the extracellular matrix (ECM).
 Properties and functions of GAGs
o GAGs are highly negatively charged and
hydrophilic molecules due to its high
density of sulfate and carboxylate
groups.
o Being highly hydrophilic, GAGs attract
water molecules to form a hydrated gel
like matrix and occupy space, thus
cushioning or lubricating the other
structures.
o Due to its extended conformations and
hydrophilic property, it imparts high
viscosity in solution which contributes low compressibility of these molecules. These molecules
are ideal for lubricating fluid in the joints.
o Their rigidity provides structural integrity to cells and provides passage ways between cells,
allowing cells migration.
 Functionally significant GAGs
o Hyaluronic acid o Heparan sulfate o Dermatan sulfate
o Heparin o Chondroitin sulfate o Keratin sulfate
 Hyaluronic acid
o It is composed of repeating units of N-acetyl-glucosamine and D-glucuronic acid.
o It is an important GAG component of ground substance in connective tissues, tendons, synovial
fluid (the fluid that lubricates the joints), and the vitreous humor of the eye.
 Heparin
o It is a variably sulfated GAG that consists predominantly of alternating linked residues of L-
iduronic acid sulfates and sulfated glucosamine.
o It is not a constituent of connective tissue, but occurs almost exclusively in the intracellular
granules of the mast cells that line arterial walls, especially in the liver, lungs and skin.
o Heparan sulfate, a ubiquitous cell-surface component as well as an extracellular substance in
blood vessel walls and brain.
o Heparin is widely used as an anticoagulant drug.
 Chondroitin sulfate
o It is composed of repeating units of glucuronic acid and N-acetyl galactosamine sulphate.
o It is present as ground substance connective tissues such as cartilage, bone, tendons and
ligaments.
 Keratan sulphate
o It contains repeating units of D-galactose and N-acetyl-D-glucosamine-6-sulfate residues (and
hence lacks uronic acid residues).
o It is a component of cartilage, bone, cornea, as well as hair, nails.
 Dermatan sulfate
o It consists of repeating units of L-iduronic acid and N-acetyl galactosamine.
o It contributes to the pliability of skin and is also present in blood vessels and heart valves.
Lipids
 Lipids are water-insoluble biomolecules that are highly soluble in organic solvents such as
chloroform. Lipids are located primarily in three compartments in the body – plasma, adipose tissues
and biological membranes.
 Lipids have a variety of biological roles – fuel molecules, highly concentrated energy stores, signal
molecules and components of membranes.
Classification of lipids
 There are many different methods of classifying lipids. The most commonly used classification of
lipids is as follows – simple lipids, complex lipids and derived lipids.
o Simple lipids - esters of fatty acids with various alcohols. Depending on types of alcohols –
 Fats are esters of fatty acids with glycerol. Oils are fats in the liquid state.
 Waxes are esters of fatty acids with higher molecular weight monohydric alcohols.
o Complex lipids – esters of fatty acids containing groups in addition to an alcohol and one or more
fatty acids. They can be divided into three groups:
 Phospholipids
 Glycolipids (glycosphingolipids)
 Other complex lipids such as sulfolipids, amino lipids and lipoproteins
o Precursor and derived lipids
 These include fatty acids, glycerol, steroids, other alcohols, fatty aldehydes, ketone bodies,
hydrocarbons, lipid-soluble vitamins and hormones.
Fatty acids
 Fatty acids are carboxylic acids with hydrocarbon chains. They are the simplest form of lipid.
 Fatty acids exist in free form (unesterified fatty acids or free fatty acids) and in esterified form as
major constituents various lipids (simple or complex lipids).
 Naturally occurring fatty acids contain even number of carbon atoms (most contain 14 to 24). This is
due to the fact that biosynthesis of fatty acids mainly occurs with the sequential addition of 2 carbon
units. Of these, most abundant are the fatty acids containing 16 or 18 carbon atoms (C-16 palmitic
acid and C-18 stearic acid).
 Fatty acids can be categorized according to their chain length (short chain, medium chain, long chain
and very long chain fatty acids) as well as bond saturation (mono/polyunsaturated fatty acids and
saturated fatty acids).
 The fatty acids with hydrocarbon chains containing one or more double bonds are called
unsaturated fatty acids, whereas those lacking any double bonds are referred to as saturated fatty
acids.
 Melting points of fatty acids increases with the chain length and decreases with the number of
double bonds.
 They produce large amount of energy on oxidation, e.g., complete oxidation of a palmitic acid
produces 106 ATPs.
 Nomenclature of fatty acids
o Saturated fatty acids
end with –anoic acid,
e.g., octanoic acid.
o Unsaturated fatty acids
with double bonds end
in –enoic acid, e.g.,
octadecenoic acid.
o Carbon atoms are
numbered from the
carboxyl carbon (carbon no.1). Carbon atoms adjacent to the carboxyl carbon, i.e., carbon no.2,
3, 4 are called α, β and γ carbons.
o The terminal methyl carbon is ω- or n- carbon.
o To indicate the position of double bonds,  is used. For example, oleic acid (C 18:1, 9 or ω9)
indicates that it contains 18 carbon atoms and one double bond. 9 indicates position of the
double bond is between carbon 9 and 10 of the fatty acid. ω-9 indicates a double bond on the 9th
position from the ω-carbon.
 Cis and trans configuration of unsaturated fatty acids

o Cis-double bond is formed if the acyl chains are on the
same side of the double bonds. The hydrocarbon chain
(acyl chain) is bent 120° at the double bond.
o Trans-double bond is formed if the acyl chains are in
opposite sides.
o Naturally occurring unsaturated fatty acids are nearly all
of the cis-configuration.
o Cis-double bonds place a kink in the linear structure of
fatty acids and thus interfere with close packing and
lower the melting point.
o Trans-fatty acids
 Partial hydrogenation of vegetable oil converts the cis-double bond into trans-form with
higher melting temperature e.g., margarine, butter fat. Many fast foods are deep fried in
partially hydrogenated vegetable oil and thus, contain high amount of trans-fatty acids.
 Increased consumption of trans-fatty acids increases the risk of cardiovascular disease by
elevating plasma triacylglycerol and LDL (“bad”) cholesterol level and lowering HDL
(“good”) cholesterol level in the blood. Trans-fatty acids

contribute as a risk factor for CVD comparable with
saturated fatty acid and cholesterol.
 Essential fatty acids
o Lipid biosynthetic capacity of the body can generate various
fatty acids needed by the body. Humans lack the enzymes to
introduce double bonds at carbon atoms beyond C9 in the
fatty acid chain.
o Hence, humans cannot synthesize polyunsaturated fatty acids
(PUFAs) such as linoleic acid and linolenic acid having double
bonds beyond C9 and thus they must be provided in the diet.
These PUFAs are called essential fatty acids.
o Arachidonic acid can be synthesized from linoleic acid.
Therefore, in deficiency of linoleic acid, arachidonic acid also
becomes an essential fatty acid.
o Biomedical importance of PUFAs or essential fatty acids
1. Membrane components: These fatty acids are important constituents of phospholipids in cell
membrane and help to maintain the structural integrity of the membrane. Because cis
configuration of double bonds, PUFAs have kinks in their hydrocarbon chain structure which
also provide to increase fluidity of membranes.
2. Synthesis of eicosanoids: Linoleic acid and linolenic acid supplied by the diet are the
precursors for the synthesis of a variety of other unsaturated fatty acids. Arachidonic acid, a
fatty acid derived from linoleic acid is an essential precursor of eicosanoids which act as local
messengers within the tissue of origin.
3. Effect on serum cholesterol: Ingestion of PUFAs increases esterification and excretion of
cholesterol, thereby lowering serum cholesterol level. Thus, essential fatty acids help to
prevent the atherosclerosis.
4. Development of retina and brain: Eicosapentaenoic acid (EPA: ω3) and docosahexaenoic acid
(DHA: ω-3) which are synthesized from α-linolenic acid is particularly needed for development
of the brain and retina during the neonatal period.
o Essential fatty acids deficiency
 Approximately 2–3% of daily calorie intake should be accounted by EFAs. Dietary deficiency is
characterized by scaly dermatitis, poor wound healing and hair loss. Impaired lipid transport
and fatty liver may also occur due to deficiency of EFAs
 They are most commonly seen in patients suffering from severe fat mal-absorption, in
patients kept on parenteral nutrition for long, and in infants fed on low-fat milk formulas.
Triacylglycerol (Triglycerides or TAG)

 Triacylglycerols are the esters of glycerol
with fatty acids. They primarily function as
fuel reserves of animals. They are
commonly known as neutral fats.
 In animals, TAGs are stored in adipose
tissues.
 It serves as thermal and mechanical insulator in the subcutaneous tissues and around certain organs.
Brown adipose tissue functions in generating heat (thermogenesis).
Phospholipids
 Phospholipids are the major lipid constituents of cell
membranes.
 These are made up of fatty acids, glycerol or other alcohol
backbone, phosphoric acid and an amino alcohol or sugar
alcohol group attached to the phosphate.
 Phospholipids are amphipathic in nature, i.e. each has a
hydrophilic or polar head (phosphate group) and a long
hydrophobic tail (containing fatty acid chains).
 Phospholipids can be further subdivided into two groups:
glycerophospholipids and sphingophospholipids. Glycerol
phosphate (phosphatidic acid) forms the backbone structure
of the glycerolphospholipids while an amino alcohol,
sphingosine is present in the sphingophospholipids.
 Glycerophospholipids
o Phospholipids derived from glycerol are called
phosphoglycerides or glycerophospholipids.
o In glycerophospholipid, the hydroxyl groups at C-1 and C-
2 of glycerol are esterified with two fatty acids. The C-3
hydroxyl group of the glycerol is esterified to phosphoric
acid and resulting compound called, phosphatidic acid.
o Glycerolphospholipids are formed from phosphatidic acid
by attachment of a polar alcohol group to the phosphate
group (of phosphatidic acid) by a phosphodiester bond.
The common alcohol groups of phosphoglycerides are
ethanolamine, choline, inositol and the amino acid serine.
o Important membrane glycerophospholipids

 Phosphatidylcholine (lecithin)
 Phosphatidylethanolamine (cephalin)
 Phosphatidylserine
 Phosphatidylinositol
 Cardiolipin (disphosphatidyl glycerol)
 Ether glycerophospholipids – plasmalogen, platelet activating factor (PAF)
o Ether glycerophospholipids
 They are generally similar to other glycerophospholipids but an unsaturated fatty alcohol,
rather than a fatty acid residue, is linked with carbon 1 of glycerol by an ether bond.
o Glycerophospholipids are the most abundant membrane lipids, accounting for more than half of
the total lipids (55–70%) in most membranes. They form the well known lipid bilayer structures
because of their amphipathic nature. The most abundant phospholipid in cell membrane is
phosphatidylcholine (lecithin).
 Sphingophospholipids
o Phospholipids derived from alcohol sphingosine
instead of glycerol are called sphingo-
phospholipids, e.g. sphingomyelin.
o In sphingomyelin, the amino group of the
sphingosine is linked to a fatty acid to yield
ceramide (sphingosine-fatty acid complex). Of the
two hydroxyl groups in ceramide, the one at C-3
remains unsubstituted, and the one at C-1 is esterified with phosphocholine in sphingomyelin.
o Sphingomyelin is one of the principal structural lipids of myelin sheath in nerve tissue.
Glycolipids (glycosphingolipids)
 The glycolipids are formed by attachment of a carbohydrate component (mono- or oligosaccharide)
to ceramide.
 Glycolipids are widely distributed in every tissue of the body, particularly in nervous tissue such as
brain.
 Depending on the nature of the carbohydrate component attached, four types of glycolipids are
recognized: cerebrosides, sulphatides, globosides and gangliosides.
 Cerebrosides (Ceramide + Monosaccharides)

o Cerebroside is the simplest glycolipid in which there
is only one sugar residue, either glucose or
galactose linked to ceramide i.e., glucosylceramide
and galactosylceramide.
o Galactosylceramide is the major glycolipid of brain
and other nervous tissues whereas glucosylcermaide
is the predominant glycolipid extra-neural tissues.
 Sulphatides (Ceramide + Monosaccharide + Sulfate)
o Sulfatides are cerebrosides in which the monosaccharide contains a sulfate ester.
 Globosides (Ceramide + Oligosaccharide)
o Globosides contain two or more sugar molecules attached to ceramide.
o These glycolipids are important constituents of the RBC-membrane and are the determinants of
the A,B,O blood group system.
 Gangliosides: (Cerebroside + Oligosaccharides + N-acetylneuraminic acid, NANA)
o Ganglioside contains oligosaccharides and one or more molecules of sialic acid, which is usually N-
acetylneuraminic acid (NANA) attached to ceramide.
 Functions of glycolipids
o Glycolipids are important constituents of the nervous tissue, such as brain.
o They are present on outer leaflets of cell membrane and play a role in cell-to-cell interaction,
cellular communication and contact.
o They also serve as cell surface antigens e.g., ABO blood group antigens on RBC membrane and
receptors for some bacterial toxins e.g., cholera toxin.
Steroids
 Steroids are compounds containing a cyclic steroid
nucleus, consisting of four fused carbon rings,
namely cyclopentanoperhydrophenanthrene (CPPP).
It consists of a phenathrene nucleus (ring A, B and C)
to which a cyclopentane ring (D) is attached.
 The alcohol derivatives of steroids, in which one or more OH groups are present in the steroid
nucleus, are termed sterols.
 In animal tissues, cholesterol is the major
sterol. Cholesterol, a 27-carbon compound, has
a single polar head group, i.e. the hydroxyl
group at the C-3 position and the rest of the
molecule is non-polar.
 Cholesterol is amphipathic, with a polar head
the hydroxyl group at C-3 and a nonpolar, the steroid nucleus and hydrocarbon side chain at C-17.
 Most of the cholesterol in the body exists as a cholesterol ester, with a fatty acid attached to the
hydroxyl group at C-3.
 In plants and yeasts a different membrane steroid, phytosterol, e.g., ergosterol, is present and most
bacteria have no steroid at all.
 Functions of cholesterol
o It is the major structural constituent of cellular membranes in which it regulates membrane
fluidity.
o Cholesterol serves as precursor for a variety of biologically important molecules such as steroid
hormones, bile acids and vitamin D.
Protein
 Proteins are the most abundant macromolecules in living cells.
 The term ‘protein’ was derived from the Greek word “protos” which means primary or holding first
place. As the name indicates, protein is the most important of cell constituents. They are responsible
for almost every function that occurs in the body.
 Proteins are primary structural and functional polymers in living system. They have a broad range of
activities, including catalysis of metabolic reactions, transport of substances and signaling processes.
Some proteins made up the structure of tissues, while others function in nerve transmission, muscle
contraction and cell motility, blood clotting, immunologic defenses, as hormones and regulatory
molecules.
 Proteins are polymers of L-α-amino acids that are linked by covalent, peptide bonds. Each protein
has specific and unique sequence of amino acids that defines both its three-dimensional structure
and its biologic function.
 There are about 300 amino acids present in nature, but only 20 different amino acids appear in
proteins.
Amino acids
 The L-α-amino acids in proteins and peptides are
composed of a carboxylic acid (–COOH) and an amino
group (–NH2) functional group attached to the
tetrahedral α-carbon of carboxylic acid. Distinct functional groups (R-group) are attached to the α-
carbon (except in the case of glycine where R-group is hydrogen). The fourth substitution on the
tetrahedral α-carbon of amino acids is hydrogen.
 Amino acids are distinguished from one
another by the distinct R-groups attached to
the tetrahedral α-carbon.
 All the amino acids found in proteins are
exclusively of the L-configuration. D-forms
occur in some bacterial peptides.
 Since amino acids contain both acidic (–COOH) and
basic (–NH2) groups, they can donate or accept
proton. Thus, amino acids are regarded as
ampholytes.
 The properties of each amino acid depend on side
chain R group which is the major determinant of the
structure, function and electrical charge of proteins.
Classification of amino acids

 Amino acids can be classified depending on;
o Polarity and charge on R groups
o Structure of the side chain of amino acids
o Nutritional requirement of amino acids
o Metabolic fate of amino acids
o Involvement in protein synthesis
 Classification based on polarity and charge on R groups of amino acids

 Classification based on structure of the side chain of the amino acid

o Aliphatic amino acids
 Simple amino acids such as glycine, alanine
 Branch-chain amino acids such as leucine, isoleucine and valine
o Aromatic amino acids, e.g., phenylalanine, tryptophan, tyrosine
o Neutral polar amino acids e.g., serine, threonine, asparagine, glutamine
o Acidic amino acids e.g., aspartate, glutamate
o Basic amino acids e.g., histidine, lysine, arginine
o Sulphur containing amino acids e.g., methionine and cysteine
o Imino acid e.g., proline
 Classification based on nutritional requirement of amino acids
o Nutritionally essential amino acids
 These amino acids cannot be synthesized by the body, therefore they must be supplied in the
diet, e.g., proline, valine, threonine, tryptophan, isoleucine, methionine, histidine, arginine,
leucine, lysine.
 Among the ten essential amino acids; arginine and histidine are known as semi-essential or
conditionally essential amino acids since these amino acids are synthesized partially in
human body. Arginine and histidine become essential in diet during periods of rapid growth
as in childhood and pregnancy.
o Nutritionally non-essential amino acids
 Non-essential amino acids can be synthesized in human body and are not required in diet,
e.g., alanine, aspartate, asparagine, glutamate, glutamine, tyrosine, serine, proline, glycine,
and cysteine.
 Classification based on metabolic fate of amino acids
o From the viewpoint of their catabolic fate, carbon skeleton of amino acids may serve as
precursors for glucose and glycogen synthesis (glucogenic) or fat and ketone bodies synthesis
(ketogenic) or for both glucose and fat synthesis (both ketogenic and glucogenic).
 Classification based on involvement in protein synthesis
o Protein amino acids – Amino acids involved in protein synthesis i.e., twenty different kinds of
amino acids in protein synthesis
o Non-protein amino acids do not involve in protein synthesis but may involve in other biochemical
processes, e.g., ornithine, citrulline, taurine, etc.
Biochemical significance of different side chains of amino acids

The side chain group of amino acid determines the properties of amino acids. The structure, chemical
properties, functions and charge of the protein are determined by its amino acid side chain.
Amino acids with charged, polar or hydrophilic side chains are usually exposed on the surface of the
protein. The non-polar hydrophobic amino acids are buried in the interior core of the protein.
However, for proteins that are located in a hydrophobic environment, such as a membrane, the
nonpolar R groups are found on the outside surface of the protein, interacting with the hydrophobic
environment.
Proteins rich in arginine and lysine are basic and have a positive charge at neutral pH while the
proteins rich in aspartate and glutamate are acidic and have a negative charge.
Proline differs from other amino acids in that its side chain pyrrolidine ring includes both the α-
carbon and the α-amino group. Chemically speaking proline is an imino (–NH) acid. Proline forces a
bend in a polypeptide chain, sometimes causing an abrupt change in direction of the chain.
Side chains as sites of attachment for other compounds or chemical groups.
o The hydroxyl groups of serine and threonine exist at the catalytic sites of certain kinds of
enzyme. The polar hydroxyl group of serine; threonine; and, rarely, tyrosine, can serve as a site
of attachment for phosphate group. Phosphorylation changes their charge from neutral to
negative, which changes the shape and function of the enzyme proteins.
o In addition, the amide group of asparagine, as well as the hydroxyl group of serine or threonine,
can serve as a site of attachment for oligosaccharide chains in glycoproteins.
Thiol group of a cysteine can form a disulphide bond with another cysteine through the oxidation of
the two thiol groups. The dimeric compound so formed, called cystine, is important in cross-linking
the adjacent polypeptide chains.
Aromatic side chains are responsible for the ultraviolet absorption of most proteins, which have
absorption maxima between 275 nm and 285 nm. Tryptophan has greater absorption in this region
than the other two aromatic amino acids. Since nearly all proteins contain aromatic amino acids, the
amount of light absorbed at 280 nm by a protein is used as an indirect measure of protein
concentration.
As amino acids have both acidic
and basic groups, they can
donate a proton or accept a
proton, hence amino acids are
regarded as ampholytes. The
acid base properties of amino
acids depend on the amino and
carboxyl groups attached to
the α-carbon. Among the 20
standard amino acids, histidine serves as the best buffer at physiological pH. Because the side chain
of histidine (imidazole group) has a pKa of 6.0 and can serve as the best buffer at physiological pH.
Knowledge on these side chains and properties is important for analysis, purification and
identification of protein.
Peptides and Proteins

 Amino acids are joined by peptide bonds to form dipeptide,
tripeptide, oligopeptide and polypeptide/protein.
 Proteins are linear chains of amino acids that are linked together
by covalent, peptide bonds. Each protein has
specific and unique sequence of amino acids
that defines both its three-dimensional
structure and its biologic function.
 Proteins are primary structural and
functional polymers in living systems.
 Structural proteins
o Cytoskeletal protein
o Collagen
o Muscle proteins
 Functional proteins
o Biocatalysts in reactions (enzymes)
o Plasma proteins
o Hormones and regulatory molecules of metabolism
o Proteins involved in immunologic defense e.g., immunoglobulins
o Coagulating factors
o Transporters
Structure of proteins (Levels of organization of protein structure)

 Every protein has a unique three-dimensional structure, which is referred to as its native
conformation which is determined by its amino acid sequence.

 Protein conformation is complex and is best analyzed by considering it in terms of the following
organizational levels; primary, secondary, tertiary, and quaternary.

 Primary structure
o Primary structure of proteins refers to the specific sequence of amino acids in a polypeptide
chain joined by peptide bonds or amide bonds. These unique amino acids sequence is specified
by the respective gene.
o Each polypeptide chain is having free amino group at one end called N-terminal and free carboxyl
group at another end, called C-terminal.
 Secondary structure
o Hydrogen bonding between amide hydrogen (-NH) of

one peptide bond and carbonyl oxygen (C=O) of
nearby peptide bond gives rise to folding or twisting
of the primary structure.
o Thus, regular folding and twisting of the polypeptide
chain brought about by hydrogen bonds is called
secondary structure of protein.
o The most important kinds of secondary structure are –
 α-Helix (helicoidal structure)
 β-Pleated sheet (stretched structure).
o α-Helix
 It is a spiral structure consisting of a polypeptide chain that is coiled around a longitudinal
axis in a helical fashion.
 It is stabilized by hydrogen bonds between the NH and

CO groups of the same chain.
o β-Pleated sheet
 It is almost fully extended rather than being tightly
coiled as in the α-helix.
 β-pleated sheet is stabilized by hydrogen bonds between
NH and C=O groups in a different polypeptide chains or
neighboring polypeptide chains (inter-chain hydrogen
bonds).
 The arrangement of polypeptide chains in β-pleated
sheet conformation can occur in two ways – parallel
pleated sheet and anti-parallel pleated sheet.
 Tertiary structure
o Three-dimensional folded compact and biologically active

conformation of a protein is referred to as its tertiary
structure, e.g. myoglobin.
o Amino acid residues which are very distant from one another
in the sequence can be brought very near due to the folding
and thus form regions essential for the functioning of the
protein, like active site or catalytic site of enzymes.
o Tertiary structure is stabilized by the bonds between amino
acid side chains. These include –
 Hydrogen bonds
 Hydrophobic interactions
 Van der Waals forces
 Disulfide bond
 Ionic (electrostatic) bonds or salt bridges.
o Small folded unit of polypeptide chain arranged into tertiary
structure is called domain.
 Quaternary structure
o Some of the proteins are composed of two or more polypeptide chains referred to as subunits or
domains. Association of similar or dissimilar subunits of individual protein held together by
covalent or non-covalent interactions is referred to as quaternary structure, e.g., hemoglobin.
o Only those proteins that have more than one polypeptide chain (polymeric) have a quaternary
structure. Not all proteins are polymeric. Many proteins consist of a single polypeptide chain and
are called monomeric proteins, e.g. myoglobin.
 Protein folding occurs in orderly and guided fashion. Folding a protein is assisted by many proteins
such as chaperones. Chaperones shield newly synthesized polypeptides from solvent and provide an
environment for elements of secondary structure to fold into globular protein. Abnormal protein
folding is associated with diseases.
Protein denaturation
 Denaturation refers to disruption of the higher order structure of the protein.
 Denaturation results in the loss of secondary, tertiary and quaternary structure of proteins.
 In this process there is only disruption of higher order structure by the rupture of ionic bonds,
hydrogen bonds and hydrophobic interaction that stabilize the structure, without breakage of any
peptide linkage.
 Denaturation of proteins leads to change in physical, chemical and biological properties, e.g.,
o Decrease in solubility and increase in precipitability
o Loss of biological activities, such as enzyme activity and antigenic properties
o Increased digestibility.
 Agents of denaturation
o Physical agents – heat, ultraviolet rays and ionizing radiations
o Chemical agents – urea, acids, alkali and certain acid solutions of heavy metals, e.g. mercury, lead,
organic solvents like alcohol, acetone, ether, etc.
Protein synthesis and post-translational modification of proteins

 Information for specific amino acid sequence for polypeptide chain is encoded by respective gene.
 These polypeptide chains are synthesized as precursor proteins which require a variety of chemical
modifications. Precursor protein undergoes some form of enzymatic modifications after the
synthesis i.e., post-translational modification. These include –
o Hydroxylation
o Covalent modification (phosphorylation,
acetylation)
o Glycosylation
o Proteolytic cleavage
Protein separation and purification

 To study and use a protein it is necessary to separate it from a biologic fluid and purify it. Several
techniques are used to separate proteins based on solubility, molecular size, molecular charge and
selective binding of proteins to specific substances.
 Separation on the basis of protein solubility
o Salting out (ammonium sulfate fractionation)
 Separation on the basis of molecular size

o Dialysis and ultrafiltration
o Ultracentrifuge
o Gel filtration (Column chromatography)
 Separation on the basis of molecular charge
o Ion exchange chromatography
o High performance liquid chromatography (HPLC)
o Electrophoresis
o Polyacrylamide gel electrophoresis (PAGE)
 Separation on the basis of affinity binding
o Affinity chromatography
o Precipitation by antibodies
 Biomedical applications of protein purification and
separation
o Generally, a combination of these techniques is
employed to separate a given protein from other
proteins and molecules.
o Purification is necessary for manufacturing of proteins
that are used for therapeutic purposes, such as insulin
and clotting factors.
o The removal of low molecular weight waste products
from blood of patients with renal failure is based on
the principle of dialysis; the process called
peritoneal dialysis, hemodialysis.
o Separation of enzyme proteins (by
electrophoresis) is useful for the diagnosis of
various disorders e.g., plasma protein
electrophoresis.
Protein characterization
 Determination of primary structure
o Amino acid sequencing of peptides and proteins

o Mass spectrometry
o Determination of amino acid composition by
HPLC
o Deduction of amino acid sequence

from the nucleotide sequence of its
corresponding gene
 Determination of higher order structure of
protein
o X-Ray Crystallography
o Ultraviolet Light Spectroscopy
 It is based on absorption of
ultraviolet radiation with two
absorption maxima: one at 190
nm is caused by peptide bonds,
and the other at 280 nm is caused
by the aromatic side chains of
tyrosine and tryptophan.
o Nuclear Magnetic Resonance spectroscopy (NMR)
o Computer Based Modeling
Nucleotides and nucleic acids

 Essential component of all living organisms is
deoxyribonucleic acid (DNA) molecule which is the physical
and chemical basis of all genetic information. Genetic
information is stored in the base sequence of DNA
molecules. DNA molecule is arranged and organized into
chromosome and packed into the nucleus in eukaryotes.
 Segment of DNA that encodes information to synthesize
the functional biological product is called the “gene”.
Genetic information is used to synthesize specific amino
acid sequence of protein by a process called gene
expression which includes two components; transcription
and translation.
 Nucleic acids are polymers of nucleotides linked by 3’ – 5’
phosphodiester bonds. There are two types of nucleic acid;
deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
 DNA is found in nucleus and mitochondria whereas RNA is
found in nucleus, mitochondria and cytoplasm.
 Nucleoside is composed of pentose sugar linked to purine or pyrimidine base at its C-1 by N-glycosidic
bond. Nudeotides are nucleosides with one or more phosphates attached to the carbon 5' position of
pentose sugar. Thus, nucleotide is composed of nitrogenous base (purine or pyrimidine base),
pentose sugar (ribose or deoxyribose) and inorganic phosphate.
 Nitrogenous bases
o They are nitrogen containing heterocyclic compounds.
o Nitrogenous bases found in nucleic acid are purine and pyrimidine bases.
o Purine contains two rings whereas pyrimidine consists of one ring.
o Major purine bases
 Adenine (6 amino purine)
 Guanine (2 amino 6 oxypurine)
o Major pyrimidine bases
 Cytosine (2 oxy 4 amino pyrimidine)
 Thymine (2, 4 dioxy 5 methyl pyrimidine)
 Uracil (2, 4 dioxypyrimidine)
o Some minor or unusual bases are found in nucleic acids, mostly in RNA. These include 5’-
methylcytosine, pseudouracil, N6 -methyladenine and N7 –methylguanine.
o Some methylated purine bases are found in plants. These include alkaloids like theophylline (1,3-
dimethylxanthine), caffeine (1,3,5-trimethylxanthine) and theobromine (3,7-dimethylxanthine).
o Because of highly conjugated double bond
systems within the ring structures of
nitrogenous bases, nucleic acids have a very
strong absorption maximum at about 260
nm. This property is used for nucleic acid
quantitation.
 Pentose sugars
o The pentose sugar is either D-ribose or D-2-
deoxyribose. DNA and RNA are distinguished
on the basis of the pentose sugar present.
DNA contains D-2-deoxyribose and RNA
contains D-ribose.
o A pentose sugar (D-ribose or D-2-
deoxyribose) is linked to a base (purine or
pyrimidine) via covalent N-glycosidic bond
to form nucleoside. This linkage joins N-9 of the purine base or N-1 of the pyrimidine base with C-1
of pentose sugar.
o The nucleosides of A, G, C, T and U are named adenosine, guanosine, cytidine, thymidine and
uridine respectively. If the sugar is ribose, ribonucleoside is produced; if the sugar is deoxyribose,
a deoxy-ribonucleoside is produced.
Structure of nucleotide
 Nucleotides are phosphorylated nucleosides. The phosphate group is attached to the nucleoside by
an ester linkage to the hydroxyl group of the pentose sugar (usually at C-5).
 Mononuleotides are nucleosides in which single phosphate group is attached to C-5 of the pentose
sugar, e.g., AMP (adenosine monophosphate) is adenine + ribose + phosphate.
 Additional phosphates are linked to by acid anhydride bond possessing high energy potential. These
bonds yield about 7 kcal/mol on hydrolysis.
Nucleic acid
 Nucleic acid a polymer of nucleotides which is polymerized through a phosphodiester linkage from
the 3’ hydroxyl group of one unit to the 5’phosphate group of the next unit.
 Two types of nucleic acids are found in the living

organisms classified according to the pentose
sugars; DNA and RNA.
Deoxyribonucleic acid (DNA)

 DNA is a polymer of deoxyribonucleotides joined by
3’ – 5’ phosphodiester bonds.
 DNA is a double stranded helical structure. The two
strands are anti-parallel (runs in opposite direction;
3’ to 5’ and 5’ to 3’).
 Deoxyribose sugar and phosphate groups
(hydrophilic backbone of DNA) are on the outside
and nitrogenous bases (hydrophobic) are faced
inside of the DNA helical structure.
 Two strands of DNA molecule are held together via
complementary base pairing between the two
strands. Adenine always pairs with thymine via two
hydrogen bonds and guanine always pair with
cytosine via three hydrogen bonds.
Thus, base sequence on one strand
defines the base sequence on the
other strand. Copying genetic
information from parental DNA
strand or a gene segment is based on
the complementary base pairing rule.
 Stability of double helical structure is maintained by hydrogen bonds between the complementary
bases and hydrophobic interactions between the stacks of base-pairs.
 Because of complementary base pairing, the amount of A equals the amount of T and the amount of
G equals amount of C. Thus, total number of purine bases equals to total number of pyrimidine
bases in a DNA molecule. This is called Chargaff’s rule.
 This three-dimensional structure of DNA was deduced by James Watson and Francis Crick in 1953.
 In prokaryotes, DNA is not separated from the rest of cellular contents. In eukaryotes, DNA is located
in the nucleus and small amount is also found in mitochondria. DNA present in the nucleus of the cell
is linear. Mitochondria DNA is small circular double stranded DNA which is similar to DNA in
prokaryotes.
 In most living organisms, except for RNA viruses, genetic

information is stored in the base sequence of DNA.
 Functions of DNA
o DNA is the chemical basis of hereditary since the
genetic information is stored in the base sequence of
DNA.
o It is the source of information for the synthesis of all
protein molecules of the cell. The information in DNA
is copied to RNA by transcription process. RNAs are
used in protein synthesis.
o It provides the genetic information possessed by the
parent cells to the daughter cells or offspring by the
process of DNA replication.
Denaturation and renaturation of DNA

 It is disruption of hydrogen bonds between
complementary bases of anti-parallel strands of DNA.
 Increasing temperature or decreasing the salt
concentration can affect the disruption of hydrogen
bonds in DNA molecules, releasing single strand DNA in
vitro. Temperature required to separate double stranded
DNA with G-C rich regions is higher than that of A-T rich
regions.
 DNA strands can be renatured, i.e., re-associated or reannealed if the denaturation condition is slowly
removed.
 In vivo, local dissociation of DNA strands is induced by DNA binding proteins and enzymes (helicase).
Ribonucleic acid (RNA)

 RNA is a polymer of ribonucleotides linked by 3’ – 5’ phosphodiester bonds. RNA is a single stranded
structure folded into α-helix.
 Nitrogenous bases found in RNA are adenine, guanine (purine bases) and cytosine, uracil (pyrimidine
bases).
 In a given RNA molecule, number of A and U, C and G are not necessarily equal.
 RNA is found in nucleus, cytoplasm and mitochondria in eukaryotes.
 Different types of RNAs in a living cell
o Messenger RNA (mRNA)
o Transfer RNA (tRNA)

o Ribosomal RNA (rRNA)
o Other RNAs
 Small nuclear RNA (snRNA)
 Small cytoplasmic RNA (scRNA)
 Functions of RNAs
o mRNA conveys genetic information from DNA to
cytoplasm to the site of protein synthesis
(ribosomes). It serves as template for translation
process in which base sequence (codons) of RNA
molecules is translated into specific amino acid
sequence of protein by translation machinery.
o tRNA carries specific amino acid to the site of protein synthesis. It also serves as an adaptor
between the specific codon (three
consecutive base sequence) and the
corresponding amino acid.
o rRNA provides site for protein synthesis. It
serves as a structural component of
ribosomes together with various proteins.
Differences between RNA and DNA

 DNA is a double stranded helical molecule
whereas RNA is a single stranded structure
folded into α-helix.
 In RNA, the sugar is ribose rather than 2'-
deoxyribose of DNA.
 RNA does not possess thymine and instead of
thymine, RNA contains uracil.
 In RNA, adenine pairs with uracil rather than
thymine.
 Unlike DNA, RNA is a single stranded structure and does not obey Chargaff’s rule.
 RNA can be hydrolyzed by alkali to 2’-3’ cyclic diester of mononucleotides but DNA cannot be
hydrolyzed by alkali because of the absence of 2’ OH group.
Central dogma of molecular biology or Flow of genetic information

An organism must be able to store and preserve its genetic information (genomic stability).
An organism must be able to pass that information to the future generations (replication).
This genetic information must be expressed to carry out all the processes of life (gene expression).
Biomembranes
Eukaryotic cells have a plasma membrane as well as a number of internal (subcellular) membranes
that encloses intracellular compartments to form various organelles. Thus, membranes define
compartments with specialized functions inside the cells.
They serve as barrier and maintain integrity of a cell.
Plasma membrane is the gate keeper of the cell; it controls not only the access of inorganic ions,
vitamins and nutrients but also the entry of drugs and the exit of waste products.
Structure of membrane
Generally accepted model of biomembrane structure is the
fluid mosaic model because it consists of a mosaic of
proteins and lipid molecules, most of which can move
laterally in the plane of membrane.
Plasma membrane consists of a continuous bilayer of
amphipathic lipids into which proteins are embedded.
Polar head groups of phospholipids are exposed on the
external surfaces of membrane and nonpolar hydrophobic
tail (fatty acid chain) oriented inside of membrane.
Lipids form the structural backbone of the membrane and
proteins are in charge of specific functions. Lipids are
distributed asymmetrically in the membrane. The fluidity of
membranes primarily depends on their lipid composition.
Cholesterol also influences membrane fluidity.
Flip-flop (transverse) movement of lipids between the
outer and inner bilayer leaflets occurs extremely slowly
without the aid of the membrane enzyme flippases and floppases.
Many membrane proteins have limited mobility and are anchored in place by attachment to
cytoskeletal proteins.
Membrane proteins and lipid composition differ markedly depending on the type of cell and
subcellular organelle. The protein to lipid ratio also differs among various biomembranes. The
protein/lipid ratio is highest in membranes with high metabolic activity such as inner mitochondrial
membrane (lipid – 20%). Lipid content is about 80% in the myelin sheath that insulates nerve cells.
Structural components of biological membrane
Components of cell membrane include membrane lipids (phospholipids and cholesterol), proteins
(integral and peripheral proteins) and smaller proportion of carbohydrates that are linked to lipids
and proteins.
Membrane phospholipids
o These are the most predominant molecular
components of all membranes.
o The principal phospholipids in the membrane are the
glycerophospholipids such as phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine and
sphingosine based lipids such as sphingomyelin.
o The lipid composition varies among the different cell
types and membranes.
 The major membrane phospholipid is
phosphatidylcholine (lecithin), which accounts
for 40–50% of the total phospholipid content.
 The inner mitochondrial membrane is especially rich in cardiolipins (diphosphatidyl glycerol)
and phosphatidylethanolamine.
 High concentration of sphingolipids is present in myelin sheaths of axons of neural tissues.
 Subcellular membranes (membranes of cellular organelles) primarily contain phospholipids
with relatively smaller amount of sphingolipids or cholesterol.
o Distribution of membrane lipids is asymmetry. Higher content of phosphatidylcholine and

sphingomyelin are found in the outer leaflet and a higher content of phosphatidylserine and
phosphatidylethanolamine are found in the inner leaflet of the bilayer. Glycolipids are mainly
found on the outside of the plasma membrane.
o Phosphatidylinositol in inner leaflets of cell membrane involves in cell signaling process in which
it serves to transfer information from hormones and neurotransmitters across the cell membrane
inside the cell.
Cholesterol
o Cholesterol is found almost exclusively in eukaryotic cells.
o Cholesterol is structurally more rigid than other membrane lipids. Cholesterol is by far the least
water-soluble membrane lipid.
o It is oriented in such a way in the membrane that its hydrophilic hydroxyl group faces the exterior
and the sterol ring and nonpolar hydrocarbon chain fits into the hydrophobic lipid phase of the
membrane.
o Cholesterol which is interspersed between the phospholipids, maintains the membrane fluidity
together with cis-configuration of unsaturated fatty acids in other membrane phospholipids.
This prevents too tight close packing of hydrophobic fatty acid chains in the membrane.
Membrane proteins
o Proteins of the membrane are classified into two major categories;
 Integral proteins or intrinsic proteins or trans-membrane proteins and
 Peripheral proteins.
o Integral proteins
 Integral proteins are either partially or totally immersed in the lipid bilayer. Many integral
membrane proteins span the lipid bilayer from one side to the other and are called
transmembrane protein whereas others are partly embedded in either the outer or inner
leaflet of the lipid bilayer.
 Non-polar amino acid side chains of integral proteins interact with the membrane lipids and
polar regions of proteins protrude into either extracellular space or cytoplasm or both.
 These proteins function as channels or transporters for transport of substances across the
membrane, as receptors for hormones and neurotransmitters, as enzymes or as structural
proteins.
o Peripheral proteins
 Peripheral proteins are bound to the polar heads groups of the lipid bilayer by electrostatic
and hydrogen bonds and/or integral membrane proteins.
 They provide mechanical support for membrane and some serve as membrane bound
enzymes.
o Some membrane proteins are attached to cytoskeleton and limited in lateral movement.
o Peripheral proteins can be released from the membrane by relatively mild treatment such as by
salt solutions of different ionic strengths, or through alteration of the surrounding pH.
o Integral proteins can be removed from membrane lipid components by using detergents or
organic solvents.
Carbohydrates component of membrane
o Carbohydrate never exists as free form in the membranes but occurs as oligosaccharide units
that form part of membrane glycolipids and glycoproteins.
o Carbohydrates are generally located towards the exterior of plasma membrane. They play an
important role in cell-cell recognition, adhesion and receptor action.
Properties of membrane
 Not rigid, highly mobile, dynamic and flexible structure
 Selectively permeable
 Many specific functions according to the protein and
lipid contents
 Membrane fluidity enables the membrane to perform
endocytosis and exocytosis.
 During cell growth and differentiation, cell membrane
can extend and deform without ever losing its
continuity and integrity.
Functions of biological membrane

 Formation of closed compartment around cytoplasm
to define cell boundaries and to protect the cells from
their environment mechanically and chemically
 Maintenance of structural integrity and barrier
function of cells and organelles
 Regulated transport of substances (selective permeability) across the membrane via transporters
and ion channels in order to maintain differences in composition between inside and outside of the
cell
 Cell membrane serves as a sensor that enables the cell to receive information about changes in the
environment as well as mediates the transfer of information between outside and inside of the cell
(cellular recognition and signal transduction).
 Interaction between the cells as well as between the cell and extracellular matrix are mediated by
the cell membrane.
TRANSPORT OF SUBSTANCES ACROSS THE MEMBRANE

P rotei n S tructure & F uncti on (Hem ogl obi n) P age |1
Protein Structure and Function (Hemoglobin)
Hemoglobin
Structure of hemoglobin
Hemoglobin is red-colored conjugated metallo-
globular protein present in RBC. Hemoglobins are
tetramers composed of four globin polypeptide chains
of two different types, each conjugated with a heme
prosthetic group.
Globin chain
There are two types of polypeptide chains
existing in pairs. The adult HbA contains two α chains
and two β chains. The α chain consists of 141 amino acids and the β chain consists of 146 amino acids. The
secondary structure of all globin polypeptides consists largely of α-helical segments. The globin chain is
folded into 7 helical segments in α-chain and 8 helical segments in β-chain joined by non-helical regions
to form a globular (tertiary) structure. Helical segments are designated as A through H starting from
amino terminal. Four polypeptide chains (two α chains and two β chains)
are held together by non-covalent interactions.
Heme
The heme group resides in a pocket between E and F helices. Heme
is a ferro-porphyrin molecule.
Porphyrin is a cyclic tetrapyrrole (protoporphyrin) consists of four
pyrrole molecules linked in a planer ring by four methenyl bridges.
Porphyrin ring contains 4 methyl (M), 2 vinyl (V) and 2 propionate (P) side
chains in the order of M,V,M,V,M,P,P,M.
The iron atom is in the ferrous (Fe2+) state in functional
hemoglobin. The ferrous atom in the heme can form five or six co-ordinate
bonds, depending on whether or not O2 is bound to the protein. Four of
them are linked to the pyrrole nitrogens and the fifth bond is linked to the
imidazole nitrogen of proximal His F 8 in the globin chain. The sixth bond is
formed by oxygen when O2 is present between the ferrous atom and
distal His E7 of the globin chain.
Normal blood hemoglobin level
Adult male = 13.0 – 18.0 g/dL
Adult female = 12.0 – 16.0 g/dL
Different forms of hemoglobin
Type of hemoglobin Chain designations Developmental stage

Glycated Hb (HbA1c)
Hb A1 α2 β2 Major Hb in adult (97%)
 Stable conjugate of glucose with
globin portion of Hb
Hb A2 α2 δ2 Adult (2-3%)
 Non-enzymatic attachment of
Major Hb in fetus glucose, glucose 6-phosphate or
Hb F α 2 γ2
(<1% in adult life) other sugars with α amino group
of the β globin chains of Hb
Hb Gower-I ζ2 ε2 Embryonic Hb
 Normal level < 7%
 Rate of formation is proportional
Hb Gower-II α2 ε2 Embryonic Hb
to blood glucose level
Hb Portland ζ2 γ2 Embryonic Hb  Level reflects blood glucose level
over a period of several weeks
Hb A1c (Glycated Hb) α2 β2- conjugated with glucose (< 7%)  Routinely used for monitoring
of glycemic control in diabetic
Nitrosylated Hb NO attached to Cys93 of β chain
patients
Abnormal hemoglobin or hemoglobinopathy
 Hemoglobin variants
 Thalassaemia
Hemoglobin variants
 Abnormalities in the primary amino acid sequence of globin chains
 Caused by mutations in genes coding α or β chains of hemoglobin e.g., HbS, HbC, HbE.
HbS
 Common in Africa
 Mutation in β globin chain leads to replacement of 6th glutamate with valine.
 Non-polar valine generated a hydrophobic sticky patch on

the surface of β subunits of both oxy and deoxyHbS.
 At low PO2, deoxyHb can polymerize to form long
insoluble fiber that stiffen and distort RBC into
characteristic sickle or crescent shape.
o Shortens life-span of RBCs
o Unable to pass though the capillaries
o Impaired oxygen delivery to peripheral tissues
o Can frequently block the capillaries causing anoxia,
ischemic pain and in severe condition can cause tissue infarct.
 In heterozygous state, HbS gene product provides some protection against malaria parasite.
 Malaria infected RBCs require large amount of oxygen so that they tend to become sickle shape
more rapidly and thus removed from the circulation.
HbE
 Common in South–East Asia region
 Mutation in β globin chain leads to replacement of 26th glutamate with lysine.
 Mild anemia even in homozygous HbE disease
 Usually no treatment required
 Asymptomatic in HbE trait (Heterozygous)
Thalassaemia
Group of hemolytic disorders characterized by decreased synthesis of globin chains
Reduced or absent production of either α or β globin gene
Caused by deletion or nonfunctional globin gene.
2 major groups – α thalassaemia and β thalassaemia
Mostly occurs in Mediterranean regions and also found in Africa, India and Far East
Heterozygous thalassaemia  anemia

Homozygous α thalassaemia  incompatible with life, results in intrauterine death
Hb Barts (4γ chains)
o Both α and β globin gene are missing
o Severe anemia and death in utero (hydrops fetalis)
Role of hemoglobin in oxygen transport

Oxygen carried in the blood in 2 forms –
1. Dissolved form < 2% (0.29 mL/dL)
2. Oxygenated hemoglobin > 98% (19.5 mL/dL)
 Total oxygen capacity of blood is 20 mL/dL.
 Amount of O2 in the blood is determined by amount of dissolved O2 and the amount that carried by
hemoglobin. Oxygen carrier is needed in the blood for transport of O2 in the body due to poor
solubility of O2 in blood plasma.
 Po2 of alveolar air is 100 mmHg and that in venous blood in pulmonary artery is 40 mmHg. Along this
pressure gradient, oxygen diffuses into the blood, dissolves in plasma, enters the red cells and
combines with hemoglobin. Diffusion capacity of O2 across the alveolar membrane at rest is 20-30
mL/min/mmHg. Partial pressure of oxygen therefore rises to 97 mmHg which falls to 95 mmHg in the
aorta because of the physiologic shunt. This is because 2% of the blood in the systemic arteries
bypassed the pulmonary capillaries.
 Deoxyhemoglobin and oxyhemoglobin
o Hemoglobin has two different forms – taut or T form and relax or R form. They differ in oxygen
affinity and strength of bonds linking the subunits.
o DeoxyHb is more
compact (a tauter or
more constrained
molecule) than oxyHb
due to the presence of 8
additional salt bridges.
o In oxyHb, the carboxy
terminal residues of all
four chains have almost complete freedom of rotation. DeoxyHb is termed T (tense or taut) form
and oxyHb is termed R (relaxed) form.
o Conformation switches between T form and R form is facilitated by binding and release of
oxygen from Hb.
 Hemoglobin undergoes a major conformational change on binding with O 2.

o Binding of O2 to ferrous atom of heme forms
6th ligand bond that pulls the ferrous atom
and proximal His F8 into the porphyrin plane
(towards distal histidine). Ferrous atom pulls
the proximal His with it when it moves into
the prophyrin plane. This motion transmits to
F helix of globin chain which subsequently
leads to conformation changes in the whole globin subunit. This motion and such conformational
changes are in turn transmitted to the subunits interfaces. Thus conformation changes in one
subunit transmit to other subunits of the same hemoglobin molecule causing the rupture of ionic
bonds (salt bridges) between 4 subunits.
o Structural changes occur at 2 of 4 contact regions, i.e., α 1β2 contact and α2β1 contact, so each pair
rotates relative to other pair by 15˚. Cleavage of interchain salt linkages switches hemoglobin
from T form to R form. Thus the structural change within one subunit is translated into structural
changes at the interfaces between the subunits switching T state to R state.
 When one subunit of hemoglobin binds oxygen, this leads to conformation changes in hemoglobin
subunits resulting in increased affinity of remaining 3 subunits for oxygen within the same
hemoglobin molecule (positive cooperativity). The reverse is true for the release of oxygen from
hemoglobin (negative cooperativity). This is due to tetrameric structure of hemoglobin and such
property is known as cooperative or allosteric property.
 Because of the cooperativity in oxygen association, dissociation and allosteric properties of
hemoglobin, the oxygen saturation curve for hemoglobin is sigmoid in shape.
 If the blood is 100 % saturated with oxygen, 1 g of hemoglobin carry 1.34mL of O 2. Since arterial blood
is 97 % saturated, 15 g of Hb/ 100 mL of arterial blood carry 19.5 mL of O2.
 At the tissue, arterial blood has PO2 of 95 mmHg whereas the PO2 in tissue is 40 mmHg. Therefore,
oxygen leaves the blood to diffuse into the tissues. Reaching the tissue, salt bridges between
hemoglobin subunits are formed on release of O 2 from one subunit of hemoglobin. This causes
switching of Hb from R to T form leading to serial dissociation of O 2 molecules from hemoglobin.
 At rest, tissues extract 4.6 mL of O2 from each 100 mL of blood and about 250 mL of O 2 are
transmitted to tissues every minute.
Oxygen hemoglobin dissociation curve

It is the curve relating the percent saturation Hb with O2 or O2 carrying power of Hb to partial pressure of
O2. Because of the cooperative or allosteric property of Hb in O2 association and dissociation, the curve is
sigmoid shape.
At normal PO2 in arterial blood, Hb is nearly

100 % saturated with O2. The saturation will
not increase even though the PO2 is more
than 100 mmHg.
Between PO2 100 to 70 mmHg, very little
change in percent saturation of Hb with O 2.
This shows the ability of hemoglobin to load
with O2 despite a large decrease in PO2 level.
This gives the upper flat part of the curve.
When PO2 level falls below 60 to 50 mmHg, a
significant reduction in percent saturation
of Hb with O2 occurs. This steep middle and
lower part of the curve shows at PO2 level of
tissues, tissues can withdraw large amount of O2 from Hb for a relatively small decrease in PO2.
P50 is the partial pressure of O2 at which 50 % of hemoglobin P50 values of different Hb
is saturated with oxygen.  P50 of HbA = 26 mmHg
P50 value is inversely related with affinity of Hb for O2.  P50 of HbF = 20 mmHg
 P50 of myoglobin = 1 mmHg
The higher the P50, the lower the affinity of Hb for O2.
Factors affecting oxygen dissociation or oxygen-hemoglobin dissociation curve

pH or Bohr effect
Temperature
Electrolytes
Effect of CO2
Effect of 2,3 bisphospho-
glycerate (2,3 BPG)
Effect of CO
Effect of pH on oxygen dissociation of hemoglobin (Bohr Effect)

Fall in pH lowers the affinity of hemoglobin for O 2 and shifts the
oxygen-Hb dissociation curve to the right. A rise in pH shifts the
curve to the left. Decrease in O2 affinity of hemoglobin when
blood pH falls is called Bohr Effect.
DeoxyHb (T form) has high affinity for H+ than oxyHb (R form).
OxyHb (R form) is more acidic and H+ dissociate. Increase in H+
concentration causes O2 release and increase in PO2 level causes
H+ release.
Effect of pH on affinity of Hb for O2 depends on degree of
protonation of several groups involved in ion pairs
between subunits. Imidazolium ion of C-terminal His 146 of
each β chain forms ion pair with carboxylate group of Asp
94 in deoxyHb. Lowering pH favors formation of ion pairs
and deoxyHb conformation (T form) is stabilized.
Metabolism in peripheral tissues produces CO2 which then
enters the blood and reaches to RBCs. In RBCs, it reacts
with water and forms H2CO3 by the action of carbonic
anhydrase. H2CO3 then dissociates into HCO3- and H+. This
leads to increase H+ concentration in RBCs. Generation of
H+ lowers pH and favors the protonation which increases
the formation of ionic pairs between the subunits of Hb.
This favors T form of Hb. O2 affinity of Hb thus becomes reduced
and O2 release is increased to meet the tissues demand.
Effect of CO2
Most of CO2 released from tissues metabolism is transported as bicarbonate form which is formed
within RBCs by the action of carbonic anhydrase.
CO2 + H2O  H2CO3  HCO3- + H+
H+ generated by this reaction is taken up by deoxyHb as part of Bohr Effect.
Some of CO2 is transported as carbamate bound to the unionized α amino group of Hb (carbamino-
hemoglobin).
Hb-NH2 + CO2  Hb-NH-COO- + H+
Carbamate forms salt bridges that stabilize T form. Hence, binding of carbon dioxide to the terminal
amino groups of Hb lowers its oxygen affinity.
Effect of temperature
A rise in temperature decreases hemoglobin saturation and
shifts the curve to the right.
Fall in temperature shifts the curve to the left.
Temperature effect is more significant in severe muscular
exercise during which rise in temperature, fall in pH and
increased PCO2 level favor oxygen liberation from hemoglobin.
Electrolytes
At low O2 tension, hemoglobin gives up O2 more readily in the presence of electrolytes. Cl - binds
more tightly to deoxyHb favouring T form.
Effect of 2, 3 bisphosphoglycerate (2, 3 BPG)

2, 3 BPG is a negative allosteric effector and stabilizes the
T form of hemoglobin, thereby reducing oxygen affinity
of Hb.
It is synthesized from the glycolytic intermediate 1, 3- BPG.
It is highly negative charged molecule and binds Hb at
central cavity surrounded with highly positive charged
amino residues (α amino group of Val, Lys 82, His 143 of β
chain). 2, 3 BPG stabilizes deoxyHb by cross-linking β
chains, so lowers the affinity for O2 and shifts the O2
dissociation curve to the right.
In the absence of 2, 3 BPG, HbA is completely saturated
with oxygen and could not release its O2 to the tissues
even at PO2 less than 20 mmHg.
Positively charged His 143 of β chain of HbA is
substituted by uncharged serine in γ chain of HbF. Thus,
2, 3 BPG binding site is changed and interaction of HbF and 2,3 BPG is weaker (P50 = 20 mmHg).
Oxygen affinity of HbF is higher than adult Hb and oxygen-Hb dissociation curve of HbF is on the
left to that of HbA.
Factors affecting 2, 3 BPG concentration

pH – acidosis inhibits glycolysis in RBC, so that 2, 3 BPG level falls when pH is low.
Hormones – thyroid hormone, growth hormone and androgens increase 2, 3 BPG concentration.
Exercise – increases 2, 3 BPG within 60 minutes, but not in athletes.
Hypoxia – 2, 3 BPG is increased in anemia and in a variety of diseases in which there is chronic
hypoxia. This facilitates the delivery of O2 to the tissues by raising the PO2 at which O2 is released in
peripheral capillaries.
Storage – In stored blood, 2, 3 BPG level falls and ability of blood to release O2 to the tissues is
consequently reduced. This decrease is less if the blood is stored in citrate-phosphate-dextrose
solution rather than the usual acid-citrate-dextrose solution.
Effect of carbon monoxide

Carbon monoxide binds tightly but reversibly to ferrous atom in
heme prosthetic group of Hb and forms carbon-monoxyHb or
carboxyHb (HbCO).
Affinity of Hb for CO is 220 times greater than O2. Isolated heme
binds carbon monoxide (CO) 25,000 times more strongly than O2.
Binding of CO to one or more subunits of Hb causes binding of
O2 to the remaining sites with high affinity. Hb becomes unable to release O2 to the tissues.
Carbon monoxide causes O2-Hb dissociation curve shifts to left and also changes to hyperbola shape.
Normally HbCO percentage in blood is about 3% of total Hb but it may exceed 10% in heavy smokers.
CO toxicity, which occurs when the concentration of carboxyhemoglobin rises above about 25%,
causes neurological impairment, usually dizziness and confusion.
Treatment of choice for severe or complicated CO
poisoning is hyperbaric O2 therapy which facilitates
dissociation of CO from Hb.
Fetal hemoglobin (HbF)

Consists of 2 α chains and 2 γ chains.
Major fetal Hb, found less than 1 % of total Hb in adults.
HbF has uncharged serine in position 143 instead of
positively charged histidine in β chain of HbA.
Cationic groups that participate in 2, 3 BPG binding is not
available in HbF so that HbF binds 2, 3 BPG less tightly; thus
binds O2 more tightly than HbA.
O2 binding affinity of HbF is higher than adult HbA and
oxygen-Hb dissociation curve is shifted to the left in
relative to that of HbA.
P r o t e i n S t r u c t u r e & F u n c t i o n ( H e m o g l o b i n ) P a g e | 10
It is adapted to the environment of the fetus in order to get O2 form the maternal blood. In order to
compete with maternal Hb for O2, HbF must bind O2 more tightly.
Pulse Oximetry
Methemoglobin
 Non-invasive method of estimating the
A form of Hb in which ferrous iron (Fe2+) in Hb is oxygen saturation of arterial Hb
3+
oxidized to ferric iron (Fe ).  Based on the principle of different visible
May be due to hereditary defect of globin chain and infrared spectral characteristics of
oxy- and deoxy-Hb and identification of
(HbM) or exposure to oxidizing drugs or
the pulsatile blood component of the signal
chemicals.
 Transmission or reflectance measurements
Methemoglobin thus can neither bind nor are made in a translucent tissue site
transport O2. with reasonable blood flow, commonly a
Enzyme methemoglobin reductase reduces the finger, toe or ear of adults and children,
or a foot or hand of infants.
Fe3+ of methemoglobin to Fe2+.
 Typically correlates within 4–6% of the
Presence of MetHb shifts the curve to the left and
value found by arterial blood gas analysis.
impairs the delivery of O2 to the tissues.
 Useful to monitor the cardio pulmonary
status during local and general anesthesia,
Sulfhemoglobin
in intensive care and neonatal units, and
Formed when a sulfur atom is incorporated into during patient transport
porphyrin ring of Hb (due to certain drugs or  Body movement, radiated ambient light,
sulfides) elevated bilirubin, artificial or painted
Presence of sulfHb shifts the curve to the right. fingernails can interfere with pulse
oximetry.
Myoglobin  Cannot discriminate among oxy-, carboxy-
Myoglobin is present in red muscle tissue for and metHb.
storage of oxygen which can be released upon deprivation of oxygen in severe exercise. It consists
of single polypeptide chain (consists of 153 amino acids) which is extremely compact.
Heme is in the crevice of Mb formed by E and F helices of globin chains. Iron atom of heme is bonded
to His F8 in deoxygenated Mb and sits out of the porphyrin plane by 0.3 Å. When O2 occupies the 6th
coordinate bond position, the iron moves to within 0.01 nm (0.1 Å) of the plane of the heme ring. It
can bind one oxygen molecule.
Oxygen dissociation curve for Mb is hyperbola shape.
Mb does not release a large fraction of oxygen even at 20 mmHg of PO2. When strenuous exercise
lowers the PO2 of muscle tissue to about 5 mmHg, Mb readily releases its bound oxygen. Oxygen
saturation curve of Mb is on the left side of that of Hb, so it can take up O2 from Hb.
It cannot serve as an effective vehicle for delivery of oxygen from lungs to peripheral tissues.
Enzymology Page |1
ENZYMOLOGY
Enzymes
Enzymes are biological catalysts with high degree of specificity
for a certain substrate or class of substrates. They increase the rate of
chemical reactions by lowering the activation energy of the reactions.
Chemical nature of enzymes
Almost all enzymes are proteins except catalytic RNAs, known

as ribozymes. The catalytic activity of an enzyme depends on integrity
of the protein structure.
Distribution of enzymes
Enzymes are found where the reactions they catalyzed are

located i.e., compartmentalization.
 Cellular enzymes
o Cytosolic enzymes
o Enzymes in cellular organelles (mitochondrial enzymes,
microsomal enzymes, lysosomal enzymes, etc.,)
o Membrane bound enzymes
 Extracellular enzymes
o Plasma enzymes
o Digestive enzymes
Nomenclature of enzymes
Most commonly used enzyme names have the suffix “-ase” attached to the substrate of the
reaction (for example, glucosidase and urease) or to a description of the action performed (for example,
lactate dehydrogenase and adenylyl cyclase). A numbering system, (Enzyme Commission or E.C number)
consisting of four numbers for each enzyme, has been developed by the International Union of
Biochemistry and Molecular Biology (IUBMB) to characterize each enzyme. The first number defines the
type of reaction that is catalyzed, followed by numbers to define details of the reaction. Example –
alcohol dehydrogenase has the IUBMB number 1.1.1.1. First digit indicates that the enzyme is involved in
an oxidation-reduction reaction. Second digit represents that the enzyme removes hydrogen by using
NAD+ as the electron acceptor whereas the third digit denotes for the substrates that the enzyme can be
catalyzed (most primary alcohols). The last digit is reserved to differentiate each enzyme that catalyzes
the same overall reaction but with different substrates.
Enzymology Page |2
Classification of enzymes
All enzymes are classified as belonging to one of six
classes, defined by the chemical reaction they catalyze.
Class 1 – Oxidoreductases
 Oxidoreductases catalyze oxidation-reduction reactions
such as electron transfer, hydrogen transfer and
reactions involving molecular oxygen. e.g.,
dehydrogenase, oxidase.
Class 2 – Transferases
 Transferases catalyze the transfer of groups (other than
electrons, hydrogen) from one substrate to another. e.g.,
kinases catalyze transfer of phosphate group from ATP to
a second substrate.
Class 3 – Hydrolases
 Hydrolases catalyze the hydrolytic cleavage of complex
molecules into individual components. e.g., digestive
enzymes and lysosomal enzymes.
Class 4 – Lyases (synthases)

 Lyases consist of a diverse group of enzymes that
catalyzes the cleavage of C–C, C–O, and C–N bonds by
means other than hydrolysis or oxidation, giving rise to
compound with double bonds or catalyze the reverse reaction, by the Velocity of a reaction:
addition of group to a double bond. In cases where reverse reaction is It is defined as the rate of
conversion of substrate to
important, then synthase, (not synthetase of group EC-6) is used in the product per unit time.
name. e.g., carbonic anhydrase, dehydratases, decarboxylases.

International Unit (IU):
Amount of enzyme that
Class 5 – Isomerases catalyzes conversion of one
 Isomerases catalyze intra-molecular structural rearrangement within a micromole of substrate to
product per unit time
molecule, not affecting the atomic composition of the molecule. They (1 IU=1μmol/min)
are called epimerases, isomerases or mutases, depending on the type of isomerism involved. e.g.,
triose phosphate isomerase, phosphoglucomutase
Class 6 – Ligases (synthetases)

 Ligases catalyze the joining of two molecules coupled with the hydrolysis of ATP. These enzymes
are often called synthetases. e.g., DNA ligase, glutamine synthetase.
Enzymology Page |3
How Enzymes Work

Virtually all chemical reactions have an energy barrier,
separating the reactants and the products. This barrier, called the free
energy of activation or activation energy, is the energy difference
between the energy of the reactant and high energy intermediates
that occurs during the formation of a product. The peak of free
energy activation represents the transition state, in which the high
energy intermediates of the reactant (substrate) are formed during
the conversion of a reactant to a product.
An enzyme lowers the energy required for activation to the
transition state. Without a catalyst, the reaction will occur only if
enough heat energy is added to the reaction system. With an enzyme
as a catalyst, the reaction may easily proceed at the normal
physiological temperature.
Enzymes lower the energy of activation for a reaction; they do
not affect the free energy change (ΔG) or the equilibrium constant for the reaction, Keq.
Active site of the enzyme molecule

An enzyme must bind to the substrates (reactants) of the reaction in order to convert them into
the products. The substrates are bound to specific substrate binding sites on the enzyme through
interactions with the amino acid residues on the enzyme. Enzyme molecules contain a special pocket or
cleft called the active site. The active site, formed by folding of the protein, contains amino acid side
chains (or cofactors or coenzymes groups) that participate in substrate binding and catalysis.
The substrate binds the enzyme, forming an enzyme–substrate (ES) complex. The functional
groups in the active site of the enzyme activate the substrate and decrease the energy needed to form
the high energy intermediates stage of the reaction known as the transition-state complex. Enzymes are
highly specific, interacting with one or a few substrates and catalyzing only one type of chemical
reaction. It is due to the spatial geometry of the active site of the enzyme molecule in which amino acid
residues and other functional groups (such as metal ions or coenzymes) are arranged complementarily to
interact with specific substrate and to catalyze specific type of reaction.
Substrate specificity is
determined by the size,
Enzyme specificity structure, charges, polarity and
Enzymes are highly specific to the type of reaction catalyzed and hydrophobicity of the substrate
the nature of the substrates. Reaction specificity and substrate binding site.
specificity are determined by the structure of the active site of the enzyme.
Enzymology Page |4
Substrate specificity
1) Absolute substrate specificity
 Certain enzymes will act on only one substrate and catalyze one reaction, e.g. glucokinase,
lactase, urease, catalase, etc.
2) Optical specificity or stereo-specificity
 Enzymes are stereospecific catalysts. Most enzymes act only on specific optical isomer form of a
substrate, e.g., mammalian enzymes of carbohydrate metabolism catalyze only on D-isomers of
sugars and those of amino acid metabolism act only on L-isomers of amino acids
3) Group specificity
 Lytic enzymes act on specific chemical groups, e.g., proteases on proteins, lipase on lipids,
nucleases on nucleic acids, etc.
 Some enzymes show high group specificity. In group specificity, an enzyme acts on more than
one substrate containing a particular chemical group, e.g., proteolytic enzymes such as trypsin,
chymotrypsin differs in bond specificity on which they hydrolyze. (Chymotrypsin acts on several
proteins by hydrolyzing peptide bonds attached to aromatic amino acids. Trypsin hydrolyzes
peptide linkages involving arginine or lysine.)
 In bond specificity, an enzyme acts on more than one substrate containing a particular kind of
bond, e.g., salivary α-amylase cleaves α-(1→4) glycosidic bonds of carbohydrates, lipase
hydrolyzes ester bonds of lipids.
 Some enzymes show broader substrate specificity, e.g., cytochrome enzymes involved in
xenobiotic metabolism can act on a variety of substrates.
Functional groups in enzyme catalysis
Functional groups on amino acid side chains

 Amino acid side chains of enzymes can participate in catalysis. Almost all of the polar amino acids
participate directly in catalysis in one or more enzymes.
 Serine, cysteine, lysine and histidine can participate in covalent catalysis. Histidine often participates
in acid-base catalysis due to the presence of side chain which can donate and accept a proton at
physiological pH. Most of the polar amino acid side chains are nucleophilic and participate in
nucleophilic catalysis by stabilizing more positively charged groups that develop during the
reactions.
Coenzymes and cofactors

 Some enzymes require an additional non-protein component for its optimum activity. This additional
component is called cofactor which may be either loosely or tightly bound to the enzyme.
Enzymology Page |5
Prosthetic groups:
 Enzymes with covalently or non-covalently bound coenzymes are  Tightly bound, often
covalently linked to an
referred to as holo-enzymes. A holo-enzyme without a coenzyme is
enzyme.
termed an apo-enzyme.
 Remain associated with
 Apoenzyme + cofactor/coenzyme = holoenzyme. the enzyme during the
 Coenzymes are heat stable, low molecular weight, non-protein organic entire catalytic cycle.
 Act as transient carriers of
compound, required for enzyme activity.
specific functional groups.
 Coenzymes are classified according to the group they transfer:
 E.g., pyridoxal phosphate,
1. Coenzymes for the transfer of group other than hydrogen FMN, FAD, Biotin
 Sugar phosphates, coenzyme A (CoA-SH), thiamin pyrophosphate Co-substrate:
(TPP), pyridoxal phosphate (PLP), Biotin, folate coenzyme,  Loosely bound coenzymes
cobalamine, Lipoic acid  Function as second

substrate or co-substrate.
2. Coenzymes for the transfer of hydrogen
 Bind reversibly to the
 Nicotinamide adenine dinucleotide (NAD+), nicotinamide adenine protein moiety of the enzyme
+
dinucleotide phosphate (NADP ), flavin mononucleotide (FMN),  Changed chemically during
flavin adenine dinucleotide (FAD), Lipoic acid, coenzyme Q the reaction.

 Chemically modified co-
Coenzyme A (CoA-SH) substrate dissociates away
 It is synthesized from vitamin pantothenic acid and contains adenine from the enzyme and is free
nucleotide in the active coenzyme structure. to participate in another

enzymatic reaction cycle
 Coenzyme A binds reversibly but tightly to a site on an enzyme. Its
 E.g., NAD+, NADP+
functional group is sulfhydryl group (-SH); thus its structure is
abbreviated as CoA-SH.
 Sulfhydryl group in CoA-SH forms energy rich thioester
bond with acyl units e.g., acetyl CoA, fatty acyl CoA
(from fatty acid).
Biotin
 Biotin is important coenzyme for carboxylation reactions
catalyzed by carboxylase enzymes. It is covalently linked
to lysine residue of carboxylase enzyme.
 It functional group is a nitrogen atom that covalently
binds to CO2 group by using energy from ATP hydrolysis.
This bound CO2 group is the activated form of carboxyl
group for addition to another molecule, e.g., pyruvate
carboxylase.
Enzymology Page |6
Pyridoxal phosphate (PLP)

 It is synthesized from the vitamin pyridoxine (vitamin B6).
The reactive aldehyde group usually functions in enzyme
catalyzed reactions by forming covalent bond with the amino
group from amino acids i.e., transamination reactions.
Nicotinamide adenine dinucleotide (NAD+)

 It is synthesized from niacin or vitamin B3 (which contributes
nicotinamide ring) and from ATP (which contributes
adenosine monophosphate).
 The functional group of NAD+ is reactive nitrogen atom in
nicotinamide ring which accepts hydride ion (a hydrogen
atom with two electrons) transferred from a specific carbon
atom on the substrate, forming NADH, e.g., lactate
dehydrogenase.
Enzymology Page |7
Metal ions
 Some enzymes require inorganic metal ions for their enzymatic
activity. Enzymes that contain a tightly bound metal ion are called
metalloenzymes.
 Metal ions participate in binding of substrates or coenzymes to the
enzyme protein. E.g., the phosphate groups of ATP are usually bound
to enzymes (kinases) through Mg2+ chelation.
 Metal ions having positive charge contribute to the catalysis process
by acting as electrophils. They promote
electrophilic catalysis at the site of bond
cleavage or by stabilizing intermediates in the
reaction pathway. E.g., the active site of
alcohol dehydrogenase contains zinc which
interacts with oxygen in the substrate allowing
the transfer of a hydride ion from alcohol to
NAD+, generating acetaldehyde and NADH.
 Some metal ions such as copper and iron can
accept or donate electrons in oxidation-
reduction reactions as well as in the activation
of molecular oxygen in the reaction process
e.g., heme iron in cytochrome enzymes, copper in dopamine β hydroxylase.
 Metal cofactors also play in structural role of the enzyme protein by stabilizing the active
conformation of the enzyme protein, e.g., zinc in cytosolic Cu-Zn superoxide dismutase, K+ in
pyruvate kinase.
Isoenzymes
Isoenzymes are groups of enzymes that have the same catalytic activity but differ in molecular
structure, physical properties, and reaction kinetics. They may have similar, but not identical amino acid
sequences. Isoenzymes can differ in;
1. tissue origin e.g., muscle and liver 4. physical properties
glycogen phosphorylase 5. chemical properties (i.e., substrate
2. intracellular distribution e.g., affinity, response to allosteric
cytosolic and mitochondrial modifiers, kinetic properties)
isocitrate dehydrogenase 6. metabolic role
3. genetic loci
Enzymology Page |8
Many enzymes have different isoforms e.g., lactate dehydrogenase, creatine kinase, and alkaline
phosphatase.
Creatine kinase
 Dimmer, consisting of both or either of two types of subunit
 Two types of subunits – M (muscle type)
and B (brain type)
 Three types of creatine kinase are –
o CK 1 (BB) – in brain
o CK 2 (MB) – in myocardium
o CK 3 (MM) – in skeletal muscle
Lactate dehydrogenase
 Lactate dehydrogenase is a tetrameric
protein, catalyzing inter-conversion of
pyruvate & lactate. It is composed of four polypeptide chains of two types, H and M.
 Different isoforms are based on the synthesis of H and M units controlled by different genetic loci in
distinct tissues according to their metabolic role. E.g., H type predominates in heart muscles and M
type predominates in liver and skeletal muscles.
 Lactate dehydrogenase isoforms –
o LD1 (H4)  heart muscles
o LD2 (H3M)  heart muscles and RBC
o LD3 (H2M2)  brain and kidney
o LD4 (HM3)  skeletal muscles
o LD5 (M4)  liver and skeletal muscles
 Five isoenzymes of LDH can be detected by electrophoresis as they have different electrophoretic
mobilities. Numbering of isoenzymes are based on the order of migration in the electrophoresis;
beginning with fastest migration type.
Biomedical significance of isoenzymes

 Isoenzymes can be separated and identified
by electrophoresis since they contain
different numbers of charged amino acids.
 Pattern of isoenzymes found in plasma can be used as a means of identifying site of tissue damage
and can provide diagnostic and prognostic information, e.g., determination and identification of
serum CK and LDH isoforms in the diagnosis of myocardial infarction.
Enzymology Page |9
 Increased CK 2 (MB) isoenzyme level is diagnostic of MI since it

appears 4-8 hours of onset of chest pain and peaks within 1 day.
 Increased plasma LDH level is indicative of MI (specifically increased
LDH1 and LDH2 isoforms pattern). But it has less diagnostic value than
the determination of serum CK 2 level in diagnosis of MI since serum
LDH level rises slowly and reaches peak at 36 – 48 hours of onset of
chest pain.
 Other clinical applications of serum LDH and CK level
 Elevation of LDH 5 occurs after damage to the liver or skeletal muscle.
 Elevated levels of CK 3 in serum occur in all types of dystrophies and myopathies.
Mechanism of enzyme action

Enzyme reduces the activation energy of a
reaction which is a potential energy barrier that
must be overcome for the reaction to proceed.
Mechanism of enzyme action in general can be
explained by the enzyme-substrate complex
theory (Michaelis-Menten Theory). According to
this theory, the reaction between enzyme and its
substrate occurs in two stages.
Stage 1 – binding of substrate to the enzyme specifically at active site or catalytic site and formation of
enzyme-substrate complex (ES)
Stage 2 – decomposition of ES complex into product and free enzyme
Mechanism of substrate binding

 Models of catalytic site
 Lock and key model  Induced-fit model
Enzymology P a g e | 10
Active site of the enzyme:
 Kinetics behavior of multi-substrate reaction  Region of an enzyme
concerned with substrate
 Ordered mechanism
binding and catalysis
 Random mechanism  Theoretically, it can be
 Ping-Pong mechanism subdivided into substrate

binding site and the catalytic
Models of catalytic site site which is determined
chemically by the amino acid
Lock and key model residues in the catalytic
Proposed by Emil Fischer in 1894. center of the enzyme.
Active site of enzyme is structurally

complementary to their substrates. Active site or
substrate binding site is pre-shaped and rigid.
It can explain high degree of specificity or
absolute specificity of enzymes. It can also
distinguish between L and D isomers of a
substrate molecule (stereospecificity).
Induced-fit model
Proposed by Daniel E.Koshland in 1958.
Active site is not rigid.
Shape of active site of enzyme is modified by the binding of
the substrate. Approach of substrate induces
conformational change in active site of the enzyme aligning
the groups correctly for substrate binding and catalysis.
Such conformational change is induced by weak interactions
between enzyme and the substrate.
Substrates analogs may cause the correct conformational change but if the attached substrate
analog is too bulky or too slim, it induces incorrect alignment of groups required for catalysis.
Kinetics behavior of multisubstrate reaction

Most enzymes catalyzed reactions involve two or more substrates.
Sequential or single displacement reaction

All substrates must be added to the enzyme before any
products can be released. Addition of substrates and
release of products from the enzyme could be in
compulsory order or random mechanism.
Double displacement reaction (Ping-Pong mechanism)

One or more products are released from the
enzyme before all the substrates have reacted with the enzyme.
Ordered mechanism
Binding of substrates to
the active site and release of products form the enzyme are in specific order. e.g., alcohol
dehydrogenase, lactate dehydrogenase
Random mechanism
Substrate binding and release of
products from the enzyme are in
random order. e.g., glycogen phosphorylase, creatine kinase
Ping-Pong mechanism
One or more
products are released
from the enzyme
before all the substrates have been added. The group undergoing transfer is displaced from the
substrate to the enzyme, converting first substrate into product and enzyme becomes modified in
the process. Subsequent group transfer from the modified enzyme to the second substrate forming
the second product and regenerate the original enzyme, e.g., aminotransferases or transaminases.
Mechanism of catalysis
1) Catalysis by proximity 3) Covalent catalysis
2) Catalysis by strain 4) Acid base catalysis
Catalysis by proximity (entropy effect)

 For the molecules to react they must come within bond-forming distance of one another. This is
achieved when the substrates are bound on the enzyme surface. Thus the enzymes increase the rate
of reaction by decreasing entropy (randomness or disorder) effect.
 When correctly positioned and bound on the enzyme surface, the
substrates may be strained towards the transition state.
Catalysis by strain
 Enzymes that catalyze the lytic reactions strains or weakens the targeted
bond of the substrate; making it more vulnerable to cleavage.
Covalent catalysis
 Covalent catalysis involves formation of covalent bond
between the amino acid residues in active site of enzyme (or
sometimes with enzyme bound coenzyme) and one or more
substrates.
 It is particularly common among enzymes that catalyze group
transfer reactions, e.g., the amino group acid substrate forms
Schiff base with enzyme bound coenzyme pyridoxal phosphate
in transaminases.
 Frequently involved amino acid residues in covalent catalysis are
cysteine or serine, and occasionally histidine. Serine proteases
such as trypsin, chymotrypsin and thrombin employ covalent catalysis.
Acid base catalysis

 Ionizable functional groups of amino acid residues
or of prosthetic groups in active site contribute to
catalysis by acting as acids or bases.
 Specific acid-base catalysis
 H3O+ and OH- serve as specific acids and bases
 They are found in some metal dependent
enzymes, e.g., carbonic anhydrase
 General acid base catalysis
 Weakly ionizable groups at the active site the
enzyme serve as general acids or general
bases.
 Most important and commonly seen residue in
histidine, e.g., active site of ribonclease
 Enzymes employ more than one catalytic
mechanisms to facilitate catalysis.
Kinetic or collision theory:
Enzyme kinetics In order to react, two molecules

 Must approach within bond-forming
It is the study concerned with quantitative measurement
distance of one another or collide
of rates of enzyme catalyzed reactions and the systematic study  Must possess sufficient kinetic
of factors that affects these rates. energy to overcome the energy
barrier for reaching the transition
Factors affecting the rate of reaction
state
1. Substrate concentration
2. Enzyme concentration Michaelis-Menten Equation
3. Coenzyme concentration
4. Temperature
5. pH
Lineweaver-Burk plot:
6. Modifiers
 Several linear transformations
of Michealis-Menten equation
Effect of substrate concentration on enzyme catalyzed reaction
have been developed.
 When all other conditions are kept constant, at low substrate  The most common graphical
concentration, the rate of enzyme catalyzed reaction is directly presentation is Lineweaver-
proportionate to the substrate concentration (First order kinetics). Burk plot or double
reciprocal plot.
Increased substrate concentration increases collision frequency of
 Obtained by taking the
reacting molecules which then possess sufficient energy to reach
reciprocal of the steady
the transition state. state Michaelis-Menten
 Initial velocity increases as the substrate concentration increase equation.
 Yields straight line enzyme
until Vmax is reached. Beyond Vmax, reaction velocity is not affected
kinetics curve.
by further increase in substrate concentration. So at high substrate
 Practically useful to determine
concentration, rate of reaction is independent of the substrate Vmax and Km value of a
concentration (Zero order kinetics). Beyond Vmax, almost all the particular enzyme from
enzymes are saturated with substrate and no free enzyme remains available data in laboratory
settings
available for forming enzyme-substrate complex.
 When change in velocity is plotted against various substrate
Michaelis constant or Km:
concentrations, a hyperbola curve is obtained. [S] to reach half Vmax
 The concentration of substrate required to achieve one half of  Indicate amount of substrate
Vmax is called Michaelis constant or Km. to be used in the reaction.

 Indicate substrate affinity of
the enzyme.
 Serve as a measure of the
dissociation constant (Kd).
 Measure of strength of E-S
complex
 Km is inversely related with
substrate affinity of the
enzyme.
 The catalytic constant, kcat, also known as the turnover

number, is a rate constant which describes how quickly
an enzyme can catalyze a reaction. The kcat is defined
as the number of substrate molecules converted to
product per enzyme molecule per unit time.
Effect of enzyme concentration on rate of reaction

 When temperature, pH and substrate concentration are kept constant, initial velocity of a reaction is
directly proportional to the enzyme concentration provided that the molar concentration of enzyme
is very much less than that of the substrate. Straight line curve is obtained when initial velocity is
plotted against various enzyme concentrations.
Effect of coenzyme concentration on rate of reaction

 Since coenzyme is regarded as second substrate, increased
coenzyme concentration will increase the rate of reaction.
Effect of temperature on enzyme catalyzed reaction

 Chemical reactions are accelerated by increased temperature. The greater the activation energy of
the reaction, the greater is its temperature dependence.
 Within certain limit, raising the temperature increases the rate of reactions by increasing the
collision frequency and kinetic energy of the reacting molecules.
 The factor by which reaction rate changes by 10°C change in temperature is called temperature
coefficient or Q10. For most enzymes, the velocity doubles with 10°C rise in temperature, thus Q 10 is
approximately two.
 The temperature at which the velocity is maximal is called optimal temperature of that enzyme.
Optimal temperature of an enzyme is the temperature of the surrounding where an enzyme exists.
For the enzymes of human body, it is 37°C.
 At very high temperature, velocity of reaction sharply declines due to denaturation of enzyme
accompanied by loss of catalytic activity. When change in velocity is plotted against different
temperatures, skew shaped curve is obtained.
 Temperature dependence nature of reaction contributes to increased metabolic rate during fever.
Human cannot tolerate body temperature higher than 42°C to 43°C.
Effect of pH on enzyme catalyzed reaction

 The rate of almost all enzyme-catalyzed reactions depends
on hydrogen ion concentration.
 Changes in pH can affect on the ionic charge of amino acid
side chains of enzymes or charged state of substrates or
both. Binding between enzyme and substrate via formation
of salt bridges (ionic bonds) can be affected by pH changes.
 For the enzyme, pH can affect the enzyme catalytic activity
by changing the conformation or by changing the charge on
a residue functional in substrate binding or catalysis. For
enzymes whose mechanism involves acid-base catalysis, the
residues involved must be in the appropriate state of
protonation for the reaction to proceed. Extreme pH can
cause denaturation of enzyme.
 Enzyme activity is maximal at their optimal pH. At the optimal pH, enzyme functions best because
the charge state of functional residues on both the enzyme and the substrate are in the required
state of ionization. Below or above this pH, enzyme activity is decreased. Optimal pH of most
intracellular enzymes ranges between 5 and 9. Optimal pH for pepsin is 1-2. Lysosomal enzymes
generally have pH optima in acidic range since lysosomes are mildly acidic with pH values between
4.5 and 5.5.
 When change in velocity is plotted against different pH values, bell-shaped curve is obtained.
Effect of modifiers on enzyme catalyzed reaction

 Modifiers are molecules that bind to the enzyme and alter the activity of
enzyme. Negative modifiers or inhibitors decrease the activity of enzyme
and positive modifiers or activators increase the activity of enzymes.
Enzyme inhibitors
 Any substance that can diminish the velocity of an enzyme catalyzed
reaction is called enzyme inhibitor.
 Many drugs (naturally occurring or synthetic), act as enzyme inhibitors.
Most enzyme inhibitors act reversibly, but there are also irreversible
inhibitors that permanently modify the enzyme.
 Based on the Lineweaver-Burk plots, it is possible to distinguish between
three forms of inhibition — competitive, non-competitive and
uncompetitive inhibitors.
Competitive inhibitor
 Resembles in chemical structure to substrate.
 Binds to active site of the enzyme, competes with substrate for the active site.
 Inhibits substrate access to the active site of the enzyme.
 Apparent increase in Km but no change in Vmax
 Inhibition can be overcome by increasing substrate concentration.
Substrate
Competitive inhibitor Target enzyme Therapeutic use
resemblance
Malonate Succinate dehydrogenase Succinate
Sulphonamide Folate synthase PABA analog Antibiotics
Hypoxanthine
Allopurinol Xanthine oxidase Treatment of gout
analog
Statin (Lovastatin) HMG CoA reductase HMG CoA analog Cholesterol lowering agent
Ephedrine
Monoamine oxidase Monoamine analog Antidepressant drug
Amphetamine
Methotrexate Dihydrofolate reductase Folate Analog Anti-cancer drug
Angiotensin converting Angiotensin I

ACEi Antihypertensive drug
enzyme analog
Antidote for methanol

Ethanol Alcohol dehydrogenase
poisoning
Non-competitive inhibitor
 Does not resemble in chemical structure to substrate.
 Binds to the site distinct from substrate binding site of enzyme-substrate
complex or free enzyme; thus there is no competition between
inhibitor and substrate.
 Does not affect substrate binding affinity but decrease the efficiency
of transforming substrate to product by inhibiting enzyme catalysis.
 Reduces Vmax but does not affect Km.
 Inhibition cannot be overcome by increasing substrate concentration.
 Irreversible non-competitive inhibitors are enzyme poisons.
Inhibitory
Enzyme poison Target enzyme
mechanism
Heavy metals Enzymes having SH Binding to SH group

(Pb, Hg, As) groups in active site of enzyme
Cytochrome c oxidase in Inhibit transport of

Cyanide
electron transport chain electrons in ETC
Binding to serine
DIPF
Acetylcholine esterase residue in active site
(nerve gas, insecticides)
by covalent linkage
Alkylating agents Modification of

Some enzymes
(iodoacetamide) cysteine residues Eggs or egg white protein
(ovalbumin)
 Antidote for accidental

Drug Target enzyme Therapeutic use
ingestion of heavy metals
Anti-inflammatory,
Aspirin Cyclooxygenase  Rich in SH groups
anti-platelet
(Acetyl salicylic acid) (Acetylation of serine)  Can trap free metal ions and
aggregation
prevent their absorption
Aldehyde Treatment of from GI tract
Disulfiram (antabuse)
dehydrogenase alcoholism
Mixed inhibition
 Affect on enzyme catalytic

activity as well as influence
substrate binding to enzyme
 Affect both Km & Vmax

Uncompetitive inhibitor
 Binds at a site distinct from the substrate binding site.
 Binds only to enzyme substrate complex and not to free enzyme and
leads to formation of inactive ESI complex
 Decrease in both Km and Vmax.
 Lithium
 Inhibits myo-inositol monophosphatase; thereby
preventing the recycling of inositol and phospho-
inositides formation.
 Is used to treat bipolar disorder (manic depression).
 Some pesticides
 Herbicide glyphosate, also known as Roundup
 Uncompetitive inhibition of enzyme in aromatic amino acids biosynthesis
Suicide inhibitor or Mechanism based inhibition

“Mechanism-based” or “suicide” inhibitors are specialized substrate analogs that mimic or
participate in an intermediate step of the catalytic reaction.
They contain a chemical group that can be transformed by the catalytic machinery of the target
enzyme. Upon binding to the enzyme, enzyme catalysis transforms the chemical group in the
inhibitor into a highly reactive group that forms a covalent bond to a catalytically essential residue
in the active site, thereby inhibiting the enzyme function.
It is called mechanism-based inactivation because they utilize the normal enzyme catalysis
mechanism to inactivate the enzyme. It is also called suicide inhibitor because the enzyme literally
commits suicide.
Kinetic analysis of mechanism-based inhibitor cannot be explained by Lineweaver-Burk plot.
E.g., allopurinol
 A drug used to treat gout (hyperuricemia) by inhibiting xanthine oxidase
 Xanthine oxidase catalyzes oxidation of hypoxanthine to xanthine and xanthine to uric acid
(urate) in purine catabolism pathway. The enzyme active site contains molybdenum-sulfide
(Mo-S) complex for catalysis.
 Xanthine oxidase oxidizes the drug allopurinol to oxypurinol (alloxanthine), a compound
that binds very tightly to a molybdenum–sulfide complex in the active site.
 As a result, the enzyme has committed suicide by converting a drug to a transition-state
analog, thereby unable to carry out its normal function.
Transition state analog

Transition state analogs are molecules that bind tightly to the enzyme by mimicking the transition
state of the substrate or resembling an intermediate stage of the reaction. They are extremely
potent and specific inhibitors of enzymes because they bind more tightly to the enzyme than do
substrates or products.
E.g., HIV protease inhibitors (saquinivir), penicillin
Penicillin
o It is a transition analog that binds tightly to and inhibits glycol-peptidyl transferase involved
in cell wall synthesis in bacteria. Penicillin contains four membered β-lactam ring that mimics
the transition state of the normal substrates.
o When penicillin binds to the active site of the enzyme, its lactam ring opens, forming a
covalent bond with a serine residue at the active site.
o Penicillin irreversibly inhibits bacterial trans-peptidase and thus is sometimes termed suicide
inhibitors. They are used as antibiotics.
Allosteric inhibitor
 Allosteric inhibitor binds to the enzyme at a site other than the active site but on a different region in
the enzyme molecule called the allosteric site. They alter the enzyme conformation and affecting
enzyme catalytic activity and/or substrate binding affinity.
 End product of a biosynthetic pathway usually acts as an allosteric inhibitor to the key enzyme of
that pathway. Product feedback inhibition is an example of allosteric inhibition. E.g., cholesterol
inhibits HMG CoA reductase, key enzyme of cholesterol synthesis.
 Allosteric inhibitors usually shift the kinetic curve (sigmoid curve) to the right thus raising Km. Some
allosteric inhibitors affect Km and some affect Vmax of enzyme depending on type of allosteric
inhibition on enzyme. E.g., phosphofructokinase-1, rate-limiting enzyme for glycolysis is allosterically
inhibited by high concentration of ATP and citrate in which ATP affects Km of the enzyme whereas
citrate affects catalytic efficiency of the enzyme (Vmax).
 Allosteric inhibition is important in regulation of metabolic pathways.
Allosteric enzyme
An allosteric enzyme is one whose activity is
modulated through the non-covalent binding of a
specific metabolite (called an allosteric effector
molecule) at a site other than the catalytic site.
Allosteric enzymes usually contain multiple subunits
and exhibit positive co-operativity in substrate
binding (the binding of substrate to one subunit facilitates the binding of substrate to another
subunit). They have distinct active sites (for binding of substrates) and one or more regulatory (or
allosteric) sites for binding regulatory metabolites called the allosteric modulators.
Allosteric enzymes do not obey Michaelis-Menten kinetics and exhibit sigmoid curve on velocity
versus substrate concentration plot due to its cooperative property.
Binding of an effector molecule to the allosteric site affects the binding of the substrate to the
catalytic site by changing the conformation of the allosteric enzyme. This effect can be either positive
(to increase the binding of substrate) or negative (to decrease the binding of substrate).
Allosteric enzymes are key or regulatory enzymes that catalyze at the committed step or rate-
limiting step of a metabolic pathway. The pathway's final product usually serves as a negative
allosteric effector that inhibits the allosteric enzyme. This mechanism is referred to as end-product,
or feedback, inhibition.
Depending on allosteric inhibition, allosteric enzymes may be of 2 types –
o K-series allosteric enzymes (increase Km but no change in Vmax)
o V-series allosteric enzymes (decrease Vmax but no change in Km)
Regulation of enzyme activity

Cellular metabolism must be regulated according to the metabolic needs of the body and the
changes in physiologic state, dietary intake, environment and age. Regulation of body metabolism is
also essential for metabolic integration and maintenance of body homeostasis. In human body,
regulation of different metabolic processes is achieved by regulating the activities of various key
enzymes, thereby fulfilling the metabolic needs of the body without wasting dietary components or
energy. Metabolic pathways are composed of sequential enzyme catalyzed reactions; only a few of
which are regulated to proceed or turn off the respective pathways. Theses reaction steps are called
regulatory step or rate-limiting step or committed step.
Regulatory enzymes are usually the enzymes that catalyze at the rate-limiting step in a
pathway. Frequently, regulatory enzymes are at or near the initial steps in a pathway, or part of a
branch-point between different pathways. Multistep metabolic pathways are regulated by controlling
the key enzymes which catalyze rate-limiting step (slowest step that limits overall rate of a metabolic
pathway) of that pathway. Controlling the regulatory enzyme is carried out by a variety of mechanisms.
1. Regulation of catalytic efficiency
a) Allosteric regulation of rate-limiting enzyme
b) Stimulation and inhibition by control protein
c) Covalent modification
d) Proteolytic activation
2. Regulation of enzyme quantity
 Control of enzyme synthesis
 Control of enzyme degradation
1. Regulation of catalytic efficiency

a) Allosteric regulation of rate-limiting enzyme
The activity of some rate-limiting enzymes can be
regulated by reversible binding of small molecules
to the enzyme at sites other than the active site.
Binding of allosteric effector induces the
conformation changes in the catalytic site, thereby
changing the catalytic activity and/or substrate
binding affinity of the enzyme.
Allosteric effectors act on the regulatory or rate- Aspartate transcarbamoylase (ATCase)
limiting enzyme in a biosynthetic pathway.  Key enzyme of pyrimidine synthesis
Feedback inhibition of the key enzyme activity in a  Feedback inhibited by end product CTP.
biosynthetic pathway by an end product of that  Allosterically activated by ATP.
pathway is usually based on the allosteric inhibition  Consists of 6 regulatory subunits and 6
catalytic subunits
mechanism. Substrates itself may exert allosteric
 Switched between inactive T (tense) state
effects (referred as homotropic effect) and the
and active R (relax) state
binding of substrate to one subunit facilitates the  Substrates binding to the catalytic
binding of substrate to another subunit. E.g., in E. subunits switch the enzyme into R state
coli, aspartate transcarbamoylase (ATCase), key from which CTP dissociates. CTP binding to
the regulatory subunits converts the
enzyme for pyrimidine synthesis is feedback
enzyme into T state and substrate
inhibited by final product CTP. dissociates.
Allosteric activators increase substrate binding affinity and enhance catalytic activity whereas
allosteric inhibitors decrease substrate binding affinity and reduce catalytic activity of the
enzyme. Allosteric enzymes switch between inactive, Tense (T) state and active, Relax (R) state
depending on the presence or absence of allosteric activator or inhibitor.
Allosteric interaction may be homotropic or heterotropic –
o If substrate itself acts as an effector, it is said to be homotropic.
o If allosteric effector is molecule other than the substrate, it is said to be heterotropic.
Some hormones mediate the regulation of metabolic pathway in the target cells via specialized
allosteric effectors called second messengers; e.g., cAMP. cAMP activates protein kinase A (PKA)
which in turn modulates the key enzyme activity by phosphorylation (covalent modification).
b) Stimulation and inhibition by control protein

It is the regulation of enzyme activity by binding of regulatory protein to the enzyme.
Calmodulin is calcium binding regulatory protein which functions as sensor for intracellular Ca2+
concentration. Ca++-calmodulin complex activates multiple protein kinases inside a cell.
Antihaemophilic factor enhances the activity of serine protease (clotting factor) and accelerates
blood clotting. Anti-thrombin binds and inhibits the activity of thrombin (serine protease)
thereby inhibiting blood coagulation.
Cyclin binds and activates cyclin dependent protein kinase which is essential for cell cycle
progression and control
c) Covalent modification
Enzyme activity can be modulated by the covalent attachment of specific chemical group (such
as phosphoryl, acetyl, methyl) to the enzyme. Regulatory covalent modification can be
reversible or irreversible. E.g., acetylation, ADP-ribosylation, phosphorylation, acetylation.
Irreversible covalent modification by drug or toxin leads to inactivation of specific enzyme, e.g.,
acetylation of COX by aspirin.
Most common covalent modification in the regulation of enzyme activity in metabolism is
phosphorylation and dephosphorylation.
o Various protein kinases such protein kinase A catalyze the phosphorylation of specific
serine, threonine or tyrosine residue on an enzyme protein under the influence of
hormones. It is a reversible process since dephosphorylation can be done by the action of
protein phosphatases.
o Hormonal regulation of metabolism can be mediated through phosphorylation and
dephosphorylation of key enzymes via activation of kinases or phosphatases.
o Phosphorylation can activate or inactivate the enzyme, depending on enzyme concerned,
e.g., phosphorylation activates glycogen phosphorylase but inactivates glycogen synthase.
d) Proteolytic activation
Some enzymes are stored in specific organelle or compartment in inactive precursor forms. A
number of proteolytic enzymes found in the blood or in the digestive tract are present in an
inactive (precursor) form, called zymogen or proenzymes. They are activated by selective
proteolytic cleavage of peptide extension that masks the active site.
Some digestive enzymes such as pancreatic proteases are secreted as zyomogens.
o Stored as inactive zymogens in the pancreas.
o Secreted in pancreatic juice as zymogens following a meal.
o Activated in the GI tract by proteolytic cleavage e.g., proteolytic activation of trypsinogen to

trypsin by intestinal enteropeptidase. Active trypsin then activates other zyomogens
Similar proteolytic cascade are seen in blood clotting and fibrinolytic processes.
Proteolytic activation is irreversible. It can only be switched off by binding of specific inhibitory
protein.
2. Regulation of enzyme quantity

 Control of enzyme synthesis
Rate of enzyme synthesis can be enhanced by increasing the rate of gene transcription
(induction) or can be reduced by decreasing rate of gene transcription (repression).
The synthesis of certain enzymes depends upon the presence of inducers, typically substrates or
structurally related compounds that stimulate the transcription of the gene that encodes them.
Synthesis can also be influenced by hormones and metabolites. Inducible enzymes of human
include HMG CoA reductase, cytochrome P450.
Conversely, an excess of a metabolite may repress the synthesis of key enzyme via repression.
 Control of enzyme degradation
Many intracellular proteins and enzymes are degraded by ubiquitin dependent proteasome
pathway. Proteins targeted for degradation are covalently attached with 4 or more ubiquitin
molecules (polyubiquitinylation) for proteasomal degradation.
Rate of degradation of various enzymes are determined by the half-life of the enzymes.
Proteasome degradation is responsible for the regulated degradation of selected cellular
proteins and for the removal of defective or aberrant protein. Dysfunction of proteasome
pathway leads to accumulation of aberrantly folded protein in the cell and causes cellular
degeneration, e.g., neurodegenerative diseases.
Biomedical importance of enzymes

Knowledge of enzyme can be useful in clinical medicine and practice by various ways;
1. Enzymes as diagnostic agents
2. Enzymes as laboratory agents
3. Enzymes as chemotherapeutic agents
4. Enzymes as site of drug action and poison action
5. Enzymes and drug sensitivity
1. Enzymes as diagnostic agents

 Analysis of plasma enzyme level is useful as marker of cellular damage and is used in the
investigation of diseases of liver, heart, skeletal muscle, the biliary tract, and the pancreas.
Restriction Fragment Length
 The enzymes that are found in plasma can be categorized into Polymorphism (RFLP)
 Cleavage of sample DNA into
two major groups: functional plasma enzyme and non-
characteristic set of smaller
functional plasma enzyme.
DNA fragments by restriction
 Functional plasma enzymes enzyme action
o They have known catalytic function in the blood and thus,  Mutation leads to deviation in
present higher concentration in plasma. Many enzymes are normal fragment pattern
called RFLP.
functional constituent of blood. These include enzymes
 Used to facilitate prenatal
involved in blood coagulation, fibrinolytic system, etc. detection of a number of
o These enzymes are clinically of interest when their hereditary disorders such as
concentration decreases in plasma. sickle cell anemia, thalassemia,

Huntington’s disease, etc.
 Non-functional plasma enzyme
Polymerase Chain Reaction
o They have no physiological function in the blood, so their
(PCR)
plasma concentration is normally very low or absent. They
 Produce thousands of copies of
are released into the circulation following cell death or defined DNA segment by using
damage to the specific tissue. Quantitative analysis of the heat stable Taq polymerase.
 Used to detect and identify
activity of released enzymes is useful for diagnosis,
pathogens and parasites.
prognosis and monitoring of treatment in some diseases.
o Tissue specific enzyme activity measurement in
plasma can identify the tissue damage site.
o Enzyme assay data is not absolutely specific for
the indicated disease and should be considered
together with other clinical presentation factors.
 Inborn errors of metabolism and inherited enzyme
deficiency can be detected by determination of
specific enzyme activity in a body fluid or in a biopsy
sample form relevant tissue.
 Enzymes facilitate diagnosis of genetic and infectious
diseases. E.g., restriction enzyme for RFLP to detect
hereditary genetic disorders, Taq polymerase for PCR
to detect and identify pathogens and parasites.
2. Enzymes as laboratory agents (analytical use of enzyme)

 Many metabolic diseases are characterized by changes in the concentrations of particular
metabolites in the blood or other body fluids. Specific and accurate measurement of
concentration of such metabolites involves the use of enzymes (enzymatic assay). For example –
o Blood cholesterol determination by cholesterol oxidase method,

o Blood glucose determination by glucose oxidase method for diagnosis and monitoring of
diabetes mellitus
o Plasma triglycerides assay by using lipase
 Enzyme linked immunosorbent assay (ELISA) employ in combination of enzyme chemistry and
immunology. This method use enzyme as indicators for detection of specific antigens. Antibody
is used as probe which is conjugated with an enzyme that will produce a colored product when
react with substrate. E.g., assay for HIV virus
3. Enzyme as chemotherapeutic agents

Some enzymes can be used as chemotherapeutic agents.
 Thrombolytic enzyme such as streptokinase in treatment of acutely occluded coronary arteries
 Serine protease (tissue plasminogen activator, tPA) for fibronolytic therapy in AMI
 Asparaginase therapy in some types of adult leukemia
 Diseases caused by deficiency of certain enzymes can be treated by giving preparations
containing the deficient enzyme. E.g., digestzyme for indigestion, anti-inflammatory enzyme for
better penetration of drugs to the site of inflammation.
4. Enzyme as site of drug action and poison action

 Enzyme inhibitors can be designed for the use as therapeutic agents that can produce specific
metabolic changes to counteract the metabolic lesion of the disease. E.g., endogenous
depression is considered to be due to reduced concentration of monoamine in specific areas of
the brain and it can be treated by monoamine oxidase inhibitors (MAOIs). MAOI inhibits
monoamine oxidase enzyme that degrades monoamines. Thus, MAOI can be used as anti-
depressant drug.
 Poisons can damage health by inhibiting one or more enzymes that are important in metabolism.
o Cyanide poisoning due to inhibition of cytochrome C oxidase in respiratory chain
o Organophosphate poisoning due to inhibition of acetylcholine esterase
5. Enzyme and drug sensitivity

 Many drugs are metabolized by enzyme catalyzed reactions in tissues (especially in liver). The
effectiveness and duration of drug action depend on activity of drug metabolizing enzyme and
rate of metabolism.
 Low activity of drug metabolizing enzyme leads to greater effect and more prolong duration of
particular drug action. Impaired liver function is associated with reduced activity of drug
metabolizing enzyme and can lead to drug toxicity.
Bioenergetics and Biologic Oxidation Page |1
Bioenergetics and Biologic Oxidation
Bioenergetics
Bioenergetics or biochemical thermodynamics is the study of the energy changes accompanying
biochemical reactions. It is concerned with the transfer and utilization of energy in biologic systems.
It is based on the basic ideas from the field of thermodynamics, particularly the concept of free
energy. Thermodynamics is the branch of science dealing with energy and its conversion from one
form to another. Hence, bioenergetics is the thermodynamics of living systems.
The three fundamental thermodynamic variables are — Laws of thermodynamics
First law of thermodynamics
o Enthalpy (H)
Total energy within the closed system
o Entropy (S)
(universe) remains constant. Thus,
o Free energy (G) energy cannot be created nor destroyed
Enthalpy (H) – heat content of a physical object or a body when it is changed from one form to
or a system. It is derived from the first law of another. This is also called the law of
conservation of energy.
thermodynamics.
Second law of thermodynamics
Change in enthalpy (ΔH) – heat absorbed or released If a process is to occur spontaneously,
during a reaction. It is expressed in kcal/mol. the total entropy of a system must
Entropy (S) – randomness or disorder of a system. It is increase. Entropy is the extent of

disorder or randomness of the system
derived from the 2nd law of thermodynamics.
and becomes maximal as equilibrium is
Change in entropy (ΔS) – degree of randomness or
approached.
disorders created during the reaction
Free energy (G) – maximum usable work that can be obtained
from a system at constant pressure, temperature and volume.
Free energy change (ΔG) – the change in free energy occurring
during biological reactions. It is related to enthalpy and
entropy.
ΔG = ΔH – T ΔS
(T = absolute temperature in Kelvin)
Standard free energy change (ΔG0) – free energy change
under standardized conditions. Standard conditions is defined
as pH 7.0, temperature 25 °C, all reactants at 1 M
concentration, all gases at pressure of 1 atm. It can be used to
predict the direction of a reaction at constant temperature
and pressure. It gives no information about reaction rate.
Exergonic and endergonic reactions

ΔG = negative or < 0
o Reaction can proceed spontaneously with net loss of energy.
o It is called exergonic reaction, e.g., hydrolysis of ATP.
ΔG = positive or > 0
o Reaction cannot go spontaneously unless energy is added to the
system.
o It is called endergonic reaction, e.g., formation of glucose-6
phosphate from glucose.
ΔG = zero
o The system is in equilibrium.
o The energies of the reactants are equal and there is no net loss
and gain of energy from the reaction.
Coupling of endergonic reaction to exergonic reaction
Several body reactions are endergonic i.e., synthesis of
biomolecules, transport of substances against concentration
gradient require energy to proceed.
An endergonic reaction can proceed in the forward direction, if it is
coupled through a common intermediate to exergonic reaction.
Example – synthesis of glucose 6 phosphate
Reaction A – Formation of glucose 6 phosphate from glucose
Glucose + Pi → Glucose 6 phosphate + H2O (ΔG = + 3.3 kcal/mol)
Reaction B – Hydrolysis of ATP to ADP
ATP + H2O → ADP + Pi (ΔG = – 7.3 kcal/mol)
In order to proceed the reaction A (endergonic reaction), it must be coupled with reaction B
(exergonic reaction).
Glucose + Pi → Glucose 6 phosphate + H2O (ΔG = + 3.3 kcal/mol)
ATP + H2O → ADP + Pi (ΔG = – 7.3 kcal/mol)
Glucose + ATP → Glucose 6 phosphate + ADP
Exergonic and endergonic reactions in metabolism

Metabolism includes catabolism and anabolism.
Catabolism comprises oxidation of carbohydrates, lipids
and proteins which are usually exergonic or energy
releasing reactions.
Anabolism comprises synthetic reactions such as biosynthesis

of macromolecules which are endergonic reactions or energy
requiring reactions.
Thus, all anabolic pathways depend on catabolic pathways in
metabolism, i.e., anabolism is coupled with catabolism.
Biologic oxidation
Biologic oxidation is the oxidation occurring in the body. It
involves the utilization of oxygen and production of carbon
dioxide at cellular level.
A cell is able to obtain energy from sugar or fatty acids or
carbon skeleton of amino acids by allowing their carbon and
hydrogen atoms to combine with oxygen to produce carbon
dioxide and water. It is also known as cellular respiration.
Oxidation – addition of oxygen or removal of electrons or dehydrogenation
Reduction – removal of oxygen or addition of electrons or hydrogenation
Oxidation-reduction potential (redox potential)

In biochemical reactions, any electrons removed
from one molecule are always passed to another.
Therefore, whenever one molecule is oxidized, another is
reduced. Tendency of such oxidation-reduction reaction,
or redox reaction, to proceed spontaneously depends on
free energy change (ΔG) for electron transfer, which in
turn depends on the relative affinities of the two
molecules for electron. Free energy change can be
expressed in terms of redox potential (E0).
Standard redox potential (E’0)

It is the electrical potential (E0) in volts, measured during the oxidation-reduction reaction under
standard conditions (at pH 7.0). The negative redox potential pair has low affinity for electrons and
positive redox potential pair has high affinity for electrons. Electrons flow from the pair with more
negative redox potential to more positive redox potential.
Oxido-reductases
 Enzymes involved in oxidation-reduction reactions are called oxido-reductases.
 These oxido-reductases or respiratory enzymes belong to enzyme class 1 (EC-1) and can be classified
into four sub-classes.
 Oxidases
o Oxidases catalyze the removal of hydrogen from a substrate using oxygen as a hydrogen
acceptor. They form water or hydrogen peroxide as a reaction product.
o Examples of oxidase enzymes
 Cytochrome oxidase
 Flavoproteins – L-amino acid oxidase (FMN linked), xanthine oxidase (FAD linked) ,
glucose oxidase
 Dehydrogenases
o Dehydrogenases catalyze the transfer of hydrogen (or electrons) from one substrate to
another, but they do not use oxygen as hydrogen acceptor. They use coenzymes such as
nicotinamide (NAD+, NADP+) or flavin groups (FMN and FAD) as hydrogen acceptors.
o Dehydrogenases are grouped into NAD+ linked dehydrogenase, NADP+ linked
dehydrogenase, FAD linked dehydrogenase, FMN linked dehydrogenase and cytochromes.
o NAD+ linked dehydrogenase catalyzes oxid0-reduction reactions in the oxidative pathways
particularly in glycolysis, CAC and in mitochondrial respiratory chain.
o NADP+ linked dehydrogenases are found in reductive synthesis such as fatty acid synthesis,
steroid synthesis and also found in pentose phosphate pathway. These enzymes are essential
for xenobiotic metabolism and biotransformation reactions.
o FAD linked dehydrogenases are concerned with electron transport in (or to) the respiratory
chain and oxidative decarboxylation of pyruvate and α ketoglutarate.
o Except for cytochrome oxidase, cytochromes are classified as dehydrogenases. In the
respiratory chain, they are involved as carriers of electrons from flavoproteins on the one
hand to cytochrome oxidase on the other.
Bioenergetics and Biologic Oxidation P a g e |5
 Hydroperoxidases
o Hydroperoxidases use hydrogen peroxide or organic peroxides as substrates.
o They protect the body against harmful peroxides.
o There are two types of hydroperoxidases — namely, peroxidase and catalase.
o Peroxidase
 Peroxidases reduce peroxides using various electron acceptors such as ascorbate,
quinone and cytochrome c. The overall reaction catalyzed by a peroxidase is as follows.
 They are found in milk, leucocytes, platelets and tissues involved in eicosanoids
metabolism. These enzymes contain heme as prosthetic group.
 In erythrocytes and some tissues, the enzyme glutathione peroxidase, containing
selenium as a prosthetic group, catalyzes the destruction of H2O2 and lipid peroxides by
reduced glutathione.
o Catalase
 Catalase uses hydrogen peroxides as electron donors and electron acceptors. It destroys
H2O2 which is produced from peroxisomes, mitochondrial and microsomal electron
transport systems and xanthine oxidase reaction. It is a hemoprotein containing four
heme groups.
 Catalase is found in blood, bone marrow, mucous membranes, kidney, and liver.
 Oxygenases
o Oxygenases catalyze the direct transfer and incorporation of oxygen into a substrate. Their
purpose is not for provision of energy but for the synthesis and degradation of different
types of metabolites.
o There are two types of oxygenases — dioxygenases and monooxygenases.
o Dioxygenases
 Dioxygenases incorporate both atoms of molecular oxygen into the substrate.
 Nearly all cleavages of aromatic rings in biologic system are catalyzed by dioxygenases.
These enzymes contain iron which may be present as heme or non-heme Fe-S protein.
A + O2 → AO2
 E.g., homogentisate oxidase in catabolism of tyrosine, cyclooxygenase in eicosanoid
synthesis
o Monooxygenases
 Monooxygenases incorporate only one atom of molecular oxygen into the substrate
while the other oxygen atom is reduced to water using an electron donor or co-substrate.
AH2 + O2 + ZH2 → A-OH + H2O + Z
 They are also called hydroxylases or mixed function oxygenases/oxidases e.g.,
phenylalanine hydroxylase and cytochrome P450 monooxygenase system
 There are two types of cytochrome P450 monooxygenase system — microsomal and
mitochondrial cytochrome P450 monooxygenase system.
 Microsomal cytochrome P450 monooxygenase system
 They use NADH or NADPH as donor of reducing equivalents.
 In the endoplasmic reticulum of the liver, cytochromes P450 together with
cytochrome b5 have a major role in drug metabolism and detoxification. In the liver,
it also catalyzes the 7α-hydroxylation of cholesterol in bile acid synthesis.
 Mitochondrial cytochrome P450 monooxygenase system
 They are found in steroidogenic tissues such as adrenal cortex, testis, ovary, and
placenta and are concerned with the biosynthesis of steroid hormones from
cholesterol.
 In the kidneys, they catalyze 1α- and 24-hydroxylations of 25-hydroxycholecalciferol in
vitamin D metabolism.
Activated carrier molecules

Activated carrier molecules are molecules that store energy in easily exchangeable form either as
readily transferable chemical group or as high energy electrons and they can serve as a dual role as
a source of both energy and chemical groups in biosynthesis reactions.
Formation of an activated carrier molecule is coupled to
an energetically favorable reaction. Coupling an
exergonic to an endergonic process is to synthesize a
compound of high-energy potential or activated carrier
molecule in the exergonic reaction and to incorporate
this new compound into the endergonic reaction, thus
affecting transference of free energy from the
exergonic to the endergonic pathway. The most
important activated carrier molecules are ATP and
some coenzymes molecules; FADH2, NADH and NADPH.
ATP
o It is the most widely used activated
carrier molecule in the cell. It serves
as an energy currency to drive a
variety of energy-requiring reactions
in the cell. Nearly half of the energy
obtained from metabolic fuel
oxidations is channeled into the
synthesis of ATP, a universal energy
transducer in living system.
o ATP is synthesized in an energetically unfavorable phosphorylation reaction in which
phosphate group is added to ADP. When energy is required ATP gives off its energy through
energetically favorable hydrolysis reaction in which ATP is hydrolyzed to ADP and inorganic
phosphate.
NADH and NADPH (important electron carriers)
o Some activated carriers are specialized to carry high energy electrons and hydrogen atoms
in oxidation and reduction reactions. Most important electron carriers are NAD+ and NADP+
which carry hydride ions.
o NAD+ is a major electron carrier in the oxidation of fuel molecules. The other major electron
carrier in the oxidation of fuel molecules is coenzyme FAD.
o NADP+ serves as an activated carrier of electrons for most reductive biosynthesis e.g., fatty
acid synthesis, cholesterol synthesis.
Other activated carrier molecules in the cell
o Coenzyme A carries an acetyl or acyl
group in a readily transferable linkage
(thioester bond). Hydrolysis of
thioester linkage in acetyl CoA molecule
is energetically favorable and thus
transfer of acetyl group is exergonic.
o Other activated carrier molecules (e.g.,
S-adenosyl methionine, UDP glucose)
involve in the transfer of methyl or
glucosyl group in biosynthesis reactions. These activated carrier molecules are usually
generated in reactions that are coupled to ATP hydrolysis.
How cells obtain energy from food

Living organisms require constant input of energy
for vital processes. Vital processes are powered by
energy stored in the chemical bonds of organic
molecules. Energy is extracted from food molecules
by a process of gradual oxidation. Energy from
foodstuffs is extracted in three stages.
Stage 1
o Carbohydrate, protein and lipids contained
in foods must be broken down into smaller
molecules (absorbable forms) before using
either as a source of energy or as building
blocks for other molecules. Macromolecules
in food are broken down into their monomer units—polysaccharides into monomer sugars,
fats into fatty acids, glycerol and protein into amino acids by the action of hydrolytic
enzymes. This process is digestion.
o The degradation products are then absorbed by the intestinal cells and distributed
throughout the body. Then, these small organic molecules enter the cytosol of the cells
where their gradual oxidation begins. This stage is strictly a preparation stage; no useful
energy is captured in this phase.
Stage 2
o In this stage, small organic molecules are degraded to a few simple units that play a central
role in metabolism.
o Glycolysis converts each glucose molecule or other monomer sugar into two molecules of
pyruvate which then subsequently passes from cytosol into mitochondria. There, pyruvate is
oxidatively decarboxylated to acetyl CoA. During these processes, ATP and NADH are
produced.
o Large amount of acetyl CoA are also produced from oxidation of fatty acids and amino acids.
o Some ATP is generated in this stage by substrate-linked oxidative phosphorylation in certain
reaction steps of glycolysis but nearly all of the energy released from these oxidation
reactions is in the form of high energy electrons or reducing equivalents available as NADH
or FADH2.
o Generated reducing equivalents are collected by the respiratory chain for oxidation and
coupled generation of ATP in the next stage.
Stage 3
o The last stage takes place entirely in mitochondria and consists of the citric acid cycle and
oxidative phosphorylation, which are the final common pathways in the oxidation of fuel
molecules.
o Acetyl CoA is completely oxidized into CO2 through citric acid cycle. High energy electron
carriers such as NADH and FADH2 are generated by oxidation of acetyl CoA in CAC.
o Finally high energy electrons from NADH and FADH2 are passed along mitochondrial electron
transport chain (ETC). Electron transport via ETC creates a proton motive force across the
inner mitochondrial membrane by using the energy released by oxidation of components in
ETC as the electrons flow. This proton gradient is used to drive a membrane located ATP
synthase to synthesize ATP.
o Thus electron transport via ETC creates a proton motive force that drives ATP synthesis and
consumes molecular oxygen. The phosphorylation of ADP to form ATP that is driven by
electron transport in inner mitochondria membrane is known as respiratory chain linked
oxidative phosphorylation.
Nearly half of the energy derived from fuels oxidation is used to drive the energetically unfavorable
reactions and the rest of the energy is released by the cell as heat to make the body warm.
Mitochondria
Mitochondria are subcellular organelles, about the size of bacteria. They are essential for aerobic
metabolism in eukaryotes. Their main function is to oxidize metabolic fuels and conserve free
energy by synthesizing ATP.
It has two membrane systems—outer mitochondria membrane (OMM) and inner mitochondria
membrane (IMM).
Outer mitochondria membrane (OMM)
o It contains enzymes, transporter proteins and pore-forming protein porin.
o It is permeable to all ions, small molecules. Large proteins must be transported via translocases.
Bioenergetics and Biologic Oxidation P a g e | 10
Inner mitochondria membrane (IMM)

o It is highly specialized and contains a high content of double phospholipid called cardiolipin
which helps to make membrane impermeable to nearly all molecules especially to ions. It is
usually highly convoluted; forming a series of infoldings called cristae that project into the matrix.
o The membrane also contains a variety of transport proteins that make it selectively permeable to
those small molecules that are metabolized or required by the mitochondrial matrix enzymes.
o The inner membrane also contains components of electron transport chain for ATP production
via oxidative phosphorylation.
There are two separate compartments in mitochondria—inter-membranous space (between inner
and outer mitochondrial membrane) and mitochondrial matrix.
Matrix
o Mitochondrial matrix is bounded by inner mitochondria membrane. It contains enzymes for
oxidative decarboxylation of pyruvate, β oxidation of fatty acids and CAC. These enzymatic
reactions generate high energy electrons carried by the activated carrier molecules NADH and
FADH2. These high energy electrons are transferred to ETC in inner mitochondrial membrane for
ATP generation.
o Part of urea cycle and heme synthesis also occurs in mitochondrial matrix.
Mitochondrial genome
 Mitochondria contain 2 to 10 copies of small circular double-stranded DNA, which encodes a variety
of proteins and functional RNAs. Mitochondrial DNA
constitutes 1% of cellular DNA. Mitochondrial DNA is
maternally inherited.
 Human mitochondrial DNA comprises 16,569 base pairs
and encodes 13 electron transport chain proteins, small
and large rRNAs, tRNAs for mitochondrial gene
expression.
 Almost all mitochondrial proteins are encoded by nuclear
DNA and must be transported into the mitochondria.
Mitochondria transport systems

 The inner mitochondrial membrane is freely permeable to
uncharged small molecules, such as oxygen, water, CO2,
NH3, and to monocarboxylic acids, such as 3-
hydroxybutyric, acetoacetic, and acetic acid.
 But small charged molecules and large molecules need transporter or mitochondrial shuttles to cross
the membrane. Dicarboxylate and tricarboxylate anions (e.g., malate, citrate) and amino acids
require specific transporter or carrier systems to facilitate their passage across the membrane.
o Long-chain fatty acids are transported into mitochondria via the carnitine system.
o The transport of di- and tricarboxylate anions is closely linked to that of inorganic phosphate,
e.g., malate transport by dicarboxylate transporter requires HPO42– in exchange.
o Pyruvate transport into matrix is by specific carrier (pyruvate-H+ symport) together with H+.
o The net uptake of citrate by the tricarboxylate transporter and α-ketoglutarate transport
requires malate in exchange.
o The adenine nucleotide transporter allows the exchange of ATP and ADP, but not AMP.
Oxidation of extra-mitochondrial NADH by substrate shuttles in mitochondria

 Glycerol phosphate shuttle and malate aspartate shuttle are used for transfer of reducing
equivalents from cytosol into mitochondria to permit oxidation of cytosolic NADH.
 Glycerol phosphate shuttle
o They are found in brain, white muscles but deficient in heart muscles.
o The transfer of reducing equivalents through the mitochondrial membrane requires substrate
pairs, linked by suitable dehydrogenases on each side of the mitochondrial membrane
(glycerol-3-phosphate dehydrogenase in cytosol and in the outer surface of IMM). The
reducing equivalents from cytosolic NADH are transported to mitochondria as FADH2.
o Since the mitochondrial enzyme is linked to the respiratory chain via a flavoprotein rather
than NAD, only 1.5 mol of ATP are formed.
 Malate aspartate shuttle

o Malate aspartate shuttle system is present in heart, liver, kidneys and is of more universal
utility. This system operates with the help of malate dehydrogenase (MDH) and aspartate
transaminase (AST).
o The reducing equivalents from cytosolic NADH are first transferred to cytosolic oxaloacetate
(OAA) to yield malate catalyzed by cytosolic malate dehydrogenase. Malate then passes
through IMM via malate/α-ketoglutarate transporter.
o Within the matrix, malate is reoxidized to OAA by transferring its electrons to NAD by the
action of matrix MDH forming NADH. NADH can pass electrons directly to the ETC. OAA is
impermeable to IMM so it is converted to aspartate by AST. Aspartate is then transported
back to cytosol in exchange with glutamate by glutamate/aspartate transporter.
Creatine-phosphate shuttle
Creatine-phosphate shuttle facilitates transport of
high energy phosphate from mitochondria in active tissues
such as heart and skeletal muscles. An isoenzyme of
creatine kinase found in the mitochondrial inter-
membranous space catalyzes the transfer of high energy
phosphate from ATP to creatine. Creatine phosphate is then
transported into cytosol via protein pores in the outer
mitochondrial membrane. Creatine phosphate becomes
available for generation of extra-mitochondrial ATP.
Electron transport chain

Respiratory chain or electron transport chain is a series
of catalysts in inner mitochondrial membrane that
collect and transport reducing equivalents and direct
them to their final reaction with oxygen to form water.
Sources of reducing equivalents

o Glycolysis pathway (from cytosol)
o Oxidative decarboxylation of pyruvate, β oxidation and CAC (from mitochondria)
Cytosolic source of reducing equivalents are transported to inner mitochondrial membrane by –
glycerol phosphate shuttle and malate aspartate shuttle.
Components of respiratory chain
o Flavoproteins
o Ubiquinone
o Iron sulfur proteins
o Cytochrome
Arrangement of respiratory chain
o Components of the respiratory chain are present in the inner mitochondrial membrane as 4
protein-lipid complexes. Cytochrome c and ubiquinone (coenzyme Q10) are mobile
components connecting these fixed complexes.
o The major components of respiratory chain are arranged in order of increasing redox
potential (from negative potential to positive potential).
o Complex I, III and IV have proton pump activity and pump protons in the number of 4, 4, 2
respectively.
Complex I – NADH-Ubiquinone oxidoreductase (NADH dehydrogenase complex)

o It contains NAHD dehydrogenase, FMN and iron-sulfur clusters.
o High energy electrons of NADH are transferred to complex I of ETC, with concomitant
oxidation of NADH to NAD+; after passing through the FMN and Fe-S components of the
complex, these electrons are transferred to cytochrome Q or ubiquinone (Q) forming QH2.
Substrate → NADH → FMN → FMNH2 → Fe-S → Q → QH2
o Free energy changes derived from electron transfer from NADH to QH 2 drive pumping of 4
protons (H+) from the matrix to the inter-membranous space.
Complex II – Succinate-Ubiquinone (CoQ) oxidoreductase
o Complex II catalyzes the reduction of CoQ by electrons removed from succinate. It contains
succinate dehydrogenase, FAD and Fe-S cluster.
Succinate → FAD → FADH2 → Fe-S → Q → QH2
o Other substrates for mitochondrial dehydrogenases such as acyl CoA dehydrogenase,
glycerol-3-phosphate dehydrogenase pass electrons into the respiratory chain at the level of
ubiquinone, but not through Complex II.
o Succinate-Q oxidoreductase complex and other enzymes that transfer electrons from FADH 2
to ubiquinone do not couple with pumping of protons because the free energy change of
the catalyzed reaction is too small. Consequently, less ATP is formed from the oxidation of
FADH2 than from NADH.
Complex III – Ubiquinol-ferricytochrome c oxidoreductase
o This complex contains Fe-S proteins, cytochrome b and cytochrome c 1. It catalyzes transfer
of electrons from CoQ to cytochrome c.
QH2 → Cyt b → Fe-S → Cyt c 1 → Cyt c
o This complex pumps 4 protons form the matrix to the inter-membranous space.
Complex IV – Ferrocytochrome c-oxygen oxidoreductase (cytochrome oxidase)
o This complex contains cytochrome a, a3, two Cu2+ ions. It catalyzes transfer of electrons from
ferrocytochrome c to molecular oxygen.
Cyt c → CuA → Cyt a → Cyt a 3 → CuB → ½ O2 → H2O
o Two electrons are transferred to O2 to completely reduce it to H2O together with pumping of
2 protons from matrix to intermembranous space.
Biomedical importance
o Electron transport chain is the main site of ATP formation in aerobic organism.
o Many chemicals, drugs, poisons and gases can inhibit respiratory chain linked oxidative
phosphorylation with fetal consequences. Lack of O2 supply or anoxia affects electron
transport along ETC subsequently ATP formation will be decreased in the cell.
o Copper deficiency can impair ATP production by inhibiting the terminal reaction of the
electron transport chain, leading to pathology in the heart e.g., cardiomyopathy.
o Inherited defects in oxidative phosphorylation cause myopathy or encephalopathy. A
number of diseases have been now identified as mitochondrial genome mutation. Organs
highly dependent on oxidative phosphorylation such as nervous system and heart are most
vulnerable to mutations of mitochondrial DNA. Aging and degenerative disorders can result
from mitochondrial mutations.
o MERRF syndrome (myoclonus epilepsy with ragged red fibers) causes myopathies in
affected persons.
o Leber’s hereditary optic neuropathy (LHON) is an inherited form of blindness associated with
mutations in NADH-Q oxidoreductase.
o MELAS (mitochondrial encephalopathy, lactic acidosis, and stroke) is an inherited condition
due to NADH-Q oxidoreductase (Complex I) or cytochrome oxidase (Complex IV) deficiency.
o Some individuals with diabetes mellitus have been found to have mutation of mitochondria
DNA which involves in oxidative metabolism or tRNA synthesis.
Oxidative phosphorylation
Oxidative phosphorylation is a process in which the energy from oxidation of fuel (foodstuff) is
converted to the high energy phosphate bonds of ATP.
Two mechanisms of oxidative phosphorylation – substrate linked and respiratory chain linked
oxidative phosphorylation
Substrate linked oxidative phosphorylation

This type of phosphorylation occurs at the substrate level (during glycolysis and TCA cycle).
Substrate linked oxidative phosphorylation in glycolysis
Phosphoglycerate kinase
1, 3 bisphosphoglycerate 3 phosphoglycerate
ADP ATP
Pyruvate kinase
Phosphoenol-pyruvate Pyruvate
ADP ATP
Substrate linked oxidative phosphorylation in TCA cycle

CoA-SH
Succinate thiokinase
Succinyl CoA Succinate
ADP + Pi ATP
Respiratory chain linked oxidative phosphorylation

It is the phosphorylation of ADP to ATP driven by free energy released from the transfer of
electrons from NADH or FADH2 to O2 by the ETC in the inner mitochondria membrane.
Most of the energy released from oxidation of metabolic fuels is in the form of reducing equivalents
(high energy electrons) carried as reduced coenzyme NADH or FADH2. These reduced coenzymes
donate high energy electrons to a specialized carrier in the respiratory chain. Cytosolic source of
reducing equivalents are transported to inner mitochondrial membrane by glycerol phosphate
shuttle or malate aspartate shuttle.
There are 4 protein-lipid complexes in respiratory chain. Components of the respiratory chain are
flavoproteins, ubiquinone, Fe-S proteins and cytochromes. These electron carriers are arranged in
order of increasing redox potential.
Electrons are conducted through ETC in a defined sequence from reduced nucleotide coenzymes to
oxygen and free energy changes drive the transport of protons from the matrix into the inter-
membranous space via three proton pumps in complex I, III and IV. This creates proton motive force
and proton flow back into mitochondria matrix via ATP synthase drives ATP synthesis.
Thus, transport of electrons down ETC release free energy and part of this energy is used to drive
synthesis of ATP from ADP and Pi and remaining free energy is liberated as heat.
When substrates are oxidized via NAD linked dehydrogenases, 2.5 ATPs are formed and when
oxidized via flavoprotein (FAD) linked dehydrogenases, only 1.5 ATPs are formed.
For the coupled formation of one mole of ATP, there must be a free energy change of approximately
37 kJ from oxidation (electron transfer) between the components of ETC. There are three sites in the
respiratory chain where the energy change is sufficient to drive ATP synthesis.
o Between NADH and flavoprotein in complex I
o Between Cyt b and c in complex III
o Between Cyt a and a3 in complex IV
Mechanisms of respiratory chain linked oxidative phosphorylation

There are three possible mechanisms postulated for the mechanism of respiratory chain linked
 Chemical coupling mechanism

 Conformational coupling mechanism
 Chemiosmotic mechanism
The latter mechanism is widely accepted to explain the ATP production via respiratory chain linked
Chemiosmotic mechanism
 Chemiosmotic theory proposed
by Peter Mitchell explains how
the free energy generated by the
transport of electrons in ETC is
coupled with ATP production. It
postulates that the two
processes are coupled by a
proton gradient across the inner
mitochondrial membrane so that
the proton motive force caused
by the electrochemical potential difference drives the mechanism of ATP synthesis.
 The flow of electrons in ETC is coupled with the translocation of protons (H+) across the inner
membrane to the intermembranous space due to free energy changes occur in complex I, III and IV.
ETC complexes I, III and IV act as proton pumps and pump protons in the number of 4, 4, 2.
 Pumping of H+ results in an electrochemical potential gradient which consists of chemical potential
(difference in pH) and an electrical potential (negative on the matrix side).
 This electrochemical potential difference or proton motive force drives the synthesis of ATP by ATP
synthase in the presence of ADP and Pi as ATP synthase is the site to reenter the proton from
intermembranous space to matrix.
 Thus electron transport via the respiratory chain creates
a proton motive force which drives the ATP synthesis
 ATP synthase is embedded in the inner membrane,
which is responsible for ATP production.
 ATP synthase is composed of
o Membrane protein complex Fo that spans the
membrane and forms proton channels (consists of
several protein subunits)
o Catalytic protein complex F1 that projects into the matrix and

catalyzes the phosphorylation (3α, 3β, 1γ, 1δ , 1ε)
o F1 is attached to membrane protein complex Fo via its γ subunit.
 Flow of protons through Fo causes rotation of γ subunit which
induces conformation changes in β subunits and drives ATP
formation by F1 complex.
 Translocation of 3H+ through ATP synthase leads to formation of 1
ATP. Another H+ is used for the entry of Pi into the matrix. Hence,
2.5 ATPs are formed per NADH oxidation and 1.5 ATPs are formed per FADH2 oxidation via ETC.
Control of respiratory chain linked oxidative phosphorylation

It is the regulation of cellular respiration through tight coupling of electrons flow with ATP synthesis.
Rate of respiration in mitochondria can be controlled by —
o Stage 1 – availability of ADP and substrate
o Stage 2 – availability of substrate only
o Stage 3 – capacity of respiratory chain itself
o Stage 4 – availability of ADP only
o Stage 5 – availability of oxygen only
Rate of oxidative phosphorylation is determined by the need for ATP. Under physiologic conditions,
electron transport is tightly coupled to phosphorylation.
Oxidative phosphorylation requires a supply of NADH or other sources of high energy electrons, O 2,
ADP and Pi. The most important factor is ADP level. The flow of electrons is reduced when ADP
becomes limited and increased when ADP becomes plentiful. This mechanism ensures that electrons
flow down the ETC only when ADP levels are high (low ATP/ADP ratio).
Inhibitors of cellular energy production

They are categorized into four —
1. Inhibitors of electron transport chain or respiratory chain
2. Inhibitors of ATP synthase
3. Inhibitors of ATP/ADP transport
4. Uncouplers
1. Inhibitors of electron transport chain or respiratory chain

They causes interruption of electrons flow through the respiratory chains thus subsequently
stops protons pumping and ATP synthesis.
Inhibitors cause reduction of all components prior to the point of inhibition whereas those after
the point of inhibition become fully oxidized.
Complex I Complex II Complex III Complex IV

Barbiturates (amobarbital) Carboxin Dimercaprol CO
Antibiotics (pericidin A) TTFA (Fe chelating agent) Antimycin A Cyanide (CN-)
Inhibitors
Insecticide Malonate Sodium azide
Fish poison (rotenone) H2 S
Complex I inhibitors
 Barbiturates such as amobarbital, antibiotics pericidin A, insecticide and fish poison rotenone
 These inhibitors prevent the oxidation of substrates through ETC via NAD-linked
dehydrogenases by blocking electron transfer from Fe-S to Q.
 Oxygen uptake is also markedly inhibited by rotenone.
Complex II inhibitors
 Carboxin and TTFA (Fe chelating agent) specifically inhibit transfer of electrons from
succinate dehydrogenase to CoQ.
 Malonate is the competitive inhibitor of succinate dehydrogenase.
Complex III inhibitors
 Dimercaprol and antibiotic antimycin A inhibits electron transfer between cytochrome b and
cytochrome c.
Complex IV inhibitors
 Cyanide (CN), carbon monoxide (CO), H2S and sodium azide
 Since complex IV is the terminal electron transfer complex, its inhibition cannot be bypassed.
 CN and sodium azide reacts with ferric form and CO inhibits the ferrous form of heme in
cytochrome a3.
2. Inhibitors of ATP synthase

Oligomycin (antifungal agent) inhibits ATP synthase activity by inhibiting proton flow through Fo
complex.
Arsenic (As) is a potent poison. It is an analog of phosphate so that it substitutes for phosphate in
phosphorylation reaction.
3. Inhibitors of ATP/ADP transport
Plant and mold toxin such as bongkrekic acid and atractyloside inhibit ATP/ADP translocase.
4. Uncouplers
Uncouplers are hydrophobic molecules (weak
acids or bases) which enhance the proton to
diffuse readily back to the mitochondrial
matrix. Uncouplers allow electron transport to
proceed but prevent the coupling of electron
transfers to the phosphorylation of ADP by
disturbing proton gradient across the
membrane.
These compounds are
proton i0nophores
(carry protons across the
inner membrane to
matix and disturb the
proton gradient). The
free energy is dissipated
as heat and maintaining
body temperature.
Examples of uncouplers
– antibiotics and K+
inophores (valinomycin and nigercin), transmembrane proton carrier (2, 4 dinitrophenol,
dinitrocresol, pentachlorophenol), natural uncouplers (bilirubin, free fatty acids, thyroxine),
thermogenin (UCP1) in brown adipose tissues
Thermogenin
 Thermogenin (or the uncoupling protein) is a physiological uncoupler found in brown adipose
tissue that functions to generate body heat, particularly for the newborn and during
hibernation in animals.
 It provides a path for the re-entry of protons into mitochondria and abolishes proton
gradient. As a result, energy released from oxidation in ETC is not conserved by ATP
formation but is dissipated as heat which contributes to maintaining body temperature.
Adult humans have relatively little brown fat, but the mitochondria of ordinary adipose tissue and
muscle appear to contain uncoupling proteins known as UCP2 and UCP3.
Role of brown adipose tissue in thermogenesis

Physiological function of brown adipose tissue (brown
fat) is heat generation. Thus, the tissue is extremely
active in some species, for example, during arousal from
hibernation, in animals exposed to cold (non-shivering
thermogenesis), and in heat production in the newborn.
The tissue is characterized by a well-developed blood
supply and a high content of mitochondria and
cytochromes, but low activity of ATP synthase. Brown
fat mitochondria contain a proton channel known as
uncoupling protein (UCP1, also called thermogenin).
The mechanism of heat generation in brown fat involves
the regulated uncoupling of oxidative phosphorylation.
Norepinephrine liberated from sympathetic nerve
endings is important in increasing lipolysis in the tissue.
The activated lipase hydrolyzes triacylglycerols to yield
free fatty acids that counteract the inhibitory effect of
the purine nucleotides on UCP1. The resulting flow of
protons through UCP1 dissipates the proton gradient
across the inner mitochondrial membrane. Oxidation
and phosphorylation are not coupled in mitochondria of
the tissue. Thus, oxidation produces much heat, and
little free energy is trapped in ATP.
Though not a prominent tissue in adult humans, it is present in normal individuals, where it could be
responsible for “diet-induced thermogenesis.”
High energy compounds or energy rich compounds

High energy compounds possess sufficient free energy to liberate 7 kcal/mol or more on hydrolysis.
High energy bond is a bond which releases free energy not less than 7 kcal/mol on hydrolysis.
Energy rich compounds or high energy compounds include —

1. Nucleoside triphosphates
 ATP, GTP, UTP, CTP
 dATP, dGTP, dCTP, dTTP
2. Compounds that contain phosphate with higher energy than that of ATP (very high energy
compounds)
 Phosphoenol pyruvate (PEP)
 1, 3 bisphosphoglycerate (1, 3 BPG)
 Phosphocreatine (creatine phosphate)
 Carbamoyl phosphate
3. Other biologically important high energy compounds
 Thioester involving coenzyme A  S-adenosyl methionine
(acetyl CoA, succinyl CoA)  UDP glucose
 Acyl carrier protein (ACP)  5-phosphoribosyl 1-pyrophosphate
 Amino acid esters involved in (PRPP)
protein synthesis (amino acyl  Guanidine phosphate
tRNA)  Arginine phosphate
Low energy phosphate containing compounds
o Free energy released on hydrolysis is less than 4 kcal/mol.
o Glucose 6 phosphate, glycerol 3 phosphate, AMP
Functions of ATP
ATP is the primary source of energy for physiological and biochemical processes.
Biochemical functions of ATP

1. Phosphorylating agent
Hexokinase
Glucose Glucose 6 phosphate
ATP ADP
2. Pyrophosphorylating agent
PRPP synthetase
Ribose 5 phosphate PRPP
ATP AMP
3. Adenylating agent
Acetic acid Acetyl adenylic acid
ATP PPi
4. Energy donating agent
Glutamine synthetase
Glutamate + NH3 Glutamine
ATP ADP + Pi
Physiological functions of ATP

1. Conduction of electrical impulses in nervous tissues
2. Muscle contraction
3. Motility of cells
4. Release of insulin from pancreatic cells
5. Transport of water and electrolytes across the cell membrane
6. Formation of urine which is higher osmotic pressure than that of blood
7. Vasodilation effect
Functions of other energy rich compounds

UTP – glycogen synthesis (UDP glucose)
GTP – protein synthesis (peptide bond formation), gluconeogenesis
CTP – phospholipid synthesis (CDP choline, CDP diacylglycerol)
ATP, GTP, CTP, UTP – RNA biosynthesis
dATP, dGTP, dCTP, dTTP – DNA biosynthesis
Creatine phosphate – regeneration of depleted ATP during continuous muscle contraction as in
strenuous exercise
Oxygen toxicity
In general, biologic oxidation ensures that oxygen is completely reduced to H2O. However, partial
reduction of oxygen generates reactive oxygen species (ROS) which are referred as free radicals.
Reactive oxygen species (ROS)
o Reactive oxygen species or oxidants are highly reactive strongly oxidizing forms of oxygen
derived molecules produced by partial reduction of molecular oxygen.
o ROS include free radicals and non-free radical compounds.
Free radicals – A free radical is an atom or molecule that has one or more unpaired electrons.
Non free radicals – ROS are highly reactive due to its tendency to acquire electrons from other
molecules, but not all ROS are free radicals. Relatively stable ROS species are called non-free radicals
e.g., H2O2.
Free radicals Non free radicals
Superoxide anion (O2•-) Hydrogen peroxide (H2O2)
Hydroxyl radical (OH•) Singlet oxygen (1O2)
Peroxynitrite (ONOO•)
Hydroperoxy free radical (HOO•)
Among ROS, H2O2 is present at highest concentration in blood and tissues. It is relatively stable but
decomposes in the presence of redox-active metal ions.
The hydroxyl radical (OH•) is the most reactive and damaging species.
HOO• and H2O2 are small, uncharged molecules and readily diffuse through cell membranes.
Sources and generation of free radicals or ROS

ROS are formed by three major mechanisms in vivo:
1. Mitochondrial leak
 Small amount of free radical intermediates
generated during electron transport via ETC

is leaked into mitochondrial matrix and
forms superoxide anions.
2. Enzymatic reaction
 Some of oxygenases and oxidases reactions produce partially reduced forms of oxygen e.g.,
reactions catalyzed by xanthine oxidase, fatty acid oxidase in peroxisomes, etc.
Hypoxanthine + H2O + O2 → Xanthine + H2O2
3. Metal dependent oxidation reactions
 ROS are produced by non-enzymatic
reactions of molecular oxygen with
decompartmentalized transition metal
ions such as iron and copper e.g., Fenton
reaction, Heber-Weiss reaction, metal
catalyzed Heber-Weiss reaction.
Formation of ROS is aggravated by environmental
insults such as cigarette smoke, pollutants, UV
light, ionizing radiation, xenobiotics, etc.
Three isoforms of NOS
Reactive nitrogen species nNOS in neuronal tissue, where
Nitric oxide (NO•) NO• serves a neurotransmitter

function.
Nitric oxide synthases (NOS) catalyze the production
iNOS in the immune system,
of the free radical nitric oxide (NO•) from the amino
where it is involved in regulation of
acid L-arginine. the immune response.
Peroxynitrite (ONOO ) – eNOS in endothelial cells, where

NO•, known as endothelium-
It is a potent reactive nitrogen species (RNS) formed
derived relaxation factor (EDRF),
when NO• reacts with O2•- in a side reaction at sites of
has a role in the regulation of
inflammation. vascular tone.
RNS produce nitrosative stress by reaction with biomolecules (nitrotyrosine in proteins,
nitrated phospholipids in membranes and nucleotides in DNA).
Simultaneous production of NO• and O2•-, with the concomitant increase in ONOO– and a
decrease in NO•, is thought to limit vasodilatation and exacerbate hypoxia and oxidative
stress in the vascular wall during ischemia-reperfusion injury.
•
NO2 is formed by eosinophil peroxidase or myeloperoxidase reaction and also by the cleavage of
ONOOH (produce two very reactive species, OH• and NO2•).
Cellular effects of ROS and free radicals

ROS are extremely reactive and their half-life is only a few milliseconds. They are both beneficial and
harmful to the cells.
Beneficial effects of ROS and free radicals
Effect in phagocytosis
 Respiratory burst in phagocytic cells is
beneficial for bactericidal activity in
phagocytes.
 Respiratory burst includes reactions catalyzed
by NADPH oxidase, superoxide dismutase and
myeloperoxidase. These reactions produce
ROS such as hypochlorus acid (HOCl), H2O2
which mediate bactericidal activity and
protects body against bacterial infection.
As substrates for enzymes, e.g., H2O2 for heme
containing thyroperoxidase involved in oxidation of
iodide and iodination steps of thyroid hormone synthesis.
Regulatory function of NO
Radiotherapy in treatment of cancer

 The radiation produces a flux of hydroxyl radicals (from water) and organic radicals at
the site of the tumor.
 This localized oxidative stress causes damage to all biomolecules in the tumor cell,
and prevents tumor cell replication, inhibiting tumor growth.
Important signaling molecules involved in regulation of metabolism particularly H2O2
 Significant changes in H2O2 concentration occur in response to cytokines, growth
factors and biomechanical stimulation.
 Insulin signaling appears to involve H2O2 as part of the mechanism for reversible
inactivation of some protein tyrosine phosphatases.
Harmful effects of ROS
Free radicals are highly reactive and can cause damage to all types of biomolecules.
ROS randomly attack cellular components such as proteins, lipids and nucleic acids and
carbohydrates.
Effect of ROS on proteins
 Attack of ROS on proteins leads to loss of protein structure and function.
 Effects include protein fragmentation, cross-linking, aggregation, and increased or
decreased susceptibility to proteolysis.
Effect of ROS on lipids
 PUFA in membrane phospholipids and lipoprotein structures are highly susceptible to
ROS attack and lead to lipid peroxidation reactions.
 Lipid peroxidation of cellular and sub-cellular membranes affects membrane integrity,
permeability and functions leading to increased membrane permeability, cell swelling,
mitochondria and endoplasmic reticulum dysfunction, etc.
Effect on nucleic acid
 Free radical attack on nucleic acid results in DNA stand break and modification of
bases.
Effect of malodialdehyde (MDA) and hydroxynonenal (HNE)
 Lipid peroxides and oxidized carbohydrate decompose into reactive carbonyl species
such as MDA and HNE. They subsequently react with proteins leading to loss of
membrane integrity, protein dysfunction and cell death.
Lipid peroxidation (autooxidation)

 It is a chain reaction providing a continuous supply of free radicals that initiate further peroxidation.
PUFAs are highly susceptible to free radical attack and lead to lipid peroxidation chain reaction.
 The process of lipid peroxidation comprises

initiation, propagation, degradation, and
termination.
 Initiation
o Initiator such as hydroxyl radical is required
to begin the chain reaction.
o Peroxidation begins by extraction of
hydrogen atoms containing one electron
from conjugated double bonds in fatty acid.
Lipid-H + OH• → Lipid• + H2O
 Propagation
o Lipid peroxidation is propagated by the
addition of oxygen to form lipid peroxy
radicals and lipid peroxides.
Lipid• + O2 → Lipid-OO•
Lipid-OO• + Lipid-H → Lipid-OOH + Lipid•
 Degradation
o Degradation of lipid peroxides results in the
formation of molecules such as
malondialdehyde (MDA).
Lipid-OOH → Lipid• + aldehydes
(reactive carbonyl species)
 Termination
o Lipid peroxidation chain reaction can be terminated by antioxidants such as vitamin E.
o Lipid• + vitamin E (tocopherol) → Lipid-H + tocopherol radical
Advanced lipoxidation end-products (ALEs) and advanced glycation end-products (AGEs)

These compounds may be formed directly in the oxidation reaction with the ROS, or by secondary
reactions between oxidation products and other biomolecules.
Lipid peroxidation reaction, producing secondary oxidation products, lipid peroxides and lipid
peroxyl radicals. The lipid oxidation products formed degrade to form reactive compounds, such as
malondialdehyde (MDA) and hydroxynonenal (HNE). These compounds react with proteins (lysine
residues) to form adducts and crosslinks, known as advanced lipoxidation end products (ALE). ALEs
are found in plasma lipoproteins and vascular wall in atherosclerosis, neuronal plaque in Alzheimer’s
disease, implicating oxidative stress and damage in the pathogenesis of these diseases.
ROS also react with carbohydrates to form reactive carbonyl compounds that react with protein to
form glycoxidation products or advanced glycation end products (AGE). AGEs are increased in tissue
proteins in diabetes as a result of hyperglycemia and oxidative stress, and contribute in the
development of diabetic vascular complications.
Nitrotyrosine, like ALEs, is increased in atherosclerotic and Alzheimer’s plaques.
ALEs and AGEs are biomarkers of oxidative stress.
Antioxidant defense system

Antioxidants are molecules that counteract the effects of oxidants.
Antioxidants can be divided into two types.
1) Enzymatic antioxidants 2) Non-enzymatic antioxidants
 Superoxide dismutase (SOD)  Alpha tocopherol
 Catalase  Ascorbic acid
 Glutathione peroxidase  Beta carotene
 Ceruloplasmin  Uric acid
 Bilirubin
 Albumin
 Ceruloplasmin
 Transferrin and ferritin
 Flavonoids
1) Enzymatic antioxidants
 Superoxide dismutase (SOD)
 It catalyzes dismutation of superoxide anion to H2O2 and O2.
O2•- + O2•- + 2H+ → H2O2 + O2
 Three forms of SOD in mammalian tissues are cytosolic SOD (Cu-ZnSOD), mitochondrial SOD
(MnSOD) and extracellular SOD (Cu-ZnSOD).
 Catalase
 Catalase converts hydrogen peroxide to H2O and O2. It is found mainly in peroxisomes.
 Glutathione peroxidase
 It catalyzes neutralization of H2O2 by
reduced glutathione to form H2O and
oxidized glutathione. Oxidized glutathione
is reduced by the action of glutathione
reductase to form reduced glutathione for further reaction of glutathione peroxidase.
 Ceruloplasmin
 Ceruloplasmin has ferrooxidase activity and acts as an antioxidant by catalyzing oxidation of
ferrous iron to ferric ion. Ferric iron is less reactive than ferrous iron in forming free radicals.
2) Non-enzymatic antioxidants
 Alpha tocopherol (vitamin E)
 It is a chain breaking antioxidant. It acts as the first line of defense against lipid peroxidation
of PUFAs in the cell membrane. Tocopherol breaks the free radical chain reaction by
transferring its phenolic hydrogen to peroxy radical.
 Ascorbic acid
 It is also one of the chain breaking antioxidants. Ascorbic acid scavenges superoxide anion,
hydroxyl radical, peroxy radicals, hydrogen peroxide and singlet oxygen.
 Beta carotene
 Beta carotene exerts its antioxidant effect by stabilizing organic peroxide free radicals within
their conjugate alkyl groups.
 Uric acid
 Uric acid efficiently scavenges free radicals and converted to allontoin during the reactions. It
forms stable non-reactive complex with iron and also directly scavenges free radicals.
 Bilirubin
 Albumin bound bilirubin also acts as an effective free radical scavenger. It is particularly
important in protecting the neonate from oxidative damage.
 Albumin
 Albumin has one reactive free cysteine group that neutralizes the peroxy radicals. It also
binds copper ions and inhibits copper dependent lipid peroxidation and hydroxyl radical
formation.
 Ceruloplasmin
 It acts as an antioxidant by binding of copper thus inhibiting copper dependent lipid
peroxidation and hydroxyl radical formation.
 Transferrin and ferritin
 They act as antioxidants by sequestering iron thereby preventing iron catalyzed free radical
formation.
 Flavonoids
 Flavonoids are large group of polyphenolic compounds and mostly found in many fruits,
vegetables and beverages such as tea and wine. Naturally occurring flavonoids include
quercetin, catechins, apigenin and genistein.
Oxidative stress
Oxidative stress is the imbalance between production of ROS and antioxidant defense mechanisms.
Oxidative stress is caused by —
 Ingestion of prooxidants (compounds that are capable of generating ROS)
 Decrease in antioxidant defense activity
 Antioxidant vitamins deficiency

 Inactivation of antioxidant enzymes
Diseases associated with oxidative stress
 Ischemic reperfusion injury  Aging
 Atherosclerosis and ischemic heart  Oxygen toxicity in premature infants
disease  Septicemic shock
 Diabetes mellitus  Degenerative diseases such as

 Cancer Alzheimer’s disease, Parkisonism,
 Ultraviolet radiation or sunburns arthritis
Oxidative stress is a characteristic feature of inflammatory disease.
Oxidative stress may be localized, e.g., in the joints in arthritis or in the vascular wall in
atherosclerosis or can be systemic, e.g., in systemic lupus erythematosus (SLE) or diabetes.
Free Radicals & the Mitochondrial Theory of Aging

Aging is a multifactorial process. Three major factors that interact in leading to aging are genetic
factors, environmental factors and life-style factors.
One endogenous process that is likely to cause significant biomolecule damage is the production of
free radicals. In 1956, Denham Harmon proposed the so-called free radical theory of aging. It
postulated that the cumulative damage associated with aging was caused by the continual and
inescapable production of ROS.
Not only is the mitochondria host to the major source of ROS in the cell, the electron transport chain,
but oxidative damage to the components of this pathway could lead to increased leakage of
hydrogen peroxide, superoxide, etc., into the cytoplasm. Damage to the mitochondria would be
likely to adversely affect its efficiency of ATP synthesis. A significant slowing in the rate of ATP
synthesis could readily lead to declines in the physiological function that occur in aging.
Mitochondria and apoptosis

In case of significant cell damage, individual cells within multicellular organisms undergo
programmed cell death or apoptosis. Mitochondria act as control centers for regulation of this
process.
One of the most potent activators of apoptosis is

cytochrome c. its presence in cytosol activates a cascade of
proteolytic enzymes called caspases which involves in
apoptosis. These activated caspases degrades important
proteins in the cell that maintain cellular structure, DNA
structure and favors the mechanisms of apoptosis.
Role of mitochondria in energy production

Mitochondria are subcellular organelles essential for the
aerobic metabolism in eukaryotes. Their main function is to
oxidize metabolic fuels and conserve free energy by
synthesizing ATP.
It has two membrane systems – outer and inner
mitochondrial membrane, and together they create two
separate mitochondrial compartments: internal matrix and
intermembrane space.
Mitochondria matrix contains many enzymes involved in
oxidation of metabolic fuels. These include pyruvate
dehydrogenase enzyme complex (for oxidative
decarboxylation of pyruvate to acetyl CoA for complete
oxidation of glucose), citric acid cycle enzymes, fatty acid β
oxidation enzymes, amino acid oxidation enzymes.
These oxidation pathways located in the mitochondrial
matrix generates high energy electrons which are carried
by the activated carrier molecules NADH and FADH2.
Inner mitochondrial membrane contains electron transport chain or respiratory chain and ATP
synthase for ATP production via oxidative phosphorylation.
Role of electron transport chain in ATP generation
 All of the energy liberated during oxidation of fatty acids and amino acids and nearly all of
energy released from oxidation of carbohydrate are available as reducing equivalents or high
energy electrons carried by NADH and FADH2.
 Finally, high energy electrons from NADH and FADH2 are passed along the electron transport
chain in inner mitochondrial membrane to their final reaction with oxygen to form water.
 There are 4 protein-lipid complexes in respiratory chain. Components of

the respiratory chain are flavoproteins, ubiquinone, Fe-S proteins and
cytochromes. These electron carriers are arranged in order of increasing
redox potential.
 Energy derived from the conductance of electrons
through the electron transport system is used by three
of the complexes (proton pumping of complex I, III and
IV in the number of 4, 4, 2) to pump protons into the
intermembrane space, creating an electrochemical
potential gradient or proton motive force. ATP synthase
that spans the inner membrane uses the potential
energy of the proton gradient or proton motive force to
synthesize ATP from ADP and Pi by rotary catalysis. In
this way, oxidation via ETC is closely coupled to
phosphorylation to generate ATP for the energy needs
of the cell.
 The ratio of ATP synthesized per ½O2 reduced to H2O
(the P/O ratio) is about 2.5 when electrons enter the
respiratory chain at Complex I (i.e., NADH) and 1.5 when
electrons enter at ubiquinone (i.e., FADH2).
 Cytosolic source of reducing equivalents are transported
to inner mitochondrial membrane by glycerol phosphate
shuttle or malate aspartate shuttle. NADH equivalents
moved in by the malate-aspartate shuttle enter the
respiratory chain at Complex I and yield 2.5 ATPs; those
moved in by the glycerol 3-phosphate shuttle enter at
ubiquinone and give 1.5 ATPs.
 Many well-known poisons such as cyanide arrest
respiration by inhibition of the respiratory chain.
 Lack of O2 supply or anoxia affects electron transport
along ETC subsequently ATP formation will be decreased
in the cell.
Carbohydrate Metabolism Page |1
Metabolism of carbohydrate
Introduction
Main purpose of
carbohydrate in
metabolism is to
supply and store
fuel molecules for
cellular metabolism.
Carbohydrate is mainly utilized by the cell as glucose. The end products of carbohydrate digestion are
monosaccharides; hexoses and pentoses.
The principle hexoses are glucose, galactose and fructose. Glucose accounts for 80 – 100% of the
total carbohydrate amount absorbed from GI tract, the remaining 20% or less are mannose from
vegetables, galactose from milk sugar (lactose) and fructose from cane sugar (sucrose). After
absorption from GI tract, essentially all mannose, galactose and fructose enter the liver for
conversion into glucose.
Transport of glucose into the cells or glucose uptake

 Glucose cannot diffuse directly into cells, but enters by one of two transport mechanisms;
o Na+ dependent co-transport system
o Na+ independent facilitated transport system
 Na+ dependent co-transport system
o It is an energy requiring process in which glucose or galactose is co-transported with Na+ into
cells. This transport process is linked to Na+ concentration gradient, to power the transport of
monosaccharides against their concentration gradient. This type of transport occurs in the
epithelial cells of the intestine, renal tubules and choroid plexus.
 Na+ independent facilitated transport system
o This system is mediated by one of among at least 14 glucose transporters (GLUTs) in cell
membranes. Each GLUT functions in the cells of specific tissues and binds to monosaccharide on
one side of the plasma membrane then undergoes a conformational change to transport and
deposit them on the other side of the plasma membrane down their concentration gradient.
o GLUT-1 – brain, kidney, erythrocytes
o GLUT-2 – liver, pancreatic cells, small intestine, kidney
o GLUT-3 – primary glucose transporter in neurons
o GLUT-4 – adipose tissues, cardiac and skeletal muscles (insulin dependent glucose transporters)
o GLUT-5 – primary fructose transporter in small intestine and testes
o GLUT-6 – mediates glucose flux across ER in liver.
Fate of glucose or glucose 6 phosphate

 Glucose entering the cell is rapidly phosphorylated
to glucose 6-phosphate and is subsequently

undergoes catabolic or anabolic fates in the cells.
 Catabolic fates
o Oxidation of glucose via glycolysis, oxidative

decarboxylation of pyruvate and citric acid cycle
o Oxidation via pentose phosphate pathway (hexose monophosphate shunt)
o Oxidation via uronic acid pathway
 Anabolic fates
o Glycogen synthesis (glycogenesis)

o Conversion to fat (lipogenesis)
o Conversion to non-essential amino acids
 Fate of glucose in the cell depends on the metabolic specialization of the tissues and metabolic state
of the cell.
Catabolic fate of glucose

 Glycolysis, oxidative decarboxylation of pyruvate and citric acid cycle
o Glucose 6-phosphate is oxidized via glycolysis which is the principle pathway for glucose
metabolism in all eukaryotic cells leading to production of end product pyruvate in aerobic
glycolysis and lactate in anaerobic glycolysis. It provides 7 molecules of ATP under aerobic
condition and 2 molecules of ATP under anaerobic condition.
o After aerobic glycolysis, end product pyruvate is oxidatively decarboxylated to acetyl CoA which
then undergoes citric acid cycle for complete oxidation to carbon dioxide and water.
o Anaerobic glycolysis is the major pathway of ATP production in some tissues e.g., RBCs, lens,
cornea, etc. It also provides ATP in the tissues in the absence of O2 e.g., in skeletal muscles during
strenuous exercise, for tissues survival during anoxic episodes.
 Pentose phosphate pathway or hexose monophosphate shunt
o It is a direct oxidative pathway of glucose in some tissues. This pathway generates NADPH for
reductive biosynthesis and ribose 5 phosphate for nucleotide synthesis. The activity of pentose
phosphate pathway is high in the erythrocytes, cornea, lens, liver, adipose tissues and
steroidogenic tissues.
 Uronic acid pathway
o It is a minor oxidative pathway in which glucose is converted to UDP glucose, glucuronic acid
and pentose sugars. UDP glucose is utilized for glycogen synthesis (liver and muscles). UDP
glucuronate is used for conjugation reactions of xenobiotic metabolism in liver and for the
synthesis of proteoglycans. Uronic acid pathway occurs mainly in liver parenchymal cells.
Anabolic fates of glucose

 Glycogen synthesis (glycogenesis)
o It is the synthesis of glycogen from glucose. UDP glucose provides glucose molecules for
glycogen synthesis. Glycogen synthesis occurs mainly in liver and muscle cells when glucose 6
phosphate and ATP are abundant in the cells.
 Conversion to fat (lipogenesis)
o Oxidative decarboxylation of glycolytic end product (pyruvate) provides acetyl CoA which can be
used as building blocks for fatty acid synthesis in liver and adipose tissues. During fed state,
excess acetyl CoA from mitochondria is exported to cytosol in the form of citrate for the
synthesis of fatty acids.
o Glycolysis intermediate DHAP also provides precursor for glycerol 3 phosphate synthesis for
esterification of fatty acids to form TAG. This process is called lipogenesis and occurs mainly in
liver and adipose tissues.
o TAG formed in liver is exported incorporated in VLDL into the blood stream but TAG synthesized
in adipose tissues is stored as lipid droplets.
o Acetyl CoA also serves as precursor for cholesterol synthesis.
 Conversion to non-essential amino acids
o Glycolytic end product pyruvate can be converted into non-essential amino acid alanine by
transamination reaction catalyzed by ALT.
o Glycolytic intermediate 3 phosphoglycerate provides precursor for serine synthesis.
Fates of glucose in red blood cells, liver parenchymal cells, skeletal and heart muscle cells and adipose
cells are illustrated in the following figures. (a=glucose uptake via glucose transporter or GLUT,
b=hexokinase or glucokinase (in hepatocytes), c=pentose phosphate pathway, d=glycolysis, e=lactate
export, f=oxidative decarboxylation (pyruvate dehydrogenase), g=citric acid cycle, h=glycogenesis,
i=glycogenolysis, j=lipogenesis, k=gluconeogenesis, l=glucose export, m=uronic acid pathway)
Glycolysis (Embden – Meyerhof pathway)

 Glycolysis is the series of reactions that oxidize glucose to pyruvate under aerobic condition or
lactate under anaerobic condition.
 Site – cytosol of all eukaryotic cells
 Stages of glycolysis
o Priming stage
 This step involves phosphorylation of glucose to glucose 6-phosphate (G6P) which is trapped
within the cells. It is catalyzed by hexokinase in extrahepatic tissues and by glucokinase in
liver.
 G6P is converted to fructose 6 phosphate (F6P) which then phosphorylated to fructose 1, 6-
bisphosphate (F-1, 6-BP) by phosphofructokinase-1 (PFK-1).
 This stage uses two molecules of ATP.
o Splitting stage
 Six carbon molecules F-1, 6-BP is cleaved to yield glyceraldehyde 3-phosphate and
dihydroxyacetone phosphate (DHAP) by aldolase. DHAP can be isomerized into
glyceraldehyde 3-phosphate by phosphotriose isomerase.
o Oxidoreduction and phosphorylation stage
 Two molecules of glyceraldehyde 3 phosphate are finally converted to 2 molecules of
pyruvate or lactate.
 NADH is produced during conversion of glyceraldehyde 3 phosphate to 1, 3-
bisphosphoglycerate (1, 3 BPG) by glyceraldehyde 3-phosphate dehydrogenase action.
 ATPs are produced at substrate level during phosphoglycerate kinase and pyruvate kinase
reactions.
 Net reaction in the transformation of glucose into pyruvate
o Glucose + 2ADP + 2Pi + 2NAD+  2pyruvate + 2ATP + 2NADH + 2H+ + 2H2O
Carbohydrate Metabolism P a g e |5
 Energy yield from glycolysis

o Two ATPs are used in the priming stage of glycolysis
and four ATPs are produced (substrate level oxidative
phosphorylation) in the conversion of glucose to two
molecules of pyruvate.
o Under aerobic conditions, two NADH yield 5 ATPs via
respiratory chain linked oxidative phosphorylation.
Therefore, there is net production of 7 ATPs in aerobic
glycolysis. Metabolic significance of hexokinase
+
o Under anaerobic condition, NAD is regenerated from and glucokinase
NADH is by conversion of pyruvate to lactate, but not  Hexokinase is present in most tissues,
whereas its isozyme, glucokinase
by respiratory chain as oxygen is not sufficient for
(hexokinase IV) is found only in liver
oxidation via respiratory chain. So net energy cells, and pancreatic β cells. They differ
production is 2 ATPs. in enzyme kinetic properties and their
sensitivity to changes in the levels of
End product of glycolysis is lactate under anaerobic
glycolytic intermediates.
conditions.  Hexokinase has a low Km value (high
End product of glucose oxidation via glycolysis is pyruvate affinity for glucose) and a low Vmax
under aerobic condition. Under aerobic condition, NADH value. This allows hexokinase to
catalyze its reaction even when blood
which is formed from glyceraldehyde 3-phosphate
glucose levels are low but it cannot
dehydrogenase reaction step is re-oxidized to NAD+ by
respond to very high glucose
oxidation through mitochondrial respiratory chain via concentration. Hexokinase is inhibited
malate aspartate or glycerol 3-phosphate shuttles. NAD+ by its product, glucose-6-phosphate

and is therefore most active when this
can then only be reutilized for glyceraldehyde 3-
product is being used up rapidly.
phosphate dehydrogenase reaction in further glycolysis.
 Glucokinase, in contrast, does not
During anaerobic glycolysis, NAD+ cannot be regenerated exhibit Michaelis-Menten kinetics
by mitochondrial respiratory chain since there is not with respect to glucose and has low
sufficient O2 for oxidation via mitochondrial respiratory affinity for glucose (high Km value).
It has higher Vmax value and cannot
chain (e.g., in exercising muscles or in tissues during
be inhibited by the product, glucose-
anoxic episodes) or mitochondria is absent in some cells, 6-phosphate. These characteristics
+
(e.g., erythrocytes). During anaerobic glycolysis, NAD is make glucokinase most active when
regenerated from NADH by lactate dehydrogenase blood glucose levels are high providing
more glucose 6-phosphate than is
reaction and NAD+ can only be then used for further
required for ATP production from glucose
glycolysis. In this reaction, pyruvate is reduced to lactate
oxidation; it is used for glycogen
and thus, the end product of glycolysis is lactate rather synthesis and lipogenesis in liver.
than pyruvate in anaerobic glycolysis.  Glucokinase can be hormonally
modulated. Elevated levels of insulin
induce glucokinase gene expression,
whereas glucagon represses it.
Biomedical importance and metabolic significance of

glycolysis
 It is the principal route for glucose metabolism
and also the main pathway for fructose and
galactose metabolism leading to production of
pyruvate which is then converted to acetyl CoA.
Acetyl CoA is oxidized in the citric acid cycle into CO2 and H2O. Inhibitors of glycolysis
Iodoacetate inhibits glyceraldehyde
 It provides 7 molecules of ATP under aerobic condition and 2
3 phosphate dehydrogenase.
molecules of ATP under anaerobic condition.
Arsenate is an analogue of inorganic
 It accounts for 90% of energy requirement in RBCs and 84% phosphate. Arsenate competes with
of that in the tissues of eye such as lens, cornea and retina. phosphate for its binding site in
glyceraldehyde 3 phosphate
 The ability to provide ATP in the absence of O2 allows
dehydrogenase (GAPDH), inhibiting
skeletal muscles to perform at very high level when aerobic
glycolysis.
oxidation becomes insufficient. This also allows tissues with Arsenite are also toxic, but have a
sufficient ability to survive anoxic episodes. However, heart different mechanism of action: they
muscles is adapted only aerobic performances, it has react with thiol groups in
sulfhydryl group of enzymes, e.g.,
relatively poor glycolytic ability and poor survival under
GAPDH, irreversibly inhibiting their
ischemic condition.
activity.
 Glycolysis rate is very high in fast growing cancer cells. Since Fluoride inhibits enolase. Fluoride
there is much higher rate of glycolysis than KCAC and ETC containing tubes are used for
collecting blood samples for glucose
oxidation, accumulated pyruvate leads to excessive
measurement.
production of lactate. Cancer cells tend to convert glucose
Fluoride in toothpaste may inhibit
into lactate, even in the presence of oxygen sufficient to the metabolism of bacteria in oral
support mitochondrial oxidative phosphorylation. This cavity by inhibiting their glycolysis
phenomenon is known as the Warburg effect. This results in and acid production (pyruvate,
lactate).
hypoglycemia and high local acidic environment which can
be applied in cancer therapy. In malignant tumor, such Biomedical implications
The high glycolytic rate in tumor cells
hypermetabolism leads to cancer cachexia.
(Warburg effect) has diagnostic
 In erythrocytes, glycolytic intermediate 1, 3 BPG can be
usefulness. The relative rates at
converted to 2, 3 BPG which binds to Hb and cause decrease which tissues take up glucose can be
in oxygen affinity of Hb; thus helping to unload O2 from Hb. used in some cases to pinpoint the
location of tumors.
 Glycolytic intermediate DHAP can be converted to glycerol 3-
In positron emission tomography
phosphate which is used for esterification of fatty acids.
(PET), individuals are injected with a
 Glycolytic intermediates can be used for synthesis of non- harmless, isotopically labeled glucose
essential amino acids e.g., conversion of 3-phosphoglycerate analog (2-fluoro-2-deoxyglucose or
to serine, pyruvate to alanine. FdG) that is taken up but not

metabolized by tissues.
Glucokinase deficiency
 The reversal steps of glycolysis are used in Heterozygous inactivating mutation in the
gluconeogenesis. glucokinase gene leads to only 50% of the
 Inherited deficiency of glycolysis enzymes such as normal glucokinase activity in liver and
pancreatic β cells.
pyruvate kinase deficiency leads to hemolytic anemia
Results in mild hyperglycemia that
and phosphofructokinase (PFK-1) deficiency leads to responds to dietary management in most
muscle fatigue. cases, diagnosed as maturity-onset
diabetes of the young (MODY).
Regulation of glycolysis
Three steps involving irreversible reactions catalyzed by hexokinase (or glucokinase in liver),
phosphofructokinase (PFK-1) and pyruvate kinase are the major sites in regulation of glycolysis.
There are three mechanisms in regulating the activity of key enzymes of glycolysis.
Allosteric mechanism
o Hexokinase is allosterically inhibited by glucose 6
phosphate.
o Pyruvate kinase is allosterically activated by F-1,6-BP
(positive feed-forward regulation) and inhibited by acetyl
CoA, citrate and ATP.
o Phosphofructokinase-1 (PFK-1) is allosterically activated by
AMP, F-2,6-BP, F6P and inhibited by ATP, citrate. PFK-1 is
also inhibited by H+ i.e., why acidosis reduces glycolysis and
2, 3 BPG formation.
o Fructose 2, 6 bisphosphate
 Fructose 2, 6 bisphosphate (F-2, 6-BP) is the most potent positive allosteric effector of PFK-1
and inhibitor of fructose 1, 6 bisphosphatase (key enzyme of gluconeogenesis).
 It relieves the inhibition of PFK-1 by ATP and increase affinity for fructose 6-phosphate.
 F-2, 6-BP is synthesized from fructose 6 phosphate by bifunctional enzyme, PFK-2/F-2,6-BPase
which has both kinase and phosphatase activities. Concentration of F-2, 6-BP is under both
substrate (allosteric) and hormonal control (covalent modification).
 In the phosphorylated state, affected by glucagon through cAMP dependent PKA, this
enzyme displays F-2,6-BPase activity which reduces the level of F-2,6-BP. Decrease in F-2,6-BP
diminish stimulation of glycolysis at PFK-1 and relieves inhibition of gluconeogenesis at F-1,6-
BPase.
 Insulin mediates dephosphorylation of PFK-2/F-2, 6-BPase turning on its PFK-2 activity. The
resultant increase in F-2, 6-BP activates PFK-1 and inhibits F-1, 6-BPase, thus glycolysis is
promoted and gluconeogenesis is inhibited.
Covalent modification by reversible phosphorylation
o Glucagon and to a lesser extent epinephrine inhibit glycolysis by phosphorylation and inactivation
of pyruvate kinase via cAMP dependent protein kinase activation. These hormones also affect the
concentration of F-2, 6-BP via cAMP signaling cascade.
o In contrast, insulin stimulates glycolysis by dephosphorylation and activation of pyruvate kinase
by activating protein phosphatases.
Induction and repression of enzyme synthesis
o Insulin induces the gene transcription of key enzymes involved in glycolysis thereby enhancing
the synthesis of key enzymes of glycolysis.
o Gene transcription and synthesis of glucokinase, phosphofructokinase and pyruvate kinase are
decreased when plasma glucagon is high and insulin is low.
Oxidative decarboxylation of pyruvate to acetyl CoA

This reaction is the link between glycolysis and
the citric acid cycle. It occurs in the mitochondrial
matrix.
Pyruvate formed in the cytosol is transported into
the mitochondria by pyruvate/H+ symporter.
Inside the mitochondria, pyruvate is oxidatively
decarboxylated to acetyl CoA by pyruvate dehydrogenase enzyme complex which consists of 3
enzymes; pyruvate dehydrogenase, dihydrolipoyl transacetylase and dihydrolipoyl dehydrogenase.
TPP, lipoamide, CoA-SH, FAD and NAD+ serve as coenzymes for the enzyme complex.
Pyruvate + NAD+ + CoA-SH  Acetyl CoA + NADH + H+ + CO2
o Inherited pyruvate dehydrogenase
deficiency results in lactic acidosis.
o Thiamin deficiency impairs glucose
metabolism and there is significant
lactic and pyruvate acidosis.
Carbohydrate Metabolism P a g e | 10
Nutritionally deprived alcoholics are thiamin deficient and glucose injection can result in
accumulation of pyruvate and lactic acidosis.
o Arsenic (arsenite) and mercury ions complex with the –SH group of lipoic acid and inhibit
pyruvate dehydrogenase enzyme complex.
Citric acid cycle (Kreb’s cycle or tricarboxylic acid cycle)

 Citric acid cycle is a series of reactions in mitochondria that oxidizes acetyl CoA to CO2 and liberates
reducing equivalents which are re-oxidized through the electron transport chain, linked to the
formation of ATP.
 This cycle operates only under aerobic conditions.
 In the TCA cycle, the oxidation of the two-carbon skeleton of acetyl CoA into CO2 occurs via eight
enzyme-catalyzed reactions. Since these reactions form a closed loop, the substrate oxaloacetate
used in the first reaction is regenerated in the last step and substrates involved in most of the
reactions steps are tricarboxylic acids, it is called tricarboxylic acid cycle.
 Site – mitochondrial matrix (Succinate dehydrogenase is found in the inner mitochondrial membrane,
whereas the remaining seven are soluble enzymes found in the mitochondrial matrix.)
 Steps involved in citric acid cycle
1) Synthesis of citrate from acetyl
CoA and oxaloacetate by citrate
synthase
2) Isomerization of citrate to
isocitrate by aconitase
3) Oxidation and decarboxylation
of isocitrate by isocitrate
dehydrogenase
4) Oxidative decarboxylation of α
ketoglutarate by α ketoglutarate
dehydrogenase enzyme complex
5) Cleavage of thioester bond of
succinyl CoA by succinate
thiokinase
6) Oxidation of succinate to
fumarate by succinate
dehydrogenase
7) Hydration of fumarate to malate
by fumerase
8) Oxidation of malate to regenerate oxaloacetate by malate dehydrogenase
Biomedical importance and metabolic significance

 Final common pathway for oxidation of metabolic fuels
o The citric acid cycle is the final common pathway for the oxidation of carbohydrate, lipid, and
protein because glucose, fatty acids, and most amino acids are metabolized to acetyl-CoA or
intermediates of TCA cycle.
o The common metabolite acetyl CoA reacts with oxaloacetate to form citrate. By a series of
oxidation (dehydrogenation) and decarboxylation reactions, citrate is degraded, liberating
reducing coenzymes (NADH, FADH2), 2CO2 and regenerating oxaloacetate.
o Three reaction steps produce NADH and another produces FADH2 which are oxidized by the
respiratory chain providing energy for ATP synthesis via respiratory chain linked oxidative
phosphorylation. One ATP is also produced in the cycle by substrate level oxidative
phosphorylation.
o Thus TCA cycle is the major pathway for formation of ATP from metabolic fuel oxidation
providing 10 ATPs per oxidation of one mole of acetyl CoA.
 Amphibolic nature of citric acid cycle
o The citric acid cycle is an amphibolic pathway and

plays in both oxidative and synthetic processes.
o The citric acid cycle is the final common pathway
for the oxidation of carbohydrate, lipid, and protein
because glucose, fatty acids, and most amino acids
are metabolized to acetyl-CoA or CAC
intermediates.
o It also has a central role in gluconeogenesis,
lipogenesis, and interconversion of amino acids.
Several TCA cycle intermediates serve as precursors
for anabolic pathways that synthesize biomolecules
such as glucose, lipids, and amino acids.
 It participates in the synthesis of glucose during starvation and fasting (gluconeogenesis) by
contributing oxaloacetate.
 It is also involved in the conversion of carbohydrates to fat following a carbohydrate-rich
meal by contributing citrate to generate acetyl CoA for lipogenesis in cytosol.
 It is a source of non-essential amino acids, such as aspartate and glutamate, which are
synthesized directly from TCA cycle intermediates, oxaloacetate and α ketoglutarate
respectively via transamination reaction.
 One TCA cycle intermediate succinyl-CoA serves as a precursor for porphyrins (heme)
synthesis in all cells, especially in bone marrow and liver.
 Disruption of the function of TCA cycle by vitamin deficiencies
o Since α ketoglutarate dehydrogenase enzyme complex require 5 coenzymes (TPP, FAD, NAD+,
CoA and lipoic acid) and some enzymes require coenzymes such as NAD + and FAD, a deficiency
in their precursor vitamins disrupts the function of the TCA cycle and causes a dramatic decrease
in ATP production e.g., Beriberi in vitamin B1 deficiency, pellagra in vitamin B3 deficiency.
 Inhibitors of TCA cycle enzymes
o Fluoroacetate is a rat poison that inhibits the TCA cycle. Fluoroacetate reacts with CoA to form
fluoroacetyl CoA, which (instead of acetyl CoA) condenses with oxaloacetate to produce
fluorocitrate. Fluorocitrate is an analogue of citrate and is a competitive inhibitor of aconitase.
Inhibition of aconitase leads to accumulation of citrate which in turn inhibits citrate synthase.
o Arsenic (arsenite) and mercury ions complex with the –SH group of lipoic acid and inhibit α
ketoglutarate dehydrogenase enzyme complex.
o Compounds that are structurally similar to succinate inhibit TCA cycle by competing with
succinate for binding to succinate dehydrogenase. These include TCA cycle intermediates malate,
oxaloacetate as well as chemical malonate, a dicarboxylic acid which is a structural analogue of
succinate and is a strong competitive inhibitor of succinate dehydrogenase once used in the
studies to map the active site of the
enzyme.
Regulation of citric acid cycle

 Regulation of the citric acid cycle depends
primarily on the supply of oxidized cofactors,

e.g., NAD+ which in turn is dependent on the
rate of utilization of ATP. Thus, when cellular
ATP levels are low, the activity of the TCA cycle
is increased to provide more NADH to serve as
a substrate for oxidative phosphorylation to
generate ATP. Conversely, when cellular ATP levels are
high, the production of NADH via TCA cycle and its
oxidation via the mitochondrial electron transport chain
are inhibited.
 Regulation of the cycle is controlled by the three
enzymes (citrate synthase, isocitrate dehydrogenase,

and α ketoglutarate dehydrogenase), which catalyze the
irreversible reactions. These enzymes are activated by
increased Ca2+ concentration during muscular
contraction when there is increased energy demand.
 ATP and long chain acyl CoA allosterically inhibit citrate synthase. It is also inhibited by NADH, citrate,
succinyl CoA and stimulated by ADP. ADP allosterically stimulates whereas ATP inhibits isocitrate
dehydrogenase. α ketoglutarate dehydrogenase is inhibited by succinyl CoA and NADH. Succinate
dehydrogenase is inhibited by oxaloacetate and malate dehydrogenase activity depends on
[NADH]/[NAD+] ratio. Malate dehydrogenase activity is inhibited by high [NADH]/[NAD+] ratio.
Hexose monophosphate shunt (Pentose phosphate pathway)

 Pentose phosphate pathway is a direct oxidative pathway of glucose. It is a multicyclic process in
which 3 moles of G6P give rise to 2 moles of G6P and one mole of glycolytic intermediate. This
pathway generates NADPH and ribose 5-phosphate but it does not generate ATP.
 This pathway is found in the cytosol of tissues like liver, adipose tissues, steroidogenic tissues,
thyroid gland, lactating mammary gland, erythrocytes, lens and cornea.
 Two phases of HMS pathway
o Irreversible oxidative phase
 Dehydrogenation of glucose 6-phosphate to 6-
phosphogluconate by NADP+ dependent glucose
6-phosphate dehydrogenase (G6PD) releasing
NADPH and H+
 Oxidative decarboxylation of 6-phosphogluconate
to ribulose 5-phosphate by NADP+ dependent 6-
phosphogluconate dehydrogenase (6PGD)
+
releasing NADPH, H and CO2
o Reversible non-oxidative phase

 It consists of a series of
reversible reactions that
start with the isomerization
and epimerization of three
molecules of ribulose 5-
phosphate, followed by
reactions in which two and
three carbon segments are
transferred between
intermediates. The end
result is the production of
two fructose 6-phosphates
and one glyceraldehyde 3-
phosphate. The following
are key features of the two
enzymes that catalyze the
transfer reactions:
 Transketolase enzyme requires TPP as a coenzyme
and catalyzes the transfer of two-carbon segments
from ketose to aldehyde carbon of aldose sugar.
 Transaldolase enzyme catalyzes the transfer of
three-carbon segments (dihydroxyacetone moiety)
from ketose sugar (sedoheptuloase 7-phosphate)
to aldose sugar (glyceraldehyde 3-phosphate)
resulting in formation of fructose 6-phosphate
(ketose) and erythrose 4-phosphate (aldose).
Sources of NADPH
Biomedical importance and metabolic significance of HMS pathway
1) Pentose phosphate pathway
 Pentose phosphate pathway does not produce ATP but produce
2) NADP+ dependent cytosolic malic
NADPH and ribose 5-phosphate intermediate. enzyme reaction
 It provides ribose 5-phosphate intermediate that reacts with ATP 3) NADP+ dependent cytosolic
isocitrate dehydrogenase reaction
to form 5-phosphoribosyl 1-pyrophosphate (PRPP) to be used in
nucleotide biosynthesis. Ribose 5-phosphate can be synthesized in virtually all tissues by reversal of
the non-oxidative phase of the pentose phosphate pathway utilizing fructose-6-phosphate.
 It also provides NADPH which serves as coenzymes for many metabolic processes and biochemical
reactions e.g., reductive synthesis such as fatty acid synthesis, steroidogenesis, cholesterol synthesis,
Importance of GSH
amino acid synthesis via glutamate dehydrogenase, xenobiotic
 GSH is used for removal of
metabolism in liver and regeneration of reduced glutathione in
hydrogen peroxide by selenium
erythrocytes, cornea, lens and leukocytes. containing glutathione peroxidase
 Metabolic significance of NADPH (Uses of NADPH) thereby preventing accumulation
of H2O2 in erythrocytes.
o Reductive synthesis such as fatty acid synthesis, steroid
 GSH is important for maintenance
hormone synthesis, cholesterol synthesis, bile acid synthesis,
of reduced sulfhydryl groups in
amino acid synthesis via glutamate dehydrogenase. proteins and antioxidant action of
o Critical importance for liver microsomal cytochrome P450 glutathione peroxidase, providing
mono-oxygenase system involved in hydroxylation reactions defense against deleterious effect

of ROS in lens and cornea.
of xenobiotic metabolism in liver
 GSH is essential for removal of
o Hydroxylation reactions by liver and kidney mitochondrial excess peroxide formed during
cytochrome P450 mono-oxygenase system in 1, 25 DHCC phagocytosis by glutathione
synthesis peroxidase in leukocytes.
o Regeneration of reduced glutathione (GSH) which is essential for antioxidant action of

glutathione peroxidase in removal of H2O2 in erythrocytes, lens, cornea, leukocytes and
maintenance of reduced sulfhydryl groups in proteins of lens, cornea providing defense against
deleterious effects of ROS
o Prevention of methemoglobin formation by maintaining iron of Hb in reduced ferrous state
o Formation of ROS by NADPH oxidase reaction for phagocytosis by WBCs
o Conversion of glucose to sorbitol by aldose reductase
o Nitric oxide synthesis by nitric oxide synthase
o Conversion of folic acid to active form tetrahydrofolate by the action of dihydrofolate reductase
o Oxidation of iodide by thyroperoxidase in thyroid hormone synthesis in thyroid gland
o Formation of deoxyribonucleoside diphosphates from ribonucleoside diphosphates by
ribonucleotide reductase enzyme complex
Glucose 6 phosphate dehydrogenase deficiency

G6PD deficiency is a hereditary disease characterized
by hemolytic anemia caused by the inability to
detoxify oxidizing agents. G6PD deficiency is the most
common disease-producing enzyme abnormality in
humans. It is a X-linked recessive genetic disorder
caused by a number of different mutations in the
gene encoding for G6PD. Diminished G6PD activity
impairs the ability to generate NADPH that is
essential for the maintenance of the G-SH pool
resulting in a decrease in the detoxification of free radicals and peroxides formed within the cell.
Although G6PD deficiency occurs in all cells, erythrocytes are particularly vulnerable because HMS
pathway is the only means of generating NADPH. In contrast, other tissues have alternate sources
for NADPH production e.g., NADP+ dependent malic enzyme, cytosolic isocitrate dehydrogenase.
In the erythrocytes, NADPH generated by pentose phosphate pathway is utilized for the reduction of
oxidized glutathione to reduced glutathione catalyzed by flavoprotein enzyme glutathione
reductase. In turn, reduced glutathione (GSH) is used for removal of hydrogen peroxide and
peroxides by a reaction catalyzed by glutathione peroxidase. Reduced activity of glutathione
peroxidase due to diminished GSH pool leads to accumulation of hydrogen peroxides in RBCs. This
may cause lipid peroxidation damage to erythrocyte cell membrane and increasing rate of oxidation
of hemoglobin to methemoglobin; all of which resulting in decreasing life-span of erythrocytes and
hemolytic anemia.
Most individuals who have inherited G6PD deficiency do not show clinical manifestations. However,
some patients with G6PD deficiency develop hemolytic anemia if they are treated with an oxidant
drug e.g., sulfamethoxazole (antibiotic), primaquine (antimalarial), acetanilide (antipyretic), ingest
fava beans, or contract a severe infection.
Regulation of pentose phosphate pathway

 Whether glucose 6-phosphate enters glycolysis or the pentose phosphate pathway depends on the
current needs of the cell and on the concentration of NADP+ in the cytosol. When a cell is rapidly
converting NADPH to NADP+ in biosynthetic reductions, the level of NADP+ rises, allosterically
stimulating G6PD and thereby increasing the flux of glucose 6-phosphate through the pentose
phosphate pathway. Insulin induces the synthesis of G6PD and 6PGD enzymes thereby promoting
pentose phosphate pathway.
Uronic acid pathway

 It is an alternative pathway for the conversion of
glucose to glucuronic acid, ascorbic acid (but not
in all primates, guinea pigs, fruit eating bats) and
pentose sugar.
 Site – occurs mainly in the cytoplasm of liver
parenchymal cells.
 Steps in uronic acid pathway
o Conversion of glucose 6-phosphate to glucose
1-phosphate by phosphoglucomutase
o Formation of UDP glucose by the action of
UDP glucose pyrophosphorylase
o Oxidation of UDP glucose to UDP glucuronate
by NAD+ dependent UDP glucose
dehydrogenase followed by hydrolytic cleavage of UDP
o Reduction of L-glucoronate to L-gulonate
 L gulonate is the direct precursor of ascorbate in animals capable of synthesizing vitamin C.
 Humans and other primates, guinea pigs and fruit eating bats lack enzyme L gulonolactone
oxidase required for ascorbate synthesis.
o Oxidation and decarboxylation of L gulonate to pentose L-xylulose
o NADPH-dependent reduction of L-xylulose to D-xylitol
o Oxidation of D-xylitol to D-xylulose by NAD+ dependent D-xylulose reductase
o Conversion of D-xylulose to D-xylulose 5-phosphate which is further metabolized in the pentose
phosphate pathway Proteoglycans
 Long chains of repeating
 Biomedical importance and metabolic significance
disaccharides consisting of
o This pathway is significant for excretion of metabolites and amino sugar and acid sugar
xenobiotic metabolism by providing UDP-glucuronate for (glucosamine-glycans/GAGs)
conjugation reaction. attached to specific core protein

 Formerly known as muco-
o UDP-glucuronate is also used for the synthesis of
polysaccharides.
glycosaminoglycans (GAGs) and proteoglycans
 Important biomolecules present
(mucopolysaccharides). in the extracellular matrix
o Xylitol naturally found in some foods such as plums, spinach and carrots or used as artificial
sweetener in some foodstuffs such as sugar less chewing gum is principally metabolized in the
liver where it is converted to glucose. Xylitol is absorbed more slowly from the intestinal tract
that other sugars and its metabolism does not depend on insulin.
o Hereditary enzyme defects in uronic acid pathway cause essential pentosuria.
o Various drugs increase the rate of glucose entry into the uronic acid pathway e.g., administration
of barbital or chlorobutanol.
Glycogen metabolism
Glycogen is a branched
polysaccharide of glucose
(homoglucan) and is a
storage form of glucose.
It contains only two types of glycosidic linkages, chains of α 1-4 linked
glucose residues with α 1-6 branches spaced about every 4 – 6 residues
along the α 1-4 chain. Its structure is closely related to starch, the storage
form of polysaccharides in plants, consisting of linear α 1-4 chain amylose
and branched chain amylopectin component with fewer α 1-6 branches
compared to glycogen.
It is stored mainly in liver and skeletal muscles. Liver glycogen is used to
maintain blood glucose level. Muscle glycogen is to provide a source of
energy upon prolonged muscle contraction.
Glycogenesis
 It is the synthesis of glycogen from
molecules of D-glucose.
 Site – occurs mainly in the cytoplasm of
liver and skeletal muscle cells.
 Steps in glycogenesis
o Synthesis of UDP glucose
 UDP glucose is the activated form
of glucose to be used as glucose
precursors for glycogen synthesis.
Synthesis of UDP glucose consists
of three steps –
1. Conversion of glucose to
glucose 6-phosphate catalyzed by glucokinase in liver and hexokinase in skeletal muscles
2. Isomerization of glucose 6-phosphate to glucose 1-phosphate by phosphoglucomutase
3. Reaction of glucose 1-phosphate and UTP to form activated form UDP glucose catalyzed
by UDP glucose pyrophosphorylase
o Formation of glycogen primer (glycogenin)
 A pre-existing glycogen primer or glycogenin is required for glycogen synthesis.
 Glycogenin is a protein and has self-glycosylating enzyme

activity using UDP glucose to link glucose to specific tyrosine
residues of its structure. Further glucose residues are linked
to the pre-existing glucose residue in glycogenin for
elongation of glycogen chain.
o Elongation of glycogen chain
 Glycogen synthase catalyzes the formation of α 1-4
glycosidic bond between C1 of activated UDP glucose and C4
of pre-existing terminal glucose residue of glycogen primer;
releasing UDP.
o Formation of branches
 It is catalyzed by the branching enzyme in which a part of α
1-4 chain (about 7 oligosaccharides) is transferred to a
neighboring chain to form α 1-6 linkage, thus establishing a
branch point. The branch grows by further addition of 1-4 glycosyl units.
Regulation of glycogen synthesis

 Key enzyme in glycogenesis is glycogen synthase. Glycogen
synthase may exist as an inactive phosphorylated form

(glycogen synthase b) and an active dephosphorylated form
(glycogen synthase a).
 It is regulated by allosteric mechanism, covalent modification
and regulation of glycogen synthase gene expression.

 Allosteric mechanism
o Glucose 6-phosphate is an allosteric activator of

glycogen synthase b causing a decrease in Km for UDP
glucose and allowing glycogen synthesis by the
phosphorylated enzyme.
o Insulin stimulates glycogenesis by increasing glucose 6-phosphate concentration.
 Covalent modification
o The active dephosphorylated form (glycogen synthase a) can be phosphorylated and inactivated
to inactive phosphorylated form (glycogen synthase b) by different protein kinases such as
Ca+/calmodulin dependent protein kinase, cAMP dependent protein kinase and glycogen
synthase kinases.
o Glucagon inhibits glycogen synthesis in liver by phosphorylating glycogen synthase via cAMP
dependent protein kinase activation. Likewise, epinephrine inhibits glycogen synthesis in both
liver and muscle via cAMP signaling cascade.
o Insulin stimulates glycogen synthesis in liver and muscle by dephosphorylating glycogen

synthase via protein phosphatase activation. Insulin also inhibits glycogen synthase kinase 3
(GSK3) and decreases cAMP level by activating cAMP phosphodiesterase thereby opposing
inhibitory action of glucagon and epinephrine on glycogenesis.
 Regulation of gene expression
o Glucocorticoids induce glycogen synthase gene expression in

liver thereby increasing hepatic glycogen synthesis.
Glycogenolysis
Glycogenolysis is the breakdown of glycogen to free glucose in liver
and to glucose 6-phosphate in muscle due to the absence of glucose
6-phosphatase in muscles.
Site – cytoplasm of liver and muscle cells
Steps in glycogenolysis
o Shortening of the chains
 The terminal glycosyl residues from the outermost chains of glycogen are sequentially
removed until approximately four glucose molecules remain on either side of 1-6 branch.
 This process is catalyzed by glycogen phosphorylase that cleaves terminal α 1-4 linkage to
release glucose 1-phosphate.
o Removal of branches
 It involves glucan transferase and debranching enzyme. Glucan transferase transfers a
trisaccharide unit from one branch to other, exposing α 1-6 branch point. Debranching
enzyme catalyzes hydrolytic cleavage of exposed α 1-6 linkage releasing free glucose.
o Conversion of glucose 1-phosphate to glucose 6-phosphate
 Glucose 1-phosphate is converted to G6P by the action of phosphoglucomutase.
 In liver, glucose 6-phosphate is then hydrolyzed by glucose 6-phosphatase yielding glucose
to increase blood glucose concentration. In muscles, the end product of glycogenolysis is
glucose 6-phosphate due to lack of glucose 6-phosphatase.
Regulation of glycogenolysis
 Key enzyme in glycogenolysis is glycogen phosphorylase. Glycogen phosphorylase may exist as an
inactive dephosphorylated form (glycogen phosphorylase b) and an active phosphorylated form

(glycogen phosphorylase a).
 It is regulated by allosteric mechanism
and covalent modification.

 Control of glycogenolysis in liver and
muscle differs in some aspects.

 Control of glycogenolysis in liver
o Allosteric mechanism
 Glycogenolysis in liver is to maintain
blood glucose level by exporting
free glucose into the blood. Thus,
free glucose and glucose 6-
phosphate inhibit active
phosphorylase. ATP also inhibits
glycogen phosphorylase in liver.
o Covalent modification
 Glucagon stimulates glycogenolysis
in liver by phosphorylating and
activating phosphorylase kinase via
activation of cAMP dependent
protein kinase. Activated
phosphorylase kinase in turn
phosphorylates and activates phosphorylase enzyme.
 Epinephrine and norepinephrine also activates glycogenolysis in liver by activating cAMP
dependent protein kinase via β adrenergic receptor. Catecholamines can also stimulate
glycogenolysis in liver by activating calmodulin component of phosphoryalse kinase via
increasing cytosolic Ca2+ concentration through α1 adrenergic receptor.
 Protein phosphatase 1 inactivates glycogen phosphorylase by dephosphorylation. Protein
phosphatase 1 activity is inhibited by inhibitor protein 1 which is active only after it has been
phosphorylated by cAMP dependent PKA.
 Insulin inhibits glycogenolysis in liver by dephosphorylating and inactivating phosphorylase
kinase and glycogen phosphorylase via activation of protein phosphatase 1. It also decreases
cAMP level by activating cAMP phosphodiesterase thereby opposing stimulatory action of
glucagon and epinephrine on glycogenolysis in liver.
 Control of glycogenolysis in muscles
 Muscles lack glucose 6-phosphatase.
Thus glycogenolysis in muscles is to
provide glucose 6-phosphate to be
used as energy fuel for muscle
contraction. Thus, AMP (potent
signal for low energy state of
muscle cell) and Ca2+ act as allosteric activator for phosphorylase and ATP, glucose 6-
phosphate allosterically inhibit active phosphorylase enzyme.
 Muscles lack glucagon receptor. So muscle glycogenolysis is activated only in response to
epinephrine through cAMP dependent PKA activation via β adrenergic receptor.
 The subunit composition of muscle phosphorylase kinase is (αβγδ)4. The catalytic activity
resides in the γ subunit. This kinase is under dual control; it is activated both by
phosphorylation via PKA and by increases in Ca2+ levels at the onset of muscle contraction. δ
subunit is identical to calmodulin and binds four Ca2+. Phosphorylase kinase attains maximal
activity only after both phosphorylation of α and β subunits and activation of the δ subunit by
Ca2+ binding.
 Insulin inhibits glycogenolysis in muscles by dephosphorylating and inactivating
phosphorylase kinase and glycogen phosphorylase via activation of protein phosphatase 1.
Biomedical importance and metabolic significance

During and immediately following a meal, glucose is converted into glycogen by glycogenesis in both
liver and muscles. Glycogen synthesis occurs mainly in the liver (up to 6%) and in muscles (up to 1%).
However, because of its greater mass, muscle represents three to four times as much glycogen store
as liver.
Metabolic significance of liver glycogen
o Liver glycogen is the first line of defense against declining blood glucose concentration. Hepatic
glycogen serves for storage of glucose during fed state and export of glucose to maintain blood
glucose particularly between meals. However, total hepatic glycogen stores are sufficient for
maintenance of blood glucose concentration during 12 to 18 hours fasting.
o High liver glycogen content also contributes the following benefits;
 Depresses of deamination of amino acids and rate of ketogenesis in liver
 Favors the detoxification by acetylation or glucuronide formation
 Protects the liver against the harmful effects of many toxins e.g., carbon tetrachloride, ethyl
alcohol.
Metabolic significance of muscle
glycogen
o Muscle glycogen is not available
for maintenance of blood
glucose since it cannot export
glucose due to the absence of
glucose 6-phosphatase.
o Muscle glycogen functions as source of glucose 6-phosphate for glycolysis within the muscles
during bursts of physical activity. It is depleted significantly after prolonged vigorous exercise.
Inherited deficiencies in specific enzymes of glycogen metabolism in liver and muscles cause
glycogen storage diseases.
Metabolic consequences of glucose 6-phosphatase deficiency in Von Gierke’s disease
Gluconeogenesis
Gluconeogenesis is the synthesis of glucose from
non-carbohydrate precursors. These precursors
include glucogenic amino acids, lactate, pyruvate,
propionate and glycerol.
Site – liver and kidney. Liver is the major site of
gluconeogenesis (about 90%) whereas glucose
amount contributed by kidney is about one-tenth
that of liver but this becomes important during
prolonged starvation.
Steps in gluconeogenesis
o Gluconeogenesis pathway is the modification
and adaptation of glycolysis and citric acid cycle
and involves both cytosolic and mitochondrial
enzymes. Except for three key reactions, the
reactions of gluconeogenesis are reversals of
glycolysis process. The reversal of glycolysis is bypassed between
 Conversion of pyruvate to PEP by mitochondrial enzyme pyruvate carboxylase and cytosolic
enzyme phosphoenol pyruvate carboxykinase (PEPCK)
 Conversion of fructose-1,6-bisphosphate to fructose 6-phosphate by fructose 1, 6-
bisphosphatase
 Conversion of glucose 6-phosphate to glucose by glucose 6-phosphatase
Synthesis of glucose from lactate

Lactate formed by anaerobic glycolysis
in skeletal muscles and RBCs is
transported to liver via blood stream.
In the liver, lactate is oxidized to
pyruvate by lactate dehydrogenase.
Pyruvate cannot be converted to PEP
due to high energy barrier. Thus
pyruvate enters the mitochondria
where it is carboxylated to
oxaloacetate by pyruvate
carboxylase; requiring biotin and ATP.
Since oxaloacetate cannot cross the
mitochondrial membrane, it is thus
reduced to malate by CAC enzyme
malate dehydrogenase and
transported to cytosol. In the cytosol,
malate is re-oxidized to oxaloacetate
by cytosolic malate dehydrogenase.
Cytosolic oxaloacetate is
decarboxylated and phosphorylated
by PEPCK using GTP and yields PEP.
PEP then enters the reversal reactions of glycolysis until it is converted to fructose-1, 6-bisphosphate
which is then hydrolyzed by fructose-1, 6-bisphosphatase enzyme to fructose 6-phosphate. Fructose
6-phosphate is further converted to glucose 6-phosphate. G6P is then hydrolyzed to free glucose by
glucose 6-phosphatase. Two moles of lactate are required to form one glucose molecule via
gluconeogenesis.
Gluconeogenesis from pyruvate is energetically expensive, requiring a net expenditure of 6 high
energy phosphate bonds (4ATPs and 2GTPs) per conversion to one molecule of glucose i.e., 2 moles
of ATP at pyruvate carboxylase reaction, 2 moles of GTP at PEPCK reaction and 2 moles of ATP at
phosphoglycerate kinase reaction. These ATPs are provided by oxidation of fatty acids.
Cori cycle or lactic acid cycle

Lactate formed by anaerobic glycolysis in skeletal muscle and erythrocytes, is transported to the liver
and kidney where it reforms glucose, which again becomes available via the circulation for oxidation
in the tissues. This process is known as the Cori cycle, or the lactic acid cycle. This cycle is especially
important for the removal of lactic acid generated from skeletal muscles during strenuous exercise.
Glucose alanine cycle

In the fasting state, there is a considerable output of alanine from skeletal muscle, far in excess of the
amount in the muscle proteins that are being catabolized. It is formed by transamination of pyruvate
produced by glycolysis of G6P derived from muscle glycogen breakdown. Alanine is exported to the
liver, where, after transamination back to pyruvate, it can serve as a substrate for gluconeogenesis.
This glucose-alanine cycle thus provides an indirect way of utilizing muscle glycogen to maintain
blood glucose in the fasting state.
Synthesis of glucose from glucogenic amino acids

All amino acids except leucine and lysine can supply carbon skeleton for the net synthesis of glucose
by gluconeogenesis.
The catabolism of glucogenic amino acids produces CAC intermediates (α-ketoglutarate, succinyl
CoA, fumarate) or pyruvate which can be converted to malate via CAC. Malate is then transported to
cytosol where it is re-oxidized to oxaloacetate; providing carbon skeleton for gluconeogenesis.
Alanine and glutamine are the major amino acids exported from muscle for gluconeogenesis. Alanine
formed in muscles by protein degradation is transaminated to pyruvate and converted to glucose in
the liver.
Synthesis of glucose from glycerol and propionate

Glycerol is derived from hydrolysis of triacylglycerol (lipolysis) in adipose tissues. Glycerol is
phosphorylated by glycerol kinase to glycerol 3-phosphate and then converted into DHAP by
glycerol 3-phosphate dehydrogenase action. DHAP enters the reversal of glycolysis to form glucose.
Conversion of glycerol into glucose requires only 2 moles of ATP per mole of glucose produced.
Acetyl CoA and even chain fatty acids cannot serve as substrates for net gluconeogenesis. However,
propionate, the end product of catabolism of odd chain fatty acids can serve as minor precursor for
gluconeogenesis. It is also produced in

the catabolism of valine, isoleucine and
conversion of cholesterol to bile acids.
Propionyl CoA is first carboxylated to
methylmalonyl CoA which undergoes
isomerization reaction to form succinyl
CoA, a CAC intermediate. Succinyl CoA
can then converted into oxaloacetate via
CAC and enter gluconeogenesis pathway.
Regulation of gluconeogenesis
Since gluconeogenesis and glycolysis
essentially use the same pathway except
for some key steps, these pathways are
reciprocally regulated so that one
pathway is relatively inactive while the
other is highly is active.
Regulation of gluconeogenesis is determined primarily by glucogenic substrates. Gluconeogenesis
can also be regulated by control of key gluconeogenic enzymes activities via allosteric mechanism,
covalent modification as well as by controlling the rate of key enzymes synthesis.
Key enzymes of gluconeogenesis are pyruvate carboxylase, PEPCK, fructose 1, 6-bisphophatase,
glucose 6-phosphatase.
Allosteric mechanism
o Pyruvate carboxylase is allosterically activated by acetyl CoA during starvation. During starvation,
rate of formation of acetyl CoA by β oxidation of fatty acids exceeds body’s ability to oxidize via
CAC resulting in accumulation of acetyl CoA leading to activation of pyruvate carboxylase.
o F-2, 6-BP formed from F6P by PFK-2 allosterically inhibits fructose 1, 6-bisphosphatase. It is also
inhibited by fructose 1, 6-bisphosphate and AMP. Under condition of glucose shortage, low
concentration of F-2, 6-BP allows gluconeogenesis to occur by releasing the inhibition of fructose
1, 6-bisphosphatase.
o Increase in ATP level with consequent decrease in AMP favors gluconeogenesis by activating
fructose 2, 6-bisphosphatase and inhibiting PFK-1 and pyruvate kinase (glycolysis).
Covalent modification
o The primary control point in reciprocal regulation of glycolysis and gluconeogenesis is at PFK-1
and F-1, 6-BPase key enzymes. Activity of these enzymes are sensitive to the allosteric effector F-
2, 6-BP. F-2, 6-BP activates PFK-1 and inhibits F-1, 6-BPase thereby counter-regulating the two
opposing pathways.
o F-2, 6-BP is synthesized by bifunctional enzyme, PFK-2/F-2,6-BPase which is under hormonal

control via covalent modification.
o In the phosphorylated state, affected by glucagon through cAMP dependent PKA, this enzyme
displays F-2, 6-BPase activity which reduces the level of F-2, 6-BP. Decrease in F-2, 6-BP diminish
stimulation of glycolysis at PFK-1 and relieves inhibition of gluconeogenesis at F-1, 6-BPase, thus
leading to liver cells in a gluconeogenic mode.
o Insulin mediates dephosphorylation of PFK-2/F-2, 6-BPase via activation of protein phosphatase,
turning on PFK-2 activity. The resultant increase in F-2, 6-BP activates PFK-1 and inhibits F-1, 6-
BPase, thus glycolysis is promoted and gluconeogenesis is inhibited.
Control of gene expression of key enzymes
o Hormonal control contributes long-term effects upon the levels of hepatic enzymes involved in
gluconeogenesis. A high glucagon/insulin ratio in the blood increases the capacity for
gluconeogenesis. Glucocorticoids, glucagon and epinephrine are the inducers of gluconeogenic
enzymes (pyruvate carboxylase, glucagon, PEPCK, fructose 1, 6-bisphosphatase, glucose 6-
phosphatase) and insulin is the repressor of these enzymes synthesis.
o Glucagon, via cAMP signaling cascade and CREB protein, increases the rate of transcription of
PEPCK gene, thus increasing the rate of synthesis of PEPCK. Insulin decreases the rate of
transcription of PEPCK gene, thus decreasing the rate of synthesis of PEPCK.
Biomedical importance and metabolic significance of gluconeogenesis

 Gluconeogenesis provides glucose to the body when carbohydrate is not available in sufficient
amount in diet, because glucose is the most important fuel for every cells especially nervous tissues
and RBCs. It can result in coma and death if blood glucose level falls below 2.2 mmol/L in adults.
Children are more susceptible due to larger brain/body weight ratio than adults since brain utilizes
disproportionately greater amount of glucose than the rest of the body. Premature and small for
gestational age neonates have a greater susceptibility to hypoglycemia than full term because of
smaller stores of liver glycogen and presence of very low amount of PEPCK during first hour after
birth.
 Gluconeogenesis also clears the products of metabolism of other tissues from the blood stream
(lactate from skeletal muscles and erythrocytes) and glycerol from adipose tissues.
 Glucose is the only fuel that supplies energy to skeletal muscle under anaerobic condition.
 Glucose is required by the adipose tissues as a source of glycerol 3-phopshate and fatty acids.
 Glucose is the precursor of lactose in the mammary gland.
Metabolism of fructose
Sources – sucrose, fruits, vegetables, honey Site – liver, adipose tissues
The entry of fructose into the cells is insulin independent.
Fructose metabolism in liver

o Fructose is metabolized in liver via
fructose 1-phosphate pathway.
o Steps in fructose metabolism
 Phosphorylation of fructose to
fructose 1-phosphate by
fructokinase
 Splitting of fructose 1-
phosphate into glyceraldehyde
and DHAP by fructose 1-
phosphate aldolase (aldolase B)
 Phosphorylation of
glyceraldehyde by triose kinase
to form glyceraldehyde 3-
phosphate which then enters
into glycolysis
o Two triose phosphates, DHAP and
glyceraldehyde 3-phosphate, may either be degraded by glycolysis or may be substrates for
gluconeogenesis in the liver.
o Alternative pathway of fructose metabolism in liver is fructose 6-phosphate pathway but little
fructose 6-phosphate is formed in liver because of abundance of glucose in relative to fructose.
o Fructose undergoes more rapid glycolysis in the liver than does glucose because it bypasses the
regulatory step catalyzed by PFK-1. Increased dietary intake of fructose significantly enhances
the formation of acetyl CoA in liver leading to increased fatty acid synthesis, esterification of
fatty acids, secretion of VLDL which may raise serum TAG and LDL cholesterol concentrations.
In extra-hepatic tissues, hexokinase catalyzes the phosphorylation of most hexose sugars, including
fructose, but glucose inhibits the phosphorylation of fructose since it is a better substrate for
hexokinase. In adipose tissues, which have much more fructose than glucose, fructose is
metabolized through fructose 6-phosphate pathway.
In hepatocytes, ovaries and seminal vesicles, fructose is generated from glucose via the polyol
pathway. Glucose is firstly reduced to sorbitol by aldose reductase. Sorbitol dehydrogenase is
responsible for the oxidation of sorbitol into fructose. This pathway is responsible for the occurrence
of fructose in seminal fluid.
Biomedical and metabolic significance
o Metabolic consequences of high dietary fructose intake
 Hypertriglyceridemia  Hypercholesterolemia  Hyperuricemia
o Inherited disorders of fructose metabolism

 Hereditary fructose intolerance (deficiency of aldolase B)
 Essential fructosuria (deficiency of fructokinase)
o Sorbitol accumulation in tissues
 Alternative pathway of glucose metabolism leads to reduction of glucose to sorbitol followed
by oxidation of sorbitol to fructose by sorbitol dehydrogenase. Increase sorbitol formation
can occur when sorbitol dehydrogenase is low or absent (especially in lens, retina,
peripheral nerves and renal glomerular cells).
 Since sorbitol does not diffuse through cell membranes and accumulates in the cells causing
osmotic damage to the cells. This type of damage manifests as retinopathy, cataracts and
peripheral neuropathy.
Galactose metabolism
Source– from lactose in milk
Entry of galactose into the
cells is not insulin dependent.
Galactose is mainly
metabolized in liver by conversion of galactose to UDP glucose which then incorporated into
glycogen synthesis as the following steps –
o Phosphorylation of galactose to galactose 6-phosphate by galactose kinase
o Formation of UDP galactose catalyzed by the action of galactose 1-phosphate uridyl transferase
o Epimerization of UDP galactose to UDP glucose by UDP galactose epimerase
The epimerase reaction is freely reversible, so glucose can be converted to galactose.
Biomedical and metabolic significance
o Galactose is required for the formation of lactose in lactation, glycolipids, glycoproteins and
proteoglycan synthesis.
In the synthesis of
lactose in the mammary
gland, UDP galactose
condenses with glucose
to yield lactose, catalyzed by lactose synthase.
o Galactose is also a substrate for aldose reductase forming galacitol. Its accumulation in eye lens
can cause cataract.
o Inherited disorders of galactose metabolism
 Galactosemia (deficiency of galactose 1-phosphate uridyl transferase or galactokinase) –
associated with failure to thrive in infants, diarrhea and vomiting on milk consumption,
hepatomegaly and jaundice, mental retardation.
Blood glucose homeostasis

Normal blood glucose level
o Fasting blood glucose level  72 – 110 mg/dL or 4 – 6.1 mmol/L
o Random blood glucose level  110 – 140 mg/dL or 6.1 – 7.8 mmol/L
Sources of blood glucose
1. Exogenous source – dietary carbohydrate
2. Endogenous source – from glycogenolysis in liver, from gluconeogenesis in liver and kidney
Blood glucose homeostasis or regulation of blood glucose level

Maintenance of blood glucose level is essential since glucose is the major fuel for the tissues and for
the fetus. The maintenance of a stable blood glucose concentration is one of the most finely
regulated of all homeostatic mechanisms, involving the liver, extra-hepatic tissues, and several
hormones.
Role of liver in blood glucose homeostasis

Liver is the only organ that can release glucose to the blood stream when blood glucose level is low
or can remove glucose from the blood stream when the concentration in the hepatic portal blood is
above normal. Uptake of glucose into liver is via insulin independent, low-affinity and high capacity
glucose transporter GLUT2.
High Km value (15mM) of GLUT-2 in the liver allows hepatocytes to uptake glucose especially
when blood glucose level is high. Glucose entering into liver is phosphorylated by liver specific
glucokinase which has high Km, high Vmax value and cannot be inhibited by G6P. Since
glucokinase is most active when blood glucose levels are high, this allows more uptake of glucose
by liver when the blood glucose level is high during fed state.
Insulin secreted in response to high blood glucose level during fed state induces glucokinase as
well as promotes glycolysis by increasing F-2, 6-BP concentration which allosterically activates
PFK-1. Glycolytic end product pyruvate is then used as a source of acetyl CoA for complete
oxidation in CAC and for fatty acid synthesis.
Glucose is also oxidized via HMS pathway providing NADPH for reductive synthesis (fatty acid and
cholesterol synthesis) and also enters uronic acid pathway for provision of UDP glucuronic acid
essential for xenobiotic metabolism in liver.
Liver can store excess glucose as glycogen. During fed state, excess glucose 6-phosphate and
insulin enhances glycogen synthesis in the liver by activating glycogen synthase. In this way, during
fed state liver uptake of glucose increases providing more glucose 6-phosphate than it is required
for ATP production from glucose oxidation; it is used for glycogen synthesis and lipogenesis in liver.
During fasting state, glucose uptake by liver is decreased due to low activity of glucokinase. Fall in
blood glucose level increases the level of glucagon, epinephrine, glucocorticoids and other
hyperglycemic hormones.
Liver release glucose into the blood by rapid mobilization of glycogen stores due to activation of
glycogen phosphorylase by glucagon via cAMP dependent PKA activation. Glycogenolysis is also
stimulated by catecholamines.
At the same time, these hormones inhibit glycolysis and glycogenesis thus reducing utilization of
glucose in the liver. Amount of liver glycogen store is not sufficient to maintain blood glucose level in
fasting especially lasting more than 12 hours.
Liver can also synthesize glucose from non-carbohydrate precursors (glucogenic amino acids, lactate
and glycerol) by gluconeogenesis. It becomes the main source of blood glucose during prolonged
fasting and starvation. Glucagon and glucocorticoids stimulate and induces key enzymes of
gluconeogenesis (PEPCK, F-1, 6-BPase) and the pathway is fully active as glycogen stores are depleted.
In this way, during fasting state, liver provides blood glucose via glycogenolysis and gluconeogenesis
for blood glucose homoestasis.
Thus, liver acts as glucostat organ maintaining the normal blood glucose level at all time.
Role of pancreas in blood glucose homeostasis

Pancreas serves to maintain blood glucose level by secreting insulin or glucagon in response to blood
glucose level changes. Insulin is the only hypoglycemic hormone in the body and glucagon secreted
in response to hypoglycemia maintains normal blood glucose level in fasting state.
Role of insulin in blood glucose homeostasis
o The rise in blood glucose stimulates the release of insulin from β cells of pancreas. It is a protein
hormone which binds and activates receptor tyrosine kinase on cell membrane of target cells and
mediates its signaling cascade and intracellular response; promoting glucose utilization in the
target cell.
o Insulin lowers the blood glucose level by the following mechanisms –
o Increasing glucose entry into the cell by stimulating translocation of GLUT4 to the plasma
membrane and increased expression of GLUT-4 in muscles and adipose tissues
o Increasing glucose utilization in the cell
 Insulin induces high Km glucokinase in hepatocytes and stimulates the activity of key
enzymes of glycolysis (PFK-1, pyruvate kinase) in all eukaryotic cells. It also promotes
glycolysis by increasing F-2, 6-BP concentration which allosterically activates PFK-1.
 Insulin enhances TAG formation in liver and adipose tissues by providing acetyl CoA for fatty
acid synthesis, DHAP for glycerol 3-phosphate in esterification of fatty acid and NADPH via
HMS pathway for lipogenesis.
 Insulin promotes glycogenesis in liver and muscle by activating glycogen synthase and
inhibits glycogenolysis by inhibiting glycogen phosphorylase. Insulin decreases intracellular
cAMP level by activating cAMP PDE thereby opposing actions of hyperglycemic hormones
glucagon and epinephrine.
o Decreasing glucose production by liver

 Insulin inhibits glycogenolysis by dephosphorylation of phosphorylase kinase and
phosphorylase via activation of protein phosphatase. It also decreases cAMP level that
mediates phosphorylation and activation of phosphorylase.
 Insulin inhibits gluconeogenesis by repression of PEPCK gene transcription.
 Insulin also decreases the activity of glucose 6-phosphatase resulting in retention of glucose
6-phosphate which enters into glycogenesis. In this way, insulin decreases hepatic glucose
output and increases glucose utilization in the liver.
o Thus, insulin increases glucose uptake and utilization in muscles and adipose tissues. It stimulates
glycogen synthesis in muscles and lipogenesis in adipose tissues. In the liver, insulin promotes
glucose utilization by stimulating glycolysis, glycogenesis and lipogenesis. It also suppresses
hepatic glucose output by inhibiting glycogenolysis and gluconeogenesis.
Role of glucagon in blood glucose homeostasis
o Glucagon is released from α cells of pancreas in response to low blood glucose level.
o Glucagon maintains adequate blood glucose level for glucose dependent tissues by enhancing
hepatic glucose output via activation of glycogenolysis and gluconeogenesis in liver.
o During fasting, glucagon promotes rapid mobilization of glycogen stores in liver by activating
glycogen phosphorylase and inhibiting glycogen synthase via cAMP dependent PKA activation. It
also inhibits glycolysis by inactivating PFK-1 and pyruvate kinase.
o Glucagon stimulates hepatic gluconeogenesis by inactivating pyruvate kinase and lowering the
level of F-2, 6-BP which is an allosteric activator of key glycolytic enzyme PFK-1 and an allosteric
inhibitor of fructose 1, 6-bisphosphatase, key gluconeogenic enzyme. Fall in F-2, 6-BP level by
glucagon action causes fructose 1, 6-bisphosphatase to be relieved from its inhibition leading to
liver cells in gluconeogenic mode.
o Glucagon also has long-term effects on hepatic glycolysis and gluconeogenesis by inducing gene
transcription of key gluconeogenic enzymes and repressing synthesis of key glycolytic enzymes.
Effect of other endocrine organs in blood glucose level

Anterior pituitary gland
o Anterior pituitary gland secreted hormones that tend to
elevate blood glucose level. These are growth hormone,
ACTH and TSH.
o Growth hormone
 It stimulates mobilization of free fatty acids (FFAs)
from adipose tissues and increases plasma FFA level
which inhibits glucose utilization in peripheral tissues. Thus, growth hormone increases
blood glucose level by indirectly decreasing glucose uptake in peripheral tissues.
 Growth hormone stimulates hepatic glycogen synthesis.

o ACTH
 Major effect of ACTH on carbohydrate metabolism is due to its stimulatory effect on
secretion of glucocorticoids from adrenal cortex. It inhibits the peripheral uptake of
glucose.
o TSH
 Its effect on blood glucose level is mediated through stimulation of thyroid hormone
secretion from thyroid gland.
Thyroid glands
o Thyroxine has diabetogenic action. It stimulates glycogenolysis in liver and decreases insulin
sensitivity of tissues.
Adrenal medulla
o Adrenal medulla secretes catecholamines (epinephrine, norepinephrine) in response to stress
including metabolic stress such as hypoglycemia during fasting or starvation.
o Epinephrine stimulates hepatic glycogenolysis through activation of glycogen phosphorylase by
cAMP dependent phosphorylation. It also stimulates gluconeogenesis in liver by decreasing
allosteric inhibitor of F-1,6-BPase, F-2,6-BP level, induction of key gluconeogenic enzymes and
providing substrates for hepatic gluconeogenesis. Net effect is increased hepatic glucose output.
o In the skeletal muscles, epinephrine stimulates glycogenolysis providing G6P. Pyruvate produced
by glycolysis of G6P is reduced to alanine or transmianted to alanine which is then transported to
liver to be used as gluconeogenic substrates (Cori cycle and Glucose Alanine cycle).
o Epinephrine opposes the action of insulin in tissues; thus decreasing insulin sensitivity of tissues.
Adrenal cortex
o Glucocorticoid increases blood glucose level by increasing hepatic glucose production via
gluconeogenesis.
o It increases hepatic gluconeogenesis by induction of key gluconeogenic enzymes and providing
carbon skeletons for gluconeogenesis from catabolism of amino acids by transamination
reaction.
o Glucocorticoids favor muscle proteolysis and increases transaminases activity to provide alanine
for hepatic gluconeogenesis.
o It also decreases peripheral uptake of glucose.
Role of kidney in blood glucose homeostasis

Normally glucose is continuously filtered by the glomeruli but is completely reabsorbed in renal
tubules so that no glucose is present in the urine. But the capacity of tubular reabsorption system to
absorb glucose is limited to a rate of about 350 mg/min. In normal individuals, glycosuria occurs when
blood glucose level exceeds 10 mmol/L or 180 mg/dL. This is called renal threshold.
When blood glucose level rises to relatively high levels (above renal threshold), kidney exerts a
regulatory effect by filtering more glucose than it can be reabsorbed; excess glucose passes into the
urine to produce glycosuria.
Kidney also maintains the blood glucose level by gluconeogenesis especially during prolong
starvation.
Role of hypothalamus in blood glucose homeostasis

Hypothalamus concerns with regulation of food intake via interaction between its two areas;
feeding center and satiety center. Feeding center is chronically active and its activity is transiently
inhibited by satiety center. Activity of satiety center depends on level of glucose utilization of the
cells in the center (glucostats).
When blood glucose level is high after meals, glucose utilization in glucostats cells is increased
resulting in activation of satiety center that decreases food intake. In this way, hypothalamus has
regulatory effect on blood glucose level by regulating food intake.
Oral glucose tolerance test

The ability of the body to utilize glucose is reflected by
measuring its glucose tolerance. The oral glucose tolerance
test (OGTT) is the reference method for the assessment of
glucose tolerance. It is indicated by the nature of blood
glucose curve following 75 g oral glucose load.
Decreased glucose tolerance is commonly seen in diabetes
mellitus. Diminished glucose tolerance is also seen in liver disease, some types of infections,
hyperactivity of pituitary gland or adrenal cortex.
Increased glucose tolerance is seen in pituitary or adrenal insufficiency.
Hypoglycemia
 Glucose is the principle fuel of many cells types of the body. RBCs and brain cells have an absolute
requirement for blood glucose for energy metabolism. Thus hypoglycemia compromises brain
function leading to confusion, disorientation and possibly life-threatening coma at blood glucose
concentration below 2.5 mmol/L or 45 mg/dL (neuroglycopenia).
 Low plasma glucose stimulates the sympathetic nervous system and leads to secretion of
epinephrine and glucagon, initiating the stress response. This manifests as sweating, tremor,
increased heart rate and a feeling of hunger.
 Causes of hypoglycemia
o Strenuous exercise, fasting and excessive alcohol drinking in healthy persons
o Excess of exogenous insulin
o Insufficient amount of counter-regulatory hormones to balance insulin effects e.g.,

adrenocortical insufficiency
o Liver diseases
o Rare tumor of β cells (insulinoma)
o Glycogen storage disease
Lipid Metabolism Page |1
Lipid Metabolism
Biomedical significance of lipids

 Lipids are an extremely heterogeneous class of compounds that serve as the following:
o Fuel stores – Lipids stored in adipose tissue as triacylglycerols (fats) are a major source of
energy. They contain 9 kcal/g of energy, which is more than twice the amount contained in
carbohydrates and proteins.
o Structural components – Lipids are the primary components of cellular membranes and are
critical for maintaining their structural integrity and function.
o Signaling molecules – Lipids serve as signaling molecules that affect pathways that control cellular
processes, such as cell growth, survival and the immune response.
o Miscellaneous - Brown adipose tissue functions in generating heat (thermogenesis), white
adipose tissue provides insulation and lipid components of bile play a role in the digestion of fat
in the small intestine.
Classification of lipids
Fatty acid derivatives
o Simple lipids – esters of lipids with various alcohols
 Fats (glycerolipids) – esters of fatty acids
with glycerol. (in liquid state – oil)
 Waxes – esters of fatty acids with high
molecular weight monohydric alcohols.
o Complex lipids – esters of fatty acids containing groups in addition to an alcohol and a fatty acid.
 Phospholipids – lipid containing fatty acids, an alcohol, a phosphoric acid residue, nitrogen
containing bases and other constituents.
 Glycerolphospholipids – backbone alcohol is glycerol.
 Sphingophospholipids – backbone alcohol is sphingosine.
 Glycolipids (glycosphinogolipids) – lipids containing a fatty acid, sphingosine and sugar
component
o Other complex lipids – sulfolipids, aminolipids, and lipoproteins.
o Eicosanoids – derivatives of C-20 PUFA (arachidonic acid) e.g., prostaglandins, thromboxanes.
Isoprene derivatives (isoprenoids)

o Composed of multiple isoprene units that are linked together to form linear chains or cyclic
structures.
o Include the lipid-soluble vitamins (A, D, E, K), ubiquinone (coenzyme Q), cholesterol and the
steroid hormones.
Fatty acids
 Carboxylic acids with hydrocarbon chains of 4 to 36 carbons long.
 Based on the Geneva system, fatty acids can be classified into saturated and unsaturated fatty acids.
 Fatty acids may also be classified based on the number of carbon atoms into four types: short-chain
fatty acids (SCFAs) contain fewer than 6 carbons, medium-chain fatty acids (MCFAs) have 6 to 12
carbons, long-chain fatty acids (LCFAs) have 13 to 20 carbons and very long chain fatty acids (VLCFAs)
contain 22 or more carbons.
 Nomenclature of fatty acids
o Saturated fatty acids end with –anoic acid, e.g., octanoic acid.
o Unsaturated fatty acids with double bonds end in –enoic acid, e.g., octadecenoic acid.
o Carbon atoms are numbered from the carboxyl carbon (carbon no.1). Carbon atoms adjacent to
the carboxyl carbon, i.e., carbon no.2, 3, 4 are called α, β and γ carbons.
o The terminal methyl carbon is ω- or n- carbon.
o To indicate the position of double bonds,  is used.
o For example, oleic acid (C 18:1, 9 or ω9) indicates that it contains 18 carbon atoms and one
double bond. 9 indicates position of the double bond is between carbon 9 and 10 of the fatty
acid. ω-9 indicates a double bond on the 9th position from the ω-carbon.
 In animals, additional double bonds are introduced only between the existing double bond (e.g., ω9,
ω6, or ω3) and carboxyl carbon.
 Saturated fatty acids contain no double bonds between the adjacent carbons.
 Unsaturated fatty acids contain one or more double bonds. They are subdivided into –
o Monounsaturated fatty acids (MUFAs) – containing one double bond e.g., oleic acid (C18:1, ω9),
palmitoleic acid (C16:1, ω7)
o Polyunsaturated fatty acids (PUFAs) – containing two or more double bonds e.g., linoleic acid
(C18:2, ω6), linolenic acid (C18:3, ω3)
 Most naturally occurring unsaturated fatty acids have cis double bonds.
 Essential fatty acids
o Some fatty acids cannot be synthesized by the body and must be supplied in the diet; thus they
are celled essential fatty acids.
o These include linoleic acid and linolenic acid.
o Linoleic acid serves as precursor of arachidonic acid which in turn is used as substrate for
eicosanoids synthesis.
o α-linolenic acid is the precursor of other ω-3 fatty acids that is important for growth and
development e.g., eicosapentaenoic acid.
o Arachidonic acid becomes essential if linoleic acid is deficient in the diet.
Sources of fatty acids in the body

Dietary TAG
o Dietary FFAs that are absorbed and re-esterified to TAG in the enterocytes are transported into
the circulation in chylomicrons. Lipoprotien lipase hydrolyzes this TAG to FFAs and glycerol.
o Fatty acids then enter into the adjacent tissues for energy production or for storage (adipose
tissues).
Endogenous de novo synthesis from excess acetyl CoA derived from glucose oxidation
Biosynthesis of fatty acids (Lipogenesis)

The majority of fatty acids required by humans are supplied in the diet. However, there is de novo
synthesis of fatty acids or lipogenesis in the body in which carbohydrate is converted to fatty acids in
the liver and stored as triacylglycerol (TAGs) in the adipose tissues.
Site – cytoplasm of many tissues such as liver, adipose tissues, mammary glands, brain, lungs and
kidneys.
Synthesis of fatty acids is linked to HMS pathway which is the main provider of NADPH. Some NADPH
is also generated by NADP+ dependent decarboxylation of malate to pyruvate by malic enzyme as
well as cytosolic NADP+ dependent isocitrate dehydrogenase reaction.
Process of lipogenesis
o Stage 1 – formation and transport of acetyl CoA to cytosol
 Fatty acids are synthesized from two
carbon compound acetyl CoA. Acetyl
CoA is generated in the mitochondria
from pyruvate. It is transported to
cytosol in the form of citrate. In the
cytosol, citrate is cleaved into acetyl
CoA and oxaloacetate by citrate lyase
in the presence of ATP and CoA.
o Stage 2 – formation of key precursor malonyl CoA from acetyl CoA
 It is the rate-limiting step of fatty acid synthesis. Carboxylation of acetyl
CoA to malonyl CoA is catalyzed by biotin-dependent acetyl CoA
carboxylase enzyme requiring ATP and bicarbonate as a source of CO2.
o Stage 3 – elongation of fatty acid in two-carbon increments by fatty acid synthase

 This step is catalyzed by fatty acid synthase enzyme complex. This enzyme complex is a
dimer comprising two identical monomers, each containing seven enzyme activities and an
acyl carrier protein (ACP). It arranges in head to tail manner. Fatty acid synthase builds fatty
acid up to 16 carbon length.
 A molecule of malonyl CoA condenses with first molecule of acetyl CoA to form a C4
molecule releasing
one carbon as CO2.
This step is followed
by two reduction
steps.
 The sequence of
reactions is repeated
six more times, each
adding 2 carbons to
the fatty acid chain
(total 7 cycles) until
a saturated 16
carbon fatty acyl
radical (palmityl) is formed. It is liberated from the enzyme complex by the action of
thioesterase (deacylase).
 The end product is 16-carbon fatty acid, palmitate.
Lipid Metabolism P a g e |5
 It must be activated to acyl CoA before using it. It can either undergo chain elongation, or
desaturation or can be esterified to acylglycerols, or esterified with cholesterol to form
cholesteryl ester.
 Synthesis of one palmitate molecule requires 8 molecules of acetyl CoA, 7 ATP and 14
NADPH. This process allows the building up of fatty acids with even number carbon atoms.
Acetyl CoA+7malonyl CoA+14NADPH+20H+  Palmitate+7CO2+14NADP++8CoA-SH+6H2O
Regulation of lipogenesis or fatty acid synthesis

 Formation of malonyl CoA from acetyl CoA catalyzed by acetyl CoA carboxylase is the committed
step of fatty acid synthesis. The rate of lipogenesis is controlled by –

 Nutritional factor
o High carbohydrate/low fat diet increases fatty acid synthesis. Diet containing high sucrose or
fructose content especially favors lipogenesis.
o Restricted calorie intake and high fat diet depress lipogenesis. Conditions where there are high
circulating fatty acids level lead to marked inhibition of lipogenesis e.g., fasting, starvation.
 Regulation of key enzyme activity
o Fatty acid synthesis is controlled in short term by allosteric and covalent modification and in long
term by gene expression regulation.
o Short term control
Allosteric regulation
 Acetyl CoA carboxylase is allosterically
activated by citrate. It increases in well-fed
state and is an indicator of plentiful supply of
acetyl CoA.
 Acetyl CoA carboxylase is inhibited by the end product,
long chain acyl CoA molecule (negative feedback
inhibition).
 Acyl CoA also inhibits the mitochondrial tricarboxylate
transporter, thus preventing export of citrate from
mitochondria to cytosol.
Covalent modification
 Acetyl CoA carboxylase is active in dephosphorylated state
and inactive in phosphorylated state.
 Insulin activates protein phosphatase which
dephosphorylates and activates acetyl CoA carboxylase.
 Glucagon and epinephrine stimulates cAMP dependent PKA which phosphorylates and
inactivates acetyl CoA carboxylase thereby inhibiting lipogenesis.
 AMP activated kinase (AMPK) also phosphorylates and inactivates acetyl CoA
carboxylase. When cellular energy level is low, AMP level increases and AMP activates
AMPK. Thus, less energy status in the cell prevents synthetic process.
o Long term control
This is achieved by regulating the rate of synthesis of enzymes involved or related in fatty
acid synthesis.
Synthesis of acetyl CoA carboxylase, fatty acid synthase, citrate lyase, glucose 6-phosphate
dehydrogenase and malic enzymes are up-regulated under high carbohydrate and low-fat
intake. But starvation or high fat, low carbohydrate diet intake leads to down-regulation of
these enzymes synthesis. This type of regulation is known as adaptive control.
Hormonal control of lipogenesis

Insulin stimulates lipogenesis by –
o Increasing glucose entry into the adipose tissues (stimulates GLUT4 expression)
o Stimulating glucose oxidation to provide acetyl CoA for fatty acid synthesis and glycerol 3
phosphate for esterification of fatty acids
o Activation of acetyl CoA carboxylase by dephosphorylation via activation of protein phosphatase
and lowering cAMP level
o Lowering plasma FFA level by inhibiting lipolysis in adipose tissues
Glucagon and epinephrine inhibits lipogenesis by phosphorylation and inactivation of acetyl CoA
carboxylase by activating cAMP dependent PKA.
Catecholamines inhibit acetyl CoA carboxylase by phosphorylation via Ca2+/calmodulin dependent
protein kinase through α1 adrenergic receptor.
Biosynthesis of long chain fatty acids

Palmitate (C16:0), the principal product of fatty acid synthesis, is the precursor of other long-chain
fatty acids. It may be lengthened to form stearic acid (C18:0) and longer saturated fatty acids by
further addition of acetyl groups, through action of fatty acid elongation systems present in the
smooth endoplasmic reticulum and in mitochondria.
Microsomal system for fatty acid chain elongation
o It is catalyzed by microsomal fatty acid elongase system. It uses malonyl CoA as the acetyl donor
and NADPH as the reductant.
o This system elongates saturated and unsaturated fatty acids of above C10. Elongation of stearyl
CoA (C18) in brain occurs in order to provide C22 and C24 fatty acids for sphingolipids synthesis in
myelination of nerve cells.
Mitochondrial system for fatty acid elongation
o This is a less active system.
o This enzyme system uses acetyl CoA as a source of carbon units and both NADH and NADPH
serve as reducing agents.
Desaturation of fatty acids

Desaturation of fatty acids requires a mixed function oxidase enzyme (desaturase) system in
smooth endoplasmic reticulum. The enzyme system is similar to a monooxygenase system involving
cytochrome b5.
Oxygen and either
NADH or NADPH
are necessary for
the reaction.
Double bonds can be introduced at 4, 5, 6 and 9 positions in most animals, but never beyond the
9 position.
Biosynthesis of monounsaturated fatty acids

Monounsaturated fatty acids can be synthesized in the body by
microsomal 9 desaturase system. Several tissues including the liver are
considered to be responsible for the formation of nonessential
monounsaturated fatty acids from saturated fatty acids.
The first double bond introduced into a saturated fatty acid is nearly always in the Δ9 position.
Microsomal 9 desaturase system catalyzes the conversion of palmitoyl-CoA to palmitoleoyl-CoA
and stearoyl-CoA to oleoyl-CoA. Thus palmitoleic and oleic acids are not essential in the diet.
Biosynthesis of polyunsaturated fatty acids (PUFAs)

Since animals have a Δ9 desaturase, they are able to synthesize the
ω9 (oleic acid) family of unsaturated fatty acids completely by a
combination of chain elongation and desaturation.
Most animals cannot introduce double beyond Δ9 the position. In
contrast, plants are able to synthesize the nutritionally essential fatty
acids by introducing double bonds at the Δ12 and Δ15 positions. Thus,
linoleic and α-linolenic acids are nutritionally essential fatty acids.
Certain long-chain unsaturated fatty acids of metabolic significance
in mammals are derived from oleic, dietary essential fatty acids such
as linoleic and α-linolenic acids by chain elongation and desaturation.
Arachidonate (eicosatetraenoic acid) can be formed from linoleic
acid (C18:2, ω6) via γ-linolenate pathway. Nutritional requirement
for arachidonate may thus be dispensed if there is adequate linoleate
in the diet.
Trans-fatty acids
Other C20, C22 and C24 PUFAs can also be synthesized from Mainly found in partially hydrogenated
essential fatty acids, e.g., eicosapentaenoic acid from α- vegetable oils (e.g., magarine).
Small amounts are also found in
linolenic acid.
ruminant fat, eg., 2–7% of butter fat.
May compete with essential fatty acids
o Desaturation and chain elongation system is greatly and may exacerbate essential fatty
diminished in starvation state, in response to glucagon, acid deficiency.
epinephrine, and in the absence of insulin in type 1 Promotes hypercholesterolemia and

atherosclerosis.
diabetes mellitus.
o Essential fatty acids are found in high concentrations in vegetable oils and in small amount in
animal carcasses.
o Essential fatty acids are required for eicosanoids synthesis. Arachidonic acid is the major
precursor for eicosanoids synthesis (prostaglandins, thromboxanes, leukotrienes and lipoxins).
o Docosahexaenoic acid (DHA; ω3, 22:6), which is synthesized to a limited extent from α-linolenic
acid or obtained directly from fish oils, is present in high concentrations in retina, cerebral
cortex, testis, and sperm. DHA is particularly needed for development of the brain and retina.
o Essential fatty acids are thought to be involved in normal growth and reproductive efficiency
based on the results of experimental research on animal models.
o Symptoms of essential fatty acid deficiency in humans include skin lesions and impairment of
lipid transport. Infants receiving formula diets low in fat and patients maintained for long periods
exclusively by intravenous nutrition low in essential fatty acids are high risk of essential fatty acid
deficiency. This can be prevented by an essential fatty acid intake of 1% to 2% of the total caloric
requirement.
o Diets with a high
PUFA/SFA ratio reduce
serum cholesterol levels
and are considered to
be beneficial for
reducing the risk of
coronary heart disease
development.
Eicosanoids
 Eicosanoids are compounds
derived from C-20 PUFA
(eicosapolyenoic acids) such
as arachidonic acid.
 Eicosanoids include prostanoids (prostaglandins, prostacyclins and thromboxanes) and

leukotrienes, lipoxins.
Biosynthesis of eicosanoids
 There are three groups of eicosanoids that are synthesized from C-20 eicosanoic acids derived from
the essential fatty acids linoleate and α-linolenate, or directly from dietary arachidonate and
eicosapentaenoate.
 Because arachidonic acid is stored in cells in sn-2 position of membrane glycerophospholipids, it can
be released by the action of an inducible cytosolic phospholipase A2 (PLA2).
 Arachidonate is the substrate for the synthesis of the PG2, TX2 series (prostanoids) by the
cyclooxygenase pathway, or the LT4 and LX4 series by the lipoxygenase pathway.
 Biosynthesis of prostanoids
o Prostanoids are synthesized from arachidonic acid via cyclooxygenase pathway. This pathway
leads to the formation of prostaglandins and thromboxanes via unstable intermediates, PGG2
and PGH2. This occurs via reactions catalyzed by prostaglandin H (PGH) synthase or
Lipid Metabolism P a g e | 10
cyclooxygenase (COX), a heme-containing dioxygenase that exhibits both cyclooxygenase and

peroxidase activities.
o PGH2 is the immediate precursor of all series-2 prostanoids e.g., PGD2, PGE2, TXA2, PGI2, etc. It is
rapidly converted into other prostaglandins, prostacyclin, and thromboxanes by additional
enzymes present in specific tissues. Each cell type produces only one type of prostanoid.
o Thromboxane synthase present in platelets (thrombocytes) converts PGH2 to thromboxane A2
(TXA2) from which other thromboxanes are derived. Vascular endothelial cells contain
prostacyclin synthase, which catalyzes the synthesis of prostacyclin I2 (PGI2), a vasodilator and
inhibitor of platelet aggregation.
o There are two forms of cyclooxygenase (COX) called COX-1 and COX-2. COX-1 is constitutively
expressed in many tissues, whereas COX-2 is inducible by oxidative stress, cytokines (TNFα, IL-1)
and platelet activating factor (PAF).
 Biosynthesis of leukotrienes and lipoxins
o The leukotrienes are a family of conjugated trienes formed from eicosanoic acids in leukocytes,
mastocytoma cells, platelets, and macrophages by the lipoxygenase pathway in response to
both immunologic and non-immunologic stimuli. Three different lipoxygenases (dioxygenases)
insert oxygen into the 5, 12, and 15 positions of arachidonic acid, giving rise to hydroperoxides
(HPETE). Only 5-lipoxygenase forms leukotrienes.
o Lipoxins are a family of conjugated tetraenes also arising in leukocytes. They are formed by the
combined action of more than one lipoxygenase.
Mechanism of action of eicosanoids

 Eicosanoids are local mediators and act in an autocrine or paracrine fashion. They act through
interaction with specific prostaglandin receptor. Many different types of prostaglandin receptors are
G-protein-coupled receptors and many of their effects are intracellularly mediated by cAMP or
cGMP, e.g., PGE2 decreases cAMP and thromboxane signaling decreases cAMP and cGMP levels.
PGF2α signaling causes an increase in cGMP levels.
Biomedical importance and metabolic significance of eicosanoids

 Thromboxanes (TXAs) are synthesized and released by platelets. They cause vasoconstriction and
platelet aggregation. Their synthesis is specifically inhibited by low dose aspirin.
 Prostacyclins (PGI2) are produced by endothelial cells. They are vasodilators and potent inhibitors
of platelet aggregation.
 Prostaglandin E (PGE2) formed by the gastric mucosa stimulates mucus secretion and suppresses
gastric acid secretion.
 PGE2 and PGF2α, synthesized in the endometrium, induce uterine contraction. Together with
oxytocin, they involve in the induction of labor.
 PGE2 and TXA2 formed by WBCs serve as inflammatory mediators.
 PGD2 is a potent sleep-promoting substance.

 Potential therapeutic uses of prostaglandins
o Prevention of conception, induction of labor and termination of pregnancy
o Prevention or alleviation of gastric ulcers
o Control of inflammation and of blood pressure
o Relief of asthma and nasal congestion
 Slow-reacting substance of anaphylaxis (SRS-A) is a mixture of leukotrienes C4, D4 and E4. This
mixture cause potent bronchoconstriction and together with leukotriene B4, they also cause
vascular permeability and attraction and activation of leukocytes. They are important regulators in
inflammatory or immediate hypersensitivity conditions, such as asthma.
 Lipoxins appear to have anti-inflammatory role in vasoactive and immune-regulatory function.
Therapeutic agents that target eicosanoid metabolism

 Several drugs for anti-inflammatory, antipyretic and analgesic action, relief of asthma and allergies
target eicosanoid synthesis.
 Steroidal anti-inflammatory drugs
o Corticosteroids act as anti-inflammatory agents by inhibiting the activity of PLA2, thereby
preventing release of arachidonic acid from membrane phospholipids for eicosanoid synthesis.
Corticosteroids such as dexamethasone and corticosterone repress tumor necrosis factor (TNF)-
induced expression of PLA2 gene and inhibit the production of all eicosanoids.
o Steroids also block leukotrienes production by inhibiting PLA2 activity and can be used for
prevention of asthmatic attacks and treatment of hypersensitivity reactions.
 Non-steroidal anti-inflammatory drugs
o They inhibit both isoezymes of COX (non-selective COX inhibitors), thus reducing inflammation,
fever and pain by decreasing prostaglandin production, e.g., aspirin, ibuprofen, indomethacin.
o They also inhibit blood clotting by inhibiting thromboxane production, e.g., low dose aspirin.
o Aspirin (acetylsalicylic acid) irreversibly inhibits cyclooxygenase activity by acetylating a serine
residue in the active site of enzyme.
o NSAIDs are relatively nonspecific and therefore can have adverse side effects, most notably
gastrointestinal ulceration, when used to treat inflammation or fever.
 Selective COX-2 inhibitors
o Selective COX2 inhibitors such as celecoxib and rofecoxib selectively inhibit COX-2 which is
associated with the synthesis of prostaglandins involved in pain and inflammation.
o These drugs do not inhibit COX-1 and are therefore less prone to gastric injury while still being
effective for treating pain and inflammation. However, some COX-2 inhibitors (e.g., rofecoxib)
have been found to cause a higher rate of incidence of thrombotic cardiovascular events due to
decreased PGI2 synthesis and uninhibited thromboxane synthesis.
 Leukotrienes blockers
o Zileuton is a 5-lipoxygenase inhibitor that blocks the production of leukotrienes C4, D4, E4
members as well as LTB4 and 5-HETE.
o Pranlukast, zafirlukast and montelukast are antagonists of leukotrienes C4, D4, E4 receptors.
Triacylglycerol metabolism
 Dietary fatty acids or endogenously synthesized fatty acids have one of two fates; incorporation into
TAG for storage of metabolic energy or incorporation into phospholipid components of membrane.
 TAGs are esters of glycerol and fatty acids. TAGs constitute the majority of lipids in the body and
serve as the body’s major fuel storage reserve; highest energy content of all stored nutrients – more
than 38J/kg.
 TAGs also have implications in lipid transport since dietary fatty acids absorbed by the enterocytes or
endogenously synthesized fatty acids in liver are transported into the blood stream after being
esterified into TAG and incorporated into lipoproteins (chylomicron or VLDL).
 In animal tissues, TAGs and glycerophospholipids such as phosphatidyl-ethanolamine share two
precursors (fatty acyl CoA and glycerol 3-phosphate) and several biosynthesis steps.
 Biosynthesis of TAG
o TAGs are synthesized in the smooth endoplasmic reticulum of liver, adipose tissues and
intestinal cells. Whenever carbohydrate is ingested in excess of the organism’s capacity to store
glycogen, the excess is converted to triacylglycerols and stored in adipose tissues.
o Phosphatidate is the common precursor in the biosynthesis of triacylglycerols, phosphoglycerols
and cardiolipins. Both fatty acids and glycerol must be converted into activated forms before
being incorporated into acylglycerols.
o Fatty acid is converted into acyl CoA by acyl-CoA synthetase, using ATP. Glycerol kinase catalyzes
the activation of glycerol to glycerol 3-phosphate. But in muscles and adipose tissues where
glycerol kinase activity is low or absent, glycerol 3-phosphate is formed from glycolytic
intermediate DHAP by glycerol 3-phosphate dehydrogenase action.
o Two molecules of acyl CoA combines with glycerol 3-phosphate to form phosphatidate (1,2-
diacylglycerol phosphate) catalyzed by specific acyl transferases. Phosphatidate is hydrolyzed to
DAG by phosphatidate phosphohydrolase. Esterification of another acyl group to DAG by DAG
acyl-transferase (DGAT) produces TAG.
o TAGs are stored in adipose tissues as large lipid droplets during fed state when glycolysis is
activated. TAGs formed in the liver are not stored but are complexed with cholesterol,
phospholipids and apolipoproteins to form VLDL for export into the blood stream.
o In the intestine, TAGs are synthesized via MAG pathway. Dietary lipid digestion end product,
MAG is esterified with fatty acids to form DAG and subsequently TAG by the action of specific acyl
transferases. These TAGs are packed in chylomicrons for export of dietary TAG into blood.
 Catabolism of TAGs (lipolysis)

o TAGs undergo hydrolysis by a hormone-sensitive lipase to form free fatty acids (FFAs) and
glycerol.
o Since glycerol cannot be utilized in the adipose tissues, it enters the blood stream and is taken
up by the tissues such as liver and kidney which possess an active glycerol kinase.
o FFAs from lipolysis can be reconverted to acyl CoA in adipose tissues by acyl CoA synthetase and
re-esterified with glycerol 3-phosphate to form TAG.
o When the rate of re-

esterification is not sufficient
to match the rate of lipolysis,
FFAs accumulate and diffuse
into the plasma, increasing
the plasma FFA level. FFAs are
transported bound to albumin
in the plasma and are taken up
by tissues like liver, heart,
kidney, muscles, testes, brain
and are subsequently oxidized
or re-esterified.
o When glucose utilization by
adipose tissues increases,
FFAs outflow decreases since
glucose oxidation provides
glycerol 3-phosophate via
DHAP for esterification of
FFAs in adipose tissues.
o Thus there is a continuous
cycle of lipolysis and re-esterification within adipose tissues.
These two processes are entirely different pathways involving
different substrates and reactants. This allows the processes of
esterification and lipolysis to be regulated separately by many
nutritional, metabolic and hormonal factors. The balance
between these two processes determines FFA pool in adipose
tissues which in turn determines the circulating plasma FFA level.
Metabolism of triacylglycerol stored in adipose tissues or adipose

tissue metabolism
 Adipose tissue is a specialized connective tissue designed for
synthesis, storage and hydrolysis of TAG. TAGs are stored in adipose

tissues as large lipid droplets and are continually undergoing lipolysis
and re-esterification. The balance between these two processes
determines FFA pool in adipose tissues which in turn determines the
circulating plasma FFA level.
 TAG formation (esterification)
o TAG is synthesized from acyl CoA and glycerol 3-phosphate. Glycerol 3-phosphate is derived from
glycolysis because glycerol kinase is not expressed in adipose tissues. Acyl CoA is derived from
chylomicron and VLDL by action of lipoprotein lipase or synthesized from acetyl CoA or from
lipolysis.
 Lipolysis
o TAG undergoes lipolysis by hormone sensitive lipase to form FFA and glycerol. Glycerol diffuses
into the plasma and is utilized by liver and kidney because they have glycerol kinase. FFA can be
converted to acyl CoA which is re-esterified to TAG or mobilizes into the blood. Fate of FFA
depends on glucose utilization of adipose tissues. When glucose utilization by adipose tissues
increases, FFAs outflow decreases since glucose oxidation provides glycerol 3-phosophate via
DHAP for esterification of FFAs in adipose tissues.
Regulation of lipolysis or FFA mobilization from adipose tissues

 In adipose tissues, there is a continuous cycle of lipolysis and re-esterification. These two processes
are entirely different pathways involving different substrates and reactants. This allows the
processes of esterification and lipolysis to be regulated separately by many nutritional, metabolic and
hormonal factors. The balance between these two processes determines FFA pool in adipose tissues
which in turn determines the circulating plasma FFA level.
 The rate of release of FFA from adipose tissue is affected by many hormones that influence either the
rate of esterification or the rate of lipolysis.

 Insulin inhibits the release of fatty acids from adipose tissues thereby lowering circulating plasma
FFA level.
o Insulin enhances lipogenesis and TAG synthesis by –
 Promoting glucose uptake via GLUT4
 Stimulating glucose oxidation (glycolysis, HMS pathway) to provide acetyl CoA, NADPH for
lipogenesis and glycerol 3-phosphate via DHAP for esterification of fatty acids
 Increasing FFA uptake by activating lipoprotein lipase which hydrolyzes TAG in lipoproteins
to release FFA and glycerol
 Increasing the activity of pyruvate dehydrogenase, acetyl-CoA carboxylase, and glycerol
phosphate acyltransferase, reinforcing the effects of increased glucose uptake on the
enhancement of fatty acid and acylglycerol synthesis
o Insulin reduces lipolysis and release of FFA by inhibiting hormone sensitive lipase activity via–
 Activation of lipase phosphatase which dephosphorylates and inactivates hormone sensitive
lipase
 Decreasing cAMP level by stimulating cAMP PDE and inhibiting adenylyl cyclase activity
 Other hormones accelerate the release of FFAs from adipose tissue and raise plasma FFA level by
increasing the rate of lipolysis. These include epinephrine, norepinephrine, glucagon, ACTH, α- and β-
MSH, TSH, growth hormone and ADH.
 These hormones mediates intracellular signaling cascade by increasing cAMP level via activation of
adenylyl cyclase. cAMP stimulates cAMP dependent PKA which phosphorylates and activates
hormone sensitive lipase. In this way, these hormones promote lipolysis.
 Processes that destroy or preserve cAMP influence lipolysis. cAMP is degraded to 5’AMP by cyclic
nucleotide phosphodiesterase. Methylxanthines such as caffeine and theophylline promotes

lipolysis by inhibiting PDE enzyme.
 Thyroid hormones and glucocorticoids act in a facilitatory or permissive manner for optimal effect
of other lipolytic endocrine factors. Glucocorticoids promote lipolysis via induction of hormone
sensitive lipase synthesis by a cAMP-independent pathway and also by promoting transcription of
genes involved in the cAMP signal cascade.
 Growth hormone promotes lipolysis by increasing synthesis of proteins involved in the formation of
cAMP.
 The sympathetic nervous system enhances mobilization of FFA by liberation of norepinephrine
which promotes lipolysis in adipose tissues.

 Prostaglandin E1 (PGE1) and nicotinic acid have anti-lipolytic effect. They decrease cAMP level by
inhibiting adenylyl cyclase enzyme via Gi protein.

Perilipin
 Perilipin, a protein involved in the formation of lipid  Protein involved in the formation of
lipid droplets in adipocytes.
droplets in adipocytes, enables the storage and breakdown
 It inhibits lipolysis in basal conditions
of TAG to be coordinated according to the metabolic needs
by preventing access of the lipase to
of the body. the stored TAGs
 Adipose tissues secrete hormones such as adiponectin  Promotes lipolysis when it is
which modulates glucose and lipid metabolism in muscle phosphorylated by hormones that
promote TAG degradation, exposing
and liver and leptin which regulates energy homeostasis.
the lipid droplet surface to hormone-
sensitive lipase.
Endocrine function of adipose tissues
Adipocytes produces hormones such as adiponectin, leptin, resistin (collectively known as
adipokines), growth factors such as vascular endothelial derived growth factors (VEGFs) and pro-
inflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6).
Leptin
o Its secretion is linked to the adipose tissues mass and size of adipocytes.
o It stimulates fatty acid oxidation and decreases lipogenesis via stimulation of AMP-activated
protein kinase (AMPK). It also decreases ectopic deposition of fat in liver and muscles.
o It crosses the blood-brain barrier and reduces appetite and causes an increase in energy
expenditure.
o Leptin increases synthesis of mitochondrial uncoupling protein thermogenin (UCP-1) in brown
adipocytes, promoting catabolism and thermogenesis.
Adiponectin
o It increases insulin sensitivity in tissues; thus stimulates glucose utilization in muscle and
increases fatty acid oxidation in muscle and liver. It also blocks fatty acid synthesis and
gluconeogenesis in hepatocytes, and stimulates glucose uptake and catabolism in muscle and
liver. These effects of adiponectin are indirect and many of them are mediated by activation of
AMPK.
o It down-regulates the secretion of pro-inflammatory cytokines; IL-6, IL-8 and monocyte
chemoattractant protein-1 (MCP-1) from adipocytes.
o Low adiponectin levels are linked with insulin resistance, hepatic steatosis and also low-grade
inflammation, oxidative stress and endothelial dysfunction.
Factors affecting plasma FFA levels

 FFAs are also known as non-esterified or unesterified fatty acids in plasma which are derived from –
o Lipolysis of TAGs in adipose tissues
o Hydrolysis of TAGs in plasma lipoproteins (chylomicron, VLDL) by the action of lipoprotein lipase
 FFAs are transported bound to albumin in plasma. Plasma concentration of FFAs varies between 0.1
to 2 μEq/mL.
 Since there is a continuous cycle of lipolysis and re-esterification within adipose tissues, the balance
between these two processes determines the circulating plasma FFA level. These two processes are
entirely different pathways involving different substrates and reactants. This allows the processes of
esterification and lipolysis to be regulated separately by many nutritional, metabolic and hormonal
factors.
 Nutritional factor
o Plasma FFA levels are low in the fully fed condition and rise to 0.7 to 0.8 μEq/mL in the
starvation state.
 Hormonal factor
o Insulin – decreases plasma FFA level by enhancing lipogenesis and inhibiting lipolysis
 It enhances lipogenesis by increasing glucose uptake and utilization in adipose tissues with
the provision of acetyl CoA and NADPH for fatty acid synthesis and glycerol 3-phosphate for
acylglycerol synthesis. It also activates enzymes involved in lipogenesis and esterification of
fatty acids to form TAG.
 It inhibits lipolysis in adipose tissues by inactivating hormone sensitive lipase (HSL) action,
thus reducing FFA release. Anti-lipolytic action of insulin is due to dephosphorylation and
inhibition of HSL via lipase phosphatase, lowering cAMP level by activating PDE and inhibiting
adenylyl cyclase.
 In uncontrolled diabetes mellitus, plasma FFA level may rise to 2 μEq/mL.
o Other anti-lipolytic factors
 Prostaglandin E1 (PGE1) and nicotinic acid inhibits lipolysis by lowering cAMP level via
inactivation of adenylyl cyclase through Gi protein.
o Hormones that accelerate FFA release from adipose tissues by increasing lipolysis
 These include glucagon, epinephrine, norepinephrine, ACTH, TSH, MSH and ADH.
 These hormones stimulate cAMP dependent PKA which phosphorylates and activates
hormone sensitive lipase thereby increasing lipolysis.
 Growth hormone promotes lipolysis by increasing synthesis of proteins involved in the
formation of cAMP.
 Thyroid hormones and glucocorticoids act in a facilitatory or permissive manner for optimal
effect of other lipolytic endocrine factors. Glucocorticoids promote lipolysis via induction of
hormone sensitive lipase synthesis by a cAMP-independent pathway and also by promoting
transcription of genes involved in the cAMP signal cascade.
o Other factors increasing lipolysis
 Methyl xanthine derivatives such as caffeine and theophylline inhibit phosphodiesterase and
thus increase cAMP level which leads to increased lipolysis and FFA release from adipose
tissues.
Role of brown adipose tissue in thermogenesis

Physiological function of brown adipose tissue (brown
fat) is heat generation. Thus, the tissue is extremely
active in some species, for example, during arousal from
hibernation, in animals exposed to cold (non-shivering
thermogenesis), and in heat production in the
newborn.
The tissue is characterized by a well-developed blood
supply and a high content of mitochondria and
cytochromes, but low activity of ATP synthase. Brown
fat mitochondria contain a proton channel known as
uncoupling protein (UCP1, also called thermogenin).
The mechanism of heat generation in brown fat involves
the regulated uncoupling of oxidative phosphorylation.
Norepinephrine liberated from sympathetic nerve
endings is important in increasing lipolysis in the tissue.
The activated lipase hydrolyzes triacylglycerols to yield
free fatty acids that counteract the inhibitory effect of
the purine nucleotides on UCP1. The resulting flow of
protons through UCP1 dissipates the proton gradient
across the inner mitochondrial membrane. Oxidation
and phosphorylation are not coupled in mitochondria of the tissue. Thus, oxidation produces much
heat, and little free energy is trapped in ATP.
Though not a prominent tissue in adult humans, it is present in normal individuals, where it could be
responsible for “diet-induced thermogenesis.” Brown adipose tissue is reduced or absent in obese
persons.
Oxidation of fatty acids

 Plasma fatty acids derived from lipolysis in adipose tissues, or from TAG breakdown in chylomicrons
and VLDL are taken up by cells and used for energy production.
 The utilization of fatty acids for energy production varies considerably from tissues to tissues and
depends to a significant extent on metabolic status, i.e., fed or fasted, exercising or resting. Most
tissues can use fatty acids as a fuel. Fatty acids are a major energy source in cardiac and skeletal
muscle, but the brain does not oxidize fatty acids because they cannot cross the blood-brain barrier.
RBCs cannot oxidize fatty acids since they lack mitochondria, the site of fatty acid oxidation.
 Major site of fatty acid oxidation is mitochondria but it can also occur in peroxisomes and
endoplasmic reticulum.
 Several ways of fatty acid oxidation are –

o β oxidation
o α oxidation
o ω oxidation and
o Peroxisomal oxidation
β oxidation
 It is the major pathway for oxidation of fatty acids in which two carbon fragments are successively
removed from carboxyl end of fatty acyl CoA producing acetyl CoA, NADH, and FADH2. Since
oxidation occurs in a series of reaction at the β carbon and the chain is broken down between the
α(2)- and β(3)- carbon atoms, it is called β oxidation.
 Site – mitochondrial matrix
 Step 1 – priming stage 0r activation of fatty acids to fatty acyl CoA
o Fatty acids are first activated by attachment to CoA via a
thioester bond to form fatty acyl CoA. This reaction is
catalyzed by acyl CoA synthetase in the presence of ATP
and coenzyme A.
 Step 2 – transport of fatty acids (acyl CoA) into the mitochondria
o Long chain fatty acids penetrate the inner mitochondrial
membrane as carnitine derivatives.
o Carnitine palmitoyl transferase I, located in the outer
mitochondrial membrane converts long chain acyl CoA to
acyl carnitine, which is able to penetrate the inner
membrane.
o Carnitine-acylcarnitine translocase acts as an inner
membrane exchange transporter. Acyl carnitine is transported in, coupled with the transport out
of one carnitine molecule.
o Then, carnitine palmitoyl transferase II, located on the inside of the inner membrane catalyzes
conversion of acyl carnitine to acyl CoA which can then enter into β oxidation in mitochondrial
matrix.
 Step 3 – oxidation of acyl CoA in mitochondrial matrix
o Typical fatty acids, such as those that have saturated carbon chains and even-numbered carbon
chains, are completely broken down by the four steps of β-oxidation.
1. Oxidation
 Acyl CoA dehydrogenase catalyzes removal of two hydrogen atoms from α (2) and β (3)
carbon atoms of acyl CoA using FAD as hydrogen acceptor. This results in the formation of
2-trans enoyl CoA and FADH2 which can be oxidized via ETC to produce 1.5 ATP.
2. Hydration
 Water is added to saturate the double bond and form 3-hydroxy-acyl-CoA, catalyzed by Δ2 -
enoyl-CoA hydratase.
3. Oxidation
 The 3-hydroxy derivative undergoes further dehydrogenation on the 3-carbon catalyzed by 3-
hydroxyacyl-CoA dehydrogenase to form the corresponding 3-ketoacyl-CoA compound using
NAD+ as hydrogen acceptor. Released NADH then enters into ETC for re-oxidation and
produces 2.5 ATP.
4. Cleavage
 Finally, 3-ketoacyl-CoA is split at the 2,3-position by thiolase (3-ketoacyl-CoA-thiolase),
forming acetyl-CoA and a new acyl-CoA two carbons shorter than the original acyl-CoA
molecule.
o The shorter acyl-CoA formed in the cleavage reaction reenters the oxidative pathway at the first
step of oxidation. In this way, a long-chain fatty acid with an even number of carbons may be
degraded completely to acetyl-CoA (C2 units). For example, after seven cycles, the C16 fatty acid,
palmitate, would be converted to eight acetyl CoA molecules. Since acetyl-CoA can be oxidized to
CO2 and water via the citric acid cycle, the complete oxidation of fatty acids is achieved.
Energetics of complete oxidation of C16 fatty acid

C16 fatty acid (palmitate) undergoes total 7 cycles of β oxidation, yielding 8 moles of acetyl CoA, 7
moles of FADH2 and 7 moles of NADH.
Reducing equivalents (one FADH2 and one NADH) are oxidized via ETC and produce 4 ATPs through
respiratory chain linked oxidative phosphorylation (4ATPs per one cycle of β oxidation). Seven cycles
of β oxidation produce 28 ATPs (7 x 4 = 28 ATPs).
Acetyl CoA formed from β oxidation cycles are completely oxidized via CAC releasing 10 ATPs per one
mole of acetyl CoA oxidation. Total 8 moles of acetyl CoA gives rise to 80 ATPs from oxidation via
CAC (8 x 10 = 80 ATPs). Thus a total of 108 ATPs (28 + 80 = 108 ATPs) is obtained from complete
oxidation of C16 fatty acid.
Since two ATPs are used in the priming stage or activation of fatty acid to acyl CoA, there is net ATP
production of 106 ATPs per mole of C16 fatty acid (palmitate) oxidation (108 – 2 = 106 ATPs).
Oxidation of odd number carbon chains fatty acids

Odd number carbon chains fatty acid undergoes multiple
rounds of β oxidation until three carbons remain in the
form of propionyl CoA.
Propionyl CoA is converted to succinyl CoA by a multi-step
process.
o Carboxylation of propionyl CoA to D-methyl malonyl
CoA by biotin dependent propionyl CoA carboxylase,
using ATP
o Epimerization of D-methyl malonyl CoA to L-
stereoisomer by methyl malonyl CoA epimerase
o Isomerization of L-methyl malonyl CoA to succinyl CoA
by methyl malonly CoA mutase requiring 5’deoxyadenosyl cobalamin as coenzyme
Succinyl CoA is oxidized via CAC producing 5 ATPs or it can also enter into gluconeogenesis or heme
synthesis.
Oxidation of unsaturated fatty acids

 Unsaturated fatty acids undergo β-oxidation until a disruptive alkene (double bond) is reached.
Double bonds in unsaturated fatty acids are in the cis-configuration and cannot be acted upon by
enoyl-CoA hydratase, as in β oxidation process since it can only catalyze addition of water to trans-
double bond. Therefore, two additional enzymes are needed for β oxidation of the common
unsaturated fatty acids; an isomerase and a reductase.
 Since unsaturated fatty acids are partially oxidized, less FADH2 and correspondingly less ATP is
produced by their oxidation.
Oxidation of long chain fatty acids (peroxisomal

oxidation)
 Atypical fatty acids, such as those with chain
lengths greater than 20 carbons, are degraded
by β-oxidation in peroxisomes rather than in the
mitochondrial matrix.
 In peroxisomes, the first step is catalyzed by a
FAD containing acyl CoA oxidase rather than an
acyl CoA dehydrogenase as seen in mitochondria.
When FADH2 is formed in peroxisomes, it is oxidized to FAD by donating its electrons directly to O2,
forming hydrogen peroxide (H2O2), in contrast to mitochondrial FADH2, whose electrons are
transferred to ETC. Therefore, in peroxisomes, the energy released in the first oxidative step of fatty
acid breakdown is not conserved as ATP, but is dissipated as heat.
 Peroxisomal oxidation is induced by high-fat diet and in some species by hypolipidemic drugs such as
clofibrate. The enzymes in peroxisomes do not attack shorter chain fatty acids. When fatty acids are
shortened to fewer than 20 carbons e.g., octanoyl CoA, they are sent to mitochondria for further
degradation via β-oxidation.
 Another role of peroxisomal oxidation is to shorten the side chain of cholesterol in bile acid
formation.
ω oxidation
 It is a minor pathway for fatty acid oxidation which occurs in
the endoplasmic reticulum of many tissues. It becomes more
important when β oxidation is defective. Medium-chain fatty
acids are the principal substrates for this pathway e.g., C10 –
C12 fatty acids.
 In this pathway, hydroxylation takes place on the methyl
carbon or the carbon next to the methyl end of fatty acids. It is
catalyzed by hydroxylases involving microsomal cytochrome
P450 mixed function oxidases requiring O2 and NADPH.
 Hydroxylated fatty acids can be further oxidized in the cytosol
to dicarboxylic acids via sequential action of cytosolic alcohol
and aldehyde dehydrogenases. Dicarboxylic acids then form
CoA esters at either carboxyl group and then undergo β
oxidation to produce shorter chain dicarboxylic acids such as
adipic acid (C6) and succinic acid (C4) which can enter CAC.
α oxidation
 α oxidation is a minor pathway
occurring in the endoplasmic
reticulum and mitochondria. Unlike β-oxidation, the function
of this pathway is not the production of any high-energy
phosphate bonds, but rather it is used for the degradation of
methylated fatty acids or branched chain fatty acids such as
phytanic acids. α oxidation oxidizes carbon 2 (the α-carbon)
and then releases carbon-1 as CO2, thereby shortening the
fatty acid by one-carbon at a time from the carboxyl end.
 In oxidation of phytanic acid, the first and essential step is α
oxidation to a pristanic acid, which then undergoes β
oxidation producing acetyl CoA and propionyl CoA alternately and in equal amounts.
Regulation of fatty acid oxidation

 Rate of β oxidation in mitochondrial matrix is primarily controlled by the availability of substrates for
β oxidation. Thus, carnitine transport system or carnitine shuttle by which fatty acyl groups are
transported from cytosol into the mitochondria is the rate-limiting step or regulatory point for fatty
acid oxidation.
 Increased malonyl CoA concentration during well-fed state inhibits carnitine shuttle at carnitine acyl
transferase I (CPT-I) step. The inhibition of carnitine acyltransferase I by malonyl-CoA ensures that
the oxidation of fatty acids is inhibited whenever the liver is amply supplied with glucose as fuel and
is actively making TAGs from excess glucose.
Biomedical importance of fatty acid oxidation

 Complete oxidation of fatty acids provides an abundant supply of energy.
 Since gluconeogenesis in liver is dependent upon energy production from fatty acid β oxidation, any
impairment in fatty acid oxidation e.g., carnitine deficiency, leads to hypoglycemia.
 Increased fatty acid oxidation during starvation and uncontrolled diabetes mellitus leads to ketone
body production by the liver (ketosis).
 Carnitine is a derivative of the amino acid lysine. Although small amounts of carnitine can be
synthesized by the liver and the kidney, the diet is a major source of carnitine found in skeletal
muscle. Carnitine deficiency may be primary (strict vegetarian diets, defects in hepatic synthesis of
carnitine, defects in carnitine uptake from the bloodstream, renal losses or loss during hemodialysis)
or due to genetic defects in carnitine transport system. Carnitine deficiency is associated with
hypoglycemia and lipid accumulation with muscles weakness and it particularly affects in the
newborn and pre-term infants. It also affects cardiac energy metabolism leading to cardiomyopathy.
 Refsum’s disease is a rare neurologic disorder, characterized by accumulation of phytanic acid

deposits in nervous tissues as a result of a genetic defect in α-oxidation.
 Hypoglycin A is a toxin in the unripe fruit of the akee tree (found in West Africa and Jamaica).
Metabolism of hypoglycin A generates products that conjugate and sequester carnitine and
coenzyme A, inactivates medium- and short-chain acyl-CoA dehydrogenase, resulting in inhibition of β
oxidation of long chain fatty acids. This leads to hypoglycemia, metabolic acidosis (accumulation of
small chain fatty acids and dicarboxylic acids), lethargy and CNS effects (due to accumulation of
octanoic acid). It causes Jamaican vomiting sickness with vomiting, generalized weakness, altered
consciousness and death.
 Zellweger (cerebrohepatorenal) syndrome occurs in individuals with a rare inherited absence of
peroxisomes in all tissues. They accumulate C26 – C38 polyenoic acids in brain tissue causing severe
neurological symptoms and most patients die in the first year of life.
 Several disorders of lipid catabolism e.g., carnitine deficiency, CPT-I deficiency, acyl-CoA
dehydrogenase deficiency, are associated with the appearance of medium-chain dicarboxylic acids
in urine. When β-oxidation of fatty acids is impaired, fatty acids undergo α-oxidation, releasing one
carbon at a time or ω-oxidation, producing dicarboxylic acid. These dicarboxylic acids undergo
peroxisomal β-oxidation, producing short-chain dicarboxylic acids which are then excreted in urine.
Metabolism of ketone bodies

 Ketone bodies are metabolic products that are produced in excess during excessive
fatty acid breakdown in liver e.g., during starvation. Ketone bodies are the water-
soluble and acidic compounds known as acetoacetate, β-hydroxybutyrate and
acetone. Their accumulation lowers the pH of blood. They serve as an important
energy source for peripheral tissues during periods of prolonged (>24 hours) fasting.
 Liver uses fatty acids as its source of energy for gluconeogenesis during fasting and
starvation. Low oxaloacetate level
in hepatic mitochondria due to
gluconeogenesis results in an
inability to metabolize acetyl CoA
efficiently in the TCA cycle.
 Free CoA is required to initiate and
continue the cycle of β oxidation
which is primary source of ATP in
liver. Because of the reduced
oxidative capacity of CAC and
needs for recycling of acetyl CoA to
CoA for repeated cycles of β oxidation, excess acetyl CoA in liver undergoes a pathway known as
ketogenesis. Thus, free CoA is regenerated and the acetate groups are converted into ketone bodies
which are then releases into the blood stream for utilization in extra-hepatic tissues.
Ketogenesis
 It is the pathway for formation
of ketone bodies from acetyl

CoA in liver. Acetyl CoA is
derived from β oxidation of
fatty acids.
 Ketogenesis occurs solely in
the liver mitochondria since

enzymes for ketogenesis are
located in liver mitochondria.
The liver is unique in its
content of HMG-CoA synthase
and HMG-lyase.
 When fatty acid oxidation is
high and carbohydrate is

depleted to provide oxaloacetate for acetyl CoA oxidation in CAC, acetyl CoA level exceeds oxidation
capacity in liver. Excess acetyl CoA enters into ketogenesis.
 Major pathway of ketogenesis is HMG CoA lyase pathway.
o Acetoacetyl CoA is formed by condensation of two acetyl CoA molecules by the action of
thiolase, releasing CoA-SH or arises directly from the terminal four carbons of acyl CoA during β
oxidation.
o Condensation of acetoacetyl CoA with one more acetyl CoA by the action of HMG CoA synthase
leads to formation of HMG CoA.
o HMG CoA is cleaved into acetoacetate and acetyl CoA by the action of HMG CoA lyase.
 Acetoacetate is reduced to β-hydroxy butyrate by β-hydroxy butyrate dehydrogenase and it may
also be non-enzymatically or spontaneously decarboxylated to acetone. Acetone is volatile and

cannot be oxidized further, it enters the bloodstream and is exhaled (detected as a fruity odor) or
excreted in urine.
 Acetoacetate can also be formed form a minor pathway (simple deacylation) by hydrolysis of
acetoacetyl CoA into acetoacetate and CoA-SH by the action of deacylase.

 Since liver lacks enzymes required for ketone bodies catabolism, ketone bodies are not oxidized in
the liver. Acetoacetate and β-hydroxybutyrate are released into the blood by the liver and are taken
up by peripheral tissues.
 The ratio of β-hydroxybutyrate to acetoacetate in the blood varies between 1:1 and 10:1. Usually β-
hydroxybutyrate is quantitatively predominant ketone body present in the blood. The ratio of β-
hydroxybutyrate to acetoacetate in blood depends on the redox state of hepatic mitochondria as
defined by the NADH/NAD+ ratio. Although the plasma β-hydroxybutyrate/acetoacetate ratio is
usually 1:1 after a meal, it can rise to 6:1 after prolonged (>72 h) fasting and it can go as high as 10:1
during acute diabetic ketoacidosis (DKA).
Utilization of ketone bodies

in extrahepatic tissues
 Ketone bodies are
utilized for energy
needs in extra-hepatic
tissues such as brain,
heart, skeletal muscles
and kidney.
 Ketone bodies are
broken down via the
following mechanism.
 β-hydroxybutyrate is
oxidized to acetoacetate by β-hydroxybutyrate dehydrogenase action, releasing NADH+H+ which
can then donate its electrons to complex I of respiratory chain to generate 2.5 moles of ATP. Formed
acetoacetate is then activated to acetoacetyl CoA for further oxidation process. β-hydroxybutyrate
may also be activated directly by synthetase.
 Acetoacetate is activated to acetoacetyl CoA by succinyl CoA-acetoacetate CoA transferase
(thiophorase). CoA is transferred from succinyl CoA to acetoacetate to form acetoacetyl CoA
releasing free succinate. (Use of succinyl CoA as CoA donor in this reaction bypasses one ATP
formation via substrate level oxidative phosphorylation by succinate thiokinase in CAC).
 In some tissues, acetoacetate may also be directly activated to acetoacetyl CoA by the action of
acetoacetyl CoA synthetase in the presence of ATP and CoA-SH.
 Acetoacetyl CoA is split into two acetyl CoA by thiolase. Acetyl CoA is oxidized in CAC and produces
10 ATPs per oxidation of one mole of acetyl CoA via CAC.
 In normal individuals, ketone body utilization or oxidation in extra-hepatic tissues is proportionate to
their blood concentration. Succinyl CoA-acetoacetate CoA transferase is not expressed significantly
during well-fed state. However, during prolong and enhanced ketosis which occurs during prolonged
starvation or uncontrolled diabetes mellitus, this enzyme synthesis is increased in the brain.
 Normal plasma ketone level is not more than 0.2 mmol/L or 1 mg/dL. Urinary loss of ketone bodies is
negligible (less than 1 mg/day).
Energy yield from ketone bodies oxidation in extra-hepatic tissues

 One acetoacetate yields 19 ATPs (20 ATPs from 2 acetyl CoA oxidation via CAC minus 1 ATP lost due to
activation of acetoacetate).
 One β-hydroxybutyrate yields 21.5 ATPs (2.5 ATP from NADH via respiratory chain linked oxidative
phosphorylation, 20 ATPs from 2 acetyl CoA oxidation via CAC minus 1 ATP lost due to activation of
acetoacetate).
Regulation of ketogenesis
 Ketogenesis is regulated at three crucial steps.
1. Control of fatty acid mobilization from adipose tissues
o Ketogenesis is increased when there is increased
circulating FFA level that arises from lipolysis of TAG
in adipose tissues. Acetyl CoA derived from β-
oxidation of fatty acids is the precursor of ketone
bodies in the liver. Liver both in fed and in fasting
conditions, extracts ~30% of the FFAs passing through
it, so that at high concentrations the flux passing into
the liver is substantial. Therefore, the factors
regulating mobilization of FFA from adipose tissue
are important in controlling ketogenesis.
2. Control of the activity of carnitine transporter system
o After uptake by the liver, FFAs either undergo β-oxidation to provide acetyl CoA or esterification
to form TAG and phospholipids. The activity of carnitine shuttle determines whether FFAs
undergo β-oxidation or esterification.
o Fatty acid flux for oxidation is controlled by carnitine palmitoyltransferase I (CPT-I). CPT-I activity
is low in the fed state, leading to depression of fatty acid oxidation because malonyl CoA formed
during fed state by acetyl CoA carboxylase is a potent inhibitor of CPT-I.
o CPT-I activity is high in starvation and this allows fatty acid oxidation to increase providing
excess acetyl CoA which then enters into ketogenesis pathway.
3. Partition of acetyl CoA between oxidation via CAC and ketogenesis pathway
o Acetyl-CoA, formed in β-oxidation in liver mitochondria, has two possible fates. It may be
completely oxidized to CO2 via CAC or it enters the ketogenesis pathway.
o As the level of serum FFA is raised, proportionately more FFA is converted to ketone bodies and
less is oxidized via the citric acid cycle.
o The partition of acetyl CoA between ketogenesis and CAC is regulated by the availability of
oxaloacetate within mitochondria. Increased β oxidation of fatty acids leads to increased
[NADH]/[NAD+] ratio which in turn leads to decrease in concentration of oxaloacetate. During
Tests for ketones

starvation, reduced oxaloacetate concentration can also  Commercial tests for ketones in
occur due to its utilization in gluconeogenesis and urine and blood are based on the
depletion of glucose for conversion of pyruvate to principle in which acetoacetate in

urine or blood sample is reacted
oxaloacetate.
with nitroprusside (nitroferricyanide)
o This results in reduced function of CAC and excess acetyl
reagent under alkaline conditions to
CoA diverts to ketogenesis pathway. produce a purple-colored complex
(Legal reaction) on a test strip.
Biomedical and metabolic significance of ketone bodies  Legal test is semi-quantitative and
Ketone bodies serve as fuel for extra-hepatic tissues and are does not measure β-hydroxy-
butyrate.
rich source of energy. Ketone bodies increase in plasma
 The blood levels of β-hydroxy-
during fasting and starvation. They are used in cardiac and
butyrate can be measured fairly
skeletal muscles in proportion to their plasma concentration. accurately in the range of 0.1 to 6.0
During starvation, brain also uses ketone bodies for more than mM using immobilized β-hydroxy-
butyrate dehydrogenase test strips.
50% of its energy metabolism.
If the blood ketone bodies level is raised, oxidation of
ketone bodies also increases until blood
concentration of approximately 12 mmol/L is reached
at which oxidative machinery is saturated. Ketonemia
occurs when hepatic ketone bodies production
exceeds utilization by extra-hepatic tissues leading
to accumulation ketone bodies in the blood.
Higher than normal quantities of ketone bodies present in the blood or urine constitute ketonemia
(hyperketonemia) or ketonuria, respectively. The overall condition is called ketosis.
The basic form of ketosis occurs in starvation that involves depletion of available carbohydrate
coupled with mobilization of FFA. Excessive mobilization of FFA and ketogenesis is also found in
diabetes mellitus.
Non-pathologic forms of ketosis are found under conditions of high-fat feeding and after severe
exercise in the post-absorptive state.
Acetoacetic and 3-hydroxybutyric acids are both moderately strong acids and are buffered when
present in blood or other tissues. This progressively depletes the alkali reserve, causing ketoacidosis.
This may be fatal in uncontrolled diabetes mellitus.
Other causes of pathological ketoacidosis include toxic ingestion of ethanol, methanol, ethylene
glycol or salicylates, toxaemia of pregnancy and prolonged labor.
Diabetic ketoacidosis
Diabetic ketoacidosis (DKA) is a common acute complication in patients with type 1 diabetes
mellitus (formerly known as insulin dependent DM). It is a medical emergency condition.
The condition is triggered by severe insulin

deficiency coupled with glucagon excess and
elevation of other stress hormones such as
catecholamines, cortisol and growth hormone.
The major metabolic derangements are –
o Marked hyperglycemia due to diminished
peripheral glucose uptake, utilization and
elevated hepatic glucose output,
o Increased plasma FFA level as a result of
excessive FFA mobilization from adipose tissues
leading to increased FFA uptake and
ketogenesis in liver,
o Ketonemia and ketouria resulting from excessive hepatic ketone bodies production higher than
utilization capacity of extra-hepatic tissues.
Blood concentration of acetoacetate and β-hydroxybutyrate is as high as 20 mmol/L. They are
moderately strong acids and are buffered by HCO3– (bicarbonate buffer). So it progressively depletes
the alkali reserve causing ketoacidosis or increased anion gap metabolic acidosis. Acetoacetate can
also be spontaneously converted to acetone, which is exhaled, giving a characteristic fruity odor.
For respiratory compensation to metabolic acidosis, there is also hyperventilation (deep and fast
breathing).
DKA is also associated with water and electrolyte imbalance due to excessive glycosuria and urinary
water, Na+, K+ loss.
Insulin is essential in treating DKA. It increases peripheral glucose uptake and utilization as well as
reduces FFA mobilization for ketogenesis. It lowers plasma glucagon level and antagonizes the
catabolic effect of glucagon and catecholamines, inhibiting the flow of ketogenic and glucogenic
substrates (FFA and amino acids) from the peripheral tissues.
Metabolism of phospholipids
 Phospholipids are polar, ionic compounds composed of fatty acids, an alcohol (glycerol or
sphingosine), a phosphoric acid residue and other constituents such as nitrogen containing bases or
sugar residues. They are amphipathic in nature.
Biosynthesis of glycerophospholipids
 Glycerophospholipids are made up of two fatty acyl residues and a phosphate group esterified to a
glycerol backbone. The phosphate residue, in turn, can be esterified to amino or sugar alcohols (e.g.,
choline and inositol, respectively) to generate specific glycerophospholipids.
 Synthesis of the various glycerophospholipids share two common features –
o Diacylglycerol (DAG) is a common substrate.

o Cytidine diphosphate (CDP) is attached to different
alcohols to help them serve as substrates e.g., CDP-
ethanolamine, CDP-choline, and CDP-DAG.
 Glycerol 3-phosphate is the primary starting material for
synthesis of phosphatidic acid. Glycerol 3-phosphate is
acylated by the transfer of two long chain fatty acids from fatty acyl CoA to the hydroxyl groups at
carbons 1 and 2, producing phosphatidic acid.
 The first fatty acid added to carbon 1 (sn-1) position of phosphatidic acid is usually a saturated fatty
acid and second fatty acid added to carbon 2 (sn-2) of phosphatidic acid is usually an unsaturated
fatty acid or PUFA e.g., arachidonic acid.
 Synthesis of glycerolphospholipids occurs
primarily through transfer reactions
catalyzed by transferases. Phosphatidate is
the substrate for some glycerophospholipids
synthesis such as phosphatidyl-inositol,
cardiolipin but it can also be hydrolyzed to
DAG which can be used for other
glycerolphospholipids synthesis such as
phosphotidyl-choline, phosphatidyl-serine, phosphatidyl-ethanolamine.
 Biosynthesis of phosphatidylethanolamine and phosphatidylserine
o Phosphatidyl-ethanolamine is produced when ethanolamine phosphotransferase transfers
phosphor-ethanolamine from CDP-ethanolamine onto the carbon 3 (sn-3) of DAG.
o Phosphatidylserine is generated when ethanolamine is removed from phosphatidyl-
ethanolamine and replaced with serine.
o Phosphatidyl-serine can be converted back to phosphatidyl-ethanolamine by the removal of CO2.
 Biosynthesis of phosphatidylcholine
(lecithin)
o Phosphatidylcholine is generated
when choline phosphotransferase
transfers phosphocholine from
CDP-choline onto the carbon 3
(sn-3) of DAG.
o Alternatively, phosphatidylcholine
is produced via a reaction in which
three methyl groups (CH3) are
added to phosphatidyl-ethanolamine. These methyl groups are provided by S-adenosyl

methionine (SAM).
 Biosynthesis of phosphatidylinositol
o Phosphatidyl-inositol is generated when inositol is transferred to the carbon 3 (sn-3) of CDP-
DAG. This reaction, which releases CMP, is catalyzed by CDP-DAG – inositol-3-phosphatidyl
transferase.
Biosynthesis of glycerol
ether phospholipids
 In glycerol ether
phospholipids, one
or more of the
glycerol carbons is
attached to a
hydrocarbon chain
by an ether linkage
rather than an ester bond. Plasmalogens and platelet activating factor (PAF) are important
examples of this type of lipid.
 The biosynthetic pathway is located in peroxisomes. DHAP is the precursor of the glycerol moiety.
Esterified fatty acyl group is replaced by a long chain alcohol to form the ether linkage.
Biosynthesis of sphingophospholipids and glycolipids

 Sphingolipids are built on the
long-chain amino alcohol
sphingosine, whose
components are derived from
serine and palmitic acid.
Addition of a fatty acyl chain
to sphingosine generates
ceramide, the precursor
molecule for the synthesis of
sphingolipids such as
sphingophospholipids and
glycosphingolipids.
 Sphingomyelin is generated
when phosphocholine is transferred from phosphatidylcholine to ceramide releasing DAG.
 Gangliosides are produced when two or more sugars and N-acetylneuraminic acid (NeuSAc or sialic
acid) are added to ceramide.
 Cerebrosides such as galactocerebroside and glucocerebroside are produced when a sugar (e.g.,
galactose or glucose, respectively) is attached to ceramide.
 Globosides such as ceramide trihexoside are formed when the amino sugar GalNAc is attached to
cerebrosides.
 Sulfatides are generated when sulfate residues are transferred to cerebrosides. PAPS serves as the
donor of these sulfate residues.
Degradation of phospholipids
 Phospholipids are in a continuous state of turnover in most membranes.
This occurs as a result of oxidative damage, during inflammation and
through activation of phospholipases particularly in response to
hormonal stimuli.
 Phospholipase A1 and A2 remove acyl groups to form lysophospholipids. Phospholipase A2 (PLA2)
releases arachidonic acid, a prescursor for eicosanoid synthesis. PLA2 is found in pancreatic fluid and
snake venom.
 Phospholipase C liberates two 2nd messengers, DAG and IP3 from PIP2. It is also one of the major
toxins secreted by bacteria.
 Phospholipase D generates phosphatidic acid from various phospholipids and it is known to be
involved in mammalian signal transduction.
 Sphingolipids are normally digested in lysosomes. Sphingomyelinase cleaves sphingomyelin into
phosphocholine and ceramide. The sugars are removed from the terminal ends of the
oligosaccharides by lysosomal exoglycosidases.
 Several genetic diseases referred to as sphingolipidoses (lipid storage diseases) result from
deficiencies in these lysosomal enzymes, e.g., Niemann-Pick disease, Tay-Sachs disease, Gaucher
disease.
Biomedical importance and metabolic significance of phospholipids

 Phospholipids are found in highest concentration in the various cell membranes and serve as
structural components of biologic membrane.
 Function significance of membrane glycerophospholipids
o Phosphatidyl-choline (lecithin) is the most abundant phospholipids of the cell membrane.
Lecithin in plasma lipoprotein provides acyl residue for esterification of cholesterol to
cholesterol ester by the action of lecithin cholesterol acyl transferase (LCAT).
o Dipalmitoyl lecithin is a major constituent of the surfactant of lungs.
o Cardiolipin (diphosphatidyl-glycerol) found in inner membrane of mitochondria is responsible for
special features of inner mitochondrial membrane. It has a key role in mitochondrial structure
and function, and is also thought to be involved in programmed cell death (apoptosis).
o Phospholipids also plays a role in activating certain enzymes e.g., activation of lipoprotein lipase
by membrane phospholipids of lipoprotein in conjunction with apoC-II.
o Inositol phospholipids in the cell membrane act as precursors of hormone second messengers.
o Phosphtidyl serine is required for activation of PKC in some signaling cascades. It also plays a role
in apoptosis.
 Platelet-activaiting factor is an alkyl phospholipid. It involves in inflammation, chemotaxis and
protein phosphorylation.
 Plasmalogen resembles phosphatidyl-ethanolamine but possess an ether link on the sn-1 carbon.
Plasmalogens are a second major class of mitochondrial lipids and are enriched in nerve and muscle
tissue.
 Functional significance of sphingophospholipids and glycolipids
o Ceramide is an important signaling molecule (second messenger) regulating pathways including
programmed cell death (apoptosis), the cell cycle, and cell differentiation and senescence. It
may act as a lipid mediator activating a protein kinase and opposing some of the action of DAG.
o Sphingomyelin is a characteristic phospholipid present in membranes of organelles involved in
secretory processes. It is also found in large quantities in brain and nerve tissues as a component
of myelin sheath.
o Glycosphingolipids are constituents of the outer leaflet of plasma membranes and are important
in cell adhesion and cell recognition. Some are antigens, for example, ABO blood group
substances. Certain gangliosides function as receptors for bacterial toxins, eg., cholera toxin.
o Cerebrosides (galactosyl-ceramide, glucosyl-ceramide) contain a number of characteristic C-24
fatty acids, e.g., cerebronic acid. Galactosyl-ceramide is a major glycosphingolipids of brain and
other nervous tissues. Its derivative sulfogalactosyl-ceramide (sulfatide) is present high amounts
in myelin. Glucosyl-ceramide is the predominant simple glycolipid of extra-neural tissues.
o Gangliosides are complex glycosphingolipids that contain oligosaccharides moiety consisting of
sialic acid, usually N-acetyl neuraminic acid and other sugar residues. They are found high
concentrations in nervous tissues. They appear to have receptor and other functions.
Clinical aspects of phospholipids metabolism

 Lung surfactant is composed mainly of lipid with some proteins and carbohydrate and prevents the
alveoli from collapsing. The phospholipid dipalmitoyl-phosphatidylcholine decreases surface tension
at the air-liquid interface and thus greatly reduces the work of breathing. Deficiency of lung
surfactant in the lungs of many preterm newborns gives rise to infant respiratory distress
syndrome (IRDS).
 Certain diseases are characterized by abnormal quantities of these lipids in the tissues, often in the
nervous system. They may be classified into two groups: (1) true demyelinating diseases and (2)
sphingolipidoses.
 Demyelinating disease e.g., multiple sclerosis
o There is loss of both phospholipids (particularly ethanolamine plasmalogen) and of
sphingolipids from white matter in multiple sclerosis. Thus lipid composition of white matter
resembles that of gray matter. The cerebrospinal fluid shows raised phospholipid levels.
 Sphingolipidoses (lipid storage diseases)
o They are a group of inherited diseases that are caused by a genetic defect in the catabolism of
lipids containing sphingosine. Several lipid storage diseases are due to deficiency of enzymes in
glycolipid breakdown in lysosomes.
Transport of lipid and lipoprotein metabolism

Lipids absorbed from diet and synthesized by liver and adipose tissues must be transported to
various tissues for storage and utilization.
Since lipids are insoluble in water, nonpolar lipids (triacylglycerol and cholesteryl esters) are
associated with amphipathic lipids (phospholipids and cholesterol) and proteins to make water-
miscible lipoproteins for transport. Free (non-esterified) short and medium chain fatty acids can
travel in plasma bound to albumin.
Plasma lipoproteins
o Lipoproteins are molecular complexes of lipids and specific
proteins (apolipoproteins) serving to solubilize lipid in
plasma for their transport.
o Lipoprotein particle consists of a core of hydrophobic lipids
(TAG, cholesterol esters) surrounded by a single surface
layer of amphipathic phospholipids, free cholesterol
molecules and one or more proteins called apolipoproteins. Apolipoproteins serve either as
enzyme activators or as ligands that bind to receptors.
o The interactions between lipids and proteins in the lipoprotein particle are of a purely non-
covalent nature.
o There are four major groups of lipoproteins, and they differ in size, density, relative amounts of
and types of lipid and protein they contain. These include —
 Chylomicrons (derived from intestinal mucosal cells for transport of absorbed dietary TAG
and other lipids)
 Very-low-density lipoproteins (VLDL or pre-β lipoproteins, derived from liver for the export
of endogenously synthesized TAGs)
 Low-density-lipoproteins (LDL or β-lipoproteins, derived from catabolism of VLDL)
 High-density-lipoproteins (HDL or α-lipoproteins, involved in reverse cholesterol transport,
chylomicron and VLDL metabolism)
o Triacylglycerol is the predominant lipid in chylomicrons and VLDL, whereas cholesterol and
phospholipid are the predominant lipids in LDL and HDL.
o The largest lipoproteins are the least dense due to a relatively high concentration of
triacylglycerols. The smaller the lipoprotein the higher is the density due to its relatively high
concentrations of proteins and phospholipids.
o Lipoprotein can be separated according to –
1. Electrophoretic mobility
 Those with higher protein content (HDL) will
move faster to the anode and those with less protein content have minimum mobility
(chylomicron and VLDL).
2. Ultracentrifugation (according to density)
 Those with higher lipid content (chylomicron) will float on centrifuge and those with less
lipid content will sediment easily (HDL) on centrifuge.
Apolipoproteins
The protein moiety of a lipoprotein is known as an apolipoprotein or apoprotein, constituting nearly
70% of some HDL and as little as 1% of chylomicrons.
Some apolipoproteins are integral and cannot be removed (e.g., apo B), whereas others are bound
to the surface (peripheral) and are free to transfer to other lipoproteins, e.g., apo C and E).
The major apolipoprotein of HDL is apo A and main apolipoprotein of LDL and VLDL is apo B-100.
Chylomicrons contain a truncated form of apo B (B-48) that is synthesized in the intestine, while apo
B-100 is synthesized in the liver.
Apo C-I, C-II and C-III are smaller polypeptides which are freely transferable between several different
lipoproteins. Apo E, found in VLDL, HDL, chylomicrons and chylomicron remnants, is also freely
transferable. Apolipoproteins are mainly synthesized in liver.
Apolipoproteins carry out several roles;
o For solubilization and transport of lipid as a structural component of lipoprotein e.g., apo B
o As enzyme cofactors and activation of enzymes e.g., C-II for lipoprotein lipase, A-I for LCAT, or as
enzyme inhibitors, e.g., apo A-II and apo C-III for lipoprotein lipase, apo C-I for cholesteryl ester
transfer protein
o As ligands for interaction with lipoprotein receptors in tissues, e.g., apo B-100 and apo E for the
LDL receptor, apo E for the LDL-receptor-related protein-1 (LRP-1) or remnant receptor and apo
A-I for the HDL receptor.
o Facilitation of the transfer of lipids between different lipoproteins (role as lipid transfer protein)
e.g., apo D as cholesterol ester transfer protein (CETP).
Metabolism of chylomicron
Chyl0microns are synthesized in the intestinal mucosal cells and carry dietary TAG, cholesterol,
cholesterol esters and other lipids to the peripheral tissues.
Major apoprotein of chylomicron is apo B-48 and TAG is the predominant lipid in chlylomicrons.
Apo B-48 which is synthesized in RER is glycosylated and incorporated with TAG, cholesterol,
cholesterol ester and phospholipids to from chylomicrons in SER. They are packaged into secretory
vesicles and secreted by reverse pinocytosis (exocytosis) into lymphatics and then into the venous
blood.
Nascent chylomicron receives apo E and apo C-II from HDL in the circulation and becomes mature
chylomicron.
Lipoprotein lipase on capillary endothelium is activated by apo C-II and phospholipids. It hydrolyzes
TAG in chylomicrons into glycerol and FFAs. FFAs are delivered to the tissues for oxidation or
esterification. After 90% of TAG from chylomicron is lost, apo C-II returns to HDL and chylomicron
becomes chylomicron remnants which is rich in cholesterol and its ester.
Chylomicron remnants are endocytosed into liver via apo E mediated interaction with LDL receptor
and remnant receptor (LRP). Then it is hydrolytically degraded in lysosomes.
Half-life of chylomicrons in plasma is less than 1 h. Thus, chylomicrons are present only after fat-
containing meals causing a milky appearance of plasma.
Metabolism of VLDL
VLDL transports TAG (mainly), cholesterol, cholesterol ester and other lipids synthesized in liver to
other tissues.
Major apoprotein of VLDL is apo B-100 and TAG is the predominant lipid in VLDL.
Apo B-100 which is synthesized in RER is glycosylated and incorporated with TAG, cholesterol,
cholesterol ester and phospholipids to from VLDL in SER. They are packaged into secretory vesicles
and released into the blood stream by reverse pinocytosis (exocytosis). Nascent VLDL contains apo
B-100 and apo A-1.
Nascent VLDL receives apo E and apo C-II from HDL in the circulation and becomes mature VLDL.
Lipoprotein lipase on capillary endothelium is activated by apo C-II and phospholipids. It hydrolyzes
TAG in VLDLs into glycerol and FFAs. FFAs are delivered to the tissues for oxidation or esterification.
As TAG in VLDL is depleted, VLDL decreases in size and density is increased. Apo C-II returns to HDL
and cholesterol esters are transferred from HDL to VLDL by CETP. VLDL then becomes VLDL
remnants or intermediate density lipoproteins (IDL).
VLDL remnants or IDLs are either taken up by the liver via apo E mediated interaction with LDL
receptor (apo B-100, apo E receptor) or IDLs lose more triacylglycerol and all apoproteins except for
apo B-100 through the action of hepatic lipase to become LDL.
LDLs are enriched in cholesterol (particularly cholesterol esters) and serve as the main carriers of
cholesterol in human blood.
Factors that enhance both synthesis and secretion of VLDL by the liver
o Fed state rather than starvation state
o Feeding of diets rich in carbohydrates (especially high sucrose or fructose content in diet)
o Elevated plasma FFA level
o Ingestion of ethanol
o High insulin/glucagon ratio
o TAGs in VLDL are hydrolyzed more slowly compared to those in chylomicrons, thus half-life of
VLDL in serum is 1 – 3 hours.
Lipoprotein lipase
 Lipoprotein lipase is located on the walls of blood capillaries, anchored to the endothelium by
negatively charged proteoglycan chains of heparan sulfate. It is found in capillary endothelium of
various tissues such as heart, adipose tissues, lungs although it is not active in adult liver.
 It is not normally found in blood; however, following injection of heparin, lipoprotein lipase is
released from its heparan sulfate binding sites into the circulation.
 Hepatic lipase bound to the sinusoidal surface of liver cells is functionally similar to lipoprotein lipase
except that this enzyme does not react readily with chylomicrons or VLDL but is involved in
chylomicron remnant and HDL metabolism.
 Both phospholipids and apo C-II are required as cofactors for lipoprotein lipase activity, while apo A-II
and apo C-III act as inhibitors. When it is activated by apo C-II and phospholipids, lipoprotein lipase
hydrolyzes TAG in lipoproteins attached to the enzyme on the endothelium.
 Hydrolysis of TAG in lipoproteins progressively releases FFA and glycerol. Some of the released FFA
return to the circulation, attached to albumin, but the bulk is transported into the adjacent tissues.
 Heart lipoprotein lipase has a low Km for triacylglycerol, about one-tenth of that for the enzyme in
adipose tissue. This enables the delivery of fatty acids from TAG to be redirected from adipose tissue
to the heart in the starved state when the plasma triacylglycerol decreases. A similar redirection to
the mammary gland occurs during lactation allowing uptake of long-chain fatty acids for milk fat
synthesis.
 In adipose tissue, insulin enhances lipoprotein lipase synthesis in adipocytes and its translocation to
the luminal surface of the capillary endothelium.
Metabolism of LDL
In general, cells outside the liver and intestine obtain cholesterol from the plasma rather than
synthesizing it de novo. Their primary source of cholesterol is low-density lipoprotein (LDL).
LDL is derived from VLDL remnant or IDL by the action of hepatic lipase or it can also be produced
directly by the liver. LDL contains large amount of cholesterol ester, some free cholesterol, apo B-
100 and apo E. They are main carrier of cholesterol in plasma and are taken up either by the liver
(approximately 70%) and by extra-hepatic tissues.
Plasma LDLs are recognized by the specific cell surface receptor proteins, LDL receptors via
interaction with apo B-100 on LDL particles. In this way, LDL particles are taken up by the cells that
need cholesterol through receptor mediated endocytosis.
After internalization into the cells, endosome containing LDL and LDL receptor fuses with lysosome.
Components of LDL are hydrolyzed by lysosomal enzymes to release free cholesterol, amino acids,
fatty acids and phospholipids. But LDL receptor escapes degradation and recycles to the cell surface
to function again in LDL uptake.
The number of LDL receptors on the cell membrane is up or down-regulated depending on
cholesterol requirement in the cells. When cholesterol is abundant inside the cell after cholesterol
influx, LDL receptors synthesis is repressed and it also affects intracellular cholesterol synthesis by
influencing the rate of HMG-CoA reductase gene transcription.
There is a positive correlation between the incidence of atherosclerosis and the plasma
concentration of LDL cholesterol.
Defective LDL receptors in familial hypercholesterolemia result in increased blood LDL cholesterol
levels which lead to premature atherosclerosis.
Metabolism of HDL
HDL transports cholesterol in reverse direction i.e., from peripheral tissues to the liver for excretion
via the bile (either as cholesterol or after conversion to bile acids) in the process known as reverse
cholesterol transport. HDLs are synthesized in the intestine and liver. The major apolipoproteins are
apo A-I and apo A-II but they also contain apo C, apo E and LCAT. HDL synthesized in intestine has no
apo E and apo C-II. Thus they are taken from HDL synthesized in liver.
Nascent HDL (discoidal

HDL) consists of
discoid phospholipid
bilayers containing apo
A and free cholesterol.
Nascent HDL accepts
cholesterol from cells
through ATP-binding
cassette transporters
(ABC-A1, ABCG-1).
This free cholesterol is
esterified by lecithin-
cholesterol acyl
transferase (LCAT) to
cholesterol ester
releasing lysolecithin which is transferred to plasma albumin whereas cholesterol ester moves into
the hydrophobic interior of HDL particle. LCAT is activated by apo A-I.
HDL becomes spherical (HDL-3) due to the increase in cholesterol esters within its core. HDL
transfers its cholesterol esters to chylomicron remnants, VLDL, IDL and LDL in exchange for TAGs and
phospholipids. These exchanges are facilitated by cholesterol ester transfer protein (CETP) and
phospholipid transfer protein (PLTP). Acquisition of TAG and further uptake of cholesterol from
tissues leads to increase in size of HDL-3 and becomes less dense forming HDL-2.
In tissues, scavenger receptor mediates cholesterol efflux from the cells to HDL. But in the liver and
steroidogenic tissues, HDL binds SR-B1 on the cell membrane via apo A-I and cholesteryl ester is
selectively delivered to the cells, although the particle itself is not taken up. HDL-3 can then be
reformed from HDL-2 either after selective delivery of cholesteryl ester to the liver via the SR-B1 or
by hydrolysis of HDL-2 phospholipid and triacylglycerol by hepatic lipase and endothelial lipase. This
interchange of HDL-2 and HDL-3 is called the HDL cycle.
Free apo A-I is released by these processes and forms pre β-HDL after associating with a minimum
amount of phospholipid and cholesterol. Pre β-HDL can then re-enter into reverse cholesterol
transport. Preβ-HDL is the most potent form of HDL inducing cholesterol efflux from the tissues.
Surplus apo A-I is destroyed in the kidney.
Another major function of HDL is to act as a repository for the apo C and apo E required in the
metabolism of chylomicrons and VLDL.
HDL activates the endothelial nitric oxide synthase (eNOS), promoting production of NO. They also
have anti-inflammatory properties.
Scavenger receptors
HDL-2 concentrations are inversely related to the incidence Can bind many different molecules
of atherosclerosis, possibly because they reflect the (non-specific).
efficiency of reverse cholesterol transport. They are not subjected to feedback

regulation.
Class A, B and CD36.
Biomedical importance of lipoprotein metabolism
Class A receptors do not bind intact
Lipoprotein metabolism links closely with the metabolism of
LDL but readily bind chemically
energy substrates. Abnormalities of lipoprotein metabolism modified or (e.g., acetylated or
are key factors in the development of atherosclerosis, a oxidized) LDL. These receptors are
process affecting arterial walls and consequently blood present on phagocytic cells such as
macrophages.
supply and oxygen delivery to heart (causing coronary heart
Class B receptors selectively delivers
disease), brain (causing stroke) and other large arteries
CE from HDL particles to liver and
(causing peripheral vascular disease). steroidogenic tissues, but in other
There is a strong direct relationship between plasma levels tissues, these receptors serves for
cholesterol efflux to HDL.
of cholesterol and LDL cholesterol (LDL-C) and an inverse
relationship between HDL cholesterol with risk of coronary
heart disease (CHD).
The Fredrickson classification of lipoprotein disorders is
based on abnormalities of concentrations of the major
classes of lipoproteins in plasma. Lipoprotein phenotyping
is carried out by measuring total cholesterol and
triacylglycerol levels, and cholesterol levels in LDL and HDL.
Another commonly used phenotypic classification in clinical
practice divides dyslipidemia into hypercholesterolemia,
hypertriglyceridemia and mixed dyslipidemia.
Hyperlipoproteinemias can be primary (genetic) or
secondary. Some causes of secondary hyper-
lipoproteinemias include diabetes mellitus,
hypothyroidism, nephrotic syndrome, chronic renal failure, cigarette smoking, ethanol abuse,
primary biliary cirrhosis, and intake of oral contraceptives.
Primary disorders can be due to a single-gene defect or to a combination of genetic defects.
Secondary hyper-lipoproteinemias, known as multifactorial or polygenic hyper-lipoproteinemias,
are affected by metabolic disorders and environmental insults such as a diet high in saturated fat and
cholesterol, obesity, diabetes mellitus and ethanol abuse.
Type I hyper-lipoproteinemia is associated with defects in apo C-II or deficiency in lipoprotein lipase
leading to extreme elevation of chylomicrons and VLDL.
Type II hyper-lipoproteinemia, also known as familial hypercholesterolemia (FH), is an inherited
metabolic disorder due to defects in the cellular uptake of LDL through the LDL receptor pathway.
Defects in this process lead to an

accumulation of LDL-cholesterol in
the blood and the subsequent
development of atherosclerosis.
Defects in apo E are linked to type
III hyperlipoproteinemia (familial
dysbetalipoproteinemia), which
disrupts hepatic uptake of
chylomicron remnants (and IDL).
Familial combined hyperlipidemia
is characterized by overproduction
apo B-100 rather than the impairment of receptor-mediated clearance. There is an increased
production of VLDL and consequently, increased generation of the LDL. This dyslipidemia presents
with variable plasma lipid patterns (either hypercholesterolemia alone or hypercholesterolemia with
hypertriglyceridemia).
Several rare mutations lead to a decreased HDL cholesterol concentration. Low HDL-cholesterol can
result from mutations in genes coding for the apoA1, the ABCA1 transporter and the LCAT. A rare
autosomal recessive disorder called Tangier disease is a type of hypo-lipoproteinemia. It is marked by
excessively low levels of HDL due to defects in the ABC-A1 transporter.
Metabolism of ethanol
Ethanol is oxidized in the liver mainly by alcohol dehydrogenase, to form acetaldehyde which is in
turn oxidized by aldehyde dehydrogenase (ALDH) to

acetate.
Alcohol dehydrogenase is a cytosolic enzyme and
aldehyde dehydrogenase is a mitochondrial enzyme.
Both enzymes use NAD+ as coenzyme.
A cytochrome P450 enzyme, CYP2E1, also oxidizes
ethanol but is quantitatively less important than the
alcohol dehydrogenase. This is also called
microsomal ethanol oxidizing system (MEOS).
CYP2E1 is induced by alcohol. Therefore an increased proportion of the alcohol is metabolized by this
route in chronic alcoholics.
Chronic alcoholism leads to hyperlipidemia, fat accumulation in the liver (fatty liver) and ultimately
cirrhosis.
Metabolic consequences of alcoholism

o Alcohol metabolism in hepatocytes leads to increased [NADH]/[NAD+] ratio, leading to –
 Decreased fatty acid β oxidation
 Decreased citric acid cycle activity due to shift in reaction equilibrium from oxaloacetate to
malate
 Increased lactate formation and lactic acidosis due to shift in reaction equilibrium from
pyruvate to lactate
 Decreased gluconeogenesis due to decreased availability of NAD+ for oxidation of lactate to
pyruvate, malate to oxaloacetate and glycerol 3-phosphate to DHAP in gluconeogenesis
o Oxidation of ethanol by alcohol dehydrogenase leads to excess production of NADH, which
competes with reducing equivalents from other substrates, including fatty acids, for the
respiratory chain. This inhibits their oxidation and causes increased esterification of fatty acids
to form triacylglycerol. Increased TAG formation can cause increased secretion of VLDL into the
blood stream leading to hyperlipidemia or if TAG formation exceeds VLDL export than, TAG will
be deposited in hepatocytes leading to fatty liver (hepatic steatosis).
o MEOS activity is increased in chronic alcoholism. Ethanol can inhibit the metabolism of some
drugs, e.g., barbiturates, by competing for cytochrome P450-dependent enzymes.
Role of liver in lipid metabolism

 The liver carries out the following major functions in lipid metabolism:
1. It facilitates the digestion and absorption of lipids by the production of bile, which contains
cholesterol and bile salts synthesized within the liver de novo or after uptake of lipoprotein
cholesterol. This process also important for excretion of excess cholesterol from the body.
2. It actively synthesizes and oxidizes fatty acids and also synthesizes TAGs and phospholipids.
3. It converts fatty acids to ketone bodies (ketogenesis) which can be used as metabolic fuel in
extra-hepatic tissues during starvation period.
4. It plays an integral part in the synthesis and metabolism of plasma lipoproteins, e.g., export of
TAG and cholesterol esters by VLDL into the blood stream, synthesis and secretion of HDL for
reverse cholesterol transport, synthesis of apolipoproteins required for plasma lipoproteins
metabolism.
Fatty liver
 Fatty liver is a condition in which there is extensive accumulation of TAG in the liver due to
imbalance in the rate of TAG formation and export.
 The fatty acids derived from de novo synthesis (synthesized from acetyl-CoA which is derived mainly
from carbohydrate) or uptake from the circulation are esterified to TAG in the liver. Normally hepatic
TAGs are transported as VLDL as soon as it is formed.
 Extensive accumulation of fatty acids in liver is regarded as a pathologic condition. When

accumulation of lipid in the liver becomes chronic, inflammatory and fibrotic changes may develop
leading to nonalcoholic steatohepatitis (NASH), which can progress to liver diseases including
cirrhosis, hepatocarcinoma and liver failure.
 Fatty livers fall into two main categories.
o Fatty liver due to raised plasma FFA levels and increased TAG synthesis
 Raised plasma FFA level resulting from mobilization of fat from adipose tissue or from the
hydrolysis of lipoprotein triacylglycerol by lipoprotein lipase leads to increased FFA uptake in
liver.
 The production of VLDL does not keep pace with the increasing influx and esterification of
free fatty acids, allowing triacylglycerol to accumulate, which in turn causes a fatty liver.
 It is seen in diabetes mellitus, chronic alcoholism, during starvation and feeding of high-fat
diets. The ability to secrete VLDL may also be impaired in starvation.
o Fatty liver due to a metabolic block in the production of plasma lipoproteins
 Defective VLDL synthesis may be due to –
1. Defective apolipoprotein synthesis or an increase in its degradation before it can be
incorporated into VLDL,
2. Defective synthesis of the lipoprotein from lipid and apolipoprotein,
3. Failure in provision of phospholipids or
4. Defective secretory mechanism itself.
Metabolism of cholesterol
 Cholesterol is present in tissues and in plasma either as free
cholesterol or combined with a long-chain fatty acid as
cholesteryl ester, the storage form. In plasma, both forms are
transported in lipoproteins.
 It is synthesized in many tissues from acetyl-CoA and is the precursor of all other steroids in the body,
including corticosteroids, sex hormones, bile acids, and vitamin D. Cholesterol is an amphipathic lipid
and serves as an essential structural component of membranes and of the outer layer of plasma
lipoproteins.
 Plasma low-density lipoprotein (LDL) is the vehicle that supplies cholesterol and cholesteryl ester to
many tissues. Free cholesterol is removed from tissues by plasma high-density lipoprotein (HDL) and
transported to the liver, where it is eliminated from the body either unchanged or after conversion to
bile acids in the process known as reverse cholesterol transport.
Sources of cholesterol in human

 Exogenous source
o Since cholesterol is a product of animal
metabolism, it is present in various
foodstuffs of animal origin such as egg yolk,
meat, and liver. Plants contain other sterols,
which are collectively called phytosterols.
Ergosterol (mostly in fungi) and beta-
sitosterol (in higher plants) are examples of
phytosterols. They are poorly absorbed from
dietary sources and therefore are present
only in small amounts in the human body.
o Average diet contains about 300 – 500
mg/day.
 Endogenous source
o About half of the body cholesterol is derived
from de novo synthesis from acetyl CoA.
Cholesterol is synthesized about 500 – 700
mg/day. All nucleated cells can synthesize
cholesterol.
o Cholesterol is synthesized in the liver (50%),
intestine (15%), skin, nervous tissues, gonads
and adrenal cortex.
Biosynthesis of cholesterol
 All 27-carbon atoms of cholesterol are derived from the acetyl-CoA. The enzyme system of
cholesterol synthesis present in cytosolic and microsomal (endoplasmic reticulum) fractions.
 Cholesterol synthesis is a reductive synthesis process consuming large amount of ATP.
 The reactions of cholesterol biosynthesis occur in 5 stages.
1. Synthesis of mevalonate from acetyl-CoA through HMG-CoA
 First, two molecules of acetyl-CoA condense to form acetoacetyl-CoA, catalyzed by a
cytosolic thiolase enzyme.
 Another molecule of acetyl-CoA condenses with acetoacetyl-CoA catalyzed by HMG-CoA
synthase to form HMG-CoA.
 This sequence of reactions in the cholesterol synthesis is similar to those for the synthesis of
ketone bodies except that ketone body synthesis occurs in mitochondria and cholesterol is in
the cytosol.
 HMG-CoA is reduced to mevalonate by HMG-CoA reductase using two molecules of NADPH as
the reducing power. This is the rate limiting step in the cholesterol synthesis.
2. Formation of isoprenoid unit by decarboxylation of mevalonate
 Mevalonate is phosphorylated by ATP and subsequently decarboxylated to form an active
five carbon isoprene unit, isopentenyl pyrophosphate (IPP).
3. Formation of squalene from condensation of six isoprenoid units
 Six isopentenyl pyrophosphate
molecules condense with loss of
their pyrophosphate groups to
yield the squalene (30-carbon
atoms compound) through the
formation of geranyl
pyrophosphate (C-10) and
farnesyl pyrophosphate (C-15).
4. Cyclization of squalene to lanosterol
 Squalene undergoes a series of
complex enzymatic reactions;
oxygenation to form squalene
epoxide and cyclization of
squalene epoxide to form lanosterol which has the four condensed rings that form the
steroid nucleus of cholesterol.
5. Formation of cholesterol from lanosterol
 The conversion of lanosterol to cholesterol is a multistep process that involves –
 Shortening of the carbon chain from 30 to 27

 Removal of the three methyl groups at C4
 Migration of the double bond from C8 to C5
 Reduction of the one double bond between C24 and C25 by NADPH.
 Essential isoprenoids derived from cholesterol synthesis pathway
o The polyisoprenoids dolichol and ubiquinone are formed from farnesyl diphosphate by the
further addition of up to 16 (dolichol) or 3–7 (ubiquinone) isopentenyl diphosphate residues.
o Some GTP-binding proteins in the cell membrane are prenylated with farnesyl or geranylgeranyl
(20 carbon) residues.
o Protein prenylation is believed to facilitate the anchoring of proteins into lipid membranes and
may also be involved in protein-protein interactions and membrane-associated protein
trafficking.
o Isopentenyl pyrophosphate is used for synthesis of isopentenyl-adenosine which is a modified
base of tRNA.
 Energy cost of cholesterol
synthesis
o In the pathway from
acetyl-CoA to squalene,
ATP is consumed only
in the steps that
convert mevalonate to
the activated isoprene
precursors of squalene.
o Three ATP molecules
are used to create each
of the six activated
isoprenes required to
construct squalene, for
a total cost of 18 ATP
molecules.
Regulation of cholesterol
synthesis
 HMG-CoA reductase is the
rate limiting enzyme in
cholesterol synthesis.
 In mammals, cholesterol production is regulated by intracellular cholesterol concentration, by the

supply of ATP, and by the hormones glucagon and insulin.
 The activity of HMG-CoA reductase is regulated by –
 End product cholesterol and intermediate product
mevalonate are negative feedback regulator on
the enzyme and acetyl CoA is a positive feed-
forward effector on the enzyme.
 It is also repressed by bile salts or bile acids, thus
decreasing the cholesterol synthesis in liver.
 HMG-CoA reductase is active in dephosphorylated
state and inactive in phosphorylated state.
 Insulin activates protein phosphatase which dephosphorylates and activates HMG-CoA
reductase.
 HMG-CoA reductase is phosphorylated and inactivated by cAMP dependent PKA and AMPK.
Glucagon inhibits HMG CoA reductase by preventing its dephosphorylation as well as
inactivation via phosphorylation.
 Conditions of low energy, marked by high AMP levels, stimulate the phosphorylation and
inhibition of HMG-CoA reductase via activating AMP-activated protein kinase (AMPK).
o Hormonal control
 Cholesterol synthesis is increased by insulin and thyroid hormone and is decreased by
glucagon and glucocorticoid by stimulating and inhibiting HMG-CoA reductase enzyme
respectively.
o Modulation of HMG-CoA reductase gene transcription
 Cellular cholesterol level affects HMG-CoA reductase gene expression. Regulation of HMG-
CoA reductase synthesis by cholesterol is mediated by transcriptional regulation of the HMG-
CoA reductase gene.
 Expression of HMG-CoA reductase gene, along with more than 20 other genes encoding
enzymes that mediate the uptake and synthesis of cholesterol is controlled by the
transcription factor, sterol regulatory element-binding proteins (SREBPs) that bind to the
sterol regulatory response element (SRE) of the target genes.
 SREBPs are embedded in the ER in a complex with the protein SREBP cleavage-activating
protein (SCAP), which is in turn bound to Insig. When bound to SCAP and Insig, SREBPs are
inactive. When sterol levels decline, sterol-binding sites on Insig and SCAP are unoccupied,
the complex migrates to the Golgi complex and SREBP is cleaved to produce a regulatory
domain. SREBP then translocates to the nucleus and increase the transcription of sterol-
regulated genes.
o Modulation of enzyme degradation
 In the long term, the level of HMG-CoA reductase is also regulated by proteolytic degradation
of the enzyme itself. High levels of cellular cholesterol are sensed by Insig, which triggers
attachment of ubiquitin molecules to HMG-CoA reductase, leading to its degradation by
proteasomes.
o Diurnal variation
 It occurs in both cholesterol synthesis and reductase activity, which is highest at midnight and
lowest at noon.
Therapeutic inhibition
 Statins (lovastatin, mevastatin, simvastatin, pravastatin, rosuvastatin) are used as cholesterol
lowering agents. They are structural analogs of HMG-CoA and reversible competitive inhibitors of
HMG-CoA reductase.
 Statins are usually taken at night to ensure maximal effect.
Function of cholesterol (Fates of cholesterol in the cell)

1. In the liver, cholesterol is used for synthesis of bile acids and bile salts which is required for dietary
lipid digestion and absorption.
2. In adrenal cortex, cholesterol is used as precursors for adrenocortical hormones (steroid hormones)
synthesis.
3. In gonads, cholesterol is used as precursors for sex hormone synthesis e.g., estrogens, progesterone
in ovaries and testosterone in testes.
4. A small fraction of cholesterol is incorporated to the cell membrane.
5. A large amount of cholesterol is precipitated in the corneum of the skin.
o Cholesterol together with other lipids makes the skin inert to acids and different solvents, thus
preventing penetration of these solutions.
o Cholesterol in the skin also helps to prevent water evaporation from the skin.
o 7-dehydrocholesterol in the skin is the precursor of vitamin D synthesis in the body.
Cholesterol transport in the body

Cholesterol is transported in plasma in
lipoproteins, with the greater part in the form
of cholesteryl ester and in humans the
highest proportion is found in LDL.
Transport of dietary cholesterol from intestine
o Cholesteryl ester in the diet is hydrolyzed
to cholesterol, which is then absorbed by
the intestine together with dietary
unesterified cholesterol and other lipids.
In the intestinal mucosal cells, much of
cholesterol is subsequently reconverted
into cholesterol esters.
o Cholesterol esters together with some
un-esterified cholesterol are incorporated into chylomicrons, which transports cholesterol and
other dietary lipids from intestine to the blood stream.
o When TAGs in chylomicrons is hydrolyzed by lipoprotein lipase in the peripheral tissue, only about
5% of the cholesterol ester is lost. The rest (more than 95%) is taken up by the liver in the form of
chylomicron remnants and is hydrolyzed to cholesterol. Most of the cholesterol is secreted by
liver in VLDL.
Transport of cholesterol from liver to peripheral tissue
o Cholesterol from liver is transported in the form of VLDL into the plasma. Like chylomicrons, TAGs
in VLDL is hydrolyzed by lipoprotein lipase in the peripheral tissues. Cholesterol is retained in the
lipoprotein as cholesteryl ester resulting in formation of IDL, and subsequently cholesterol rich
LDL. LDL is the major carrier of cholesterol to the tissues.
o LDL cholesterol is taken up by the liver or extra-hepatic tissues by LDL receptors.
Transport of cholesterol from peripheral tissue to liver
o HDL picks up excess cholesterol from the peripheral tissues and converts it to cholesterol esters
by LCAT enzyme. These cholesterol esters are ultimately returned to the liver by HDL. This
process is called “reverse cholesterol transport.”
Degradation of cholesterol
 Sterol ring structure in cholesterol cannot be metabolized to CO2 and H2O in human. It can only be
metabolized in the liver to bile acids and eliminated into the bile.
 About one gram of cholesterol is eliminated from the body daily. About half is excreted in the feces
through the bile after conversion to bile acids and the remainder is directly secreted into the bile as
neutral sterol which is then ultimately excreted into the intestine.
 In the intestine, some cholesterol is modified by bacteria enzymes to coprostanol and cholestanol.
These reduced derivatives and cholesterol constitute the bulk of neutral sterols in feces.
Factors affecting blood cholesterol level

 Normal blood cholesterol level of a healthy adult is 150 – 250 mg/dL or 3.8 – 6.7 mmol/L.
 Plasma cholesterol level is influenced by several factors.
 Hereditary factor
o They play the greatest role in determining individual blood cholesterol level.
o Cholesterol level is markedly increased in familial hyperlipidemia such as –
 Type II-a ( defective LDL receptor)
 Type II-b (over-production of apo B)
 Type III (apo E abnormality)
 Dietary factors
o Dietary cholesterol
 Reducing amount of cholesterol in the diet produce variable results and has relatively little
effects on plasma cholesterol level due to feedback control of endogenous cholesterol
synthesis at HMG-CoA reductase step.
 Generally, a decrease of 100 mg in dietary cholesterol causes a decrease of approximately
0.13 mmol/L (5 mg/dL) of plasma cholesterol.
o Dietary polyunsaturated fatty acids (PUFAs)
 PUFAs lowers plasma cholesterol level by –
1. Stimulating oxidation of cholesterol to bile acids for excretion
2. Stimulation of cholesterol excretion into the intestine
3. Increased LDL cholesterol uptake in tissues by up-regulation of LDL receptor.
 PUFAs lower both LDL and HDL cholesterol levels, while monounsaturated fatty acids such as
oleic acid appears to lower LDL without affecting HDL levels.
 ω-3 PUFA (α-linolenic acid, eicosapentoenoic acid, docosahexaenoic acid) is beneficial in
preventing atherogenesis and thrombosis by modulating prostaglandin metabolism and
decrease plasma cholesterol level.
o Dietary saturated fatty acids (SFAs)
 SFAs raise plasma cholesterol level by –
1. Inhibiting conversion of cholesterol to bile acids
2. Down-regulating LDL receptors thereby decreased uptake of LDL cholesterol by tissues
3. Formation of smaller VLDL particles with more cholesterol content which are utilized
by tissues at a slower rate than larger particles.
 Changing dietary PUFA/SFA ratio from 0.3 to 1 can reduce the plasma cholesterol level.
o Garlic – lowers plasma lipid level. It also reduces blood pressure and inhibits platelet aggregation.
o Dietary fibers
 Reasonable amount of dietary fiber (especially water soluble fibers) lowers 5% of plasma
cholesterol level due to its inhibition on intestinal cholesterol absorption.
 Hormonal factors
o Estrogens
 Estrogens decreases blood cholesterol level by increasing HDL and decreasing LDL
cholesterol level. It reduces plasma LDL cholesterol level by enhancing its uptake via up-
regulation of LDL receptors in the liver.
 Due to cholesterol lowering action of estrogens, females have relatively lower
atherosclerosis and cardiovascular risk as compared to males.
o Thyroid hormone
 Thyroid hormones decrease plasma cholesterol level by increasing LDL receptor formation in
the liver.
 Life-style
o Nicotine from smoking, male gender, obesity, lack of exercise, sedentary life style, excessive
consumption of soft drinks, emotional stress, excessive alcohol consumption and partaking of a
few large meal rather than more continuous feeding elevates plasma FFA. Diabetes mellitus also
increases plasma FFA level. It will lead to increased VLDL secretion by liver, subsequently more
TAG and cholesterol output into circulation.
o Moderate alcohol drinking elevates HDL concentration. Red wine is beneficial perhaps due to its
anti-oxidants content.
Cholesterol balance in tissues (Regulation of intracellular cholesterol)

 In tissues, cholesterol balance is regulated as follows.
 An increase in cellular cholesterol is caused by –
o Uptake of cholesterol-containing lipoproteins by receptors, e.g., LDL receptor or scavenger

receptor,
o Uptake of free cholesterol from cholesterol-rich lipoproteins to the cell membrane,
o Endogenous cholesterol synthesis and
o Hydrolysis of cholesteryl esters by cholesteryl ester hydrolase.
 A decrease in cellular cholesterol is caused by –
o Reduced uptake of LDL cholesterol by down-regulation of LDL receptor,

o Decreased endogenous cholesterol synthesis,
o Efflux of cholesterol from the membrane to HDL via the ABCA1, ABCG1, or SR-B1,
o Esterification of cholesterol by ACAT (acyl-CoA:cholesterol acyltransferase) and
o Utilization of cholesterol for synthesis of steroids hormones in steroidogenic tissues and for bile
acids synthesis in the liver.
 There are two sources of intracellular cholesterol; de novo synthesis and uptake of cholesterol from
plasma lipoproteins. The exogenous cholesterol reaches cells predominantly within lipoproteins: as a
component of chylomicron remnants, VLDL remnants, or LDL.
 The intracellular cholesterol concentration is a key factor regulating both cellular cholesterol
synthesis and the expression of LDL receptors. Under normal circumstances, there is an inverse
relationship between dietary cholesterol intake and the rate of cholesterol biosynthesis. Thus, an
increase in the free cholesterol concentration results in the following –
o Reduction in both the activity and expression of HMG-CoA reductase limiting cholesterol
synthesis,
o Down- regulation of LDL receptors limiting cellular uptake of cholesterol,
o Activation of ACAT, which increases esterification of cholesterol for storage,
o Increase in cholesterol and phospholipid efflux from cell to HDL promoted by induction of ABC-
A1, ABC-G1 transporters, and
o Increase in the rate of conversion of cholesterol to bile acids increasing cholesterol excretion
from liver.
 Regulation of intracellular cholesterol concentration involves sterol mediated transcription
regulation of HMG-CoA reductase, LDL receptor, 7α-hydroxylase and a network of nuclear

receptors.
 Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate the
genes coding for proteins involved in cholesterol synthesis, cellular cholesterol uptake (LDL
receptors) and fatty acid synthesis. SREBPs bind to SRE of the target genes and modulate the gene
transcription.
o When cholesterol is depleted, the SCAP/SREBP complex dissociates from Insig-1 and travels to
the Golgi complex where SREBPs are cleaved by a protease, releasing the active transcription
factors. Then active SREBP translocates to the nucleus and activate all the genes in the
cholesterol synthetic pathway. In addition to the effect on cholesterol synthesis, SREBPs increase
the expression of LDL receptor gene and affect fatty acid synthesis.
o When cholesterol is abundant in the cell, cholesterol binding to SCAP stabilizes the
SCAP/SREBP/Insig-1 complex which is bound to ER membrane, blocking its movement to the
Golgi. Consequently, there is a decrease in nuclear SREBP and the transcription of genes
associated with cholesterol synthesis remains repressed. The synthesis of cholesterol is inhibited.
Cholesterol excess also decreases the level of Insig-1 mRNA. Uptake of cholesterol from the
blood is also diminished due to repression of transcription of the gene that encodes the LDL
receptor(via SREBP), thus reducing production of the receptor.
 Liver X receptor (LXR) is a nuclear transcription factor activated by oxysterol ligands (reflecting high
cholesterol levels), which integrates the metabolism of fatty acids, sterols, and glucose.
o When bound to an oxysterol ligand, LXRs form heterodimers with a second type of nuclear
receptor, the retinoid X receptors (RXR), and the LXR-RXR dimer activates transcription from a
set of genes including those for acetyl-CoA carboxylase, fatty acid synthase, CYP7A1 required for
sterol conversion to bile acid, apolipoproteins involved in cholesterol transport, the ABC
transporters ABCA1 and ABCG1 involved in reverse cholesterol transport, GLUT-4 and SREBP1C.
o High concentration of cholesterol in the hepatocyte induces, through LXR-SREBP1c mechanism,
genes coding for cholesterol transporters that control its efflux from cells to HDL particles (ABC-
A1, ABC-G1 transporters).
 The transcriptional regulators LXR and SREBP therefore work together to achieve and maintain
cholesterol homeostasis; SREBPs are activated by low levels of cellular cholesterol, and LXRs are
activated by high cholesterol levels.
 PPARα also regulates cholesterol efflux, acting through LXR. It is affected by lipid lowering drugs
known as fibrates.
Biochemical basis of atherosclerosis

Atherosclerosis is a process that leads to the narrowing, or a sudden complete occlusion, of the
arterial lumen. The narrowing is due to slow-growing atherosclerotic plaques whereas a sudden
complete occlusion is caused by the thrombus that forms over a ruptured plaque.
The development of atherosclerotic plaque is termed atherogenesis.
Atherogenesis involves lipid transport and deposition in the subendothelial layer of the arterial wall
(intima). It is accompanied by endothelial damage and inflammatory reaction that affects the intima.
Endothelial dysfunction,
lipid deposition and
inflammatory reaction
in the vascular wall are
the key processes in
atherogenesis.
Endothelial dysfunction
precedes formation of
atherosclerotic lesions.
Atherogenesis is
initiated by endothelial
damage. Damage is
functional rather than
structural in which
endothelium becomes
pro-atherogenic, inflammatory type by losing its cell-repellent quality, and admits inflammatory cells
into the vascular wall. It also becomes more permeable to lipoproteins, which subsequently deposit
in the intima.
Plaque formation in blood vessels is initiated when LDL containing partially oxidized fatty acyl
groups adheres to and accumulates in the extracellular matrix of epithelial cells lining arteries.
Immune cells (monocytes) are attracted to regions with such LDL accumulations, and they
differentiate into macrophages, which take up the oxidized LDL and the cholesterol they contain.
Macrophages cannot limit their uptake of sterols, and with increasing accumulation of cholesteryl
esters and free cholesterol, the macrophages become foam cells.
As excess free cholesterol accumulates in foam cells and their membranes, they undergo apoptosis.
Over long periods of time, arteries become progressively occluded as plaques consisting of
extracellular matrix material, scar tissue formed from smooth muscle tissue and foam cell remnants
gradually become larger.
Protein and Amino Acid Metabolism Page |1
Protein and amino acid metabolism
Introduction
 Proteins are macromolecules produced by the living things. Proteins are functionally and structurally
fundamental component of all living things.
 Proteins serve as –
Selenocysteine (21st amino acid)
o Biocatalyst, structural components
 At least 25 human selenoproteins
o Hormones and receptors  Selenocysteine is present at the
o Source of energy during scarcity of metabolic fuels active site of several human enzymes
 Proteins can be classified into globular (soluble, functional that catalyze redox reactions.
 Selenocysteine is not the product of a
proteins e.g., enzymes) and fibrous proteins (insoluble,
post-translational modification, but is
structural proteins e.g., collagen) depending on the
inserted directly into a growing
structure. polypeptide during translation.
 Depending on usability, proteins may be inert e.g., hair, nail  Incorporation of selenocysteine is
specified by a large and complex
or labile (can be degraded to amino acids and can be
genetic element for the unusual tRNA
reutilized) e.g., muscle proteins, plasma proteins.
called tRNASec which utilizes the UGA
 Building blocks for proteins and peptides are L-α-amino anti-codon that normally signals
acids. There are twenty different kinds of amino acids are STOP.
involved in protein synthesis.  Impairments in human selenoproteins

have been implicated in tumorigenesis
 The biologic properties of proteins are determined by the
and atherosclerosis, and are
kind of amino acid present, the order in which they are
associated with selenium deficiency
linked together in a polypeptide chain. cardiomyopathy (Keshan disease).
Chemistry of amino acids

 The L-α-amino acids in proteins and peptides (excluding proline) are composed of a carboxylic acid (–
COOH) and an amino group (–NH2) functional group attached to the tetrahedral α-carbon of
carboxylic acid. Distinct functional groups (R-group) are attached to the α-carbon (except in the case
of glycine where R-group is hydrogen). The fourth substitution on the tetrahedral α-carbon of amino
acids is hydrogen.
 Amino acids are distinguished from one another by the
distinct R-groups attached to the tetrahedral α-carbon.
Biomedical importance of amino acids

 Building blocks for peptides and proteins
 Transmission of nerve impulse in the nervous system e.g., glutamate as excitatory neurotransmitter,
glycine as inhibitory neurotransmitter
 Biogenic amines are derived from decarboxylation of amino acids e.g., histamine from histidine, γ-
amino-butyric acid (GABA) from glutamate.
 When amino acids are catabolized, amino group is released as ammonia which is converted into urea
and excreted in urine. Carbon skeleton of amino acids can be used as metabolic fuel for energy
production or it may also be used to produce glucose, acetyl CoA, ketone bodies or fatty acids
depending on nature of carbon skeleton derived from amino acid catabolism.
 Some amino acids can be synthesized in the body but some must be supplied in the diet. Essential
amino acids cannot be synthesized in the body and thus must be supplied in the diet.
 A number of diseases are due to abnormalities of the transport of amino acids into cells. Many of
these conditions are characterized by the presence of greatly increased amount of one or more
amino acids in the urine.
Protein turnover
 Protein turnover is the continuous degradation and synthesis of all cellular proteins. Adults degrade
1 – 2% of their total body protein daily, especially muscle protein. Of the liberated amino acids, 75 –
80% is utilized for new protein synthesis whereas the remaining 20 – 25% is catabolized.
 Nitrogen (amino group) of catabolized amino acids are released as ammonia and converted to urea
in liver which is then excreted by kidneys. Carbon skeletons of degraded amino acids form
amphibolic intermediates.
 Proteins are synthesized from expression of specific gene that encodes information for specific
amino acid sequence of the protein. Gene expression includes two steps; transcription of specific
gene segment into mature mRNA and translation of the transcribed mRNA into polypeptide chain.
 The susceptibility of a protein to degradation depends on its half-life. Rate of protein degradation
varies widely for individual proteins. Some are short half-lived proteins (5 to 20 minutes) and some
have half-lives of many hours or days. Proteins with short half-lives have PEST sequences, regions
rich in amino acids proline (P), glutamate (E), serine (S) and threonine (T) which target them for rapid
degradation.
 Rate of protein degradation varies according to physiologic demand. High rates are seen in tissues
undergoing major structural rearrangement e.g., uterine tissues during pregnancy. Rate of muscle
protein degradation is high during periods of fasting and released amino acids are used for
gluconeogenesis.
 The rate of protein turnover is also influenced by hormones. Growth hormone, insulin, testosterone
and thyroid hormone increase protein synthesis whereas 11-oxysteroid and excess thyroid hormones
enhance protein degradation.
 Examples of proteins that undergo extensive synthesis and degradation are hemoglobin, muscle
proteins, digestive enzymes and cellular proteins of intestinal mucosal cells.

 Damaged intracellular proteins due to oxidation or other modifications, misfolded proteins are non-
functional and thus they are degraded to amino acids. Dietary amino acids in excess of body
requirement are not stored but degraded and carbon skeletons from such amino acids catabolism
are converted to glycogen and triacylglycerol for storage.
 Proteins are degraded by lysosomal system or proteasomal system.
 Lysosomal degradation
o Lysosomal system degrades extracellular proteins and membrane associated proteins.

o Extracellular or membrane associated proteins internalized into the cells by a process known as
autophagy to form autophagosomes. These vesicles fuse with lysosomes and its contents are
degraded by lysosomal acid hydrolases.
o Within lysosomes, ingested proteins are denatured by acidic pH of lysosomes and polypeptides
are hydrolyzed by lysosomal acid proteases (cathepsins) into individual amino acids.
 Ubiquitin dependent proteasomal system
o Proteasomal system degrades

short-lived intracellular proteins,
damaged proteins, misfolded or
denatured proteins. Proteins
targeted for proteasomal
degradation must be tagged with
four or more ubiquitin residues.
o Ubiquitin
 Small protein containing 76
amino acids
 Ubiquitinylation of protein targeted for proteasome degradation requires three enzyme
complex consisting of –
 ATP-dependent ubiquitin activating enzyme (E1)
 Ubiquitin conjugating enzyme (E2)
 Ubiquitin-protein ligase (E3).
 Three enzyme system catalyzes formation of covalent linkage between C-terminal of
ubiquitin and the ε amino group of lysine residue in target protein.
o After polyubiquitinylation is completed, targeted protein is released from three enzyme complex
and is directed to the proteasome.
o 26S Proteasome
 26S Proteasome is a barrel shaped structure consisting of two types of subunits – 19S and 20S
subunits.
 19S subunits are on the outer ends of the proteasome complex.
 They act as gate keepers allowing only target polyubiquitinylated proteins to enter the
proteasome; preventing inappropriate degradation of cytoplasmic proteins.
 It cleaves ubiquitin residues from target proteins which are then recycled to the cytosol
for reutilization.
 Target protein is denatured by ATP dependent hydrolysis process and then resulting
polypeptide chain is pushed into inner core portion.
 20S subunits in inner core portion contain three different types of threonine proteases which
degrade target protein to small peptides (6 to 7 amino acids length). Resultant peptides are
released into the cytoplasm for further degradation.
 Amino acids released from protein degradation join the intracellular amino acid pool. Amino acids are
re-utilized for new protein synthesis and excess amino acids are degraded.
 Biomedical importance
o Some abnormal proteins resist proteasomal degradation. Accumulation of such proteins can
cause damage to the cell e.g., Alzheimer disease, Parkinsonism, prion disease.
Metabolism of amino acids
Biosynthesis of non-essential amino acids

Of 12 nutritionally non-essential amino acids, 9
are formed from amphibolic intermediates
of glycolysis and CAC. The remaining 3
(cysteine, tyrosine, hydroxylysine) are
formed from nutritionally essential amino
acids.
Carbons for cysteine synthesis are derived
from glucose (3-phosphoglycerate  serine
 cysteine) and sulfur is contributed by
essential amino acid methionine.
Tyrosine is derived from hydroxylation of
phenylalanine catalyzed by phenylalanine hydroxylase.
Lysine is hydroxylated to hydroxylysine during post-translational
processing of collagen.
Carbon skeleton of four amino acids (alanine, serine, glycine,
cysteine) are produced from glucose through components of
glycolytic pathway.
TCA cycle intermediates provide carbon skeleton for synthesis of six
nonessential amino acids (aspartate, asparagine from oxaloacetate
and glutamate, glutamine, proline, arginine from α-ketoglutarate).
The main reactions in non-essential amino acid synthesis are –
Protein and Amino Acid Metabolism P a g e |5
o Transamination by transaminases (aminotransferases) e.g., alanine transaminase, aspartate

transaminase
o Reductive amination by glutamate dehydrogenase (conversion of α-KG to glutamate)
o Amidation by glutamine synthetase (glutamate to glutamine)
Transport of amino acids into the cell

 Amino acids in the blood stream are transported across the
cell membrane of various tissues by Na+ dependent co-
transporters and facilitated transporters.
 Gamma glutamyl cycle
o Amino acids can also be transported via γ-glutamyl cycle.
This type of transport requires glutathione (tripeptide consisting of glycine, cysteine and
glutamate) and γ-glutamyl transpeptidase.
This transport mechanism is more prominent
in liver, renal tubules, RBC and brain.
o Extracellular amino acid reacts with
glutathione to form γ-glutamyl amino acid by
the action of γ-glutamyl transpeptidase on the
cell membrane. Formed γ-glutamyl amino acid
enters into the cell and is cleaved to release
free amino acid. Glutathione is regenerated
from glutamate, cysteine and glycine.
 Amino acid uptake is enhanced by insulin and growth hormone.

 Normal plasma amino acid level during fasting is 35 – 60 mg/dL.
 Plasma amino nitrogen level is 4 – 6 mg/dL.
Amino acid catabolism or catabolic fate of amino acid

 Excess amino acids are not stored but degraded by the following processes;
o Removal of amino group or α-amino nitrogen

1. Transamination
2. Oxidative deamination or non-oxidative deamination
3. Ammonia metabolism
4. Urea synthesis
o Catabolism of carbon skeleton
Removal of amino group

1. Transamination
o Transamination is the transfer of an amino group (–NH2)
from an amino acid to α-keto acid. This process involves the
inter-conversion of a pair of amino acids and a pair of α-keto
acids, catalyzed by transaminases (aminotransferases).
o All transaminases require pyridoxal phosphate (PLP) as
coenzyme which acts as a carrier of amino group. There is no
free ammonia liberated in the process; only the transfer of
amino group occurs.
o Transamination is the principal mechanism of removal of amino groups from amino acids.
Amino group of most amino acids are transferred to suitable α-keto acid acceptor which is almost
always α-ketoglutarate, forming glutamate. Thus, amino acids that undergo transamination
finally concentrate nitrogen as glutamate since glutamate is the only amino acid that undergoes
oxidative deamination to a significant extent to liberate α-amino nitrogen as free ammonia for
urea synthesis.
o All amino acids except lysine, threonine, proline and hydroxyproline can undergo transamination.
o Excess amino acids can be diverted into energy generation via
transamination. Transamination is also important for the
synthesis of non-essential amino acids.
2. Deamination
o Oxidative deamination
 Oxidative deamination results in the liberation of amino
group as free ammonia. These reactions occur primarily in
the liver and renal tubular cells, releasing α-keto

acids and ammonia.
 Major pathway for oxidative deamination is
catalyzed by glutamate dehydrogenase. The
amino groups of most amino acids are ultimately
funneled to glutamate by transamination with α-
ketoglutarate. Glutamate undergoes oxidative deamination catalyzed by glutamate
dehydrogenase to liberate ammonia. Thus, sequential action of transamination and
oxidative deamination of glutamate is the major pathway for removing amino group of most
amino acids to be released as ammonia.
 Oxidative deamination can also be done by the
action of L-amino acid oxidase in liver and
kidney but this is a minor pathway. L-amino
acid oxidase is an auto-oxidizable flavoprotein
enzyme i.e., reduced FMN and FAD in the
reaction are directly re-oxidized by molecular
oxygen forming hydrogen peroxide without
using any cytochromes or other electron carriers. H2O2 is then split to O2 and H2O by catalase.
o Non-oxidative deamination
 In addition to glutamate, several amino
acids release their nitrogen as NH4+.
 The α-amino groups of serine and
threonine can be directly deaminated into NH4+ ion. These amino acids contains hydroxy
group attached to its β-carbon atom. These direct deaminations are catalyzed by serine
dehydratase and threonine dehydratase, in which PLP is the coenzyme.
 Glutaminase acts on glutamine, forming glutamate and NH4+. Histidine may be directly
deaminated to form NH4+ and urocanate. Asparagine is deamidated by asparaginase, yielding
aspartate and NH4+.
3. Metabolism of ammonia
o Ammonia is formed in a number of reactions in the body and quantitatively the most important
source is catabolism of amino acids derived from protein metabolism. Ammonia is produced in
most tissues. Since ammonia is extremely toxic, it is immediately converted to non-toxic
metabolites such as, glutamate or glutamine or alanine and ultimately to urea. For the ultimate
conversion of ammonia to urea, ammonia is transported to the liver.
o Transport of ammonia in circulation
 Although ammonia is constantly produced in the tissues, it is present at very low levels in
Sources of ammonia
blood. This is due to rapid removal of 1. Amino acids
ammonia from the blood by the liver and Quantitatively most important source
release of amino nitrogen from the tissues Many tissues particularly liver, form
ammonia from amino acids by
to the blood stream in the form of amino
transamination to glutamate followed
group in amino acid i.e., alanine (especially by oxidative deamination of glutamate.
muscles) or glutamine rather than free 2. Glutamine
ammonia. Ammonia is derived from hydrolysis of

glutamine by glutaminase action in
 Normal blood ammonia is very low and
intestine and kidney.
ranges from 10 – 20 μg/dL.
3. Bacterial action in intestine
o Transport of ammonia in the form of glutamine Considerable amount of ammonia is
 In many tissues (liver, kidney and brain), produced in intestine by the action of
intestinal bacterial enzymes upon
ammonia is enzymatically combined with
dietary protein and urea secreted into GI
glutamate to yield glutamine by the action
tract.
of glutamine synthetase. Glutamate is This ammonia is absorbed from intestine
formed from ammonia and α-ketoglutarate into portal venous blood. Under normal
by the action of glutamate dehydrogenase. conditions, liver promptly removes ammonia

from portal blood so that blood leaving the
 The glutamine, so formed is a neutral
liver is virtually ammonia free.
nontoxic major transport form of 4. Amines
ammonia. The glutamine is transported by Amines obtained from diet and monoamines
blood to the liver, where it is cleaved by (hormones or neurotransmitters) release

ammonia upon amine oxidase action.
glutaminase to yield glutamate and free
5. Purine and pyrimidine
ammonia. Glutamate undergoes oxidative
Amino groups attached to purine and
deamination to release free ammonia. The pyrimidine ring are released as ammonia
ammonia so formed is converted by the during catabolism of purine and

pyrimidine.
liver into urea.
o Transport of ammonia in
the form of alanine
 Alanine transports
ammonia from
muscles to the liver
through glucose
alanine cycle.
 In muscle, glutamate
is formed from
ammonia and α-
ketoglutarate by the
Protein and Amino Acid Metabolism P a g e | 10
glutamate dehydrogenase reaction. L-glutamate then transfers its α-amino group to pyruvate
by transamination reaction to form alanine.
 Alanine so formed in muscle is transported to liver where it is converted to pyruvate and
glutamate again by transamination reaction.
 In the liver, glutamate undergoes oxidative deamination to release free ammonia, which is
converted to urea. Whereas, pyruvate is converted to glucose by gluconeogenesis.
4. Urea synthesis
o It is the major pathway for nitrogen excretion in the body. This cycle converts toxic ammonia into
less toxic urea. Amino groups for urea synthesis are collected in the form of ammonia and
aspartate. Urea synthesis occurs partly in mitochondria and partly in cytoplasm. Raw materials
for urea synthesis include NH4+, CO2, 3 ATP (4 high energy phosphate bonds), amide nitrogen of
aspartate. Six amino acids are involved in the process; one functions as enzyme activator (N-
acetyl glutamate) and remaining five function as carrier of atoms in urea cycle (ornithine,
citrulline, aspartate, argininosuccinate, arginine).
o Urea is synthesized in the liver and released into the blood. Finally it is excreted by the kidneys
into the urine. Ammonia removal mechanism
o There are five major steps in urea biosynthesis. (Ammonia fixation mechanism)
Ammonia in the blood stream is
1. Synthesis of carbamoyl phosphate
removed by –
2. Synthesis of citrulline o Synthesis of glutamate
3. Synthesis of argininosuccinate o Synthesis of glutamine
4. Cleavage of argininosuccinate into arginine and fumarate o Synthesis of urea

o Urinary excretion of NH4+
5. Cleavage of arginine into urea and ornithine
1. Synthesis of carbamoyl phosphate

 Condensation of NH4+, CO2 and phosphate to form
carbamoyl phosphate is catalyzed by mitochondrial
enzyme carbamoyl phosphate synthetase I (CPS-I). Two
ATPs are used in the process. N-acetyl glutamate is
required as an enzyme activator.
2. Synthesis of citrulline
 Carbamoyl moiety of carbamoyl phosphate is transferred
to ornithine to form citrulline and phosphate is released.
It is catalyzed by ornithine transcarbamoylase in
mitochondria. Citrulline is then transported to
cytoplasm.
3. Synthesis of argininisuccinate
 Citrulline and aspartate are linked together via the amino
group of aspartate. This is catalyzed by
argininosuccinate synthetase and ATP is required.
4. Cleavage of argininosuccinate to arginine and fumarate
 Argininosuccinate is cleaved into arginine and fumarate
by argininisuccinase. Fumarate may be converted to
oxaloacetate by fumerase and malate dehydrogenase
reactions and then transaminated to regenerate
aspartate.
5. Cleavage of arginine to urea and ornithine
 Hydrolytic cleavage of arginine to urea and ornithine is catalyzed by arginase. Ornithine can
then enter into mitochondrial part of urea cycle.
 Arginase is activated by Ca2+ or Mn2+ and competitive inhibitors are ornithine and lysine.
Urinary excretion of NH4+
o Ammonia is formed from glutamine by the action of glutaminase and from glutamate by the
action of glutamate dehydrogenase in renal tubular cells. Most of ammonia is excreted into the
urine as NH4+ which can serve as a urinary buffer.
o Urinary excretion of NH4+ is an important mechanism for maintaining body’s acid-base balance.
Catabolism of carbon skeleton of amino acid (fate of carbon skeleton of amino acid)
After removal of α-amino groups, catabolism of carbon skeletons of 20 different types of amino acid
converges to form seven products – oxaloacetate, α-ketoglutarate, pyruvate, fumarate, succinyl
CoA, acetyl CoA and acetoacetyl CoA.
They may follow one of the following fates –

o Utilization for energy production
o Synthesis of non-essential amino acids
o Synthesis of TAG and ketone bodies
o Synthesis of glucose
Regulation of urea cycle

Rate limiting enzyme is carbamoyl phosphate synthetase I (CPS-I).
Essential allosteric activator of CPS-I is N-acetyl glutamate which is synthesized from acetyl CoA and
glutamate. The intrahepatic concentration of this compound increases after ingestion of protein rich
meal leading to an increased rate of urea synthesis.
Arginine is allosteric activator of N-acetyl glutamate synthase and also a source of ornithine (via
arginase) for the urea cycle.
Concentration of urea cycle enzymes increases or decreases in response to high or low protein diet.
Urea synthesis and excretion are decreased and NH4+ excretion is increased during acidosis as a
mechanism to excrete protons into the urine.
Metabolic significance of urea cycle

The toxic ammonia is converted into the harmless nontoxic urea.
It disposes off two waste products, ammonia and CO2.
It forms semi-essential amino acid, arginine.
Ornithine which is formed in urea cycle can form nonessential amino acid proline. Ornithine is a
precursor for the formation of polyamines like putrescine, spermidine and spermine.
Metabolic disorders of urea cycle

Disorder of urea cycle cause ammonia intoxication.
The most common cause is hepatic failure.
Rare causes include genetic defects in any of the six enzymes (including N-acetyl glutamate
synthase) associated with urea cycle or in three distinct transporters (mitochondrial ornithine
carrier, mitochondrial aspartate/glutamate carrier and dibasic amino acid carrier).
Their symptoms are more severe when early steps in the cycle are affected. Ornithine
transcarbomoylase is the most common of urea cycle defects and X-linked inheritance pattern. The
remainder of the known defects associated with urea cycle is autosomal recessive.
Clinical symptoms common to all urea cycle disorders include vomiting in infancy, avoidance of high
protein diet, intermittent ataxia, irritability, lethargy and mental retardation.
Management of the urea cycle defects involves limiting protein intake while ensuring adequate
calorie consumption and the use of agents that can conjugate certain amino acids into excretable
metabolites. For example, sodium benzoate combines with glycine to form hippuric acid and
phenylacetate can conjugate glutamine. In some cases, the diet may be supplemented with citrulline
or arginine.
Hyperammonaemia
Elevated ammonia concentration in blood is called hyperammonaemia.
Symptoms of ammonia intoxication includes:
o Impaired neuronal activity o Convulsions o Vomiting
o Tremor o Lethargy o Slurred speech
o Ataxia o Nausea o Blurred vision
In severe cases, it can lead to coma and death.
There are two types of hyperammonaemia.
o Hereditary hyperammonaemia
 Genetic defects in enzymes and mitochondrial transporters associated with urea cycle can
cause hyperammonaemia. Inherited urea cycle enzymes deficiency result in mental
retardation.
o Acquired hyperammonaemia
 Liver failure can lead to reduced capacity in conversion of ammonia to urea which could
result in hyperammonaemia.
 Portal blood had characteristically higher ammonia level than systemic blood due to ammonia
directly absorbed from intestine which is derived from the action of intestinal bacterial
enzymes upon dietary proteins and urea excreted into the intestine. Under normal condition,
liver promptly removes ammonia from portal blood so that blood leaving the liver to the
systemic circulation is ammonia free. In cirrhosis of liver (which may be caused by chronic
alcoholism or hepatitis or biliary obstruction), there is shunting of portal blood directly to
systemic circulation as a result of formation of collateral circulation around the liver. This
leads to hyperammonaemia.
Ammonia toxicity
Excess ammonia, due to either a disorder of the urea cycle or liver failure, can have highly toxic
effects on the brain and the CNS. Ammonia readily crosses the blood-brain barrier, so any condition
that raises the level of ammonia in the bloodstream will expose the brain to high concentrations.
Symptoms of ammonia intoxication include tremors, slurring of speech, blurring of vision, reduced
consciousness and in severe cases, can lead to coma and death.
Biochemical basis of ammonia toxicity
o Removal of excess ammonia from brain cells requires reductive amination of α-ketoglutarate to
glutamate by glutamate dehydrogenase and conversion of glutamate to glutamine by
glutamine synthetase.
o High levels of ammonia lead to increased levels of glutamine, which acts as an osmotically active
solute (osmolyte) in brain astrocytes of CNS. This triggers an uptake of water into the astrocytes
to maintain osmotic balance, leading to cerebral edema and increased intracranial pressure
which in turn can lead to coma.
o The activity of the TCA cycle is also inhibited due to a depletion of α-ketoglutarate as a result of
formation of glutamate and glutamine for removal of ammonia. This depresses energy
generation of neurons and subsequently the neuronal activity.
o Depletion of glutamate results in a disruption of neurotransmitter activity since glutamate and
its derivative γ-aminobutyrate (GABA) are important neurotransmitters.
Normal blood urea level

A normal healthy adult on an average diet has 21 – 58 mg/dL or 3.5 – 9 mmol/L of blood urea level.
Factors affecting blood urea level
o Age
 Blood urea level increases with advancing age.
o Sex
 Males have a slighter higher blood urea than females, especially in young age, because of
higher protein turnover in the males.
o Diet
 Over a period, high protein diet increases blood urea level.
o Pregnancy
 Blood urea is low in pregnancy due to positive N2 balance and hemodilution.
o Pathological factors
 Urea is excreted through the kidneys into the urine.
 Pre-renal causes (due to reduced renal blood flow or increased protein breakdown)
 Dehydration due to diarrhea, severe vomiting
 Heavy blood loss
 Severe burn
 Increased protein degradation in prolong starvation
 Renal causes
 Blood urea is increased in all forms of kidney diseases because of impaired excretion.
 Post-renal causes (due to renal outflow obstruction)
 Kidney, bladder or urinary stones
 Benign prostate hypertrophy
 Stricture urethra
o Liver diseases lead to decreased blood urea level due to defective urea synthesis.
Inborn errors of amino acid metabolism

They occur due to genetically determined deficiency or absence or modification of a specific protein.
The affected protein may be an enzyme or a transport protein.
In majority of the cases, the deficient protein is an enzyme of a metabolic pathway. As a result, the
metabolic pathway is blocked which causes abnormalities in the normal metabolism.
In some other cases, the transport proteins responsible for the renal or the intestinal absorption of
amino acids, is defective. For example, renal tubular reabsorption of cystine is impaired in cystinuria;
renal tubular and intestinal transport of the neutral amino acids is impaired in Hartnup disease.
Disorders in amino acid
Disease Site of metabolic block
catabolism
Disorder in proline Hyperprolinemia type I Proline dehydrogenase
catabolism Hyperprolinemia type II Glutamate semialdehyde dehydrogenase
Disorder in
hydroxyproline Hydroxyprolinemia 4-hydroxy proline dehydrogenase
catabolism
Disorder in arginine
Hyperargininemia Liver arginase
catabolism
Disorder in histidine
Histidemia Liver histidase
catabolism
Aminoadipate semialdehyde synthase
Disorder in lysine Periodic hyperlysinemia with
Competitive inhibition of liver arginase by
catabolism associated hyperammonemia
elevated lysine lead to hyperammonemia
Cystine, lysine, arginine and ornithine are

excreted in cystine-lysinuria (cystinuria) due to
Cystinuria
defect in renal reabsorption of these amino
Disorder in cysteine,
acids.
cysteine and
Cystathionine β-synthase defect
methionine metabolism
Homocysteinemia is thought to be related
Homocystinuria
with increased risk of cardiovascular
disease.
Tyrosinemia type I Fumarylacetoacetate hydrolase
(tyrosinosis) Maleylacetoacetate hydrolase
Tyrosinemia type II
Hepatic tyrosine transaminase
(Richner-Hanhart syndrome)
Disorder in tyrosine
Neonate tyrosinemia Hydroxyphenylpyruvate hydroxylase
metabolism
Alkaptouria Homogentisate oxidase
Hypomelanosis due to tyrosinase defect in eye

Albinism
and skin melanocytes
Phenylalanine hydroxylase defect
Incidence – 1 : 10,000 live births
Disorder in
Elevated phenylalanine level in blood
phenylalanine Phenylalkaptouria (PKU)
Phenylketones (phenylpyruvate,
catabolism
phenyllactate, phenylacetate) are excreted
in urine.
Impaired intestinal and renal transport of
Disorder in tryptophan tryptophan and other neutral amino acids
Hartnup disease
metabolism Increased urinary excretion of indole
acetate and tryptophan
Maple syrup urine disease
Disorders in branched- α-keto acid decarboxylase complex
(branched-chain ketouria)
chain amino acid
catabolism Isovaleric acidemia Isovaleryl CoA dehydrogenase
Synthesis of specialized products from some amino acids

 Amino acids serve as precursors of a large number of biologically important compounds; for
example, amines, porphyrins, nitrogenous bases of phospholipids, creatine phosphate, polyamines,
etc. Compounds like purines and pyrimidines are derived in part from amino acids.
Amino acid Compounds
Serotonin  Melatonin
Tryptophan
Niacin
Histidine Histamine
An excitatory neurotransmitter
Glutamate
Gamma amino butyric acid (GABA)
Ethanolamine  Choline  Acetylcholine

Serine
Sphingosine
Catecholamines
Tyrosine Thyroid hormones
Melanin
An inhibitory neurotransmitter
Glycine Bile acid conjugation, conjugation reaction in xenobiotic metabolism
Heme, Creatine, Purine
Creatine, creatinine
Arginine Nitric oxide
Arginine  ornithine  spermine, spermidine
Aspartate An excitatory neurotransmitter, Purine and pyrimidine synthesis
Glycine, arginine and methionine Creatine  creatinine by non-enzymatic dehydration
Glycine, cysteine, glutamate Glutathione
Coenzyme A (CoA)
Cysteine
Taurine for bile acid conjugation
Methionine S-adneosyl methionine as methyl donor
Glutamine Purine and pyrimidine synthesis
β-alanine CoA, cytosine, carnosine
Proteins of extracellular matrix

Extracellular matrix (ECM) is a complex network of macromolecules that fills the spaces between the
cells and binds the cells together.
It plays in determining the shape of tissues, in regulating basic cellular processes including
proliferation, differentiation, migration and even survival.
Major components of ECM
o They are secreted by a variety of cell types including fibroblasts, chondrocytes and osteoblasts.
o Fibrous structural proteins
 Collagen
 Elastin
 Fibrillin
o Specialized proteins e.g., adhesion proteins linking matrix components to each other and to cells.
 Laminin
 Fibronectin
o Proteoglycans
Collagen
o Most abundant protein in body (nearly 25% of total protein mass of mammals)
o At least 28 distinct types of collagen (most common – type I collagen)
o Stretches of triple helix with repeating structure of (Gly-X-Y)n in each helix

o Complex biosynthesis process which requires many post-translational modification including
hydroxylation of proline and lysine.
o Long-life span, rigid protein
o Main fibril forming collagens in skin and bone – type I and in cartilage – type II
o Collagen I and hydroxyappetite are the major constituents of bone.
o Collagen II and certain proteoglycans are major constituents of cartilage.
o Diseases associated with impaired synthesis of collagen
 Scurvy
 Osteogenesis imperfect
 Ehlers-Danlos syndrome
 Menkes disease
Elastin
o Confers extensibility and elastic recoil on tissues such as lungs, blood vessels and ligaments.
o Does not contain repeat Gly-X-Y sequences, hydroxylysine, triple helical structure, or sugar
moieties.
o Contains desmosine and isodesmosine cross-linkages that are not found on collagen.
o Highly insoluble, extremely stable and has a very low-turnover rate.
o Diseases associated with fragmentation or decrease of elastin
 Pulmonary emphysema (deficiency of α1 anti-trypsin which inhibits elastase activity)
 Cutis laxa
 Aging of skin
Fibrillin
o Glycoproteins and major structural component of microfibrils
o Provides a scaffold for the deposition of elastin in the ECM.
o They are secreted into ECM by fibroblasts.
o Mutations in fibrillin-1 gene cause Marfan syndrome (tall stature, long arms and legs, lens
dislocation, arachnodactyly, hyperextensibility of the joints, dilation of ascending aorta).
Fibronectin
o Major glycoprotein of the ECM, also found in a soluble form in plasma.
o Consists of two identical subunits (dimer) joined by two disulfide bridges near their carboxyl
terminals.
o At least 20 different tissue-specific isoforms which are produced by alternative mRNA splicing.
o Play an important role in adhesion of the cells to ECM and involve in cell migration by providing
a binding site for cells.
Laminin
o Major protein component of basal laminas
o Large heterotrimeric molecule which is composed of α, β, and γ chains.
o Non-collagenous glycoproteins found in
basement membranes and expressed in
variant forms in different tissues.
o At least 15 different laminin variants
o It is found in renal glomerulus and has an
important role in glomerular filtration.
Proteoglycans
o Proteins that contain covalently linked
glycosaminoglycans (GAGs)
o Contain larger amount of carbohydrate
(>95% of its weight) than glycoprotein.
o GAGs – repeating disaccharides containing
acid sugar/uronic acid (glucuronic or
iduronic acid) or hexose (galactose) and amino sugar/hexosamine (glucosamine or
galactosamine)
o Major GAGs – hyaluronic acid (hyaluronan), chondroitin sulfate, keratan sulfates I and II,
heparin, heparan sulfate, and dermatan sulfate
o Ground substance of ECM or gel-forming components of ECM

o Can act as sieves, restricting the passage of large macromolecules into the ECM but allowing
relatively free diffusion of small molecules.
o Proteoglycans are synthesized by a series of glycosyl transferases, epimerases and
sulfotransferases. They are degraded by the sequential action of lysosomal hydrolases.
o Diseases associated with proteoglycan metabolism
 Mucopolysaccharidoses – deficiencies of enzymes that degrade glycosaminoglycans e.g.,
Hurler and Hunter syndromes
 Proteoglycans are associated with major diseases and with aging.
 Hyaluronic acid may be important in permitting tumor cells to migrate through the ECM.
 Dermatan sulfate binds plasma LDLs and appears to be the major GAG synthesized by
arterial smooth muscle cell that proliferate in atherosclerotic lesions. Dermatan sulfate
may play an important role in development of the atherosclerotic plaque.
 The amount of chondroitin sulfate in cartilage diminishes with age, whereas the
amounts of keratin sulfate and hyaluronic acid increase. These changes may contribute
to the development of osteoarthritis.
Amino acid pool

The amino acids derived from
Inert Protein
ingested dietary protein (hair,nail)
digestion and absorption and

from endogenous protein
breakdown form a common
amino acid pool that supplies
the needs of the body. Body
proteins are being continuously
hydrolyzed to amino acids and
re-synthesized to proteins.
Amino acids are taken up by
the liver and other tissues and
are used for various purposes.
Free amino acids in excess of cellular use or body requirement are not stored but catabolized.
Anabolic fate of amino acids - Amino acids are used for –
o Synthesis of body protein (300 – 400 g/day) which includes structural proteins such as
cytoskeletal protein, collagen and functional proteins such as enzymes, receptors, hormones,
immunoglobulins, plasma proteins
o Synthesis of specialized nitrogen containing compounds such as heme, purine, pyrimidine,

biogenic amines, neurotransmitters, choline, glutathione, creatine, etc
Excess amino acids are not stored in the body and they are catabolized providing 10 – 15 % of energy
requirement of the body.
Catabolic fate of amino acids
o The α-amino groups of amino acids are removed by transamination followed by oxidative
deamination with the liberation of free ammonia and carbon skeleton (α-keto acids).
o Ammonia is transported in the form of alanine and glutamine in circulation to the liver where it is
converted to less-toxic compound urea, for excretion. Urea is excreted in urine by kidney. Urea is
the end product of protein metabolism.
o The remaining carbon skeleton (α-keto acids) are catabolized to amphibolic intermediates such
as pyruvate, oxaloacetate, α-ketoglutarate, fumarate, succinyl CoA, acetoacetyl CoA and acetyl
CoA.
o These compounds are either completely oxidized in CAC to provide ATP or used for synthesis of
glucose or ketone bodies or lipids depending on metabolic state of the body. Carbon skeletons
except acetoacetyl CoA and acetyl CoA can undergo glucoconeogenesis whereas acetoacetyl
CoA and acetyl CoA mainly enter into ketogenesis or lipogenesis.
Level of most amino acids in plasma does not remain constant throughout the day and varies in a
circadian rhythm. Plasma amino acid level is lowest at 4 a.m and rise to 15 – 35% by noon or early
afternoon.
Amino acids present at highest mean concentration such as Glu, Val, Ser change the least. Amino
acids present at lowest mean concentration such as Tyr, Trp, Phe, Met, Cys show the most striking
change. The rhythm responds within 48 hours to an inversion of sleeping-eating pattern but physical
activity has little effect on amino acid level.
Between meals, normal plasma nitrogen level is 4 to 6 mg/dL and plasma amino acid level is 35 – 60
mg/dL. Aminoaciduria occurs when plasma amino acid level exceeds 60 mg/dL.
Role of kidney in regulation of plasma amino acid level
o The filtered amino acids are actively reabsorbed in proximal tubules and thus urinary loss of free
amino acid is normally less than 5 mmol/day or 0.9 – 1 g/ day (negligible).
o Amino acids are renal threshold substances. When a particular amino acid level rises too high in
plasma, subsequently in glomerular filtrate, the excess cannot be actively reabsorbed and is lost
into the urine. This condition is termed as aminoaciduria.
Hormones influencing body protein metabolism
o Growth hormone – increases cellular uptake of amino acids and protein synthesis, causing
positive nitrogen balance.
o Testosterone – has anabolic effect on muscle protein synthesis.
o Insulin – has anabolic effect on protein metabolism by stimulating protein synthesis and
inhibiting protein degradation.
o Thyroid hormone – has anabolic effect on protein metabolism. It also stimulates growth
hormone production. However, excess thyroid hormones cause negative nitrogen balance.
o Glucocorticoids (11-oxysteroids) – promote protein degradation thereby increasing plasma amino
acids level. These amino acids are used as glucogenic substrates for gluconegensis in liver.
Nucleotide and Heme Metabolism Page |1
Nucleotide Metabolism
Introduction
Nucleoproteins are conjugated proteins made up of a non-protein
prosthetic group, nucleic acid and one or more simple basic
proteins, protamines and histones. They constitute a large part of
nuclear material of the cell.
Nucleic acids are polymers of nucleotides linked by 3’ – 5’
phosphodiester bonds. There are two types of nucleic acid; DNA
and RNA. They can be broken down to nucleosides and ultimately
to nitrogenous bases and pentose sugars.
Nucleoside is composed of pentose sugar linked to purine or
pyrimidine base at its C-1 by N-glycosidic bond. Nudeotides are
nucleosides with one or more phosphates attached to the carbon
5' position of pentose sugar.
Nucleotides are dietary non-
essential since human tissues
can synthesize purines and
pyrimidines nucleotides from
amphibolic intermediates.
Ingested nucleic acid and
nucleotides are degraded in the
GI tract to mononucleosides,
purine and pyrimidine bases
which are absorbed into
enterocytes. In enterocytes, purine bases are then oxidized to uric acid which are then transported
into the blood stream and excreted in the urine.
Functions of nucleotides (Biomedical importance of nucleotides)

Nucleotide consists of a nitrogenous base, a pentose sugar and phosphate. Nucleotides play key role
in nearly all biochemical processes.
1. Nucleoside triphosphates serve as monomeric precursors of DNA and RNA synthesis.
2. Nucleotides are components of activated carriers in synthesis of biomolecules.
 UDP glucose in glycogen synthesis,
 UDP glucuronic acid as conjugating agent,
 CDP diacyglycerol, CDP choline in phospholipid synthesis,
 S-adenosylmethionine as methyl group donor in methylation reaction.
3. Nucleosides triphosphates serve as energy rich compounds.

 ATP is a universal energy currency in biologic system driving many metabolic processes,
 GTP is utilized in protein synthesis and gluconeogenesis
 UTP is utilized in glycogen, glycoprotein and glycolipids synthesis,
 CTP is utilized in phospholipid synthesis.
4. Nucleotides are important for many biochemical reactions.
 Adenine nucleotides are components of active coenzyme forms of some vitamins e.g.,
NAD+,NADP+, FAD, coenzyme A and thus important in many oxidation-reduction reactions.
 Adenosine triphosphate (ATP) serves as phosphate group donors for some biochemical
reactions, e.g., phosphorylation, pyrophosphorylation reactions.
5. Nucleotides are important in signal transduction cascades.
 Cyclic nucleotides such as cAMP, cGMP act as second messengers for signal mediation of the
actions of many hormones.
 GTP powers the activation of signal coupling protein (G-protein) in signaling pathway of many
hormones.
6. Adenine nucleotides act as modulators of metabolic pathways by regulating the key enzymes
activity.
 Concentration of ADP controls the rate of respiratory chain-linked oxidative phosphorylation.
 ATP and AMP serves as allosteric effectors in regulation of some enzymes activities; AMP inhibits
fructose 1,6-bisphosphatase whereas it activates PFK-1. ATP allosterically inhibits PFK-1 and
muscle glycogen phosphorylase.
 Cellular AMP level also influences the activity of some metabolic pathways by activating AMP
activated kinase (AMPK).
7. Synthetic nucleotides are used as drugs in the treatment of cancer, viral infections, auto-immune
diseases and gout.
 Nucleoside reverse transcriptase inhibitors e.g., AZT in treatment of HIV infection
 5’ flurouracil in treatment of cancer
 Allopurinol (purine analog) in treatment of gout.
Biosynthesis of purine nucleotides

 Purine nucleotides are synthesized from simple
building blocks or from basic metabolites (de novo
synthesis) or by recycling of preformed bases or
nucleosides (salvage synthesis).
 The atoms of purine are contributed by a number of compounds; one carbon units carried by THF,
CO2 and amino acids (aspartate, glycine and glutamine).
Nu cl e o t i de a nd He m e Me t a b o l ism Pa g e |3
De novo synthesis
 The purine ring is constructed by a series of reactions
that add the donated carbons and nitrogen to a
preformed ribose 5-phosphate which is derived from
HMS pathway.
 Steps in de novo synthesis of purine nucleotides
1. Synthesis of 5-phosphoribosyl 1-pyrophosphate
(PRPP)
2. Synthesis of 5-phosphoribosylamine
3. Synthesis of inosine monophosphate (IMP)
4. Conversion of IMP to AMP and GMP
5. Conversion of nucleoside monophosphates to
nucleosides diphosphates and triphosphates
6. Formation of deoxyribonucleoside diphosphates
from ribonucleoside diphosphates
 11 enzymes catalyzed reactions convert ribose 5-
phosohate to inosine monophosphate.
 The first step is the formation of PRPP from ribose 5-
phosphate and ATP by the action of PRPP synthetase.
It is the regulatory step in de novo synthesis of purine
nucleotides.
 Purine ring is constructed on the carbon-1 of PRPP. It
starts with the reaction of PRPP with glutamine to form
5-ribosylamine by the action of amidotransferase.
 After a series of reactions in which glutamine, glycine,
methenyl-THF, CO2, aspartate and formyl-THF donate
respective carbon and nitrogen atoms to construct
purine ring on carbon-1 of ribose 5-phosphate, the first
purine nucleotide formed is inosine monophosphate
(IMP).
 IMP is the precursor for both AMP and GMP synthesis.
Conversion of IMP to either AMP or GMP is energy-
requiring process. AMP synthesis requires GTP as
energy source whereas GMP synthesis requires ATP.
 Nucleoside monophosphate (AMP, GMP) is converted
to nucleoside diphosphate (ADP, GDP) by specific
nucleoside monophosphate kinases and then to

nucleoside triphosphate (ATP, GTP) by specific
nucleoside diphosphate kinases. ATP is generally the
phosphate donor for these reactions because it is
present in higher concentration than other nucleoside
triphosphates.
 Deoxyribonucleoside diphosphate is formed from the reduction of 2-hydroxyl group of
ribonucleoside diphosphate by the action of ribonucleotide reductase enzyme complex. This
reaction requires thioredoxin, thioreducin reductase and NADPH. The enzyme complex is active only
when cells are actively synthesizing DNA.
 Regulation of de novo synthesis of purine nucleotides

o De novo synthesis of purine nucleotides is controlled by:
 Concentration of PRPP
 Feedback regulation at several sites.
o Major determinant of rate of purine nucleotide synthesis is
the concentration of PRPP.
o Concentration of PRPP depends on:
 Availability of ribose-5-phosphate
 Activity of PRPP synthetase (rate of synthesis) and
 Rate of utilization and degradation of PRPP.
o Three major feedback mechanisms regulate the

overall rate of de novo purine nucleotide synthesis.
 The activity of PRPP synthetase is feedback
inhibited by purine nucleotides, AMP, ADP and
GMP, GDP.
 The committed step in purine nucleotide
biosynthesis is the conversion of PRPP into
phosphoribosylamine by PRPP glutamyl-amido
transferase. This enzyme is feedback inhibited by
AMP, GMP and stimulated by PRPP.
 AMP and GMP feedback regulate their formation
from IMP.
Salvage pathway for purine nucleotide synthesis

 Purines that are derived from the normal turnover of cellular
nucleic acids, nucleotides or that are obtained from the diet are not
degraded but converted into nucleotides and used by the body.
This is called the ‘salvage pathway’ of purine nucleotides synthesis.
 The salvage pathway requires far less energy than does de novo
synthesis.
 The major mechanism in salvage pathway is phosphoribosylation
of free purine base to form a purine nucleotide. Two enzymes
involved are adenine phosphoriosyl transferase (APRT) and
hypoxanthine-guanine phosphoribosyl transferase (HGPRT). Both
enzymes use PRPP as the source of ribose 5-phosphate group. The
release of pyrophosphate makes these reactions irreversible.
Free purine + PRPP  Purine nucleotide + PPi
 A second salvage mechanism involves phosphorylation of a purine
nucleoside to form purine nucleotide, e.g., adenosine kinase
catalyzes phosphorylation of adenosine and deoxyadenosine to
AMP and dAMP.
Purine nucleoside + ATP  Purine nucleotide + PPi
 Salvage pathway provides purine nucleotides for
tissues, which are incapable to synthesize purine
nucleotides by de novo pathway, e.g. human brain,
erythrocytes and polymorphonuclear leukocytes.
Catabolism of purine nucleotides

Purine nucleotides, nucleosides and bases degrade to form uric acid in humans. But mammals other
than primates oxidize uric acid to allantoin by uricase which is absent in humans.
AMP, IMP and GMP are converted into their nucleoside forms adenosine, inosine and guanosine by
the action of 5’ nucleotidase.
Amino group is removed from AMP to produce IMP and from adenosine to produce inosine by AMP
or adenosine deaminase.
Purine nucleoside phosphorylase converts inosine and guanosine into their respective purine bases,
hypoxanthine and guanine.
Guanine is deaminated to xanthine by the action of guanase.
Hypoxanthine is oxidized by xanthine oxidase to xanthine,
which is further oxidized by xanthine oxidase to uric acid.
Uric acid is the final product of purine degradation in humans.
Uric acid is excreted in the urine. The uric acid is freely
filtered by the glomerulus and 98% is reabsorbed in the
proximal convoluted tubules. Distal tubules secrete uric acid
which is excreted in urine. Net urinary excretion of uric acid
averages 400 – 600 mg/24 hr.
Uric acid is sparingly soluble in water. Water solubility of uric
acid depends on its ionization state. Ionized form, urates, are
more water soluble than uric acid. Proportion of uric acid
and urate salt depends on pH. At urinary pH 5, it can dissolve
only one tenth of total uric acid (urate) dissolved at pH 7.
Normal blood uric acid level

Blood uric acid level of normal adults varies with sex.
o Males  0.2 – 0.5 mmol/L or 3 – 9 mg/dL
o Females  0.1 – 0.4 mmol/L or 2.5 – 7.5 mg/dL
Sources of uric acid

o Endogenous – purine catabolism
o Exogenous – high intake of diet containing nucleic acids such as Hilsa eggs, mushroom, organ
meats, young germinating plants and seeds, methyl xanthine derivatives such as coffee, tea.
Factors affecting blood uric acid level

Sex
o Men have higher incidence of hyperuricemia due to slighter lower urinary uric acid excretion in
males than females.
o Urinary excretion of uric acid in women after menopause decreases towards that of men.
Pregnancy
o Blood uric acid level rises near term of pregnancy.
Dietary intake
o High intake of diet rich in nucleotides and nucleic acids such as organ meats, eggs, mushroom,
coffee and tea leads to increase in blood uric acid level (secondary hyperuricemia).
Pathological factors
o Primary hyperuricemia (Inherited disorders of purine metabolism)
 PRPP synthetase over-activity
 Resistance to feedback inhibition leads to over-production and degradation of purines.
 HGPRT deficiency (Lesch-Nyhan syndrome)
 Rise in cellular PRPP results in over-production and degradation of purines.
 APRT deficiency may lead to purine over-production and hyperuricemia.
 Von Gierke’s disease (glucose 6-phosphatase deficiency)
 Purine over-production and hyperuricemia is due to enhanced generation of PRPP
precursor, ribose 5-phosphate secondary to oxidation of accumulated glucose 6-
phosphate via HMS pathway.
 Associated lactic acidosis also elevates renal threshold for uric acid excretion, increasing
plasma uric acid level.
o Secondary hyperuricemia
 Due to increased production of uric acid (increased cell breakdown)
 Cancers such as polycythemia, leukemia,
 Chronic hemolytic anemia
 Hyper catabolic states such as starvation, trauma
 Due to decreased urinary excretion of uric acid
 Renal disease  Thiazides diuretics
 Lactic acidemia  Renal outflow obstruction
 Lead poisoning  Deficit in renal tubular transport of uric acid
Nu cl e o t i de a nd He m e Me t a b o l ism Pa g e |8
Diseases associated with purine metabolism
Gout
Gout is a metabolic disorder associated with an elevated serum uric acid level. The increased serum
uric acid is due to either increased formation of uric acid or its decreased renal excretion.
Gout is associated with hyperuricemia but hyperuricemia is not always associated with gout.
Gout is classified into two broad types: primary gout and secondary gout.
Primary gout
o Primary gout is an inborn error of purine metabolism in which there is increased uric acid level as
a result of increased synthesis of purine nucleotides and its breakdown.
o PRPP synthetase over-activity
 It is a X-linked inherited disorder due to mutations in PRPP synthetase gene that results in
lower Km for ribose 5-phosphate, increased Vmax for PRPP production of the enzyme or
decreased sensitivity to feedback inhibition by purine nucleotides.
 Due to loss of allosteric feedback regulation, abnormally high activity of PRPP synthetase
results in excessive production of PRPP, which in turn accelerates the rate of de novo
synthesis of purine nucleotides. Increased synthesis is associated with increased break down
to uric acid.
o Lesch-Nyhan syndrome
 Lesch–Nyhan syndrome is an X-linked recessive disorder caused by deficiency of HGPRT, an
enzyme of purine salvage pathway.
 In the absence of HGPRT, the salvage pathway is inhibited and purines cannot be reconverted
to nucleotides; instead they are degraded to uric acid. The lack of HGPRT also causes an over-
production of PRPP, which stimulates purine nucleotide biosynthesis. Increased synthesis is
associated with increased break down to uric acid, resulting in increased uric acid level in
plasma and urine.
 It is characterized by neurological symptoms, including intellectual disability, motor
disorders, aggressive behavior and compulsive self-mutilation (particularly chewing on their
fingers and lips).
o von Gierke’s disease
 Von Gierke’s disease is due to deficiency of glucose-6-phosphatase. This results in
accumulation of glucose-6-phosphate which is then oxidized via HMS pathway, generating
large amounts of ribose 5-phosphate.
 There is over-production of purine nucleotides and hyperuricemia secondary to enhanced
generation of ribose 5-phosphate.
 Associated lactic acidosis elevates renal threshold for urate excretion and increase blood
uric acid level.
Secondary gout
o Secondary gout results from a variety of conditions that cause over-production of uric acid i.e.,
elevated destruction of cells or decreased excretion of uric acid.
o Elevated destruction of cells is accompanied by increased degradation of nucleic acids to uric
acid, which occurs in cancers (leukemia, polycythemia), chronic hemolytic anemia, psoriasis and
hyper catabolic states (starvation, trauma, etc.).
o Decreased urinary excretion of uric acid may result from chronic renal disease, renal outflow
obstruction, thiazide diuretics, lactic acidosis.
Consequences of hyperuricemia
o In hyperuricemia, salt of uric acid (urates) are deposited in synovial joints, kidney and other
tissues.
o Uric acid crystals deposited in joints can lead to inflammatory changes resulting in pain, swelling,
stiffness and limitations of joint movement. The most commonly affected joint is
metatarsophalangeal joint of big toe. This is called gouty arthritis.
o Uric acid crystals deposited in kidneys lead to uric acid stone formation, especially after the site
of urine acidification e.g., collecting duct. In severe cases, renal stone formation can lead to renal
outflow obstruction which may ultimately cause chronic kidney disease and renal failure.
o Nodular masses of monosodium urate crystals (tophi) may be deposited in soft tissues, resulting
in chronic tophaceous gout.
Treatment of gout
o Gout can be treated by a combination of nutritional therapy and drug therapy. These include –
 Restriction of intake of foods especially rich in nucleotides and nucleic acids such as organ
meats, eggs, coffee and tea, and alcohol consumption
 Allopurinol
 It is an analog of hypoxanthine which competitively inhibits xanthine oxidase. This leads
to reduced formation of uric acid and accumulation of xanthine and hypoxanthine, which
are more soluble and thus easily excreted.
 Xanthine oxidase hydroxylates allopurinol, yielding alloxanthine, which remains tightly
bound to the enzyme, thereby inactivating it. Allopurinol is therefore a suicide inhibitor
of xanthine oxidase.
 Colchicine decreases the activation, degranulation and the movement of neutrophils to the
affected areas, thereby producing anti-inflammatory action.
o Water solubility of uric acid depends on its ionization state. Ionized form, urates, are more water
soluble than uric acid. Proportion of uric acid and urate salt depends on pH. At urinary pH 5, it can
dissolve only one tenth of total uric acid dissolved at pH 7. Urates becomes crystals after the site
of urine acidification, i.e., in the collecting duct. They can be removed by alkalinization of urine.
N u c l e o t i d e a n d H e m e M e t a b o l i s m P a g e | 10
Adenosine deaminase and purine nucleoside phosphorylase deficiency

ADA deficiency causes severe combined immuno-deficiency (SCID) in which both thymus-derived
lymphocytes (T-cells) and bone marrow derived lymphocytes (B-cells) are dysfunctional.
Purine nucleoside phosphorylase deficiency is associated with a severe deficiency of T cells but
apparently normal B cell function. Immune dysfunction appears to result from accumulation of dGTP
and dATP which inhibit ribonucleotide reductase, thereby depleting DNA precursors for the cells.
Xanthinuria
Deficiency of xanthine oxidase, due to either genetic
defect or severe liver damage results in hypouricemia
and increased urinary excretion of xanthine and
hypoxanthine. Xanthine lithiasis (formation of
stones) and secondary renal damage may occur in
severe xanthine oxidase deficiency.
Biosynthesis of pyrimidine nucleotides

 Unlike purine ring synthesis in which the ring is
constructed on a pre-existing ribose 5-phosphate,
the pyrimidine ring is synthesized before being
attached to ribose 5-phosphate.
 The sources of carbon and nitrogen atoms in
pyrimidine ring are aspartate, glutamine and CO2.
 Steps in pyrimidine nucleotide synthesis
o Formation of carbamoyl phosphate
o Synthesis of orotic acid
o Formation of pyrimidine nucleotides
o Synthesis of UTP and CTP
o Synthesis of dTMP from dUMP
 Pyrimidine biosynthesis starts with the formation
of carbamoyl phosphate from glutamine, ATP
and CO2. This reaction is catalyzed by cytosolic
carbamoyl phosphate synthase-lI (CPS-II).
 Condensation of carbamoyl phosphate with
aspartate forms carbamoyl aspartate in a reaction
catalyzed by aspartate transcarbamoylase and is
the committed step in the biosynthesis of
pyrimidine.
 The first pyrimidine nucleotide formed is orotidine monophosphate (OMP) which is decarboxylated
to form uridine monophosphate (UMP). UMP is further phosphorylated to form UTP. Amination of
UTP produces CTP and glutamine is the amino group donor for this reaction.
 dTMP is formed from methylation of dUMP by the action of thymidylate synthase which uses
methylene THF as the source of methyl group.
 Pyrimidine deoxyribonucleotides are formed from respective ribonucleoside diphosphates by the
action of ribonucleotide reductase enzyme complex.
Salvage pathway for pyrimidine nucleotides synthesis

 Few pyrimidine bases are salvaged in human cells.
 Pyrimidine nucleosides, uridine and cytidine, can be converted to UMP, CMP by uridine-cytidine
kinase. Deoxycytidine can be salvaged by deoxycytidine kinase and thymidine can be salvaged by
thymidine kinase.
Regulation of pyrimidine nucleotide biosynthesis

 The first two enzymes carbamoyl phosphate synthetase-II and aspartate transcarbamoylase are key
enzymes for pyrimidine nucleotide synthesis.
 Carbamoyl phosphate synthase-II is feedback inhibited by UTP and activated by PRPP.
 Aspartate transcarbamoylase is feedback inhibited by CTP and activated by ATP.
Catabolism of pyrimidine nucleotides

 Unlike purine rings which are not cleaved in human cells, pyrimidine ring can be cleaved and
degraded to highly water soluble metabolites such as β-alanine and β-aminoisobutyrate, which can
serve as precursors of acetyl CoA and succinyl CoA respectively.
 Catabolism of all pyrimidine releases CO2 and NH3.

Specifically, end product of cytosine and uracil degradation
is β-alanine whereas that of thymine is β-aminoisobutyrate.
All these products are water soluble.
Inherited disorders of pyrimidine nucleotide metabolism

 Since the end products of pyrimidine catabolism are highly
water soluble, overproduction of pyrimidine nucleotides is
rarely associated with clinically significant abnormalities.
 Hyperuricemia is associated with severe over-production of
PRPP and subsequently, there is over-production of
pyrimidine nucleotides and increased excretion of β-alanine.
 Since methylene THF is required for dTMP synthesis, folate
and vitamin B12 deficiency result in deficiencies of dTMP.
 Orotic aciduria
o Orotic aciduria is a hereditary disorder which can result
from a defective enzyme in pyrimidine synthesis.
o A defect in the conversion of orotic acid to UMP results
in the excretion of orotic acid in the urine.
o There are two types of orotic acidemia:
 In type-I, there is deficiency of both enzymes;
orotate phosphoribosyltransferase and orotidylate
decarboxylase.
 In type-II, only orotidylate decarboxylase is deficient.
o The deficiency in UMP and other pyrimidine nucleotides
results in inhibition of DNA and RNA synthesis, which causes megaloblastic anemia and failure to
thrive (growth retardation).
 Reye’s syndrome
o Reye’s syndrome is a secondary orotic aciduria, which may be due to inability of severely
damaged mitochondria to utilize carbamoyl phosphate (e.g., ornithine transcarbamoylase
defect) in the formation of urea which then may be diverted for cytosolic overproduction of
orotic acid.
Clinical uses of inhibitors that target nucleotides biosynthesis

 Anti-cancer agents
o Many chemotherapeutic agents for cancer treatment target enzymes in the nucleotide
biosynthetic pathways.
o 5-flurouracil
 It is a thymine analog and can be used as inhibitor for pyrimidine synthesis.
 In the body, 5-flurouracil is metabolically converted to 5-fdUMP which becomes permanently
bound to the inactivated thymidylate synthase, irreversibly inhibits the enzyme activity.
 It is a suicide inhibitor.
o Azaserine and Acivicin
 They are glutamine analogs which inhibit glutamine PRPP amidotransferase in purine
nucleotide de novo synthesis. Glutamine is a nitrogen donor in many reactions of nucleotide
biosynthesis pathways.
 It is a mechanism-based enzyme inhibitor or suicide inhibitor.
o Azathioprine (6-mercaptopurine)
 It is an anti-cancer agent that interferes with de novo synthesis of purines. By utilizing PRPP
and by the action of HGPRT, it is converted into 6-mercaptopurine ribonucleotide which acts
as a negative allosteric effector to glutamyl PRPP amidotransferase, thereby inhibiting
purine nucleotide synthesis required for cell proliferation.
 It also inhibits the conversion of IMP to GMP and IMP to AMP.
 6-Mercaptopurine and azathiopurine therapy are effective treatments for leukaemia and
several autoimmune conditions, including psoriatic arthritis and inflammatory bowel disease
(e.g. Crohn’s disease).
o Hydroxyurea
 It is an anti-cancer agent that blocks DNA synthesis indirectly by inhibiting ribonucleotide
reductase, which catalyzes formation of deoxyribonucleotides from ribonuceloside
diphosphates.
 But its clinical use is limited due to its rapid rate of clearance and high drug concentration
required for effective inhibition.
o Some anti-cancer agents are folate analogs which inhibit formation of active THF required as one
carbon-unit carrier for purine nucleotide synthesis as well as coenzyme of thymidylate synthase in
formation of dTMP from dUMP.
o Methotrexate
 Methotrexate and related compounds inhibit the reduction of dihydrofolate to
tetrahydrofolate catalyzed by DHF reductase. These compounds decrease the amount of
THF available for use in purine nucleotide synthesis as well as conversion of dUMP to dTMP.
 They are useful in treating rapidly growing cancers but are toxic to all dividing cells.
 Antibiotics and anti-viral agents
o Sulfonamides
 Structural analogs of PABA that competitively inhibit bacterial synthesis of folic acid.
 Since purine nucleotide synthesis requires THF as a coenzyme, sulfonamide slows down the
growth of bacteria. Humans cannot synthesize folic acid; therefore it does not interfere with
human purine synthesis.
o Trimethoprim
 It is a folate analog which has potent antibacterial activity because it selectively inhibits
bacterial DHF reductase.
o Acyclovir
 Acyclovir (acycloguanosine) is a purine nucleoside analog which is used for treatment of
herpes virus (HSV) infection.
 Viral thymidine kinase rapidly converts acyclovir to acyclo-GMP. Cellular kinases
subsequently convert acyclo-GMP to acyclo-GTP. This compound is a surrogate for GTP and is
incorporated into rapidly dividing viral cells. Since acyclovir lacks a 3'·hydroxyl group, it leads
to the termination of viral DNA replication.
o Azido-thymidine (AZT)
 It is a nucleoside reverse transcriptase inhibitors (NRTIs) used for inhibition of HIV virus
replication.
 It is phosphorylated by cellular kinases to AZT-triphosphate which blocks HIV replication by
inhibiting viral reverse transcriptase enzyme. Similar NRTIs include stavudine (thymidine
analog), lamivudine (cytosine analog).
Heme metabolism
Biosynthesis of heme
1. Formation of δ-aminolevulenate (ALA)

 It is the rate-limiting step of
heme synthesis and occurs in
mitochondria.
 Condensation of succinyl CoA
with activated glycine followed
by decarboxylation
 Catalyzed by δ-aminolevulenate
synthase (Pyridoxal phosphate
as coenzyme)
2. Formation of porphobilinogen
 Condensation of two molecules of ALA catalyzed by ALA dehydratase (zinc as cofactor)
3. Formation of linear tetra-pyrrole (hydroxymethylbilane)
 C0ndensation of four molecules of porphobilinogen catalyzed by uroporphyrinogen I synthase
4. Conversion to uroporphyrinogen III
 By combined actions of uroporphyrinogen I synthase and uroporphyrinogen III synthase
5. Conversion of uroporphyrinogen III to coproporphyrinogen III
 Decarboxylation of all acetate groups to methyl groups catalyzed by uroporphyrinogen
decarboxylase
6. Conversion of coproporphyrinogen III to protoporphyrinogen IX
 Decarboxylation and oxidation of two propionate side chains to vinyl groups catalyzed by
coproporphyrinogen oxidase
7. Conversion of protoporpyrinogen IX to
protoporphyrin IX
 Catalyzed by protoporphyrinogen oxidase
8. Formation of heme
 Incorporation of ferrous (Fe2+) into
protoporphyrin IX by the action of
ferrochelatase or heme synthase
Regulation of heme synthesis
 Key enzyme  ALA synthase
 ALA synthase enzyme synthesis can be repressed by heme and aporepressor protein
 Translation of ALA synthase mRNA can be inhibited by heme via binding of heme-sensitive
regulatory protein to 5’ untranslated region of mRNA.
 Transport of enzyme into mitochondria can be blocked by increased heme level.
Porphyrias
 Genetic disorder of heme biosynthesis due to mutations of gene encoding enzymes involved in
heme synthesis leading to inherited deficiency of enzymes in the heme biosynthetic pathway
 Porphyrinogens are spontaneously oxidized to porphyrins leading to photosensitivity.
Defective heme synthesis

 Can lead to hypochromic microcytic anemia
 Due to deficiency of precursors required for heme synthesis e.g., iron
 Due to decreased activity of enzymes in heme synthesis
o Inherited deficiency of enzymes involved in heme biosynthesis (porphyria)
o Lead poisoning  inhibit ALA dehydratase and ferrochelatase
o Deficiency of enzyme cofactors or coenzyme  zinc deficiency affects ALA dehydratase activity,
vitamin B6 deficiency affects ALA synthase activity
Catabolism or breakdown of hemoglobin
Under physiological conditions, a 70 kg man turns over

nearly 6 g of Hb/ day. The normal human erythrocytes
have life-span of about 120 days. Aged RBCs are
recognized by their membrane changes and removed
from the circulation by reticulo-endothelial system (Bone
marrow, spleen and liver) where they are degraded by
the cells of RE system.
In the RE cells, the globin chains of hemoglobin are
denatured, releasing heme into the cytoplasm. Globin
chains are hydrolyzed into its constituent amino acids. These amino acids are either reutilized for general
metabolic needs or degraded.
Catabolism of heme
 Occurs in the microsomal fraction of the RE cells.
 Oxidative cleavage of ring structure of heme
o Cleavage of α methenyl bridge between pyrrole I and II, releasing Fe3+ and CO
o Heme is converted into biliverdin (linear tetrapyrrole).

o Catalyzed by microsomal heme oxygenase system which requires NADPH and molecular O2.
o Ferric iron enters into iron pool and reutilized by the body.
 Reduction of biliverdin to bilirubin
o Catalyzed by biliverdin reductase which requires NADPH.
o Bilirubin is secreted into the blood.
 Bilirubin, yellow colored water insoluble compound is transported bound to plasma albumin in the
blood. It must be further metabolized to water soluble form before excretion. This occurs in the
liver.
 One gram of Hb yields 35 mg of bilirubin; 250-350 mg/day of bilirubin are formed in an adult.
Bilirubin metabolism in the liver

Uptake of bilirubin
 Via facilitated transport system
 Carried by intracellular Z and Y
proteins.
Conjugation of bilirubin in smooth endoplasmic
reticulum
 Conversion of bilirubin to polar form
 Esterification of carboxylic side chains of bilirubin with glucuronic acid
 Glucuronic acid donor – UDP glucuronic acid
 Catalyzed by UDP glucuronyl transferase (can be induced by phenobarbituates).
Secretion of conjugated bilirubin into the bile
 Active process
 Rate limiting step for the entire process of hepatic bilirubin metabolism
 Active transport system is inducible by phenobarbitol.
Metabolism in the intestine (terminal ileum and large intestine)

 Deconjugation of bilirubin glucuronides by intestinal bacterial enzyme β glucuronidase
 Reduction of bilirubin to colorless urobilinogen by intestinal bacterial enzymes
 Most of urobilinogens are oxidized to urobilins (colored compounds) and excreted in feces (40-280
mg/day).
 Small amount of urobilinogen is reabsorbed into entero-hepatic circulation and re-excreted through
liver (entero-hepatic urobilinogen cycle).
 Small fraction of urobilinogen reaches to the kidneys via systemic circulation and excreted into the
urine (0 – 4 mg/day).
Hyperbilirubinemia
Blood bilirubin level higher than normal is
called hyperbilirubinemia.
Bilirubin is the end product of heme
catabolism in the RE cells. The main source of
heme is from Hb breakdown.
Hyperbilirubinemia can be caused by –
1) Increased Hb breakdown,
2) Impaired uptake and conjugation in liver disease,
3) Impaired secretion due to bile duct obstruction.
Depending on the type of bilirubin present in the serum – i.e., unconjugated or conjugated,
hyperbilirubinemia can be classified as –
o Retention hyperbilirubinemia (increased unconjugated bilirubin due to overproduction) and
o Regurgitation hyperbilirubinemia (reflux of conjugated bilirubin of into the bloodstream
because of biliary obstruction).
Normal blood bilirubin level
Jaundice  < 17.1 μmol/L or < 1mg/dL
Jaundice (icterus) is the yellowish  Direct or conjugated bilirubin = 0.1 – 0.4 mg/dL
discoloration of skin, sclera and mucous  Indirect or unconjugated bilirubin = 0.2 – 0.7 mg/dL
membrane due to increased serum bilirubin level more than 50μmol/L or 3mg/dL.
Classification of jaundice
1. Pre-hepatic jaundice
2. Hepatic jaundice
3. Post-hepatic jaundice
1. Pre-hepatic jaundice
Results from excessive production of
bilirubin as a result of hemolysis or a genetic
abnormality in hepatic uptake of
unconjugated bilirubin.
Increased unconjugated bilirubin level in the
blood (indirect test positive)
Increased urinary urobilinogen
Absent urine bile pigments or bilirubin
E.g., hemolysis due to thalassaemia, sickle cell anemia, G6PD deficiency, autoimmune disease,
malaria infection, etc.
Determination of serum bilirubin

2. Hepatic jaundice
(Van den Bergh Test)
Results from generalized hepatocytes dysfunction.  Important in clinical study of
Elevation of both conjugated and unconjugated jaundice
bilirubin in the serum (both direct and indirect tests  Quantitative assaying of serum
bilirubin by application of Erhlich’s
positive or biphasic reaction)
reagent.
Absent or low urinary urobilinogen  Coupling of diazotized sulfanilic acid
Presence of urine bile pigments or bilirubin (Ehrlich’s reagent) and bilirubin to
It is accompanied by other abnormalities in produce a reddish-purple azo

compound.
biochemical markers of hepatocellular function
 Colored compounds formed on
(e.g., increased plasma level of liver enzymes; ALT,
addition of serum to the reagent 
AST). detection of water soluble
E.g., viral hepatitis, alcoholic hepatitis, conjugated bilirubin (Direct test)
 Colored compounds formed only on
hepatotoxicity by drugs and chemicals
addition of methanol  detection of
water insoluble unconjugated or free
3. Post-hepatic jaundice
bilirubin (Indirect test)
Caused by bile duct obstruction.
Elevation of conjugated bilirubin and other biliary metabolites in the plasma (direct test -
positive)
Pale color stool (due to absence of fecal bilirubin and urobilin)
High color/dark color urine (presence of water soluble conjugated bilirubin in the urine)
Absence of urobilinogen in the urine
E.g., bile duct obstruction by gall stone, pancreatic tumor
Neonatal jaundice
 About 50% of normal babies become jaundiced 48 hours after birth.
 Caused by temporary inefficiency in bilirubin conjugation (due to immaturity of the enzymes involved in
bilirubin conjugation), and resolves in the first 10 days.
 Can be precipitated by increased hemolysis conditions.
 Unconjugated bilirubin can cross blood brain barrier into CNS when its plasma concentration exceeds
20 – 25 mg/dL which can be tightly bound to albumin.
 Results in toxic encephalopathy or kernicterus which can cause permanent neurological damage and
mental retardation.
 Exposure to visible light (phototherapy) blue-white light, which isomerizes bilirubin to more soluble
non-toxic form that might be excreted with bile, or exchange blood transfusion can remove the excess
bilirubin.
Xenobiotics Metabolism P age |1
Xenobiotics metabolism
 It is the metabolism of foreign chemicals (xenobiotics) exposed to the body such as drugs, food
additives, pollutants, certain insecticides, potential carcinogens. It is the conversion of toxic water-
insoluble chemicals into non-toxic water soluble forms for excretion. The biochemical processes
whereby the noxious substances are rendered less harmful and more water soluble are known as
detoxification.
 Xenobiotics – are compounds that are foreign to the body. Xenobiotic is a substance that does not
occur naturally but interferes with the metabolism of any organism.
 Xenobiotic metabolism occurs in two phases.
o Phase I – reaction increases the polarity of the xenobiotics which can either be excreted or can
undergo conjugation reactions in phase II.
o Phase I reactions include – hydroxylation (major reaction), oxidation, reduction and hydrolysis.
o Phase II – conjugation reactions
 Conjugation with glucuronic acid  Sulfation
 Conjugation with glutathione  Acetylation
 Conjugation with glycine  Methylation
Phase I
Phase I reactions
result in the formation
of compounds with
decreased toxicity
(detoxification).
Sometimes this may
result in increased
toxicity
(entoxification).
a) Hydroxylation
 Hydroxylation is the major reaction in phase I, catalyzed by microsomal cytochrome P450
monooxygenases enzyme system. It requires molecular O2 and NADPH.
RH + O2 + NADPH + H+  R–OH + NADP+ + H2O
 RH can represent drugs, carcinogens, pesticides, petroleum products and endogenous
compounds such as steroids, eicosanoids and retinoids. These substrates are generally lipophilic
and are rendered hydrophilic by hydroxylation reactions.
 In some cases, products of cytochrome P450 can be mutagenic or carcinogenic e.g., aflatoxin B 1
in the mold is converted to epoxide compound which can bind to guanine base in DNA.
b) Oxidation
 Oxidation and detoxification of alcohol is an important
function of the liver. Two enzymes are involved in the
process; alcohol dehydrogenase oxidizes alcohol to aldehyde
and aldehyde dehydrogenase oxidizes aldehyde to acid.
Alcohol dehydrogenase is located in the cytosol whereas
aldehyde dehydrogenase is located in mitochondria.
 Oxidation of some compounds may result in production of
more toxic substances. E.g.,
Methanol  formaldehyde  formic acid
Halogenated alcohol  halogenated acid (fluroacetate)
Ethylene glycol  glycolate  oxalic acid
Minor reactions
c) Reduction
 Major group of compounds which are reduced and detoxified by the liver are nitro compounds.
 Nitro compounds are reduced to amines while aldehydes and ketones are reduced to alcohols.
 Reduction is catalyzed by reductases located in endoplasmic reticulum.
 E.g., conversion of picric acid to picramic acid
d) Hydrolysis
 Hydrolysis reaction splits the toxicant into
two fragments or smaller molecules by
addition of water. Esters, amines, hydrazines,
amides, glycosidic bonds are generally susceptible to hydrolysis, e.g., aspirin, acetanilide,
aliphatic ester, procaine, xylocaine, di-isopropyl fluro phosphate (DFP), etc.,
Acetyl salicylic acid (aspirin) + H2O  acetic acid + salicylic acid
 Epoxides produced by the action of monooxygenase upon certain procarcinogenic substrates
are highly reactive. They are converted to less reactive dihydrodiols by epoxide hydrolase.
Phase II
Phase II reactions convert the xenobiotics and the hydroxylated or other compounds derived from
phase I reactions into various polar metabolites by conjugation with a polar group. Conjugation
increases the water solubility of the xenobiotic and decreases its biologic activity.
Conjugation reactions include – conjugation with glucuronic acid, glutathione, glycine or glutamine
and sulfation, acetylation and methylation.
a) Conjugation with glucuronic acid

 Glucuronide conjugation is the
most common phase 2 reaction
and also the major pathway of
drug metabolism.
 Compounds excreted as
glucuronide conjugates are
bilirubin, hydroxyl compounds
(phenolic and alcoholic), carboxyl, sulfhydryl and amino compounds.
 UDP glucuronic acid is the glucuronyl donor and enzyme catalyzing the reaction is UDP
glucuronyl transferase which is present in the endoplasmic reticulum.
b) Conjugation with glutathione Metabolism of glutathione conjugates
 Glutathione is a tripeptide consisting of Glutathione conjugates are subjected to further
metabolism.
glutamic acid, cysteine and glycine.
o Removal of glutamyl and glycine groups
 Compounds that are detoxified by
from glutathione
conjugation with glutathione include alkyl o Addition of acetyl group to remaining
or aryl halides, epoxides, alkenes and cysteinyl moiety forming mercapturic acid
some potentially toxic metabolites. o Excretion of N-acetyl-cysteinyl derivative

(mercapturic acid) in the urine
 The reaction is catalyzed by glutathione S
transferase.
 If potentially toxic
xenobiotics are not
conjugated to glutathione,
they would be free to
combine covalently with
DNA, RNA or proteins Glutathione S transferase
leading to serious cell R–X + GSH (glutathione) GS–R + XH
damage. Conjugation with glutathione is therefore, an important defense mechanism against

certain toxic compounds like drugs and carcinogens.
c) Conjugation with amino acids,
glycine or glutamine
 Many aromatic carboxylic acids
are conjugated with glycine.
The most common example is conjugation of benzoic acid with glycine to form hippuric acid
which is excreted in urine.
d) Sulfation (conjugation with sulfate)

 Some alcohols, phenols,
acrylamines, indole are
conjugated with sulfate. Sulfate
donor is active 3’-phosphor-
adenosine-5’-phospho-sulfate
(PAPS).
 The reaction is catalyzed by sulfotransferase.
Phenol + PAPS  Phenyl sulfate + PAP
Indole + PAPS  Indoxyl sulfate + PAP
e) Acetylation (conjugation with acetate)
 Some drugs are conjugated with acetate, e.g., isoniazid, sulfanilamide, para-amino salicylic acid
(PAS).
 Acetyl group donor is acetyl CoA.
 The reaction is catalyzed by acetyl transferase.
Isoniazid + Acetyl CoA  Acetyl isoniazid + CoA-SH
f) Methylation
 Amino, hydroxyl or thiol groups are methylated by methyl transferase enzymes.
 Catechol-O-methyl transferase (COMT) catalyzes the O-methylation of norepinephrine,
dopamine, epinephrine, dopa and a number of drugs. COMT converts epinephrine to
metanephrine.
 S-adenosyl methionine serves as methyl group donor.
Cytochrome P450
Cytochrome P450 enzymes are integral membrane protein enzymes, containing a single heme
prosthetic group. It catalyzes mono-oxygenation of a large variety of structurally diverse
compounds (broad substrate specificity). The overall reaction catalyzed by a cytochrome P450 is:
RH + O2 + NADPH + H+ → R-OH + H2O + NADP+
These enzymes act on a broad range of Designation of Cytochrome P450 isoforms
substrates including endogenous compounds Cytochrome superfamily is classified

according to relative identity of amino
such as steroids, cholesterol, fatty acids and
acid sequences of the enzymes.
exogenous compounds such as drugs, food Superfamily is designated by the prefix
additives, components of cigarette smoke, ‘CYP’, followed by an Arabic numeral
pesticides, and chemicals that are ingested, (e.g., CYP1, CYP2, etc.).
Subfamily is identified by additional
inhaled or absorbed through the skin.
capital letter (e.g., CYP1A, CYP1B,
They are present in highest amount in liver and
CYP1C, etc.)
small intestine but probably present in all Individual members of each subfamily
tissues. In liver and most other tissues, they are are numbered according to the order of
present mainly in the membrane of smooth identification (e.g., CYP1A1, CYP1A2, etc.).
endoplasmic reticulum. In the adrenals, they are found in mitochondria as well as in the endoplasmic
reticulum.
There is a large number of cytochrome
P450 isoforms (about 150) designated as
CYP1, CYP2, CYP3, etc. Most isoforms
of cytochrome P450 can be induced by
certain drugs such as phenobarbital.
Such induction has important
implications on drug metabolism and
drug interactions.
Deamination, dehalogenation, desulfuration, peroxygenation, epoxidation and reduction are also
catalyzed by cytochrome P450 monooxygenase enzymes. In some cases, their products are
mutagenic or carcinogenic, e.g., conversion of aflatoxin in the mold to epoxide compound.
Some exhibit genetic polymorphism which can result in atypical drug metabolism. Genotyping of
cytochrome P450 profile may permit individualization of drug therapy in the future.
Cytochrome P450 activities may be altered in cirrhosis of liver, affecting drug metabolism.
Functions of cytochrome P450
o Xenobiotics metabolism in liver
 Variety of exogenous substances including drugs, chemicals, environmental contaminants
and food additives are metabolized by microsomal cytochrome P450 monooxygenase
system in the liver.
 Approximately 50% of the common drugs that humans ingest are metabolized by isoforms of
cytochrome P450 (CYP1, CYP2, and CYP3 families).
o Metabolism of endogenous
compounds and synthesis of
steroid hormones
 Cytochromes P450 are also
important for the metabolism of
a number of physiological
compounds—e.g., the synthesis
of steroid hormones and the
conversion of vitamin D to its
active metabolite, calcitriol.
Both mitochondrial and
microsomal cytochrome P450
systems are required to convert
cholesterol to aldosterone and
cortisol in adrenal cortex,
testosterone in testes, and
estradiol in ovaries.
 In liver, CYP7A1 (7α-
hydroxylase) catalyzes first and
rate limiting step in bile acid
synthesis. In kidney, CYP27B1
(1α-hydroxylase) catalyzes the
production of 1, 25 DHCC (calcitriol).
Entoxification
In certain situations, metabolism of xenobiotics may result in the production of substances which
are more toxic and harmful. Such process is called entoxification reactions.
Some examples of entoxification processes are epoxidation reactions (such as formation of
aflatoxin epoxide from aflatoxin B1, benzpyrene epoxide formation), oxidation reactions of
methanol, halogenated hydrocarbon (halogenated alcohol) and ethylene glycol.
Methanol poisoning
 Methanol is metabolized in the liver by the subsequent
actions of alcohol dehydrogenase and aldehyde
dehydrogenase enzyme, producing formaldehyde and
ultimately formate. Subsequent formate metabolism in
human is comparatively slow due to relatively small
folate pool in the primates. Thus, methanol poisoning
leads to accumulation of formate in the cells and blood
which is responsible for methanol toxicity.
 Formate accumulation leads to metabolic acidosis in the
early stage. In late stages, inhibition of respiratory chain
by formate results in lactate accumulation. Inhibition of
respiratory chain may explain the ocular and general toxicity due to methanol poisoning.
 Therapeutically, formate formation can be prevented by inhibiting methanol access to alcohol
dehydrogenase by administration of ethanol in methanol poisoning.
Halogenated hydrocarbon toxicity

 Halogenated hydrocarbons represent a large group of aromatic and aliphatic compounds with
diverse industrial, agricultural, medical applications. It comprises haloalkanes, haloalkenes,
haloalkynes and halogenated alcohols such as carbon tetrachloride (CCl4), chloroform (CHCl3),
iodoform (CHI3), fluorocarbons and tetrachloroethylene. These halogenated hydrocarbons can
induce toxicity to many organs after biotransformation.
 In the liver, halogenated alcohol is oxidized to halogenated acid, e.g., fluroacetate which can
inhibit citric acid cycle.
Halogenated alcohol  halogenated acid (fluroacetate)
 CCl4 is activated to trichloromethyl radical (CCl3•) by the action of cytochrome P450 isoforms
CYP2E1, CYP2B1, CYP2B2. This radical can bind to various cellular molecules (nucleic acid, protein
and lipids), impairing their functions and cellular processes. CCl3• adducts to DNA is mutagenic,
leading to DNA damage and initiates hepatic cancer. This radical can also react with oxygen to
form the trichloromethyperoxy radical (CCl3OO•). CCl3OO• is highly reactive and initiates the
lipid peroxidation chain reaction of PUFA in cell membrane phospholipids. This affects the
integrity and permeability of mitochondrial, endoplasmic reticulum and plasma membrane,
resulting in cellular dysfunction and subsequent cell damage.
Ethylene glycol toxicity
 Ethylene glycol is a chemical used for the production of polyester fibers and in chilled water air-
conditioning system.
 In the liver, it is oxidized more rapidly than methanol by the action of alcohol dehydrogenase to
glycoaldehyde. Glycoaldehyde is rapidly metabolized to glycolate which is responsible for the
metabolic acidosis in ethylene glycol poisoning. Glycolate is further metabolized by various
pathways; one of which leads to formation of oxalate. Oxalate precipitates calcium in various
tissues and urine by forming calcium oxalate crystals.
 Ethylene glycol toxicity is complex and not fully understood, but it is mainly due to the severe
metabolic acidosis caused by glycolate and due to calcium oxalate precipitation.
Ethylene glycol  glycolate  oxalic acid
Alcohol metabolism in liver

Ethanol is oxidized in the liver mainly by alcohol
dehydrogenase, to form acetaldehyde which is in
turn oxidized by aldehyde dehydrogenase (ALDH) to
acetate. Alcohol dehydrogenase is a cytosolic enzyme
and aldehyde dehydrogenase is a mitochondrial
enzyme. Both enzymes use NAD+ as coenzyme.
A cytochrome P450 enzyme, CYP2E1, also oxidizes ethanol but is quantitatively less important than
the alcohol dehydrogenase. This is also called microsomal ethanol oxidizing system (MEOS). CYP2E1
is induced by alcohol. Therefore an increased proportion of the alcohol is metabolized by this route
in chronic alcoholics.
Alcohol induced hepatotoxicity

Ethanol may cause excessive fat deposition in the liver (alcoholic steatosis), hepatitis or finally
fibrosis (known as cirrhosis), which in turn leads to liver failure.
Liver damage in chronic alcoholics may arise from toxicity of acetaldehyde, increased ratio of NADH
to NAD+ (alteration of redox potential of hepatocytes) and ethanol-induced decrease in proteasome
activity.
Many of the toxic effects of chronic ethanol consumption result from accumulation of acetaldehyde
which forms Schiff base adducts with other macromolecules. This could enhance free radical
damage to hepatocytes.
Alcohol metabolism in hepatocytes leads to increased NADH/NAD+ ratio, leading to –
o Inhibition of oxidation of lactate to pyruvate—increased NADH/NAD+ ratio shifts lactate
dehydrogenase reaction towards lactate formation. Lactate accumulation may lead to lactic
acidosis.
o Decreased gluconeogenesis—due to depletion of hepatic gluconeogenic substrate pyruvate and
reduced availability of NAD+. Therefore, alcoholism carries risk of hypoglycemia. Risk of
hypoglycemia is further increased in alcoholics during fasting because of low hepatic glycogen
stores as a consequence of poor nutrition in alcoholics.
o Inhibition of β oxidation of fatty acids and increased TAG synthesis—synthesized TAGs are
secreted into plasma as VLDL, but excess is deposited in the liver leading to fatty liver or hepatic
steatosis.
Ethanol-induced decreased proteasome activity
o Chronic alcohol consumption decreases activity of ubiquitin dependent proteasome system of
protein degradation. This prevents the degradation of CYP2E1 which is involved in peroxidation
reactions, thus increasing oxidative stress and hepatocellular damage. Inhibition of proteasome
activity may also lead to increased apoptosis, a feature of alcoholic liver disease.
Liver damage in alcoholic liver disease is associated with elevation in serum levels of transaminase
enzymes.
Drugs-induced hepatotoxicity
 Drug toxicity may occur in all individuals exposed to a sufficient concentration of a particular drug.
 A drug may be toxic in some individuals at concentrations normally tolerated by most other patients.
This phenomenon is known as idiosyncratic drug toxicity.
Xenobiotics Metabolism P a g e | 10
 Toxic effects of drugs on liver may occur through the

hepatic production of toxic metabolites.
Acetaminophen (paracetamol) toxicity

 Paracetamol is widely used as a pain killer.
 In usual therapeutic dose, it is conjugated with
glucuronic acid or sulfate, which is then excreted by
the kidneys.
 In overdose, the capacity of these conjugation
pathways is overwhelmed and acetaminophen is
oxidized by liver cytochrome P450 enzyme CYP3A4 to
N-acetyl benzoquinoneimine (NABQI). Since NABQI is toxic, it may be detoxified by conjugation
with glutathione. But in acetaminophen overdose, glutathione stores become exhausted, causing
accumulation of NABQI which can cause a free radical-mediated peroxidation of membrane lipids,
and consequently hepatocellular damage.
 N-acetyl cysteine (NAC) is used as an antidote to acetaminophen poisoning. It promotes
detoxification of NABQI by the glutathione pathway and also scavenges free radicals (but cannot
reverse hepatocellular damage).
 The risk of hepatotoxicity can be predicted from measurement of the plasma concentration of
acetaminophen in relation to the time which has elapsed since the overdose.
Solubilization and excretion of cholesterol in bile

 Cholesterol cannot be oxidized or
catabolized and can only be excreted from
the body in the bile as cholesterol or bile
acids (salts).
 Cholesterol is insoluble in water however it
can be incorporated into mixed phospholipid-
bile acid micelles and thereby solubilized.
Large quantities of cholesterol present in the
bile are solubilized in these water soluble mixed micelles, allowing cholesterol to be transported in
bile via biliary tract to the small intestine.
 Solubility of cholesterol in bile depends on the relative proportions of bile salts, phosphatidyl choline
(lecithin), cholesterol and water.
Biochemical basis of gall stone formation

Cholesterol is excreted from the body in the bile as cholesterol or bile acids (salts). Solubility of
cholesterol in bile depends on the relative proportions of bile salts, phosphatidyl choline (lecithin),
cholesterol and water.
Excessive cholesterol excretion or decreased bile acid synthesis in the liver can produce bile that is
supersaturated with cholesterol. In patients with gall stones, the activity of the key enzyme in bile
acid formation (7α-hydroxylase) is reduced leading to diminished bile acid pool. The bile then
becomes overloaded with cholesterol, which is unable to dissolve completely in the mixed micelle.
Supersaturated bile is considered lithogenic (tendency to stone formation).
With time, various factors like infection will serve as seeding agent to cause the supersaturated bile
to precipitate the excess cholesterol as crystals. The newly formed crystals must be promptly
excreted into the intestine with the bile, otherwise, the crystals will grow to form gall stones.
Crystal formation usually occurs in the gall bladder, rather than in the hepatic bile ducts, because of
more concentrated bile and prolonged contact time between bile in the gall bladder.
The tendency to secreted bile supersaturated with cholesterol is inherited, occurs more frequently
in females than in males and is associated with obesity.
Chenodeoxycholic acid is used as specific medical treatment for asymptomatic radiolucent gall
stones in functioning gall bladder. It is used as an attempt to dissolve gall stones or to prevent
further gall stones formation by inhibiting HMG CoA reductase, the key enzyme for cholesterol
synthesis.
In case of bile duct obstruction, impairment of dietary fat digestion and absorption will lead to
steatorrhea (fatty bulky stool) and increased risk of fat soluble vitamins deficiency.
Bile acids
Primary bile acids are cholic acid and chenodeoxycholic acid synthesized from cholesterol in the
liver. Secondary bile acids are deoxycholic and lithocholic acids formed by intestinal bacterial
enzyme action.
Bile salts
Bile salts are sodium or potassium salts of bile acids conjugated to glycine or taurine.
Bile acids metabolism

Bile acids (C-24) are synthesized in hepatocytes from cholesterol (C-27). Primary bile acids
synthesized in the liver are cholic acid and chenodexoycholic acid.
First and principal regulatory step in the biosynthesis of bile acids is conversion of cholesterol to 7α-
hydroxy cholesterol catalyzed by 7α hydroxylase. It is a microsomal enzyme which requires O 2,
NADPH and cytochrome P450.
After that, bile acid synthesis is divided into two pathways—cholic acid synthesis and
chenodeoxycholic acid synthesis. Both pathways involve similar steps (hydroxylation, shortening of
side chain, saturation of steroid nucleus with OH group at position 3 and 7)
The primary bile acids are secreted as glycine or taurine conjugates (glycocholic or taurocholic acid
and glycochenodeoxycholic or taurochenodeoxycholic acid) into the bile together with water,
phospholipids, cholesterol and excretory product such as bilirubin. Ratio of glycine or taurine
conjugates is normally about 3:1.
In the alkaline pH of bile, bile acids and their conjugates exist in Na + or K+ salt forms hence it is
termed as bile salts.
In the intestine, a portion of primary bile acids and their conjugates undergo deconjugation and 7α
dehydroxylation by intestinal bacterial enzymes forming secondary bile acids—deoxycholic and
lithocholic acids.
Up to 30 g of bile acids is secreted into the intestine each day. Only 2% (0.5 g) is lost through the
feces and most of them are reabsorbed in the ileum into enterohepatic circulation. Cholate,
deoxycholate, chenodeoxycholate and their conjugates participate in the enterohepatic circulation.
Most of lithocholic acid is sulphated during passage through the liver. These sulphate esters of
lithocholic acid is not reabsorbed and so excreted in the feces. Body maintains bile acid pool of only
3 – 5 g.
About 1 g of cholesterol is eliminated from the body each day into the feces. Approximately 50% of
the loss is excreted as bile acids and the remainder as neutral sterols, coprostanol and cholesterol.
Choleretics – substances which stimulate the bile secretion by liver

 Bile salts, hormone secretin, vagal stimulation
Cholagogues – stimulate the release of bile from the gall bladder.
 Cholecystokinin – most powerful cholagogue

 Release of CCK is stimulated by fatty acids and amino acids in duodenum.
Functions of bile salts

In the liver
1. Micelle formation for cholesterol excretion
 Bile salts together with phospholipids and cholesterol form micelles which solubilize and
transport cholesterol in the bile.
2. Inhibition of hepatic cholesterol synthesis
 Bile salts inhibit cholesterol synthesis in liver by inhibiting HMG CoA reductase.
3. Stimulation of choleresis
 Bile salts stimulates the process of bile secretion—choleresis.
In the gut lumen

1. Emulsification of lipids for digestion
 Bile salts solubilize lipids into smaller lipid droplets due to its surface tension lowering action
and detergent action. Emulsified lipid droplets are readily hydrolyzed by the enzymes.
2. Micelle formation of lipids for absorption
 Lipids are water insoluble and therefore for their absorption, they need to form micelles
(aggregates of lipid digestion products and bile salts). Micellar formation allows the
transport of digestive products of lipids from intestinal lumen to the brush border of the
mucosal cells for absorption.
3. Activation of enzymes
 Bile salts activate lipase, co-lipase, and enterokinase enzymes.
4. Absorption of fat soluble vitamins
 Proper digestion and absorption of dietary fat aided by bile salts also facilitates the
absorption of fat soluble vitamins.
In the mucosal cell

1. Stimulation of re-esterification
 Bile salts stimulate the enzymes required for re-esterification of fatty acids forming
triacyglycerol, phospholipids and cholesterol ester.
2. Stimulation of lipoprotein (chylomicron) formation
 Bile salts stimulate incorporation of TAG with small amounts of phospholipids, cholesterol
ester and apoprotein B48 to form chylomicron. It is then released from the cell into the
lymphatics and subsequently to the blood.
3. Inhibition of cholesterol synthesis
 Bile acid pool controls cholesterol synthesis. When bile acid level is reduced, cholesterol
synthesis is increased.
4. Stimulation of cholecystokinin (CCK) release
 CCK is released in response to long chain fatty acids and bile salts.
 Its major action is to stimulate pancreatic secretion.
Role of bile salts in lipid digestion and absorption

Bile salts are sodium or potassium salts of bile acids conjugated to glycine or taurine. They are
amphipathic compounds synthesized in the liver and are secreted as the component of bile via the
gallbladder into the intestinal lumen. Bile secretion is stimulated by the hormone cholecystokinin.
Role of bile salts in lipid digestion
o Hydrophobic nature of lipids excludes water-soluble digestive enzymes limiting the surface area
for enzyme action. It is overcome by emulsification process (conversion of dietary fat globules
to very small lipid droplets for enzymatic digestion).
o Lipids are emulsified into smaller droplets by –
 Detergent action and surface tension lowering action of bile salts
 Surfactant action of degraded lipids
 Mechanical mixing due to peristalsis
o Bile salts solubilize lipids into smaller lipid droplets by its surface tension lowering action and
detergent action. It also acts as detergents, by binding to dispersed lipid droplets formed by
intestinal peristaltic movements, thereby preventing re-association of lipid droplets.
o Emulsification of lipid droplets increases surface area for pancreatic digestive enzymes action.
Emulsified lipid droplets are readily hydrolyzed by the enzymes such as pancreatic lipase,
colipase, phospholipase A2, cholesterol esterase and other non-specific lipid esterases. End
products of lipid digestion include 2-MAG, glycerol, free fatty acids, cholesterol,
lysophospholipids. Bile salts also activate the action of lipase, colipase and enterokinase.
Role of bile salts in lipid absorption
o Lipids are water insoluble and therefore for their absorption, they need to form micelles.
o Bile salts emulsify products of lipid digestion into micelles (aggregates of lipid digestion
products and bile salts). Other dietary lipids such as cholesterol, lysophospholipids and fat-
soluble vitamins are also packaged in these micelles.
o Micellar formation provides the major vehicle for the transport of lipid digestion products from
the intestinal lumen to the brush border of the mucosal cells for absorption.
o In the mucosal cells, bile salts also stimulate the enzymes required for re-esterification of lipids
forming triacyglycerol, phospholipids and cholesterol ester. Thus bile salts help to maintain the
concentration gradient for passive diffusion of lipids into enterocytes.
o Esterified lipids such as TAGs and cholesterol esters are hydrophobic. So hydrophobic TAGs and
cholesterol esters are coated with a layer of amphipathic phospholipids, free cholesterol and
apolipoprotein B48 to form chylomicron and secreted into the lymphatics, finally entering into
the blood stream via the thoracic duct. In this process, bile salts also stimulate incorporation of
TAG with phospholipids, cholesterol and apoprotein B48 to form chylomicron.
Proper digestion and absorption of dietary fat aided by bile salts also facilitates the absorption of fat
soluble vitamins.
Bile salts stimulate the release of CCK from the intestinal mucosa. CCK stimulates gall bladder
contraction for bile secretion and also increases pancreatic digestive enzymes secretion.
Integration of Metabolism Page |1
Overview of intermediary metabolism
General principles of intermediary metabolism

Metabolism is composed of many coupled, interconnecting pathways. Metabolism consists of energy
yielding and energy requiring reactions. Fuels are degraded and large molecules are constructed step
by step in a series of linked reactions called metabolic pathways. The oxidation of carbon fuels
powers the formation of ATP. An energy currency common to all life forms, adenosine triphosphate
(ATP), links energy-releasing pathways with energy-requiring pathways.
Metabolic pathways fall into three categories –
o Catabolic pathways
 These pathways involve the breakdown of larger molecules and oxidation of fuel molecules.
 Oxidative pathways are exothermic and produce reducing equivalents and ATP.
o Anabolic pathways
 These pathways involve synthesis and formation of larger and more complex compounds
from smaller precursors. Anabolic pathways are endothermic and use up the energy or
reducing power to proceed the reactions.
o Amphibolic pathways
 These pathways are cross-roads or junctions of metabolism, acting as links between the
anabolic and catabolic pathways.
Although there are many metabolic pathways, a limited number of types of reactions and particular
intermediates are common to many pathways. Metabolic pathways contain many recurring motifs
e.g., activated carriers of electrons for fuel oxidation (NAD+/NADH, FAD/FADH2), activated carriers
of electrons for reductive synthesis (NADPH), activated carrier of two-carbon fragments (CoA).
General principles in regulation of metabolic pathways

Complex network of metabolic reactions must be regulated to adjust metabolic activity to the
constantly changing external environments of cells.
Concept of regulation of metabolic pathways at rate-limiting steps

o The metabolic pathway is carried by the series of reaction with several enzymes. However, some
enzymatic reactions determine the continuation of the flow of this pathway. They are called
committed or rate limiting steps. Rate limiting steps are reactions in which a small change of
rate will affect the flux through the whole pathway. Thus, regulation of metabolic pathway is
achieved by control of one or more key reactions or rate limiting steps in the pathway, catalyzed
by regulatory enzymes.
o In human cells, most pathways are interconnected with other pathways and have regulatory
enzymes for every branch points. Regulation usually occurs at the first (committed step) steps of
a pathway or at metabolic branch points.
Many pathways have feedback regulation, i.e., the end product of the pathway controls the rate of
its own synthesis. Feedback regulation many involve inhibition of an early step in the pathway
(feedback inhibition) or regulation of gene transcription.
Metabolism is regulated through control of (1) the amounts of enzymes, (2) catalytic activities of
enzymes, and (3) the accessibility of substrates.
Control of amount of enzymes
o The amount of a particular enzyme depends on both its rate of synthesis and its rate of
degradation. The level of many enzymes is adjusted primarily by a change in the rate of
transcription of the genes encoding them. Such regulation of gene transcription of key enzymes
of metabolic pathway can be influenced by metabolites or hormonal control.
Control of the catalytic activity of enzymes
o The catalytic activity of enzymes is controlled in several ways. These include reversible allosteric
control and reversible covalent modification of key enzymes.
o Reversible allosteric control
 The first reaction in many biosynthetic pathways is allosterically inhibited by the ultimate
product of the pathway, e.g., inhibition of aspartate transcarbamoylase by CTP.
o Reversible covalent modification
 Covalent attachment of a chemical group to an enzyme protein can affect the functional
conformation changes of that enzyme, ultimately the catalytic activity of the enzyme. Most
common form of covalent modification seen in regulation of metabolism is phosphorylation
and dephosphorylation. This type of regulation can be mediated through a variety of protein
kinases and protein phosphatases which in turn may be under the influence of hormonal
signaling cascade. Thus, hormones coordinate metabolic relations between different tissues,
often by regulating the reversible modification of key enzymes. For instance, epinephrine
triggers a signal-transduction cascade in muscle, resulting in phosphorylation and activation
of key enzymes and leading to the rapid degradation of glycogen to glucose, which is then
used to supply ATP for muscle contraction.
o Many reactions in metabolism are controlled by the energy status of the cell, e.g., ATP/ADP ratio
or redox potential of the cell, e.g., NADH/NAD+ ratio.
Control of the accessibility or availability of substrates
o When the intracellular concentration of an enzyme’s substrate is near or below Km, the rate of
reaction depends strongly upon substrate concentration. This is the most immediate regulation
of metabolic pathways.
o Human cells use compartment system to control the access of substrates and activators or
inhibitors to different enzymes. Localization of pathways in separate subcellular compartments
or organelles permits integration and regulation of metabolism.
o Metabolic regulation and flexibility are enhanced by compartmentalization. For example, fatty
acid oxidation takes place in mitochondria, whereas fatty acid synthesis takes place in the
cytoplasm. Compartmentalization segregates opposed reactions.
o Controlling the flux of substrates is an important means of regulating metabolism. Glucose
breakdown can take place in muscles and adipose tissues only if insulin is present since insulin
promotes the entry of glucose into the cell. The transfer of substrates from one compartment of
a cell to another (e.g., from the cytoplasm to mitochondria) can serve as a control point.
Regulation of metabolism in higher eukaryotes is enhanced by the existence of organs with different
metabolic roles. Metabolic specialization of an organ is the result of differential gene expression.
Hormonal regulation in metabolism

 Metabolic pathways and fuel metabolism can be influenced by many hormones such as insulin,
glucagon, catecholamine, glucocorticoids, etc. Hormonal regulation integrates responses in

pathways requiring more than one tissue.
 Hormonal regulation in metabolism can be mediated through –
o Changing the phosphorylation state of enzymes e.g., glucagon and catecholamines via activation
of PKA and insulin via activation of protein phosphatases
o Changing the amount of enzymes by modulating the rate of synthesis of enzyme proteins
(induction or repression of specific gene transcription)
o Changing the concentration of an activator or inhibitor e.g., hormonal control of F 2, 6 BP
concentration which is an important allosteric effector for PFK-1 and fructose 1,6 bisphosphatase
o Changing the availability of substrates, e.g., insulin dependent glucose entry in adipose tissues
and muscles.
Integration of Metabolism
Introduction
 The various anabolic and catabolic pathways by which carbohydrates, lipids and proteins are
processed for energy supply or as precursors for the biosynthesis of compounds required by the cell
for maintenance or growth are closely co-ordinated. This coordination between these metabolic
pathways and metabolites is called integration of metabolism.
 Integration of metabolism is considered at two levels; at the cellular level and at tissues or organ
level.
 Integration of metabolism at cellular level includes the different metabolic pathways of glucose, fatty
acids and amino acids that results in inter-conversion between different biomolecules. The
metabolic processes involving various biomolecules are interconnected through certain branch
compounds which lie at junctions of the major metabolic pathways. Some important branch
compounds are acetyl CoA, pyruvate and glucose 6-phosphate.
 Inter connections between major pathways via such branch compounds make inter-conversions of
major foodstuffs possible. For example, pyruvate, the degradation product of carbohydrates and
glycogenic amino acids can be converted to acetyl CoA, thereby providing its carbons for
biosynthesis of cholesterol, fatty acids or other compound lipids.
 Interrelationships and coordination of various organs is of crucial significance in maintaining
metabolic homeostasis. Metabolic flow between these organs, each of which has a specialized role,
occurs in well defined pathways. For example, following a meal, glucose, amino acids and fatty acids
are directly available from the intestine. Later, when these fuels are exhausted, the liver and adipose
tissue supply various organs with fuel molecules: liver provides glucose and ketone bodies and
adipose tissue provides fatty acids.
 Biosynthesis and degradation pathways are almost always distinct. Biosynthetic pathway
(endergonic) is always coupled with degradation pathway (exergonic).
Integration of Metabolism P a g e |5
Strategies of metabolism
 Formation of ATP
 Generation of reducing power
 Formation of building blocks for biosynthesis
Formation of ATP
 During fed state, ATP is obtained from
o Glycolysis
o Oxidative decarboxylation of pyruvate
o Complete oxidation of acetyl CoA through citric acid cycle.
 During fasting state, ATP is obtained from

o Fatty acid oxidation
o Ketone bodies oxidation
o Amino acid oxidation by glutamate dehydrogenase
 Most of the energy derived from fuel oxidation is obtained as reducing equivalents, NADH, FADH2;
only a few is directly generated as ATP during the fuel oxidation process by substrate level oxidative
phosphorylation.
 ATP is produced by respiratory chain linked oxidative phosphorylation through oxidation of high
energy electrons via respiratory chain.
Generation of reducing power

 Most of the biosynthetic pathways require reducing power as well as ATP.
 NADPH is the major electron donor in reductive synthesis.
 NADPH is supplied mainly by pentose phosphate pathway.
 Other sources of NADPH include malic enzyme and cytosolic isocitrate dehydrogenase reactions.
Formation of building blocks for biosynthesis

 Biomolecules are constructed from a relatively small set of building blocks.
 Small carbon skeleton compounds (amphibolic metabolites) serve as building blocks, e.g.,
o Acetyl CoA, common intermediate in breakdown of most fuels, supplies a two-carbon unit in a
wide variety of biosynthesis such as fatty acids, cholesterol synthesis.
o Formation of α-ketoacid for non-essential amino acid synthesis or glucose synthesis via
gluconeogenesis.
Metabolic regulation
 Anabolism and catabolism must be precisely co-ordinated. Metabolic pathways are regulated
according to metabolic needs and changes in the body by the following mechanisms –
o Allosteric regulation
o Control of enzyme synthesis (regulation of
gene expression) and degradation
o Compartmentalization
o Organ specialization
Metabolic junction or cross-roads molecules

 The metabolic junction is the reaction that connects
the molecules from catabolic to anabolic reaction
and from one metabolism to another.
 Three important metabolic cross-road molecules are glucose 6-phosphate, pyruvate and acetyl CoA.
Common metabolic pool

 Common metabolic pool is composed of short chain carbon compounds (amphibolic intermediates)
which are derived from catabolism of carbohydrate, fat and protein and can be used for synthesis of
carbohydrate, fat and non-essential amino acids.
 Amphibolic intermediates
o Glyceraldehyde 3-phosphate
o Pyruvate
o α-ketoglutarate
o Oxaloacetate
o Fumarate
o Succinyl CoA
o Acetyl CoA
Metabolic fate of pyruvate

 Sources
o Aerobic glycolysis (most common source)

 Aerobic oxidation of one mole of glucose provides 2 moles of pyruvate.
o Catabolism of some amino acids – glycine, serine, alanine, cysteine, threonine, tryptophan
o Oxidation of lactate to pyruvate
 Lactate transported in circulation is
taken up by liver for gluconeogenesis. In
the liver, lactate is oxidized by the action
of lactate dehydrogenase to pyruvate
which then enters into gluconeogenesis.
 Fates of pyruvate
o Formation of acetyl CoA by oxidative

decarboxylation
 Oxidative decarboxylation of pyruvate to acetyl CoA by pyruvate dehydrogenase enzyme
complex provides route for complete oxidation of glucose as well as building blocks for
lipogenesis.
o Formation of oxaloacetate by carboxylation
 Carboxylation of pyruvate to oxaloacetate by the action of pyruvate carboxylase leads to
gluconeogenesis or replenishes CAC intermediates for complete oxidation of acetyl CoA.
o Transamination of pyruvate to alanine
 Transamination of pyruvate to alanine by alanine transaminase (ALT) provides linkage
between α-keto acids and amino acids; thus it is important for catabolism of alanine as well as
biosynthesis of non-essential amino acids.
o Reduction of pyruvate to lactate

 During anaerobic glycolysis in RBC and skeletal muscles (O2 insufficiency during strenuous
exercise), pyruvate is reduced to lactate by lactate dehydrogenase. This process allows
regeneration of NAD+ for glyceraldehyde 3-phosphate dehydrogenase to proceed glycolysis.
o Fermentation to ethanol (in yeast)
Metabolic fates of acetyl CoA

 Sources
o Oxidative decarboxylation of pyruvate (from glucose oxidation)

 Acetyl CoA can be derived from oxidative decarboxylation of pyruvate which is the end
product of glucose oxidation.
o Fatty acid β-oxidation
o Oxidation of ketone bodies
 Acetyl CoA can be derived from ketone bodies oxidation in extra-hepatic tissues during
prolong starvation.
o From catabolism of ketogenic amino acids – leucine, lysine, phenylalanine, isoleucine, tyrosine
and tryptophan
 Fates of acetyl CoA
o Complete oxidation via CAC for energy generation

o Precursor for fatty acid synthesis (lipogenesis)
o Precursor for cholesterol synthesis
o Ketone bodies formation in liver during fasting, starvation
o Acetylcholine synthesis
o Acetyl group donor in acetylation reaction
Glyceraldehyde 3-phosphate
 It is an intermediate
product of glycolysis.
 It can be oxidized to CO2
and H2O through glycolysis
and TCA cycle.
 It can be utilized for
formation of glycerol 3-
phosphate for esterification
of fatty acids to form TAG
and glycerophospholipids.
Oxaloacetate, α-ketoglutarate, fumarate and succinyl CoA

 They are CAC intermediates and involve in inter-conversion of carbohydrate, fat and amino acids
through these four-carbon molecules. They are regarded as amphibolic intermediates.

 Oxaloacetate
o Oxaloacetate is derived from carboxylation of pyruvate by pyruvate carboxylase in

mitochondria. It is required as starting material for complete oxidation of acetyl CoA in CAC.
o It also provides the carbon skeleton for gluconeogenesis as well as non-essential amino acid
(alanine) synthesis via transamination.
 Succinyl CoA
o It is formed from α-KG in CAC or from catabolism of branch-chain amino acids (valine, isoleucine)
or from oxidation of odd number carbon fatty acids (propionyl CoA  methyl malonyl CoA 
succinyl CoA).
o It is oxidized via CAC to provide energy. It can be utilized in heme synthesis and can also provide
carbon skeleton for gluconeogenesis.
Metabolic profile of organs

 Each tissue of the human body has a unique
metabolic profile, reflected in its metabolic
activity and specialized function.
 Due to different metabolic functions and
capabilities of different tissues and organs,
integration of metabolism at tissue or organ
level includes the inter-relationship of
different tissues and organs to meet
metabolic demands for the whole body.
Metabolic profile of brain

 Brain tissue has a remarkably high respiration rate. For instance, the human brain constitutes only
~2% of the adult body mass but is responsible for ~20% of its resting O2 consumption. This
consumption, moreover, is independent of the state of mental activity.
 Most of the brain’s energy production serves to power Na+-K+–ATPase which maintains the
membrane potential required for nerve impulse transmission and propagation of nerve impulse.
 Brain is an organ with no significant fuel reserves—no glycogen, expendable protein, or fat.
Normally, the brain uses only glucose as a fuel and is totally dependent on the blood for a
continuous, incoming supply. Fatty acids cannot be transported across the blood-brain barrier.
 Glucose entry into brain cells is via GLUT-3. Interruption of glucose supply for even brief periods of
time (as in a stroke) can lead to irreversible losses in brain function.
Integration of Metabolism P a g e | 10
 During prolonged fasting or starvation, brain adapts to use ketone bodies as source of fuel,
converting it to acetyl-CoA for energy production via CAC. The brain’s other potential source of fuel
during starvation is glucose obtained from gluconeogenesis in the liver. The adaptation of the brain
to use ketones bodies from fat spares protein from degradation until lipid reserves are exhausted.
Metabolic profile of muscles

 Skeletal muscle can use free fatty acids,
ketone bodies, or glucose as fuel,
depending on the degree of muscular
activity.
 Rested, well-fed muscle, in contrast to
brain, synthesizes a glycogen store
comprising 1 to 2% of its mass. The
glycogen serves muscle as a readily
available fuel depot since it can be
rapidly converted to G6P for entry into
glycolysis.
 In resting muscle, the primary fuels are
free fatty acids in circulation mobilized from adipose tissues or derived from plasma lipoproteins, are
the major fuel, meeting 85% of the energy needs.
 Moderately active muscle uses blood glucose in addition to fatty acids and ketone bodies. Glucose is
the preferred fuel for bursts of activity.
 In maximally active fast-twitch muscles, the demand for ATP is so great that the blood flow cannot
provide O2 and fuels fast enough to supply sufficient ATP by aerobic respiration alone. Under these
conditions, stored muscle glycogen is broken down to glucose 6-phosphate then oxidized to lactate
by anaerobic glycolysis. Formed lactate is transported to the liver via circulation and converted to
glucose (Cori cycle or lactic acid cycle).
 The relatively small amount of glycogen (about 1% of the total weight of skeletal muscle) limits the
amount of energy available from glycolysis during all-out exertion. Moreover, the accumulation of
lactate and consequent decrease in pH in maximally active muscles reduces their efficiency. Skeletal
muscle, however, contains another source of ATP, phosphocreatine which can rapidly regenerate
ATP from ADP by the creatine kinase reaction.
 Muscle phosphocreatine can generate enough ATP to power about 4 seconds of exertion. During
strenuous exertion, such as a 100-meter sprint, once the phosphocreatine is depleted, muscle relies
solely on its glycogen reserves, making the ATP for contraction via glycolysis.
 Muscle cannot export glucose because it lacks glucose 6-phosphatase. Nevertheless, muscle serves
the body as an energy reservoir because, during the fasting state, its proteins are degraded to amino
acids, many of which are converted to pyruvate, which in turn, is transaminated to alanine. The
alanine is then exported via the bloodstream to the liver, which transaminates it back to pyruvate, a
glucose precursor. This process is known as the glucose–alanine cycle.
Metabolic profile of cardiac muscles

 Heart muscle differs from skeletal muscle in that it is continuously active in a regular rhythm of
contraction and relaxation and it has a completely aerobic metabolism at all times. Heart functions
as a completely aerobic organ and it is very rich in mitochondria; roughly half the cytoplasmic volume
of heart muscle cells is occupied by mitochondria.
 Under normal working conditions, the heart prefers fatty acids as fuel. Heart tissue has minimal
energy reserves: a small amount of phosphocreatine and limited quantities of glycogen. As a result,
the heart must be continually nourished with oxygen and free fatty acids, glucose, or ketone bodies
as fuel.
 Because the heart is normally aerobic and obtains its energy from aerobic oxidation, the failure of O2
to reach part of the heart muscle when the blood vessels are blocked by lipid deposits
(atherosclerosis) or blood clots can cause ischemic heart disease and myocardial infarction.
Metabolic profile of adipose tissues

 White adipose tissue is widely
distributed in the body: under the
skin, around the deep blood vessels,
and in the abdominal cavity. It is
specialized for lipogenesis,
esterification of fatty acids to form
TAG and serves as enormous
reservoir of metabolic fuel.
 Adipocytes are spherical cells, completely filled with a single large lipid (TAG) droplet that constitutes
about 65% of the cell mass. Adipocytes need glucose for the synthesis of TAG.
 During periods of high carbohydrate intake, adipose tissue can convert glucose (via pyruvate and
acetyl-CoA) to fatty acids, convert the fatty acids to TAGs, and store the TAGs as large fat globules.
Adipocytes also store fatty acids derived from plasma lipoproteins.
 Glucose plays a pivotal role for adipocytes. If glucose levels are adequate, glycerol 3-phosphate is
formed in glycolysis and the free fatty acids liberated in triacylglycerol breakdown are re-esterified to
triacylglycerols. However, if glucose levels are low, glycerol-3-phosphate is not enough for re-
esterification and free fatty acids are released to the blood stream. Thus, during fasting state,
adipose tissues provide FFAs to be used as fuel source in the tissues. Glycerol exported into the
blood stream is used as precursor for gluconeogenesis in the liver.
 In addition to its central function as a fuel depot, adipose tissue plays an important role as an
endocrine organ, producing and releasing hormones that signal the state of energy reserves and
coordinate metabolism of fats and carbohydrates throughout the body.
 Brown adipose tissues is specialized to generate heat; non-shivering thermogenesis.
Metabolic profile of kidney

 Kidney requires a large amount of energy for reabsorption of substances in renal tubules (to drive
active transport). Kidney uses glucose, fatty acids and ketone bodies as metabolic fuel.
 Kidney may contribute as much as half of blood glucose by gluconeogenesis during prolong
starvation.
Metabolic profile of liver

 Metabolic activities of liver are essential for providing fuels to the brain, muscle and other peripheral
tissues.
 Most compounds absorbed by the intestine first pass through the liver, which is thus able to regulate
the level of many metabolites in the blood.
Carbohydrate metabolism in liver

 Major function of liver in carbohydrate metabolism is blood glucose homeostasis. Liver acts as a
glucostat, maintaining the normal blood glucose level at all time. Liver metabolizes two-third of
glucose and all remaining monosaccharides from the blood.
 Glucose is taken up by hepatocytes via insulin independent GLUT-2 transporter. Glucose is then
converted into glucose 6-phosphate by glucokinase. Since liver glucokinase has a much higher Km
for glucose than do hexokinases in other cells, it allows hepatocytes to continue phosphorylation of
glucose when blood glucose level rises and it also ensures glucose uptake is minimal when blood
glucose is low, sparing glucose for other tissues.
 Glucose 6-phosphate is at the crossroads of
carbohydrate metabolism in the liver. It may

take any of several major metabolic routes,
depending on the metabolic needs of the body.
 During the fed state when the blood glucose
level is increased;
o Excess glucose 6-phosphate is stored as
glycogen.
o Some are metabolized by glycolysis and
pyruvate dehydrogenase reaction and
acetyl-CoA so formed can be oxidized for
ATP production by CAC for immediate
energy needs. (Normally, however, fatty
acids are the preferred fuel for ATP
production in hepatocytes.)
o Acetyl-CoA can also serve as the precursor of fatty acids, which are incorporated into TAGs and
phospholipids, and of cholesterol. Much of the lipid synthesized in the liver is transported to
other tissues by blood lipoproteins.
o Alternatively, glucose 6-phosphate can enter HMS pathway, yielding both reducing power
(NADPH), needed for the reductive biosynthesis and ribose 5-phosphate, a precursor for
nucleotide biosynthesis.
 In the fasting state, when the blood glucose level is decreased;
o Glucose 6-phosphate is formed from mobilization of glycogen stores or gluconeogenesis from

non-carbohydrate precursors such as lactate, glycerol, glucogenic amino acids and it is then
dephosphorylated by glucose 6-phosphatase to
yield free glucose which is exported to replenish
blood glucose.
o These processes are stimulated by glucagon,
epinephrine and glucocorticoids.
Lipid metabolism in liver

 In the fed state, liver synthesizes fatty acids from
excess glucose and these endogenously synthesized

fatty acids as well as uptake of FFAs derived from
plasma lipoproteins are esterified to form TAG,
phospholipid and cholesterol ester. TAG synthesized is
not stored in the liver but exported to tissues via VLDL.
 Liver synthesizes cholesterol from acetyl CoA and also receives cholesterol from plasma lipoproteins
which are then exported via plasma lipoproteins (VLDL  IDL  LDL) to the extra-hepatic tissues.
Some cholesterol is used for bile acids synthesis and secreted into the bile. Bile acids are essential
for dietary lipid digestion and absorption and they also serve to solubilize excess cholesterol to be
excreted into the bile.
 Under most circumstances, fatty acids are the primary oxidative fuel in the liver.
 Liver synthesizes apolipoproteins which are required for plasma lipoprotein metabolism e.g., apo C
and apo E needed for chylomicron metabolism are exported to the blood stream bound to HDL. Liver
also produces HDL to scavenge excess cholesterol from extra-hepatic tissues for excretion into bile.
 In prolonged starvation, liver synthesizes ketone bodies from excess acetyl CoA derived from β
oxidation. These ketone bodies serve as efficient energy source for extra-hepatic tissues as an
alternate fuel to spare the use of amino acids from protein degradation.
Protein metabolism in liver

 Amino acids that enter the liver follow several
important metabolic routes. They are precursors for

protein synthesis. Most plasma proteins except γ
globulins are synthesized by the liver.
 Alternatively, amino acids pass in the bloodstream to
other organs to be used in the synthesis of tissue

proteins. Other amino acids are precursors in the
biosynthesis of nucleotides, hormones and other
nitrogenous compounds in the liver and other
tissues.
 Liver serves as a labile protein storage organ.
Hepatic protein turnover is highly regulated which

allows metabolic pathways to adapt to physiologic
changes. Proteins to be degraded enter the liver cells
by pinocytosis and degraded by lysosomal system.
 Amino acids not needed as biosynthetic precursors
are transaminated, deaminated and degraded to

yield amphibolic intermediates and amino groups
are released as ammonia which is then converted to
excretory product urea.
 Liver is the site of removal of ammonia which is derived from amino acid catabolism or from portal
blood, into less toxic product urea for urinary excretion. Ammonia derived from amino acid
catabolism in tissues is transported to the liver in the form of alanine or glutamine.
 Carbon skeletons from amino acid degradation can be oxidized via CAC for energy production, or
can be converted to glucose via gluconeogenesis and then glycogen for storage, or it can be
converted to lipids for storage during fed state. They can also be utilized for non-essential amino
acid synthesis.
 Carbon skeletons from amino acid degradation can be used as precursors for gluconeogenesis
(glucogenic amino acids) and ketogenesis (in case of ketogenic amino acids) during fasting and
prolong starvation.
Metabolic changes in fed state, fasting state and prolong starvation
Metabolic changes in well-fed state

Well-fed state or absorptive state – within 2 to 6
 After meal, glucose and amino acids are
hours after meal
transported from the intestine to the blood. The Fasting state or post-absorptive state – 10 to 12
dietary lipids are packaged into chylomicrons and hours after having last meal
transported to the blood via the lymphatics. Starvation – fasting state for days to month
 During fed state, glucose is the major fuel for oxidation in most tissues. Glucose uptake and
oxidation is enhanced by insulin secreted from β cells of pancreas in response to elevated blood
glucose level during fed state. Insulin signals the fed state, stimulating the storage of fuels and
anabolic processes in the body.
 In muscles and adipose tissues, insulin stimulates GLUT-4 expression on plasma membrane, thereby
enhancing glucose uptake by the cells. It also promotes glucose utilization in these tissues.
 Glucose is taken up by hepatocytes via insulin independent GLUT-2 transporter. Glucose is then
converted into glucose 6-phosphate by glucokinase. Since liver glucokinase has high Km for glucose,
it continues phosphorylation of glucose when blood glucose level rises, allowing more glucose entry
into the liver. Glucose 6-phosphate in excess of energy requirement is used for glycogen synthesis in
liver and muscles. Insulin stimulates glycogen synthase and inhibits glycogen phosphorylase in liver
and muscles; thus promoting glycogen synthesis and suppressing glycogenolysis. Insulin also
inhibits gluconeogenesis in the liver. In the liver, some of the excess glucose may be used for
lipogenesis and subsequently TAG synthesis.
 In adipose tissues, insulin stimulates glucose uptake, oxidation and conversion to fatty acids and
their esterification to TAG. It also inhibits lipolysis, limiting fatty acids mobilization from adipose
tissues.
 Dietary lipids are transported in the blood stream by chylomicrons. TAGs in the chylomicrons are
hydrolyzed by lipoprotein lipase on capillary endothelium, releasing FFAs and glycerol. Lipoprotein
lipase synthesis and expression are activated by insulin. FFAs are taken up by the adjacent tissues
whereas glycerol is taken up by the liver and used for either gluconeogenesis and glycogen synthesis
or lipogenesis. Fatty acids remaining in the blood stream and TAG-depleted chylomicron remnants
are taken up by the liver. In the liver, fatty acids are re-esterified to TAG and remaining lipids derived
from chylomicron remnant are exported together with endogenously synthesized lipids via VLDL into
the blood stream.
 Under normal conditions, rate of tissues protein catabolism is relatively constant throughout the day
except in conditions such as cachexia associated with cancer and hyper-metabolic states in some
diseases. There is increased net protein synthesis during the fed state due to increased availability
of amino acids, increased amino acids uptake and cellular protein synthesis in tissues in response to
anabolic action of insulin. Protein synthesis is an energy requiring process, consuming up to 20% of
resting energy expenditure in fed state but only 9% in the fasting state.
Metabolic changes during fasting

 Blood glucose level begins to drop several hours after meal, leading to decrease in insulin secretion
and rise in glucagon secretion (increased glucagon/insulin ratio).
 The main target organ of glucagon is the liver. Glucagon inhibits glycogen synthesis and activates
glycogen breakdown in the liver. Resulting glucose 6-phosphate is hydrolyzed by glucose 6-
phosphatase and glucose is released to bloodstream. The liver glycogen is only capable of
maintaining the blood glucose concentration for 12 to 16 hours of fasting. In addition, glucagon
blocks glycolysis and stimulates

gluconeogenesis generating
glucose from substrates such as
lactate, amino acids and glycerol.
 Increased glucagon/insulin ratio
suppresses lipogenesis, lipoprotein
lipase expression and activity and
activates hormone sensitive lipase,
enhancing FFAs and glycerol
release. Released glycerol is used
as glucogenic substrate for
gluconeogenesis in liver whereas
FFAs released to the blood stream
can be used by liver, skeletal and
cardiac muscles as their preferred
fuel, sparing glucose. Besides,
decreased insulin level diminishes glucose entry and utilization in peripheral tissues (muscles, adipose
tissues) and shifts the fuel use from glucose to fatty acids.
 In the fasting state, there is considerable protein degradation from skeletal muscle, providing
amino acids that are used by the liver for gluconeogenesis. Muscle glycogen cannot directly
contribute plasma glucose since muscles lack glucose 6-phosphatase. It can only be used for energy
generation in muscles itself. Pyruvate derived from glycolysis of muscle glycogen is accumulated in
muscle cells due to inhibition of pyruvate dehydrogenase by acetyl CoA from fatty acid oxidation.
Pyruvate is then transaminated to alanine, at the expense of amino acids derived from muscle
protein degradation. Alanine is transported to the liver where it can be used as glucogenic substrate
for gluconeogenesis. This glucose-alanine cycle provides an indirect way of utilizing muscle
glycogen to maintain blood glucose in the fasting state.
 Increased fatty acid oxidation in liver forms more acetyl CoA than it can be oxidized and excess
acetyl CoA are used to synthesize ketone bodies which are major fuels for skeletal and heart
muscles. Ketone bodies can provide up to 20% of brain’s energy needs.
 Blood glucose level is kept above 80 mg/dL (4.4 mmol/L) during early fasting state which decreases
as fasting state is prolonged but still must be kept above 40mg/dL (2.2 mmol/L). As the fasting state
is prolonged, plasma FFAs level increases and plasma ketone bodies level increases markedly.
Metabolic changes during prolong starvation

 Prolonged starvation is a state of more than 24 hours of fasting which may extend to several days
and months.
 Even under starvation conditions, the blood glucose level must be maintained above 40 mg/dl (2.2
mmol/L). The first priority of metabolism in starvation is to provide sufficient glucose to glucose
dependent tissues such as brain and RBCs. This is achieved by –
o Repressing glucose uptake and utilization by other tissues due to increased glucagon/insulin
ratio and elevated catecholamines and glucocorticoids in response to metabolic stress
o Elevated plasma FFA level by enhancing lipolysis, shifting fuel use from glucose to fatty acids
o Increased gluconeogenesis in the liver and kidney by using glycerol, amino acids from protein
breakdown, pyruvate and lactate as glucogenic substrates.
 The processes which take place in early starvation cannot go on indefinitely, because although
gluconeogenesis provides glucose efficiently for the body’s energy requirements, it will soon deplete
the substantial proportion of body protein and death can ensue when 30 to 50% of the body protein
is lost. Adjustments to metabolism are made after 24 to 48 hr, which conserve body protein.
 Second priority of metabolism in starvation is to preserve protein. Conservation of body proteins is
accomplished by reduction in glucose production by gluconeogenesis. In prolonged starvation,
glucose may represent less than 10% of whole body energy-yielding metabolism.
 During prolonged fasting, lipolysis in adipose tissues continues

to release fatty acids and glycerol. These fatty acids serve as the
major source of fuel for the body. The glycerol is converted to
glucose while fatty acids are oxidized by muscle and in the liver
where they are converted into ketone bodies.
 Increased fatty acid oxidation in liver forms more acetyl CoA
than it can be oxidized and excess acetyl CoA are converted to
ketone bodies which become major fuels for skeletal and heart
muscles. The synthesis of ketone bodies from acetyl-CoA
increases markedly as the plasma FFA level increases with ongoing starvation period, producing the
state of ketosis. Ketosis is a metabolic adaptation to starvation to minimize protein degradation
during prolonged starvation.
 As the supply of ketone bodies increases and the supply of glucose diminishes, the brain reduces its
utilization of glucose and begins to consume appreciable amounts of ketone bodies in place of
glucose. After 3 days of starvation, about a third of energy needs of brain are provided by ketone
bodies and after several weeks of starvation, ketone bodies become the major fuel of the brain.
 Thus, during the first few days of starvation, there is a rapid breakdown of muscle protein, providing
amino acids for gluconeogenesis. However, in prolonged starvation, less muscle is degraded than in
the first days of starvation due to decreased need of glucose as a fuel for brain which has begun
using ketone bodies as a source of energy.
 Because of these adaptive mechanisms the duration of starvation of the adult human is determined
by the size of TAG depot. When TAG stores are completely exhausted, body proteins become the
only source of energy. At that time, the protein stores once again enter a stage of rapid depletion.
Because proteins are also essential for the maintenance of cellular function, death ordinarily ensues
when the proteins of the body have been depleted to about half their normal level.
Diabetes mellitus
Diabetes is a disorder of fuel metabolism characterized by hyperglycemia and vascular damage. It is
caused by a relative or absolute deficiency of insulin action.
Classification of diabetes
Type 1 diabetes mellitus – autoimmune destruction of β cells
Type 2 diabetes mellitus – insulin resistance and β cells failure.
Gestational diabetes
Other types (secondary diabetes)
 Genetic defects of β cells
 Endocrine diseases (acromegaly, Cushing syndrome)
 Drugs and chemical induced diabetes
 Diabetes accompanying with genetic diseases (e.g., Down’s syndrome)
Type 1 diabetes mellitus

Type 1 diabetes typically starts in childhood or
adolescence. It is caused by insulin deficiency due to
autoimmune destruction of β cells. Without
endogenous insulin production, patients depend on
insulin injections for life. Persons with type 1 DM are
prone to development of ketoacidosis.
Type 2 diabetes usually develops in obese patients
especially affecting middle-aged and older individuals.
It is caused by either reduced insulin secretion or
insulin resistance of the target tissues or a combination of both.
Type 2 diabetes has a strong hereditary component (multigenic disorder). Two important risk factors
are family history and obesity which is closely linked to insulin resistance.
Insulin resistance
 Insulin resistance is a condition in which a
given dose of insulin produces a less than
expected response in the cell. Insulin
resistance is a major factor in the
development of type 2 diabetes mellitus.
 The most important cause of insulin resistance
is defective insulin signaling. Within a cell,
insulin resistance may be caused by defects at
several levels which could be defective insulin binding to receptor or anti-receptor autoantibodies or

most importantly, defects in the insulin signaling pathways.
 Peripheral insulin resistance particularly in

muscle is induced by the presence of excess
fatty acids. Fatty acids can also exert a direct
harmful effect on the β cell. The elevated fatty
acids concentrations contribute to insulin
resistance by inhibiting peripheral glucose
disposal, enhancing hepatic glucose output and
damaging β cell function.
 Mildly insulin resistant individuals have normal plasma glucose concentration but have
hyperinsulinemia since more insulin is required to produce normal insulin effect. As the resistance
becomes more severe, plasma glucose concentration increases and in later stages, insulin secretion
decreases as a result of β cells exhaustion and failure.
 Clinically, it is seen first as impaired glucose tolerance (IGT) and subsequently type 2 diabetes. Insulin
resistance is common in obesity which is a major risk factor for the development of type 2 diabetes.
 Determination of insulin resistance can be done by calculating HOMA IR index.
Metabolic changes in diabetes mellitus

The metabolic abnormalities of diabetes are the result of insulin resistance expressed primarily in
liver, muscle, and adipose tissue.
Hyperglycemia is caused by increased hepatic glucose production via glycogenolysis and
gluconeogenesis, combined with diminished peripheral use. Due to insulin deficiency or insulin
resistance, liver generates rather than utilizes glucose whereas there is less glucose uptake in
peripheral tissues like adipocytes and muscles.
In adipocytes, there is excessive lipolysis which leads to elevated plasma FFA level. Oversupply of FFA
reduces glucose utilization in liver and skeletal muscles, e.g., ATP generated by fatty acid oxidation
reduces glycolysis by inhibiting PFK-1.
Excess acetyl CoA derived from fatty

acid oxidation in liver enters
ketogenesis in case of insulin
deficiency in type 1 diabetes mellitus
or severe uncontrolled diabetes
mellitus. Ketosis is usually minimal or
absent in patients with type 2 DM
because the presence of insulin, even
in the presence of insulin resistance,
restrains hepatic ketogenesis.
In the liver, FFAs are converted to
TAGs, which are packaged and
secreted in VLDL. Because lipoprotein
degradation catalyzed by lipoprotein
lipase in adipose tissue is low in diabetics, the plasma chylomicron and VLDL levels are elevated,
resulting in hypertriacylglyceridemia. Elevated VLDL and plasma triglycerides contribute to the
accelerated development of atherosclerosis and coronary heart disease in diabetes.
Acute complications of diabetes mellitus
The acute complications of diabetes include diabetic ketoacidosis in type 1 diabetes and non-ketotic
hyperosmolar coma in elderly patients with type 2 diabetes. Both are associated with excessive
glycosuria with osmotic diuresis resulting in severe dehydration.
Ketosis results from increased mobilization of fatty acids (FAs) from adipose tissue, combined with
accelerated hepatic FA β-oxidation and synthesis of 3-hydroxybutyrate and acetoacetate.
Metabolic and clinical abnormalities in Diabetic Ketoacidosis
Late complications of diabetes mellitus

Morbidity associated with long
standing diabetes results from
vascular complications caused by
accelerated atherosclerosis. Diabetic
complications include –
 Microvascular complications such
as retinopathy, nephropathay and
neuropathy
 Macrovascular complications such
as coronary heart disease, stroke
and peripheral arterial diseases
All these complications are the consequences of the metabolic derangements,

mainly hyperglycemia.
Proposed mechanisms linking hyperglycemia to the complications of long
standing diabetes are non- increased activity of polyol pathway, enzymatic
glycosylation and oxidative stress.
Increased activity of polyol pathway
 Aldose reductase (AR) which is the key enzyme of polyol pathway has a
high Km for glucose. It becomes active in hyperglycemia and leads to
increased sorbitol formation.
 Since sorbitol is osmotically active, it increases intracellular osmolarity and influx of water leading
to osmotic cell injury e.g., development of diabetic cataract in lens.
 High concentration of sorbitol in nerve tissues decreases the cellular uptake of myoinositol
resulting in decreased phosphor-inositide metabolism, DAG, PKC and Na+/K+ ATPase activity. This
mechanism is responsible for damage to Schwann cells and to pericytes of retinal capillaries with
resultant peripheral neuropathy and retinal microaneurysms respectively.
Non-enzymatic glycosylation
 This is the process in which glucose attaches to the terminal amino groups of proteins non-
enzymatically. It interferes with normal function or turnover of proteins.
 First it forms labile or reversible form known as Schiff base which transforms into more stable
Amadori type early glycation products which are still reversible. Then it undergoes slow chemical
rearrangements such as oxidation, dehydration to form irreversible advanced glycation end
products (AGEs). Formation and accumulation of AGE lead to development of microvascular
diseases and atherosclerosis.
Oxidative stress
 Hyperglycemia increases the flux of reducing equivalents into electron transport chain, causing
increased electrochemical potential difference across the inner membrane and consequently
increased ROS generation by respiratory chain. Glucose can also undergo auto-oxidation in the
presence of transitional metal ions (Cu+ and Fe2+) and generates ROS.
 ROS interferes with signaling cascades e.g., activation of PKC. It also impairs endothelium
dependent relaxation of vascular smooth muscle cells. Thus, ROS production is one of the
primary causes of long-term diabetic complications.
Laboratory assessment of diabetes mellitus

Determination of plasma glucose concentration
 Random blood glucose level
 Fasting blood glucose level
 2 hours post-prandial blood glucose level

Oral blood glucose tolerance test

 Recommended test for all individuals
whose fasting plasma glucose falls
into impaired fasting glucose
category (6.1 – 7.0 mmol/L or 110 – 126
mg/dL)
Determination of glycated hemoglobin
(HbA1c) level for glycemic control
Urine sugar determination (Benedict’s
test) – not a standard diagnostic test for
diabetes
Screening tests for complications
(periodic assessment)
 Urinary albumin excretion
measurement for microalbuminuria,
determination of serum urea,
creatinine level to detect diabetic nephropathy
 Plasma lipid profile
Other periodic assessments
 Eye examination
 Neurologic examination
 ECG, etc.
Gastroenterology Page |1
Gastroenterology
The well-orchestrated functioning of many organs of the gastrointestinal system is required for
efficient digestion and absorption of the essential nutrients in foods.
Gastrointestinal system consists of gastrointestinal tract (oral cavity, esophagus, stomach, small
intestine, large intestine) and its associated glands or organs (salivary glands, liver, pancreas, gall
bladder).
Digestion is the process by which food is broken down into components simple enough to be
absorbed in the intestine. Digestion process consists of mechanical and chemical changes.
Mechanical digestion is the break-down of foodstuff by motility of GI tract. Chemical digestion is
carried out by hydrolytic action of digestive enzymes.
Absorption is the uptake of the products of digestion by intestinal cells (enterocytes) and their
delivery to blood or lymph.
Digestion is a sequential, ordered series of processes which involve –
1. Lubrication and mechanical homogenization of food with fluids secreted by the glands of the GI
tract.
2. Secretion of digestive enzymes that hydrolyze macromolecules to oligomers, dimers, or
monomers.
3. Secretion of electrolytes, hydrogen ions, or bicarbonate to provide an appropriate environment
for optimal enzymatic digestion.
4. Secretion of bile acids to solubilize dietary lipids, thus facilitating their digestion and absorption.
5. Further hydrolysis of nutrient oligomers and dimers by membrane bound enzymes.
6. Specific transport of digested material and electrolytes into the enterocytes and then to the
blood or lymph.
Digestion and absorption of dietary carbohydrates
Digestion of dietary carbohydrates

Dietary carbohydrates – mainly polysaccharides, starch (amylose, amylopectin), and glycogen,
disaccharides (sucrose and lactose), monosaccharides (glucose, galactose, fructose)
Only monosaccharide form is absorbed by the GI tract.
Digestion in the oral cavity

 Carbohydrate digestion begins in the oral cavity by mechanical digestion and by the action of
salivary α amylase.
 Salivary glands secrete saliva into the oral cavity. Saliva contains salivary α amylase, water and other
organic, inorganic ions. Hydration is required for lubrication, homogenization of foods and also for
the action of amylase.
 Salivary α amylase (optimal pH – 6.8) is

an endoglycosidase which randomly
hydrolyzes internal α 1-4 glycosidic
bonds yielding dextrins.
Digestion in the stomach

 No digestion occurs since salivary
amylase is inactivated by the acidic pH
of the stomach.
Digestion in the intestine

 Pancreas secretes pancreatic juice into
the duodenum via pancreatic duct. It
contains digestive enzymes including pancreatic α amylase and bicarbonate
ions (HCO3¯).
 Bicarbonate ions neutralize the acidic chyme entering into the duodenum
from the stomach and also provide optimal pH for the action of pancreatic α
amylase.
 Pancreatic α amylase is a α 1-4 endoglycosidase which hydrolyzes starch and
glycogen forming disaccharide (maltose), trisaccharide (maltotriose), non-
branching oligosaccharides (dextrin) and branching oligosaccharides (α-limit
dextrins).
 Both salivary and pancreatic α amylase do not act on α 1-6 glycosidic bonds,
terminal α 1-4 glycosidic bonds and α 1-4 bonds next to branch point.
 Oligosaccharides, disaccharides and trisaccharides are further digested by
brush border enzymes (intestinal mucosal membrane bound glycosidases).
 Brush border enzymes include oligosaccharidases, disaccharidases such as
maltase, isomaltase, lactase, sucrase and trehelase.
o Oligosaccharidase (exo α 1-4 glycosidase) removes single glucose residue
from oligosaccharides by hydrolyzing the terminal α 1-4 bonds and also
digests α limit dextrin down to isomaltose.
o Isomaltase (α 1-6 glycosidase, α limit dextrinase) hydrolyzes α 1-6 bonds of
isomaltose and α limit dextrin.
o Maltase (α 1-4 glycosidase) hydrolyzes maltose and maltotriose into
glucose.
o Lactase (β 1-4 galactosidase) hydrolyzes lactose into glucose and
galactose.
o Sucrase (α 1-2 glycosidase) hydrolyzes sucrose into glucose and fructose.

o Trehelase (α 1-1 glycosidase) hydrolyzes trehelose into two molecules of glucose.
Absorption of dietary carbohydrates

Digestive end products of dietary carbohydrates are
monosaccharides primarily glucose, galactose and fructose.
Absorption of monosaccharides is by means of specific
carrier-mediated mechanisms.
At least two carrier-mediated transport mechanisms for
monosaccharides –
o Na+ dependent co-transporter
o Na+ independent facilitated transporter
In addition to carrier-mediated transport mechanisms, all the
monosaccharides can be absorbed by simple diffusion
process but this is extremely low.
Carbohydrate absorption occurs in the jejunum and upper part of small intestine.
Sodium-dependent glucose transporter (SGLT-1) Molecular configuration of sugar
o Glucose is co-transported along sodium into the enterocytes transported by SGLT-1
along with Na+ concentration gradient.  Same configuration of OH group

on carbon 2 as glucose
o Low intracellular sodium concentration is maintained by Na⁺-K⁺
 Presence of a pyranose ring
ATPase pump on the serosal side of intestinal mucosal cells.
 Methyl or substituted group on
o Galactose is also transported by SGLT-1 but fructose is not. carbon 5
Sodium-independent facilitated transporter (GLUT-5)

o Luminal membrane associated glucose transporter (GLUT-5) transports fructose, as well as
glucose and galactose down to their concentration gradients.
Pentoses are absorbed by simple diffusion.
These monosaccharides are transported out of enterocytes into the portal venous system by GLUT-2
facilitated transporter.
Lactose intolerance
It is a clinical condition characterized by diarrhea,
 Congenital deficiency of lactase occurs rarely in abdominal pain, and flatulence after ingestion of
infants, leading to lactose intolerance and milk or milk-containing products due to body’s
failure to thrive when fed on breast milk or inability to digest lactose.

It can be divided into primary lactase deficiency
normal infant formula.
and secondary lactase deficiency
 Congenital deficiency of sucrase-isomaltase Primary lactase deficiency
occurs among the Inuit, leading to sucrose o Congenital lactase deficiency
intolerance, with persistent diarrhea and failure o Primary low lactase deficiency (decreasing
lactase activity with advancing age which is
to thrive when the diet contains sucrose.
genetically determined and also varied by
 Flatulence after ingestion of leguminous seeds
ethnicity)
(beans, peas and soya) is due to presence of Secondary lactase deficiency
raffinose which cannot be hydrolyzed by o Occurs as a consequence of disorders which
human intestinal enzymes. damage the jejunal mucosa, such as viral

gastroenteritis, bacterial overgrowth and
 SGLT is inhibited by phlorizin and sodium
mucosal injury of the GI tract.
independent transport is inhibited by Lactase deficiency leads to dietary lactose
cytocholasin B. accumulation in the intestinal lumen. Since lactose
 An incomplete digestion of carbohydrates (the is osmotically active, accumulation of lactose draws

water into the lumen causing diarrhea after
components of fiber) leads to their conversion
consumption of relatively large amounts of milk.
to short-chain fatty acids (acetate, propionate,
Bacterial fermentation of lactose to lactate, and
butyrate) by the colonic bacteria. SCFAs subsequent production of gas results in abdominal
increase intestinal motility. distension, abdominal discomfort and flatulence.
Digestion and absorption of dietary proteins
Digestion of protein
Different types of peptidases
Protein load in the GI tract derived from two sources –
involved in dietary protein digestion
o Dietary protein (75 – 100 g)  Endopeptidase – hydrolyze internal
o Endogenous protein (35 – 200 g) peptide bonds.
 Proteins secreted into the gut (mostly digestive  Exopeptidase – hydrolyze peptide
bonds next to carboxyl or amino
enzymes)
ends and release free amino acids.
 Shedding from intestinal epithelium  Dipeptidase and tripeptidase
Only 1 – 2 g of nitrogen is lost in the feces daily so digestion  Intracellular peptidase –
and absorption of protein is extremely efficient. hydrolyze dipeptides and tripeptides

absorbed into the enterocytes.
Digestion in the stomach  Most peptidases are secreted as
 Protein digestion begins in stomach. inactive zymogens which are

activated by proteolysis cleavage.
 Gastric juice contains HCl, protease (pepsinogen) and rennin.
 Gastric acid HCl

o Provides acidic pH (1 – 2) in stomach which is required for denaturation of dietary proteins.
o Activates pepsinogen to pepsin.
 Pepsinogen
o It is secreted by the chief cells of the stomach.
o Pepsinogen is activated to pepsin by high H+ concentration (auto-activation) or by pepsin itself
(auto-catalysis)
o Pepsin is an endopeptidase which hydrolyze peptide bond formed by carboxyl group of acidic
(Asp, Glu) and aromatic (Phe, Tyr, Trp) amino acids.
o Pepsin digests proteins into smaller polypeptides (proteoses or peptones).
 Rennin (Chymosin, Rennet)
o Rennin causes milk coagulation and prevents rapid passage of milk from stomach thereby
prolonging exposure time of milk proteins to pepsin for digestion.
o Rennin irreversibly changes casein of milk to
calcium paracaseinate in the presence of calcium.
o This process is found in infants.
Digestion in the intestine

 Digestion of protein in the intestine is carried out by
pancreatic proteases and intestinal peptidases.
 Gastric contents entering into small intestine
encounter with secretions from exocrine pancreas.
 Bicarbonate ions (HCO3⁻) in pancreatic juice neutralize
acidic chyme and also provide optimal pH for
pancreatic protease action.
 Pancreatic proteases
o Secreted as inactive zymogens from pancreas.
o Includes trypsinogen, chymotrypsinogen,
proelastase and procarboxypeptidase.
o Trypsinogen is activated to trypsin by
enteropeptidase (enterokinase) secreted by
duodenal epithelial cells. Trypsin further activates
other trypsinogen (auto-catalysis) as well as other
zymogens (chymotrypsinogen to chymotrypsin,
proelastase to elastase, and procarboxypeptidase
to carboxypeptidase).
o Trypsin, chymotrypsin and elastase are endopeptidases.

o Trypsin hydrolyzes peptide bond formed by carboxyl group of basic amino acids (His, Lys, Arg).
o Chymotrypsin hydrolyzes peptide bond formed by carboxyl group of acidic (Asp, Glu) and
aromatic (Phe, Tyr, Trp) amino acids.
o Elastase hydrolyzes peptide bond formed by carboxyl group of small amino acids (Ala, Gly, Ser).
o Carboxypeptidase is an exopeptidase which hydrolyzes carboxyl terminal peptide bonds
releasing single amino acid until the dipeptide stage is reached.
o Pancreatic proteases cleave polypeptides into oligopeptides, dipeptides and amino acids.
 Further cleavage of oligopeptides and dipeptides into amino acids is accomplished by intestinal
proteases.
 Intestinal proteases
o Includes membrane-bound intestinal endopeptidase, dipeptidase, aminopeptidase and
intracellular peptidases.
o Cleaves oligopeptides and dipeptides into amino acids.
o Aminopeptidase – exopeptidase which hydrolyzes amino terminal peptide bonds of peptide
chain releasing single amino acids
o Dipeptidase – cleaves dipeptides into amino acids.
o Intracellular peptidase – hydrolyzes small peptides absorbed by enterocytes.
Absorption of protein
Digestive end-products of protein – amino acids
Mechanisms of amino acid absorption from the intestinal lumen
o Na+ dependent transport
o Facilitated diffusion
o Gamma-glutamyl cycle
Na+ dependent transport
o Similar to sodium dependent glucose transporter
o Amino acid is co-transported along sodium into the
enterocytes along with Na+ concentration gradient.
o Low intracellular sodium concentration is maintained by Na⁺-K⁺ ATPase pump on the serosal
membrane.
o At least six different Na+ dependent amino acid carriers in apical brush border membrane
 For acidic amino acids (Asp, Glu)
 For basic amino acids (His, Arg, Lys), cystine and ornithine
 For small neutral amino acids (Ala, Gly, Ser, Thr)
 For branch chain amino acids (Val, Ile, Leu) and aromatic amino acids (Phe, Tyr, Trp)
 For imino acids (Pro, Hyp)

 For β amino acids (β Ala, Taurine)
Absorbed amino acids are then transported out of enterocytes into the blood through facilitated
transporters in the serosal membrane. These transporters can also transport amino acids from the
blood into the enterocytes for use in the enterocytes for energy production. So, amino acid
transport across serosal membrane is bidirectional.
In addition to Na+ dependent
transporters, some amino acids are
transported across luminal membrane
via facilitated transporters down their
concentration gradient.
Most amino acids are transported by
more than one transport system.
Gamma-glutamyl cycle
o Involves in the transport of amino
acids into the cells of the intestine
and kidney.
o Membrane bound γ-glutamyl
transpeptidase catalyzes reaction of
extracellular amino acids with
glutathione (γ-glutamyl-cysteinyl-glycine)
o Formed γ-glutamyl amino acid is transported across the mucosal membrane.
o Amino acid is released into the cell and glutathione is reformed.
o This process is termed group translocation. All the amino acids except proline can be
transported by group translocation.
H+-dependent symporter is present on the brush border surface for di- and tripeptide transport into
the cell. Relatively large peptides may be absorbed intact, either by uptake into mucosal epithelial
cells (transcellular) or by passing between epithelial cells (paracellular).
 Large peptides absorbed intact into enterocytes often stimulate antibody formation causing allergic
reactions to foods.
 IgA molecules from the colostrum of maternal milk are absorbed intact (via transcytosis) providing
passive immunity to the infants.
 Hartnup disease – congenital defect of neutral amino acid transport system in intestinal and renal
epithelial cells
 Cystinuria – congenital defect of basic amino acid transport system
Digestion and absorption of dietary lipids
Dietary lipids – TAG (> 90 %), cholesterol (C), cholesterol esters (CE), phospholipids (PL) and non-
esterified/ free fatty acids (FFA)
Digestion of lipids
Hydrophobic nature of lipids excludes water-soluble digestive enzymes limiting the surface area for
enzyme action. It is overcome by emulsification process (hydrolysis of dietary fat globules to very
small lipid droplets for enzymatic digestion). Fat soluble vitamins are also absorbed, dissolved in lipid
micelles.
Digestion in the oral cavity

 During mastication, fat is emulsified with the help of dietary phospholipids and proteins.
 Lingual glands secrete lingual lipase for lipid digestion. But no digestion takes place in the mouth.
Digestion in the stomach

 The core body temperature within the stomach helps to liquefy dietary lipids.
 Lingual lipase (optimal pH 2.5 – 5) becomes active at low pH in the stomach. Chief cells of the
stomach secrete gastric lipase for lipid digestion. Gastric lipase secretion is stimulated by gastrin.
 Hydrolysis of TAGs is initiated by lingual and gastric lipases, which attack the sn-3 ester bond
forming 1, 2- DAGs and free fatty acids, which act as emulsifying agents. Only limited digestion of
lipids (about 30% of dietary TAGs with shorter chain fatty acids e.g., milk fats) occurs in the stomach
due to low solubility of substrates.
Digestion in the small intestine

 The lipid emulsion passes from the stomach into the duodenum where the further digestion occurs,
driven by pancreatic enzymes, aided by the emulsification of lipid droplets by bile salts.
Emulsification process is essential for lipid digestion since it disperse lipid globule into smaller
droplets and increase the surface area of lipid droplets for digestion process.
 Lipids are emulsified into smaller droplets by –
o Detergent and surface tension lowering action of bile salts

o Surfactant action of degraded lipids, phospholipids
o Mechanical mixing due to peristalsis
 Action of bile salts
o Bile salts are amphipathic compounds synthesized in the liver and secreted as the component of
bile via the gallbladder into the intestinal lumen.
o Bile secretion is stimulated by the hormone cholecystokinin.
o They are essential for solubilizing lipids during the digestion process in which they act as
detergents, by binding to dispersed lipid droplets formed by intestinal peristaltic movements.
o These emulsified lipids have increased surface area for pancreatic digestive enzymes action.
 Action of pancreatic enzymes
o Pancreatic juice contains pancreatic lipase, proco-lipase,
prophospholipase A2, cholesterol esterase and other less
specific lipid esterase e.g., retinyl esterase.
o Bicarbonate ions provide optimal pH for digestive enzymes.
o Proco-lipase and prophospholipase A2 are converted to
their active forms by trypsin.
o Pancreatic lipase – specifically hydrolyzes sn-1 and sn-3
ester bonds in TAGs releasing 2-MAGs and free fatty acids.
Pancreatic lipase requires co-lipase for its activity.
o Co-lipase binds to both the water–lipid interface and to
pancreatic lipase, simultaneously anchoring and activating
pancreatic lipase.
o 2-MAGs are poor substrates for hydrolysis. Its removal requires isomerization and subsequent
hydrolysis by pancreatic lipase. This is a relatively slow process, as a result 2-MAGs are major end
products of TAG digestion and only less than 25% of ingested TAG is completely hydrolyzed to
glycerol and fatty acids.
o Phospholipase A2 – hydrolyzes the sn-2
ester bonds in phospholipids yielding free
fatty acids and lysophospholipids.
o Cholesterol esterase – hydrolyzes
cholesterol esters into free fatty acids
and free cholesterol.
o Pancreatic non-specific lipid esterase –
acts on cholesterol esters, MAGs, or other lipid esters such as retinyl esters.
Gastroenterology P a g e | 10
Absorption of lipids
Digestive end products of lipid digestion – 2-MAGs, glycerol, free fatty acids, lysophospholipids, free
cholesterol
Solubilization and micelle formation of digestive end products of lipids
o Bile salts emulsify products of lipid digestion into micelles. Other dietary lipids such as
cholesterol, lysophospholipids and fat-soluble vitamins are also packaged in these micelles.
o Micelles provide the major vehicle for transport of lipids from the intestinal lumen to the cell
surface where absorption occurs.
Uptake of products of lipid digestion into enterocytes and re-esterification of lipids
o Micelles move down their concentration through the unstirred water layer to the brush border
of the intestinal mucosal cells.
o Lipids diffuse out of the micelles and enter the mucosal cells by passive diffusion and rapidly
esterified inside the cells to maintain the concentration gradient.
o Bile salts are left behind in the lumen where they are available for formation of new micelles.
Most of bile salts are absorbed only in the ileum into the entero-hepatic circulation.
Re-esterification of absorbed lipids in the enterocytes
o 1-MAGs are hydrolyzed to fatty acids and glycerol.
o 2-MAGs are re-esterified to TAG via the monoacylglycerol pathway.
o Glycerol released in the lumen is not reutilized and directly transported into the portal vein.
o Glycerol released in the enterocytes is re-esterified to TAG via normal phosphatidic acid
pathway.
o Short and medium
chain fatty acids (<
10 carbon atoms)
pass directly through
the enterocytes into
the hepatic portal
vein as FFAs where it
is transported bound
to albumin.
o Fatty acids of more
than 12 carbon
atoms are re-
esterified to TAG in
the mucosal cells.
o Some of the cholesterol absorbed is re-esterified to cholesterol ester.

o Some of the lysophospholipids absorbed are also re-esterified to phospholipids.
Packaging of esterified lipids into chylomicrons and secretion into the lymphatics
o Hydrophobic TAGs and cholesterol esters are coated with a layer of amphipathic phospholipids,
free cholesterol and apolipoprotein B48 to form chylomicron and secreted into the lymphatics,
finally entering into the blood stream via the thoracic duct.
Almost 100 % of fatty acids and MAGs are absorbed whereas only 30-40% of cholesterol is absorbed.
Plant sterols and stanols inhibit cholesterol absorption, hence lowering the plasma cholesterol
concentration.
Pancreatic disorders, bile duct obstruction, liver diseases causes defective fat digestion and
absorption leading to steatorrhea (clay-colored, fatty, bulky stool) and increasing the risk of fat-
soluble vitamin deficiency.
Orlistat – non-hydrolyzable analog of TAG and powerful inhibitor of pancreatic lipase which is
pharmacologically used to prevent fat absorption and obesity.
Digestion and absorption of dietary nucleoprotein

Dietary nucleic acids – organ meats, eggs, young plants
No digestion occurs in the oral cavity.
Dietary nucleic acids are denatured by gastric HCl in the stomach.
Digestion mainly occurs in the intestine, primarily by the action of pancreatic enzymes.
Ribonucleases and deoxyribonucleases hydrolyze RNA and DNA to oligonucelotides. Pancreatic 3’ –
5’ phosphodiesterase degrades oligonucleotides into mononucleotides. Nucleotidase and
phosphatase hydrolyze mononucleotides into nucleosides. Nucleosides are either absorbed intact
or further degraded to free bases and pentose sugars by nucleosidase.
Some of unabsorbed purines are metabolized by intestinal bacteria. Dietary purines and pyrimidines
are not used for synthesis of tissues nucleic acids since they are oxidized in the intestinal cells.
Absorbed purine bases are oxidized to uric acid and pyrimidine bases are oxidized to CO 2 and NH3
which are then excreted into the urine.
Absorption of water and electrolytes
Total fluid entry into the intestine – 2 liters of ingested fluid plus 7 liters of secretions from mucosa
of GI tract and associated glands
98% of fluid is reabsorbed, with a daily fluid loss of about 200 mL in stools.
Absorption of water
Only a small amount of water moves across gastric mucosa.
Water movement across the intestinal mucosa is bidirectional in response to osmotic gradient.
Water moves into or out of the intestine until the osmotic pressure of intestinal contents equals to
that of plasma.
Water is absorbed passively along with the absorption of the osmotically active particles.
Osmolality of duodenal contents may be hypotonic or hypertonic depending on ingested meals.
Osmolality of jejunum is equal to that of plasma.
In the colon, Na+ is pumped out and water moves passively with it along with the osmotic gradient.
Absorption of sodium
Na+ movement into and out of the small intestine depends on its concentration gradient.
Luminal membrane of all enterocytes in the small intestine and colon are permeable to sodium and
their basolateral membrane contains Na+/K+ ATPase pump. Sodium is actively absorbed through out
small and large intestine.
Active transport of sodium is important for absorption of glucose, some amino acids and some
vitamins. Conversely, the presence of glucose in the intestinal lumen facilitates the reabsorption of
sodium.
Absorption of chloride
Chloride from the interstitial fluid enters enterocytes across
basolateral membrane via Na+/K+/2Cl- cotransporters.
Chloride is then secreted into intestinal lumen via Cl- channels that
are regulated by various protein kinases (e.g., cAMP dependent
PKA).
In the ileum and colon, Cl- is actively absorbed in exchange for HCO3-
resulting in alkalinity of intestinal contents.
Absorption of potassium
Potassium moves across GI mucosa by diffusion via K+ channels in
both luminal and basolateral membrane. It is mainly absorbed in the
jejunum.
Some potassium is secreted into the intestinal cells as a component
of mucus.
K+ channels are present both in the luminal and basolateral
membrane, so K+ are secreted into the colon. The accumulation of K+
in the colon is offset by H+/K+ ATPase in the luminal membrane of
cells in the distal colon, resulting active transport of K+ into the cells.
Excessive loss of ileal and colonic fluids in chronic diarrhea can lead
to severe hypokalemia.
Absorption of bicarbonate
Large quantities of bicarbonate ions must be reabsorbed from the
upper small intestine (duodenum and jejunum) because large amounts of bicarbonate ions have
been secreted into the duodenum in both pancreatic secretions and bile.
In the ileum and colon, Cl⁻ is actively reabsorbed in exchange for HCO3⁻ resulting in the alkalinity of
the intestinal contents.
Diarrhea
Diarrhea is a condition in which there is excessive loss of sodium and water into feces.
Diarrhea can be caused by the non-absorbable solutes present in the gut (osmotic diarrhea), by the
failure to either digest or absorb nutrients, and also by the secretory agonists (secretory diarrhea).
Severe diarrhea results in the loss of alkaline intestinal contents leading to dehydration and
metabolic acidosis. It also results in the loss of sodium, potassium and other minerals.
Osmotic diarrhea – Caused by mal-absorption, digestive enzyme deficiencies short bowel
syndrome, and inflammatory disease.
Secretory diarrhea – Caused by infections, mal-absorption of bile salts, mal-absorption of fat or by
endocrine causes such as carcinoid syndrome or Zollinger–Ellison syndrome.
Compositions of WHO
Main causes of inflammatory diarrhea (associated impaired
Recommended ORS
absorption and increased secretion) are Crohn’s disease, ulcerative
Total osmolality – 200 to 310
colitis, and irritable bowel syndrome.
mmol/L
Secretory diarrhea but not osmotic one, persists on fasting.
Equimolar concentrations of
Biochemical basis of cholera
glucose and sodium
o Cholera bacillus produce cholera toxin. It causes ADP Glucose concentration not in
ribosylation of α subunit of Gs protein inhibiting its GTPase excess of 20 g/L (111
activity. The result is prolonged stimulation of adenylyl cyclase mmol/L)

[Na+] – 60 to 90 mEq/L
and resultant increase in intracellular cAMP concentration.
[K+] – 15 to 25 mEq/L
⁻
o Since Cl channels in the luminal membrane are regulated by
[Citrate] – 8 to 12 mmol/L
⁻
cAMP dependent PKA, increased cAMP increases Cl secretion [Cl⁻] – 50 to 80 mEq/L
into the lumen. Consequently, the function of mucosal carrier for Fluid replacement in diarrhea
+
Na is reduced, impairing NaCl absorption. Fluids with high sugar
o Na+/glucose cotransporter and Na+/K+ ATPase pump are not content (e.g., fruit juices and
soda beverages) will increase
affected and thus coupled absorption of Na+ and glucose bypass
diarrheal losses. The higher
the effect. unabsorbed glucose load will
Biochemical basis of ORS therapy in diarrhea increase the osmolality in the
o Water absorption in intestine is passive depending upon the lumen, decreasing water
absorption.
osmotic gradient that is dictated by sodium transport via the
Fluids with excess Na+
following three principal mechanisms:
concentration as compared
 Na+/H+ exchangers to glucose (e.g., chicken
 Electrochemical gradient broth) also will increase
diarrheal losses, as there is
 Sodium-dependent transport with carrier organic solutes
no organic solute for
(e.g., glucose, amino acids)
facilitated transport of
o In diarrheal diseases, disruption of many of these processes sodium.
occurs; however, the sodium-coupled co-transport with glucose and other carrier organic
solutes remains intact, even with viral enteritis associated with epithelial damage. The
preservation of this facilitated co-transport of glucose and sodium is the basis of ORS therapy
in diarrhea.
o ORS (oral rehydration salts) or solutions containing NaCl and glucose is given orally as a
treatment because presence of glucose in the intestinal lumen facilitates the absorption of Na+.
Digestion and absorption of vitamins and minerals

Vitamins and minerals are released from food during digestion, although this is not complete, and
the availability of vitamins and minerals depends on the type of food and, especially for minerals, the
presence of chelating compounds.
The fat-soluble vitamins are absorbed in the lipid micelles that are the result of fat digestion.
Most water-soluble vitamins are absorbed from the small intestine either by Na+ dependent co-
transporter or active transport or by carrier-mediated diffusion followed by binding to intracellular
proteins to achieve concentrative uptake.
Most vitamins are absorbed in the upper part of small intestine but vitamin B12 is absorbed in ileum,
complexed with intrinsic factor secreted by the stomach.
Absorption in large intestine

The unabsorbed digested food residues from the small intestine pass into the large intestine where
considerable absorption of water takes place.
In the colon, there is considerable intestinal bacterial activity. By fermentation and putrefaction,
bacteria produces various gases such as CO2, methane, hydrogen, nitrogen, H2S which cause
flatulence as well as short chain fatty acids such as acetic, propionic and butyric acids which serve as
energy source of intestinal cells.
Many amino acids undergo decarboxylation by action of intestinal bacterial enzymes and produce
toxic amines.
Large intestine is the source of ammonia as a product of bacterial enzymes activity on nitrogenous
substrates. Ammonia is absorbed into the portal circulation and removed from the liver as urea. In
liver diseases, ammonia may rise to toxic level in the blood (hyperammonemia).
Neomycin reduces ammonia formation in the intestine due to its antibacterial action to intestinal
flora. So, it is beneficial for patients with liver disease for prevention of ammonia intoxication.
Prolonged vomiting causes loss of water, hydrogen and chloride ions, and a further loss of
potassium due to the body’s compensatory mechanisms.
Saline cathertics such as magnesium sulfate are poorly absorbed salts that retain in the intestinal
lumen. Since these substances are osmotically active and draws water into the lumen, increasing the
intestinal volume and consequently exerting laxative effect.
Energy Metabolism Page |1
ENERGY METABOLISM
 Calorie intake provides energy that can be used to perform various body functions or stored for
later use. Energy balance is a state in which calorie intake equals energy expenditure. Persistent
energy balance state is required to maintain stable body weight and composition over long periods.
 When a person is overfed and energy intake persistently exceeds expenditure, most of the excess
energy is stored as fat, and body weight increases. When energy intake is insufficient to meet the
metabolic needs, loss of body mass occur as a result of mobilization of stored metabolic fuels.
 In order to balance the energy intake and output of the body, the regulatory mechanism of food
intake should be in accordance with metabolic needs of the body. Unbalancing these mechanisms
causes the different metabolic diseases such as underweight, obesity, and metabolic syndrome.
Energy metabolism
 It is sometimes referred to as general metabolism and is concerned with gross energy change, i.e.,
heat production and energy change for maintenance of vital functions of the body. It deals with the
overall energy production and energy requirement of the organisms.
 Metabolism – chemical changes occurring in the living tissues
 Catabolism – metabolic reactions concerned with degradation (breakdown) of large molecules into
simple units to release energy
 Anabolism – energy requiring metabolic reactions concerned with synthesis (building up) of complex
compounds
 Integration of these reactions is metabolism and leads to —
1. Growth
2. Production of heat required to maintain body temperature
3. Energy supply for performance of other vital activities such as –
 Mechanical work e.g., pumping of heart, body movement
 Electrical work e.g., production of electrical potential
 Osmotic work e.g., production of urine with higher osmotic pressure than blood
 Chemical work e.g., glycogen synthesis
 Biological work e.g., maintenance of living structures of cells and tissues
 When the animal dies, not only the production of heat and work ceases, but also the capacity for
doing so is universally lost, and the cells and tissues disintegrate.
 Energy metabolism can be studied from various aspects such as;
1. Sources and utilization of energy 4. Metabolic rate
2. Balance between energy input and output 5. Energy cost – calorific requirement for
3. Calorimetry various activities
Energy sources
 Potential sources of biological energy – thermal or heat energy, light, electrical energy, mechanical
energy, chemical energy
 Given proper circumstances, any form of energy may be completely converted into any other form,
but conditions in the body are not suitable for such universal inter-conversion. Simple form of
energy which animal or human can utilize is the chemical free energy of food inherent in specific
molecular configuration of certain ingested substances.
 This ability is due to the development of the biochemical structural and physiological apparatus in
the body, which permit the transformation of chemical free energy into other energy forms essential
for life.
Energy unit
 Energy unit that usually employed in human metabolism is the kilocalories or large calorie written as
kcal or C.
 1 kcal = quantity of heat energy necessary to raise one kilogram or one liter of water by one °C
 1 kcal = 1 C = 1,000 common or small calories
 1 kcal = 4.184 KJ or 4184 Joules
Internal utilization of energy

 If the maximum amount of useful or free energy is extracted from food when it is metabolized, some
heat must be produced. Such fraction of energy obligated to heat is not large, e.g., in the
metabolism of glucose, entropy change accounts for about 5% of the total energy.
 Thus, 95% of the ingested energy is potentially available as free energy. Such energy must be
converted into the appropriate chemical form for utilization of energy in the body, e.g., ATP. Thus,
ATP is the energy currency of the cell linking energy producing and energy requiring process.
 Conversion of food energy into a high energy biochemical compound is far from perfectly efficient.
For example, combustion of one molecule of glucose in the body produces 32 molecules of ATP (1
molecule of ATP produce 7.6 kcal of free energy on hydrolysis), and hence, 32 x 8 = 256 kcal. But its
free energy when oxidized outside the body in a bomb calorimeter is 686 kcal/mol. So, the energy
amount trapped is approximately 40%. More than half of the potentially available free energy is
wasted because of biochemical inefficiency. This wasted energy appears as heat which is lost to the
environment.
 Only 45% of the available free energy is trapped in the form of ATP in the body for utilization in a
various body processes such as –
o Performing internal work of various organs systems like blood circulation, ventilation, nerve
conduction, etc.
o Maintenance of structural and biochemical integrity of the body

o Performing external work like skeletal muscle contraction; energy utilized for this purpose is
about 25% of the total energy available to the body.
 All the energy utilizing processes produce common energy product – heat.
Food energy (100%)
Entropy change
5% Heat
Potentially available free energy (95%)
Biochemical inefficiency
50% Heat
Free energy pool (45%)
Maintenance of structural and

biochemical integrity of body
Internal work Heat
External work 0 – 25%

(Skeletal muscle contraction)
Energy cost
Calorie requirement during various activities
Food provide energy for three main reasons
1. For basal metabolism to perform vital functions of the body
2. For energy expenditure in simple activities of everyday life, such as sitting, walking, dressing, etc.
3. Diet induced thermogenesis (specific dynamic action of food)
Energy balance
Energy balance is a state in which calorie intake equals energy output.
Positive energy balance
o Calorie of food intake more than the energy expenditure
o Excess calories are stored as fat in the fat depots and the individual gains weight.
Negative energy balance

o Calorie content of food ingested is less than the energy output
o Endogenous stores of glycogen, body fat and protein are catabolized to provide the energy
deficit and the individual loses weight.
The relationship between energy balance and stored fat is the basis of dietary control of body
weight.
Child requires an excess of energy intake over expenditure to furnish substrates for natural
increase in body mass. If the child’s energy intake exceeds his energy output plus growth
requirement, the excess, as in the adult, will be deposited as body fat.
Supposing a person has energy output of 2,888 kcal/day and energy intake of 2,500 kcal/day, there
will be energy deficit of 388 kcal daily. Energy deficit 388 kcal will be provided by oxidation of stored
fat. This means catabolism of 43 g of stored fat per day (388 kcal/9 kcal per gram = 43 g of stored
fat). On this diet, this person’s body weight would be expected to decrease linearly at the rate of 43 g
(1/10 lb) per day. On the short interval, retention of body water may obscure body weight changes
due to stored fat catabolism. If such energy deficit is maintained for a sufficient time, the body water
will eventually return to its normal value and weight loss will follow.
Regulation of food intake and energy storage

Only about 45% of the energy ingested is extracted as ATP and much of this is eventually converted
to heat generated as a result of protein metabolism, muscle activity and activities of the various
organs and tissues of the body. Excess energy intake is stored mainly as fat where a deficit of energy
intake causes a loss of total body mass.
Energy balance state is required to maintain stable total body mass and composition over long
periods. Maintenance of adequate energy supply is necessary for survival which is determined by
amount of food intake and amount of energy storage (i.e., fat mass).
There are multiple short-term and long-term control systems that regulate not only food intake but
also energy expenditure and energy stores.
Energy balance is regulated by –
1. Short-term control and
2. Long-term control
 Short-term control
 Small weight-fluctuation that occurs within the acceptable weight range is regulated by short-
term regulator, appetite.
 Hypothalamus plays highly critical roles in the regulation of energy metabolism and the control
of feeding behaviors. Food intake is regulated by two centers in the hypothalamus; one is
feeding center situated in the lateral hypothalamus, and another is satiety center located in the
ventromedial hypothalamus. Feeding center is chronically active and its activity is transiently
inhibited by the activity of satiety center.
 The activity of satiety center is governed by nutrient signals (i.e., by means of the level of
glucose utilization in by cells within the center called glucostats), and a variety of neural and
hormonal signals.
 There are two different types of neurons in the arcuate nucleus that influence the activity of
satiety center. Anorexigenic neurone that expresses catabolic neuropeptides pro-
opiomelanocortin (POMC) and orexigenic neurone that expresses anabolic neuropeptide Y (NPY).
 Anorexigenic neurone – activation causes expression of POMC which is cleaved, yielding
melanocortins such as α-MSH that decrease food intake.
 Orexigenic neurone – activation leads to expression of NPY which links to neurons expressing
melanin-concentrating hormone (MCH) and orexins A and B which enhances food intake and
decreases thermogenesis.
 These hypothalamic nuclei also influence the secretion of several hormones that are important in
regulating energy balance and metabolism, including those from the thyroid and adrenal glands,
as well as the pancreatic islet cells.
 These neurons of the arcuate nuclei appear to be a site of convergence of many of the neural
and hormonal signals that
regulate energy stores.
 The hypothalamus receives neural
signals from the gastrointestinal
tract that provide sensory
information about stomach filling;
chemical signals from nutrients in
the blood (glucose, amino acids,
and fatty acids) that signify satiety;
signals from gastrointestinal
hormones; hormonal signals from
adipose tissue; and signals from
the cerebral cortex (sight, smell,
and taste) that influence feeding
behavior.
 Endogenous opiate or morphine –
stimulates feeding. Opiate receptor
blockers can reduce appetite.
 Endogenous cannabinoid system – Endocannabinoids synthesized from membrane

phospholipids stimulate food intake. Its hypothalamic level is increased during food deprivation.
 Gamma-amino butyric acid (GABA) – enhances appetite by inhibiting the satiety center and
reduces appetite by inhibiting the dopaminergic system.
 5-Hydroxytryptamine, Serotonin – reduce food intake.
 Corticotrophin releasing factor (CRF) – reduces appetite and stimulates thermogenesis.
 Various peripheral neural signals and hormones act on central neural centers concerned with
regulation of food intake.
 Gastrointestinal filling inhibits feeding by
stretch inhibitory signals. Distension of GI tract
produce stretch inhibitory signals that are
transmitted mainly by way of the vagi to
suppress the feeding center, thereby reducing
the desire for food.
 Gut peptides like somatostatin, bombesin or
gastrin releasing peptide are released from
stomach by the presence of food. They reduce
food intake. Other peptides like glucagon,
cholecystokinin (CCK), peptide YY (PYY),
substance P also reduce food intake.
 Ghrelin – secreted by stomach upon fasting,
gastric emptying, hypoglycemia and leptin. The major effect of ghrelin is exerted within the CNS
at the level of the arcuate nucleus. It stimulates the release of neuropeptide Y (NPY) and enhance
food intake. Its secretion is regulated by ingestion of nutrients and is the inverse of insulin
secretion. Secretion is inhibited by food intake, hyperglycemia and obesity.
 Circulating FFAs released from adipose tissues by lipolysis are the regulators of satiety (lipostat
theory).
 Amino acids and biogenic amines - tryptophan, tyrosine and choline affect the formation of
serotonin. Acetylcholine and catecholamines are critical to central regulation of feeding and
satiety center.
 Insulin enhances the uptake of tryptophan into CNS leading to production of serotonin by
neurons which reduces calorie intake.
 Glucagon-like peptide 1 (GLP1) – potentiate glucose dependent insulin secretion, inhibits gastric
emptying, leading to increased satiety with reduced food intake.
 Long term control

 Hormones released by adipose tissues and adiposity signals involve in long term control of
energy intake.
 Insulin – fasting plasma insulin concentration is directly related to adiposity and fat cells size.
Insulin can act on the anorexigenic neurons and reduces food intake. Hyperinsulinemia is
associated with hyperphagia and weight gain.
 Leptin – a hormone derived from adipose tissues (adipokines). It inhibits insulin secretion,
suppresses food intake and stimulates thermogenesis by inhibiting orexigenic neurons and
stimulating anorexigenic neurons.
 Glucocorticoids – enhances food intake by inhibiting CRF and stimulating NPY synthesis.
Energy value (kcal/g)

Calorific value or energy content of food
Foodstuff Physical Physiological Thermal
Energy content of the food can be determined
Heat Heat Equivalent
by combusting the food in a bomb calorimeter
Carbohydrate 4.1 4.1 5.04
in the presence of O2.
The energy liberated in kcal when one liter of Lipids (Fat) 9.4 9.4 4.68
O2 is utilized in the combustion is known as Protein 5.4 4.1 4.5
thermal equivalent or calorific value of O2. Alcohol 7.1 7 4.86

Mixed diet 4.18
Calorimetry
Calorimetry is the study of estimation of the energy output or energy expenditure by the
measurement of heat production by the body since all the energy produced in the body is ultimately
dissipated as heat.
Two methods of determination – direct calorimetry and indirect calorimetry

Direct calorimetry
o Combustion of food stuff in the presence of O2 results in the production of heat. The amount of
heat produced is then converted to energy content.
o Heat production is measured by using Atwater-Benedict Respiratory Chamber (Sealed insulated
chamber supplied by a system of copper pipes, through which water is circulated).
o Large, elaborate and expensive
Indirect calorimetry
o It is a method of calculating energy production based on
respiratory quotient since release of energy from food is
associated with the consumption of O2 and production of CO2 in
the steady state.
o The ratio of the volume of CO2 liberated to the volume of O2
utilized in oxidation of particular food stuff in unit time is called
respiratory quotient (RQ).
o RQ is different for different foodstuffs due to the different ratio
of carbon and oxygen present in the foodstuffs. Significance of RQ
o RQ for carbohydrate (glucose as an example) is 1. RQ  RQ gives information about type of
for fat (palmitic acid C16 FA as an example) is 0.7. In foodstuff being metabolized.
 RQ of an organ gives information about
case of protein, it is necessary to consider that some
metabolic process occurring in them,
of its end products of protein catabolism are
e.g., brain has an RQ of 0.97 – 0.99
eliminated in the urine instead of being completely indicating that its main fuel is
oxidized to H2O and CO2. RQ of protein is found to be carbohydrate.
0.802. RQ of an ordinary mixed diet is about 0.85.  RQ can be useful in calculating

metabolic rate or energy expenditure
o This method of calculating energy production is only
by indirect calorimetry method.
of approximation because volume of CO2 expired and Thermal equivalent of oxygen can be seen
volume of O2 inspired may vary with other factors. in normogram from respective RQ value.
Determination of O2 consumption in indirect calorimetry

Open circuit method
o The subject inspires room air and expires into a container. O2 and CO2 content of the expired air is
analyzed by means of gas-air analysis apparatus called Haldane or Lloyds gas analyzer.
o This method is Ideal for subjects either at rest or exercising on a treadmill or bicycle ergometer.
Close circuit method
o This method requires Benedict-Roth Spirometer.
o The subject rebreathes air contained in a closed system.
o It can be used when a subject is at rest or doing light exercise. CO2 eliminated cannot be
measured. Therefore, RQ cannot be determined.
SAMI (Socially Acceptable Monitoring Instrument)
o This method is based on the principle of close relation between O2 consumption and heart rate
at different level of exercise.
o Small heart rate counting apparatus (SAMI) is attached to the subject’s chest and each
electrocardiographic deflection is converted to a standard size pulse which is applied to an
electrochemical integrator for conversion to energy expenditure.
Metabolic Rate (MR)

It is the amount of energy expenditure in unit time.
It can be calculated by multiplication of rate of O2 consumption by thermal equivalent of mixed diet.
Factors influencing metabolic rate

1. Size of body or body surface area
 Body surface area or body size is directly related
with metabolic rate. Large person has higher
metabolic rate than a small person because of
larger body surface area.
2. Activity
 The degree of activity or the rate of exercise
markedly affects the rate of energy output or
metabolic rate. The lowest metabolic rate is
seen during sleep. Exercise causes further rise in
MR depending upon the severity of work.
 Mental work does not require energy.
 Maximum steady state energy output for a
person in good physical condition is 350kcal/hr.
3. Age and sex
 Metabolic rate is high in children, reaches peak
at puberty and then declines with advancing
age.
 Females generally have about 10% lower
metabolic rate under standardized conditions than males of the same age and size. This may be
due to greater subcutaneous fat mass in the females.
Energy Metabolism P a g e | 10
4. Temperature
a) Body temperature
 Body temperature affects metabolic rate by direct effect on all body functions. Decrease in
body temperature leads to a lower metabolic rate whereas rise in body temperature causes
increased metabolic rate.
 1°C change in temperature causes change in metabolic rate of 14% and 1°F change in
temperature causes 7% change in MR.
b) Environmental temperature
 In cold environment, chemical energy is converted to heat by shivering. Energy expenditure
by shivering depends upon the severity of cold stress (250kcal/m2/hr).
 When external temperature is high, the metabolic rate is decreased because of low
metabolism. During heat stress, there may be slight increase in metabolism to provide energy
needed for increased cutaneous blood flow and sweat secretion.
5. Specific dynamic action of food (SDA)
 It is the obligatory energy expenditure that occurs during assimilation of food in the body. The
amount of calories available from the foods is thus reduced by this amount. The energy used in
the assimilation of food comes from the food itself or the body stores. The effects of SDA are not
additive.
 SDA of carbohydrate – 6% , SDA of fats – 4%, SDA of proteins – 30%, SDA of mixed diet – 6 to 10%
6. Race and climate
 People living in tropic regions have a resting metabolic rate about 10% below than those living in
the temperate region. Indian, Myanmar and Chinese have lower BMR than Europeans.
7. Pregnancy
 Since 6th month of pregnancy, the mother’s metabolic rate begins to increase and continues to
rise until delivery (20% above the pre-pregnancy level). Increased metabolic rate is due to the
additional metabolism of the fetus.
8. Endocrine factors
 Thyroid hormones increase the basal metabolism of most tissues.
 Catecholamines have a direct effect on the rate of heat production and thus increase MR.
 Testosterone increases metabolic rate about 10-15%. Female sex hormones may increase
metabolic rate by small amount but not so significant.
9. Emotional state
 Anxiety and tension increase metabolic rate due to increased tension of muscles and secretion
of catecholamines.
 Apathy and depression states has low metabolic rate.
Basal Metabolic Rate

o It is the amount of energy expenditure in unit time measured under standardized conditions.
o The term ‘basal’ denotes a set of standardized and accepted conditions since metabolic rate during
sleep is lower than the basal rate.
o The standardized conditions are
 Complete physical and mental rest as possible
 Measurement in a comfortable and thermally neutral environment (20 – 25°C)
 12 – 14 hours after the last meal (post-absorptive state)
 Subject should be awake and in recumbent position during the test.
o BMR of an adult of 1.8m2 body surface area is 40 kcal/m2/hr for males and 37 kcal/m2/hr for females.
BMR is expressed as the percentage above and below the accepted normal standard for the
individual. The normal range of BMR may vary by ± 15%.
o Abnormal BMR is associated with thyroid disease. Hyperthyroidism is considered if BMR is increased
by +50% to +75% and hypothyroidism when BMR is decreased by -30% to -60% of normal value.
Factors influencing BMR

Age – The rate is high
in children, reaches
peak at puberty, and
then declines with
advancing age.
Sex – BMR of females
is slightly lower than that of males due to larger subcutaneous fat mass in females.
Climate – BMR is relatively lower in people living in tropics region and is relatively higher in people
living in temperate regions.
Body temperature – 1°C change in temperature causes 14% change in BMR, and for every 1°F change,
BMR changes by 7%.
Racial variation – Indian, Myanmar and Chinese have lower BMR than Europeans.
Pregnancy – After 6th month of pregnancy, BMR rises because of additional metabolism of the fetus.
Hormones
o Thyroid hormones – increase O2 consumption in almost all tissues. BMR is increased in
hyperthyroidism and decreased in hypothyroidism.
o Catecholamines – direct effect on the rate of heat production and thus increase BMR.
o Male sex hormones (testosterone) – increases metabolic rate about 10-15%. It is related to its
anabolic effect to increase skeletal muscle mass.
o Female sex hormones – may increase BMR slightly but not significantly.
o Growth hormone – increases BMR 15 – 20% because of direct stimulation of cellular metabolism.
Applications of BMR in clinical conditions

Formerly it is useful in aiding diagnosis of doubtful cases of hyper or hypothyroidism, evaluation of
effectiveness of medication in thyroid disease and determination of timing for operation in
hyperthyroid patients. Nowadays it has no significant diagnostic values in evaluating thyroid
disorders.
BMR is used in calculating caloric requirements of an individual and planning of diets. It may help in
prescribing an adequate diet for diabetic and obese patients.
BMI (kg/m2) Interpretation
Obesity
< 18.5 Malnourished
 It is a state of excessive accumulation of fat in the body.
 Surrogate marker for body fat content is body mass index (BMI). In 18.5 - 20 Underweight
clinical terms, BMI between 25 and 29.9 kg/m2 is called overweight and 21 - 25 Desirable
2
BMI greater than 30 kg/m is called obese. BMI is not a direct estimate
26 – 30 Overweight
of adiposity. High BMI may be due to large muscle mass.
> 30 Obese
 Body fat percent measurement is better indicator to define obesity.
Percentage of body fat can be estimated by various methods such as skin-fold thickness, bioelectrical
impedance or underwater weighing or calculation from BMI values. Obesity is usually defined as 25%
or greater total body fat in men and 35% or greater in women. In clinical practice, BMI is commonly
used to assess obesity.
Energy balance and obesity

 When calories intake exceeds body energy expenditure, most of the excess calories is stored as fat
and the body weight increases. Excessive adiposity is caused by energy intake in excess of energy
output for a prolonged duration. For each 9.3 kcal of excess energy entering the body,
approximately 1 gram of fat is stored.
 Fat is stored mainly in adipocytes in subcutaneous tissues and in the intra-peritoneal cavity but
significant amount of lipids can also be deposited in viscera, liver and other body tissues in some
obese persons.
 Hyperplastic obesity is associated with increased number of adipocytes and only small increases in
adipocyte size whereas hypertrophic obesity is associated with increase only in adipocyte size.
Causes of obesity
 Abnormal feeding behavior
o It leads to excessive energy intake and obesity. Environmental, social and psychological factors
contribute to abnormal feeding such as abundance of calorie-dense foods (especially fatty
foods), stressful conditions, etc.
Medical problems associated
 Sedentary lifestyle
with obesity
o About 20 to 30% of the energy used each day by average person is Metabolic syndrome
for muscular activity. Regular physical activity and physical Non-alcoholic fatty liver
training increases muscle mass and decrease body fat mass, disease (NAFLD), Gall stones
whereas inadequate physical activity is typically associated with
Dyslipidemia
decreased muscle mass and increased adiposity. Increased
Hypertension and stroke
physical activity is an effective means of reducing fat stores. Some cancers – breast,
 Childhood over-nutrition endometrial cancer, colon,
ovary, gall bladder
o Rate of formation of new fat cells is especially rapid in the first
Respiratory disorder
few years of life, and the greater the rate of fat storage, the
Musculoskeletal disorder
greater the number of fat cells. Psychological problem
o It has been suggested that over-nutrition of children, especially in infancy, and to a lesser
extent, during the later years of childhood, can lead to lifetime of obesity.
 Neurogenic abnormalities
o Lesions in ventromedial nuclei of hypothalamus lead to excessive food intake and obesity. (e.g.,
hypophyseal tumors).
o Abnormalities in neurotransmitters or receptors mechanisms in the neural pathways of the
hypothalamus that control feeding can lead to obesity. “Set-point” of an obese person’s feeding
control system is at a much higher level of nutrient storage than that of a non-obese person.
 Genetic factors
o Genetic abnormalities concerning with one or more pathways that regulate the feeding centers,
energy expenditure and fat storage can lead to obesity.
o Monogenic causes of obesity
1. Mutations of MCR-4
2. Congenital leptin deficiency due to mutations of leptin gene
3. Mutations of leptin receptor
o Many genetic variations can interact with environmental factors to influence the amount and
distribution of body fat.
 Endocrine disorders
o Some endocrine disorders are associated with weight gain and obesity e.g., hypothyroidism,
acromegaly, Cushing’s syndrome etc.
o Leptin resistance is seen in obese persons.
Methods of determining Caloric needs

 Basal metabolic rate (BMR) can be estimated for healthy individuals from knowledge of age, sex,
height and weight using the Harris–Benedict equation.
 The Harris-Benedict equation

o Calorie formula using the factors of height,
weight, age, and sex to determine basal
metabolic rate (BMR)
o More accurate than determining calorie
needs based on total bodyweight alone
 Estimated TDEE (Total Daily Energy
Expenditure) can be derived by multiplying the
BMR with activity factor or from calculating
estimated energy requirements (EER).
 For estimation of calorie needs in hospitalized
patients or for sick patients, suitable adjustment
can be made for disease state, pyrexia, mobility
etc., i.e., by multiplying with disease factor.
 Estimation of calorie needs is important for
preparation of adequate
diet in hospitalized
patients, especially
patients on nutritional
support such as naso-
gastric tube feeding,
parenteral nutrition, etc.
 It is also useful for
prescribing adequate
diet for diabetic and
obese persons.
Nutrition Macronutrients Page |1
Macronutrients
Nutrition
Nutrition is the science concerned with chemical constituent of food, their action and interaction
in relation to health and disease.
Nutrients
Nutrients are chemically defined as components of food that are required by the body for
energy, growth and regulation of cellular processes.
Six basic Nutrients

1. Carbohydrate
2. Lipids (Fats) Macronutrients
3. Proteins
4. Vitamins Micronutrients
5. Minerals
6. Water
Energy content of foods (calorific value of food) Energy value (kcal/g)

Foodstuff
Food is the fuel source of the body. The ingested In Bomb calorimeter In the body
food undergoes metabolism to liberate energy required Carbohydrate 4.1 4
for the vital activities of the body. The calorific value of
Lipids (Fat) 9.4 9
food is calculated from the heat released by the total
Protein 5.6 4
combustion of food in a Bomb calorimeter.
 Carbohydrate and fat are completely oxidized to CO2 and water. Factors influencing nutritional
status of an individual
 Proteins are not completely burned because urea (end product of
 Genetic background
protein and amino acid catabolism) still contains some energy, which
 Functional status of GI tract
is not available to body.
 The environment
 Vitamins and minerals have no calorific value. They are involved in
 Age and phase of life cycle
the energy generation process by serving as coenzymes or cofactors  The level of physical activity
of respective enzymes involved during the processes.  Psychological factors
Nutritional deficiencies may result from –  Social factors
Dietary inadequacies  The presence of disease
Maldigestion and malabsorption Unit of energy – calorie
Genetically determined metabolic errors One calorie represents amount of

heat required to raise the
Nutritional status assessment temperature of one kilogram of
 No single definitive marker of nutritional status water by 1˚C.
 Relies on the interpretation of a range of variables 1 Cal (1 kcal) = 4.128 kJ

1 Cal = 1000 cals
Dietary Recommended Intake (DRI)
1. Clinical assessment on state of nutrition
 Dietary intake that meets the requirements of 97
2. Assessment of dietary habits and dietary history
to 98 % of healthy individuals in a category
 24-hour dietary recall
 Recommendations on nutrient intake are based
 Food frequency questionnaire on DRI.
 Food diary  Quantitative estimates of nutrient intakes can be
3. Anthropometric measurements used in evaluating and planning diets for healthy
 Body weight people.
 Body Mass Index 1. Recommended Dietary Allowance (RDA)
 Body fat percent  Average daily nutrient intake level to meet

requirement of nearly all healthy individuals
 Waist/Hip Ratio
(minimum dietary requirement + added
 Mid-arm circumference
amount to compensate incomplete digestion
 Skin-fold thickness
and absorption)
4. Biochemical and hematologic laboratory tests
2. Estimated Average Requirement (EAR)
 Common biochemical markers  Average daily nutrient intake to meet the
 Urinary nitrogen excretion for N2 balance requirement of half of the healthy individuals
 Total and differential plasma protein level in a particular gender group & phase of life
 Hemoglobin concentration, etc. 3. Adequate Intake (AI)
 Recommended average daily nutrient intake
Body Mass Index based on estimates of nutrient intake by a
 It is a measure of nutritional index which determines group of healthy people (limited data source)
whether one is taking enough, too much or too little 4. Lower Reference Nutrient Intake (LRNI)
food to supply the total energy requirements (TER) of  Lower end of intake distribution in a
the body. population
 Measure of body proportions and composition, 5. Tolerable Upper Intake Level

 Highest average daily intake likely to pose no
thinness or under-nutrition.
health risk to almost all individuals
BMI (kg/m2) Interpretation
< 18.5 Malnourished
18.5 - 20 Underweight
21 - 25 Desirable
26 – 30 Overweight
> 30 Obese
𝑊𝑒𝑖𝑔ℎ𝑡 𝑖𝑛 𝑘𝑖𝑙𝑜𝑔𝑟𝑎𝑚
𝐵𝑀𝐼 =
(𝐻𝑒𝑖𝑔ℎ𝑡 𝑖𝑛 𝑚)2
Nutrigenomics
Energy balance
 Study of how individual’s
 State of energy balance is needed to maintain health. genotype influence on
 Daily energy requirement for a normal healthy Myanmar adult doing nutritionally related
sedentary work is about 2400 kcal/day for a male and 2000 kcal/day for diseases
 Study of genetic influence
a female.
of digestion, absorption
 Energy balance is a state in which the caloric intake and energy output of nutrients and their
are equal. metabolism and excretion
 Negative energy balance is seen when caloric content of food ingested is less than the energy
output. Endogenous stores are utilized; glycogen, fat and body protein are catabolized to meet the
energy requirement thereby leading to weight loss.
 Positive energy balance is seen when caloric value of food intake exceeds energy output. Excess
calories are stored as fat in the body and the individual gains weight.
Malnutrition
Markers of malnutrition risk
Gradual decline in nutritional status, which in its more advanced stages  BMI of below 18.5 kg/m2
leads to a decrease in functional capacity and other complications.  Unintentional loss of 10%
Key public health issue in developing countries of body weight within

preceding 3–6 months
Also a problem in hospitalized patients (stroke, cancer, GI problems) in
 Inability to eat for more
developed countries than 5 days in the course
Reduced food intake  protein-energy malnutrition (PEM) of acute illness
Chronic excessive food intake  obesity
Under-nutrition
 Can be resulted from decreased intake of nutrients,
impaired absorption, impaired storage, increased
demand or increased excretion.
Protein calorie malnutrition (PEM)

Most common form of malnutrition worldwide
Inadequate intake of protein and carbohydrate are
especially common in infants and young children in
developing countries.
Can be classified either as marasmus or kwashiorkor.
Marasmus – inadequate intake of both protein and
energy (carbohydrate)
Kwashiorkor – inadequate protein intake with adequate
calorie intake
Often diets that lead to marasmus and kwashiorkor are similar.

Kwashiorkor can be precipitated by conditions of increased protein demand such as infection.
Marasmus infants will have thin, wasted appearance and will be small for age.
Kwashiorkor can present as plump appearance in patients due to oedema.
Prolonged duration of PEM can lead to permanent stunting in physical and mental development in
children.
Other signs of kwashiorkor – dry brittle hair, diarrhea, dermatitis of various forms, retarded growth
Over-nutrition
 It is resulted from excessive calorie intake for
prolonged duration, reduced physical activity and

sedentary life style.
Obesity
Defined as excessive accumulation of fat in the
body.
Can be classified based on
o BMI (mild, moderate, severe)
o Fat distribution (general and central obesity)
Associated with increased risk of medical and
surgical problems.
Balanced diet (Adequate diet)

Group Food Nutritive value
A balanced diet contains adequate amount
Protein, vitamin B
of all six basic nutrients in suitable Group I Meat, organ meat,
complex, iron,
proportion that is required to maintain Body building fish, poultry, milk,
mineral, calcium,
the health and efficiency of an individual. foods eggs, beans, nuts
phosphorous
Essential nutrients are carbohydrate, lipid,
protein, vitamin, mineral and water. Starch, cereals,
Group II
A nutritionally sound diet includes the four butter, margarine Calories, small
Energy rich
basic food groups Vegetable oil, amount of protein
foods
o Milk and milk products animal fats
o Cereals and bread Green leafy

Group III
o Vegetables and fruits vegetables Carotene, iron,
Protective
o Meats, fish, poultry Yellow fruits and calcium, fiber, vitamin
(Regulatory)
Plans or preparation of balanced diet are vegetables C, vitamin B complex
food
differed by various countries. Citrus fruits
Food plan varies with underlying diseases, food habits, and socio-economic status.
Three-food group plan can be used for preparation of balanced diet and it is applicable to Myanmar
diet.
Principles in planning a balanced diet

1. The diet must contain various foodstuffs such as body building, energy yielding and protective food
in correct proportion.
2. The constituents of a balanced diet can differ according to age, sex, physical activity, economic
status and the physiological states.
3. The approximate constituents of food, i.e., carbohydrate, fat and protein should be in ideal
proportion.
Carbohydrate – 55 to 70 % of total calorie intake
Sample menu
Fat – 20 to 30 % of total calorie intake  Breakfast – Fried rice with steamed peas
Protein – 10 to 15 % of total calorie intake  Lunch – Rice and fish, Nga-pi-ye, boiled
4. It should be prepared based on locally available foods. string beans, dandalun-ywet soup
5. It should be with the economic means of the people.  Snacks – Plain tea with roasted ground
6. It should fit within local food habits. nuts or jiggery
7. Diet should be easily digestible and palatable.  Dinner – Rice and dried fish, egg curry,
fried vegetables, ngapi, kazun ywet and
8. It must be hygienic.
tomato salad
9. It should contain enough roughage materials.
Dietary carbohydrate
 Constitutes monosaccharides such as glucose and fructose, disaccharides such as lactose and sucrose
and polysaccharides such as starch and glycogen.
 Sources
o Monosaccharides – fruits, grapes, honey
o Disaccharides – cane sugar, beet sugar, milk
o Polysaccharides – rice, potatoes, oat, barley
 Daily requirement – 55 to 70 % of total calorie intake (250-500 g)
 Minimum 5 g of carbohydrate/ 100 kcal of total diet or 100 g/day is required to prevent ketosis.
Biochemical functions
Most efficient and cheapest source of energy for vital processes (4kcal/g)
Chief metabolic role of dietary carbohydrate is energy production.
Any carbohydrate in excess of energy requirement is converted to glycogen and TAG for storage.
Exert protein sparing action (spares protein for anabolic purposes) by decreasing catabolism of
amino acids and increasing tissue protein synthesis.
Glycemic index
Aids in complete assimilation of fat by providing
The effect of 50 g of a particular food on blood
oxaloacetate. glucose levels compared to 50 g of glucose
 Fatty acids mobilized from triacylglycerol in Reflect the effect of carbohydrate in particular
adipose tissues are oxidized to acetyl CoA which food stuff on blood glucose level.
System of ranking the carbohydrate-containing
then enters CAC for complete combustion to CO2
foods according to the degree of increase in
and H2O. For complete oxidation of acetyl CoA blood glucose which takes place after their
derived from fatty acid oxidation, oxaloacetate ingestion
must be provided from carbohydrate, otherwise GI is expressed on a scale of 1 to 100 (low GI

is 0–55, moderate 56–69, and high >69).
excess acetyl CoA can lead to ketone bodies
Foods that are rapidly digested and
formation in liver.
absorbed have a high GI.
Dietary lactose favors intestinal synthesis of Foods that are slowly digested and
bacteria and absorption of calcium. absorbed yield low GI.
Structural and functional constituents of Pastries, refined cereals, rice and starchy
vegetables have high glycemic index.
biomolecules
Non-starchy vegetables, fruits, legumes, nuts
o Pentose sugars are important constituent of have low glycemic index.
nucleotides, nucleic acids and many coenzymes. Low GI foods control post-prandial glycemia
o Monosaccharides such as galactose and glucose and insulinemia and are beneficial for
people with diabetes and appropriate for
are important constituent of lactose, glycolipids
weight control.
and glycolproteins.
o Amino sugar and acid sugar are Glycemic Index of selected foods
important constituents of
mucopolysaccharides or GAG.
Deficiency – Pure carbohydrate deficiency

is rare. Protein and carbohydrate deficiency
in children leads to marasmus.
Excess – Excess carbohydrate is stored as

fat and can lead to obesity.
Dietary fiber
 Components of food that cannot be
broken down by human digestive enzymes
 Sources
o Cellulose – unrefined cereals, beans, wheat o Lignin – woody parts of vegetables
o Hemicellulose – unrefined cereals, fruits o Pectin – fruits
and vegetables o Gums – dried beans, oats
Cellulose and hemicellulose increase stool bulk and decrease transit time, thus it is beneficial for
constipation and reducing risk of colon cancer.
Fibers decrease intra-colonic pressure and appear to play a beneficial role in diverticular diseases.
Lignins adsorb cholesterol and decrease dietary cholesterol absorption thereby decreasing serum
cholesterol level.
Mucilaginous fibers such as pectin and gums tends to form viscous gel in the stomach and slow the
rate of gastric emptying and the rate of absorption of many nutrients especially glucose. This effect
is beneficial for diabetics and dieters by improving glucose tolerance.
Colonic fermentation of fibers may contribute to human energy requirement (2 – 7 %).
Binding properties of fibers decrease the absorption of toxic compounds.
Adverse effects
o Adverse effect on dietary protein digestion and absorption
o Decreased intestinal absorption of certain minerals
o Flatulence and discomfort on bacterial fermentation of fibers
Dietary lipids
 Major dietary lipid is triacylglycerol (95-98%). Others are phospholipids, cholesterol, fatty acids and
lipid soluble vitamins.
 Main source of saturated fat in the human diet is the meat of ruminants, diary products and hard
margarine. Cholesterol is found only in food of animal origin and particularly in egg yolk.
 Sources
o Animal sources – butter, fat from meat, lard
o Vegetable sources – oil of peanut, sesamum, coconut, sunflower, corn
 Daily requirement – 20 to 30 % of total calorie intake per day (66 – 83 g)
 One % of total calorie should be provided daily from essential fatty acids.
High fuel value and the richest source of energy (9 kcal/g)
Exert protein sparing action.
PUFA lowers plasma cholesterol level.
Essential fatty acids serve as precursors of important compounds called eicosanoids
(prostaglandins, thromboxanes, leukotrienes and lipoxins). Linoleic acid is essential is infants, leads
to local formation of prostaglandin E1, which is potent inhibitor of platelet aggregation.
Phospholipids are major component of cellular, subcellular membrane, myelin sheath, brain and
nervous tissues.
Phosphatidyl choline activates microsomal enzyme.

Cholesterol is the precursor of bile salts, vitamin D and steroid hormones.
Serve as thermal, electrical and mechanical insulator in the body.
Act as vehicle for lipid soluble vitamins.
Increase palatability of food and produces a feeling of satiety.
Essential amino acids
Deficiency – Essential fatty acids deficiency leads to dermatitis in infants. Arginine
Leucine
Excess – High dietary fats are associated with high plasma cholesterol level and Lysine
development of atherosclerosis and coronary heart disease. Histidine
Isoleucine
Dietary protein Threonine

Valine
 Protein in human tissues is made up of 20 different kinds of amino acids which
Methionine
can be classified according to nutritional requirement as essential and non-
Phenylalanine
essential amino acids. Dietary protein provides both essential and non- Tryptophan
essential amino acid. Essential amino acids are those which Nitrogen Balance
cannot be synthesized by the body, so they must be supplied in The relationship between intake
the diet. of nitrogen, primarily in the form

of protein, and excretion of
 Sources
nitrogen, chiefly in the form of
o Animal sources – egg, milk, fish, meat, visceral organs
undigested protein in feces and
o Vegetable sources – pulses, beans, legumes, rice and corn urea and ammonia in the urine
 Daily requirement – 10 to 15 % of total calorie intake (65 – 90 Positive nitrogen balance
g/day)  Net increase in body protein

synthesis
Biochemical functions  E.g. growing children, pregnant
New tissue formation during growth and pregnancy women and in convalescence
Negative nitrogen balance
Formation of tissue proteins which are constantly undergoing
 Results from inadequate intake of
destruction and re-synthesis, “the wear and tear quota”.
protein
Formation of structural proteins in every cell  E.g., starvation, old age, net
Formation of functional proteins such as enzymes, certain destruction of tissues after injury
hormones, plasma proteins and hemoglobin or operation, adaptive response

to major trauma or illness by
Production of milk proteins (lacalbumin, casein) during lactation
increasing protein catabolism
Protein supply specific amino acids and nitrogen for the
synthesis nitrogen containing compounds such as purine, pyrimidine, heme, etc.
Formation of antibodies to build up resistance to disease
Production of energy under certain condition (4kcal/g) but this is not their primary function.
Deficiency – Protein deficiency leads to kwashiorkor in weaning infants and muscle wasting in adults.
Quality of protein
 Depends on the content and amount of essential amino acids
 Determined by biochemical method and chemical method
Complete or reference Protein Efficiency Ratio

Nutritional value of protein
protein (PER)
1. Biological value (BV)
Protein in which all the
𝑮𝒂𝒊𝒏 𝒊𝒏 𝒃𝒐𝒅𝒚 𝒘𝒆𝒊𝒈𝒉𝒕 (𝒈)
2. Protein efficiency ratio (PER) essential amino acids 𝑷𝑬𝑹 =
𝑷𝒓𝒐𝒕𝒆𝒊𝒏 𝒊𝒏𝒈𝒆𝒔𝒕𝒆𝒅 (𝒈)
3. Net Protein Utilization (NPU) are present in adequate
concentration for protein PER of egg protein = 4.5
4. Chemical score
synthesis. e.g., whole egg PER of milk protein = 3.0
Biological Value (BV) protein PER of rice protein = 2.2
Percentage of absorbed
Chemical score
nitrogen retained in the body
Ratio between the quantity of the most limiting essential amino
𝑵𝒊𝒕𝒓𝒐𝒈𝒆𝒏 𝒓𝒆𝒕𝒂𝒊𝒏𝒆𝒅 acids in the test protein to the quantity of the same amino acid
𝑩𝑽 = × 𝟏𝟎𝟎
𝑵𝒊𝒕𝒓𝒐𝒈𝒆𝒏 𝒂𝒃𝒔𝒐𝒓𝒃𝒆𝒅 in the egg protein, which is expressed in percentage
Most limiting essential amino acid = essential amino acid
Net Protein Utilization (NPU) present in the lowest amount in the test protein
Better nutritional index than High correlation with the biological value
BV Animal proteins have high chemical score and plant proteins
Takes into account of have low chemical score.
digestibility factor
𝒎𝒈 𝒐𝒇 𝒍𝒊𝒎𝒊𝒕𝒊𝒏𝒈 𝒂𝒎𝒊𝒏𝒐 𝒂𝒄𝒊𝒅
𝒈 𝒑𝒓𝒐𝒕𝒆𝒊𝒏
𝑵𝒊𝒕𝒓𝒐𝒈𝒆𝒏 𝒓𝒆𝒕𝒂𝒊𝒏𝒆𝒅 𝑪𝒉𝒆𝒎𝒊𝒄𝒂𝒍 𝒔𝒄𝒐𝒓𝒆 = × 𝟏𝟎𝟎
𝒎𝒈 𝒐𝒇 𝒍𝒊𝒎𝒊𝒕𝒊𝒏𝒈 𝒂𝒎𝒊𝒏𝒐 𝒂𝒄𝒊𝒅
𝑵𝑷𝑼 = × 𝟏𝟎𝟎 𝒈 𝒆𝒈𝒈 𝒑𝒓𝒐𝒕𝒆𝒊𝒏
𝑵𝒊𝒕𝒓𝒐𝒈𝒆𝒏 𝒊𝒏𝒈𝒆𝒔𝒕𝒆𝒅
Limiting amino acids

 The essential amino acid found most inadequate in a given protein is called the limiting amino acid
for that particular protein.
 E.g., lysine in rice protein, methionine in legumes, tryptophan in maize protein
Grade I protein – proteins of high biological value and high chemical score
 Contain all essential amino acids in proportion suitable for the synthesis of human proteins e.g.,
animal proteins.
Grade II protein – proteins of low biological value and low chemical score
 Supply different proportions of amino acids and some lack one or more essential amino acids e.g.,
plant proteins except soya bean and peanut.
Nutrition M a c r o n u t r i e n t s P a g e | 10
Parental Nutrition for hospitalized patients
Parental Nutrition for hospitalized patients
Least invasive method is to use peripheral vein as for
Parental nutrition or intravenous any other IV infusion
nutrition is needed in patients who are  Limitation – not suitable for long term due to
severely catabolic or unable to digest hypertonicity

 Solution of 5% glucose and 4.25 % amino acids can be
and absorb food normally.
used safely.
Infusion of nutrients solution via
Most aggressive method is total parenteral nutrition
peripheral vein or via indwelling catheter (TPN) via indwelling catheter inserted into large fast-flow
in SVC (Total Parenteral Nutrition) vessel such as SVC
 Suitable for infusion of hypertonic solutions for long term
 Solution up to 60% glucose and 4.25% amino acids
Micronutrients Vitamins Page |1
Micronutrients
Vitamin
Vitamins are organic micronutrients that are required by the body, in small amount for a variety
of biochemical functions. They are generally cannot be synthesized by the body in sufficient amount, so
they must be provided in the diet.
Classification of vitamins (depending on solubility)

I. Fat soluble vitamins
1. Vitamin A (retinol, retinoic acid, retinal) 3. Vitamin E (tocopherol)
2. Vitamin D (cholecalciferol) 4. Vitamin K (phylloquinone)
II. Water soluble vitamins
1. Vitamin B1 (thiamin) 6. Vitamin B5 (pantothenic acid)
2. Vitamin B2 (riboflavin) 7. Vitamin B6 (pyridoxine)
3. Vitamin B3 (niacin) 8. Vitamin B12 (cobalamin)
4. Biotin 9. Folic acid
5. Lipoic acid 10. Vitamin C (ascorbic acid)
Fat soluble vitamins

 Fat soluble vitamins are non-polar molecules which are all isoprene derivatives since they are made
up of one or more of five carbon units namely isoprene units (—CH=C.CH3—CH=CH—).
 They can only be absorbed efficiently when there is normal fat absorption. Lipid mal-absorption
accompanied by steatorrhea usually results in poor uptake of all the fat-soluble vitamins.
 Deficiency disease (except in the case of vitamin K) is difficult to produce in adults because large
amounts of most fat-soluble vitamins are stored in the liver and in adipose tissues. Thus they can be
mobilized as needed during long periods of deprivation.
 They are not readily excreted in urine.
Excessive consumption of these
vitamins (particularly A and D) leads to
their accumulation and toxic effects.
Vitamin A (retinol, retinal, retinoic acid)
Structure – long chain of alcohol with

cyclohexenyl ring
Sources
 Retinoids or preformed vitamin A (retinol,
retinaldehyde and retinoic acid) Carotene dioxygenase
 Foods of animal origin; liver, egg yolk,

cod liver oil, milk, cheese, etc.
 Carotenoids or pro-vitamin A (carotenes and
(Lipoprotein Lipase)
related compounds)
 All dark green and yellow pigmented
fruits and vegetables (papaya, pumpkin,
tomato, carrot, mango and green leafy
vegetables)
Metabolism (Retinol Binding Protein)
 Retinoids such as retinol, retinal and retinoic

acid are found in foods of animal origin. The
α-, β-, and γ-carotenes and cryptoxanthin are
quantitatively the most important pro-vitamin A carotenoids and are found in plant sources. Pro-
vitamin A (β carotene) consists of two molecules of retinal linked at their aldehyde ends.
 Most abundant dietary forms of vitamin A are retinyl ester and β-carotene.
 Retinyl esters are hydrolyzed into retinol and FFAs in the small intestine.
 β-Carotene and other pro-vitamin A carotenoids are cleaved in the intestinal mucosa by carotene
dioxygenase, yielding retinaldehydes which are then reduced to retinols. Retinols are esterified with
fatty acids and secreted in chylomicrons together with esters formed from dietary retinol.
 The intestinal activity of carotene dioxygenase is low, so that a relatively large proportion of
ingested β-carotene may appear in the circulation unchanged.
 The retinol esters of chylomicrons are taken up by the liver and stored. More than 90% of the body’s
supply of vitamin A is usually stored in the liver cells, which contain a year’s supply of the vitamin.
 When needed, retinol is released from the liver and transported to extra-hepatic tissues by retinol-
binding protein (RBP) in the plasma.
 Active forms of vitamin A are retinol, retinal and retinoic acid.
Biochemical functions of vitamin A

 Role of vitamin A in vision
o Vitamin A is essential for
vision as component of
visual pigments.
o Retina contains two types of light receptors cells: rods and cones. Rods Dark adaptation time
are involved in dim light vision (scotopic vision) whereas cones are Upon entry into dark or dim
responsible for bright light and color vision (photopic vision). light place, rhodopsin stores
are depleted and vision is
o Both rod and cone cells of retina contain a photoreceptor pigment in
impaired. However, within a
their membrane and 11-cis retinal is a component of these pigments.
few minutes, known as dark
o The cyclic events occur in the process of vision, known as rhodopsin adaptation time, rhodopsin is
cycle or Wald’s visual cycle. resynthesized and vision is
improved. Dark adaptation
 The visual pigment in the rod cells is rhodopsin which contains 11-
time depends on vitamin A
cis retinal conjugated to the apoprotein, opsin. The 11-cis retinal
status of the person and is
serves as the light absorbing part of rhodopsin, and opsin is an increased in vitamin A
integral membrane protein with seven transmembrane helices. deficient individuals.
 The absorption of light by rhodopsin causes isomerization of the 11-cis retinal to all-trans
retinal, and a conformational change in opsin.
 This results in the generation of a nerve impulse that is transmitted by the optic nerve to the
brain which is followed by dissociation of all-trans retinal from the opsin.
 Some of all-trans retinal is reconverted to 11-cis retinal by retinal isomerase, but most are
reduced to retinol in the rod outer segment. It is transported into pigmented epithelium
where a specific isomerase converts it to 11-cis retinol that is then oxidized to 11-cis retinal
and transported back into the rod outer segment. It can then recombine with opsin to form
rhodopsin and visual cycle can begin again.
o The key to initiation of the visual cycle is the availability of 11-cis-retinal, and hence vitamin A. So,
continuous supply of retinol is needed for rapid regeneration of rhodopsin for dark adaptation.
In deficiency, both the time taken to adapt to darkness and the ability to see in poor light are
impaired.
o Color vision is mediated by three cone receptors that are homologous to rhodopsin viz.,
porphyropsin (red), iodopsin (green) and cyanopsin (blue). Any one cone cell contains only one
type of opsin and is sensitive to only one color. The photoreceptors of cone cells also contain 11-
cis retinal as a component of their photoreceptor pigments.
o Vitamin A also involves in growth, differentiation and maintenance of the integrity of

specialized epithelial cells of eyes. Its deficiency produces defective epithelialization and
keratomalacia – corneal softening and opacity. Severe vitamin A deficiency leads to permanent
blindness.
 Maintenance of the integrity of epithelial cells and glycoprotein synthesis
o Vitamin A is necessary for maintenance of the structural integrity and normal permeability of
mucous membranes of the eyes, respiratory tract and lining epithelia of the gland ducts.
Vitamin A deficiency causes squamous metaplasia. Retinoid X receptors
o Retinol and retinoic acid down-regulates keratin formation in  RXR—9-cis retinoic acid
complex forms dimers with
epithelial cells.
vitamin D, thyroid, and
o Retinyl phosphate appears to serve as glycosyl donor in cell other a nuclear acting
membrane glycoprotein and GAG synthesis. hormone receptors.
 Role in regulation of gene expression and tissue differentiation  Deficiency of vitamin A

impairs vitamin D and
o Retinoic acids regulate growth, development, and tissue
thyroid hormone function
differentiation by modulating the synthesis of proteins involved
because of lack of 9-cis-
in cell growth and differentiation. Retinoic acid binds to nuclear retinoic acid to form active
receptors which then bind to response elements of DNA and receptor dimers.
 Unoccupied retinoid X
regulate the transcription of specific genes. There are two
receptors form dimers with
families of nuclear retinoid receptors: the retinoic acid receptors
occupied vitamin D and
(RAR) and the retinoid X receptors (RXR). thyroid hormone receptors
and may repress gene
expression.
 It facilitates growth of skeleton and soft tissues by promoting mucopolysaccharides synthesis,
stimulating vessel growth and production of collagen and elastic fibers.
 Retinoic acid accelerates skin cell turn over, so it is used for sunburn, acne, and wrinkles.
 It has a role in immune function as part of innate immunity by maintaining structural integrity of
mucosal membranes
 Anti-oxidant action
o β carotene acts as anti-oxidant by trapping peroxyl free radical in its conjugated alkyl structure.
o Complements anti-oxidant activity of vitamin E in membranes and other lipid rich structures.
 All-trans retinoic acid is used in the treatment of acute promyelocytic leukemia (PML) to induce
complete remissions by promoting the conversion of leukemic blast cells into mature cells.
Deficiency state
Eye manifestation
Night blindness (nyctalopia) – impaired dark adaptation
time due to reduced formation of rhodopsin
Severe vitamin A deficiency – total blindness
Xerophthalmia and corneal xerosis (squamous
metaplasia, conjunctival and corneal keratinization)
Bitot’s spot (white triangular plaques on conjunctiva)
Keratomalacia (destruction of cornea causing blindness)
Skin manifestation
Follicular hyperkeratosis or toad’s skin (rough keratinized skin
resembling ‘goosebumps’)
Increased susceptibility to infection and cancer
Impaired reproduction and skeletal growth in laboratory animals
Excess state
Hypervitaminosis A – toxic effect on CNS and liver
Hypercarotenemia – yellowish coloration of skin but not sclera
Both vitamin A excess and deficiency are teratogenic.
Daily requirement of vitamin A

Recommended dietary allowance of vitamin A is expressed as retinol equivalents (RE).
RDA of vitamin A for adults is 800 – 1000 retinol equivalents.
One retinol equivalent = 1 μg retinol = 6 µg β-carotene
Vitamin D (cholecalciferol)
Structure – sterol
1. Vitamin D2 (ergocalciferol) – derived from ergosterol
in plants by the action of UV light
2. Vitamin D3 (cholecalciferol) – derived from 7-
dehydrocholesterol in animals by the action of UV light
Sources
 Fish liver oil (cod liver oil), milk and
fortified foodstuffs, small bony fish,
butter, eggs yolk
Metabolism
 In animals, 7-dehydrocholesterol in the
epidermis layer can be converted into
cholecalciferol by UV light. It is a pro-
hormone which is converted to active
hormone 1, 25 dihydroxycholecalciferol
(1, 25 – DHCC) or calcitriol. It is done by
microsomal 25 hydroxylase in liver and
1-α hydroxylase in the kidney.
 Exogenous or dietary vitamin D is
absorbed in the duodenum along with other lipids and circulated as a constituent of chylomicrons
and finally transported to the liver in chylomicron remnants.
 Storage form in liver is mainly as 25 – hydroxycholecalciferol. It is also the major form in circulation.
 It is transported in the plasma bound to vitamin D binding globulin.
Biochemical functions of vitamin D

 1, 25 DHCC (calcitriol) is a
hormone of steroid class.
It regulates gene
expression of specific
proteins through binding
of intracellular receptors.
 It is an essential hormone
that regulates body
calcium and phosphorous
metabolism.
 Actions on intestine
o It promotes intestinal calcium absorption by inducing the calcium binding protein (calbindin D)
synthesis in intestinal mucosal cells.
o Calcitriol-receptor complex interacts with DNA and causes transcription of the gene that code
for calbindins, the calcium-binding proteins that increase intestinal absorption of calcium, thus
elevating serum calcium levels. So, it provides adequate calcium for bone mineralization.
o It facilitates phosphate absorption by stimulating phosphate transport mechanism.
 Actions on bone
o It promotes bone mineralization indirectly by provision of Ca2+ and PO43-.
o It acts on osteoblasts and osteoprogenitor cells and promotes the mineralization of bones by
deposition of calcium and phosphorus.
 Calcitriol stimulates calcium uptake for deposition as calcium phosphate.
 It also induces the expression of bone matrix proteins such as osteocalcin and osteopontin
and increases cross linking of collagen and alkaline phosphatase activity.
o It mediates osteoclastic bone resorption indirectly since these cells lack vitamin D receptors.
 Calcitriol induces the expression of a cytokine called receptor activator of NFκβ ligand
(RANKL) in osteoblasts. The RANKL released from the osteoblasts then interacts with the
receptor protein RANK, found on the cell surface of immature osteoclast precursors (also
called pre-osteoclasts), to stimulate the differentiation and maturation of the osteoclasts.
 The mature osteoclasts in turn release hydrochloric acid, alkaline phosphatase, collagenase,
and other hydrolytic enzymes and substances which dissolve and catabolize the bone
matrix. The net effect on the bone is bone demineralization and calcium mobilization.
 Actions on kidney
o Vitamin D increases reabsorption of calcium and phosphorous by distal renal tubules and
decreases its excretion.
o It inhibits its own synthesis by suppressing 1-α hydroxylase activity.
Deficiency state
Vitamin D deficiency is caused by inadequate
exposure to sunlight, insufficient dietary
intake, or impairments in the metabolic
activation (hydroxylations) of the vitamin.
Deficiency may also develop in liver disease,
kidney disease, fat mal-absorption.
Hypoparathyroidism impairs the conversion
of 25-HCC to active 1, 25 DHCC.
Certain drugs may interfere with vitamin D metabolism, e.g., prolonged use of corticosteroids,
sedatives, anti-convulsants.
Deficiency in children leads to rickets. It is characterized by soft, poorly mineralized and pliable
bones.
o Clinical features of rickets
 Delayed development  Enlargement of epiphysis at lower
 Muscle hypotonia end of radius
 Craniotabes (small unossified area  Rickety rosary (swelling of the rib at
in membranous bone of skull) costochondrial junctions)
 Frontal bossing  Bow leg, knock-knee
 Delayed anterior fontanelle closure  Pot belly
Deficiency in adults leads to osteomalacia (characterized by muscular weakness, bone tenderness
and pain and demineralization of preexisting bones resulting in softening of the bones with
increasing susceptibility to fractures).
Abnormalities of biochemical parameters in both conditions are low serum calcium and phosphate,
and elevated serum alkaline phosphatase (bone isoenzyme) activity.
Excess state
Vitamin D is stored mostly in liver and slowly metabolized. Among the vitamins, vitamin D is the
most toxic in overdoses (10 to 100 times RDA).
Toxic effects of hypervitaminosis D include demineralization of bone (resorption) and increased
calcium absorption from the intestine, leading to elevated calcium in plasma (hypercalcemia).
Hypercalcemia can impair renal function and early signs include polyuria, polydipsia and nocturia.
Prolonged hypercalcemia can result in calcium deposition in soft tissues (metastatic calcium
deposition), notably in the kidney (nephrocalcinosis), producing irreversible kidney damage.
Daily requirement of vitamin D

The daily requirement (RDA) of vitamin D is 400 International Units or 10 mg of cholecalciferol.
In countries with good sunlight, the RDA for vitamin D is 200 IU (or 5 mg cholecalciferol).
Toxicity is seen especially in those with vitamin D intakes of 10,000 IU (or more) per day for several
months.
Vitamin E (tocopherol)
Structure – 6-hydroxy chromane (tocols) with isoprenoid

side chain
 Vitamin E family includes α, β, γ, δ tocopherols
depending on the nature of side chain R1, R2 and R3. The most potent is α tocopherol.
Sources
 Richest sources are vegetable oils, e.g., wheat germ oil, sunflower oil, cotton seed oil, peanut oil,
corn oil, and also present in nuts.
Metabolism
 Among different tocopherols, 90 % of vitamin E present in humans is in the form of natural isomer, α
tocopherol.
 Vitamin E is absorbed from intestine together with dietary lipids and delivered to liver via
chylomicron. Liver can export vitamin E to the target cells via very low density lipoprotein (VLDL).
 Vitamin E is enriched in all lipid-laden structures, with highest concentrations in fat depots of
adipose tissue.
 It is mainly excreted in feces via hepato-biliary route, after the chromane ring is oxidized followed
by its conjugation with glucuronic acid.
Biochemical functions of vitamin E

 Vitamin E is lipophilic and is found in association with cellular membranes, lipoproteins and fat
deposits.
 Maintenance of the cohesion and stability of the cell membrane
o Methyl groups of tocopherols fit into pockets of phospholipids and stabilize the membrane by
physiochemical juxtaposition.
 Anti-oxidant activity
o Vitamin E serves as lipid-soluble, chain-
breaking antioxidants in cell membranes and
plasma lipoproteins. It prevents the
peroxidation of PUFA in cell membrane
phospholipids.
o It breaks the free radical chain reaction by
transferring its phenolic hydrogen to peroxyl
free radical and itself becomes phenolic free
radical (Toc-O•).
o This phenolic free radical or tocopheroxyl
radical can react with a further peroxyl radical
to form non-free radical product (tocopherylquinone) or is reduced back to active tocopherol
by vitamin C and reduced glutathione. Non-free radical product of tocopherol is then
metabolized in the liver by conjugation with glucuronic acid and excreted in the bile.
ROO• + Toc-OH  ROOH + Toc-O•
ROO• + Toc-O•  ROOH + Non-free radical product of vitamin E
Micronutrients V i t a m i n s P a g e | 10
o Antioxidant action of
tocopherol is effective at
high PO2 and thus it is
concentrated in RBC
membrane, membranes
of respiratory tracts and
the retina. So it protects
RBC from hemolysis by
oxidizing agents as well as
lung tissues from damage
by oxidants such as ozone,
nitrogen dioxide from
polluted air.
o As an antioxidant, vitamin E protects against the development of cancer, atherosclerosis and
aging process by preventing oxidative stress.
o Selenium complements the antioxidant effects of vitamin E and reduces the requirement of
vitamin E in the diet.
 Vitamin E prevents lipid peroxidation of LDL, thereby contributing in the reduction of
cardiovascular disease risk since oxidized LDL is atherogenic. Anti-atherogenic role vitamin E is also
contributed by inhibiting signaling pathway via protein kinase-C that affects cell adhesion,
archidonic acid metabolism and proliferation of vascular smooth muscle cells.
 It protects the skin against damaging effects of UV rays.
 It is required for cellular respiration– through electron transport chain (by stabilizing coenzyme Q).
 It is involved in the immune function, and also in cellular signaling and gene expression.
 It inhibits the activity of protein kinase C (PKC) which results in inhibition of proliferation of smooth
muscle cells, platelet adhesion and aggregation, and function of adhesion molecules and affects cell
adhesion as well as arachidonic acid metabolism (decrease the synthesis of leukotrienes and
increase synthesis of prostacyclins by upregulating phospholipase A2 and cyclooxygenase).
Deficiency state – It is seen in premature infants and adults with defective fat absorption. Increased
fragility of erythrocyte membranes as a result of lipid peroxidation leads to hemolytic anemia.
Deficiency may also cause thrombocytosis, peripheral neuropathy, myopathy, ataxia and neurological
symptoms such as spinocerebellar degeneration.
Excess state – Vitamin E is least toxic of all fat soluble vitamins—even 50 times the recommended
intake has been reported non-toxic. Extremely high doses of vitamin E may interfere with the blood
coagulation and enhance the effects of drugs used to oppose blood clotting, causing hemorrhage.
Daily requirement of vitamin E
Daily consumption of about 10 mg (15 IU) of α-tocopherol for man and 8 mg (12 IU) for woman is
recommended. (One mg of α-tocopherol = 1.5 IU)
Vitamin K (phylloquinones)
Structure – quinones, containing isoprenoid side chain

1. Vitamin K1 (phylloquinone) – plants
2. Vitamin K2 (menaquinone) – synthesized by intestinal bacteria
3. Vitamin K3 (menadione, menadiol) – synthetic form
Sources
 Dark green leafy vegetables, vegetable oil, cereals
 Egg yolk, meat, liver, dairy products
 It may be obtained from intestinal bacterial synthesis. But colonic
bacteria do not make a significant contribution because vitamin K is absorbed mostly in the small
intestine.
Metabolism
 The intestinal absorption of vitamin K is dependent on appropriate fat absorption and it requires
bile salts. Chylomicrons carry the absorbed vitamin to liver where it is stored. From liver, it is
released into blood circulation where it is transported in association with lipoproteins (VLDL).
 Unlike the other fat-soluble vitamins, the body stores of vitamin K are insignificant (50–100 μg), so
vitamin K is the first fat-soluble vitamin to be deficient in acute fat mal-absorption.
Biochemical functions of vitamin K

 Vitamin K is required for carboxylation of glutamate in post-translational modification of several
proteins in blood coagulation cascade, protein C, protein S and some bone matrix proteins.
Vitamin K is required to introduce Ca2+ binding sites in these calcium-dependent proteins.
 Role in post-translational modification of coagulation factors Vitamin K dependent
o Vitamin K is required for post-translational modification of carboxylation mechanism

Epoxidation of vitamin K
several proteins in coagulation cascade (clotting factor II,
hydroquinone activates a
VII, IX, X and protein C, protein S).
glutamate residue in the protein
o All these proteins are synthesized by the liver as inactive substrate to carbanion.
precursors and are activated by carboxylation of glutamate Glutamate carbanion reacts non-
residues (Glu) to form γ-carboxyglutamate (Gla) residues by enzymically with CO2 to form γ-
carboxyglutamate.
vitamin K dependent enzyme.
Vitamin K epoxide is reduced to
o These Gla residues are essential for chelation of calcium the quinone by a warfarin-
2+
during its function in coagulation since Ca binds with the sensitive reductase and quinone
negatively charged phospholipids present on the platelet is then reduced to active

hydroquinone by either warfarin
cell membrane, thereby bridging of the phospholipids to the
sensitive reductase or warfarin
Gla residue of pro-thrombin. Thus, vitamin K is essential for
insensitive quinone reductase.
synthesis of clotting factors in liver. In the presence of warfarin,
 Vitamin K is also required for the synthesis of protein C and vitamin K epoxide cannot be
reduced, but accumulates and is
protein S which require carboxylated glutamate residue for their
excreted.
function.
A high dose of vitamin K is the
 Role in the synthesis of bone and other Ca2+ dependent proteins antidote to an overdose of
o A number of proteins undergo vitamin K dependent warfarin.
carboxylation of glutamate to γ-carboxyglutamate.

 Osteocalcin and the matrix Gla protein of bone
involved in the calcification of osteoid matrix
 Nephrocalcin in kidney
 Vitamin K can be therapeutically useful as an antidote to
dicumarol and warfarin overdose and accidental
ingestion of rat poison since these drugs inhibit epoxide
reductase and warfarin sensitive vitamin K quinone
reductase which regenerates active vitamin K.
Deficiency state
Conditions predisposing to vitamin K deficiency include —
o Prolonged and excessive treatment with broad-
spectrum antibiotics (eliminate intestinal bacteria)
o Fat mal-absorption (pancreatic dysfunction, liver diseases, biliary tract obstruction, intestinal
mucosa atrophy or resection)
o Newborn infants (inefficient placental transfer, little intestinal flora, very low vitamin K in breast
milk), especially in a home-birth where a postnatal injection of vitamin K may not be given
Certain drugs such as warfarin, dicumarol can impair vitamin K function.
Deficiency of vitamin K affects blood coagulation and this leads to hemorrhagic conditions.
Prothrombin level is lowered and prothrombin time is prolonged.
It can cause hemorrhagic disease of newborn especially in premature infants.
Excess state
Ingestion of large amounts of phylloquinone and menaquinone has not been shown to cause
toxicity. The synthetic form menadione, however, is toxic if consumed in large amounts, causing
hemolytic anemia and liver damage (indicated by hyperbilirubinemia and severe jaundice).
Menadione is thought to combine with sulfhydryl groups such as those in glutathione, resulting in
glutathione oxidation and ultimately membrane damage induced by phospholipid oxidation.
Large doses of vitamin K by parenteral administration produce hyperbilirubinemia in infants.
Daily requirement of vitamin K

Vitamin K can be adequately synthesized in the gut. It is however, this source provides only about
half of a person’s needs. Suggested RDA for an adult is 70 – 140 μg/day.
Water soluble vitamins

 Water soluble vitamins are a heterogeneous group of compounds since they differ chemically from
each other. The only common character shared by them is their solubility in water.
 Water soluble vitamins serve as biosynthetic precursors of coenzymes for various enzymes,
thereby playing pivotal role in cellular metabolism. Most of the water-soluble vitamins are
converted to coenzymes which are utilized either in the pathways for energy generation or
hematopoiesis.
 Water soluble vitamins are not stored extensively in the body except vitamin B12.
 Unlike fat soluble vitamins, most water
soluble vitamins are readily excreted into
urine once their concentration surpasses the
renal threshold. Thus toxicities are rare. Their
metabolic stores are labile and depletion can
often occur in a matter of weeks or months.
Vitamin B1 (thiamin)
Structure – contains pyrimidine ring and thiazole

ring linked by a methylene bridge. Active
coenzyme form is thiamin pyrophosphate (TPP).
Sources
 Plant source – Mostly concentrated in outer coats of cereal grains. Good sources are unrefined
cereals, pulses and nuts.
 Animal source – liver kidney, lean cut pork
Metabolism
 Dietary thiamine is absorbed readily from jejunum and proximal ileum. It is absorbed predominantly
by passive diffusion and also by Na+ dependent secondary active transport, carrier mediated active
transport.
 After cellular uptake it is converted into its active form, thiamine pyrophosphate (TPP) in an ATP-
dependent reaction catalyzed by thiamine pyrophosphokinase.
 Some ferns, shellfish, fish, and species of bacteria contain thiaminase, which cleaves the pyrimidine
ring from the thiazole ring.
Biochemical functions of vitamin B1

 Active coenzyme form of thiamin is thiamin pyrophosphate (TPP).
 TPP serves as a coenzyme in several enzyme catalyzed reactions, which involve aldehyde group
transfer. These include oxidative-decarboxylation reactions and transketolase reactions.
 Central role in energy-yielding metabolism (especially carbohydrate metabolism)
o Thiamin pyrophosphate serves as coenzyme for three multi-enzyme complexes that catalyze
oxidative decarboxylation reactions which are important for energy generation:
 Pyruvate dehydrogenase in carbohydrate metabolism,
 α-ketoglutarate dehydrogenase in the citric acid cycle and
 Branched-chain keto-acid dehydrogenase involved in the catabolism of branched chain
amino acids.
o Oxidative decarboxylation reactions play a key role in energy generation of the cells. Pyruvate
which is the end product of glucose oxidation is converted to acetyl CoA which can only then
enter into CAC for complete oxidation of glucose in the cells. α-ketoglutarate dehydrogenase
reaction is part of the CAC cycle which is the final common oxidation pathway in oxidation of
metabolic fuel. TPP acts as coenzyme for respective enzymes in the oxidative decarboxylation
of α-keto acids. Thus, vitamin B1 deficiency will affect energy generation process in the cells,
particularly affecting the tissues with high energy requirement such as brain, heart and kidney.
 TPP serves as coenzyme for transketolase reaction in pentose phosphate pathway or hexose
monophosphate shunt (concerned with production of ribose and NADPH).
Assessment of Vitamin B1 status

Determination of erythrocyte transketolase activity
Determination of plasma lactate or pyruvate after oral
glucose load
High risk persons for vitamin B1

deficiency
Elderly persons
Malnourished persons
Persons relying exclusively on
polished rice for food
Chronic alcoholics
Infants born to mother suffering
from thiamin deficiency
Deficiency states
Since TPP serves as coenzyme for pyruvate dehydrogenase enzyme complex and α-ketoglutarate
dehydrogenase reaction of CAC, loss of these enzyme complex activities compromises cellular
energy generation. Deficiency will affect tissues with high energy requirement such as brain, heart
and kidney.
Loss of pyruvate dehydrogenase activity also causes accumulation of pyruvate and lactate.
The early symptoms of thiamine deficiency are anorexia (loss of appetite), nausea, constipation,
mental confusion, peripheral neuritis, muscle fatigue and irritability. Peripheral neuropathy, mental
confusion and ataxia develop in moderate deficiency conditions and severe deficiency presents with
severe neuromuscular and cardiovascular disorders.
Severe vitamin B1 deficiency results in a condition called beriberi.
Dry beriberi
o It is characterized primarily by chronic peripheral neuritis, severe muscle weakness and fatigue
which may or may not be associated with heart failure and edema.
o In longstanding cases, there is degeneration and demyelination of sensory and motor nerves
and severe wasting of muscles.
Wet beriberi
o It is severe thiamin deficiency state in which cardiovascular system is affected in addition to
neurological symptoms (heart failure and metabolic abnormalities predominate).
o It is characterized primarily by edema of extremities, heart enlargement and cardiac failure.
Wernicke encephalopathy with Korsakoff psychosis
o It is mostly seen in chronic alcoholics. In chronic alcoholics, the nutritional deficiencies result
from either poor intake of food or mal-absorption of nutrients from intestine.
o Wernicke-Korsakoff syndrome is characterized by anorexia, nausea, vomiting, depression,
ophthalmoplegia (paralysis of the ocular muscles), nystagmus (constant, involuntary eye-ball
movement), ataxia (impaired muscle coordination), loss of recent memory, mental confusion,
peripheral paralysis, muscular weakness, etc.
Infantile beriberi
o It is seen in breast-fed infants born to mothers suffering from thiamin deficiency.
o In acute condition, the infant may die due to cardiac failure.
The role of TPP in pyruvate dehydrogenase means that thiamin deficiency impairs the conversion of
pyruvate to acetyl CoA. In subjects on a relatively high carbohydrate diet, this results in increased
plasma concentrations of lactate and pyruvate, which may cause life-threatening lactic acidosis.
Thiamin deficiency due to thiaminase and pyrithiamine
o Thiamin can be destroyed if the diet contains thiaminases. These enzymes are present in raw
fishes and sea foods, and they are thought to contribute to thiamin deficiency.
o Pyrithiamine, a structural analogue and an antimetabolite of thiamin is found in certain plants
like ferns. Horses and cattle often develop thiamin deficiency (fern poisoning) due to the
overconsumption of the plant fern.
Daily requirement of vitamin B1
Thiamin is related to energy metabolism; therefore its dietary requirement depends on the caloric
intake. The recommended daily allowance is 0.4 mg/1000 kcal for adults with the recommendation
for a minimum absolute intake of 0.5 mg/day.
Recommended daily intake of thiamin is 1.0 to 1.5 mg/day for adults. Requirement is increased with
increased muscular activity, dietary carbohydrates and in pregnancy and lactation.
Vitamin B2 (riboflavin)
Structure
 Heterocyclic isoalloxazine ring attached
to ribitol (a sugar alcohol)
 Active coenzyme form
o Flavin adenine dinucleotide (FAD)
o Flavin mononucleotide (FMN)
Sources
 Plant source – germinating seeds and
young plants, peas, beans
 Animal source – egg yolk, liver, organ meat, muscles
 Main dietary sources of riboflavin are milk and dairy products.
Metabolism
 Riboflavin is a heat-stable, colored, fluorescent molecule which decomposes in visible light.
 Riboflavin is ingested in the form of flavoproteins from which FAD and FMN components are
released in the stomach. Free riboflavin is released in the intestine which is then absorbed by
sodium dependent active transport system.
 Activation of riboflavin is via an ATP-dependent enzyme system resulting in the production of active
form FAD and FMN.

 Riboflavin is a precursor of active coenzymes form FMN and FAD, which serve as prosthetic group
for oxido-reductase enzymes involved in carbohydrate, protein, lipid, nucleic acid metabolism and
electron transport chain. Enzymes containing riboflavin as prosthetic group are called flavin-
dependent enzymes or flavoprotein enzymes.
 Riboflavin has a central role in energy yielding metabolism because flavin coenzymes serve as high
energy electron carriers in oxidation-reduction reactions involved in energy metabolism.
 FMN-dependent enzymes
o L-amino acid oxidase in deamination of amino acids
in amino acid catabolism
o NADH-Ubiquinone oxidoreductase or NADH
dehydrogenase enzyme complex (as a component
of complex I of electron transport chain)
 Role of FAD-dependent enzymes in energy metabolism
o Dihyrolipoyl dehydrogenase of pyruvate
dehydrogenase enzyme complex in oxidative decarboxylation of pyruvate to acetyl CoA for
complete oxidation of glucose
o Succinate dehydrogenase and α-ketoglutarate dehydrogenase enzyme complex in CAC
o Acyl CoA dehydrogenase in β-oxidation of fatty acids
o FADH2 derived from oxidation reactions (succinate dehydrogenase and acyl CoA dehydrogenase
reactions) donate its high energy electrons to the electron transport chain for ATP production
via respiratory chain linked oxidative phosphorylation (1.5 ATP per one molecule of FADH2
oxidation in ETC).
High risk persons for riboflavin
 Other FAD dependent enzymes
deficiency
o Xanthine oxidase in purine nucleotide catabolism Chronic alcoholics
o Glutathione reductase in reduction of oxidized glutathione Inadequate diet
o Involved in hydroxylation reactions as a constituent of Newborn infants with jaundice who

are treated with phototherapy (due
microsomal cytochrome P450 monooxygenase enzyme
to light sensitivity)
system (as prosthetic group of NADPH-cytochrome P450 Patients with hypothyroidism
reductase enzyme) (affecting the conversion of
riboflavin to FMN and FAD)
Deficiency states
Riboflavin deficiency is quite rare as it has a wide distribution in food stuffs. It is usually associated
with deficiency of other vitamins.
Characteristic symptoms of riboflavin deficiency
o Angular stomatitis (fissures at the angles of the mouth)
o Cheilosis (swollen and cracked lips)
o Glossitis (tender and magenta colored tongue)
o Seborrheic dermatitis (rough and scaly skin around nasolabial and scrotal areas)
o Corneal vascularization and inflammation
Assessment of riboflavin status – determination of erythrocyte glutathione reductase activity

Daily requirement of riboflavin for an adult is 1.2 – 1.7 mg.
Higher intakes (by 0.2-0.5 mg/day) are advised for pregnant and lactating women.
Vitamin B3 (niacin)
Structure
 Nicotinic acid is monocarboxylic acid
derivative of pyridine.
 Nicotinamide is amide derivative of the
nicotinic acid.
 Active coenzyme forms are nicotinamide
adenine dinucleotide (NAD+) and
nicotinamide adenine dinucleotide
phosphate (NADP+).
Sources
 Animal sources – liver, meat, eggs, milk,
fish, yeast
 Plant sources – whole grains, cereals, pulses, soya beans, peanut, leafy vegetables
 Body can synthesize small quantities of niacin coenzymes from amino acid tryptophan.
Metabolism
 Coenzymes of niacin are hydrolyzed in intestinal tract
and both nicotinic acid and nicotinamide forms are
absorbed readily by passive diffusion.
 Conversion of nicotinamide and nicotinic acid to active coenzyme forms NAD+ and NADP+ requires
ATP. NAD can be converted to niacin by various reactions and thus, niacin is recycled.
 Excess nicotnamide and nicotinic acids are metabolized in the liver (methylation, oxidation, etc.)
and excreted into the urine as methyl nicotinamide or its oxidation products, pyridones.
In the process of electron/hydrogen transfer in oxidation-reduction reaction, NAD+ or NADP+ accepts

only one hydrogen atom fully. The other hydrogen is ionized. Only the electron is accepted. Thus NAD +
accepts one H atom and one electron (e-) (hydride ion transfer), to form NADH or NADPH. The
hydrogen ion (H+) is released.
NAD+/NADP+ + H2  NAD+/NADP+ + e- + H + H+  NADH/NADPH + H+

 Niacin is the precursor of coenzymes, NAD+ and NADP+ which act as coenzymes for many oxido-
reductases.
 Role of NAD+ in energy yielding metabolism
o NAD+ serves as coenzyme in various dehydrogenase enzymes in oxidative pathways of
carbohydrate, lipid and protein metabolism. They carries high energy electrons derived from
these oxidation pathways and donate its electrons to the electron transport chain for ATP
production via respiratory chain linked oxidative phosphorylation. (2.5 ATP per one molecule of
NADH oxidation in ETC).
o NAD+ linked dehydrogenase enzymes in oxidative pathways
 Glyceraldehyde-3-phosphate dehydrogenase in glycolysis
 Lactate dehydrogenase in anaerobic glycolysis
 Pyruvate dehydrogenase enzyme complex in oxidative decarboxylation of pyruvate to
acetyl CoA
 Isocitrate dehydrogenase, α-ketoglutarate dehydrogenase enzyme complex and malate
dehydrogenase in citric acid cycle
 β-Hydroxy acyl-CoA dehydrogenase in β-oxidation of fatty acids
 Glutamate dehydrogenase in oxidative deamination of amino acid catabolism
 NADP+ linked dehydrogenase enzymes (e.g., glucose 6-phosphate dehydrogenase in pentose
phosphate pathway) produces NADPH which in turn serves as coenzyme in NADPH linked reductase
enzymes in reductive synthesis pathways such as fatty acid synthesis, cholesterol synthesis, steroid
hormones synthesis etc.
 NAD+ is the source of ADP-ribose for the ADP-ribosylation of proteins and polyADP-ribosylation of
nucleoproteins involved in the DNA repair mechanism.
 Niacin can be used in treatment of hyperlipidemia. Niacin at doses of 1.5 g/day strongly inhibits
lipolysis in adipose tissue, the primary producer of circulating free fatty acids (FFAs).
Deficiency states
Niacin deficiency initially produces superficial glossitis but may
progress to pellagra, which is characterized by the three Ds:
o Diarrhea
o Dermatitis (sunburn-like skin lesions in areas of body
exposed to sunlight and to pressure)
o Dementia (neurological symptoms due to actual
degeneration of nervous tissues)
Untreated pellagra is fatal.
Pellagra is mostly seen among people whose staple diet is corn
or maize. Niacin present in maize is unavailable to the body as it
is in bound form, and tryptophan content is low in maize.
Certain drugs, e.g. the anti-tuberculosis drug isoniazid,
predispose to niacin deficiency.
Very high doses of niacin can cause hepatotoxicity.

The daily requirement of niacin for an adult is 15-20 mg and for children, around 10-15 mg.
Limited quantities of niacin can also be obtained from the metabolism of tryptophan. For every 60
mg of tryptophan, 1 mg equivalent of niacin can be generated. Tryptophan can only provide about
10% of the total niacin requirement.
Biotin (Vitamin B7)
Structure – heterocyclic sulfur

containing monocyclic acid. It
contains fused imidazole and
thiophene ring with a valeric acid
side chain.
Sources – widely distributed in both animal and plant foods. Rich

sources are liver, kidney, egg yolk, milk, tomatoes, grain.
Metabolism
 Biotin can be synthesized by the intestinal flora.
 Biotin is mostly bound to dietary protein by an amide linkage.
The linkage is cleaved prior to absorption of biotin from
intestine.
 Raw egg white contains glycoprotein avidin, which binds the imidazole group of biotin tightly;
thereby preventing its absorption from the intestine.
 Enzyme-bound biotin, biocytin is an active form of biotin. Biotin is covalently bound to ε-amino
group of lysine of the enzyme to form biocytin.
Biochemical functions of biotin

 Biotin is covalently bound to
ε-amino group of lysine of
carboxylase enzyme to form
biocytin which serves as
coenzyme for carboxylation
reactions.
 Biotin serves as a carrier of the activated CO2 (carboxyl group) in carboxylation reactions,
transferring it to various acceptor molecules, e.g. acetyl CoA, pyruvate and propionyl CoA, etc.
 Biotin-dependent carboxylation reactions
o Conversion of acetyl-CoA into malonyl-CoA catalyzed by acetyl-CoA carboxylase in fatty acid
synthesis
o Conversion of pyruvate into oxaloacetate, catalyzed by pyruvate carboxylase in
gluconeogenesis pathway and replenishment of oxaloacetate for CAC
o Conversion of propionyl-CoA to D-methyl malonyl-CoA catalyzed by propionyl-CoA carboxylase
in the pathway of conversion of propionate to succinate and in the branched chain amino acid
catabolism
 Non-coenzymes roles
o Biotin also has a role in regulation of the cell cycle, acting to biotinylate key nuclear proteins.
o Biotin influences multiple cellular functions through biotinylation of proteins, especially non-
histone and histone proteins.
Deficiency state
 Biotin deficiency is uncommon, since it is well distributed in foods and also supplied by the intestinal
bacteria. But deficiency can occur due to high consumption of raw eggs. The raw egg white contains
a glycoprotein–avidin, which tightly binds with biotin and blocks its absorption from the intestine.
An intake of about 20 raw eggs per day is needed to produce biotin deficiency symptoms in humans.
 Deficiency symptoms include nausea, anorexia, glossitis, alopecia (loss of hair), depression,
hallucination, muscular pain and dermatitis.
 Certain inherited single or multiple carboxylase deficiencies can also lead to apparent biotin
deficiency syndrome.
Daily requirement of biotin

 A daily intake of about 150–300 µg is recommended for adults.
Lipoic acid
Structure – sulfur containing fatty acid. It exists in oxidized and reduced forms.
Sources – widely distributed. Liver and yeast are rich sources.
Biochemical functions of lipoic acid

 Lipoic acid serves as coenzyme of the multienzyme complexes involved in oxidation pathways such
as, pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. Active coenzyme form is
lipoamide. It involves in transfer of acyl group and
hydrogen.
Deficiency – not found in humans.
Vitamin B5 (pantothenic acid)
Structure – consists of pantoic acid and β-alanine held

together by peptide linkage.
Sources – widely distributed in plant and animal tissues.

Vitamin occurs in nature as a component of coenzyme A and
acyl carrier protein.
Active forms - Coenzyme-A (CoA-SH) and Acyl carrier protein (ACP). Its reactive group is sulfhydryl
group or thiol group (—SH ).

 Pantothenic acid is the precursor for active coenzyme forms coenzyme A and ACP. Thiol group (-SH)
in CoA and ACP act as a carrier of acetyl and acyl groups.
 Coenzyme A plays a central role in metabolism of carbohydrate, lipid and proteins. It is regarded as
a coenzyme of metabolic integration since it involves in both oxidative pathways and biosynthesis
reactions.
 Role of coenzyme A in energy metabolism
o CoA acts as coenzyme in some enzymes involved in fuel oxidation pathways. Acetyl and acyl
groups are accepted by CoA molecules during these oxidation pathways (oxidative
decarboxylation of pyruvate, oxidative decarboxylation of α-ketoglutarate in CAC and thiolase
reaction in fatty acid oxidation). Formed acetyl CoA molecules then enter into CAC for complete
oxidation and energy production. Acetyl CoA, succinyl CoA and fatty acyl CoA are very
important molecules which produce ATP by being metabolized.
 The thio ester bond in acyl-CoA is a high energy bond. These acyl groups are transferred to other
acceptors thereby involving in many biosynthetic pathways. CoA plays important role in
biosynthesis of various biomolecules like fatty acid, phospholipids, cholesterol, acetylcholine, etc.
 CoA is essential for acetylation, acylation (myristoylation) and isoprenylation of proteins. These
reactions are crucial for some functions of proteins like membrane anchoring, signaling etc.
 ACP is a component of fatty acid synthase enzyme complex and participates in fatty acid synthesis
reactions.
Deficiency state
 Deficiency is rare. Deficiency of
pantothenic acid is thought to
occur more often in conjunction
with multiple nutrient
deficiencies, as in malnutrition.
It is seen in chronic alcoholics
and renal dialysis patients.
 Clinical features of deficiency
state includes burning foot
syndrome, paresthesia of hand
and foot.
Daily requirement of pantothenic acid
 The requirement of pantothenic acid for humans is not clearly known. A
daily intake of about 5-10 mg is advised for adults.
Vitamin B6 (pyridoxine)
Structure – pyridine derivatives, pyridoxine (major), pyridoxal and

pyridoxamine.
Source
 Animal source – liver, fish, milk, meat
 Plant source – whole grains, nuts, legumes, wheat, corn, cabbage, leafy
vegetables
 Intestinal bacteria synthesis also provide vitamin B6.
Metabolism
 Pyridoxine is the major form in the diet and phosphorylated pyridoxine
vitamers are hydrolyzed by intestinal membrane alkaline phosphatase and dephosphorylated forms
are absorbed. Most tissues contain pyridoxal kinase which re-synthesizes the phosphorylated forms
required for biochemical reactions.
 Pyridoxic acid is the principal excretory form of the vitamin in urine.

 Pyridoxal phosphate is a coenzyme for many enzymes
involved in amino acid metabolism.
 Transamination reaction
o Transamination is the transfer of an amino group (—NH2)
from an amino acid to α-keto acid. It is catalyzed by
transaminases and PLP acts as coenzyme, e.g., aspartate
transaminase (AST) and alanine transaminase (ALT).
o Transamination reaction is important for
the synthesis, catabolism and
interconversion of amino acids. Pyridoxal
phosphate (PLP) acts as a carrier of amino
group during the transamination reaction.
 Decarboxylation reaction
o PLP acts as coenzyme in the decarboxylations of some amino acids in which amino acids are
decarboxylated to corresponding amines. This reaction is important for the synthesis of some
neurotransmitters. Some important biogenic amines whose synthesis require PLP are given
below —
 Glutamate → GABA (Gamma Amino Butyric acid), GABA is an inhibitory neurotransmitter,
and hence in B6 deficiency, especially in children, convulsions may occur.
 Dopa → dopamine which is the catecholamine neurotransmitter in the nervous system.
 5-hydroxy tryptophan → Serotonin
 Histidine → Histamine, which is the mediator of allergy and anaphylaxis
 Condensation reaction
o ALA synthase that catalyzes the condensation of glycine and
succinyl CoA in the rate-limiting step of heme biosynthetic
pathway requires PLP as a coenzyme. This accounts for anaemia seen in vitamin B6 deficiency.
o PLP is required for the synthesis of sphingosine (a component of sphingolipids) in which the
amino acid serine condenses with palmitoyl-CoA in a reaction catalyzed by a PLP-dependent
transferase.
 PLP is required for niacin coenzyme (NAD+ /NADP+ ) synthesis from tryptophan.
 As an integral component of muscle glycogen phosphorylase
o PLP is required for glycogen phosphorylase activity which involves in glycogen breakown. PLP
is covalently linked to a lysine residue and stabilize the enzyme glycogen phosphorylase, where
the phosphate group is catalytically important. More than 70% of total PLP content of the body
is in muscles, where it is a part of the phosphorylase enzyme.
 PLP is important in steroid hormone action. PLP removes the hormone-receptor complex from DNA
binding, terminating the action of steroid hormones. In vitamin B6 deficiency, there is increased
sensitivity to the actions of low concentrations of estrogens, androgens, cortisol, and vitamin D.
 Vitamin B6 at pharmacological dose (100 – 150 mg) can produce anti-emetic effect.
Deficiency state
Because of its role in amino acid metabolism, vitamin B6 requirement increases with protein intake.
High risk persons for deficiency include elderly persons, infants, chronic alcoholics, persons taking
anti-TB drug isoniazid (INH), contraceptives.
Mild form of deficency causes irritability, nervousness, and depression; progressing to peripheral
neuropathy, convulsion and coma in severe deficiency. These symptoms are due to decreased
production of neurotransmitters, such as catecholamines, GABA and serotonin. Severe deficiency of
pyridoxine causes epileptic seizures (convulsions) in infants due to reduced production of GABA.
Demyelination of nerves causes peripheral neuropathy. Since vitamin B6 is required for synthesis of
sphingolipids needed for myelin sheath formation.
Severe deficiency is also associated with sideroblastic anemia. Vitamin B6 deficiency causes
hypochromic microcytic anemia due to decreased heme synthesis since PLP is required for the
synthesis of heme.
Assessment of pyridoxine status

Measurement of erythrocyte aspartate aminotransferase activity
Tryptophan loading test involves measurement of urinary excretion of xanthurenic acid following a
loading dose of tryptophan. Xanthurenic acid excretion is increased in vitamin B6 deficiency.

The requirement of pyridoxine for an adult is 1.6 – 2.2 mg/day.
Requirements increase during pregnancy and lactation.
Vitamin B12 (cobalamin)
Structure – contains corrin ring with a cobalt ion at the centre.

Dimethyl benzimidazole group is attached to cobalt. Cobalt can
be attatched to substituent groups forming: cyanocobalamin,
hydroxycobalamin, methyl cobalamin, 5-deoxy adenosyl
cobalamin.
Sources
 It is synthesized only by microorganisms. Foods of
animal origin are the only source for vitamin B12.
 The rich sources are liver, kidney, meat, milk and eggs.
Metabolism
 Gastric acid and pepsin release the vitamin from protein
binding in food and make it available to bind to
cobalophilin, a binding protein secreted in the saliva. In
the duodenum, cobalophilin is hydrolyzed, releasing the
vitamin for binding to intrinsic factor secreted by
parietal cells of the stomach.
 Cobalamin bound to intrinsic factor is absorbed in the
ileum. After absorption, it is transported to the tissues
via blood stream bound to a plasma protein known as
transcobalamin II.
 It is stored in the liver bound to transcobalamin I.
Vitamin B12 is the only water soluble vitamin that is stored in significant amounts in the liver.

 Active coenzyme forms are methyl cobalamin and 5’ deoxyadenosyl cobalamin. Only two human
enzyme systems are known to require vitamin B12 coenzyme.
 Conversion of homocysteine to methionine
o Methyl cobalamin acts as coenzyme for methionine synthesis and recycling of
tetrahydrofolates. In this reaction, methyl cobalamin serves as coenzyme for methionine
synthase in which methyl cobalamin donates a methyl group to homocysteine to form
methionine. Cobalamin then receives the methyl group from methyl tetrahydrofolate to form
active tetrahydrofolate (THF) which becomes available for
purine, pyrimidine nucleotide and nucleic acid synthesis.
 Isomerization of methylmalonyl-CoA to succinyl-CoA
o 5’ deoyadenosyl cobalamin is coenzyme for methyl
malonyl CoA mutase which converts methyl malonyl CoA
to succinyl CoA. This reaction involves in catabolism of
propionyl CoA derived from catabolism of odd number
chain faty acids and branch-chain amino acids. Succinyl
CoA formed may enter into CAC or used in heme synthesis.
Deficiency states
Pernicious anemia
Liver can store up to a 6-year supply of vitamin B12; thus B12
It is caused by failure of the
deficiencies are extremely rare. absorption of vitamin B12 due to
Deficiency can be occasionally seen in strict vegans, patients failure of intrinsic factor
with ileal dysfunction or ileal resection, intrinsic factor secretion caused by auto-
immune disease affecting parietal
deficiency due to autoimmmune parietal cells destruction
cells or from production of anti-
or partial or total gastrectomy or due to old age. intrinsic factor antibodies.
Vitamin B12 deficiency leads to reduction in both methyl Functional folic acid deficiency
malonyl CoA mutase and methionine synthase activity, occurs as a result of impaired folic
acid metabolism due to vitamin B12
leading to methyl malonyl aciduria, homocysteinuria, and
deficiency.
trapping of folate as methy THF (folate trap) thereby
It leads to disturbed erythropoiesis,
reducing active THF concentration. causing immature RBC precursors to
Vitamin B12 deficiency gives rise to hemopoietic and be released into the circulation
neurological symptoms. (megaloblastic anemia).

It also causes irreversible
Neurological symptoms (Neuropathy)
degeneration of the spinal cord
o Neurological disorders seen in B12 deficiency are due to as a result of failure of methylation
progressive demyelination of nervous tissue. of one arginine residue in myelin
o Accumulation of methylmalonyl CoA interferes with basic protein due to methionine

deficiency in CNS.
myelin sheath formation in 2 ways:
 Methylmalonyl CoA is a competitive inhibitor of malonyl CoA in fatty acid biosynthesis.
Since myelin sheath is continually turning over, any severe inhibition of fatty acid synthesis
will lead to its eventual degeneration.
 In the residual fatty acid synthesis that does occur, methylmalonyl CoA can substitute for
malonyl CoA in the reaction sequence, leading to branched-chain fatty acids, which might
disrupt normal membrane structure.
o Subacute combined degeneration of the cord can occur probably secondary to a relative
deficiency of methionine in the cord.
Hematological symptoms
o Megalobalstic anemia associated with B12 deficiency is thought to be due to the effect of B12 on
folate metabolism. Vitamin B12 dependent homocysteine to methionine conversion appears to
be the only major pathway by which methyl THF can return to the active THF pool. Essentially all
the folate becomes trapped as methyl THF. Thus, in B12 deficiency, there is deficiency of THF
derivatives (functional folate deficiency) for purine and dTMP biosynthesis and subsequently
decreased DNA synthesis, arrest of cells in S phase and a characteristic megaloblastic change in
nuclei of rapidly dividing cells. The block in DNA synthesis slows down cell division, maturation
of RBC and cause megaloblastic macrocytic anemia.
Giving folate alone in a case of megaloblastic anemia due to vitamin B12 deficiency can over the
anemia associated with B12 deficiency but may trigger or aggrevates neurological symptoms. Thus,
vitamin B12 must be supplemented when folate treatment is given in megaloblastic anemia.
Assessment of vitamin B12 status

The measurement of urinary methylmalonic acid (elevated) level and estimation of serum B12 level
are used to assess B12 deficiency.

A daily intake of about 3 μg of vitamin B12 is adequate to meet the adult requirements. For children,
0.5 – 1.5 μg/day is recommended. During pregnancy and lactation, the requirement is 4 μg/day.
Folic acid
Structure – contain pteridine ring, para-

amino benzoic acid (PABA) and
glutamin acid residues (1 – 7). Pteroyl
glutamic acid contain one glutamic acid residue.
Sources – widely distributed and rich sources are green leafy

vegetables, whole grains and cereals, yeast, liver, kidney.
Metabolism
 The folates in foods may have up to seven additional glutamate residues linked by γ-peptide bonds.
Before polyglutamates can be absorbed, they must be hydrolyzed by glutamyl hydrolyase in the
small intestine.
 It can also get from degradation of histidine, serine, choline, glycine and methionine. Folic acid is
stored in liver as pentaglutamyl conjugate.
 Folic acid is physiologically inactive until it is reduced to tetrahydrofolic acid (THF) by the action of
folate reductases which uses NADPH as hydrogen donor. The main circulating form is
monoglutamate-N5-methyl THF derivative.
Biochemical functions of folic acid
 Active coenzyme form of folic acid is tetrahydrofolate
(THF).
 THF is the carrier of activated one carbon unit (formyl,
formimino, methenyl, methylene, methyl) in one carbon
metabolism. The one carbon unit binds with THF at N5 or
N10 or on both N5 and N10 of pteroyl ring.
 THF serve as coenzymes for transfer of one carbon units in
a variety of reactions involved in amino acid and nucleic
acid metabolism.
 Role of THF in nucleic acid metabolism
o Methenyl THF and formyl THF contribute carbon atom
for C-2 and C-8 of purine ring in purine nucleotides
synthesis. Purine nucleotides are used for DNA and RNA
synthesis.
o Synthesis of pyrimidine nucleotide dTMP from dUMP
requires methylene THF as a coenzyme for thymidylate
synthase enzyme. This reaction provides dTMP for DNA
synthesis.
o Thus, folate coenzyme plays a central role in the
biosynthesis of nucleic acids. Rapidly dividing cells have high
requirement for folic acid due to its role in DNA synthesis.
 Role of THF in amino acid metabolism
o Synthesis of methionine from homocysteine - Together with
B12, it takes part in synthesis of methionine.
o Catabolism of histidine – During the catabolic pathway of
histidine to α-ketoglutarate, THF is required to accept
formimino group from formimino-glutamate (FIGLU). In case
of folic acid deficiency, FIGLU accumulates and is excreted in
urine.
o Serine-glycine interconversion
Deficiency states
Folate trap or functional folate deficiency
Folic acid deficiency is seen in the elderly as a
 During methionine synthesis, methyl group
result of poor diet and poor absorption. It is also bound to cobalamin is transferred to
seen in chronic alcoholics, women taking oral homocysteine to form methionine and the
cobalamin then removes the methyl group from
contraceptives and persons taking drugs that
methyl THF to form THF.
interfere folic acid metabolism e.g.,
 This step is essential for the liberation of
methotrexate, anticonvulsants. There are many free THF and for its repeated use in one carbon
causes of folate deficiency which include metabolism.
inadequate intake, impaired absorption,  In vitamin B12 deficiency, conversion of

methyl THF to free THF is blocked and thus,
impaired metabolism secondary to drug or
most of folic acid of the body is irreversibly
vitamin B12 deficiency and increased demand “trapped” as methyl THF. There is functional
(e.g., pregnancy, lactation). folate deficiency due to the lack of free THF.
Megalobalstic macrocytic anemia This is called folate trap.
o Folic acid deficiency causes decreased DNA synthesis due to Structural analogues of folate
decreased availability of purine nucleotides and dTMP. This They competitively inhibit
leads to megaloblastic dysplasia of cells in bone marrow. dihydrofolate reductase and

block the formation of THF,
The block in DNA synthesis also slows down cell division,
thereby reducing the biosynthesis
thus the production of erythrocytes, and cell maturation,
of purines, thymine nucleotides
causing the appearance of macrocytic erythrocytes with and hence DNA synthesis is
fragile membranes with increased susceptibility to impaired. This results in the
blockage of cell proliferation.
hemolysis.
Since rapidly dividing cells have
Neural tube defects
high requirements for folate, they
o Since, folate is required for the formation of neural tube in exhibit selective toxicity towards
early stage of gestation, the folate deficiency during early rapidly growing cells such as
stage of pregnancy increases the risk of neural tube defect. bacteria and cancer cells. Thus,
folic acid antagonists are used as
anti-cancer agents (methotrexate)
and antibiotics (trimethoprim).
o This includes anencephaly, spina
bifida. Folate supplementation
during the periconception period
(4 weeks before and until 8
weeks after conception) prevents
neural tube defects since closure of neural tube occurs between 22-28 days after conception.
Hyperhomocysteinemia
o Due to folic acid deficiency, methylation of homocysteine to methionine is impaired which leads
to hyperhomocysteinemia. Increased level of homocysteine is a risk factor for cardiovascular
disease (increase risk of thrombosis, atherosclerosis, hypertension, etc.).
Assessment of folate status

Histidine loading test or FIGLU excretion test
o Histidine is normally metabolized to formimino glutamic acid (FIGLU) from which formimino
group is removed by THF. Therefore in folate deficiency, FIGLU is excreted in urine.
Daily requirement of folic acid

The daily requirement of folic acid is around 200 μg .
Higher intakes are recommended during pregnancy (400 μg/day) and lactation (300 μg/day).
Vitamin C (ascorbic acid)
Structure – it is a derivative of hexose sugar, L-

glucose. Only the L form possesses the vitamin
activity.
Sources – citrus fruits (e.g., lemons, guava,

tomatoes), growing point of vegetables.
Metabolism
Animals can synthesize ascorbic acid from
uronic acid pathway except primates (human,
chimpanzee), guinea pigs, fruit-eating bats due
to the lack of L-gulonolactone oxidase.
Vitamin C is labile and easily destroyed by redox metal ions, increased pH, heat and light. Dietary
ascorbic acid is absorbed by sodium-dependent transporter in intestine.
Dehydroascorbic acid is spontaneously hydrated to inactive 2,3 diketogulonic acid. This compound is
metabolized to oxalic acid and excreted in urine.
Biochemical functions of vitamin C
 Active forms of vitamin C are ascorbic acid (reduced) and dehydroascorbic acid (oxidized). It is a
powerful oxidizing and reducing agent. Ascorbic acid functions as a reducing agent in many
metabolic processes.
 Vitamin C functions as external reductant in the hydroxylation reactions by maintaining the metal
cofactors of enzymes in reduced state. It serves as cofactors for the copper-containing and iron-
containing hydroxylases involved in collagen synthesis, catecholamine synthesis, steroid hormone
synthesis and bile acid synthesis.
 Role in collagen synthesis
o Newly synthesized collagen must undergo
extensive post-translational modification to
become mature collagen fiber. This include
hydroxylation of proline and lysine in
procollagen to hydroxyproline and
hydroxylysine by prolyl hydroxylase and lysyl
hydroxylase.
o These hydroxylase enzymes are iron-containing, ascorbate-requiring hydroxylases in which
ascorbic acid acts as an reductant to reduce ferric iron to ferrous iron for the functional
catalytic activity of these hydroxylases.
o Hydroxyproline and hydroxylysine are essential for the proper collagen cross-linking to form
collagen triple helix that maintains collagen strength and stability.
o Thus, vitamin C plays a role in the formation of intercellular matrix of bones, cartilages,
connective tissue, wound healing and capillary integrity. In absence of vitamin C, newly
synthesized collagen cannot form triple helix properly, which accounts for the prominent
connective tissue abnormalities of scurvy.
 Catecholamine synthesis
o Dopamine β-hydroxylase is a copper-containing
enzyme involved in the synthesis of norepinephrine
from tyrosine in the adrenal medulla and central
nervous system. During hydroxylation, Cu+ is oxidized to
Cu2+; reduction back to Cu+ specifically requires
ascorbate.
 Steroid hormone synthesis
o Adrenal cortex contains large amount of vitamin C
which are depleted upon stimulation by ACTH. Cortisol
synthesis involves mutiple hydroxylation steps that utilizes vitamin C.
 Bile acid synthesis from cholesterol
o During bile acid synthesis in liver mitochondria, 7α-
hydroxylase reaction requires ascorbic acid.
 Ascorbate also participates in tyrosine catabolism and carnitine synthesis.
 It enhances intestinal absorption of non-heme iron, by reducing it to ferrous state.
 It participates in immune functions.
 Anti-oxidant activity of vitamin C
o It is involved in scavenging free radicals
(peroxy free radical ROO˚). Thus, it
functions to protect lipids against
peroxidation.
o It regenerates other antioxidants such
as vitamin E and glutathione by reducing phenolic radical of vitamin E and oxidized glutathione
to functional tocopherol and reduced glutathione (GSH) respectively.
o Since vitamin C protects lipids peroxidation reactions, it reduces the risk of atheroscelrosis,
cardiovascular disease by preventing the oxidation of LDL.
o Vitamin C reduces superoxide anion to less reactive H2O2 and it also neutralizes hydroxyl
radical to water.
o It inhibits formation of potentially carcinogenic nitrosamines from dietary nitrite and nitrate
during digestion.
o It reduces ferric iron in methemoglobin to ferrous iron.
Deficiency state
Deficiency of ascorbic acid causes scurvy. Symptoms of scurvy are related to deficient collagen
formation as a result of defective hydroxylation of lysine and proline residues in procollagen which
is required for proper collagen cross-linking to maintain collagen
strength and stability.
Scurvy is characterized by swollen bleeding gums, loosening of
teeth, bleeding into the skin and deep tissues, poor wound healing,
easy fracturability of bones and anemia.
Inability to maintain bone matrix in association with demineralization results in osteoporosis.
Milder forms of deficiency are more common and the manifestation of such includes easy brusing
and the formation of petechiae, both due to increased capillary fragility.
The reduction in immunity also results because of its role in normal leukocyte function.
Daily requirement of vitamin C

About 60-70 mg vitamin C intake per day will meet the adult requirement.
Excess
Excessive intake of ascorbic acid can lead to calcium oxalate stones formation in kidneys.
Megadoses of vitamin C may have pro-oxidant activity when it reacts with O2 or Cu2+ ion.
Anti-vitamins
Antivitamins are substances that interfere with normal function of a vitamin by the one or more of
the following mechanisms;
Competitive inhibition of active vitamin Inhibition of intestinal absorption
Inactivation of vitamin Chemical destruction
Interfering with normal vitamin metabolism or inhibiting synthesis of active vitamin form.
Vitamins Anti-vitamins Mechanism of interfering with normal vitamin function
Cleavage of linkage between pyrimidine and thiazole ring

Thiamine Thiaminase
Found in raw fish, fish viscera, shell fish, etc.
Biotin Avidin (raw egg white) Binds with biotin and inhibit absorption.
Anti-TB drug, combines with pyridoxal phosphate to form inactive

Pyridoxine Isoniazid (INH)
hydrazone derivatives which inhibit PLP dependent enzymes
Methotrexate, Anti-cancer drugs, stuctural analogue of folate that competitively

Folic acid
aminopterin inhibit dihydrofolate reductase and block the formation of THF
Inhibit regeneration of vitamin K hydroquinone by competitive
Vitamin K Dicumarol, warfarin inhibition of vitamin K epoxide reductase and vitamin K quinone
reductase.
Role of vitamins in energy-yielding metabolism (Energy Releasing Vitamins)

 Vitamins have no calorific value but are essential for energy generation process in the body by
serving as coenzymes for enzymes involved in fuel oxidation pathways. These include – vitamin B1,
vitamin B2, vitamin B3, vitamin B5, lipoic acid and biotin.
Role of thiamin in energy metabolism

TPP serves as coenzyme for multi-enzyme complexes that catalyze oxidative decarboxylation
reactions in fuel oxidation pathways. TPP involves with aldehyde group transfer in these reactions.
These include pyruvate dehydrogenase enzyme complex and α ketoglutarate dehydrogenase
enzyme complex.
Pyruvate dehydrogenase catalyzes oxidative decarboxylation of pyruvate (end product of glycolysis)
to acetyl CoA. Acetyl CoA formed then enters into CAC for complete oxidation. This reaction is
important for complete oxidation of glucose in the cells. α ketoglutarate dehydrogenase reaction is
part of TCA cycle which is the final common oxidation pathway in oxidation of metabolic fuels.
TPP also serves as coenzyme for transketolase reactions in pentose phosphate pathway of glucose
oxidation.
Vitamin B2
Active coenzyme forms are FMN and FAD which serve as prosthetic groups for many oxido-
reductase enzymes in fuel oxidation pathways.
FMN serves as a component of complex I of electron transport chain.
FAD dependent enzymes in energy yielding metabolism
o Dihydrolipoyl dehydrogenase of pyruvate dehydrogenase enzyme complex in oxidative
decarboxylation of pyruvate to acetyl CoA
o Succinate dehydrogenase and α ketoglutarate dehydrogenase in CAC
o Acyl CoA dehydrogenase in β oxidation of fatty acids
FADH2 derived from oxidative reactions (succinate dehydrogenase, acyl CoA dehydrogenase
reactions) donate its high energy electrons to the electron transport chain for ATP production.
Vitamin B3
NAD+ serves as coenzymes for oxido-reductase enzymes in fuel oxidation pathways.
NAD+ dependent enzymes in energy yielding metabolism
o Glyceraldehyde 3-phosphate dehydrogenase in glycolysis
o Pyruvate dehydrogenase in oxidative decarboxylation of pyruvate to acetyl CoA
o Isocitrate dehydrogenase, α ketoglutarate dehydrogenase and malate dehydrogenase in CAC
o β hydroxyl acyl CoA dehydrogenase in β oxidation of fatty acids
o Glutamate dehydrogenase in oxidative deamination of amino acid catabolism
NADH carries high energy electrons derived from these oxidative reactions and donates these
electrons to complex I of electron transport chain for ATP production.
NADP+ serves as coenzymes for oxidation reactions e.g., glucose 6-phospahte dehydrogenase in
pentose phosphate pathway of glucose oxidation.
Lipoic acid
It serves as coenzyme for multienzyme complexes (pyruvate dehydrogenase, α ketoglutarate
dehydrogenase) that catalyze oxidative decarboxylation of α keto acids in fuel oxidation pathways.
It involves in transfer of acyl group and hydrogen.
Vitamin B5 or pantothenic acid
Active coenzyme form is coenzyme A (CoA-SH). It serves as carrier of acetyl and acyl groups.
It plays a central role in metabolism of carbohydrate, lipid and protein. It takes part in the reactions
of TCA cycle and fatty acid oxidation. Acetyl and acyl groups are accepted by CoA molecules during
these oxidation pathways (pyruvate dehydrogenase, α-ketoglutarate dehydrogenase in TCA cycle
and thiolase reactions in fatty acid oxidation).
Formed acetyl CoA molecules then enter into TCA cycle for complete oxidation and energy
production. Acetyl CoA, succinyl CoA and fatty acyl CoA are important molecules which produce
ATP by being metabolized.
Biotin
Biotin serves as carboxyl group carrier in carboxylation reactions.
It serves as coenzyme for pyruvate carboxylase that catalyzes conversion of pyruvate into
oxaloacetate. Oxaloacetate formed then replenishes CAC for complete oxidation of acetyl CoA.
Role of vitamins in hemopoiesis (hemopoietic or anti-anemic vitamins)

 Vitamins required for hemopoiesis process include – vitamin B6, folic acid, vitamin B12 and vitamin C.
Folic acid
THF serves as coenzymes for transfer of one carbon units in a variety of reactions involved in purine
nucleotide and dTMP synthesis. Methenyl THF and formyl THF contribute carbon atom for C-2 and C-
8 of purine ring in purine nucleotide synthesis. Synthesis of dTMP from dUMP requires methylene
THF as a coenzyme for thymidylate synthase. Purine nucleotides and dTMP are used for DNA and
RNA synthesis which is important for normal cell division and maturation process in
erythropoiesis.
Decreased availability of purine nucleotides and dTMP due to folic acid deficiency leads to block in
DNA synthesis and arrest of cell cycle in S phase during erythropoiesis. This leads to megaloblastic
dysplasia of bone marrow cells and also slows down cell division, cell maturation, causing
appearance of macrocytic erythrocytes with fragile membranes.
Vitamin B12
Effect of vitamin B12 on erythropoiesis is related to its effect on folate metabolism. Vitamin B12
dependent homocysteine to methionine conversion is the only pathway by which methyl THF can
return to the active THF pool. In vitamin B12 deficiency, most of folic acid of the body is irreversibly
“trapped” as methyl THF. There is functional folate deficiency due to the lack of free THF. This is
called folate trap.
Vitamin B12 also serves as coenzyme for methyl malonyl CoA mutase which catalyzes conversion of
methyl malonyl CoA to succinyl CoA. Succinyl CoA can be used as precursor for heme synthesis.
Vitamin B6
PLP serves as coenzyme for ALA synthase which is the key enzyme of heme synthesis pathway.
Vitamin B6 deficiency causes hypochromic microcytic anemia due to decreased heme synthesis.
Vitamin C
It enhances intestinal absorption of non-heme iron by reducing it to ferrous state.
Other nutrients required for hemopoiesis

Dietary proteins – provide amino acids for globin chain synthesis
Minerals
o Iron – as a structural component of heme in hemoglobin synthesis
o Zinc – serves as cofactor for ALA dehydratase enzyme in heme biosynthesis pathway
o Copper – plays an important role in iron absorption. Ceruloplasmin, copper containing protein in
plasma, has ferroxidase activity that oxidizes ferrous ion to ferric form in order to be
transported bound to transferrin in the circulation.
Anti-oxidant vitamins
Antioxidants are molecules that counteract the effects of oxidants. Some vitamins serve as non-
enzymatic antioxidants in the body. These include – vitamin E, vitamin C and pro-vitamin A (β
carotene).
Vitamin E (α tocopherol)
Vitamin E serves as lipid-soluble chain-breaking antioxidants in cell membranes and plasma
lipoproteins by reacting and trapping the lipid peroxide radicals formed by peroxidation of
polyunsaturated fatty acids. It acts as first line of defense against peroxidation of PUFA in cellular
membrane phospholipids.
It breaks lipid peroxidation chain reaction by transferring its phenolic hydrogen to a peroxyl free
radical formed by peroxidation of PUFA.
Its antioxidant action is effective at high O2 concentration and thus it is concentrated in erythrocyte
membrane, membranes of respiratory tract and the retina. So it protects RBC from hemolysis by
oxidizing agents as well as prevents damage of lung tissues by oxidants such as ozone, nitrogen
dioxide from polluted air. Vitamin E prevents lipid peroxidation of LDL, thereby contributing in the
reduction of cardiovascular disease risk since oxidized LDL is atherogenic.
As an antioxidant, vitamin E protects against the development of cancer, atherosclerosis and aging
process by preventing oxidative stress.
Vitamin C (ascorbic acid)

Vitamin C is involved in scavenging free radicals (peroxy free radical ROO˚). Thus, it functions to
protect lipids against peroxidation.
It regenerates other antioxidants such as vitamin E and glutathione by reducing phenolic radical of
vitamin E and oxidized glutathione to functional tocopherol and reduced glutathione (GSH)
respectively.
Vitamin C reduces the risk of atheroscelrosis, cardiovascular disease since it protects lipids
peroxidation reactions thereby preventing the oxidation of LDL.
Vitamin C reduces superoxide anion to less reactive H2O2 and it also neutralizes hydroxyl radical to
water.
It inhibits formation of potentially carcinogenic nitrosamines from dietary nitrite and nitrate
during digestion.
Pro-vitamin A (β carotene)
β carotene act as anti-oxidant by trapping peroxyl free radical in its conjugated alkyl structure. It
complements anti-oxidant activity of vitamin E in membranes and other lipid rich structures.
Other anti-oxidant nutrients

Flavanoids – a variety of polyphenols derived from plant foods act as water-soluble free radical
trapping antioxidants. They are mostly found in many fruits, vegetables, beverages such as tea and
wine.
Anti-oxidant minerals – Some minerals are involved in antioxidant defense mechanisms by serving
as cofactors for enzymatic antioxidants such as SOD, glutathione peroxidase, etc. These include –
zinc, copper, manganese, selenium, iron.
o Zinc – serves as structural role in cytosolic SOD which catalyzes dismutation of superoxide
anions.
o Copper – as a cofactor in cytosolic Cu-Zn SOD and catalytically important for enzymatic
antioxidant activity of SOD. Copper containing ceruloplasmin has ferrooxidase activity and acts
as antioxidant by catalyzing oxidation of ferrous to ferric iron. Ferrous iron can generate free
radical by Fenton reaction and ferric iron is less reactive in forming free radical.
o Manganese – as a cofactor in mitochondrial Mn-SOD.
o Selenium – as a cofactor in glutathione peroxidase which catalyzes neutralization of H2O2 into
water. It complements the antioxidant effects of vitamin E.
o Iron – as a component of heme containing antioxidants enzyme catalase.
Nutrients involved in bone formation

Bone is a modified connective tissue consisting of a cellular component, an organic matrix, and an
inorganic (mineral) phase. It is composed of an orderly collagen matrix on which calcium and
phosphate are deposited in the form of hydroxyapatite crystals.
Bone consists of approximately 10% water, 20% organic materials, and 70% inorganic salts. The
organic matrix is mainly type I collagen, and the inorganic salts are derived from calcium phosphate.
Hydroxyapatite is the major inorganic component. The calcium-rich crystals of hydroxyapatite (Ca10
[PO4]6 [OH]2) are found on, within and between the collagen fibers.
Dietary proteins – provide amino acids for collagen and bone matrix protein synthesis.
Minerals required for bone formation – calcium, phosphate, magnesium, fluorine as structural
component of bone matrix and iron, copper, manganese as cofactors of enzymes involved in bone
matrix protein and collagen synthesis
o Calcium – 99% of body calcium is in the bone as hydroxyl apatite crystals.
o Phosphate – found as amorphous calcium phosphate forms and in more crystalline forms such
as hydroxyapatite, which is laid down on collagen in the ossification process of bone formation.
o Magnesium – about 50 – 60 % of body magnesium together with calcium and phosphate form as
organic salt in bone and teeth.
o Fluoride – about 99 percent of total body fluoride is contained in bones and teeth.
o Iron – as a cofactor in iron-containing hydroxylases involved in hydroxylation reactions which
are essential for collagen triple helix formation
o Copper – as a cofactor of lysyl oxidase enzyme which is important for collagen cross-linking
o Manganese – as a cofactor of manganese-dependent transferases which are important for
connective tissue synthesis and GAG synthesis
Vitamins required for bone formation – vitamin D, vitamin A, vitamin K, vitamin C
o Vitamin D – increases serum calcium and phosphate level required for bone mineralization by
enhancing intestinal calcium and phosphate absorption and reducing their urinary excretion. It
promotes bone mineralization indirectly by provision of Ca2+ and PO43- and also by increasing
osteoblastic activity.
o Vitamin A – facilitates growth of skeleton and soft tissues by promoting mucopolysaccharides
synthesis, stimulating vessel growth and production of collagen and elastic fibers.
o Vitamin K – promotes calcification of osteoid matrix. Osteocalcin and the matrix Gla protein in
bone involved in calcification of osteoid matrix require vitamin K dependent carboxylation of
specific glutamate residues to γ-carboxyglutamate for chelation of Ca2+.
o Vitamin C - functions as coenzyme in post-translational hydroxylation reactions of proline and
lysine in procollagen to hydroxyproline and hydroxylysine. Hydroxyproline and hydroxylysine
are essential for the proper collagen cross-linking that maintains collagen strength and stability
in the formation of bone matrix.
Vitamin deficiency states that can cause neurological symptoms

Vitamin B1 deficiency
o TPP serves as coenzyme for multi-enzyme complexes that catalyze oxidative decarboxylation
reactions which are important for energy generation. Deficiency affects tissues with high energy
requirement including brain and nervous tissues.
o Dry beriberi is characterized primarily by chronic peripheral neuritis, and severe muscle
weakness. In longstanding cases, there is degeneration and demyelination of sensory and motor
nerves and severe wasting of muscles.
o Wernicke encephalopathy with Korsakoff psychosis is characterized by neurological symptoms
such as ataxia, loss of memory, mental confusion, peripheral paralysis, etc.
Vitamin B3 deficiency – leads to pellagra which is characterized by diarrhea, dermatitis and
dementia. Dementia is characterized by the neurological symptoms due to actual degeneration of
nervous tissues.
o PLP is required for synthesis of sphingosine, a component of sphingomyelin, needed for myelin
formation. Deficiency will lead to demyelination of nerves causing peripheral neuropathy.
o PLP serves as coenzyme for decarboxylation reactions in biogenic amines or neurotransmitter
synthesis such as conversion of glutamate to GABA, dopa to dopamine, etc. Severe deficiency of
pyridoxine causes epileptic seizures (convulsions) in infants due to reduced production of GABA.
o Neurological disorders seen in B12 deficiency are due to progressive demyelination of nervous
tissue. Since vitamin B12 is required as a coenzyme for methylmalonyl CoA mutase action,
vitamin B12 deficiency leads to accumulation of methylmalonyl CoA which contributes the
pathogenesis of neuropathy.
o Accumulation of methylmalonyl CoA interferes with myelin sheath formation in 2 ways:
 Methylmalonyl CoA is a competitive inhibitor of malonyl CoA in fatty acid biosynthesis.
Since myelin sheath is continually turning over, any severe inhibition of fatty acid synthesis
will lead to its eventual degeneration.
 In the residual fatty acid synthesis that does occur, methylmalonyl CoA can substitute for
malonyl CoA in the reaction sequence, leading to branched-chain fatty acids, which might
disrupt normal membrane structure.
o Subacute combined degeneration of spinal cord can occur probably secondary to a relative
deficiency of methionine in spinal cord.
Vitamin B5 deficiency – Coenzyme A plays important role in biosynthesis of fatty acids,
phospholipids which are important for the myelin sheath formation of nervous tissues. Coenzyme A
also involves in neurotransmitter acetylcholine synthesis. Thus, deficiency state can cause burning
foot syndrome and paresthesia of hand and foot.
Folic acid deficiency - Since, folate is required for the formation of neural tube in early stage of
gestation, folate deficiency during early stage of pregnancy increases the risk of neural tube
defects, e.g., anencephaly, spina bifida. Folate supplementation during the peri-conception period
(4 weeks before and until 8 weeks after conception) prevents neural tube defects.
Micronutrients M i n e r a l s P a g e | 46
Minerals
Macrominerals
 Abundant in the body
 Daily requirement > 100 mg per day
 Calcium, phosphorus, magnesium,
sodium, potassium, chloride
Microminerals or trace elements

 Daily requirement < 100 mg per day
 Iron, iodine, copper, chromium,
cobalt, manganese, molybdenum,
zinc, selenium, fluorine
Calcium
 Calcium is the most abundant divalent cation in the body, representing about 40% of body’s mineral
mass. It represents 1.5 – 2% of total body weight (1 kg calcium in 70 kg adult body).
 About 99% of the body’s calcium is in the bone as hydroxyl apatite crystals.
Sources
 Dairy products especially milk, cheese, and yogurt
 Certain sea foods such as salmon and sardines, clams and oysters
 Vegetables such as turnip, mustard greens, broccoli, cauliflowers
Requirement – adult  800 mg/day, children  700 mg
Metabolism
Calcium is present in the
foodstuffs as relatively
insoluble salts.
It can be solubilized (ionized
to form free Ca2+ from
calcium salts) at acidic pH in
the stomach.
Solubilization does not
ensure better absorption
because free calcium can bind
to other dietary constituent,
limiting its bioavailability.
Calcium absorption in small intestine involves two main transport systems

 Saturable, carrier–mediated active transport (by binding with calbindin D)
 Diffusion
Factors influencing Ca2+ absorption
Factors increasing Ca2+ absorption Factors decreasing Ca2+ absorption
1, 25 DHCC or vitamin D High concentration of dietary Mg2+
Acidic pH Alkaline pH
Organic acid, lactose and basic amino acids in diet Phytic acid, oxalic acid, fatty acids
High dietary protein intake Excess dietary fiber
Ca2+ : P ratio – 1:1 Old age
Parathyroid hormone (indirect action) High dietary intake of Ca2+
Circulating forms of calcium

 Protein bound forms (mainly albumin and prealbumin) – nearly 40%
 Complex with organic anions such as sulfate, phosphate, or citrate – up to ~ 10%
 Free or ionized calcium – about 50% (most important physiologically active form)
Regulation of calcium concentration

 Calcium concentration is tightly controlled both intracellularly and extracellularly.
 Body calcium homeostasis is controlled by 1, 25 DHCC, parathyroid hormone (PTH) and calcitonin.
 Normal serum/plasma calcium concentrations = 8.5 – 10.5 mg/dL (2.12 – 2.63 mmol/L)
Functions
 Calcium along with phosphate is essential for the formation and development of bone and teeth.
Bone and teeth contains about 99% of the body’s calcium.
 Ionized calcium involves in
o Neuromuscular excitability
o Blood coagulation
o Secretory process
o Release of hormones and neurotransmitters
o Intracellular signaling mechanism and action of a number of hormones
o Membrane integrity and plasma membrane transport
o Enzyme reactions (e.g., ATPase)
Deficiency
Factors contributing to calcium deficiency
o Inadequate calcium intake
o Poor calcium absorption
o Excessive calcium looses

o Deficiency of hormones involved in calcium homeostasis e.g., vitamin D deficiency,
hypoparathyroidism
Calcium deficiency most profoundly affects on bone and muscle. Decreased body calcium level
causes poor bone mineralization, leading to rickets in infants and children; osteomalacia,
osteoporosis in adults.
Low levels of free ionized level Ca2+ in the blood (hypocalcemia) may result in tetany (characterized
by intermittent muscle contractions that fail to relax).
Inadequate calcium intake has been associated with hypertension, colon cancer, type 2 diabetes
mellitus and obesity.
Toxicity
It is mostly found in hyperparathyroidism, cancer, multiple myeloma.
Toxicity increases the risk of developing calcium stone formation in kidneys and urinary tract.
Hypercalcemia can cause cardiac arrhythmias.
Phosphorus
 Phosphorus is the second most abundant mineral in the body.
 Human body contains about 560 – 850 g of phosphorous, representing about 0.8% to 1.2% of body
weight.
 85% of body’s phosphorus is in the skeleton, 14% is associated with soft tissues such as muscle and
the remaining 1% is in the blood and body fluids.
Sources
 Widely distributed in foods
 Rich sources include protein–rich foods like meat, poultry, fish, eggs, milk and dairy products.
Requirement – 700 mg/day
Metabolism
Dietary phosphorus occurs as inorganic phosphates as well as organic forms bound to proteins or
sugars or lipids. Most phosphorus is absorbed from the GI tract as inorganic phosphate ions. Organic
forms must be digested enzymatically to release inorganic phosphates for absorption.
Phosphate absorption occurs throughout the small intestine but primarily in the duodenum and
jejunum. Phosphorus absorption occurs by two processes –
o Saturable, carrier mediated active transport
o Diffusion
Phosphate absorption is enhanced by vitamin D and decreased by phytic acid, excessive intake of
magnesium, aluminium, calcium.
Functions
 Phosphorus is important in the development of skeletal tissue. In bone, phosphorus is found in
amorphous calcium phosphate forms and in more crystalline forms such as hydroxyapatite, which is
laid down on collagen in the ossification process of bone formation.
 Phosphate serves as the constituent of important biomolecules
o Component of the nucleic acids DNA and RNA, alternating with pentose sugars to form linear
backbone of these molecules
o Component of cAMP, cGMP and IP3 which function as intracellular second messengers
o In the form of high–energy phosphate bonds in ATP
o Component of phospholipids which serve as structural component of lipid bilayer in cell
membrane
o Plays important role as phosphoproteins and as phosphorylated active forms of vitamins such
as thiamin pyrophosphate and pyridoxal phosphate
o Component of 2, 3-bisphosphate glycerate which is an important allosteric effector for oxygen
delivery from hemoglobin Causes of Causes of
 Phosphorylation and dephosphorylation of hypophosphatemia hyperphosphatemia
enzymes modify the activity of many Decreased absorption of Increased absorption of
enzymes; thus involve in the regulation of PO43- PO43-
enzyme activity in metabolic pathways. Intracellular shift Increased cell lysis
 Ionized phosphate functions in acid–base Increased urinary Decreased phosphorus
balance. It is an important buffer in the ICF excretion of phosphates excretion
+
and in renal handling of H . Vitamin D deficiency Hypocalcemia
Deficiency Massive blood transfusion
Rare Thyrotoxicosis
Seen in uncontrolled metabolic acidosis Renal failure
Initial symptom is muscle weakness. Hypoparathyroidism
Magnesium
Substances enhancing Mg2+ Substances inhibiting
 th
6 most abundant mineral in the body, 2 nd
absorption Mg2+ absorption
major intracellular cation
Vitamin D Phytic acid
 The human body contains about 25 g of
Carbohydrate – Excessive unabsorbed
magnesium (close to 1% of body weight), of
oligosaccharides, fructose fatty acids
which approximately 50% to 60% is located
Protein Dietary fiber
in bone, another 39% to 49% in soft tissues,
and about 1% in extracellular fluids.
Sources
 Found in wide variety of foods.
 Foods particularly high in magnesium content – nuts, legumes, whole grain cereals (especially oats
and barley)
Requirement – men  420mg/day

women  320mg/day
Metabolism
Dietary magnesium does not require digestion prior to absorption.
Absorption occurs throughout the small intestine, mainly in jejunum and ileum.
Functions
 About 50 – 60 % of body magnesium together with calcium and
phosphate form as organic salt in bone and teeth.
 About 25% of magnesium is found in soft tissues primarily in
skeletal muscles.
 Up to 90% of intracellular magnesium may be associated with
ATP or ADP. In ATP, magnesium is linked to the oxygen atoms
of the phosphate groups, forming a complex that assists in the
transfer of an ATP phosphate group in a variety of biochemical Magnesium activates a number of
enzymes, particularly those in which
processes such as
ATP–Mg2+ (a complex of ATP and
o Glucokinase, hexokinase and phosphofructokinase actions magnesium) is a substrate. The
in glycolysis magnesium ion is chelated between
o Oxidative decarboxylation in TCA cycle the β- and the γ-phosphates of ATP,

thus diminishing the anionic
o Creatine phosphate formation by creatine kinase reaction
character of the phosphates. This
o Nucleic acid synthesis
permits smooth interaction of the
o Insulin action (such as tyrosine kinase activity) ATP with specific protein sites.
o Inhibition of thrombus formation
 Essential for cardiac and smooth muscle contractibility, ion channel regulation and propagation of
nerve impulse
Deficiency
Magnesium deficiency is seen in alcoholism, diuretic therapy, metabolic acidosis, diarrhea, acute
pancreatitis.
Main symptoms are cardiac arrhythmias, tremors and muscle weakness.
Low magnesium status or poor magnesium intake is associated with hypertension, cardiovascular
disease and diabetes mellitus.
Sodium
 About 30% of sodium in the body (of a 70-kg human) is located on the surface of bone crystals. The
remainder of the body’s sodium is found in the extracellular fluid, primarily plasma; in the interstitial
fluid; and, in lower levels, intracellularly in nerve and muscle tissue.
 Sodium constitutes about 93% of the cations in the body fluids.
Sources – main source is table salt (sodium chloride) used in cooking. Rich sources are bread, cheese,
wheat germ, whole grains, and oyster. Carrots, cauliflower, eggs, milk, nuts, spinach, turnips, etc. are
also good sources.
Requirement – 5 to 15 g/day
Functions
 Sodium is the major extracellular cation and exists in the ECF in association with the anions chloride,
bicarbonate, phosphate and lactate.
 Sodium is necessary for the maintenance of normal neuromuscular excitability and the permeability
of the cells.
 Sodium is required for the maintenance of the osmotic pressure of the body fluids, thus
maintenance of the normal water balance and distribution.
 Sodium plays important role in absorption of glucose, galactose and amino acids from the small
intestine (sodium dependent secondary active transport system).
 Sodium regulates the body’s acid–base equilibrium in association with the anions chloride,
bicarbonate.
Normal plasma sodium concentration  135 – 145 mmol/L
Disturbances of Serum Sodium

Hyponatremia
o Hyponatremia may occur due to excess sodium loss form GI tract (e.g., diarrhea), chronic renal
disease and adrenocortical insufficiency (Addison’s disease).
o It may be secondary to excessive water retention or over-hydration (dilutional hyponatremia).
Hypernatremia
o Hypernatremia may occur due to excessive loss of water relative to the sodium loss in the body.
o It can be seen in Cushing’s disease, hyperaldosteronism, prolonged corticosteroid therapy,
dehydration and diabetes insipidus.
Potassium
 Potassium is the major intracellular cation. In contrast to sodium, about 95% to 98% of the body’s
potassium is found within cells.
Sources – high content is found in bananas, juices of orange, pineapple, yam, and winter squash,
chicken, beef liver, and potatoes.
Requirement – 4 g/day
Functions
 Potassium is the major intracellular cation.
 It is important for neuromuscular excitability especially for cardiac muscles.
 It has an important role in acid–base balance as well as maintenance of intracellular osmotic
pressure and hence, intracellular fluid volume.
 It is required for maximal activity of pyruvate kinase enzyme.
Normal plasma potassium concentration  3.5 – 5.0 mmol/L
Disturbances of Serum Potassium

Hypokalemia
o It may occur due to loss of K+ via GI tract, in renal tubular acidosis, hyperaldosteronism, etc.
o It may be due to redistribution of K+ caused by insulin as insulin induces K+ to move into the cells.
o Hypokalemia manifests as muscular weakness, confusion, irregular heart beat, tachycardia, and
altered ECG pattern (flattening of ECG waves).
Hyperkalemia
o It can be caused by failure of the kidneys to excrete potassium, as in Addison’s disease, renal
failure, redistribution of potassium, which occurs in acidosis and crush injuries.
o Hyperkalemia clinically manifests as irregular heartbeat, palpitations, ECG changes, etc. The
greatest danger is the possibility of cardiac arrest at levels greater than 7.0 mmol/L.
Chlorine
Chloride is the most abundant anion in the ECF, with approximately 88% of chloride found in the ECF
and just 12% intracellularly. Its negative charge neutralizes the positive charge of the Na+ with which
it is usually associated.
Sources – mainly available as sodium chloride
Requirement – depends on the climate, occupation, and salt content of the diet
Functions
 Chloride is the major extracellular anion.
 As a component of sodium chloride, it is involved in osmotic pressure regulation, water balance,
acid–base equilibrium.
 Chloride acts as the exchange anion for HCO3− in red blood cells, in a process sometimes called the
chloride shift in carbon dioxide transport from the tissues to the lungs.
 The formation of gastric hydrochloric acid requires chloride, which is secreted along with protons
from the parietal cells of the stomach.
 It serves as an activator of salivary amylase enzyme.
Normal plasma chloride concentration  95 – 105 mmol/L
Alterations of serum chloride level

Hyperchloraemia
o It is seen in dehydration, diarrhea, respiratory alkalosis, metabolic acidosis and renal tubular
acidosis.
Hypochloraemia
o It is seen in severe vomiting, prolonged gastric suction, respiratory acidosis and metabolic
alkalosis.
Sulfur
Sulfur of the body is mostly present in the organic form. Methionine and cysteine are sulfur-
containing amino acids present in the proteins. Generally, proteins contain about 1% sulfur by weight.
Sources – Plant and animal proteins, legume, eggs, cereals and cauliflower
Functions
Sulfur is a constituent of —
o Protein
o Glycosaminoglycans, e.g. heparin and chondroitin sulfate
o Vitamins, e.g. thiamine, biotin, lipoic acid, CoA of pantothenic acid
o Bile acids, e.g. taurocholic acid.
o Active form of sulfate, phosphoadenosine phosphosulfate (PAPS) is involved in detoxication
reactions, e.g. some phenolic compounds are detoxified by conjugating with PAPS and
eliminated from the body in the form of sulfate conjugate.
o Non-heme iron enzyme such as mitochondrial NADH dehydrogenase and Fe-S protein
o Compounds like glutathione and insulin
Iron
 A normal adult possesses 3–5 gm of iron. This small amount is used again and again in the body. Iron
is called a one way substance, because very little of it is excreted.
Sources
 Endogenous source – breakdown of RBCs and other hemoproteins
 Exogenous source – dietary iron
o Two forms – heme iron and non–heme iron
o Heme iron is derived mainly from hemoglobin and myoglobin.

o About 50 – 60% of the iron in meat, fish and poultry is heme iron, the rest is non–heme iron.
o Non–heme iron is found primarily in plant foods (nuts, fruits, vegetables, grains, tofu) and dairy
products (milk, cheese, eggs).
 Different iron distribution forms in the body –
o Heme iron (hemoproteins)
 Hemoglobin (60 – 70 % of total iron)
 Myoglobin (3 – 5% of total iron)
 Heme containing enzymes – cytochromes, catalase, peroxidase
o Non–heme iron
 Transferrin (iron transport protein in plasma)
 Ferritin (iron storage protein in tissues)
 Hemosiderin
 Iron – sulfur complexes
Daily requirement
 Adult men and post-menopausal women – 10 mg
 Premenopausal women – 15–20 mg
 Pregnant women – 30–60 mg
Iron metabolism
Source
Exogenous source – dietary iron (10 – 20 mg/day)
Endogenous source – mostly from breakdown of aged RBCs
Dietary iron absorption

Dietary iron can be either of two forms – heme iron or non-heme iron.
Normally, about 5–10% of dietary iron is absorbed. Most absorption occurs in the duodenum.
Iron is absorbed in the ferrous form but heme iron can be absorbed intact into the enterocytes by a
separate mechanism.
Heme iron digestion and absorption
 Dietary form of heme iron is as a component of hemoproteins such as Hb, Mb.
 Hemoproteins (Hb and Mb) must be hydrolyzed into heme and globin portion by proteases in
stomach and small intestine.
 Released free heme iron can then be absorbed.
 Heme iron is readily absorbed intact into the enterocytes by heme carrier protein 1.
 Within the enterocytes, absorbed heme is hydrolyzed into ferrous iron and protoporphyrin by the
action of heme oxygenase.
Non–heme iron digestion and absorption

 Dietary non–heme iron is typically bound to components of foods.
 Gastric secretions (including HCl) and proteases in the stomach and small intestine aid in the release
of non-heme iron from some food components mostly as Fe3+.
 Fe3+ is reduced to Fe2+ by form by a brush border enzyme ferri–reductase. Vitamin C, gastric acid and
a number of other reducing agents present in food also favors reduction of ferric iron.
 Fe2+ is then absorbed into enterocytes across the apical membrane via the divalent metal ion
transporter (DMT 1).
Factors favoring non-heme iron absorption Factors reducing non-heme iron absorption
Gastric HCl Phosphate, phytate in cereals
Dietary protein Oxalate in diet
Reducing substances in diet Tannate in tea, coffee
Sugars especially fructose and sorbitol Betel nut
Acids such as ascorbic, citric, lactic, etc. Pancreatic juice (alkaline secretion)
Copper deficiency
Divalent cations such as calcium, zinc, and manganese
Iron transport in the plasma

 Within the enterocytes, absorbed ferrous iron can either be stored as ferritin or transported across
the basolateral membrane into the circulation by the iron exporter protein ferroportin.
 Excess iron is stored in the enterocytes bound to ferritin which is lost when the enterocytes are
sloughed off.
 Only ferric iron is incorporated into transferrin and transported in the blood stream. So, ferrous iron
transport into the circulation occurs in conjunction with hephaestin, copper containing ferrooxidase
which oxidizes Fe2+ to Fe3+ or ferrous iron is oxidized to the ferric state by the ferrooxidase activity of
ceruloplasmin in the circulation in order to be transported bound to transferrin.
 Free iron is extremely toxic because it involves in the formation of harmful oxygen free radicals via
Fenton reaction. So in the plasma iron is transported bound to transferrin. Each mole of transferrin
can carry 2 moles of ferric ions.
 Normal serum iron level is about 120 μg/dL.
 The concentration of transferrin is approximately 300 mg/dL, which can bind a total of
approximately 300 μg of iron/ dL of plasma. This represents total iron binding capacity (TIBC) of
plasma. (TIBC – 240 to 250 μg/dL)
 Normally transferrin is about 30% saturated with iron.
Utilization
About 35 mg of iron is utilized daily (75 % for hemoglobin synthesis).
Storage forms of iron (ferritin and hemosiderin) can act as iron reserves which can be used when the
iron requirement is increased.
The site of storage is mainly in the liver, spleen and bone marrow.
Cells can adjust their iron uptake by regulating the number of transferrin receptors.
Output
There is no specific excretory mechanism for iron.
Iron is lost from the body by desquamation of skin, nails, hair and especially shedding of intestinal
mucosal cells (about 1mg/day).Females may have additional losses – 1 mg/day through menstruation,
200 mg in parturition.
Regulation of iron
status in the body
Body iron content
is regulated by
control of iron
absorption since
there is no proper
excretory route
for excess iron in
the body.
Normal diet contains 10 – 20 mg of iron of which 10% is absorbed.
Iron absorption is determined by
o Iron store in the body
o Rate of erythropoiesis or bone marrow activity
Normally iron taken up into the enterocytes is transported via ferroportin which regulates iron
passage across the cell into the blood. Iron in excess of binding capacity of carrier is oxidized to
ferric and deposited bound to ferritin in the enterocytes.
The main regulator of iron absorption is the protein hepcidin, which is released from the liver when
body iron stores are adequate or high.
Role of hepcidin in systemic iron regulation
o Hepcidin is synthesized and secreted from liver when iron is plentiful.
o Hepcidin down-regulates expression of the ferroportin gene (and possibly also that for DMT1).
o It also binds to and triggers the internalization and degradation of ferroportin expressed on
the surface of enterocytes and macrophages thereby decreasing intestinal iron absorption and
inhibiting iron release from macrophages.
o In response to hypoxia, anemia, or hemorrhage, the synthesis of hepcidin is reduced, leading to
increased synthesis of ferroportin and increased iron absorption.
In iron deficiency or increased erythropoiesis, absorbed iron is rapidly delivered into plasma, so little
or no ferritin is formed. The amount of transferrin is increased and TIBC is increased.
In iron overload, iron absorption is decreased. More iron is trapped in ferritin and lost when the
mucosal cells sloughed off. The amount of transferrin is decreased and TIBC is decreased or normal.
Functions
Iron serves as constituent of heme iron in many functionally important hemoproteins in the body
o Hemoglobin for transport of respiratory gases
o Myoglobin for supply of oxygen at low O2 tension in muscles
o Cytochromes in respiratory chain for electron transport reaction

o Mitochondrial cytochrome P450 monooxygenase for hydroxylation in steroid hormone synthesis
o Microsomal cytochrome P450 monooxygenase for xenobiotic metabolism in the liver
o Catalase and peroxidase for reduction of H2O2
o Myeloperoxidase for phagocytosis in macrophages
Iron serves as component of intracellular iron containing flavoprotein and iron sulfur protein for
transfer of reducing equivalents in respiratory chain.
Iron acts as a cofactor in iron-containing hydroxylases (prolyl hydroxylase and lysyl hydroxylase)
involved in hydroxylation reactions which are essential for collagen triple helix formation
Deficiency
May result from inadequate dietary intake, poor
absorption or chronic blood loss.
Worm infestation, menstruating or pregnant
women, lactating mothers, growing children are
high risk conditions for iron deficiency.
Iron deficiency can cause hypochromic microcytic
anemia and general weakness.
Excess Decreased
Surplus iron may cause excessive formation and
accumulation of hemosiderin, the condition is
termed hemosiderosis.
Massive deposits of hemosiderin may develop,
which may cause functional impairment of the involved organs. The condition is then called
hemochromatosis. Accumulation in liver can result in cirrhosis. In the pancreas, the excess iron can
damage the β cells to cause diabetes mellitus (bronze diabetes).
Zinc
 The human body contains about 1.5 to 3.0 g of zinc. Zinc is found in all organs, tissues, and body
fluids. In blood, RBCs contain very high concentration of zinc as compared to plasma.
Sources – meat, liver, egg, milk, sea food (oyster) and whole grain products
Requirement – men  11mg/day, women  8mg/day
Metabolism
Dietary zinc is found in foods complexed with nucleic acids or with amino acids as parts of peptides
and proteins.
Dietary zinc needs to be hydrolyzed

Enhancers of zinc absorption Inhibitors of zinc absorption
from amino acids and nucleic acids
Organic acids like citric acid Phytic acid
before it can be absorbed.
Zinc absorption occurs primarily in Prostaglandins Oxalic acid
duodenum and upper jejunum. Glutathione Polyphenols in tea and coffee
Zinc is absorbed bound to cysteine
Amino acids such as
Folate
rich protein, metallothionein, histidine and cysteine
associated with other divalent metal Iron
ion, copper. It is transported in the Calcium
blood in association with albumin.
Functions
 Zinc serves as structural component for many important biomolecules.
o Zinc containing metalloenzymes such as carbonic anhydrase,
carboxypeptidase, alcohol dehydrogenase, cytosolic superoxide
dismutase, DNA and RNA polymerase, ALA dehydratase, etc. Zinc
participates in the catalytic role (e.g., carbonic anhydrase) or structural
role (e.g., cytosolic SOD) in zinc containing metalloenzymes.
o Zinc serves as structural role in cytosolic SOD which is important for
antioxidant defense in the body.
o As component of salivary peptide, gustin, necessary for normal taste bud
development.
o As component of zinc finger motif in transcription factor proteins in
which zinc stabilizes the structure of these proteins to interact with
specific DNA sequence. Zinc plays a major role in regulation of gene
expression. Thus, zinc is required for growth, differentiation and normal wound healing.
o Bound to the storage protein metallothionein in the
tissues.
 Zinc is essential for normal growth, reproduction and life
expectancy of mammals.
 Zinc is important for storage and release of insulin from
β cells of pancreas and for prolongation of insulin action.
 Zinc supplementation reduces the severity and duration
of diarrhea in children and prevents further episodes of
diarrhea.
Deficiency – seen in major burns, chronic renal disease, long–term parenteral nutrition
Growth retardation Decreased taste acuity
Hypogonadism Alopecia (hair loss)
Dwarfism Acrodermatitis enteropathica – severe skin
Neuropsychiatric impairments lesion, diarrhea and hair loss
Delayed wound healing
Excess
Seen in welders due to inhalation of zinc oxide fumes
Cause nausea, vomiting, pancreatitis and excessive salivation.
Copper
 The copper content of the human body ranges from about 50 to 150 mg. Copper is found in all body
tissues and most secretions. In the aqueous environment of the body, copper is found in either of
two valence states, the cuprous state (Cu1+) or cupric state (Cu2+).
Sources
Enhancers of copper Inhibitors of
 Richest sources – meat (especially organ meat
absorption copper absorption
like liver) and shell fish (especially oysters and
Organic acids like citric
lobsters) Antacids
acid, lactic acid, acetic acid
 Plant food sources – nuts, seeds, legumes, dried
Amino acids such as H2 receptor
fruits, potatoes, whole grains, and cocoa
histidine and cysteine blockers
Requirement – 900 μg/day Proton pump
Glutathione
Metabolism inhibitors
High pH
Dietary copper is found as bound to organic
Phytic acid
components especially amino acids of dietary
Zinc
proteins. Gastric HCl and pepsin facilitate release
of copper in the stomach. Copper is absorbed primarily in its reduced state from the duodenum. It is
absorbed associated with metallothionein. Copper absorbed in the intestine is transported in portal
blood to the liver bound loosely to albumin and α2 macroglobulin.
In the hepatocytes, copper first binds to glutathione and/or metallothinein and is then transferred to
copper requiring enzymes (cuproenzymes) such as ceruloplasmin. Ceruloplasmin is released into the
blood from the liver, constituting about 60 to 70% of circulating copper. The remainder circulates
bound to albumin and α2 macroglobulin for delivery to the tissues. Excess copper is excreted through
bile. Liver is the main organ that stores copper and controls copper homeostasis in the body.
Functions
 Copper serves as cofactor for many enzymes such as
o Ceruloplasmin (ferrooxidase activity)
o Cytochrome C oxidase (electron transport chain)
o Dopamine β hydroxylase (norepinephrine
synthesis)
o Lysyl oxidase (collagen cross–linkage)
o Cytosolic Cu-Zn SOD (disproportionation of
superoxide)
o C18, ∆9 desaturase (addition of double bonds to long chain fatty acids)
 Pro–oxidant role – copper reacts with hydrogen peroxide and
catalyzes formation of hydroxyl radicals through Fenton reaction.
Deficiency
Causes – excessive zinc intake (compete for absorption), Menkes’s syndrome (a rare X–linked
recessive hereditary disease associated with a defect in copper transport), reduced intake, excess
loss (e.g., during renal dialysis), total parenteral nutrition
Clinical features
o Anemia (due to reduced ceruloplasmin activity)
o Hypercholesterolemia (increased ratio of SFA to MUFA due to reduced desaturase activity)
o Demineralization of bones, fragility of large arteries (Due to defective collagen and elastic
formation)
o Leucopenia, demyelination of neural tissues
Excess
Wilson’s disease – autosomal recessive disease associated with abnormal accumulation of copper in
various tissues.
Accumulation of copper occurs in tissues especially liver (cirrhosis), brain (neurological symptoms)
and cornea.
Low plasma ceruloplasmin, high urinary copper excretion
Iodine
 The human body contains about 15 to 20 mg of iodide, most (70–80%) of which is found in the thyroid
gland.
Source – sea foods such as sea fish, and sea weeds.
Requirement – 150 – 200 μg/day

Functions
 Most of the body’s iodine (70 – 80%) is found in the thyroid gland. Iodine is used for synthesis of
thyroid hormones.
 Thyroid hormones, triiodothyronine (T3) and tetraiodothyronine (T4), play a major role in regulating
many metabolic functions and basal metabolic rate of adults as well as growth and development of
the child.
Deficiency
Iodine deficiency can cause non–toxic goitre. This can be prevented by intake of iodide fortified salt
and foods.
Iodine deficiency in children can lead to cretinism.
Goitrogens are substances that can cause goitre by blocking of iodide uptake of the body.
o Cabbage, cassava, cauliflower, sprouts and turnips
Pollution of water supply can also be goitrogenic.
Fluorine
Sources – drinking water, china tea, sea food
Requirement – 1ppm in water (ppm – parts per million, 1 ppm = 1 gram of fluoride in million grams of
water, ~ 1mg/1000 mL)
Functions
 The superficial layer of teeth contains a high concentration of fluoride.
 High fluoride content in enamel of teeth provides more resistance to erosion by acid production from
bacteria.
 Fluoride decreases incidence of osteoporosis in older adults.
Deficiency – dental caries
Excess – fluorosis, resulting in mottling and discoloration of enamel of teeth and bone changes such as
increased bone density with bone overgrowth (exostosis)
Selenium
Sources
 Dietary forms – selenomethionine and selenocysteine in organ
meats, fish, and cereals
 Content in plant foods depends on mineral content in the soil.
Requirement – 50 to 150 μg/day
Functions
 Selenium serves as a component of antioxidant enzyme glutathione peroxidase.
 It is also a part of deiodinase enzyme which converts T4 thyroxine to T3.
 Thioredoxin reductase is a flavoprotein enzyme (containing FAD) which contains selenocysteine at
its active site. It is important in the conversion of ribonucleotides to deoxyribonucleotides in DNA
synthesis.
Deficiency – cardiomyopathy (Keshan disease) and osteoarthropathy (Kashin-Beck disease) in China,

central Africa and parts of Europe.
Excess – selenosis
Manganese
 It serves as a component of pyruvate carboxylase and mitochondrial SOD.
 It also acts as cofactor of hydrolase, decarboxylase, and transferase enzymes.
 Two manganese-dependent transferases important for connective tissue synthesis are xylosyl
transferase and glycosyl (also called galactosyl or galacto) transferase. Glycosyl transferase is
especially important for the synthesis of glycosaminoglycans.
Deficiency – impaired growth, skeletal deformities
Excess – Psychosis (manganese madness), Parkinsonism
Molybdenum
 It serves as a component of xanthine oxidase and
aldehyde oxidase enzyme.
Chromium
 It acts as a component of glucose tolerance factor
(GTF). GTF potentiates the effects of insulin by
facilitating its binding to cell surface receptor.
Deficiency – impaired glucose tolerance resulting in

decreased insulin sensitivity
Cobalt
 Found as a constituent of vitamin B12.
 Deficiency can lead to anemia.
Biochemical functions of minerals

1. Role in enzymatic reactions
 Minerals act as enzyme cofactors or activators of enzyme. They function as a prosthetic
group in the catalytic or structural role of the enzymes.
 Iron – Heme containing enzymes such as catalase, peroxidase, cytochrome P450
 Zinc – constituent of over 100 metalloenzymes e.g., carbonic anhydrase, alcohol
dehydrogenase, cytosolic SOD, DNAP, RNAP, etc.
 Copper – cytochrome C oxidase, dopamine β hydroxylase, cytosolic SOD, lysyl oxidase, etc.
 Selenium – glutathione peroxidase, deiodinase
 Manganese – pyruvate carboxylase, mitochondrial SOD
 Molybdenum – xanthine oxidase, aldehyde oxidase
 Calcium – ATPase
 Magnesium – kinases
 Potassium – required for maximal activity of pyruvate kinase
 Chlorine – activator of salivary amylase
2. Role in bone and teeth formation
 Calcium, phosphorus, magnesium as constituent of bone matrix formation
 Fluorine in the formation of enamel layer of teeth
 Iron – as a cofactor in iron-containing hydroxylases involved in hydroxylation reactions which
are essential for collagen triple helix formation
 Copper – as a cofactor of lysyl oxidase enzyme which is important for collagen cross-linking
 Manganese – as a cofactor of manganese-dependent transferases which are important for
connective tissue synthesis and GAG synthesis
3. As a component of functional biological compounds
 Phosphate as a component of nucleotide in DNA and RNA, cAMP, cGMP, IP3, phospholipids,
phosphoproteins, active coenzyme forms of vitamins such as TPP, PLP, etc.
 Heme iron in hemoglobin, myoglobin
 Non-heme iron in iron sulfur complexes of electron transport chain
 Zinc as a component of zinc finger motif in transcription factor proteins and also as a
constituent of gustin, salivary peptide
 Iodine is used in the synthesis of thyroid hormones.
 Chloride in gastric HCl synthesis
 Chromium as a component of glucose tolerance factor (GTF)
 Cobalt as a constituent of vitamin B12
4. Regulatory role or role in hormone action

 Ca2+ – release of hormones and neurotransmitters, serve as intracellular signaling molecule
for some hormone action
 Phosphate – as a component of cAMP, cGMP, IP3 which function as intracellular signal
mediator
 Iodine – as a constituent of thyroid hormone
 Chromium – as a component of GTF, potentiates insulin action
 Others – magnesium, manganese, sodium, potassium
5. Role in neuromuscular excitability
 Na+, K+, Cl-, Ca2+, Mg2+
6. Role in transport of substances across cell membrane
 Na+ dependent secondary active transport (glucose, amino acids)
7. Maintenance of membrane stability, integrity and permeability
 Sodium, potassium, calcium
 Phosphate as a component of cell membrane phospholipids
8. Role in antioxidant defense
 As cofactors in many antioxidant enzymes such as selenium in glutathione peroxidase,
copper and zinc in cytosolic SOD, manganese in mitochondrial SOD
 Ferrous iron can generate free radical by Fenton reaction and ferric iron is less reactive in
forming free radical. Copper containing ceruloplasmin has ferrooxidase activity and acts as
antioxidant by catalyzing oxidation of ferrous to ferric iron.
9. Role in body homeostasis processes
 Role in body pH homeostasis
 Phosphate serve as important intracellular and urinary buffer
 Na+, K+, Cl-
 Role in hemostasis process – Ca2+ in blood coagulation process
 Role in maintenance of osmotic pressure, body fluid balance and distribution
 Na+, Cl- – maintenance of ECF osmolarity
 K+ – maintenance of ICF osmolarity and ICF volume
Biochemical Endocrinology & Cell Signaling Page |1
BIOCHEMICAL ENDOCRINOLOGY & CELL SIGNALING
Introduction
 Adaptation to a constantly changing
environment is critical for the survival of

multicellular organisms. Intercellular
communication mechanisms; the nervous
system and endocrine system are
necessary for such adaptation.
 The nervous system can be regarded as a
fixed communication system, whereas

the endocrine system is mediated
through hormones which serve as mobile
messages. These two systems are
converged at some points e.g., neural
regulation of production and secretion of hormones from the endocrine system, synthesis of some
hormones in the nervous system.
 Hormones control not only different aspects of metabolism but also many other functions such as
cell and tissue growth, heart rate, blood pressure, kidney function, motility of gastro-intestinal tract,
secretion of digestive enzymes, lactation and the reproductive system.
Hormone
 A hormone is a chemical messenger, secreted in
trace amount by specific cells that carries a signal

to generate some alteration at the cellular level.
 The word hormone is derived from the Greek
word meaning to stir up or to excite.

 Hormones that act on distant targets (via blood stream) may be
regarded as systemic or general hormones and those acting

locally (mainly in the tissues and sites in which they are produced)
are called local hormones e.g., eicosanoids, endothelin, nitric
oxide, histamine, etc.
 In endocrine signaling, hormones that are produced by specific
tissues or gland act on distant targets cells via the circulation.

 In paracrine signaling, hormones that are produced by specific tissues act on the adjacent cells.
 In autocrine signaling, hormones act on the cells in which they were synthesized.
 In juxtacrine signaling, the molecule stays attached to the signaling cell and binds to a receptor
on an adjacent target cell, establishing physical contact between the two cells.
Chemical nature of hormones

 Lipid derivatives
o Steroids – adrenocortical hormones (cortisol,
aldosterone), sex hormones, calcitriol
o Fatty acid derivatives – eicosanoids (prostaglandins,
thromboxanes, leukotrienes, lipoxins)
o Phospholipid derivatives – platelet activating factor
o Retinol – 9-cis-retinoic acid
 Protein and amino acid derivatives
o Protein – insulin, prolactin, growth hormone, PTH,
chorionic somatomammotropin
o Glycoprotein – FSH, LH, hCG, TSH, erythropoietin
o Peptide – TRH, ACTH, ADH, MSH, oxytocin, endorphin, lipotrophin, ANP, glucagon, calcitonin
o Amino acid derivatives
 Tyrosine derivatives – catecholamines, thyroid hormones
 Arginine derivative – nitric oxide (EDRF)
Biosynthesis and modification of hormones

 Some hormones are synthesized and secreted as
“active forms” e.g., oestriol, aldosterone,
cortisol, catecholamines.
 Some hormones are synthesized as “prepro-
hormones” and secreted as “active forms” after
modification in the secreting cells prior to
secretion e.g., insulin, PTH. ACTH, β-lipotrophin,
γ-MSH are obtained from cleavage of POMC
(pro-opiomelanocortin) peptide.
 Some hormones are synthesized and secreted as “prohormones” and converted to “the active or
more active forms” in the peripheral tissues which may be;
 Target tissues e.g., conversion of T4 to T3 in liver and pituitary, conversion of testosterone to
DHT in secondary sex organs.
 Non-target tissues e.g., conversion of DHEA to androstenedione and testosterone in liver.

 Both target and non-target tissues e.g., hydroxylation of cholecalciferol in liver and kidney to
form active hormone 1, 25 DHCC.
Transportation of hormones
 Hormones are transported as free form or bound form in the circulation.
 Water soluble hormones (protein, peptide hormones) do not need carrier proteins in the plasma
and thus are transported as free forms.

 Lipid soluble hormones bind with specific carrier proteins or plasma proteins and are transported as
bound forms.
o Glucocorticoids are transported by corticosteroid binding protein (CBG) or transcortin and
small amount is transported bound to albumin.
o Estrogen and testosterone (97-99%) is transported bound to sex hormone binding globulin
(SHBG) or testosterone-estrogen binding protein (TEBG).
o Progestin is transported bound to CBG.
 Only unbound or free form of hormone has biologic activity.
 Significance of bound form
o Provision of reservoir of hormone so that a ready supply is always available.

o Buffer against sudden changes in the plasma level of hormones due to its large binding
capacity.
o Prolongation of half-life of hormone since bound proteins cannot be destroyed.
Interaction of hormones
 Inhibitory interaction
o Hormonal interaction in which two hormones have opposite effects on a target tissue, e.g.,
glucagon and insulin, PTH and calcitonin
 Synergistic interaction
o Hormonal interaction in which simultaneous administration of two hormones give an effect
greater than the sum of either of them given alone or a new effect which neither of them can
produce, e.g., FSH and LH on follicular growth, growth hormone, T3 and insulin on optimal
growth.
 Permissive interaction
o Hormonal interaction in which presence of a small quantity of one hormone allows full response
of another hormone on a target tissue, e.g., small amount of corticosteroids facilitates the
pressor effect of catecholamines.
Concentration of hormones in body fluids

 Hormones are present at very low concentrations in the ECF generally in the range of 10-15 to 10-9
mol/L.
Control of hormone secretion and control of plasma hormone level

 Circulatory hormone concentrations are determined by glandular secretory patterns and hormone
clearance rate.
 Hormone secretion is induced by multiple specific
biochemical and neural signals.
 If the circulating hormone level increases,
hormone secretion can be controlled by negative
feedback regulation. Conversely, decreased
circulating hormone level will positively regulate its
synthesis by positive feedback regulation. E.g.,
PTH secretion is induced by fall in serum calcium
level and is inhibited by rising serum calcium levels.
 Hormone secretion from some endocrine organs is
regulated via the regulatory axis, hypothalamo-pituitary-target organ axis. CNS signals such as stress,
afferent stimuli and neuropeptides affect the synthesis and secretion of hypothalamic hormones and
neuropeptides.
 Hypothalamo-pituitary-target endocrine organ axis
o Hypothalamic-releasing hormones (GHRH, CRH, TRH, and GnRH) transverse the hypothalamic
portal vessels and elicit their action on trophic hormone secreting cells of anterior pituitary.
These cells express growth hormone (GH), ACTH, TSH, and gonadotrophins (FSH, LH).
o In contrast, somatostatin and dopamine suppress GH, prolactin or TSH secretion.
o Trophic hormones then act on target endocrine organs and maintain the structural and functional
integrity of endocrine organ e.g., ACTH stimulate glucocorticoids synthesis and secretion from
adrenal cortex, TSH act on thyroid gland stimulating thyroid hormones synthesis and secretion,
and gonadotrphins act on gonads stimulating synthesis and secretion of sex hormones.
o Target hormones in turn serve as powerful negative feedback regulators of their respective
trophic hormone and also suppress secretion of hypothalamic releasing hormones.
o In certain circumstances (e.g., during puberty), sex hormones may positively induce the
hypothalamo-pituitary-target gland axis, e.g., LH induces ovarian estrogen secretion and
secreted estrogen induces further LH secretion by positive feedback stimulation.
Biochemical Endocrinology & Cell Signaling P a g e |5
Trophic hormones – are hormones that can stimulate the secretion of hormones from other endocrine
glands or tissues; they also promote the synthesis of hormones and increase the vascularity and the
growth of the target gland or tissue.
Target cell – is the cell that is able to recognize specific hormone due to the presence of specific receptor.
Hypothalamo-anterior pituitary-target organ regulatory axis

 The hypothalamo-anterior pituitary
regulatory system comprises 5
separate endocrine axes.
 Three axes form a complex of three-
level endocrine system and cause
the release of trophic hormones
from anterior pituitary which then
subsequently stimulate specific
hormones secretion from target
endocrine organ; hypothalamo –
anterior pituitary – thyroidal axis,
hypothalamo – anterior pituitary –
adrenal axis and hypothalamo – anterior pituitary – gonadal axis.
 The 4th axis is a hybrid; growth hormone (GH) is both a trophic hormone and has its action in its own
right.
 The 5th axis mediates secretion of prolactin which is not a trophic hormone.
Hypothalamo-posterior pituitary regulatory axis

 Hormones are synthesized in the cell bodies of the supraoptic and paraventricular nuclei of the
hypothalamus. Then they are packaged and transported along axons to the posterior pituitary
where they are secreted.
 Hormones in this regulatory system include oxytocin and arginine vasopressin (AVP) or ADH.
Clinical conditions associated with hormonal disorders

 These comprise two categories – hormone overproduction (hormonal excess) and underproduction
(hormonal deficiency).
 When hormone overproduction is associated with an increase in the total number of hormone-
producing cells itself, it is said to be primary e.g., in Graves’ disease, hyperthyroidism is due to
activation of TSH receptors by antibodies that mimic TSH which leads to increased thyroid cell
proliferation as well as increased synthesis and release of thyroid hormones from thyroid gland.
 Hormonal disorders may be secondary if the pathology lies in anterior pituitary or tertiary if the
pathology lies in the hypothalamus in case of hormonal disorders in hypothalamo – anterior pituitary
– target endocrine organ axis, e.g., reduced TSH release from anterior pituitary leads to low thyroid
hormone release from thyroid gland due to the lack of its trophic factor TSH.
 Underproduction of hormones can result from a variety of processes —
o Surgical removal of parathyroid gland during thyroid surgery
o Destruction of adrenal glands by tuberculous infection
o Defective insulin secretion from β cells of pancreas due to iron deposition in hemochromatosis
o Autoimmune destruction of β cells of pancreas in type 1 diabetes mellitus
 Hormonal disorders might be the result of defects in hormone signaling pathway in the target
tissues leading to altered tissue response to hormonal action rather than the consequence of defects
in hormone secretion or synthesis. Altered tissues response to hormonal activity can be caused by a
variety of genetic disorders —
Pituitary hormone disorders
o Mutation in the growth
Hormone Deficiency Excess
hormone receptor in Laron
TSH Hypothyroidism Hyperthyroidism, thyrotoxicosis
dwarfism
ACTH Hypoadrenalism Cushing’s disease
o Insulin resistance in muscle and
FSH, LH Hypogonadism Precocious puberty
liver in type 2 diabetes mellitus
GH Growth retardation Gigantism/acromegaly
o Activation mutation in LH
receptor in precocious puberty Prolactin None Galactorrhea/infertility
Major types of endocrine pathology

Autoimmunity
o Autoimmune destruction with detectable endocrine gland specific antibodies is a potential cause
of loss of function of a particular endocrine gland and less commonly may also be the cause of
gland hyper-function, e.g., type 1 diabetes mellitus, Grave’ disease.
Endocrine neoplasia
o This may be associated with increased or decreased secretion of hormones from the affect
endocrine organ. It may be either benign or malignant, e.g., adrenocortical tumor.
Exogenous causes
o Use of hormones for a desired therapeutic action can lead to clinical problem as a side effect of
hormonal therapy e.g., excessive and prolonged use of corticosteroids in inflammatory disease
or asthma can lead to Cushing’s syndrome.
o Inappropriate self-administration of hormones can also lead to clinical problem e.g., use of
thyroid hormone to induce weight loss can lead to hyperthyroidism.
Non-classic endocrine organs

Apart from classic ductless endocrine glands, many tissues are now known to be active
endocrinologically, and produce hormonal signals that mediate important metabolic cross-talk
between different tissues. Some examples are –
o Production of leptin from white adipose tissues which signals body energy stores to the brain
o Secretion of erythropoietin from the kidney in response to hypoxia
Assessment of endocrine function

Biochemical tests in the assessment of endocrine function are used to determine whether the
specific endocrine system is functioning abnormally and to localize the functional defect.
Assessment of endocrine function can be done by measuring levels of basal circulating hormones,
hormone binding proteins, evoked or suppressed hormones and peripheral hormone receptors. To
assess a particular hormonal axis, level of hormone that elicits the target tissue response and levels
of one or more upstream trophic hormones are usually determined.
Assessment of at least two points in such an endocrine feedback loop is essential and permits
focusing of later diagnostic imaging on the relevant gland.
Commonly used laboratory methods in hormone level assessment
o Radioimmunoassay (RIA)
o Enzyme linked immune sorbent assay (ELISA)
Factors to be considered in appropriate sampling for specific hormonal assay
o Stability of hormones
o Secretion pattern of hormone (circadian or pulsatile or other rhythm), e.g., for thyroxine
which has very long half-life, timing of sample is not critical, while for cortisol timing of
sample is essential as peak secretion of cortisol occur in early morning.
Some hormones are measured after a relevant stimulus has been applied and stimulus may be high
dose of trophic hormone or metabolic challenge e.g., insulin induced hypoglycemic stress to induce
cortisol
secretion.
Target cell concept

 A given hormone can affect several different cell types that express its cognate receptors and more
than one hormone can affect a given cell type.
 Hormones can exert many different effects in one cell or in different cells.
 Response of a target cell to a specific hormone can be influenced by several factors. These include
factors affecting the concentration of hormone at the target cell and factors affecting the actual
response of a target cell to the hormone.
 Factors affecting the concentration of hormone at the target cell
o Rate of synthesis and secretion of hormones
o Proximity of target cells to the hormone source (dilution effect)
o Affinity of hormones with specific carrier proteins
o Rate of conversion of inactive or sub-optimally active hormones into fully active forms
o Rate of clearance of hormones from the plasma (degradation or metabolism or excretion)
 Factors affecting target tissue response to the hormone
o Number, relative activity and state of occupancy of the hormone specific cell membrane
receptors or intracellular receptors
o Up or down-regulation of receptors as a consequence to the interaction with its ligand e.g.,
receptor desensitization or internalization or regulation receptor protein synthesis
o Presence of other factors within the target cell that are necessary for the hormone response
or signaling
o Metabolism (activation or inactivation) of the hormones in the target cell
Cell signaling
All living cells respond to their environment through a set of
mechanisms known as cell signaling. This ability is the basis for
normal cellular homeostasis, cell growth, division and differentiation.
Errors in cell signaling contribute to the development of diseases
such as cancer, autoimmunity and diabetes. Hence, a clear
understanding of cell signaling is vital to effectively treating such
diseases.
General outline of cell signaling or signal transduction

Cell signaling operates through an intricate network of biochemical pathways. Signal-transduction
pathways follow a broadly similar course that can be viewed as a molecular circuit. Key steps
involved all signaling pathways are –
o Release of the primary messenger

 In response to a stimulus, a signaling cell synthesizes and secretes a
signaling molecule (primary messenger). These are typically small lipid
soluble or water soluble molecule e.g., hormones, growth factors, etc.
o Reception of the signaling molecule
 The signaling molecule is transported to the target cell via the blood
stream. Reaching to the target cells, it binds to a specific receptor protein
which may be cell membrane receptor or intracellular receptor. Receptor
transfers the information that the signaling molecule carries into the cell’s
interior, initiating the intracellular signaling pathways.
o Delivery of the message inside the cell by intracellular signal mediators
 The signaling molecule-receptor complex activates one or more
intracellular signaling pathways involving intracellular signal mediators
such as G proteins, second messengers, protein kinases, DNA binding
proteins, etc.
o Activation of effectors that directly alter the physiological response
 Intracellular signaling proteins alter the activity of effector proteins via the
intracellular signaling pathway. Effector proteins may be pumps, channels,
enzymes, transcription factors, etc. The ultimate effect of the signaling pathway is to activate
(or inhibit) metabolic pathways, gene expression, and behavior of the target cells.
o Termination of the signal
 After a cell has completed its response to a signal, the signaling process must be terminated
or the cell loses its responsiveness to new signals. The signal is terminated by removal of the
signaling molecule or receptor or inactivation of the signaling events triggered by the
signaling molecule-receptor complex.
Signal transduction
 Signal transduction is the process by which the message carried by
signaling molecule is accepted by the specific receptor and is
transmitted via intracellular signal mediators to generate the
appropriate responses within the cells.
Signaling molecules
 Signaling molecules are molecules that can generate specific response
in its target cell to adapt the changes in its environment. These include –
o Hormones (major factor)
Biochemical Endocrinology & Cell Signaling P a g e | 10
o Growth factors and cytokines o Gases (nitric oxide, CO and free

o Neurotransmitters radicals)
o Cellular antigens and extracellular matrix o Other physicochemical factors (pH,
o Special senses (sight, sound, taste, smell O2 tension, temperature, osmolarity,
and touch) etc)
Receptors
 The first step the action of hormone is binding to a specific molecule called the hormone receptor.
 Receptors are cell associated recognition proteins that are able to recognize and bind specific
extracellular signaling molecule (ligand) and initiate intracellular signaling pathway to generate the
appropriate response in the cell.
 Receptors have very high specificity and affinity to its hormone.
 The receptors are located on the cell surface or in the cytosol/nucleus of the target cell.
 Chemical nature of hormone receptor
Protein or glycoprotein in nature
Contains two functional domains
1. Recognition domain that recognizes and binds the specific hormones with high affinity
2. Signal transduction domain that generate a signal which couples hormone recognition to
some intracellular signaling pathways
 Types of receptor
There are two type of receptors –

1. Intracellular receptors
2. Cell membrane receptors
1. Intracellular receptors
Receptors for lipophilic signaling molecules such as
steroid/thyroid hormones constitute a large superfamily
called intracellular receptors which can be found in the
cytoplasm or in the nucleus of the cell.
Many members of this family have no known ligand,
hence are called orphan receptors.
Most intracellular receptors act as gene specific transcription factors. Hormone receptor
complex mediate intracellular responses by influencing gene expression.
These receptors have several functional domains;
1. Hormone binding region (C-terminal portion)
2. Adjacent DNA binding region
3. Specific region for high affinity binding to proper region of DNA (N-terminal half)
4. One or more regions that activate or repress gene transcription
5. Cofactor binding domain
6. Regions for translocation of receptor from the cytoplasm to the nucleus
2. Cell membrane receptors

Receptors for hydrophilic signaling molecules such as neurotransmitters, protein hormones,
peptide hormones and growth factors are cell membrane receptors.
Cell membrane receptors act as signal transducers. They bind the specific hormone with high
affinity and convert extracellular signal into one or more intracellular signals that mediate
intracellular response in the target cell.
Cell membrane receptors have three functional domains;
1. Hormone binding domain
2. Signal transduction or catalytic domain
3. Trans-membrane domain spanning the cell membrane
Cell surface receptors are classified into three classes based on the mechanism used for signal
transduction. These include ion channel linked receptors, G protein coupled receptors and
enzyme linked receptors.
Ion-channel linked receptors

 They are also known as transmitter gated
ion channels and are involved in rapid

synaptic signaling between electrically
excitable cells.
 Neurotransmitters released from pre-
synaptic cells bind to the ion-channel linked
receptors on the post-synaptic cells and transiently open or
close the ion-channels associated with the receptor proteins.
Thus, upon binding with neurotransmitters, these receptors
mediate synaptic signaling by changing the ion permeability
and excitability of the post-synaptic cells, e.g., receptors for
acetylcholine.
 Ion-channel linked receptors belong to a family of
homologous, multi-pass trans-membrane proteins.
G protein coupled receptors (GPCRs)

 These receptors act indirectly to regulate the activity of a
separate plasma membrane bound target
protein (enzyme or ion channel). The
interaction between the receptor and the
membrane bound target protein is mediated
by G protein, thus it is called GPCR, e.g.,
receptor for catecholamines.
 GPCRs belong to a family of homologous, seven-pass trans-
membrane proteins.
Enzyme linked receptors

 When activated, enzyme linked receptor functions directly as
enzymes or in association with enzymes. Intracellular domain of
the receptor has catalytic activity which is activated upon binding
of ligand to extracellular domain.
 Enzyme linked receptors are heterogenous, although the majority
are protein kinases and most are single pass trans-membrane
proteins. E.g., receptors for insulin and growth factors.
Regulation of receptors
Receptors for chemical messengers are not static component of the cells. Their number increase or
decrease in response to various stimuli and their properties change with change in physiological
conditions.
When the chemical messenger is present in excess, the number of active receptors generally
decreases (down-regulation) whereas deficiency of the chemical messenger leads to increased
number of active receptors (up-regulation).
Up-regulation of receptors can be achieved by inducing receptor protein gene expression and
enhancing translocation of receptors to the membrane.
Down-regulation of receptors can be achieved by internalization of receptors by receptor mediated
endocytosis followed by recycling or degradation, desensitization of receptors (making less
responsive by chemical modification) and repression of receptor protein gene expression.
Drugs targeting the receptor activity can be used as therapeutic agents. These include receptor
agonists (stimulates the receptor activity) and receptor antagonists (inhibits the receptor activity).
Natural and synthetic androgen receptor agonists bind to androgen receptor and stimulate gene
expression that enhances the development of lean body mass. β adrenergic receptor agonists are
used for relaxation of bronchial smooth muscles in bronchial asthma.
Estrogen receptor antagonists (tamoxifen, xaloxifen) are used in the treatment and prevention of
breast cancer because it can inhibit estrogen mediated pathways in growth of some breast tumors.
Angiotensin receptor blockers (losartan) inhibit the angiotensin II mediated vasoconstriction and
can be used in the treatment of hypertension.
Classification of signaling molecules according to the mechanism of action

1. Signaling molecules that bind to intracellular receptor (lipophilic signaling molecules with long plasma
half-life)
 Glucocorticoids  Progestins
 Mineralocorticoids  Calcitriol (1, 25 DHCC)
 Androgens  Thyroid hormones (T3 and T4)
 Estrogens  Retinoic acid
2. Signaling molecules that bind to cell surface receptors (hydrophilic signaling molecules with short
plasma half-life)
a) Signaling molecules that use G protein coupled receptors (GPCRs)
 They can be differed according to 2nd messenger used or directly acting on the ion channels.
1. Signaling molecules that directly act on ion channels via GPCRs – light, smell
2. Signaling molecules that use cAMP as 2nd messenger
 ADH (V2 receptor)  Glucagon
 Angiotensin II  PTH
 Acetylcholine  Calcitonin
 α2 adrenergic catecholamines  ACTH, TSH, FSH, LH, MSH
 β adrenergic catecholamines  hCG
 Somatostatin  CRH
3. Signaling molecules that use calcium or phosphatidylinositol derivatives as 2 nd messenger
 ADH (V1 receptor)  TRH, GnRH
 Angiotensin II  Oxytocin
 Acetylcholine (muscarinic)  Substance P
 α1 adrenergic catecholamines  Gastrin, CCK
b) Signaling molecules that use ion-channels linked receptors
 Acetylcholine (nicotinic receptors)  GABA

 Glycine  Serotonin
c) Signaling molecules that use enzyme-linked receptors
Signaling molecules that use receptor guanylyl cyclase Atrial natriuretic peptide (ANP)
Insulin
Insulin like growth factor (IGF)
Epidermal growth factor (EGF)
Signaling molecules that use receptor tyrosine kinase Platelets derived growth factor
(PDGF)
Nerve growth factor (NGF)
Vascular epidermal growth factor

(VEGF)
Growth hormone
Erythropoietin
Signaling molecules that use tyrosine kinase associated
Prolactin
receptor
Cytokines
Certain antigens
Signaling molecules that use receptor serine-threonine Transforming growth factor β (TGF β)
kinase
Signaling molecules that use receptor tyrosine phosphatase

Mechanism of action of lipophilic

signaling molecules
 Lipophilic signaling molecules
such as steroid hormones,
thyroid hormones, calcitriol
and retinoic acid elicit their
mechanism by binding to
intracellular receptors.
 After secretion, these
hormones associate with
transport proteins. At the
target cells, free hormones
readily transverse the plasma
membrane and encounter with their specific receptors in either
Peroxisome proliferation
cytosol or nucleus. The hormone-receptor complex serves as activated receptors (PPARs)
intracellular messenger in signaling mechanism of lipophilic  Regulated by intracellular
metabolites rather than
hormones.
secreted signal molecules
 Cytoplasmic receptors exist in the cytoplasm as an inactive complex
e.g., fatty acids or fatty
with inhibitory protein (heat shock protein). Hormone binding alters acid derivatives
conformation of the receptor protein, causing dissociation of heat  Act in the nucleus by
shock protein. forming heterodimers with

another nuclear receptor,
 Activated hormone-receptor complex then translocates to the
RXR, binding to regulatory
nucleus and binds to specific DNA sequence called hormone regions of DNA near the
response element (HRE). HRE is part of the regulated expression genes under their control
region of gene and behaves as enhancer or silencer. affecting the rate of

transcription of those
 Thus, hormone-receptor complex acts as gene regulatory protein or
genes.
specific transcription factor by binding to HRE at the regulatory
 The three PPAR isoforms
region of specific genes to alter the rate of transcription, regulate lipid and glucose
subsequently in rate of protein synthesis encoded by these genes. metabolism in liver, muscle,
Change in the amount of target proteins leads to alteration of and adipose tissue.
 PPARα and PPARδ (its
cellular activity, metabolism in response to hormonal signals in the
related isoform PPARβ)
target cells. regulate lipid utilization.
 The intracellular receptors for lipophilic signaling molecules are all  PPARγ regulates insulin
structurally related and constitute a large nuclear receptor family. sensitivity and lipid
storage.
 Signaling pathway via nuclear receptor has cross-talk with other signaling pathways. Some hormones
and cytokines via cell surface receptors signaling pathway also regulate gene transcription through
phosphorylation and changing the functional conformation of nuclear receptors.
 Examples of signaling via intracellular receptors
o Action of calcitriol or 1, 25 DHCC on intestinal calcium absorption
o Actions of aldosterone on renal tubular reabsorption of sodium
o Actions of thyroid hormone on general metabolism
Mechanism of action of hydrophilic signaling molecules

 These signaling molecules are water soluble and have no transport proteins and thus shorter plasma
half-life. They bind to receptors present on the plasma membrane of the target cell. Signaling
mechanisms of these hormones can be classified into three groups based on the types of receptors
they use (ion-channel linked receptors or GPCRs or enzyme linked receptors).
 Signaling mechanism of hydrophilic hormones can also be described by the intracellular signals they
generate. These intracellular signals are called second messengers which are generated upon ligand-
receptor interaction and function to mediate the signaling effect of hormones in the cell.
 During signaling, hormone acts as the first messenger carrying the information from the endocrine
gland to the cell and intracellular intermediary molecules carry information from the cell membrane
to the target effector protein, thus it is called second messenger.
 The second messengers are cyclic nucleotides, calcium and phosphoinositides derivatives.
Mechanisms of action of signaling molecules via ion-channel linked receptors.

Ion-channel linked receptors have highly selective binding sites for
neurotransmitter and they are concentrated on post-synaptic cell
membrane.
Neurotransmitters released from presynaptic cells bind directly to ion-
channel linked receptors on post-synaptic cell membrane. Ion-channels
associated with receptors transiently open or close in response to
binding of neurotransmitter, leading to ion permeability and
subsequent excitability changes in post-synaptic cells. In this way, ion-
channel linked receptors convert chemical signals from
neurotransmitters into electrical potential changes at synaptic junction.
Excitatory neurotransmitters such as acetylcholine, glutamate and serotonin bind and open cation
channels, causing influx of Na+ that leads to depolarization and action potential in the post-synaptic
cell membrane.
Inhibitory neurotransmitters such as γ amino butyric acid (GABA) and glycine bind and open Cl-
channels and cause hyperpolarization of post-synaptic membrane.
Some toxins block ion-channel linked receptors and inhibit neurotransmitter signaling at synaptic
junction e.g., strychnine binds to glycine receptors and blocks inhibitory neurotransmission of
glycine causing muscle spasm, convulsions and death.
Mechanism of action of signaling molecules via G protein coupled receptors

Many hormones bind GPCRs that couple to effectors through GTP binding regulatory protein (G-
protein).
G protein
G proteins are GTP binding regulatory proteins that couple hormone-receptor interaction to its
target enzymes or ion channels in the cell membrane.
G-proteins involve in the regulation of a diverse range of biological processes, including signal
transduction, protein synthesis, intracellular trafficking (targeted delivery to the plasma membrane
or intracellular organelles) and exocytosis, as well as cell movement, growth, proliferation and
differentiation.
G-protein superfamily comprises two major subfamilies: the small, monomeric Ras-like G-proteins
(involve in growth factor and insulin signaling pathway) and the heterotrimeric G-proteins. Hetero-
trimeric G-proteins involve in transduction of trans-membrane signals from cell surface receptors to a
variety of intracellular effectors, such as adenylyl cyclase, phospholipase C (PLC), cGMP-
phosphodiesterase (PDE) and ion channel effector systems.
Role of G protein in signaling mechanisms via GPCRs

Heterotrimeric G-proteins regulate transmembrane signals by acting
as molecular switches, linking cell surface GPCRs to one or more
downstream signaling molecules. Trimeric G protein contains three
distinct subunits (α, β, γ). Effector specificity is conferred by α
subunit which contains GTP binding site and intrinsic GTPase activity.
G protein act as on-off molecular switches that are active when GTP
is bound to α subunit and inactive when GDP is bound to α subunit.
When hormone binds to a GPCR, the activated receptor switches on
the G protein by exchanging GDP for GTP in Gα subunit. This induces
a conformational change in Gα, which results in a decrease in its
affinity for both the receptor and the βγ-subunits, leading to
dissociation of the receptor–G protein complex.
The activated GTP bound Gα subunit or released βγ subunits or

both can then interact with one or more effectors to generate
intracellular second messengers, which activate downstream
signaling cascades. Target effector proteins that interact with Gα
subunit is differed by different G protein types and subsequently
2nd messengers generated and downstream signaling pathways
will be differed.
Signaling of G protein is terminated by intrinsic GTPase activity
of Gα subunit which hydrolyzes bound GTP to GDP, reforming
inactive hetero-trimeric G protein (G αβγ).
Different types of G proteins based on different Gα subunits are Gs, Gi, Gq, Gt and G12.
o Signaling via Gs (stimulatory
Gα) activates adenylate
cyclase activity and increase
2nd messenger cAMP that
mediates cellular response,
e.g., signaling by glucagon, β
adrenergic, odorant, tastants.
o Signaling via Gi (inhibitory
Gα) inhibits adenylate
cyclase activity and lowers cAMP concentration. Some Gi activates K+ channels, e.g., signaling via
M2 cholinergic receptor, α2 adrenergic receptor.
o Signaling via Gq activates phospholipase C (PLC) and leads to production of 2nd messengers
inositol triphosphates (IP3), diacylglycerol (DAG) and Ca2+, e.g., signaling via M1 cholinergic
receptor, α1 adrenergic receptor.
o Signaling via Gt activates cGMP phosphodiesterase (PDE) and lowers cGMP level, e.g., signaling
in vision via photon activated receptor (visual receptors).
o Signaling via G12 stimulates intracellular small monomeric G protein, Rho protein, e.g., signaling
by thrombin.
o Various bacterial toxins irreversibly modulate G protein function by covalent modification.
o Cholera toxin (from Vibrio cholerae) catalyzes ADP-ribosylation of Gs-α subunit thereby
inhibiting intrinsic GTPase activity of Gs-α subunit. This results in permanent activation of G
protein and subsequently prolonged increase in cAMP concentration leading to opening of Cl-
channels in intestinal epithelial cells. Large efflux of electrolytes and water into the gut results in
severe diarrhea in cholera.
o Pertussis toxin (from Bordetella pertussis) catalyzes ADP-ribosylation of Gi-α subunit thereby
preventing Gi-α subunit to interact with activated receptors and βγ subunits. This results in
permanent inactivation of G protein which cannot inhibit adenylyl cyclase causing uncoupling of
hormone receptors from their signaling cascades. Pertussis toxin is the causative agent of
whooping cough.
Mechanism of signaling molecules via cAMP cascade

 Cyclic adenosine monophosphate (cAMP) is a cyclic nucleotide that acts as the second messenger.
cAMP plays a crucial role in the action of a number of hormones. The intracellular level of cAMP is
increased or decreased by various hormones and this effect varies from tissue to tissue, e.g.,
epinephrine causes large increase in cAMP concentration in muscle and relative small changes in
liver.
 Formation of cAMP
o cAMP or 3’, 5’ cyclic adenylic acid is
derived from ATP by the action of
enzyme adenylyl cyclase. Adenylyl
cyclase system is composed of two
parallel systems; stimulatory (s)
and inhibitory (i) systems which
converge on a single catalytic
molecule (adenylyl cyclase). Each
system consists of a receptor Rs or
Ri and a G protein Gs or Gi.
o When a hormone binds to Rs
receptor, hormone receptor
complex induces activation of Gs
protein by exchanging GDP for GTP in Gs-α subunit. Gs-α – GTP subunit dissociates from βγ
subunits and activates adenylate cyclase causing increase in intracellular cAMP level, e.g.,
signaling of epinephrine via β adrenergic receptor.
o When a hormone binds to Ri receptor, hormone receptor complex induces activation of Gi
protein by exchanging GDP for GTP in Gi-α subunit. Gi-α – GTP subunit inhibits adenylate cyclase
and intracellular cAMP level is decreased, e.g., signaling of epinephrine via α2 adrenergic
receptor.
 Mechanism of cAMP action

o cAMP binds to cAMP dependent
protein kinase (PKA). PKA is hetero-
tetrameric molecule consisting of 2
regulatory subunits (R) and 2
catalytic subunits (C). Normally R2C2
complex has no enzymatic activity.
4cAMP + R2C2  R2–4cAMP + 2C
o cAMP binding to R subunits in R2C2 complex
causes dissociation of R2C2 complex, thereby
activating the catalytic subunit (C). The active
C subunit catalyzes phosphorylation of serine
or threonine residues in a variety of proteins.
o Covalent phosphorylation of specific amino
acids regulates the activity of target protein.
The target protein may be enzyme or ion
channels or DNA binding protein or
cytoskeletal proteins, e.g., regulation of
glycogen metabolism in skeletal muscle by
cAMP dependent phosphorylation on key enzymes of
glycogen metabolism.
o Effects of cAMP on gene transcription are mediated by
cAMP response element binding protein (CREB).
Phosphorylation of CREBs by cAMP dependent protein
kinase (PKA) leads to dimerization of phosphorylated
CREB which then act as a transcription factor.
o cAMP also regulate calcium and chloride channels in
the plasma membrane and alter the membrane
permeability to these ions.
o The effect of cAMP include diverse processes such as
steroidogenesis, secretion, ion transport,
carbohydrate and fat metabolism, enzyme induction,
gene expression regulation, cell growth and
replication.
 Termination of cAMP actions

o Action of hormones whose
actions are mediated through
cAMP signaling cascade can
be terminated by —
 Receptor desensitization
(phosphorylation of GPCR
by G protein receptor
kinase)
 Deactivation of Gs-α
subunit through hydrolyzing bound GTP to GDP and Pi by its intrinsic GTPase activity; thus
adenylyl cyclase is no longer activated.
 Decreasing cAMP concentration by hydrolysis of cAMP to 5’ AMP by phosphodiesterases
(PDE). Inhibitors of PDE such as methylated xanthine derivatives (caffeine) increase cAMP
level and prolong the hormonal action.
 Dephosphorylation of target proteins by phosphoprotein phosphatases
 Some examples of signaling mechanism of hormones via cAMP cascade
o Action of TSH on thyroid hormone secretion from thyroid gland
o Action of epinephrine (via β adrenergic receptor) on glycogen metabolism in muscles and liver
o Action of PTH on bone and kidney in calcium homeostasis
o Action of ADH (vasopressin) on water reabsorption in renal tubular cells
o Action of glucagon on glycogen metabolism in liver
Mechanism of signaling molecules

via phosphor-inositide cascade
 Signaling mechanism of
hormones and neurotransmitters
via phosphoinositide cascade
generate two intracellular
second messengers; DAG and IP3.
 In this signaling cascade,
hormone binding to GPCR
activates Gq protein which in turn activates the membrane bound enzyme phospholipase C (PLC).
PLC cleaves phosphotidyl inositol 4, 5 biphosphate (PIP2) to yield two intracellular signal mediators –
1, 2 diacyglycerol (DAG) and inositol 1, 4, 5 triphosphate (IP3).
 Action of IP3
o IP3 released from cleavage of PIP2 acts as a

hydrophilic intracellular signal mediator. IP3
directly binds and opens IP3 gated Ca2+ channels
on endoplasmic reticulum membrane, allowing stored Ca2+ to be released into the cytoplasm.
o The resulting elevated cytosolic Ca2+ level causes –
 Translocation of soluble enzyme protein kinase C (PKC) from cytosol to the cell membrane
where it is activated by membrane bound DAG and phosphatidyl serine
 Activation of Ca2+ - calmodulin dependent protein kinases (CaM kinases) and many other
Ca2+ - calmodulin dependent enzymes and proteins
 Fate of IP3
o IP3 is a short-lived messenger (half-life less than a few seconds). It can be degraded into inositol
by the sequential action of phosphatases. Inositol can be reutilized for synthesis of PIP2.
o Lithium ion, widely used to treat bipolar disorder, may act by inhibiting the recycling of IP3.
 Action and fate of DAG
Protein kinase C and cancer
o DAG, a cleavage product of PIP2, has two potential signaling
PKC is a family of
roles –
serine/threonine kinase
 It can be cleaved to release arachidonic acid (C20 PUFA) involved in the regulation of
which either can act as second messenger (regulating normal cellular proliferation.
phospholipase C isoforms and protein kinases) or be used as Dysregulation of PKC has

implications in promoting a
precursor in eicosanoid synthesis.
range of pathologic
 It can activate protein kinase C (PKC) which is calcium
conditions including cancer,
dependent. Activated PKC then phosphorylates specific cardiovascular disease
serine/threonine residues of target proteins. Depending on and inflammation.

Tumor-promoting phorbol
cell types, target proteins may be ion channels or other
esters (an ingredient in
protein kinases (such as mitogen activated protein kinases)
croton oil that promotes skin
or gene regulatory protein, thus it has several consequences cancer) can directly
on cellular metabolism, growth and differentiation. activate protein kinase C,
o DAG is removed by hydrolysis to monoacylglycerol and free mimicking DAG and

bypassing the receptors.
fatty acid.
2+
 Action of ionized calcium (Ca )
o Ionized calcium is an important regulator of a variety of cellular processes. It is also an

intracellular second messenger for hormone action.
o Normal intracellular concentration of free or ionized calcium is very low (0.05 – 10 μmol/L).
o Intracellular Ca2+ concentration can be increased by –

2+ 2+
 Altering Ca permeability of cell membrane via Ca channels (either ligand gated or voltage
gated)
2+
 Releasing intracellular Ca stores in ER and possibly from mitochondria by the action of IP3
o Many downstream signaling cascades of

elevated cytosolic calcium level is mediated by
calcium dependent regulatory protein,
calmodulin. Calmodulin is a Ca2+ sensing and
binding protein which has four Ca2+ binding
sites. Full occupancy of these sites leads to
conformational change which activates
calmodulin to activate enzymes and ion-channels.
o Ca2+ - calmodulin complex regulates the activity of many structural elements in the cell such as
actin-myosin complex of smooth muscle and various proteins involved in cell motility, cell
conformation changes, mitosis, granule release and endocytosis in non-contractile cells.
o Sometimes, calmodulin can be found as a regulatory subunit of complex protein such as various
kinases and cAMP phosphodiesterase.
o Most of the effects of Ca2+ in cells are mediated by a family of Ca2+ - calmodulin dependent
protein kinase (CaM kinases). These kinases phosphorylate serine or threonine residues in target
proteins and regulate metabolic processes.
o Examples of such kinases are specific CaM kinase and multifunctional CaM kinases. Specific CaM
kinase targets only specific proteins, e.g., myosin light chain kinase activates smooth muscle
contraction and phosphorylase kinase activates glycogen breakdown. Multifunctional CaM
kinase (CaM-kinase II) can phosphorylate and modulate a wide range of protein substrates. It is
found in nervous tissues. Upon activation, it phosphorylates and activates tyrosine hydroxylase
thereby increases catecholamine synthesis.
2+
 Termination of Ca signaling
o Most of the effects of Ca2+ in cells are mediated by CaM kinases. CaM kinase activation can
function as a molecular memory device because it remains active even after Ca2+ is withdrawn.
So termination of Ca2+ signaling is mainly achieved by lowering of cytosolic Ca2+ level and
dephosphorylation of target proteins.
o Intracellular Ca2+ level can be lowered by –
2+ 2+
 Sequestration of Ca into ER by Ca - ATPase or into mitochondria
2+
 Chelation with Ca binding proteins such as calsequestrin
o Some of the effects of Ca2+ mediated target protein phosphorylation can be negatively
modulated by a phosphatase activated by Ca2+ and calmodulin known as calcineurin.
 Some examples of signaling mechanisms of hormones via phosphoinositide cascade
o Action of catecholamines via α1 adrenergic receptor

o Action of TRH on TSH secretion from anterior pituitary
o Action of acetylcholine via M1 muscarinic receptor
Mechanism of action of signaling molecules via enzyme linked receptor

Signaling cascade can be varied according to type of enzyme linked receptor used. There are five
different types of enzyme linked receptor – receptor guanylyl cyclase, receptor tyrosine kinase,
tyrosine kinase associated receptor, receptor serine-threonine kinase, receptor tyrosine
phosphatase.
Mechanism of signaling molecules via

receptor guanylyl cyclase
 Signaling mechanism of atriopeptide (e.g.,
atrial natriuretic factor ANF or atriopeptin)
is via receptor guanylyl cyclase. Binding of
ANF to receptor activates the guanylyl
cyclase activity of cytosolic domain of the
receptor resulting in an increase of cGMP.
 Cyclic guanosine monophosphate (cGMP) or 3’, 5’ cyclic guanylic acid is a nucleotide derived from
GTP by the action of enzyme guanylyl cyclase. This enzyme may exist as membrane bound form (as a
component of cytosolic domain in receptor protein) and soluble form in the cytoplasm.
 Then, cGMP activates cGMP dependent protein kinase (PKG) which in turn phosphorylates a number
of target proteins at serine/threonine residues e.g., myosin light chain in smooth muscle. Presumably,
this is involved in relaxation of smooth muscle and vasodilation. In this way the effects of ANF such
as natriuresis, diuresis, vasodilation, and inhibition of aldosterone secretion are mediated by cGMP.
 Signaling transduction via cGMP can be terminated by the action of cGMP phosphodiesterase which
degrades cGMP to 5’GMP thereby decreasing cGMP concentration.
 The soluble form of guanylyl cyclase in the cytosol is activated by nitric oxide (NO). NO can directly
modulate the activity of cytoplasmic guanylyl cyclase and increase cGMP level which mediates the
signaling effects of NO in the cell (relaxation of vascular smooth muscle cells in the blood vessels).
 Compounds like nitroprusside, nitroglycerin, sodium nitrite and sodium azide cause smooth muscle
relaxation and are potent vasodilators. These agents increase cGMP by activating soluble form of
guanylyl cyclase and inhibiting cGMP phosphodiesterase e.g., sildenafil (Viagra). These agents
enhance and prolong effects of cGMP signaling e.g., vasodilation and increased blood flow to the
tissues.
 Similar guanylyl cyclase receptors present in the intestinal mucosal cells are the target of endotoxin
produced by E.coli and other gram-negative bacteria. The elevation in cGMP level caused by
endotoxin binding to receptor guanylyl cyclase increases Cl- secretion resulting in decreased water
reabsorption and diarrhea.
Mechanism of signaling molecules via receptor tyrosine kinase

 Signaling mechanisms of growth and differentiation factor such as epidermal growth factor (EGF),
platelet derived growth factor (PDGF), fibroblast growth factor (FGF), hepatocyte growth factor
(HGF), insulin, insulin like growth factor (IGF-1), nerve growth factor (NGF), vascular endothelial
growth factor (VEGF) and macrophage colony stimulating factor (M-CSF) are mediated via receptor
tyrosine kinase (RTK).
 Common steps involved in signaling via RTKs
o A signaling molecule binds to
the extracellular domain of
RTK inducing the conformation
change in receptor that leads
to receptor dimerization.
o Tyrosine kinase activity in
cytosolic domain of RTK
becomes activated upon
ligand binding and dimerized
receptor phosphorylates
specific tyrosine residues on
itself (autophosphorylation or
cross phosphorylation).
o These phosphor-tyrosine residues are recognized and bound by protein having SH2 domains
which may be adaptor or docking protein or enzyme which activate downstream signaling
pathway. Downstream signaling pathway is either dependent or independent of small,
monomeric G protein Ras.
o RAS dependent signaling is facilitated by members of mitogen-activated protein kinase (MAPK)
cascade and RAS independent signaling is facilitated by other types of kinases e.g., PI3K.
o Both signaling pathway trigger phosphorylation of target protein in the nucleus, plasma
membrane, and cytoplasm leading to alterations in the rate of gene transcription and protein
activity.
o Signaling via RTKs is terminated via multiple mechanisms –
 Degradation of the signaling molecules by extracellular proteases
 Endocytosis of receptors the followed by lysosomal degradation
 Accelerated RAS inactivation
 Dephosphorylation of target proteins by phosphoprotein phosphatases
Mechanism of signaling molecules via tyrosine

kinase associated receptors
 Growth hormone, prolactin, erythropoietin, cytokines and antigen-specific receptors on T and B
lymphocytes mediate their signaling via tyrosine kinase associated receptors.
 These receptors contain an extracellular ligand binding domain but lack a catalytic domain. Thus,
they depend on non-receptor tyrosine kinase (soluble TK or cytosolic TK) such as Src and Janus
family for their signaling mechanism.
 For many tyrosine kinase associated receptors, ligand binding causes assembly of two or more
different trans-membrane receptor. Hormone receptor interaction promotes binding and activation
of cytoplasmic tyrosine kinase e.g., Janus kinases (JAKs). Theses kinases phosphorylate itself and
intracellular domain of the receptor at tyrosine residues. JAKs provide a direct link between
receptor activation and gene transcription.
 Downstream targets of JAKs are transcription factors called signal transducers and activators of
transcription (STATs). JAKs mediated phosphorylation leads to dimerization of STATs. Dimerized
STATs then translocate to the nucleus where they bind specific DNA sequence in the regulatory
region of target genes and thus directly modulate gene transcription.
 JAKs also phosphorylate other cytoplasmic proteins through binding to SH 2 domains. This may
result in activation of PI3K, MAPK cascade, or PLC isoforms and activation of PKC. Thus there is a
potential of cross talk when different hormones activate these various signal transduction pathways.
 Biomedical importance
o Mutation in protein tyrosine kinase gene can cause defective antigen dependent T cell activation
resulting in severe combined immunodeficiency (SCID).
Mechanism of signaling molecules via receptor

serine/threonine kinases
 Transforming growth factor β (TGF β) regulate
cell proliferation and differentiation, extracellular
matrix production, tissue repair immune
regulation and cell death via receptor
serine/threonine kinase.
 Hormone binding causes dimerization and
activation of kinase domain. The activated
receptor directly binds and phosphorylates gene
regulatory proteins (Smads) and regulates
transcription of specific target genes.
Mechanism of signaling molecules via receptor tyrosine phosphatase

 Receptor tyrosine phosphatase removes the phosphate group from phosphotyrosine residues on
particular types of proteins. These phosphatases are found in both soluble and membrane bound
forms and are related to an activator protein 1 (AP 1) which regulates transcription.
Signaling mechanisms by special senses
Signaling mechanism in vision
Rod cells and cone cells on the retina are responsible for vision. These cells contain photoreceptors
called rhodopsin. Rhodopsin is a seven pass trans-membrane protein that functions as a GPCR. Each
rhodopsin molecule contains a covalently attached non-protein prosthetic group 11-cis retinal.
Rhodopsin constitutes the photo-transduction apparatus in the outer segment of the rod cells. The
plasma membrane surrounding the outer segment contains cGMP gated ion channels. cGMP bound
to these channels keep them open in the dark.
Upon exposure to photons, chromophore 11-cis retinal isomerizes to all-trans retinal and this alters
the shape of retinal forcing a conformational change in conjugated protein opsin.
Photoactivated rhodopsin molecule then switches G protein transducin (Gt) into active GTP bound
form. Light activated transducin then stimulates cGMP phosphodiesterase. Thus, cGMP level in the
cytosol decreases, resulting in the closure of cGMP gated cation channels and hyperpolarization of
the membrane. In this way, a light signal is converted into an electrical signal through
hyperpolarization of rod cell plasma membrane.
Rapid restoration to dark state in the rod cells is achieved by several negative feedback loops –
o Receptor desensitization (through phosphorylation on cytosolic tail of rhodopsin and binding of
inhibitory protein, arrestin to phosphorylated receptor)
o Formation of cGMP (by the action of guanylyl cyclase that is activated upon decreased
intracellular Ca2+ level due to closure of cation channels)
o Since 11-cis retinal requires as a component of rhodopsin, vitamin A deficiency leads to night
blindness as a consequence of slow regeneration of rhodopsin required for visual signaling.
Signaling mechanism in olfaction

The receptors in olfactory sensory neurons are coupled to heterotrimeric G protein (Golf).
When the odorant binds to the receptor, Golf is activated by exchanging GDP for GTP. Golf then
stimulates adenylyl cyclase, resulting in increased cAMP concentration. This leads to opening of
cAMP gated cation channels allowing an influx of Na+ and Ca2+ which depolarizes the olfactory
receptor neuron and initiates a nerve impulse.
Signaling mechanism by tastants
 The receptors in gustatory sensory neurons are coupled to heterotrimeric G protein, gustducin.
Binding of tastants to the receptor activates gustducin which then stimulates cAMP production by
adenylyl cyclase.
Mechanism of growth factor signaling

 Growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and
nerve growth factor (NGF) mediate their signaling via receptor tyrosine kinase.
 General steps involved in growth factor signaling pathway –
o Binding of growth factor to extracellular domain of specific RTK
o Receptor dimerization and activation of cytosolic tyrosine kinase activity of RTK upon GF binding
o Auto-phosphorylation or cross-phosphorylation on specific tyrosine residues in cytosolic domain
of dimerized receptor
o Recognition and binding of specific phosphotyrosine residues by proteins having SH2 domain
o Activation of downstream signaling pathway (Ras dependent or Ras independent)
o Termination of signaling via RTKs
 RAS dependent signaling cascade

o Grb2 binds to phosphor-tyrosine residues of RTK through SH2
domain. This is followed by binding of Sos to Grb2 and then to
Ras, causing GDP release and GTP binding to Ras.
o Activated Ras activates MAPK cascade (Raf-1  MEK  ERK).
o ERK moves into the nucleus and phosphorylates nuclear
transcription factors such as Jun, Fos, Elk1, activating them.
o Activated nuclear transcription factors stimulate the
transcription and translation of a set of genes needed for cell
division such as E2F, cyclins, Cdks.
 RAS independent signaling cascade
o Enzymes having SH2 domain can bind to phosphotyrosine
residues of RTKs and mediate downstream signaling pathways
e.g., PI3K, PLC γ.
o PI3K is allosterically activated by binding to the auto-
phosphorylated growth factor receptor or sometimes by Ras-
GTP. PI3K phosphorylates inositol lipids in the plasma
membrane in position 3 e.g., PIP2 to PIP3. PIP3 recruits and
activates protein kinase B or Akt by phosphorylating at serine
and threonine residues. Activated PKB phosphorylates a large
number of target proteins in cytoplasm and nucleus.
o PLC γ binds phosphor-tyrosine residues on growth factor
receptors via SH2 domain and becomes activated by tyrosine
phosphorylation. Activated PLC γ initiates signaling cascade
through phosphoinositol derivatives (DAG, IP3). Through this
pathway, growth factor signaling via RTKs can increase cytosolic
Ca2+ levels and activate PKC.
 Termination of GF signaling via RTKs
o Degradation of signaling molecules by extracellular proteases
o Receptor internalization followed by lysosomal degradation
o Accelerated RAS inactivation
o Dephosphorylation target proteins by phosphoprotein
phosphatases
Signal termination mechanisms

Some signals, such as those that modify
the metabolic responses of cells or that
transmit neural impulses need to turn off
rapidly when the hormone is no longer
being produced. Other signals, such as
those that stimulate proliferation, turn off
more slowly. In contrast, signals that
regulate differentiation may persist
throughout the lifetime. Many chronic
diseases are caused by failure to terminate
a response at the appropriate time.
Within each pathway of signal
transduction, the signal may be turned off
at specific steps.
Signal transduction pathways can be terminated by various means –
o Negative feedback control on secretion of signaling molecules
o Degradation and clearance of signaling molecules e.g., uptake and degradation of insulin by
liver, termination of acetylcholine signal by acetylcholine esterase
o Down-regulation of receptors (desensitization, internalization and degradation)
o Termination of intracellular signal mediators action at specific steps
 Inactivation of G protein through hydrolysis of GTP in Gα to GDP by intrinsic GTPase activity
which is accelerated via interaction with GAPs (GTPase activating proteins)
 Degradation of second messengers e.g., degradation of cAMP,

cGMP by specific phosphodiesterases (PDEs)
 Lowering cytosolic Ca2+ level by sequestration of Ca2+ into ER and
chelation with calsequestrin
 Degradation of intracellular signal mediating proteins e.g., proteins
kinases, DNA binding proteins
 Reversing the action of protein kinases through dephosphorylation
of target protein by the action of protein phosphatases.
Biomedical importance of signal transduction

1. Growth factor signaling pathway abnormalities and cancer development
o Abnormalities in growth factors, their receptors and signaling pathways play major roles in
cancer development. Cancer is strongly associated with defect in signal transduction proteins.
o The products of proto-oncogenes may
be one of the proteins involved in
signal transduction of growth factors.
Activation of proto-oncogenes to
oncogenes by gene amplification,
point mutation, promoter insertion,
enhancer insertion or chromosomal
translocation may lead to cancer.
Products of oncogenes may lead to
abnormal signal transduction pathway
leading to uncontrolled cell growth
and proliferation.
o Examples of products of oncogenes
 Sis oncogene is growth factor (PDGF-β chain).
 Erb-2 oncogene is growth factor receptor (EGF receptor).
 Ras oncogene is G-protein (Ras).
 Raf oncogene is serine/threonine kinase (kinase in MAPK cascade).
 Abl and src oncogenes are cytosolic tyrosine kinases.
 Myc, fos, jun oncogenes are transcription factors.
2. Signaling pathway defect and diseases
o Defect in insulin signaling pathway is associated with insulin resistance and type 2 diabetes
mellitus.
o Vitamin A deficiency leads to night blindness as a consequence of slow regeneration of

rhodopsin (visual receptors) since 11-cis retinal is required as a prosthetic group of rhodopsin.
o Mutated V2 receptor in renal tubular cells in CD can lead to nephrogenic diabetes insipidus.
3. Protein kinase inhibitors are used as anticancer agents.
o Mutated or overexpressed receptor tyrosine kinases and cytosolic TK are frequently observed in
tumors. For instance, the epidermal-growth-factor receptor (EGFR) is overexpressed in some
human epithelial cancers, including breast, ovarian, and colorectal cancer. Thus, monoclonal
antibodies that target EGFR can be used as a therapeutic approach in treating these cancers.
o Cetuximab (Erbitux) has effectively targeted the EGFR in colorectal cancers. Cetuximab inhibits
the EGFR by competing with EGF for the binding site on the receptor.
o Trastuzumab (Herceptin) inhibits another EGFR family member, Her2 that is overexpressed in
approximately 30% of breast cancers. So, it is used in treatment of breast cancer.
o Bcr-Abl kinase inhibitor (Gleevec) is used in treatment of chronic myeloid leukemia.
4. Receptor blockers or agonists are used in treatment of various diseases.
o Beta adrenergic receptor agonists are used in asthma.
o Beta blockers and angiotensin receptor blockers (losartan) are used for treatment of
hypertension.
o H1 receptor blockers (anti-histamines) are used for treatment of allergic reactions, motion
sickness e.g., cetirizine, loratidine, meclizine.
Hormones of hypothalamo-pituitary-thyroidal axis
Thyrotropin-releasing hormone (TRH)

 It is a modified tripeptide synthesized by hypothalamus and transported to anterior pituitary by
portal vessels.
 TRH binds to GPCR on pituitary thyrotroph cell membrane which stimulates Gq protein resulting in
activation of phospholipase C. Resulting increase in intracellular IP3 stimulates the release of
calcium from ER leading to secretion of TSH.
 More chronic actions of TRH include stimulation of TSH biosynthesis and TSH glycosylation.
Thyroid-stimulating hormone (TSH)

 TSH or thyrotropin is a small glycoprotein synthesized by pituitary thyrotrophs. It consists of two
non-covalently linked subunits; α chain which is identical to other glycoprotein hormones (FSH, LH
and β hCG) and β chain which confers the specificity of TSH.
 TSH binds to GPCR on follicular cells in thyroid gland. Binding of TSH to GPCR stimulates Gs protein
which couples to activation of adenylyl cyclase leading to increased cAMP concentration. cAMP
activates PKA which phosphorylates and regulate the activity of target proteins involved in thyroid
hormone synthesis and secretion e.g., thyroidal iodide pump, thyroperoxidase, microvilli formation
on the apical membrane, thyroglobulin synthesis. PKA mediated phosphorylation also leads to
dimerization and activation of CREB which then binds to CRE in the regulatory region of target gene
resulting in increased thyroglobulin gene transcription.
 TSH influences virtually every aspect of thyroid hormone synthesis and secretion. TSH also stimulates
growth of thyroid gland.
Thyroid hormone
Chemistry
 Two iodo-amino acid hormones called 3, 5, 3’ – triiodo-thyronine (T3) and 3, 5, 3’, 5’ – tetraiodo-
thyronine (T4) or thyroxine. T4 is more abundant than T3 which is the biologically active form.
Biosynthesis of thyroid hormones

 Thyroid hormone synthesis requires trace element iodine. Thyroid hormone synthesis involves
thyroglobulin and iodide metabolism.
 Steps in thyroid hormone biosynthesis are –
1. Concentration of iodide (I⁻)
2. Oxidation of iodide
3. Iodination of tyrosyl residues
4. Coupling of iodotyrosyls
5. Secretion
1. Concentration of iodide
 In the thyroid follicular cells,
I⁻ is concentrated against a
strong electrochemical
gradient via thyroidal iodide
pump. This process is energy
dependent and linked to
Na+/K ATPase pump activity.
The activity of the pump is
controlled by TSH.
 Inhibitors of thyroidal iodide
pump
 Perchlorate (ClO4⁻), perrhenate (ReO4⁻) and pertechnetate (TcO4⁻) which compete with I⁻ for
its carrier and are concentrated by the thyroid.
 Thiocyanate (SCN⁻) which is a competitive inhibitor of iodide transport and is not

concentrated by the thyroid.
2. Oxidation of iodide
 Thyroid gland is the only tissue that can oxidize iodide to a higher valence state.
 This step is catalyzed by heme-containing thyroperoxidase enzyme and uses H2O2 as a substrate.
It occurs at the luminal (apical) surface of the follicular cells.
 Iodide oxidation is inhibited by thiourea drugs.
3. Iodination of tyrosyl residues
 Oxidized iodide reacts with tyrosyl residues in thyroglobulin on luminal side. The 3 position of
aromatic ring in tyrosine is iodinated first and then 5 position to form monoiodotyrosine (MIT)
and diiodotyrosine (DIT) respectively. This reaction is called organification and involves
thyroperoxidase enzyme.
 Thyroglobulin is synthesized in the basal portion of follicular cell which then moves to the lumen
across the luminal membrane and it is stored in the extracellular colloidal space. It is the
precursor of T4 and T3. It is a glycosylated protein and contains 115 tyrosine residues.
4. Coupling of iodotyrosyls
 Coupling of 2 DIT molecules to form T4 or of MIT and DIT to form T3 occur within the
thyroglobulin molecule.
 This is an oxidative process and assumed to be catalyzed by thyroperoxidase enzyme.
 Coupling is inhibited by the thiourea drugs.
 About 70% of iodide in thyroglobulin exists in the inactive precursors, MIT and DIT, while 30%
exists in T4 and T3 forms.
5. Secretion
 Thyroglobulin is a storage form of T3 and T4 in the colloid. Several weeks supply of hormones
exists in the colloidal spaces of thyroid gland.
 Upon stimulation by TSH and increased cAMP concentration leads to marked increased in
microvilli on the apical membrane. This microtubule–dependent process entraps thyroglobulin
and subsequently pinocytoses into the follicular cell.
 Endocytosed thyroglobulin is then hydrolyzed by lysosomal enzymes into amino acids, MIT, DIT,
T3 and T4. T3 and T4 (80 – 90%) are discharged from the basal portion of the cell into the blood
stream. About 50 μg of thyroid hormone is secreted each day.
Transport of thyroid hormones

T3 and T4 circulate in bound form. Transport of thyroid hormones in the blood is by binding with
thyroxine binding globulin (TBG) and thyroxine binding prealbumin (TBPA).
TBG binds T3 and T4 100 times more than TBPA. T3, T4 binding capacity of TBG is 20μg/dL under
normal circumstances.
Metabolism
About 80 to 95 % of thyroid hormone
secreted from the thyroid gland is T4.
T4 is converted into T3 or reverse T3
(rT3) by deiodination in target
tissues. Deiodinase type 1 and 2
catalyze 5’ deiodination and type 3
catalyze 5 deiodination leading to formation of more active T3 and inactive rT3 respectively.
Remaining T4 (prohormone) is conjugated with sulfate or glucuronic acid and deactivated by
deamination or decarboxylation.
Biochemical action of thyroid hormone

 T3 and T4 are transported bound to
TBG or TBPA in the circulation. At the
target cells, free hormone transverse
the cell membrane and bind to
specific nuclear receptor.
 Thyroid hormone acts by binding to
a specific nuclear DNA-bound thyroid
hormone receptor (TR), usually as a
heterodimer with retinoid X receptor
(RXR) at specific sequences called
thyroid hormone response elements
(TREs) in target genes to regulate
the rate of synthesis of specific
mRNA. This results in a change in the
amount and activity of the protein
which in turn alters the rate of a
metabolic process.
 Cellular changes which result from regulation of gene expression by thyroid hormone include
increase ATP utilization by increasing sodium-potassium (Na+/K+)-dependent ATPase activity, and
increase mitochondrial oxidative metabolism by direct up-regulation of key mitochondrial
biogenesis factors, increased rate of synthesis of many structural proteins and enzymes.
 All these responses attribute to the effects of thyroid hormones on regulation of metabolism,
growth and development.
Hormones from adrenal medulla

Adrenal medulla is part of the sympathetic nervous system since pre-ganglionic fibers from the
splanchnic nerve terminates in the adrenal medulla where they innervate chromaffin cells that
produce catecholamines (dopamine, norepinephrine, epinephrine). These hormones are required
for adaptation to acute and chronic stress.
Biosynthesis of catecholamines
Catecholamines are synthesized in the chromaffin cells of the adrenal
medulla. Biosynthesis occurs in four sequential steps.
1. Ring hydroxylation
 Tyrosine is the immediate precursor of catecholamines.
 L-tyrosine is converted into L-dopa (dihydroxyl phenylalanine) by
tyrosine hydroxylase, rate-limiting enzyme containing
tetrahydropteridine as a cofactor.
 Tyrosine hydroxylase is competitively inhibited by a series of tyrosine
derivatives such as α methyl tyrosine which is used to treat
pheochromocytoma.
2. Decarboxylation
 L-dopa is converted into dopamine (3, 4 dihydroxyl phenyl
ethylamine) by the action of dopa decarboxylase which requires
pyridoxal phosphate as coenzyme.
 Compounds resemble L-dopa such as methyldopa competitively
inhibits dopa decarboxylase and can be used as anti-hypertensives.
3. Side chain hydroxylation
 Dopamine is converted into norepinephrine by the action of
dopamine β hydroxylase. Dopamine β hydroxylase is a mixed
function oxidase and requires ascorbate as an electron donor,
copper at catalytic site, and fumarate as a modulator.
4. N-methylation
 N-methylation of norepinephrine to form epinephrine is catalyzed by phenylethanolamine N-
methyl transferase (PNMT).
 PNMT synthesis is induced by glucocorticoids hormones.
Storage and release of catecholamines

Catecholamines enter the chromaffin granules via an ATP dependent transport mechanism and they
are stored in the granules bound with ATP in 4:1 (hormone: ATP) ratio.
Neural stimulation of the adrenal medulla results in exocytotic release of catecholamines. This
process is calcium dependent. Cholinergic and β adrenergic agents stimulate catecholamines
secretion whereas α adrenergic agent inhibits the secretion.
Metabolism of catecholamines
The most prominent removal mechanism for released catecholamines (almost 90% of catecholamines
released at sympathetic synapses) is reuptake into the presynaptic nerve endings. Cocaine, tricyclic
antidepressants and ouabain inhibit this uptake. They can also be removed by extra-neuronal uptake.
Metabolism of catecholamines occurs through two enzyme pathways (COMT & MAO) followed by
conjugation with sulfate and renal excretion.
Catechol-O-methyltransferase (COMT) is found primarily in extra-neuronal tissue (especially in kidney
and liver) and converts epinephrine to metanephrine and norepinephrine to normetanephrine by
methylation. S-adenosylmethionine is used as the methyl donor, and Ca2+ is required.
Monoamine oxidase (MAO) is present in both neural and non-neural tissue and oxidizes
metanephrine and normetanephrine to vanillylmandelic acid (VMA) by oxidative deamination.
Monoamine oxidase inhibitors (MAOIs) increase catecholamines concentration by inhibiting
breakdown of catecholamines and are used to treat depressive disorders.
Mechanism of action of catecholamines

There are 2 major classes of receptors – α adrenergic and β adrenergic; each consists of 2 subclasses
i.e., α1, α2, β1, β2. Epinephrine at physiological concentration primarily binds to both α and β
adrenergic receptors. Norepinephrine binds primarily to α receptors.
Adrenergic receptors belong to the family of GPCR. Upon binding with hormones, β adrenergic
receptors mediate signaling via Gs to activate adenylyl cyclase whereas α2 adrenergic receptors
mediate signaling via Gi, decreasing cAMP concentration. Hormone binding to α1 adrenergic
receptors activates Gq which in turn stimulates PLC β and mediate signaling via phosphoinositol
derivatives (IP3, DAG) and elevated cytosolic Ca2+ concentration.
Catecholamine cannot cross the blood brain barrier; hence they must be synthesized locally in the
brain. In Parkinson disease, there is a localized deficiency of dopamine synthesis and it can be treated
by L-dopa.
In adrenal medulla tumor (pheochromocytoma), there is abnormal excessive production of
catecholamines.
Steroid hormones
Steroid hormones are produced in specific cells of adrenal cortex, testes, ovaries and placenta
(steroidogenic tissues). All mammalian steroid hormones are formed from cholesterol via
pregnenolone through a series of reactions that occur in either the mitochondria or endoplasmic
reticulum of the producing cell.
Biologically active steroids particularly androgens and estrogens are formed also in non-endocrine
tissues such as skin, liver, brain, mammary gland and adipose tissues.
Hormones of hypothalamo-pituitary-adrenal axis
Corticotrophin releasing hormone (CRH)

CRH is a 41-amino acids peptide synthesized and secreted by paraventricular nuclei of hypothalamus.
CRH acts via GPCR on pituitary corticotroph cells via cAMP second messenger system to stimulate
both synthesis and secretion of ACTH.
Adreno-corticotrophic hormone (ACTH)

ACTH is synthesized as 241-amino acids precursor molecule called pro-opiomelanocortin (POMC).
POMC is cleaved at multiple sites to release several hormonally active peptides including endorphins,
MSH and ACTH.
ACTH is a 39-amino acids

peptide with the biological
activity residing in N terminal
24 residues. ACTH is secreted in
stress related bursts
superimposed on a marked
diurnal rhythm, with a rapid
surge in production from
around 3 am and a peak plasma
concentration at about 5 am.
ACTH stimulates synthesis and
release of glucocorticoid
hormones by interacting with
GPCR on adrenal cortex which
stimulates cAMP production.
Acute increase in the adrenal cortisol synthesis
is principally mediated by stimulating the
activity of cholesterol esterase. Long term
effects of ACTH include induction of
transcription of genes that encode
steroidogenic enzymes (cytochrome P450 SCC
enzyme, StAR protein and hydroxylases).
Adreno-cortical hormones
These are grouped into 3 classes of hormones
o Glucocorticoids (C21) – cortisol
o Mineralocorticoids (C21) – aldosterone
o Androgens (C19) –
dehydroepiandrosterone (DHEA),
androstenedione
Adrenal steroidogenesis
Steroid hormones are synthesized from
cholesterol. Much of cholesterol in the
adrenals is esterified and stored in cytoplasmic
lipid droplets. Upon stimulation of adrenal by
ACTH, stored cholesterol esters are hydrolyzed by an esterase.

Free cholesterol formed is transported into mitochondria by ACTH dependent transport protein
called steroidogenic acute regulatory protein (StAR). It is the rate limiting step in adrenal
steroidogenesis.
Cytochrome P450 side chain cleavage enzyme (P450 scc) converts cholesterol (C27) to
pregnenolone, C21 steroid. Pregnenolone is then utilized for synthesis of different steroid hormones.
Adrenal cortex has three distinct layers or zones. The sub-capsular area called zona glomerulosa is
associated with production of mineralocorticoids. Next are the zona fasciculata and zona reticularis
which are associated with production of glucocorticoids and androgen precursors.
Biosynthesis of mineralocorticoids
Mineralocorticoids are synthesized in the zona glomerulosa since 18-hydroxylase and 18-
hydroxysteroid dehydrogenases, which are required for aldosterone synthesis, are found only in the
zona glomerulosa cells.
Pregnenolone is converted to progesterone by the action of microsomal 3β hydroxysteroid
dehydrogenase and Δ5, 4 isomerase.
Progesterone then undergoes three sequential hydroxylations at carbon 21, 11 and 18 positions.
Synthesis and secretion of aldosterone is stimulated through RAA system.
Angiotensin II act on the zona glomerulosa to stimulate the synthesis and secretion of aldosterone in
which signaling effect is mediated by GPCR (via Gq) which utilizes phosphoinositol derivatives,
cytosolic calcium ions as intracellular signal mediators. The effect of AT II on aldosterone secretion
involves an activation of 18-hydroxylase and 18-hydroxydehydrogenase (aldosterone synthase).
Biosynthesis of glucocorticoids
Cortisol synthesis requires three hydroxylases that act sequentially on carbon 17, 21 and 11 positions.
The first two hydroxylases (17 hydroxylase and 21 hydroxylase) are smooth endoplasmic reticulum
enzyme and 11β hydroxylase is a mitochondrial enzyme.
The most potent glucocorticoid hormone in human is cortisol. It shows pronounced diurnal rhythm;
10 times higher at 08:00 h than 12:00 h (midnight).
Biosynthesis of androgens
The major androgen or androgen precursor produced by the adrenal cortex is
dehydroepiandrosterone (DHEA).
Most 17-hydroxypregnenolone follows the glucocorticoid pathway, but a small fraction undergoes
conversion to DHEA by removal of the two-carbon side chain through the action of 17,20-lyase.
Adrenal androgen production increases markedly if glucocorticoid biosynthesis is impeded by the
lack of one of the hydroxylases (adrenogenital syndrome).
Weak androgen DHEA is converted into the more potent androstenedione by the actions of 3β-
OHSD and Δ5,4 –isomerase.
Reduction of androstenedione at the C 17 position results in the formation of testosterone, the
most potent adrenal androgen, thus small amounts of testosterone are produced in the adrenal by
this mechanism.
Biomedical importance of adrenocortical hormones

Glucocorticoids (cortisol) are essential component in
adaptation to severe stress. It also promotes
gluconeogenesis. Glucocorticoid analogs can be used
therapeutically as potent anti-inflammatory agents
(inhibition of NF-κB, phospholipase A2).
Mineralocorticoids (aldosterone) regulate normal Na+ and K+ balance (promote renal Na+
reabsorption and K+ excretion).
Adrenal androgens (DHEA and androstenedione) are converted into more potent androgens in
extra-adrenal tissues. Estrogens are not made in the normal adrenal but adrenal androgens are
important precursors of estrogens in post-menopausal women.
Adrenocortical hormone disorders
o Addison’s disease - primary adrenocortical failure due to autoimmune disease or infection e.g., TB
o Cushing’s syndrome – glucocorticoid excess due to exogenous cause or disorders of
hypothalamus or pituitary (Cushing’s disease) or adrenal gland
o Conn’s syndrome – aldosterone excess
o Adrenogenital syndrome – increased adrenal androgen production due to blockage of
glucocorticoids biosynthesis by lack of one of hydroxylases e.g., 21 hydroxylase (hirsutism,
muscularization and menstrual irregularity in females)
Signaling mechanism in anti-inflammatory action of glucocorticoids

Anti-inflammatory actions of
glucocorticoids are explained in
part by the inhibition of NF-κB and
its consequent actions.
The transcription factor NF-κB
binds to a number of gene
regulatory sequences and activates
transcription of genes particularly
involved in the inflammatory
response. NF-κB is normally sequestered in the cytoplasm in a transcriptionally inactive form by

binding with IκB (inhibitor of NF-κB) family of proteins. Extracellular stimuli such as pro-
inflammatory cytokines, ROS and mitogens lead to activation of IKK (IκB kinase) complex which
phosphorylates IκB and targets for polyubiquitylation and subsequent degradation by proteasome.
Following release of IκB and its degradation, free NF-κB translocates to nucleus and activates
transcription of genes involved in inflammation.
Cortisol binds to the cytoplasmic glucocorticoid receptor (GR). Conformational changes in the
receptor-ligand complex result in dissociation from heat shock proteins and migration to the nucleus.
Cortisol-GR complex binds to specific DNA sequence—glucocorticoid response elements—in
association with the activator protein 1 (AP1). Glucocorticoids mediate their anti-inflammatory effects
through several mechanisms:
1. Induction of inhibitory protein IκB which binds and inactivates NF-κB
2. Direct binding of GR-cortisol complex to the p65 subunit of NF-κB thereby preventing its
activation
3. Competitive binding of GR to the limited
available coactivators (CREB, steroid receptor
coactivator-1) required for NF-κB action
Biosynthesis of gonadal hormones

Gonads are bifunctional organs that produce germ
cells and sex hormones. Ovaries produce ova and
estrogens and progesterone. Testes produce
spermatozoa and testosterone.
Immediate precursor of gonadal steroids is
cholesterol. The conversion of cholesterol to
pregnenolone is identical in adrenal, ovary and testis
but the reactions are promoted by LH in gonads
unlike ACTH in adrenals.
Biosynthesis of testicular hormones (testosterone)

Testicular androgens are synthesized in the Leydig
cells of interstitial tissues. Immediate precursor of
gonadal steroids is cholesterol.
Conversion of pregnenolone to testosterone occurs
through two pathways which are called Δ5 or DHEA
Reactions sequence of pregnenolone in Δ5
pathway (mostly used in human testes) and Δ4 or DHEA pathway
progesterone pathway. These pathways requires 5  17α hydroxylase  C17, 20 lyase (DHEA) 
17β hydroxy-steroid dehydrogenase (Δ5
enzymes activities contained in three proteins –
androstenediol)  3β hydroxysteroid
o 3β hydroxysteroid dehydrogenase (3β OHSD) 5,4
dehydrogenase & Δ isomerase (testosterone)
o Δ5,4 isomerase Reactions sequence of pregnenolone in Δ4
o 17α hydroxylase progesterone pathway
o C 17, 20 lyase  3β hydroxysteroid dehydrogenase & Δ5,4

isomerase (progesterone)  17α hydroxylase
o 17β hydroxysteroid dehydrogenase (17β OHSD)
 C17, 20 lyase (androstenedione)  17β
Transport and metabolism of testosterone hydroxy-steroid dehydrogenase (testosterone)
Over 97% of circulating testosterone is bound to albumin and sex hormone binding globulin (SHBG).
It is metabolized to potent metabolite dihydrotestosterone (DHT) via 5α reductase in target tissues
and to inactive metabolite 17 ketosteroids in many tissues including liver.
Biosynthesis of ovarian hormones

Estrogens are a family of
hormones synthesized in a
variety of tissues.
The general pathway and
subcellular localization of
enzymes involved in the early
steps of estradiol synthesis is
the same as those involved in
androgen biosynthesis.
Estrogens (C18) are formed
by the aromatization of
androgens by 3
hydroxylation steps, each of
which requires O2 and NADPH
and catalyzed by the aromatase enzyme complex (P450 mixed function oxidase).
17β estradiol (E2) is formed from aromatization of testosterone and it is the primary estrogens of
ovarian origin.
Estrone (E1) is formed form aromatization of androstenedione (androgen precursor from adrenal
cortex) and it can be synthesized in numerous tissues such as adipose tissues, liver, skin. This
pathway is the major source of estrogens in post-menopausal women.
Estriol (E3) is produced from placenta during pregnancy. It is derived from estrone by 16α
hydroxylase action.
In male, 80% of E2 is formed by peripheral aromatization of testosterone. In female, adrenal
androgens are important substrates because as much as 50% of E2 produced during pregnancy comes
from aromatization of androgens.
Aromatase activity is present in adipose tissues, liver, skin and other tissues. Increase in activity of
aromatase leads to estrogenization in cirrhosis of liver, hyperthyroidism, aging and obesity.
Aromatase inhibitors can be used as therapeutic agents in breast cancer and possibly in other
female reproductive tract malignancies.
Hormones from endocrine pancreas
Insulin
 Insulin was the first protein
o Proved to have the hormonal action
o That have been crystalized
o That have been sequenced
o Synthesized by chemical technique
o Shown to be synthesized as a large precursor molecule
o Prepared by recombinant technology.
 Biochemistry and structure
o Insulin is heterodimeric polypeptide composed of 2 chains; A (21 amino acids) and B (30 amino
acids). Two chains are linked by 2 inter-chain disulfide bridges that connect A7 to B7 and A20 to
B19. One intra-chain disulfide bridge connects residues 6 and 11 of A chain.
o Insulin and proinsulin combines with zinc to form hexamer.
Biosynthesis of insulin
 Insulin gene is located on the short arm of chromosome 11 in humans. It has 2 introns and 3 axons.
Insulin is synthesized as a preprohormone (signal peptide, chain B, chain C chain A arranged from N
to C terminal) in polyribosomes on RER in β cells of pancreas.
 Signal peptide or leader

sequence (hydrophobic 23
amino acids) in preproinsulin
directs the molecule into the
cisternae of RER where it is
removed. Disulfide bridges
formation occurs in ER
lumen and formed proinsulin
(B chain, C peptide and A
chain from N to C terminal)
is then transported to the
Golgi apparatus where it is
cleaved into equimolar amount of mature insulin and C peptide.
 Insulin and C peptide are packed in secretory granules and secreted by exocytosis in fed state when
blood glucose level is high.
Mechanism of insulin secretion

 Increased blood glucose level after meal stimulates insulin secretion from β cells of pancreas.
 The sequence of events of insulin secretion coupled to glucose entry into β-cells consists of
o Glycolysis, oxidative phosphorylation and ATP production
o Inhibition of the ATP-sensitive K+ channel
o Membrane depolarization
o Opening of voltage gated Ca2+ channels and Ca2+ influx into β cells
o Fusion of secretory granules with cell membrane and insulin release via exocytosis mediated
by elevated cytosolic Ca2+ level
 Neurotransmitter acetylcholine stimulates and norepinephrine inhibits insulin secretion.
 Glucagon-like peptide-1 (GLP-1, incretin) promotes insulin release via Gs and 2nd messenger cAMP.
 Sulfonamides has direct effects on sulfonylurea receptors (SURs) promoting insulin release.
Regulation of insulin secretion

 Insulin secretion is increased by –
o Rise in blood glucose level after ingestion of glucose or carbohydrate rich meal
o Rise in amino acid level after ingestion of proteins
o Gastrointestinal hormones like secretin released upon ingestion of food
o Rise in blood glucose level by glucagon
 Insulin secretion is inhibited by scarcity of dietary fuel and trauma (release of epinephrine).
Transport and metabolism of insulin

 Insulin has no carrier protein.
 The metabolism occurs in liver (50%), kidney and placenta.
 It involves 2 enzyme systems – insulin specific protease and glutathione insulin transhydrogenase
(reduces disulfide bonds).
Metabolic effects of insulin

 Effect on carbohydrate metabolism
o It depends on the tissue
 Inhibition of gluconeogenesis, glycogen breakdown and increase glycogen synthesis in liver
 Increased glucose uptake (increased GLUT-4 expression) and glycogen synthesis in muscles
 Increased glucose uptake and utilization in adipose tissues
 Effect on lipid metabolism

o Increased glucose entry and provision of raw materials for fatty acid synthesis by oxidation of
glucose in adipose tissues
o Increased esterification of fatty acids and storage as triacylglycerol
o Inhibition of lipolysis by inactivating hormone sensitive lipase
 Effect on protein metabolism
o Stimulation of entry of amino acids into cells and protein synthesis in most cells
Mechanism of action of insulin

 Insulin binds to specific
glycoprotein receptor on the
surface of the target cell.
Insulin receptor is a
dimerized receptor in which
two heterodimers (α and β) in
the configuration of α2β2 are
linked by disulfide bonds. α
subunit is entirely
extracellular and binds
insulin. β subunit is a trans-
membrane protein which
functions as a signal transducer. The cytoplasmic portion of β subunits has tyrosine kinase activity
and auto-phosphorylation sites.
 Binding of insulin to α subunit

activates cytoplasmic tyrosine
kinase domain leading to
auto-phosphorylation of
tyrosine residues on cytosolic
domain of receptors. The
phosphor-tyrosine residues
on receptors are recognized
and bound by a docking
protein called the insulin
receptor substrate 1 (IRS – 1).
 IRS-1 is then phosphorylated on its tyrosine residues by the insulin receptor. Phospho-tyrosine
residues on IRS-1 is recognized and bound by proteins having SH2 domain such as adaptor or docking
proteins, kinases, phosphatases that are involved in mediating different effects of insulin.
 RAS dependent insulin signaling
o Phosphorylated IRS-1 is recognized and bound by the adaptor protein GRB-2, initiating the
activation of RAS and the MAPK cascade. This results in the phosphorylation of nuclear
transcription factors that alters the transcription of specific genes involved in cellular
differentiation and growth, diverse metabolic processes.
 RAS independent insulin signaling
o Phosphorylated IRS-1 recruits phosphoinositide 3-kinase (PI3K), an enzyme that phosphorylates
phosphoinositides (PIP2) to generate phosphatidylinositol3,4,5-triphosphate (PIP3).
o Membrane-bound PIP3 acts as a second messenger that recruits and activates protein kinase B
(PKB) via phosphorylation. Active PKB (also known as Akt) phosphorylates at specific
serine/threonine residues of target proteins thereby altering the activity of many intracellular
proteins, which have stimulatory effects on glucose uptake and storage, e.g., translocation of
GLUT-4 from cytoplasm to the plasma membrane of muscle and adipose cells, stimulation of
glycogen synthesis by phosphorylation and inactivation of glycogen synthase kinase-3 (GSK-3).
 Termination of insulin signaling occurs by receptor internalization and dephosphorylation of receptor
by protein tyrosine phosphatases.
Glucagon
 A small peptide containing 29 amino acids, synthesized as prohormone in α cells of pancreas
 Glucagon acts on GPCR family and mediates its signaling via 2nd messenger cAMP.
Metabolic effects of glucagon

 Effect on carbohydrate metabolism
o Stimulation of liver glycogen breakdown and
gluconeogenesis leading to increased hepatic
glucose output
o Rise in blood glucose level
 Effect on lipid metabolism
o Activation of lipolysis (TAG breakdown) in
adipose tissues releasing FFA into the blood
stream
o Uptake and oxidation of FFA in liver releasing acetyl CoA which is used in ketone bodies synthesis
 Effects on protein metabolism
o Increased uptake of amino acids thereby increasing availability of carbon skeletons for
gluconeogenesis in liver
Hormonal regulation of calcium homeostasis

 Maintenance of ionized plasma calcium level is important because deviation of ionized calcium from
normal range can lead to many disorders and can be life-threatening.
 Hormones involves in calcium homeostasis are
o Parathyroid hormone (PTH)
o Calcitriol (1, 25 DHCC) or vitamin D
o Calcitonin (CT)
Parathyroid hormone
 Parathyroid hormone (PTH) is a polypeptide of 84 amino acids synthesized as preproPTH in
parathyroid gland.
 PTH synthesis and secretion is enhanced by prolong hypocalcemia or vitamin D deficiency.
 PTH secretion is inversely related to the ambient concentration of ionized calcium. Serum PTH
declines in relation to serum calcium level between 7.5 to 10.5 mg/dL.
Mechanism of action of PTH

 PTH binds to its specific receptor in target cells which belong to the family of GPCR. Hormone
receptor interaction activates Gs protein which in turn stimulates the action of adenylyl cyclase
initiating cAMP signaling cascade. Increased cAMP leads to binding and activation of PKA. PKA
phosphorylates at specific serine/threonine residues of target protein and alters the activity of target
proteins.
 PTH regulates calcium homeostasis

by acting directly on bone, kidney
and indirectly on intestinal mucosal
cells.
Biochemical action of PTH on bones

 In bones, PTH receptors are present
in pre-osteoblasts, osteoblasts, bone
lining cells and osteocytes but not on
osteoclasts. In bone, PTH promotes
bone resorption and new bone formation. Bone dissolution predominates at high concentrations of
PTH, while formation is more important at physiological levels.
 PTH binds to GPCR on osteoblasts and
stimulate cAMP signaling cascade which leads
to ion and amino acid transport, collagen
synthesis and release of local mediators and
growth factors. This signaling effect is
responsible for bone formation action of PTH.
 Since osteoclasts lack of PTH receptors, PTH
activates bone resorption activity of
osteoclasts indirectly via osteoblasts. PTH-
stimulated osteoblasts express RANK ligand
(RANKL) on their cell membrane, and M-CSF
which, in turn, interacts with RANK expressing
osteoclast and their precursor cells. Thus,
RANKL-RANK interaction and action of M-CSF
on osteoclasts result in signaling events
leading to increased number of osteoclasts and their bone resorption activity. PTH also inhibits
production of osteoprotegerin (OPG) by osteoblast lineage cells since OPG can bind RANK-ligand,
thus reducing its binding to RANK and so the stimulation of osteoclastogenesis.
Biochemical action of PTH on kidney

 PTH action on kidney is mediated via GPCR and increased cAMP concentration.
 PTH reduces proximal tubular reabsorption of phosphate so that urinary phosphate excretion
increases (phosphaturic action).
 It increases distal tubular reabsorption of calcium, decreasing urinary calcium excretion.
 It stimulates the activity of 1α-hydroxylase enzyme in proximal tubular cells thereby increasing
formation of active vitamin D (1, 25 DHCC) which increases Ca2+ absorption from the intestine.
Calcitriol (Vitamin D or 1, 25 DHCC)

 Calcitriol is steroid in nature and it is the active
metabolite of vitamin D3. Formation of 1, 25
DHCC requires two hydroxylation steps in liver
(25 hydroxylation) and in kidney (1α
hydroxylation).
Biochemical action of calcitriol on intestine

 Calcitriol enters the small intestinal mucosal cell
and binds to a specific nuclear vitamin D
receptor. Hormone-receptor complex interacts
with RXR to form heterodimer. Activated
hormone-receptor complex-RXR heterodimer binds to specific DNA sequence called vitamin D
response element in regulatory region of target genes (calbindin D).
 In this mechanism, calcitriol induces genes encoding calcium binding proteins (calbindin D), Ca2+-
ATPase, other ATPases and membrane components that facilitate increased calcium uptake from
the intestine.
Biochemical action of calcitriol on kidney

 Calcitriol induces genes encoding proteins that facilitate renal tubular Ca2+ and phosphate
reabsorption via interacting with its specific nuclear receptor and binding to vitamin D response
element of target genes.
Biochemical action of calcitriol on bone

 Vitamin D promotes bone formation by inducing bone matrix proteins such as type I collagen and
osteocalcin in osteocytes and osteoblasts. Vitamin D provides proper microenvironment for bone
mineralization (by increasing calcium level via intestinal calcium absorption).
 It also promotes differentiation of osteoclasts from hemopoietic stem cells.
 It also indirectly stimulates osteoclast formation and its bone resorption activity by increasing RANKL
production on osteoblasts or osteoblast progenitor cells.
Calcitonin
 Intermediate size peptide containing 32 amino acids which is synthesized in parafollicular C cells of
thyroid gland. Its secretion is stimulated by increase in serum calcium level.
Biochemical actions of calcitonin

 Calcitonin binds to GPCR and activates adenylyl cyclase via Gs leading to increase in cAMP level which
mediates the signaling effects of calcitonin on target cells.
 Calcitonin inhibits bone resorption by acting directly on the osteoclasts (decreasing the number and
activity of osteoclasts).
 It increases calcium and phosphate excretion in renal tubules by decreasing its reabsorption.
Molecular Biology Page |1
Molecular Biology
Introduction
Cellular nucleic acids exist in two forms; deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
Approximately 90% of nucleic acid within the cells is RNA and the remainder is DNA.
DNA is the repository of genetic information. DNA is the chemical basis of heredity and is organized
into genes, the fundamental units of genetic information.
In eukaryotes, DNA is found in nucleus (linear DNA) and mitochondria (circular DNA) and RNA is
found in nucleus, cytoplasm and mitochondria.
Central dogma of molecular biology

The flow of information from DNA to RNA to protein is termed the “central
dogma” of molecular biology.
An organism must be able to
 store and preserve its genetic information,
 pass that information to future generation, and
 express that information to carry out all the processes of life.
Genetic information is stored in the base sequence of DNA molecules in
most organisms and of RNA molecules in some viruses (e.g., retro virus).
To pass the genetic information to future generation, DNA in each
chromosome is duplicated by replication in S phase of cell division.
Replication is the process by which each strand of the parental DNA duplex
is copied precisely by base pairing with complementary nucleotides to form
new DNA strand. In retro virus, the genetic information stored in RNA is
copied to DNA by reverse transcription for new viral generation.
The genetic information is used to synthesize all proteins made by an organism. The process is
called gene expression which includes transcription and translation.
Transcription is a process by which the information contained in DNA is copied to form
complementary sequence of ribonuclotides (RNA) chain.
Translation is the process by which the information in the nucleotide sequence of mRNA molecule
directs the ordered polymerization of specific amino acids into protein.
Structure and functions of nucleic acids

Nucleic acids – polymers of nucleotides joined by 3’ – 5’ phosphodiester bonds
Nucleotide – consists of three components: a nitrogenous base, pentose sugar and phosphate
Pentose sugars – ribose sugar in RNA and deoxyribose sugar in DNA (differ in carbon number 2)
Bases – two types: purine and pyrimidine bases

o Purine base – adenine, guanine
o Pyrimidine base – cytosine, thymine, uracil
Nucleoside – nitrogenous base covalently linking to carbon
number 1 of sugar by N-glycosidic bond
Mononucleotide – single phosphate added to 5’ end of
sugar moiety of nucleoside e.g., AMP, GMP
Addition of 2nd and 3rd phosphates – nucleoside
diphosphate or nucleoside triphosphate e.g., ADP, ATP
Structure of DNA
DNA is the chemical basis of heredity and is organized into
genes, the fundamental units of genetic information.
Watson, Crick and Wilkins proposed a model of DNA as
double stranded helix.
DNA is the double stranded helical structure in which two

polydeoxyribonucleotides strands twisted around each
other in common axis. These two strands are anti-parellel
i.e., one strand runs in 3’ to 5’ direction, the other 5’ to 3’
direction.
Each strand is a polymer of deoxyribonucleotides
(deoxyribose, N-base & phosphate) in which nucleotides
are linked by 3’ – 5’ phosphodiester bonds. Monomeric
deoxyribonucleotide units of DNA are dAMP, dGMP, dCMP
and dTMP.
In a DNA molecule, hydrophilic sugar-phosphate forms
backbone structure on the outside while hydrophobic bases are
stacked inside of the molecule. Genetic information is stored in the
base sequence of the DNA molecule.
The two strands are held together by hydrogen bonds between
complementary purine and pyrimidine bases and hydrophobic
interaction between stacks of base pair maintaining the stability
of double helix structure.
Base pairing between the two strands is complementary; adenine always pairs
with thymine by two hydrogen bonds and guanine always pair with cytosine by
three hydrogen bonds. Thus, base sequence of one strand defines the base
sequence on the other strand. According to number of hydrogen bonds, GC rich
regions are more stable than AT rich regions.
Because of complementary base pairing, total purine content equals to total
pyrimidine content in a DNA molecule. Specifically number of adenine equals that
of thymine and number of guanine equals that of cytosine in a double stranded
DNA. This is called Chargaff’s rule.
Under physiological conditions, most DNA is in the B form (Watson-Crick DNA
model).
DNA contains two grooves of different widths called major groove and minor
groove, which may facilitate binding with specific proteins.
Different forms of DNA

DNA exists in at least 6 different forms — A to E and Z.
B-DNA (10 base pairs per turn) is the major form of DNA in a cell and also the
most stable structure under physiological conditions.
A form (11 base pairs per turn) is also a right handed helix. When relative
humidity of B-DNA falls to less than 75%, B-DNA undergoes reversible
transition into A-DNA form.
Z form (12 base pairs per turn) is favored at high ionic concentration and also
by DNA methylation. It is a left-handed helix yielding a form of DNA with
zigzag configuration.
These alternative forms of DNA may help to regulate gene expression.
Functions of DNA
Genetic information is stored in the nucleotide sequences of DNA.
It serves as the template for replication to provide progeny (daughter cells) with the genetic
information possessed by the parent cell.
DNA is the source of information for synthesis of all proteins of cells and organism. Thus, it serves as
the template for synthesis of RNA by means of transcription. RNA is then translated into specific
protein.
Denaturation and renaturation of DNA

Separation of double stranded DNA into two single strands is called denaturation.
It can be done by increasing temperature or decreasing the salt concentration in solution (in vitro).
Denaturation of DNA (by heating) is one of the processes involved in DNA analysis procedure.
Mid-point of the temperature range at which DNA strands separate is called melting temperature or
Tm. Melting temperature is influenced by base composition of DNA and salt concentration of the
solution.
DNA rich in G-C pairs requires higher melting temperature than DNA rich in A-T pairs. Increasing the
salt concentration of the solution increases Tm.
Re-association or re-annealing of separated DNA strands will occur due to interactions between the
complementary nucleotide sequences when appropriate physiologic temperature and salt
concentration are achieved. This is called renaturation.
In vivo, DNA strands separation is done by the action of helicase which is induced by DNA binding
proteins. That facilitates the unwinding of DNA during replication and transcription processes.
The human genome

 Genome is the total DNA content of a cell. In other words, genome is the complete set of genes or
genetic material present in a cell or an organism.
 Cellular DNA contains genes and inter-genic regions, both serve important functions to the cell.
 Only about 1.5% of human DNA is “coding” or exon DNA, carrying information for protein products.
 Most of the DNA is non-protein coding. Some of the excess DNA sequences serve to regulate gene
expression during development, differentiation and adaptation to the environment, either by serving
as binding sites for regulatory proteins or by encoding regulatory RNAs.
 Gene – it is a segment of DNA sequence that encodes information required to produce functional
biological product. The human genome contains 20,000–25,000 different protein coding genes
spread over 23 chromosomes.
 Human genome can be divided into different “sequence classes.” These are unique-sequence DNA,
or non-repetitive DNA and repetitive-sequence DNA.
 In the haploid genome, unique-sequence DNA generally
includes the single copy genes that code for proteins.
 In human DNA, at least 30% of the genome consists of
repetitive sequences. Repetitive-sequence DNA can be
broadly classified as moderately repetitive or as highly
repetitive.
 Highly repetitive sequence (simple-sequence repeats or SSRs) – consists of 5 to 500 base pair
lengths repeated many times in tandem. These sequences are often clustered in centromeres and
telomeres of the chromosome and some are present in about 1 to 10 million copies per haploid
Molecular Biology P a g e |5
genome. The majority of these sequences are transcriptionally inactive and some of these
sequences play a structural role in the chromosome.
 Moderately repetitive sequence (<106 copies per haploid genome)
o Not clustered but are interspersed with unique sequences along the genome.
o Some consists of genes that specify tRNA and histone proteins.
o Some may participate in DNA strand association and chromosomal rearrangements during
meiosis.
o Depending on their length, moderately
repetitive sequences are classified as
long interspersed repeat sequences
(LINEs) (6 to 7 kbp) or short interspersed
repeat sequences (SINEs) (70 to 300 bp).
o Alu sequence (about 300 bp) is present in about 500,000 copies per haploid genome and
accounts for ~10% of the human genome.
o Microsatellite sequences – consist of 2 to 6 bp repeated up to 50 times and occur at 50,000 to
100,000 locations in the genome. The number of these repeats may vary on the two
chromosomes, thus providing heterozygosity of the number of copies of a particular
microsatellite number in an individual. This is a heritable trait. Trinucleotide sequences that
increase in number (microsatellite instability) can cause disease, e.g., unstable (CGG)n repeats
in fragile X syndrome.
o Transposons – mobile DNA elements that have contributed to the evolution.
Mitochondrial genome
Human mitochondria contains 2 to 10 copies of small circular 16 kbp dsDNA that constitutes less than
1% of total cellular DNA.
It contains 16, 569 bps encoding 37 genes— 13 proteins, 22 tRNAs and 2 rRNA (16s and 12s mt rRNA).
Specific features of mitochondrial DNA
o Dense gene packing (no introns, very few noncoding region)
o Relax codon usage (relax codon-anticodon pairing rules)
o Variant genetic code (drifted from the universal code)
 UGA for tryptophan
 AGA and AGG as stop codons
 AUG for isoleucine instead of methionine
o Maternal non-mendelian inheritance
o High mutation rate (5 to 10 times higher than that of nuclear DNA)
DNA replication, transcription and protein synthesis

takes place in the matrix. Mitochondrial DNA replication
can occur many times throughout interphase.
Ribosomes in mitochondria gene expression are of
prokaryotic 70s type, not eukaryotic 80s type.
Mitochondrial protein synthesis starts with N-formyl
methionine as in bacteria unlike eukaryotes. So,
mitochondria are sensitive to many antibiotics that
inhibit bacterial gene expression such as streptomycin,
chloramphenicol, rifampicin.
Comparison of mtDNA sequences provides evidence
about evolutionary origins of the species.
Mitochondrial DNA mutation
o Could be due to ROS (peroxides and superoxides) derived from mitochondrial leak.
o Consequences can be wide-ranging due to central role of mitochondria in metabolism.
o Possible association with heart diseases, type 2 diabetes mellitus, Parkinson’s disease,
Alzheimer’s disease and many of the effects of aging.
o Inherited disorders include MERRF, LHON, MELAS, etc.
Structure of chromosome
Every cell of a multicellular organism contains the same genetic materials. In eukaryotes, DNA is
arranged in linear segments called chromosomes in nucleus.
Genomes of eukaryotes are divided between a set of different chromosomes. For example,
approximately 3.2 x 109 nucleotides are distributed over 24 different chromosomes in human
genome.
Each human somatic cell contains two copies of each chromosome, one inherited from the mother
and one from father, thus a total of 46 chromosomes – 22 pairs are autosomes and one pair is sex
chromosome.
The maternal and paternal chromosomes of a pair are called homologous chromosomes (homologs).
The only non-homologous chromosome pairs are sex chromosomes (XY in male).
Regions of chromosomes – DNA replication origin (ORI region), centromere and telomere
DNA replication origin (ORI region) – the location at which replication begins.
Centromere – specialized DNA sequences (highly conserved GGAAT repeats) participates in DNA
strand association and chromosomal rearrangements during cell division. It serves as genomic sites
of spindle attachment that are essential for proper chromosome segregation during cell division.
Telomere – sequences (short TG rich tandem repeats) at the ends of

eukaryotic chromosomes that are essential for chromosome stability and
cell division. Shortening of telomeres after many cells replications has
been linked to the development of cellular senescence, malignant
transformation and aging.
Metacentric chromosome – centromere is near the middle of
chromosome.
Acrocentric chromosome – centromere is near the telomere.
Sub-metacentric chromosome – centromere is between the middle and
telomere.
DNA sequences on both side of centromere are designated as arms of
chromosome. Long arm is labeled as ‘q’ and short arm as ‘p’.
Genes are located on chromosome. Site of a gene in a chromosome is
called locus. The alternated form of a gene is alleles.
Structural organization of eukaryotic DNA

The entire sequence of nucleotides in human genome
comprises approximately 3.2 x 109 bps. They are
distributed among different sizes of 23 distinct
chromosomes. Each chromosome of eukaryotic cell
contains a single, large, duplex DNA molecule which
contains 48 – 240 million bps.
To fit within the nucleus, DNA is condensed more
than 8000 folds into an organized structure. Thus,
DNA is arranged in orderly and densely packed
structure termed chromosomes.
DNA in the chromosome is tightly associated with
‘histones’ proteins and forms into structural units
called nucleosomes.
Histones are proteins involved in the packing and
folding of DNA within the nucleus. There are five
classes of histones termed; H1, H2A, H2B, H3 and H4.
They all are rich in positively charged, basic amino
acids (lysine, arginine) and hence they can readily
Heterochromatin and Euchromatin
bind the negative charges of the sugar-phosphate Chromatin containing active genes (i.e.,
backbone of DNA molecules thereby reducing transcriptionally active chromatin) has

been shown to differ in several ways
electrostatic repulsion and permit tighter DNA packing.
from that of inactive regions.
Each individual nucleosome core particle consists of the
This is the basis of differential gene
octamer core histones complex (two copies of each of expression of the common genetic
histones H2A, H2B, H3, H4) which is encircled by about information between different cell types
which contain the same genetic
146 bps of DNA segment. H1 protein associates with the
information.
outside of nucleosome core to stabilize the complex.
Euchromatin
Nucleosome particles or beads are separated by an about o Transcriptionally active chromatin
20 – 90 bps of linker DNA. o Stains less densely
Nucleosomes are the fundamental organizational units of Heterochromatin

o Transcriptionally inactive chromatin
chromatin. Nucleosome particles plus connecting DNA
o Densely packed during interphase
that links to next particle gives “beads-on-string
as observed by electron microscopic
appearance” under electron microscope. Nucleosomes studies
cores are packed and organized into a structure called the o Two types — constitutive and
facultative
30 nm chromatin fiber.
o Constitutive heterochromatin is
At the next level, 30 nm fiber is folded into loops. Loops
always condensed and thus
are bound to a protein scaffold consisting of H1 histone essentially inactive.
and several non-histone proteins, Sc-1 (topoisomerase II) o Facultative heterochromatin is at
and Sc-2, condensing into additional layers of organization times condensed, but at other times
it is actively transcribed and, thus,
and finally chromatin filaments are compacted into the
uncondensed and appears as
mature chromosome. euchromatin e.g., heterochromatic X
chromosome in females.
Modification of histones proteins
 Histones are subjected to covalent modifications such as acetylation, methylation, phosphorylation,
ADP-ribosylation, glycosylation, ubiquitylation.
 Such modifications affect the charge, shape
and other properties of histones, as well as
structural and functional properties of the
chromatin. Thus, histone modification plays a
role in the regulation of gene expression.
 For example, acetylation of histones leads to
reduced binding affinity of histones to DNA and
transcription of nearby genes can be initiated.
Cell cycle
 Cell cycle is the orderly sequence of events by which a cell
duplicates its chromosomes and other cell contents
followed by division of a cell into two genetically
identical daughter cells.
 The cell cycle of eukaryotes consists of four phases.
1. G1 phase – period of cell growth and differentiation
2. S phase (S for DNA synthesis) – during which DNA is
synthesized.
3. G2 phase – preparatory phase for cell division
4. M phase (M for mitosis) – nuclear division and
cytoplasmic division occur.
 The first three phases (G1, S, and G2) constitute interphase. Cells spend most of their time in these
three phases, carrying out their normal metabolic activities. Late in G1, the cells prepare to duplicate
their chromosomes (e.g., by producing nucleotide precursors). Gene expression occurs throughout
the interphase but not in M phase.
 G1 and G2 phases are responsible for cell growth and for providing checkpoints at which cell cycle
progress is controlled. Each phase is under close regulation at multiple biochemical checkpoints
with stop/go signals before proceeding to the next phase.
 After mitosis, a cell may reenter G1 and either continues through phases of cell cycle and dividing
repeatedly or ceases to divide, entering a quiescent phase (G0) that may last hours, days, or the
lifetime of the cell. If extracellular conditions are unfavorable, cells delay progress through G1 and
may even enter the G0 phase. On the appropriate signal such as growth factor, a quiescent cell (Go
phase) can be induced to reenter the cycle and divide.
 In human body, many cells divide frequently, spending very little or no time in G0 phase e.g.,
fibroblasts, epithelial cells, hair follicles, etc. Some cells such as hepatocytes, adult brain cells and
myocytes are normally quiescent and may spend most of their time in Go phase.
Regulation of cell cycle

Morphogens and mitogens (growth factors and cytokines) play as external signals in cell cycle
progression. Mammalian cells in G0 phase are stimulated by growth factors, which trigger them to
enter into G1 phase. Growth factors bind to their cell surface receptors and initiate signaling cascade;
inducing proteins essential for cell cycle progression.
Cyclins and cyclin-dependent kinases (CDKs) family are important proteins in cell cycle control. Cell
cycle control system has three check points— one in late G1 phase during G1 to S transition (G1
Molecular Biology P a g e | 10
restriction point), one during S phase and one in late

G2 phase (G2-M boundary). Each of different phase
specific cyclin-CDK complexes serves as molecular
switch that triggers cell cycle progression.
Cyclin turns on at appropriate time and activates its
specific CDK. Activated CDK phosphorylates target
proteins essential for progression through cell cycle.
Different cyclins and CDKs involved in cell cycle
progression
o G1 phase cyclin-CDK (cyclin D – CDK4/CDK6) drives
G1 to S phase progression.
o S phase cyclin-CDK complexes (cyclin E/cyclin A –
CDK2) initiates DNA replication once per cycle.
o Late G2 phase cyclin-CDK complexes (cylcin B –
CDK1) triggers entry into mitosis.
In the case of DNA damage, DNA damage checkpoints
sense these changes and activate tumor suppressor
protein p53 and signaling pathways that mediate cell
cycle arrest and DNA repair. Cell cycle arrest is achieved
by several ways of inactivation of cyclin-CDK
complexes. These include –
o Inhibition by CDK inhibitor proteins (CKIs) e.g., p21
o Desensitization by inhibitory phosphorylation to the complex
o Degradation of cyclins by ubiquitin dependent proteasomal system
Mechanism of cell cycle progression

Mitogenic growth factors exert their effects on late G1 phase called
restriction point. The overall rate of cell proliferation is controlled by G1.
Growth factors stimulation results in the induction of proteins involved
in regulating the cell cycle such as cyclins, CDKs. Progression through
the cell cycle is regulated by the cyclins and CDKs.
At each point in the cycle, specific CDKs associate with phase specific
cyclins. Activation of specific CDK requires binding of specific cyclin and
phosphorylation by CDK activating kinase (CAK). Activated cyclin-CDK
complexes in turn phosphorylate and activate target proteins for progression of cell cycle.
Retinoblastoma (Rb protein) is the

product of oncosuppressor gene. Rb
protein is a cell cycle regulator of G1
restriction point. It binds and
inactivates transcription factor E2F
necessary for transcription of certain
genes (histone genes, enzymes for
DNA replication, etc.).
When cell is stimulated by external
signal (growth factor), cyclin D –
CDK4/CDK6 complex phosphorylates Rb. Phosphorylated E2F is released from Rb protein, resulting
in transcription of genes required for cell cycle progression.
DNA damage and cell cycle check points control

Tumor suppressor protein p53 plays a key role in both G1
and G2 check point control. It is a DNA binding transcription
factor that is activated by damaged DNA.
One of the induced protein p21 is a potent cyclin-CDK
inhibitor (CKI) that can inhibit the action of all CDKs. Thus
p21 acts as G1-CDK inhibitor causing cell cycle arrest which
allows the cell to repair its DNA damage before proceeding
into S phase.
However, if the DNA damage is too extensive to be
repaired, p53 level highly increases which initiates cell
death or apoptosis.
Retinoblastoma, a cancer of the developing retina that
affects infants and young children, is associated with the loss of the Rb gene, which encodes the
tumor suppressor pRb. Certain tumor antigens derived from viruses such as SV40, HSV, HPV may
combine with Rb and interferes with inhibitory effect of Rb on the cell cycle; leading to continuous
cell division and cancer.
The p53 gene is the most commonly altered gene in human cancers; over 50% of human cancers is
associated with a mutation or deletion of p53 gene.
Many of the cancer causing viruses and oncogenes are capable of alleviating or disrupting the
apparent restriction that normally control the entry of cells from G1 to S phase.
Apoptosis
Apoptosis is the process of programmed cell
death, to limit the growth and proliferation of
cells.
It is a fundamentally important biologic process
to maintain the integrity and homeostasis of
multicellular organisms. Apoptosis is the normal
part of early human development. In adults, it is
important as a response to cellular damage, viral
infections, somatic mutations, hormonal
influence or lack of extracellular survival factors.
Mechanism of apoptosis
o Apoptosis is mediated by proteolytic enzymes called caspases family. They are present as
inactive procaspases in the cell that have to be activated by a proteolytic cascade.
o Stimuli for apoptosis are extrinsic signals such as tumor necrosis factor (TNF), Fas ligand and
intrinsic signals such as highly increased p53 level due to extensive DNA damage, ROS, etc.
o The extrinsic pathway is triggered by external agents (TNF, Fas ligand) that bind and activate
death receptors on the cell surface. They initiate apoptosis by directly recruiting procaspases
resulting in activation of caspases cascade that mediate apoptotic cell death in a number of cell
types especially in cells of immune system.
o The intrinsic pathway is triggered by stimuli that arise within the cell. Apoptotic stimuli trigger the
release of cytochrome c from mitochondria. Cytochrome c interacts with other proteins and
procaspases to initiate proteolytic caspases activation cascade that mediate apoptosis.
o Activated caspases trigger cell death by cleaving specific proteins in cytoplasm and nucleus
resulting in — cell cycle arrest, disabling homeostatic and repair mechanisms, detachment of cell
from its surrounding tissues structures, dismantling cytoskeletal structure, flagging the dying cell
for phagocytosis. It is characterized by dramatic morphological changes in the cell such as
fragmentation of DNA, shrinkage of cytoplasm, membrane changes and finally cell death without
lysis or damage to neighboring cells. The apoptotic cells are phagocytosed by macrophage.
B cell lymphoma protein 2 (Bcl 2) and inhibitors of apoptosis protein (IAP) family prevent apoptosis
by regulating mitochondrial integrity and inhibiting cytochrome c release. Tumor suppressor protein
p53 activates apoptosis in response to DNA damage and it also represses the expression of Bcl2.
Disruption and subversion of apoptosis machinery can result in cancer or autoimmune disease.
Inappropriate apoptosis can lead to neurodegenerative disorders (Alzheimer, Parkinson disease).
DNA replication
 Replication is the process by which each strand of the parental DNA duplex is copied precisely by
base pairing with complementary nucleotides to form new DNA strand.
 Characteristic features of DNA replication
o The primary function of DNA replication is the provision of progeny with the genetic information
possessed by the parent cells.
o Each strand of parental DNA duplex serves as template to provide sequence information.
Sequences on templates are copied by complementary base pairing rule.
o Replication begins from specific site called DNA replication origin.
o Replication of DNA must be complete and carried out with high fidelity to maintain genetic
stability within the organism and the species.
o DNA replication has proofreading mechanism that correct initial mispairings.
o It occurs only at a specific time, at the “S” phase of cell cycle in eukaryotic cells.
o The nuclear DNA is completely replicated once and only once per cell cycle.
o Simultaneous and bidirectional - Both strands of DNA are replicated at the same time and to
both directions of the replication bubbles.
o Semi-discontinuous – Because of the unidirectional 5’ to 3’ synthetic activity of the DNA
polymerase and the antiparallel nature of the two strands, one new strand is continuously
polymerized (leading strand) and the other new strand is discontinuously polymerized (lagging
strand) giving rise to short DNA segments (Okazaki fragments).
o Semi-conservative – One strand of parent DNA molecule is conserved in each new double helix
paired with a newly synthesized complementary strand.
DNA replication requires –

1. Template DNA (both strands serve as template)
2. Four activated precursor deoxyribonucleoside
triphosphates (dNTPs) – dATP, dGTP, dCTP, dTTP
3. DNA polymerase enzyme
4. RNA primer
5. Many other protein factors to facilitate the process
DNA replication in prokaryotes

DNA polymerases in prokaryotic DNA replication
o DNA Pol I – replication and repair of faulty DNA during replication, gap filling
o DNA Pol II – repair damaged DNA, DNA proofreading
o DNA Pol III – chain elongation
Initiation
o Identification of replication origin
 Replication of circular dsDNA begins at specific
site called the replication origin (ori-C in E coli).
 Binding of specific DNA binding proteins (dnaA)
to origin of replication leads to local denaturation
and unwinding of DNA.
o Unwinding
 DnaB protein has helicase activity and catalyzes
ATP dependent unwinding of double helical DNA.
 Single stranded DNA binding proteins (SSBs)
binds to the separated strands to prevent re-
association and stabilizes DNA unwinding.
 DNA unwinding forms replication bubble with
replication forks formed on both ends of the
unwinding area.
 DNA gyrase relieves positive supercoiling due to
unwinding of circular DNA by introducing the
negative supercoils. This process is ATP
dependent.
o Primer synthesis
o DNA primase forms primosome complex and synthesizes RNA primer.
Elongation
o DNA polymerase polymerizes growing two daughter strands.
o The short stretch of RNA serves as primer for DNA polymerase and then DNA polymerase III
polymerizes according to base pairing with complementary deoxyribonucleotide using dNTPs as
precursors. DNA replication occurs in both directions of replication bubble.
o DNA polymerase only synthesizes DNA in 5’ to 3’ direction.
o Due to unidirectional synthetic activity of polymerase and antiparallel nature of two strands, one
strand is synthesized continuously by keeping pace with replication fork (leading strand) and one
strand is synthesized discontinuously as short segments called Okazaki fragments (lagging
strand).
o DNA replication has proofreading mechanism that correct initial mispairings. By using 3’ to 5’
exonuclease activity, DNA Pol III removes the mismatched residue.
Termination
o DNA polymerase has exonuclease activity and
catalyzes RNA primer removal and gap filling.
o DNA ligase catalyzes final linking step.
o Antibiotics quinolones and fluroquinolones
are DNA gyrase inhibitors which are used to
control the spread of bacterial infection, e.g.,
nalidixic acid, ciprofloxacin, norfloxacin.
o Acyclovir is used as antiviral agent for the herpes simplex virus. It inhibits viral DNA polymerase.
Replication in eukaryotes
DNA polymerases in eukaryotic DNA replication
o DNA polymerase α – primase activity, initiate DNA replication
o DNA polymerase δ – primary enzyme of DNA synthesis, lagging strand synthesis, DNA repair
o DNA polymerase ε – leading strand synthesis, DNA repair, gap filling
o DNA polymerase β – DNA repair
o DNA polymerase γ – mitochondrial DNA replication and repair
Nuclear DNA replication occurs in S phase of the cell cycle.
Initiation
o Identification of the origins of replication (ORI)
 In late G1 phase of cell cycle, the cells prepare to duplicate their chromosomes. Eukaryotic
cells initiate DNA replication at multiple origins. Chromosome must be unpacked into
chromatin during replication. Nucleosomes on chromatin must be disassembled into bare
DNA strands ad histones.
 Initiation of replication depends on the origin recognition complex (ORC), a protein complex
that binds to replication origins. ORC recruits DNA helicase and other proteins forming pre-
replication complexes (pre-RCs) on origins of replication.
 Initiation of replication also requires the
synthesis and activity of S-phase cyclin-CDK
complexes.
o Unwinding of dsDNA
 DNA helicase (hexameric protein complex)
catalyzes ATP-mediated progressive unwinding of DNA strands. Single stranded DNA binding
proteins (SSBs) coat the separated strands to prevent their reassociation. Unwinding at
multiple replication origins generates replication bubbles with a replication fork at each end
of the bubbles.
o Primer synthesis
 Since DNA polymerase cannot initiate DNA polymerization
itself, but can only add nucleotides to an existing primer
that provides 3’ OH group. So, at the replication fork, DNA
replication is primed by a short stretch of RNA that is
synthesized by primase (DNA polymerase α).
 Supercoiling ahead of the replication fork is relieved by
DNA topoisomerase I and II.
Elongation
o New DNA is synthesized by DNA polymerases. Polymerization
occurs according to base pairing rule using four different
dNTPs as precursors.
o DNA polymerase catalyzes formation of 3’ – 5’
phosphodiester bond between 3’ OH of
preexisting nucleotide and 5’ phosphate of next
incoming complementary nucleotide, releasing
pyrophosphate in the reaction.
o DNA replication is occurred in both directions in
each replication bubble.
o Since DNA polymerases only synthesize DNA in 5’ to 3’
direction, one new strand is continuously synthesized
(leading strand) and the other new strand is
discontinuously synthesized as Okazaki fragments
(lagging strand).
o High fidelity of DNA replication depends on
proofreading activity. It is carried out by 3’ to 5’
exonuclease activity of DNA polymerases that
removes mispaired nucleotides from 3’ end of growing
chain and incorporates the correct nucleotide.
Termination
o RNA primers are removed by RNaseH and gap filling is done by DNA polymerase.
o These DNA segments are joined by DNA ligase.
o Telomere replication
 Synthesis of lagging strand encounters a special problem when the replication fork reaches
an end of a linear chromosome (telomere). There is no place to produce the RNA primer
needed to start the last Okazaki fragment.
 Shortening of telomere end is prevented by an
enzyme telomerase. Telomerase has RNA
dependent DNA polymerase activity like reverse
transcriptase. The telomerase use internal RNA
template for extension of parental DNA. Only
after that the lagging strand can be synthesized
at the chromosome end.
 Most normal human somatic cells have low
telomerase activity, thus telomeres shorten with
each successive cell division finally leading to
replicative senescence. Some normal cells
(normally regenerating tissues, stem cells, and
progenitor cells) express full telomerase activity.
o Reconstitution of chromatin structure
 Newly replicated DNA is rapidly assembled into
nucleosomes behind the advancing replication
forks and organized into chromatin structure.
Post-replication modification of DNA
o After DNA is synthesized, it is glycosylated and
methylated by specific enzymes. Methylation
protects the host DNA from destruction by
restriction endonuclease that attack foreign DNA.
o Doxorubicin and etoposide inhibit eukaryotic type II topoisomerases and are therefore widely
used in cancer chemotherapy.
o Several anti-cancer agents target the biosynthesis of the nucleotide precursors for DNA, thus
starving DNA polymerase for new building blocks e.g., 5-fluorouracil and 6-mercaptopurine.
o Cancer cells appear to reactivate the telomerase activity and consequently chromosome length
equilibrium is maintained, leading to continued cell division. Thus, telomerase is a potential target
for development of newer anticancer drug.
Comparison between prokaryotic and eukaryotic DNA replication
Steps in replication Prokaryotes Eukaryotes
Timing of replication Prior to cell division S phase of cell cycle
Replication origins Single ORI Multiple ORI
Recognition of ORI dnaA protein Unknown
Helicase
Unwinding of DNA Helicase (dnaB protein)
(hexameric MCM protein complex)
Stabilization of separated strand SSBs SSBs
RNA primer synthesis Primase Primase (DNA polymerase α)
Synthesis of DNA
Leading strand DNA polymerase III DNA polymerase ε
Lagging strand DNA polymerase III DNA polymerase δ
RNaseH
Primer removal DNA polymerase I
Flap endonuclease 1 (FEN1)
Gap filling DNA polymerase I DNA polymerase β and ε
Joining of Okazaki fragments DNA ligase DNA ligase
Removal of positive supercoils DNA gyrase DNA topoisomerase II
Telomerase
Telomere synthesis Not required
(RNA dependent DNA polymerase)
Inhibit bacterial proliferation Inhibit DNA topoisomerase II

Drugs affecting DNA replication
Used as antibiotics Used as anticancer agents
DNA gyrase inhibitors
(Quinolones, fluroquinolones) (Etoposide, Doxorubicin)
Reverse transcription
Reverse transcription is RNA
directed synthesis of DNA, catalyzed
by reverse transcriptase.
Some virus contain single stranded
RNA as the genome e.g., retro virus.
They are not capable of reproducing
independently and must invade host
cells to reproduce e.g., HIV virus.
Once it infects the cell, viral RNA is used as a template to produce a double stranded DNA (cDNA) by
the action of virally encoded reverse transcriptase (RNA dependent DNA polymerase). The resulting
dsDNA transcript can integrate into the host genome and subsequently serves as template for gene
expression from which new viral RNA genomes ad viral mRNAs can be transcribed.
o Retro virus is an oncogenic virus. The insertion of viral dsDNA into the chromosome of
infected host cell can transform it into a cancerous cell.
o Human immunodeficiency virus is a retro virus. HIV enters into helper T lymphocytes and
eventually kills the infected helper T cells which are important in the defense against
infection. It is the causative agent of acquired immune deficiency syndrome. (AIDS).
o Retro virus can be used in gene therapy.
o For genetic engineering purpose, reverse transcriptase has been useful for making dsDNA
copies of various mRNAs.
o Reverse transcriptase activity of retroviruses can be inhibited by nucleoside analogs (lack
3’OH group) such as azidothymidine (AZT), dideoxycytidine (ddC), dideoxyinosine (ddI) and
non-nucleoside compounds such as nevirapine. These agents are used as antiretroviral
therapy for HIV infection.
DNA damage and repair

DNA can be damaged by
numerous types of endogenous and
exogenous agents that cause nucleotide
modifications, deletions, insertions,
sequence inversions and transpositions.
Causal agents for DNA damage
Spontaneous damage by chemical reactions or cellular metabolism
o Hydrolysis
o Oxidation (by free radical, oxidative agents in inflammation)
o Non-enzymatic methylation from endogenous metabolism
o Replication errors
o Spontaneous decay of DNA (major factor in mutagenesis and aging)
Environmental factors
o Physical agents – UV rays, radiation
o Chemical agents – smoking, pollutants, chemical compounds in diet and drugs
o Biological agents – viral infection, aflatoxin
Types of DNA damage

I. Single base alteration
a) Depurination
b) Deamination of bases
 Adenine to hypoxanthine,
guanine to xanthine
 Cytosine to uracil
c) Alkylation of base
d) Insertion or deletion of nucleotides
e) Base analog incorporation
II. Two base alteration
a) UV light induced thymine-thymine dimer
b) Bifunctional alkylating agent cross-linkage
III. Cross linkage
a) Between the bases in same or opposite strands
b) Between DNA and protein molecules
IV. Chain breaks
a) Ionizing radiation
b) Radioactive disintegration of backbone element
c) Oxidative damage by free radicals
Mechanisms of DNA repair

DNA is the only macromolecule that can be repaired.
There are four mechanisms of repair of damaged region of DNA.
1. Mismatch repair
2. Base excision repair
3. Nucleotide excision repair
4. Strand break repair
General steps in DNA repair mechanism

Recognition and identification of damage site
Removal of damaged region
Filling the gap
Sealing with DNA ligase
1. Mismatch repair (MMR)

 Mismatch repair system corrects replication errors.
 Template strand is methylated and specific proteins scan the
newly synthesized DNA strand using adenine methylation within
a GATC sequence of template strand as the point of reference.
 If the mismatch base is found, GATC endonuclease cuts the DNA
segment bearing the mismatch base (corresponding to GATC
sequence) followed by exonuclease digestion thus removing
the faulty DNA.
 The gap is filled by DNA polymerase and sealed by DNA ligase.
2. Base excision repair (BER)

 Base excision repair is responsible for repair of single base lesions
such as spontaneous deamination (cytosine to uracil, adenine to
hypoxanthine), depurination, base modification (oxidation, alkylation).
 Specific DNA glycosylases can recognize specific single base lesion
and remove the affected base by cleaving the N-glycosidic bond. This
cleavage creates an apurinic or apyrimidinic site in the DNA,
commonly referred to as an AP site or abasic site.
 Sugar phosphate backbone of AP site is excised by the sequential
action of AP endonuclease and phosphodiesterase.
 Then the gap is filled by DNA polymerase and sealed by DNA ligase.
3. Nucleotide excision repair (NER)

 DNA lesions that cause large distortions in the helical structure of
DNA are generally repaired by the nucleotide-excision system. These
include UV light induced thymine-thymine dimer,
DNA damage due to smoking (guanine adducts),
strand break and cross-linkage damages.
 In this repair mechanism, DNA damage is removed as
a part of an oligonucleotides fragment (about 30
nucleotides) by the action of specific excinuclease
enzyme. This is followed by the replacement with
new DNA using the intact strand as template by the
action of DNA polymerase and finally sealed by ligase.
4. Strand break repair

 Single strand break induced by ionizing radiation
is repaired by direct ligation or by excision repair
mechanism.
 Double strands break (DSB) is induced by ionizing
radiation, ROS, and some chemotherapeutic
agents. These are repaired by two systems;
homologous recombination (HR) and non-
homologous end-joining (NHEJ). The choice
between the two depends upon the phase of the
cell cycle (NHEJ in G0/G1 and HR in S, G2, M phase)
and the exact type of DSB breaks to be repaired.
 Homologous recombination (HR) uses sequence
information available from the unaffected
homologous chromosome for proper repair of
breaks. It uses the enzymes that normally
perform genetic recombination between
homologous chromosomes during meiosis. BRCA
1 and 2 proteins normally play a role in the
homologous recombination process.
 Non-homologous end-joining (NHEJ)
o Two proteins are involved; Ku protein which
has latent ATP dependent helicase activity
and DNA dependent protein kinase (DNA-PK).
o Ku binds to the free DNA ends which then
recruits DNA-PK. Binding of DNA-PK allows
for the approximation of the two separated
ends. The free end DNA-Ku-DNA-PK complex
activates DNA-PK which then phosphorylates
Ku and the other DNA-PK molecule, on the opposing strand. DNA-PK then dissociates from
the DNA and Ku, resulting in activation of the Ku helicase.
o After unwinding of two ends, the unwound, approximated DNA forms base pairs. Extra
nucleotide tails are removed by an exonuclease; and the gaps are filled and sealed by ligase.
o Since some DNA is lost in the process, this mechanism of repair is error prone and mutagenic.
Many cancers are caused by defective repair system.
Mutation in gene that produces protein that participate in mismatch repair leads to predisposing to
hereditary cancer e.g., hereditary non-polyposis colorectal cancer or Lynch syndrome.
Xeroderma pigmentosum (XP) is the result of a defect in repair of UV-induced thymine dimers in
DNA due to excinuclease defect. The clinical syndrome includes marked sensitivity to sunlight
(ultraviolet) with subsequent formation of multiple skin cancers and premature death.
Patients with Fanconi anemia, characterized also by an increased frequency of cancer and by
chromosomal instability, probably have defective repair of cross-linking damage (NER defect).
In patients with ataxia-telangiectasia, there is defective repair system for double strand break.
About 10% of breast cancer cases are associated with inherited defects in two genes, BRCA1 and
BRCA2. Women with defects in either the BRCA1 or BRCA2 gene have a greater than 80% chance of
developing breast cancer.
Many anticancer drugs cause DNA damage, e.g., cisplatin, used for treatment of several forms of
cancer particularly effective against testicular tumor forms two intrastrand adducts with DNA.
Functional eukaryotic gene

 Gene is a segment of DNA which encodes the information required to produce a functional
biological product (protein or one of the several classes of RNA).
 Genes are located on chromosome. Site of a gene in a chromosome is called locus. The alternative
form of a gene is called allele. Direction of a gene is 5’ to 3’ direction.
 Gene expression is the synthesis of proteins under the guidance of a gene. It is the process in which
the genetic information in DNA is transcribed into RNA which then directs the polymerization of
amino acids to synthesize a protein. Gene expression occurs in two stages; transcription of DNA to
RNA and translation of mRNA to amino acid chain, protein.
 Genes do not function autonomously. The transcription initiation is a consequence of specific
protein-DNA interaction. Transcription factor proteins bind specific elements. Many transcription
factors act positively and promote transcription while some can cause gene silencing. The protein-
DNA interaction at the regulatory sites regulates gene expression.
Transcription
start site
Other Enhancer Proximal

Upstream elements
5’ regulatory & Silencer element E I E I E 3’
CAAT box or GC box
elements elements TATA box
Structural region
Regulated expression Basal expression
Regulatory region
Functional Eukaryotic Gene (E= exon, I= intron)

 A functional eukaryotic gene can be divided into the structural and regulatory regions.
 Structural region
o It consists of exons and introns.
o Exons are coding regions which carries the genetic information for protein synthesis.
o Introns are non-coding intervening sequences. Although introns do not encode for proteins, they
are important for the structural and regulatory role of a gene. Introns must be removed from
precursor mRNA (primary RNA transcript) before transported into the cytoplasm for translation.
 Regulatory region
o It consists of two classes of elements; basal expression region and regulated expression region.
o Basal expression region (promoter elements)
 It is composed of proximal component or TATA box and upstream element (CAAT box or GC
box). These elements are essential for gene expression at basal condition. They are located at
5’ end of the gene and thus called 5’ flanking sequence.
 TATA box (–25 to –30 bp upstream from the transcription stat site) serves to bind and direct
RNA polymerase to the transcription start site. Binding of TATA-binding protein (TBP)
complex to the TATA box sequence is thought to be the first step in the formation of the
transcription complex on the promoter.
 CAAT box or GC box (–40 to –110 bp upstream from the transcription start site) binds with
the transcription factors and specifies the frequency of transcription initiation.
o Regulated expression region
 It is composed of enhancer elements (increase rate of transcription), silencer elements
(decrease rate of transcription) and other regulatory elements e.g., HRE.
 They are found in both upstream and downstream of the gene.

 These regions mediate responses to various signals such as hormones, chemicals, metals and
various metabolites.
 These sequences are cis-acting elements and they interact with trans-acting protein during
transcription.
 Gene expression is tissue specific in nature e.g., insulin gene is
present in all somatic cells but only expresses in β cells of
pancreas.
 According to the expression pattern, there are two types of
genes; inducible gene that responds to the regulatory signal and
the constitutive gene which expresses at a reasonably constant
rate and does not respond to regulation. Products of
constitutive gene are always necessary for cellular metabolism,
so these genes are also called house-keeping genes.
Functional prokaryotic gene

 It is made up of two regions—
1. Structural region which code for protein and functional RNA (gene cluster)
2. Regulatory region which consist of two elements
 Basal expression region containing a promoter locus (TATA box or Pribnow box) at –
10bp and operator locus (binding site for repressor molecule)
 Regulatory gene – which produce a repressor molecule
 In bacteria, the genes for proteins that perform a specific function are grouped together in units
called operons. The structural genes in an operon are regulated by a single promoter.
Transcription
start site
5’ Regulatory Promoter locus Operator 3’

Gene Cluster
gene locus
Structural region
Regulated
expression Basal expression
Regulatory region
Functional Prokaryotic Gene

Transcription (RNA synthesis)

 Transcription is a process by which information contained
in DNA is copied by base pairing to form a complementary
sequence of ribonucleotides (RNA chain).
 Genetic information in DNA is transferred from DNA to
protein via the several RNA molecules. Different classes of RNA molecules are synthesized from DNA
by a complex process involving DNA dependent RNA polymerase enzyme and a number of proteins.
 Transcription proceeds along the antisense or template strand (3’ to 5’ direction), producing a
complementary RNA that is identical to the sense strand or coding strand (5’ to 3’ direction) of the
DNA. With the exception of T for U changes, coding strand corresponds exactly to the sequence of
primary RNA transcript.
 Characteristics of transcription
o Transcription occurs in active
genes or transcriptionally active
euchromatin of chromosomes.
o RNA strand is copied from the
template strand of DNA (anti-
sense strand). The opposite
DNA strand is called coding strand (sense strand) as it has the same sequence of primary RNA
transcript with the exception of T for U.
o RNA strand is copied from template strand by complementary base pairing.
o General steps of transcription are similar in eukaryotes and prokaryotes.
o Polymerization of the RNA strand is from 5’ to 3’ direction.
o RNA polymerase can perform de novo synthesis of RNA chain, so primer synthesis is not
required in transcription.
o RNA polymerase has no exonuclease activity and cannot carry out proofreading.
 Requirement for transcription
o Template – antisense strand of dsDNA
o RNA polymerase (DNA dependent RNA polymerase)
o Activated precursors – four different ribonucleoside triphosphates (ATP, GTP, CTP, UTP)
o Transcription factors and DNA binding proteins that facilitate the process.
Components and functions of prokaryotic RNAP

Transcription in prokaryotes
Subunit Role or function in transcription
All prokaryotic genes are transcribed by a
single form of RNA polymerase, although α Binds the regulatory sequence
different sigma factors may be involved in β Catalyzes phosphodiester bond formation

initiation of different genes. RNA β’ Binds to DNA template
polymerase from E.coli is a multi-subunit
Recognizes promoter and initiates RNA
σ
enzyme (α2ββ’σ). synthesis
General steps of transcription in prokaryotes

Identification of promoter elements, formation of initiation complex and chain initiation
o Prokaryotic transcription begins with binding of RNAP to the promoter region of a gene.
o Special sequences termed promoters recruit the RNAP to the transcription start site. Promoters
are usually located in front (upstream) of the gene to be transcribed. Many prokaryotic
promoters have two common conserved regions, located about 10 nucleotides and 30
nucleotides upstream (-10 and -35 sequences) from the transcription start site.
o Prokaryotic transcription does not require transcription factors because sigma σ subunits of
RNAP can recognize promoter sites. E.coli contains multiple σ factors. σ70 recognizes the
consensus sequence in most housekeeping genes, σ32 recognizes promoters of heat shock gene
and σ54 is required for the expression of genes that assimilate nitrogen.
o RNA polymerase (holoenzyme consisting of α2ββ’σ) locates the specific promoter regions and
binds to DNA.
o RNAP has an intrinsic ‘helicase’ activity that opens the DNA helix to form a pre-initiation complex.
o RNA synthesis does not require primer since RNAP can perform de novo synthesis. RNAP
catalyzes the coupling of the first ribonucleotide to the incoming second ribonucleoside
triphosphate to form a dinucleotide releasing pyrophosphate.
Chain elongation
o Elongation of RNA molecule occurs in 5’ to 3’ direction, antiparallel to its template.
o RNAP catalyzes successive addition of complementary ribonucleotides to the 3’ OH terminus of
the nascent RNA molecule. RNAP uses four different types of ribonucleoside triphosphates (ATP,
GTP, CTP, UTP) as substrates. Elongation takes place at transcriptional bubbles.
o Since RNAP lacks 3’ – 5’ exonuclease activity and thus do not have proofreading function.
Termination
o The transcribed region of DNA template contains stop signals that initiates termination of RNA
synthesis. Transcription termination can occur in either of two models; rho dependent
termination and rho independent termination.
1. Rho dependent termination

 It requires a protein factor called rho (ρ) which
recognizes the termination signal and has an ATP
dependent helicase activity that displaces the RNA
polymerase from template resulting in termination of
RNA synthesis.
2. Rho independent termination
 Rho-independent termination brought
about by the formation of a secondary
structure (hair-pin loop) in the newly
synthesized RNA, which removes the
RNA polymerase from DNA template, resulting in the release of the transcript. This
hairpin loop structure which is followed by several U residues leads to termination of
transcription. RNA-DNA hybrid helix produced after the hairpin is unstable due to rich
A:U pairs and it leads to dissociation of nascent RNA and RNAP from the DNA template.
 Rifampicin inhibits the β subunit of RNA polymerase and
thus interferes with transcription in prokaryotes.
 Actinomycin D binds tightly and specifically to dsDNA and
prevents it from serving as effective template for
transcription process.
Transcription in eukaryotes
 RNAs in eukaryotic cells are synthesized by three types of
DNA dependent RNA polymerases called RNAP I, II and III.
Each polymerase recognizes a different type of promoter.
 RNA polymerase I is located in nucleoli, where it
transcribes genes for 18s, 5.8s and 28s ribosomal RNA.
 RNAP II is located in the nucleoplasm and synthesizes precursors of mRNA and several small RNA
molecules such as U1 snRNA of splicing apparatus.
 RNAP III is located in the nucleoplasm and synthesizes 5s rRNA and all transfer RNA molecules.
General steps of transcription in eukaryotes (RNAP II)

 Chromosomal relaxation
o The binding of trans-activator proteins and co-regulator
proteins can control the composition and/or covalent
modification status of the DNA and histones within the
nucleosomes in and around the promoter
and enhancer thereby increasing the
availability of all other components
required for initiation complex.
o Specific transcription factors bind to
enhancer DNA and recruit chromatin
remodeling and modifying co-regulatory
proteins.
o ATP dependent chromatin remodelers and
chromatin-modifying co-regulators release
histones from DNA leading to opening of the promoter region
and facilitate initiation complex formation and transcription.
 Formation of basal transcription complex and chain initiation
o Since RNA polymerase II cannot recognize the specific
transcription initiation site or promoters, several transcriptional
factors are required to interact with enhancers and other cis-
acting sequence elements forming transcription initiation
complex, recruiting RNA polymerase to the promoter site.
o TATA box binding proteins (TFIID) and many other transcription
factor proteins (TFIIA, B, E, F, H) assemble on the promoter in
specific order by protein-DNA and protein-protein interactions.
o RNA polymerase with the help of transcription factor TFIIB
protein locates and specifically binds the promoter. TFIIH has
ATP dependent helicase activity unwinding the DNA in and
around the transcription start site.
o TFIIH phosphorylates and activates RNA polymerase enzyme.
The first and second nucleotides are attached to the initiation
site by complementary base pairing and RNAP catalyzes the
formation of the first phosphodiester bond.
 Chain elongation
o RNA chain is synthesized in 5’ to 3’ direction. Successive nucleotides complementary to the
template are added to the 3’ OH terminus of the nascent RNA molecule by the action of RNAP.
o Four different types of ribinucleoside triphosphates (ATP, GTP, CTP, UTP) are used as substrates.
o Elongation takes place at the transcription bubbles (region containing RNAP, DNA and nascent
RNA) that move along the DNA template.
o RNAP has no proofreading activity.
 Chain termination
o Signals for termination of transcription by RNAP II are poorly understood. Termination signals
and processes in eukaryotes are more complex than prokaryotes. Termination signals exist far
downstream of the coding sequence of eukaryotic genes. Formation of phosphodiester bond is
ceased when the termination signal is reached. RNA–DNA hybrid dissociates and melted region
of DNA strand rewinds. Then RNAP is released from the template DNA.
 Alpha amanitin toxin from Amanitin phalloides inhibits the eukaryotic RNA polymerase II thereby
inhibiting mRNA and ultimately protein synthesis.
RNA processing or post-transcriptional modification

A newly synthesized RNA molecule is called primary RNA transcript or heterogenous nuclear RNA
(hnRNA) or nascent RNA. Primary RNA transcript is needed to transform into mature RNA or
functional RNA. This process is called RNA processing and it occurs in the nucleus.
RNA processing or post-transcriptional modification can include —
o Removal of extra nucleotides
o Addition of nucleotides e.g., addition of polyadenylate residues
o Splicing
o Separation of different RNA sequences by action of specific nucleases
o Base modification
Formation of mature messenger RNA (mRNA)

 Primary RNA transcript or pre-mRNA or hnRNA is modified by three different mechanisms to form
mature mRNA. These include capping, tailing

and splicing.
 Capping
o Addition of 7 methyl guanosine triphosphate

residue (7mGTP) to the 5’ end of hnRNA.
 Polyadenylation or formation of 3’ poly A tail (tailing)
o About 20 nucleotides downstream from AAUAA recognition

sequence is cleaved from hnRNA.
o Poly A polymerase adds as many as 200 adenine nucleotide
residues (poly A tail) to the 3’ end of mRNA. ATP is used as
nucleotide precursor for poly A tail formation. Poly A polymerase
does not require template for its polymerization action.
 Splicing
o The primary RNA transcript contains coding

regions (exons) interspersed with non-coding
regions (introns). The intron sequences are
removed and the remaining exons are ligated
through the process of RNA splicing. This
process is carried out by a large RNA protein
complex called the spliceosome.
o Spliceosome
 Spliceosomes consist of the primary
transcript, five snRNAs (U1, U2, U4, U5 and
U6) and more than 60 proteins.
 U1, U2, U4, U5 and U6 snRNAs form complex
with protein subunits to form snRNPs (small
nuclear ribonucleoproteins). These snRNPs
form the core of spliceosome and catalyze
the pre-mRNA splicing.
o Consensus sequences for RNA splicing in higher
eukaryotes are GU and AG dinucleotides at
either ends of the intron sequence.
o The splicing reaction starts with a cut at the junction of the 5’ exon and intron. The free 5’
terminal then forms a loop or lariat structure that is linked to the 3’ splice site. A second cut is
made at the junction of the intron with the 3’ exon. The lariat structure containing the intron is
then released and hydrolyzed. The 5’ and 3’ exons are ligated to form a continuous sequence.
The autoimmune disease systemic lupus erythematosus (SLE) is characterized by the body's
production of antibodies against its own snRNPs.
Mutations at splice sites can lead to improper splicing and the production of aberrant proteins. For
example, mutations that cause the incorrect splicing of β-globin mRNA are responsible for some
cases of β-thalassemia in which the production of the β-globin protein is defective.
Alternative mRNA splicing yields multiple polypeptides/proteins from a single gene.
Formation of mature transfer RNA (tRNA)

 Both prokaryotic and eukaryotic tRNAs are transcribed as large precursors.
 Cleavage of extra sequences at both 5’ and 3’ ends
o The 5' end of the primary
transcript is removed by
ribonuclease P (RNaseP)
which is a ribozyme or
catalytic RNA associated with
proteins. The extra nucleotides
at the 3' end are removed by
an exonuclease, ribonuclease
D (RNase D).
 Modification of nucleotides
o Nucleotides of tRNA are the most highly modified of all nucleic acids.
o These include methylations, deaminations, thiolations and reductions. Pseudouridine,
however, is formed when uracil is removed and reattached to ribose through a C-5 linkage.
 3’ end addition
o The trinucleotide sequence CCA is added to the 3' end by tRNA nucleotidyltransferase.
This sequence is essential for tRNA to accept amino acid.
 Removal of an intron at the anticodon loop
o The intron is removed by an endonuclease and a ligase (similar to the DNA ligase) that
reseals the tRNA chain.
Formation of ribosomal RNA (rRNA)

 The primary transcript of eukaryotic RNA
polymerase I is the 45S pre-rRNA, which contains
sequences for 28S, 18S, and 5.8S rRNAs. The
processing includes cleavage reactions mediated
by endo- or exo-ribonucleases and nucleoside
modification reactions.
 Processing of the primary rRNA transcript

in the nucleolus involves the following —
o Modifications of nucleoside bases (e.g.,
pseudouridine formation and base
methylation)
o Cleavage, which is performed by large
pre-ribosome complexes that contain
small nucleolar ribonucleoproteins
(snoRNPs), protein components of
ribosomes, and a combination of endo- and exonucleases
 The 5S rRNA is transcribed separately by RNA polymerase III in the nucleoplasm.
 rRNA containing the group I intron undergo self-splicing in the absence of protein. This rRNA is thus a
ribozyme.
 5S, 5.8S and 28S rRNAs assemble with variety of proteins and form 60S ribosomal subunit.
 18S rRNA together with many proteins forms 40S ribosomal subunit.
Structure and functions of RNA

RNA is a single stranded structure in the form of alpha helix. It is a polymer of ribonucleotides (ribose
sugar, N-base and phosphate) joined by 3’ – 5’ phosphodiester bond.
Nitrogenous bases found in RNA are purine (adenine and guanine) and pyrimidine (cytosine and
uracil).
In a given RNA molecule, number of adenine and uracil, number of guanine and cytosine are not
necessarily equal; thus it does not obey Chargaff’s rule.
Different types of RNA
o Messenger RNA (mRNA)
o Transfer RNA (tRNA)
o Ribosomal RNA (rRNA)
o Small RNAs
 Small nuclear RNA (snRNA)
 Small nucleolar RNA (snoRNA)
 Micro RNA (miRNA)
 Silencing RNA (siRNA)
mRNA, tRNA and rRNA involve in protein synthesis while small RNAs involve in RNA splicing and
regulation of gene expression by altering mRNA function.
RNA forms the genetic material in some viruses e.g., retro viruses.
Messenger RNA (mRNA)

It constitutes about 5% of total eukaryotic cellular RNAs.
In eukaryotic mRNA, 5’ end is capped with 7 methyl gluanosine
triphosphate and 3’ terminal has polyadenylate sequence of about 20 to
250 nucleotides in length known as poly A tail.
There are loop structures at 5’
and 3’ untranslated regions (3’
UTR and 5’ UTR). Coding region
contains information for
protein synthesis.
Functional significance of 5’ cap
o Protects mRNA from attack of 5’ exonucleases.
o Involve in formation of ribonucleoprotein complex necessary for the splicing reaction.
o May involve in mRNA transport.
o Enhance the translation of mRNA by eukaryotic protein synthesizing systems.
Functional significance of 3’ poly A tail
o Protects mRNA from attack of 3’ exonucleases hence it determines the life span of mRNA.
Functions of mRNA
o It conveys genetic information from the nucleus to
protein synthesizing machine (ribosomes) in
cytoplasm.
o It serves as template for translation process.
Transfer RNA (tRNA)

It constitutes about 15% of total eukaryotic RNA.
There are at least 20 species of tRNA molecules, at least one
corresponding to each of 20 different amino acids.
Different species of tRNA differ in primary structure but all
have a common secondary structure of clover leaf shape and
tertiary structure of L shape.
All tRNA molecules have five main arms —
1. Acceptor arm – 3’ end of tRNA molecule which is
attached by CCA residues and serves as point of
attachment of an amino acid.
2. Anti-codon arm – recognizes the letter codon on mRNA (has nucleotide sequence
complementary to codon of mRNA) and is responsible for the specificity of tRNA.
3. ‘D’ arm (contains dihydrouracil) – proper recognition site by amino acyl tRNA synthetase.
4. T Ψ C arm (contains pseudouridine) – involve in binding of tRNA to ribosomes.
5. Extra arm – most variable feature of tRNA and provides basis for tRNA classification.
Functions of tRNA
 It carries specific amino acid to the site of protein synthesis.
 It serves as an adaptor between the codon of mRNA and their respective amino acid.
Ribosomal RNA (rRNA)

It constitutes about 80% of total RNA in eukaryotic cells.
There are three species of rRNA in prokaryotes (23S, 16S and 5S)
and four in eukaryotes (18S, 28S, 5.8S and 5S). They are found in
association with different proteins as components of ribosomes.
Functions of rRNA
o It serves as site for protein synthesis.
o 28S rRNA of 60S ribosomal subunit has peptidyl
transferase activity and thus it is a ribozyme.
Small RNAs
Small nuclear RNAs (snRNAs) involve in mRNA processing
(splicing) and are constituents of spliceosome.
Small nucleolar RNAs (snoRNAs) involve in the cleavage of long
pre-rRNA into its functional subunits (18S, 28S and 5.8S rRNA).
Micro RNAs (miRNAs) are 22 – 24
nucleotides in length and involve in
regulation of gene expression. They down
regulate gene expression by attaching
themselves to mRNAs and preventing
them from being translated into proteins.
Small interfering RNA (siRNA) is a class of
small RNA molecules that binds to mRNA
and facilitates its degradation.
Comparison between replication and transcription
Replication Transcription
Occur in S phase of cell cycle. Occur throughout interphase of cell cycle.
Both strands of parental DNA duplex serve as Anti-sense strand (opposite to coding strand or
template. sense strand) serves as template.
The whole genome sequence is copied completely by Only the DNA segment complementary to the
complementary base pairing rule. coding sequence is copied by complementary base
pairing rule.
Recognition of replication origin is unknown. Recognition of transcription start site by RNAP is
facilitated by assembly of transcription initiation
complex (c0mposed of various transcription factors)
on promoter region.
Five types of DNAP for eukaryotic DNA replication Three types of RNAP for eukaryotic transcription
DNAP α – primer synthesis, initiation of replication RNAP I – synthesis of all rRNA except 5S rRNA
DNAP δ – lagging strand synthesis RNAP II – synthesis of mRNA and snRNA
DNAP ε – leading strand synthesis, proofreading, repair RNAP III – synthesis of all tRNAs and 5S rRNA
DNAP β – proofreading and repair
DNAP γ – mitochondrial DNA replication
DNAP cannot perform de novo synthesis of DNA chain, RNAP can perform de novo synthesis of RNA chain,
so RNA primer is needed for initiation of replication. so primer is not needed for initiation of
transcription.
DNA polymerization is from 5’ to 3’ direction. RNA polymerization is from 5’ to 3’ direction.
Precursors for DNA synthesis – four different Precursors for RNA synthesis – four different
deoxyribonucleoside triphosphate precursors (dATP, ribonucleoside triphosphate precursors (ATP, GTP,
dGTP, dCTP, dTTP) CTP, UTP)
DNAP has 3’ to 5’ exonuclease activity and thus can RNAP has no exonuclease activity and thus cannot
carry out proofreading. carry out proofreading.
Post-replication modification is very few as compared Post-transcription modification is complex and
to transcription. includes splicing, cleavage of nucleotides, capping,
polyadenylation, base modification, etc.
Newly replicated DNA is assembled into nucleosomes Newly synthesized RNA molecule called hnRNA
behind the advancing replication forks and organized undergoes various steps of RNA processing to form
into chromatin structure. functional RNAs; mRNA, tRNA and rRNA.
Comparison between prokaryotic and eukaryotic transcription
Steps or components Prokaryotes Eukaryotes
Site of transcription Occur in the cytoplasm. Occur in the nucleus.

Polycistronic (genes for proteins Always monocistronic (one gene
performing the specific process segment codes only for one
Gene regions are clustered in one segment) polypeptide information)
Continuous coding regions Exons (coding regions) intervened
No introns by non-coding regions (introns)
Single form of RNAP Three classes of RNA polymerase
Core enzyme – α2ββ’ associated RNAP I – all rRNAs except 5S rRNA
RNA polymerase
with different sigma subunits RNAP II – mRNA, snRNA
RNAP III – tRNA, 5S rRNA
Promoters  – 10 bp (TATA) and Promoters  – 25 bp (TATA box)
–35 bp sequences and – 70 bp (CAAT box)
Different sigma factors required Transcription factors can
Initiation of transcription
to recognize specific promoter recognize promoter (e.g., TFIID to
sequence of a particular gene TATA box) and facilitate binding of
RNAP to promoter.
RNA polymerase synthesizes RNA from 5’ to 3’ direction by
RNA synthesis complementary base pairing to anti-sense strand or template strand (3’
to 5’ direction) of DNA.
Four different ribonucleoside triphosphates precursors
Precursors for RNA synthesis
ATP, GTP, CTP, UTP
Rho independent (hair-pin loop Not well understood.
formation followed by several U
Termination of transcription
residues) or
Rho dependent termination
Post transcriptional modification is more complex in eukaryotes than in
Post-transcriptional modification
prokaryotes.
None 5’ capping (7-methyl GTP)
Transcribed mRNA undergoes 3’ poly A tail (3’ polyadenylation)
translation process before being Splicing (removal of introns and
Post-transcriptional processing
synthesized completely. ligation of exons)
of hnRNA (pre-mRNA)
Only mature mRNA after complete
RNA processing is transported to
cytoplasm for translation process.
Three species of rRNA (23S, 16S Four species of rRNA (18S, 28S,
and 5S) 5.8S and 5S)
Formed into 30S ribosomal Formed into 40S ribosomal subunit
Ribosomal RNA subunit (16S rRNA + proteins) and (18S rRNA + proteins) and 60S
50S ribosomal subunit (23S, 5S ribosomal subunit (28S, 5.8S, 5S
rRNAs + proteins) which then rRNAs + proteins) which then
associate to form 70S ribosomes. associate to form 80S ribosomes.
Differences between RNA and DNA

1. RNA is a single stranded alpha helix structure whereas DNA is a double stranded helical structure.
2. RNA is a polymer of ribonucleotides and DNA is a polymer of deoxyribonucleotides.
3. In RNA, sugar moiety is ribose rather than 2’ deoxyribose in DNA.
4. As pyrimidine component, uracil is used in RNA instead of thymine in DNA.
5. Purine and pyrimidine content are not necessarily equal in single stranded RNA. Unlike DNA, RNA
does not obey Chargaff’s rule.
6. RNA can be hydrolyzed by alkali to 2’-3’ cyclic diester of mononucleotides but DNA cannot be
hydrolyzed by alkali because of the absence of 2’ OH group.
7. Different types of RNA involve in translation process (mRNA, tRNA, rRNA) and some involve in RNA
splicing (snRNA, snoRNA), regulation of gene expression (miRNA, siRNA). DNA serves as repository
of genetic information in most organisms and serves as template for replication and transcription
processes.
Genetic code
Genetic code is the relationship between
sequence of DNA or mRNA transcripts and the
amino acid sequence of protein.
Nucleotides on mRNA (A, G, C, U) are organized
into three-letter code words called codons. Each
group of three consecutive nucleotides in mRNA
is called codon. Collection of codon is called
genetic code.
Understanding of genetic code is the foundation
for explanation of translation process, mutation
and genetic errors of diseases.
There are 64 possible combinations to make codons. Out of 64 codons, 61 represent 20 different
amino acids which are found in proteins.
Three codons (UAA, UAG, UGA) do not code for any amino acids and they are termed non-sense
codons or chain termination codons. These codons are used as termination signals during protein
synthesis.
UGA codon has an additional function, coding for selenocysteine in at least 25 human proteins.
One codon AUG (code for methionine) is always used as initiator signal in protein synthesis in
eukaryotes and is known as chain initiation codon.
Third nucleotide in a codon is usually wobble, i.e., change in third nucleotide position of a codon is
least sensitive to change the amino acid sequence than first and second nucleotide sequence of
codon in translation.
Characteristics of genetic code
o Universal
 All prokaryotes and eukaryotes use the same genetic code to specify the same amino acid
except that minor differences are found in some bacteria and mitochondria.
o Degeneracy
 Since 61 codons code for 20 different kinds of amino acids, multiple codons must encode the
same amino acid. In other words, some amino acids can be represented by more than one
codon.
 E.g., GGG, GGA, GGU, GGC are codons for glycine.
 Methionine and tryptophan are encoded by only one codon; AUG and UGG respectively.
o Unambiguity
 One codon specifies only one amino acid.
o Non-punctuation (comma-less)
 The genetic message is read sequentially and continuously from a fixed starting point,
without interrupting or pause until the stop codon is reached.
o Non-overlapping
 The genetic code is read without repeating the previously read bases.
Genomic stability
Genomic stability is important for health of an individual as well as for maintenance of the species.
Since DNA is the repository of genetic information, permanent DNA damage can lead to deleterious
consequences.
Genomic instability can be caused by damage to DNA or chromosome by chemical reactions of
metabolism, drugs, chemicals, physical agents (UV rays, ionizing radiation) and biologic agents like
viruses.
Consequences of genomic instability
o Genomic instability due to mutation in germ cells gives rise to inherited genetic diseases such
as sickle cell anemia (HbS), thalassaemia, hemophilia, etc.
o Genomic instability due to chromosomal abnormalities gives rise to congenital disorders like
Down syndrome though it is not inherited.
o Genomic instability and change in genetic material in somatic cells can cause deviations in cell
growth and metabolism and can lead to cancerous changes.
Maintenance of genomic stability

o DNA, chromosome, and chromosome segregation integrity is continuously monitored
throughout the cell cycle.
o High fidelity in DNA replication is due to proofreading mechanism carried out by 3’ – 5’
exonuclease activity of DNAP that removes mispaired nucleotide from 3’ end of growing
chain and corrects initial mispairings.
o During cell cycle, DNA damage is sensed by DNA damaging check points which then activate
tumor suppressor protein p53 and signaling pathways that mediate cell cycle arrest and DNA
repair.
o If DNA damage is detected, or if the genome is incompletely replicated, or if normal
chromosome segregation machinery is incomplete, cells will not progress through the phases
of the cycle until the DNA damage is repaired by specific repair mechanisms.
o In some cases, if the damage cannot be repaired, such cells undergo programmed cell death
(apoptosis) which is mediated by tumor suppressor protein p53 activated pathways.
Genetic disorders
A genetic disorder is a disease caused by abnormalities in genetic material (genome).
There are four different types of genetic disorders —
o Single gene disorder
o Multifactorial or polygenic disorder
o Chromosomal disorder
o Mitochondrial gene disorder
Single gene disorder (Mendelian or monogenic disorder)
o Changes or mutations that occur in the DNA sequence of a particular gene
o Protein product of the affected gene may no longer carry out its normal function or there
may be reduced or absence of protein production.
o There are more than 6,000 known single gene disorders (1 in out of every 200 births) e.g.,
sickle cell anemia (HbS), Marfan syndrome, hereditary hemochromatosis, hemophilia, G6PD
deficiency, etc.
o Single gene disorders are inherited in recognizable patterns – autosomal dominant or
autosomal recessive or X linked.
Multifactorial or polygenic disorders
o This type is caused by a combination of environmental factors and mutations in multiple gene
e.g., different genes that influence breast cancer susceptibility have been found on
chromosome 6, 11, 13, 14, 15, 17 and 22.
o Some of the most common chronic diseases are multifactorial disorder e.g., hypertension,
Alzheimer’s disease, arthritis, diabetes mellitus, cancer, obesity, etc.
o Complicated nature of polygenic disorders makes it more difficult to analyze than single gene
or chromosomal disorders.
Chromosomal disorders
o Abnormalities in chromosomal structure such as missing, extra copies or gross breaks and
rejoining (translocations) can result in disease.
o Trisomy – disorder in which an individual has three copies of a particular chromosome e.g.,
trisomy 21 or Down syndrome (~ 1 in 800 live births). Trisomies of three different autosomes
(13, 18 and 21) are compatible with life.
o Monosomy – disorder in which there is only one copy of specific chromosome usually cause
by non-disjunction of chromosome during meiosis. All autosomal monosomies are lethal.
o Chromosomal translocation - structural abnormalities of chromosome arising from improper
chromosomal translocation e.g., translocation of long arms of chromosome 9 and 22 in
Philadelphia chromosome alters the activity of encoded protein in hemopoietic cells resulting
in chronic myeloid leukemia (CML).
Mitochondrial gene disorder
o Relatively rare type of genetic disorder caused by mutations in mitochondrial DNA e.g.,
MELAS, LHON, MERRF, etc.
o Inheritance of mitochondrial genetic disorder is strictly maternal.
Gene mutation
 Any permanent heritable change in the DNA base sequence of the gene is called mutation.
 Mutation affects on the pattern of gene expression or on the function of proteins encoded leading to
change in enzyme activity or phenotypes or properties of the cell.
 When mutations occur in germ cells, they can be transmitted to future generation as inherited
disease.
 Mutations in somatic cells are not transmitted to the progeny but are important in causation of
cancer and some congenital malformation.
 Mutations occur in coding regions of a gene can lead to altered amino acid sequence of the encoded
protein which could affect the function of protein.
 Splice site mutation can lead to improper splicing and production of aberrant protein, e.g., some
cases of β thalassemia.
 Mutations in promoter or other regulatory regions of a gene can lead to decrease or increase in rate
of protein synthesis, e.g., some forms of β thalassemia.
 Types of mutations – point mutation, frame shift mutation, trinucleotide repeat mutation
Point mutation
Single base substitution mutation
Single-base changes (point mutations) may be transitions or transversions.
Transitional mutation – change of purine to another
purine or change of pyrimidine to another pyrimidine
Transversional mutation – change of purine to
either of two pyrimidines or change of pyrimidine to
either of two purines
Point mutation can be classified according to the effect on translated protein— silent, missense or
nonsense mutations.
Silent mutation
o There may be no detectable effect if the changed base falls on 3rd nucleotide of the codon
which specifies the same amino acid (due to degeneracy of the codon).
o The amino acid sequence of the protein is not altered and will perform normal function.
o E.g., codon for valine at 67th position of β chain of hemoglobin is not identical in all persons
with normal functional β chain of hemoglobin.
Missense mutation
o A missense effect will
occur when a different
amino acid is incorporated
at the corresponding site
in the protein molecule.
Depending upon the
location of mistaken
amino acid in the specific
protein, the effect might
be acceptable, partially
acceptable, or unacceptable to the function of that protein molecule.
o Acceptable missense mutation
 Resulting protein does not change important functions and will be functionally similar to
the normal protein.
 E.g., in Hb Hikari, lysine at 61st position in the β chain is substituted by asparagine but
such change does not alter the normal function of β chain.
o Partially acceptable mutation

 Resulting proteins still retain some normal functions.
 E.g., in Hb S, glutamate at 6th position in the β chain is substituted by valine and this
substitution causes sickle cell anemia. This change is considered as partially acceptable
because even though HbS is abnormal, it still can bind and release oxygen.
o Unacceptable mutation
 Resulting protein cannot perform its functions at all and the individual cannot survive.
 E.g., in HbM, histidine at 58th position of α chain is substituted by tyrosine resulting in
oxidation of ferrous iron (Fe2+) in heme of Hb to ferric (Fe3+), producing methemoglobin
which cannot transport oxygen to the tissue.
Nonsense mutation
o If the substitution of nucleotide results in the formation of termination codon e.g., AAA 
UAA, there will be premature termination of peptide chain. Prematurely terminated protein
molecule or peptide fragment will not function properly e.g., some cases of β thalassemia.
Frameshift mutation
Insertion or deletion of one or
more nucleotides in coding
strand of a gene results in
altered in reading frame of the
mRNA sequence.
This leads to garbled translation
of the mRNA distal to point of
deletion or insertion, e.g., some
proteins produced in cancer
cells by such mutation leads to
abnormal functions. It can also
result in prematurely terminated
or over-translated peptide chain.
Trinucleotide repeat mutation

The human genome contains tandem repeats of trinucleotides. Normally they occur in groups of 5–35
repeats. When their number exceeds a certain threshold and they occur in a gene or close to it, they
cause diseases.
Huntington disease is caused by expansion of the trinucleotide repeat CAG in the coding sequence of
a brain-expressed gene (huntingtin). In the normal gene, CAG repeats encodes for a polyglutamine
tract in the protein huntingtin. Increased

CAG repeats lead to glutamine-expanded
huntingtin proteins which form
abnormal complexes with other proteins
ultimately leading to neuronal cell death.
The unstable (CGG)n repeat sequence is associated with the fragile X syndrome.
Protein synthesis (translation)

It is a complex process by which the information contained in nucleotide sequence of mRNA directs
the ordered polymerization of specific amino acids into protein.
Translation occurs in ribosomes found in cytoplasm (free ribosomes) or rough endoplasmic reticulum
(membrane associated ribosomes).
mRNA is translated from 5’ to 3’ end and peptide chain is synthesized from amino terminus to
carboxyl terminus.
Translation is generally divided into five steps —
1. Activation of amino acids and formation of amino-acyl tRNA
2. Initiation
3. Elongation
4. Termination
5. Post-translational processing and protein folding
This process involves mRNA, tRNA, rRNA, initiation factors,
elongation factors, releasing factors, amino acids, amino acyl
tRNA synthetase, GTP and ATP.
Protein synthesis in prokaryotes

1. Activation of amino acid and formation of amino acyl tRNA
(charging of tRNA)
o Linking of an amino acid to its corresponding tRNA is
catalyzed by amino acyl tRNA synthetase and it requires
ATP.
Amino acid + tRNA + ATP  Amino acyl tRNA + AMP + PPi
2. Initiation
o 70S ribosome dissociates into 30S and 50S ribosomal
subunits.
o mRNA binds to 30S ribosomal subunit with the aid of

initiation factors. The start codon 5’ AUG of mRNA binds to
its correct position on ribosomes guided by the Shine-
Dalgano sequence of mRNA which is recognized by 16S
rRNA.
o The IF2-GTP initiator tRNA (formyl methionyl tRNA) binds
with mRNA and forms 30S initiation complex.
o 50S ribosomal subunit then binds to form 70S initiation
complex and this binding requires hydrolysis of GTP bound
to IF2.
o At the end of initiation phase, A site (amino acyl tRNA site),
P site (peptidyl tRNA site) and E site (exit site) appear on
ribosomes and formyl methionyl tRNA is occupied at P site.
3. Elongation
o It is a cyclic process. Repeated cycles of amino acyl tRNA
binding to A site, peptide bond formation and translocation
of ribosomes occur.
o Binding of amino acyl tRNA to A site
 According to proper codon recognition, corresponding
amino acyl tRNA binds to codon of mRNA on A site with
the aid of EF and GTP.
o Peptide bond formation
 Peptide bond is formed between amino acid on tRNA in
A site and P site catalyzed by peptidyl transferase action
of 23S rRNA of 50S ribosomal subunit. Growing peptide
chain is attached to tRNA in A site.
o Translocation
 Ribosome moves over mRNA by one codon which is
mediated by EF2 (translocase) with hydrolysis of GTP.
 Peptidyl tRNA now occupies P site and leaving A site
open for another cycle of elongation. Uncharged tRNA
leaves ribosome from E site.
o The repeated cycles of elongation occur until stop codon
appears in A site.
4. Termination
o Once a termination codon (UAA or UAG or UGA) occupies the
ribosomal A site, releasing factors and peptidyl transferase
activity of 23S rRNA contribute to
 Hydrolysis of the terminal peptidyl tRNA bond
releasing free polypeptide from tRNA and ribosomes
 Dissociation of tRNA, mRNA from ribosome
General steps in protein synthesis of eukaryotes

1. Activation of amino acid and amino acyl tRNA synthesis
o Specific amino acid is attached to corresponding tRNA
catalyzed by amino acyl tRNA synthetase and this process
requires ATP.
2. Initiation
o Dissociation of 80S ribosome into 40S and 60S ribosomal
subunits with the help of eIF 1A and eIF3 which bind to 40S
subunit to prevent re-association
o Binding of ternary complex (methionyl tRNA, eIF2 and GTP) to
40S ribosomal subunit to form 43S pre-initiation complex
o Binding of mRNA to the 43S pre-initiation complex with the
help of its 5’ cap to form 48S initiation complex which requires
ATP hydrolysis
o Binding of 60s ribosomal subunit to 48S initiation complex to
form 80S initiation complex (by hydrolyzing GTP bound to eIF2
by eIF5)
o Release and recycling of eIFs
o Initiation results in
 Appearance of 3 sites on ribosome; A site (amino acyl
tRNA site), P site (peptidyl tRNA site) and E site (exit

site) at the end of initiation phase.

 Binding of initiator tRNA (methionyl
tRNA) to start codon (AUG) of mRNA

in P site
3. Elongation
o This step involves several steps and requires
proteins called elongation factors (EFs).
1. Binding of amino acyl tRNA to A site
 According to proper codon
recognition, specific amino acyl tRNA
binds to the corresponding codon of
mRNA on A site with the aid of eEF1A and GTP.
2. Peptide bond formation
 Peptide bond is formed between the amino group of
incoming amino acyl tRNA on A site and carboxyl

group of methionyl tRNA (pre-existing amino acyl
tRNA) on P site.
 It is catalyzed by peptidyl transferase activity of 28S
rRNA of 60S ribosomal subunit (ribozyme). Growing

peptide chain then attaches to tRNA on A site and
tRNA on P site becomes deacylated or uncharged.
3. Translocation
 Ribosome moves over mRNA by one codon which is
mediated by eEF2 (translocase) with hydrolysis of

GTP.
 Peptidyl tRNA now occupies P site and leaving A site
open for another cycle of elongation. Uncharged

tRNA leaves ribosome from E site.
o The repeated cycles of elongation occur until stop codon
appears in A site.
o Generally for each peptide bond formation, 4 high energy
phosphate bonds are used (2 ATPs in amino acyl tRNA
synthesis, 1 GTP in binding of amino acyl tRNA to A site and 1
GTP in translocation of mRNA).
Protein misfolding
4. Termination  Misfolded protein is rapidly degraded
o Termination occurs when a termination codon of by ubiquitin dependent proteasome

system.
mRNA (UAA or UAG or UGA) appears on A site.
 Unfolded protein forms aggregate
o There is no tRNA with an anticodon recognizing such
with other normal proteins and such
a termination codon. Releasing factor recognizes the protein aggregate is resistant to heat
stop codons. and proteolytic enzymes.
 Misfolded aggregate form of proteins
o With the help of GTP, peptidyl transferase activity of
(prions protein) causes severe cellular
28S rRNA hydrolyzes the bond between the peptide
damage and death.
chain and tRNA occupying P site.
Prion disease
o Finally tRNA, peptide chain and mRNA are released
 Proteinaceous infective particles
from ribosome.  Stanley Prusiner first described prion
5. Post-translational modification proteins (PrP) in 1982.
o ER and golgi are essential organelles for modification.  PrP is a normal protein (253 amino
acids), found in leucocytes and nerve
o Post-translational modification reactions include —
cells.
1. Removal of N-terminal amino acid residues by  PrP can undergo conformation change
specific aminopeptidase (abnormal tertiary structure) and
2. Hydroxylation and oxidation of specific amino acids becomes resistant to heat and
proteolytic enzymes. This abnormal
for cross-linkage e.g., lysine, proline in collagen
protein is called PrPsc (Prion protein
3. Covalent modification of amino acids e.g.,
scrapie).
glycosylation, phosphorylation, etc  Prion disease is seen when the gene
 Gamma carboxylation of glutamate residues e.g., is mutated or if abnormal PrPsc is
injected or ingested.
prothrombin
 PrPsc convert normal protein into
 Glycosylation (attachment of sugar residues to
abnormal varieties and producing
serine/threonine residues) e.g., hormone receptor, protein aggregates which grow larger
immunogloblins with time and leads to neurological
4. Proteolytic cleavage for conversion of prohormone defect.

 Slowly progressing and fatal disease
to mature hormone e.g., insulin
 Examples of prion disease
5. Protein folding
o Creutzfeld-Jacob disease (CJD)
 To become functionally active, newly synthesized o Neuro-degenerative disorders
protein must be folded into its unique three (Hungtinton disease, Alzheimer’s
disease)
dimension formation and complete functional
structure with the help of a class of proteins called chaperones. It is an ATP dependent
process.
 Protein folding for some proteins requires N-linked glycosylation in ER.
Protein targeting or signal hypothesis

 Intracellular proteins are synthesized on
free ribosomes in cytosol. Those proteins
that lack a translocation signal remain in
the cytoplasm where others contain signal
sequences that target them to specific
organelles (mitochondria, nucleus,
peroxisomes).
 Export proteins (secretory proteins),
membrane associated proteins and
lysosomal proteins are synthesized in
polyribosomes on rough endoplasmic
reticulum (RER). These proteins are
transferred from RER to golgi complex for
further modification and targeted to their
final location.
 Cellular fate of proteins is determined by
their signal peptide sequences.
 Proteins synthesized on membrane-bound polyribosomes contain signal peptides at their amino
terminals which mediate their attachment to ER membranes.
 Signal peptide is recognized by signal recognition particle (SRP) and SRP-ribosome-protein complex
travels to ER membrane where it binds to SRP receptor.
 SRP is then released and ribosome binds to the translocon (protein conducting channel) and signal
peptide inserts into the channel.
 Signal peptide is cleaved by signal peptidase in ER.
 The growing polypeptide is then fully translocated across the membrane driven by its ongoing
protein synthesis.
 The peptide is released into the lumen of ER where protein folding and modification occur.
Subsequently protein is transferred to the golgi complex where further changes and packaging into
vesicles for secretion. Then proteins are distributed intracellularly or secreted.
 Proteins in secretory vesicles are extruded into the extracellular space by exocytosis.
Regulation of rate of translation initiation

eIF-2 is one of two control points for protein synthesis initiation in eukaryotic cells.
o Free eIF2 is active but phosphorylated eIF2 is inactive.
o A subunit of eIF-2 (eIF-2 α) is phosphorylated by at least four different protein kinases that
are activated when a cell is under stress such as amino acid or glucose starvation, virus
infection, intracellular presence of large quantities of misfolded proteins.
o Phosphorylated eIF-2α binds tightly to and inactivates the GTP–GDP recycling protein eIF-2B.
Thus preventing formation of the 43S preinitiation complex and blocking protein synthesis.
The regulation of eIF-4E controls the rate of translation initiation.
o A cap-binding protein complex or eIF-4F complex (eIF-4E,
4G and 4A complex) is particularly important in
controlling the rate of protein translation.
o The 4F complex binds to the 5’ cap of mRNA through the
4E protein. 4E is responsible for recognition of the mRNA
cap structure, a rate-limiting step in translation.
o 4E binding protein (such as 4E-BP1, 4E-BP2 and 4E-BP3)
binds with high affinity to 4E and prevents 4E binding to
4G to form 4F complex thereby inhibiting translation
initiation.
o Insulin and mitogenic growth factors activate eIF-4E in two ways.
 By phosphorylation of 4G to facilitate its binding with other IFs and enhance binding
of 4E to the cap, thus enhancing the rate of initiation
 By phosphorylation of 4E-BPs resulting in its dissociation from 4E
Drugs and toxins that inhibit protein synthesis

Many chemicals that inhibit various aspects of protein synthesis have been identified. These
chemicals are powerful experimental tools and clinically useful drugs.
Antibiotics Actions
Streptomycin and Interferes with binding of f-Met-tRNA to ribosome, inhibits intiation and cause
other aminoglycosides misreading of mRNA.
Tetracycline Binds to 30S subunit and inhibits binding of aminoacyl tRNAs.
Chloramphenicol
Inhibits the peptidyl transferase activity of 23sRNA in 50S ribosomal subunit.
Clindamycin
Inhibits the peptidyl transferase activity of 28sRNA in 50S ribosomal subunit.
Cycloheximide
A useful laboratory tool for blocking protein synthesis in eukaryotic cells
Erythromycin and
Binds to 50S subunit and inhibits translocation.
macrolides
Causes premature chain termination by acting as an amino acyl tRNA analog.

Puromycin
Utilized mainly as an experimental tool for the investigation of protein synthesis.
Some toxins and viruses inhibit eukaryotic translation process. Viruses replicate by using host cell
processes, including those involved in protein synthesis. Some viral mRNAs are translated much more
efficiently than those of the host cell by competing limited translation factors.
Toxins Actions
Diphtheria and pseudomonas toxin Inhibits eEF2 by ADP-ribosylation.
Inactivates eukaryotic 28S ribosomal RNA by catalyzing the N-

Ricin (Isolated from castor beans)
glycolytic cleavage or removal of a single adenine.
Shiga toxin Binds to 60S subunit and prevents amino acyl-tRNAs from
(produced by Shigella dysenteriae) binding to ribosomes.
Inhibits host cell protein synthesis by preventing the association
of mRNA with the 40S ribosome via disrupting the formation and
Poliovirus and other picornaviruses function of the eIF-4F complex.
Also promotes dephosphorylation of 4E-BPs thereby decreasing
cap (4E)-dependent translation.
Mitochondrial protein synthesis

Mitochondria have their own translational machinery that
includes mitochondrial ribosomes, mRNA, and a set of tRNAs.
The genetic code of mitochondrial DNA differs from the standard
genetic code. The process of translation in mitochondria bears
several similarities to that in prokaryotes.
Peptidyl transferase in mitochondria is sensitive to chloramphenicol.
Comparison between prokaryotic and eukaryotic genetic code, mutation and translation
Steps or components Prokaryotes Eukaryotes
Start codon – AUG (codes for Met), Start codon – AUG (Met)
Genetic code sometimes GUG (codes for Val) Stop codons – UAG, UAA, UGA
Stop codons – UAG, UAA, UGA
Point mutations – silent, missense, nonsense
Frameshift mutation
Mutations Deletion
Splice site mutation
Trinucleotide repeat mutation
Translation of mRNA may occur Translation of mRNA occurs only after
Timing of gene
simultaneously with transcription (mRNA complete transcription and RNA
expression
synthesis). processing (mature mRNA synthesis).
Polycistronic mRNA (contains multiple Monocistronic mRNA (contains single
mRNA
protein coding regions) polypeptide coding region)
Three species of rRNA – 16S, 23S and 5S Four species of rRNA – 18S, 28S, 5.8S and
rRNAs 5S rRNAs
Ribosome
70S ribosome (consists of 30S and 50S 80S ribosome (consists of 40S and 60S
ribosomal subunits) ribosomal subunits)
Amino acyl tRNA Catalyzed by amino acyl tRNA synthetase and use two high energy phosphate bonds (2
synthesis ATPs) for charging of tRNA.
30S subunit binds to Shine-Dalgano 40S subunit associates with 5’ cap on
sequence on mRNA. mRNA.
Initiation
Initiator tRNA (fMet-tRNA) binds to P site. Initiator tRNA (Met-tRNA) binds to P site.
Requires hydrolysis of one GTP. Requires hydrolysis of one GTP.
Amino acyl tRNA binds to A site (1 GTP). Amino acyl tRNA binds to A site (1 GTP).
Peptide bond formation is catalyzed by Peptide bond formation is catalyzed by
peptidyl transferase activity of 23sRNA in peptidyl transferase activity of 28sRNA in
Elongation 50S subunit. 60S subunit.
Ribosome translocation (EF+ 1 GTP) Ribosome translocation (eEF-2 + 1 GTP)
Protein is synthesized from N terminus to Protein is synthesized from N terminus to
C terminus. C terminus.
Releasing factor (RF1 or RF2) recognizes Releasing factor (eRF1) recognizes stop
stop codon. codon.
Termination RF-GTP complex facilitates dissociation eRF-1-GTP complex facilitates dissociation
of the ribosomal complex. of the ribosomal complex.
Release of protein Release of protein
Regulation of gene expression

Genetic content of somatic cells of an
organism is the same but are not
expressed in all tissues. At any give
times, only a small number of genes in a
cell are expressed. This is called tissue-
specific gene expression.
Genes encoding proteins essential for very basic cellular functions are expressed in all cells all the
time, e.g., glycolysis enzymes. Other genes are expressed in particular cell types or at particular
stages of development (growth and differentiation).
This differential gene expression is achieved by regulating transcription and translation.
Moreover, organisms adapt to environmental changes by altering gene expression. Environmental
and metabolic state of the cell affect on rate of gene expression. Gene expression can be influenced
by developmental signals, hormones, heavy metals, growth factors and chemicals.
Regulation of gene expression occur at all levels of gene expression
1. Transcriptional regulation
2. Post-transcriptional regulation
3. Translational regulation
4. Post-translational regulation
Among all these levels, transcriptional regulation is the major control site of gene expression and can
be influenced by hormones, heavy metals, chemicals and drugs.
Regulation of gene expression in prokaryotes

 The major step in control of gene expression in prokaryotes is at transcriptional level;
o By activation and repression of transcription (positive and negative control gene expression)
o Multiple sigma factors in RNAP
 In prokaryotes, the genes involved in a specific process or
metabolic pathway are clustered in units called operons.

 An operon is a genetic unit in which transcription of several
genes is controlled by a single promoter. The operon is

composed of a regulatory gene, an operator site, a promoter site and a set of structural genes.
 The regulatory gene produces repressor protein that interacts with the operator site.
 Jacob and Monad described transcription regulation by E.coli lac operon model in 1961.
 Examples of operon systems in E.coli are lac operon, arabinose operon, tryptophan operon, histidine
operon.
Lac operon model

The lactose (lac) operon contains the structural genes that encode enzymes required for lactose
metabolism in E.coli, promoter and operator sites.
Structural gene cluster contains Z, Y and A. Z gene encodes for β galactosidase, Y gene for lactose
permease (lactose entry into the cell) and A gene for transacetylase.
A regulatory gene (lacI gene) encodes for lac repressor protein. It is located in a separate locus for
the lac operon and constitutively expresses repressor protein that binds to operator site.
When E. coli is exposed to both lactose and glucose or lactose is depleted in the medium, lac operon
is repressed. Lac operon is de-repressed only when lactose is present and glucose is depleted in the
medium.
Repression of lac operon
o Lac operon is
negatively controlled
by the repressor
proteins (products of
lacI gene).
o The lac repressors tightly bind to the operator site and interfere with binding of RNAP to
promoter and prevent transcription of structural genes.
De-repression of lac operon
o Lactose acts as an
inducer. Binding of
inducer to allosteric site
of repressor induces
conformational change
in the repressor and
decrease its binding affinity to operator site. With the release of repressors from operator
site, RNAP can now bind to operator locus and carry out transcription of the structural genes.
o Although the presence of inducer is necessary for transcription of lac operon, its presence
alone is not sufficient. The lac operon also requires a positive control element.
o The positive control element is composed of two parts – cAMP and CAP (catabolite gene
activator protein) that binds to cAMP to form cAMP-CAP complex. With the depletion of
glucose, adenylyl cyclase is activated and cAMP level is increased. cAMP-CAP complex binds
DNA just upstream of the promoter and facilitates the binding of RNAP to the promoter.
cAMP-CAP complex acts as a positive regulator and results in full transcription of lac operon.
Thus, the repressor protein is a

negative regulator and cAMP-
CAP complex acts a positive
regulator for the lac operon.
For the full expression of the lac
genes requires the presence of
lac inducer (lactose) which cause release of repressor from the operator locus and increased cAMP
level (due to depletion of glucose) that forms cAMP-CAP complex which binds to DNA and facilitates
binding of RNAP to the promoter.
Regulation of gene expression in eukaryotes

Regulation of gene expression in
eukaryotes is highly complex. In
eukaryotes, gene expression can be
regulated at different levels of gene
expression process and the major
control site is at transcriptional level.
Level of regulation of gene expression in eukaryotes
I. Transcriptional control
1. Gene amplification
2. Chromatin remodelling
3. DNA methylation
4. Gene regulatory protein (DNA cis-elements and trans-activator proteins)
5. Hormonal control
6. Gene rearrangement
II. Post-transcriptional regulation
1. Alternate mRNA splicing
2. mRNA editing
3. mRNA transport
III. Translational regulation
1. Regulation of mRNA stability
2. Regulatory mechanisms during translation
3. Regulation by RNA interference
IV. Post-translational regulation
I. Transcriptional control
1. Gene amplification
In order to produce large numbers of specific proteins, many copies of genes can be
generated. This process is called gene amplification. Under certain conditions, single copy
genes are amplified to many folds during development or in response to drugs.
Cancer cells “drug resistance” is due to gene amplification, e.g., malignant cells can develop
drug resistance to anti-cancer agent methotrexate by gene amplification of dihydrofolate
reductase.
2. Chromatin remodelling
Chromatin structure provides an additional level of eukaryotic gene expression control.
The presence of nucleosome structure

(complex between histone and DNA) provides
a barrier against the association of
transcription factor with promoter region.
In genome, large regions of chromatin are
transcriptionally inactive (heterochromatin)
while others are either active or potentially
active (euchromatin).
Histone modification regulates the chromatin
structure and accessibility of DNA by gene
regulatory proteins.
Two important enzymes for histone
modification are histone acetyl transferase
(HAT) and histone deacetylase (HDAC).
The reversible acetylation of lysine residues in core histone proteins results in disruption of
nucleosomal structure and opening of promoter and regulatory regions. This allows
increased access of transcriptional factors, gene regulatory proteins and DNAP to specific
elements of DNA and commence transcription.
Conversely, histone deacetylation promotes the condensation of chromosomes and inhibits
transcription.
3. DNA methylation
Methylated cytosine at C5
position in CG (CpG) is
found among 2 – 7% of
cytosines in eukaryotic DNA.
CpG are found all over the
nuclear DNA (average 1 CpG
per 100 bp). Some nuclear
DNA regions have a high
density of CpG called “CpG
island”, e.g., promoters of house keeping gene and some tissue-specific genes.
There is a correlation between DNA methylation and gene expression. Methylated DNA
regions are transcriptionally inactive while non-methylated genes are transcriptionally
active.
Methylation of base (cytidine) is generally

associated with inactivation of gene
expression. Methylation of cytosine in CpG
may interfere with binding of transcription
factors or can be a binding site for some
repressors.
DNA methylation can lead to chromatin
remodelling and heterochromatic formation.
Methylation is a mechanism for regulating
gene expression during differentiation,
particularly in fetal development.
4. Gene regulatory proteins
Certain DNA elements such as enhancer elements, silencer elements and response elements
regulate transcription of some genes by interacting with specific gene regulatory proteins.
Gene regulatory protein contains two regions –
 DNA binding domain that binds specific DNA sequences
 Cofactor binding domain (coactivators, corepressors,
etc) that aids the assembly of the transcriptional
complex at the TATA box
Four common classes of DNA-binding domain that account
for these specific protein-DNA interaction are the helix-turn-
helix, helix-loop-helix, zinc fingers and leucine zipper
motifs. Via these specific DNA binding domains, gene
regulatory proteins bind with high affinity and specificity to
the correct region of DNA.
After binding to specific regulatory sequences (cis-acting
elements), gene-specific regulatory proteins bind and
interact with mediator proteins such as coactivators or
corepressors.
Coactivators, corepressors, and other mediator proteins
generally do not bind directly to DNA but interact with the basal transcription complex and
can influence its assembly at the initiation site on the promoter. Many gene regulatory
proteins promote transcription (activators) while some promote gene silencing
(repressors).
Structural motifs of gene regulatory proteins

 Four special protein motifs for specific protein-DNA interactions – leucine-zipper motif, zinc finger
motif, helix-turn-helix
motif and helix-loop-
helix motif.
 Each of these motifs
use their α helices or
β sheets to bind to
the major groove of
DNA.
 These structures bind DNA sequences with strong and highly
specific interactions by hydrogen bonds, ionic bonds and
hydrophobic interactions.
 Leucine-zipper motif (α-helix of 30 to 40 amino acid residues
that contains a leucine every seven amino acids)
o Functions as dimers to regulate gene transcription, e.g.,
cAMP response element binding protein
o CREB mediates the effect of cAMP on transcription of certain
genes by binding to cAMP response element (CRE) of DNA.
PKA phosphorylates CREB and promotes dimerization of
CREB to function as efficient gene regulatory protein.
 Zinc finger motif (zinc molecule chelated at four positions with
either histidine or cysteine in amino acid sequence)
o Major eukaryotic motif
o Zinc is required to maintain the tertiary structure of this
domain in order to bind with specific response element e.g.,
steroid hormone receptors.
o Eukaryotic transcription factors generally have two to six
zinc-finger motifs that function independently.
 Helix-turn-helix motif
o Stretch of three short helices of amino acids separated from each other by turns, one helix fits into
the major groove of DNA.
o E.g., homeodomain proteins (regulation of gene expression during development)
 Helix-loop-helix motif (HLH)
o Functions as dimers that fit around and grip DNA in a manner geometrically similar to leucine-
zipper proteins
o Transcription factors containing the helix-loop-helix motif are involved in cellular differentiation
e.g., myogenin, neurogenin.
5. Hormonal control
Major signal for gene expression is hormones. Steroid, peptides, protein hormones and other
morphogens activate DNA-binding proteins that control gene expression.
Steroid hormones bind to

specific cytosolic steroid
hormone receptors that
function as specific gene
regulatory proteins. Activated
hormone-receptor complexes
form homodimers which then
migrate to the nucleus where they bind to hormone response
elements (HREs) on DNA thereby regulating the expression of
nearby target genes. HREs for steroid receptors are palindrome
consisting of a pair of 6bp sequences separated by a 3bp spacer.
Vitamin D, thyroid hormone and retinoic acid receptors are
members of nuclear receptor superfamily. Specific hormone-
receptor complexes then forms heterodimers, e.g, the thyroid
hormone receptor forms a heterodimer with the retinoid X
receptor (RXR). They act as morphogen that induce
differentiation of the cell by controlling of gene expression.
Peptide and protein hormones act through which activate gene
regulatory proteins to regulate expression of target genes, e.g.,
growth factors via MAPK cascade.
6. Gene rearrangement
Segments of DNA can move from one location to another in the genome, associating with
each other in various ways so that different proteins are produced. This is called gene
rearrangement.
Gene rearrangement occurs in presursor B cells during its differentiation into mature B
lymphocytes to produce specific antibodies.
The heavy-chain gene from which lymphocytes produce immunoglobulins is generated by
combining specific segments from among a large number of potential sequences in the DNA
of precursor cells.
II. Post-transcriptional regulation

1. Alternate mRNA splicing
Cells can splice the primary
transcript in different ways
and thereby make different
polypeptide chains from
the same gene by a process
known as alternative RNA
splicing.
Alternative splicing is seen
in cells or specific tissues or
at certain stages of
development or under certain conditions.
For example, in parafollicular cells of thyroid gland, the calcitonin gene produces an mRNA
that encodes for calcitonin whereas in the brain the same gene is spliced differently and uses
a different polyadenylation site producing a protein which is involved in taste sensation.
2. mRNA editing
RNA editing involves the enzyme mediated alteration of
RNA before translation. It involves the insertion, deletion or
conversion of nucleotides in RNA molecule.
It can result in tissue-specific differences in transcript.
An example of RNA editing occurs in the production of
apoprotein B (apoB) in the intestinal cells which is produced
from the same gene encoding the apoB in the liver. In the
intestine, codon CAA (codes for glutamine in liver) is edited
to UAA (stop codon) and produces protein which is of only
48% of amino acid composition produced from the same
gene in the liver. Both apoproteins are encoded by the same
gene and apoB100 synthesized in the liver contains over
4000 amino acids while the other one synthesized in the
intestinal cells, apoB48, contains 48% of amino acid composition of ApoB100.
3. mRNA transport
Mature mRNAs are transported from the nucleus through the nuclear pores to the cytoplasm
in order to be translated.
During the transport, mRNA is bound to proteins that help to prevent its degradation by
nucleases. Signals that direct mRNA localization are typically located in 3’ UTR of mRNA.
III. Translational regulation

1. Regulation of mRNA stability
Sequences at 3’ end of the mRNA appear to be involved in
determining its half-life.
The 5′ cap and 3′ poly(A) tail protect the mrNa against
exonuclease attack and are bound by specific proteins
that interact to facilitate translation.
Other sequences at the 3’ end may also involve in
protecting mRNA from degradation, e.g., stem-loop
structures in 3’ UTR may serve as binding sites for specific
proteins that modulate mRNA stability.
Transferrin receptor mRNA contains hair pin loop (iron response element) at 3’ end. When
iron level is low, iron response element binding protein (IRE-BP) binds to IRE at 3’ end and
prevents transferrin receptor mRNA degradation, leading to increased transferrin receptor
synthesis. When iron level is high, IRE-BP binds iron and becomes decreased affinity for IRE
leading to rapid degradatino of transferrin receptor mRNA.
2. Regulatory mechanisms during translation
Most eukaryotic translational control affect at the level of translation initiation. eIF2 and eIF-
4E (component of eIF4F complex) are the targets of eukaryotic translation initiation control.
The action of eIF2 can be inhibited by phosphorylation of its subunit eIF-2α.
 For example, heme regulates translation of globin
mRNA in reticulocytes by controlling the
phosphorylation of eIF-2α. In reticulocytes, globin is
produced when heme levels in the cell are high but
not when they are low. Heme binds and inactivates a
specific kinase (heme-regulated inhibitor kinase)
which phosphorylates eIF-2α; thus, favoring active state of eIF2 for globin chain synthesis.
 In other cells, conditions such as starvation, or viral infections may result in activation of a
specific kinase that phosphorylates and inactivates eIF-2α.
A cap-binding protein complex or eIF-4F complex (eIF-4E, 4G and 4A complex) is particularly
important in controlling the rate of protein translation. The 4F complex binds to the 5’ cap of
mRNA through the 4E protein.
 4E binding protein (4E-BP) binds 4E and prevent its binding

with 4G to form 4F complex. Phosphorylation of 4E-BP
releases eIF-4E to form 4F complex.
 Insulin and growth factors stimulates protein synthesis by
inducing the phosphorylation of 4E-BP which leads to
dissociation of 4E from 4E-BP.
Sequences at the 5’ end may also involve in regulation of
translation initiation, e.g., hair pin loop structure in 5’ UTR (IRE)
of ferritin mRNA serves as binding site for specific regulatory
protein (IRE-BP) that modulate its translation initiation.
 When IRE-BP does not contain bound iron, it binds to the IRE and prevents initiation of
translation. When iron levels increase and IRE-BP binds iron, it changes to a conformation
that can no longer bind to the IRE on the ferritin mRNA. Therefore, the mRNA is
translated and ferritin is produced.
3. Regulation by RNA interference
In higher eukaryotes, including
nematodes, fruit flies, plants, and
mammals, a class of small RNAs called
microRNAs (miRNAs) mediates the
silencing of many genes.
The miRNAs function by interacting with
mRNAs, often in the 3’ UTR, resulting in
either mRNA degradation or translation
inhibition. Therefore, the mRNA and
thus the protein that will be produced
from this gene will be silenced.
Thousands of different miRNAs have
been identified in higher eukaryotes, and
they may affect the regulation of a third
of mammalian genes. They are
transcribed as precursor RNAs about 70
nucleotides long, with internally
complementary sequences that form
hairpin-like structures. The precursors are recognized and cleaved by endonucleases such as
Drosha and Dicer to form short duplex RNA fragments about 20 to 25 nucleotides long,
termed as small interfering RNAs (siRNAs) or microRNAs (miRNAs). While siRNAs generally
arise from virally produced dsRNAs, miRNAs are transcribed from introns of normal genes or
from other nonprotein coding genes.
miRNAs interact with a multiprotein complex, termed the RNA-induced silencing complex,
RISC. Using the incorporated si/miRNA, the RISC complex can bind to target mRNA
sequences which are complementary to its RNA, leading to inhibition of translation (miRNAs)
or rapid degradation of mRNA (siRNAs).
RNA interference has very interesting and useful practical application. It could be used in
treatment of cancer and viral infection. Introduction of a duplex RNA corresponding in
sequence to virtually any mRNA into an organism produces small interfering RNA (siRNAs) by
the action of Dicer in the cell. These bind to the mRNA and silence the target mRNAs.
Laboratory-produced siRNAs have been used to block HIV and poliovirus infections in
cultured human cells. Epigenetics
 Literally means “on top of or in
IV. Post-translational regulation
addition to genetics”.
Post-translational regulation can be achieved by regulation
 Epigenetic regulatory mechanisms
of protein activity and proteolytic degradation. do not change or act through the
Activity of synthesized protein can be affected by covalent regulated DNA sequence, but the
modification e.g., phosphorylation. expression patterns of DNA.

 Epigenetic control mechanisms
After proteins are synthesized, their life-span is regulated
include DNA methylation, and
by proteolytic degradation. Proteins have different half- histone modification such as
lives according to their amino acid sequences. Extracellular acetylation.
proteins, membrane associated proteins are degraded by lysosomal enzymes and intracellular
proteins are degraded by ubiquitin dependent proteasome system.
Recombinant DNA and genomic technology

Molecular genetics becomes the most interesting and most active field in biomedical research and in
clinical practice. Recombinant DNA technology has revolutionized biology and is having an ever-
increasing impact on clinical medicine. The potential uses of these techniques for the diagnosis and
treatment of disease are vast.
It helps in the diagnosis of genetic susceptibilities for disease prevention, diagnosis of diseases and
choice of treatment. Understanding of the basic principles of the methods and some commonly used
applications are essential for understanding the interpretation of DNA analysis for forensic, genetic
and diagnostic service in clinical settings.
Recombinant DNA
 Recombinant DNA is an altered DNA due to insertion of a sequence of deoxyribonucleotides which is
not previously present in existing DNA molecules by enzymatic or chemical means.
Recombinant DNA technology

 Recombinant DNA technology is the technique that is used in formation of recombinant DNA and
genetic analysis of organisms.
Methods used or general steps involved in recombinant DNA technology

Cleavage of the desired DNA fragment from the large DNA molecule by restriction endonucleases at
specific sequences
Separation and isolation of DNA fragments by gel electrophoresis
Identification and visualization of a specific sequence of DNA or RNA by nucleic acid hybridization
Amplification of desired DNA segment by polymerase chain reaction or cloning
o Polymerase chain reaction is the in vitro (test tube) method of amplifying target DNA
sequence within a relatively short time interval.
o DNA fragment is joined to vector DNA (usually a bacterial plasmid or phage or cosmid) and
then chimeric DNA is multiplied within the bacteria along with the endogenous DNA of the
host cell. This process is referred to as cloning.
Collection of different recombinant clones (library)
Quantitative analysis of nucleic acids
DNA microarrays for genetic screening and analysis of gene expression
Cleavage of desired DNA fragment by restriction endonuclease

The key tool is recombinant DNA technology is restriction endonuclease enzyme obtained from
bacteria. These enzymes cut specific palindrome sequence within the DNA molecule.
In bacteria, these enzymes restrict the growth of certain virus called bacteriophages by cleaving the
viral genome and thus these enzymes are called restriction endonucleases.
Restriction enzymes are named after the bacterium from which they are isolated, e.g., EcoR1 from
E.coli.
Cleaved DNA results in blunt end or sticky end depending on the mechanism used by the enzyme.
When DNA is digested by a particular restriction enzyme, the ends of all the fragments have the same
DNA sequence. The DNA fragments can be isolated or separated by gel electrophoresis.
Diagrammatic representation of NDA molecules indicating the site of cleavage by various restriction
enzymes is called restriction map.
Separation and isolation of DNA

DNA fragments of various sizes can be separated on
agarose gel by electrophoresis as they carry negative
charges.
The rate of migration of DNA fragments depends on
their size, with the smallest fragments moving
furthest and the largest moving least.
Complementary DNA (cDNA)

It is mostly used as probe for hybridization
techniques and making of gene library.
cDNA probe is made from mRNA by reverse
transcriptase.
Probe
It is a synthetic single stranded DNA (or RNA)
sequence that is complementary the target DNA.
To be detectable, the probe must be labeled with either a radioactive isotope or a fluorescent group.
Nucleic acid hybridization or blotting

It is a method of using oligonucleotides probe, marked by
radioisotopes or chemicals to hybridize to the DNA or RNA
(Southern blot for DNA and Northern blot for RNA). This is
used to identify presence or absence of a particular gene,
amount of RNA transcribed and mutated DNA.
Western blot rely on the ability of a specific antibody to bind to
a protein of interest.
DNA cleaved with restriction enzymes or RNA isolated from
the cells is applied on agarose gel and is electrophorectically
separated by size. The nucleic acids in the agarose gel are transferred to a nitrocellulose membrane.
DNA or RNA fragments on the membrane where they stick tightly are hybridized with a radioactive
labeled probe.
Unbound probe is washed off and the membrane is exposed to X-ray film. The position of a DNA or
RNA fragment complementary to the probe appears as a band on the film.
Fluorescence in situ hybridization (FISH) is a procedure of detection of deleted gene or mutant gene
in intact chromosome. It is widely used in clinical practice e.g., detection of HER2/neu gene in breast
cancer.
Amplification of DNA fragment

 Amplification of DNA is central to the study of molecular biology and genetics. Two important
approaches to the amplification of DNA are —
 Cell-based DNA cloning
 Cell-free enzyme-based DNA amplification by the polymerase chain reaction (PCR)
Cloning
 Large population of identical molecules or cells that arises from a common ancestor. DNA fragment
of interest is joined to vector DNA and then the chimeric vector is inserted to host bacteria e.g.,
E.coli. The chimeric DNA is multiplied within the bacteria as bacteria grow and divide.
 Cloning vectors – plasmid, phage, cosmid, bacterial artificial

chromosome (BAC), yeast artificial chromosome (YAC)
 Plasmids – small, circular, non-chromosomal duplex DNA
molecules in bacterial cells which confer antibiotics resistance
to the host cell. It can accept 6 to 10 kb long foreign DNA.
 Phages – organisms that infect the bacteria. It can accept 10 to 20 kb long foreign DNA.
 Cosmids – circular DNA molecule with combination of features of plasmids and phages. It can accept
35 to 50 kb long DNA. It can replicate like a plasmid.
 Bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC) can accept larger
pieces of DNA (more than 50 kb).
Gene library
 Collection of amplified different recombinant clones constitutes library.
 Genomic library is prepared from the total DNA of a cell line or tissue (in which both introns and
exons are represented).
 cDNA library comprises complementary DNA copies of the population of mRNAs in a tissue (in which
only exons are represented).
Polymerase chain reaction (PCR)

PCR is the in vitro (test tube) method of amplifying a target sequence of DNA molecule.
PCR permits the synthesis of millions of copies of a specific DNA sequence in a few hours. It can
amplify the sequence even when the targeted DNA makes up less than one part in a million of the
total initial sample.
The process is base on the replication but many steps are overcome by different ways.
Materials required for PCR
DNA of interest (which can be extracted from blood, hair follicle, buccal smears, body fluid,
fetal blood, chorionic villi, amniotic fluid, paraffin embedded tissues or fossil, etc.)
Synthetic oligonucleotide primers (20 – 25 bp) that are complementary to the target DNA
sequence (DNA primers)
Heat stable DNA polymerase (Taq polymerase from Thermaus aquaticus)
Mixture of all four dNTPs
Reaction buffer containing Mg2+ and enhancer
Thermal cycler
Steps involved in PCR
1. Heat denaturation of template dsDNA
 The reaction mixture containing the interested dsDNA is heated at about 94 to 98°C for 5
minutes by thermal cycler to separate the dsDNA.
2. Primer annealing
 The temperature is lowered to 50 to 65°C to allow the primers binding to complementary
sequence in the template DNA strands.
3. Primer extension
 Temperature is raised to 72°C which is the optimal temperature for the heat stable Taq
polymerase to extend the new DNA strands from the annealed primers by using
complementary dNTPs.
This cycle of denaturation, annealing and extension are repeated for 30 – 40 cycles to obtain
the desired amount of DNA.
Advanced PCR systems for determination of quality and quantity of nucleic acids
Reverse transcriptase PCR (RT-PCR)
Real-time PCR (quantitative PCR)
Advantages of PCR
Amount of DNA needed for PCR is very small compared to conventional cloning method.
It can be done within a short time without requirement of biological materials such as cloning
vector and bacterial host.
Disadvantages of PCR
Template DNA sequence information is always required otherwise primers cannot be
synthesized to amplify the target DNA fragment.
Size of DNA to be amplified is limited, usually up to 5 kb. The amplification of longer DNA
sequence requires specialized “long-PCR method”.
Uses of PCR
The amplified target DNA by PCR method can be used for further manipulation for various
purposes. These include —
Diagnostic and clinical uses
 Detection of infectious agents (virus, bacteria) especially latent viruses
 Prenatal diagnosis of genetic diseases
 Diagnosis of genetic diseases in adults e.g., detection of mutation, genetic
susceptibility of cancers
 Molecular diagnosis of infectious diseases, hemoglobinopathies and cancer
 For precise tissue typing for organ transplantation
Uses in biomedical research
 Detection of allelic polymorphism, detection of mutation and restriction length
polymorphism (RFLP)
 Preparation to synthesize probe in hybridization technology and DNA sequencing
from PCR products
 Study of gene expression by RT-PCR (for RNA analysis after RNA copying and mRNA
quantitation by real-time RT-PCR)
Uses in forensic medicine
 Person identification by DNA fingerprinting
 For pedigree analysis
Uses in archeology
 Analysis of DNA from archeological samples to study evolution and people’s
migration
DNA microarrays
 It provides a wide picture of the genes that a cell is expressing. Microarray technology can be used
for the study of differential gene expression. It can also used to identify genomic deletions and
duplications associated with developmental delays, mental retardation.
Restriction fragment length polymorphisms (RFLPs)

 Differences in DNA sequence can result in variations of restriction sites that make different length of
DNA when cut by a particular restriction enzyme.
 RFLPs can occur due to –
o Mutation or single nucleotide polymorphism that renders a restriction site unrecognizable by
the restriction endonuclease
o Generation of a new recognition site
o Insertion of repeats
 RFLPs are currently utilized to facilitate prenatal detection of a number of hereditary disorders,
including sickle cell trait, β thalassemia, etc.
Applications of recombinant DNA technology

1. Gene mapping or localization
o Gene localization can be defined by a map of human genome. Somatic cell hybridization and
In situ hybridization are used to localize the gene on chromosome.
o Chromosome walking is used to search for gene when its position on a chromosome is
approximately known. It is used to clone human diseased gene.
2. Molecular analysis of disease
o Analysis of genetic variation in normal genes and disease causing genes
o Analysis of mutations, DNA rearrangements
o Prenatal diagnosis of genetic diseases
o RFLP is useful as diagnostic tool for genetic diseases.
o Study of gene expression of tumor tissues by DNA microarrays
o Pharmacogenomics for production of “personalized medicine”
3. Protein production for research, diagnostic and therapeutic purposes
o A particular goal of recombinant DNA technology is to supply large amount of proteins for
biomedical applications.
o For therapeutic uses e.g., interferon, insulin, tPA, growth hormone
o For diagnostic tests e.g, HIV tests, HbS antigen tests
o Production of vaccines for disease prevention e.g., hepatitis B vaccine
o Application in agriculture e.g., genetic engineering of plants for drought resistance and
greater nutritional value
4. Uses in forensic medicine
o Variable number tandem repeats (VNTR) serves as molecular fingerprint for person
identification (scan 13 DNA regions that vary from person to person).
o Helpful in solving of criminal problems, paternity disputes.

5. Gene therapy
o Introduction of normal gene into individuals with defective genes
o First successful gene therapy was adenosine deaminase (ADA) gene therapy for severe
combined immunodeficiency disease (SCID) in 1990.
6. Production of transgenic animals for research purpose
o Study of tissues specific effect of gene expression
o Discovery of genes involved in disease process
o Analysis of effects of overproduction of gene products
Stem cells
 Stem cells are unspecialized cells that have two important properties that distinguish them from
other cells in the body; self-renewal and differentiation.

 Self-renewal means they can replenish their numbers for long periods through cell division.
 With exposure to appropriate chemical signals, they can differentiate into specialized cells.
 Different types of stem cells
o Totipotent stem cells – can differentiate into embryonic and extra embryonic cell types
(placenta).
o Pluripotent stem cells – can differentiate into cells derived from three germ layers.
o Multipotent stem cells – can only differentiate into a limited number of cell types e.g.,
hemopoietic stem cells, neural stem cells.
o Unipotent stem cells (progenitor cells) – can only differentiate into one cell type e.g., erythroid
progenitor cells differentiate into only erythrocytes.
o Induced pluripotent stem cells (iPS cells) – induction of adult stem cells into embryonic stem
cells by introduction of active genes for four transcription factors
 Commonly used stem cells in research are embryonic stem cells (embryo’s inner cell mass) and adult
stem cells (collected from bone marrow or cord blood and umbilical cord stem cells).
 Potential uses of stem cells
o Adult stem cells (hemopoietic stem cells) are used in stem cell therapy for leukemia, bone
and blood cancers through bone marrow transplants.
o Stem cells (embryonic and adult stem cells) are extensively used in research purposes to
understand the disease pathogenesis e.g., embryonic stem cells are critical in developing
transgenic animals.
o Ongoing research for potential stem cell therapy in some chronic diseases e.g., Parkinson
disease, heart failure, myocardial infarction, spinal cord injuries, etc.
Genetic basis of cancer

 Cancer cells are associated with
genetic alterations in a large
number of genes that encode
proteins involved in the control
of cell proliferation. These can
be classified into proliferation
gene (proto-oncogene) and
anti-proliferation gene (tumor
suppressor gene).
 Products of proto-oncogene
involves in promoting cell
growth, differentiation and the assembly of the cell cycle control system that drives the cells to pass
the cell cycle control check points e.g., growth factors, growth factor receptors, intracellular signal
mediators of growth factors signaling such as Ras, various tyrosine kinases, cyclin D and cdk 4, cdk 6
for progression of G1 checkpoint, etc.
 Products of tumor suppressor genes involve in cell cycle arrest for repair of DNA damage during cell
cycle phases and removal of cells with extensive DNA damage by apoptosis.
 Therefore two classes of genes are critical in the causation of cancer – tumor suppressor gene and
proto-oncogenes. Gain of function mutation of proto-oncogenes acts in a dominant manner to
stimulate cell growth and division leading to increased cell proliferation. Loss of function mutation of
tumor suppressor gene relieves the cells from inhibitions that normally control abnormal cell growth
and it behaves in a genetically recessive manner.
Oncogene
 Oncogene is defined as an altered gene whose product acts in a dominant manner to accelerate cell
growth or cell division, contributing to cancer development.
 Oncogenes are generally derived from mutation from normal cellular proto-oncogenes. Factors that
cause mutation include chemicals and radiation. A single mutation is not sufficient to convert a
healthy cell into tumor cell. Several mutations have to occur together.
 Mutations in proliferation gene could result in overexpression of protein products or aberrant over-
activity of protein products leading to uncontrolled cell division; thus it is classified as oncogene.
 Products of tumor suppressor gene normally suppress cell growth or cell division. Mutations in tumor
suppressor gene result in diminished or loss of the inhibitory effect on cell proliferation leading to
increased cell growth and/or cell division e.g., Rb (retinoblastoma protein), p53 and p21.
 Mechanisms of transformation of
proto-oncogenes into oncogenes
o Point mutation e.g., Ras
oncogene
o Promoter insertion
o Enhancer insertion
o Chromosomal translocation
e.g., Philadelphia
chromosome in CML
o Gene amplification e.g.,
genes involved in tumor cell
chemotherapy resistance
 Mechanisms of actions of oncogenes
o General mechanisms by which the products of oncogenes (oncoproteins) may stimulate
growth and division of cells are —
1. Imitating the action of a growth factor e.g., sis oncoprotein
2. Imitating an occupied receptor for a growth factor e.g., erb-B oncoprotein
3. Acting as key intracellular signaling molecules involved in growth control, uncoupling
them from the need for exogenous/upstream stimulus e.g.,
 Protein tyrosine kinases (src protein kinase)
 G protein (monomeric G protein Ras)
 DNA binding protein (myc)
4. Acting as nuclear transcription factors that control the gene expression essential for cell
division e.g., Jun, Fos.
o One of common examples of oncogenes is ras
oncogene which is associated with 30 to 50%
of lung and colon cancers and more than 90%
of pancreatic cancers.
o Examples of tumor suppressor genes
mutations involved in uncontrolled cell
proliferation are Rb protein, p53 and p21.
o Mutations in oncogenes and tumor suppressor genes do not have all-or-none effect.
Progression of a normal cell to a malignant tumor cell requires an accumulation of mutations
in many genes; none of which alone is responsible for such transformation.
Multiple genetic changes associated with the development of colorectal cancers
Biochemical tests in the management of cancer

patients
Many cancers are associated with abnormal
molecules production (enzymes proteins or
hormones) which can be measured in the
plasma or serum. These are called tumor
markers.
These markers can be used in screening,
diagnosis, staging, detecting of metastasis,
monitoring response to treatment and detection of recurrence.
There is no single marker that is useful for all types of cancers or for all patients with a given type of
cancer.
Markers are more often detected in advanced stages of cancer rather than early stages.
Human genome project

 HGP is an international scientific research project that began in 1989 by the researchers of
Department of Energy and the National Institute of Health in US.
 Complete reference sequence for the human genome was announced in 2003.
 A parallel project was conducted by non-governmental Celera Corporation which was formally
launched in 1998.
 Primary goals of the project
o Construction of genetic map and storage of information in databases
o Identification of all human genes
o Sequencing of the entire human genome
o Improvement of tools for data analysis
o Transfer of related technologies to the private sector
o Addressing the ethical, legal and social issues arising from the project
 Sequence and analysis of the human genome working draft was published in February 2001 and April
2003 issues of Nature and Science.
 Genome sequencing was also focused on several other non-human organisms such as E.coli, fruit fly
and laboratory mouse.
 Findings obtained in HGP
o Approximately 20,500 genes in human, the same range as in mice
o 1.1 to 1.4% of the genome sequence encodes for protein.
o Catalogue of 1.4 million single nucleotide polymorphism (SNP) and specification of their exact
locations in human genome
 Benefits of human genome research in medicine and biomedical research
o Advances in genetic engineering techniques (isolation, cloning and sequencing of any DNA
segment)
o Major advances in computing technology for determining the DNA sequence
o Knowledge about the effects of DNA sequence variations among individuals
o Evolvement of new ways for disease diagnosis, treatment and prevention
o Development of rational drug design
o Gene therapy and control systems for drugs
o Emergence of pharmacogenomics
Pharmacogenomics
Study of the interaction between genetics and therapeutic drugs
It is for developing individualized medicine according to patient’s genotype since understanding an
individual’s genetic makeup is thought to be the key to creating personalized drug with greater
efficacy and safety.
DNA variations in genes encoding for cytochrome P450 (CYP) family of liver enzymes can influence
their ability to metabolize some drugs.
Anticipated benefits of pharmacogenomics
o More powerful medicine
o Better safer drugs
o More accurate methods of determining appropriate drug dosage
o Advanced screening for disease
o Better vaccines
o Improvement of drug discovery and approval process leading to decrease in overall cost of
health care
Role of nucleic acids in gene expression

Genetic information is stored in the base sequences of DNA molecules in most organisms or of RNA
molecules in some viruses. During the process of gene expression which includes transcription and
translation, this information is used to synthesize all proteins made by an organisms.
Nucleic acids are polymers of nucleotides joined by 3’ – 5’ phosphodiester bonds.
DNA is the repository of genetic information. During transcription the genetic information contained
in the base sequence of DNA is copied by complementary base pairing to form ribonucleotides
sequence, RNA chain. Transcription of genetic information from template DNA strand (opposite to
protein coding DNA strand) into primary RNA transcript is catalyzed by RNAP II.
DNA also contains sequences encoding genetic information for other functional RNA molecules
involved in translation process such as tRNAs and rRNAs. Synthesis of all rRNAs except 5S rRNA is
catalyzed by RNAP I whereas synthesis of all tRNAs plus 5S rRNA is catalyzed by RNAP III. Various
proteins called transcription factors are required for transcription of DNA to RNA. Primary RNA
transcripts are then processed to form mature functional RNA molecules for translation process. This
is also called post-transcriptional modification and this process requires small RNA molecules such as
snRNAs (splicing) and snoRNAs (cleavage of pre-rRNA into different rRNAs). All these processes
occur in the nucleus. Mature functional RNA molecules are then transported to cytosol for
translation process.
Next step of gene expression is translation in which the information in nucleotide sequence of mRNA
molecule directs the ordered polymerization of specific amino acid sequence into protein.
RNA molecules directly involve in translation process include mRNA, different types of tRNAs and
rRNAs.
There are four different types of rRNA molecules (18S, 28S, 5.8S and 5S rRNAs) involved in eukaryotic
translation process. Together with protein components, 18S rRNA forms 40S ribosomal subunit and
28S, 5.8S and 5S rRNAs combine with respective protein component to form 60S ribosomal subunit.
mRNA carries the genetic message from the nucleus to the protein synthesizing site, ribosomes.
Different types of tRNA molecules act as carriers of specific amino acids to protein synthesis site.
Steps involved in translation process include amino acyl tRNA synthesis, initiation, elongation and
termination.
Attachment of specific amino acid to corresponding tRNA is catalyzed by amino acyl tRNA synthetase
and this process uses two ATPs.
Initiation process involves ribosomal dissociation, formation of 43S pre-initiation complex (binding of
met-tRNA–eIF-2–GTP ternary complex to 40S ribosomal subunit), formation of 48S initiation complex
(binding of mRNA to 43S complex) and formation of 80S initiation complex (binding of 60S
ribosomal subunit to 48S complex).
During the translation process, mRNA serves as the template for translation process in the initiation
complex. tRNA serves as a carrier of specific amino acid to protein synthesis site and acts an adaptor
between the codon of mRNA and corresponding amino acid for protein synthesis. rRNAs serve as
structural component of ribosomes which acts as the site of protein synthesis.
During the elongation process, peptide bond formation is catalyzed by 28S rRNA of 60S ribosomal
subunit which has the peptidyl transferase activity. It also involves in the termination process for the
cleavage of peptide chain from tRNA facilitated by releasing factor binding to stop codon.
Small cytoplasmic RNA molecules such as miRNA do not directly involve in gene expression but
involve in regulation of gene expression process at the translational level. Such regulation is called
RNA interference.

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Chemical Basis of Life Page |1

Chemical Basis of Life

 Biochemistry can be defined as the chemical basis of life.

reactions and processes occurring in the cells.

and functional units

into one of three large

It is enclosed by a plasma membrane. The cytoplasm

which sunlight drives the synthesis of ATP in the process of photosynthesis).

cells) contain a nucleus (nuclei) where the cell’s genomic

envelope which is a double-layered phospholipid

particularly rRNA. It is also rich in enzymes in DNA and RNA synthesis.

Mitochondria, like gram-negative bacteria, have two membranes.

cristae that protrude into the mitochondrial lumen (space) known

is often observed to surround the nucleus. The outer layer of the

membrane are called rough endoplasmic reticulum (rough ER or

site of synthesis of steroid hormone, phospholipids and metabolism of foreign compounds

organelle appears as flat, stacked, membranous sacs.

them and contain hydrolytic enzymes.

a toxic by-product of many metabolic reactions, is detoxified in peroxisomes by catalase.

Cytoskeleton and cytosol

Molecular composition of the human body

o Pyranose and furanose ring structures

Biochemical significance of monosaccharides

Derivatives of simple sugars

o Xylitol is widely used to sweeten sugarless gums and mints.

 Streptomycin is an aminoglycoside which is used as antibiotics.

Formation of glycosidic bond

carbonyl carbon is oxidized to a carboxyl group. color yield produced is directly

Disaccharides and oligosaccharides

 Oligosaccharides consist of short chains of monosaccharide residues (3 – 10) joined by glycosidic

Mucopolysaccharides or glycosaminoglycans (GAGs)

 Cis and trans configuration of unsaturated fatty acids

(“good”) cholesterol level in the blood. Trans-fatty acids

Triacylglycerol (Triglycerides or TAG)

o Important membrane glycerophospholipids

 Cerebrosides (Ceramide + Monosaccharides)

Classification of amino acids

 Classification based on polarity and charge on R groups of amino acids

 Classification based on structure of the side chain of the amino acid

Biochemical significance of different side chains of amino acids

Peptides and Proteins

Structure of proteins (Levels of organization of protein structure)

conformation which is determined by its amino acid sequence.

organizational levels; primary, secondary, tertiary, and quaternary.

o Hydrogen bonding between amide hydrogen (-NH) of

 It is stabilized by hydrogen bonds between the NH and

o Three-dimensional folded compact and biologically active

Protein synthesis and post-translational modification of proteins

Protein separation and purification

 Separation on the basis of molecular size

o Amino acid sequencing of peptides and proteins

o Deduction of amino acid sequence

Nucleotides and nucleic acids

 Two types of nucleic acids are found in the living

Deoxyribonucleic acid (DNA)

 In most living organisms, except for RNA viruses, genetic

Denaturation and renaturation of DNA

Ribonucleic acid (RNA)

o Transfer RNA (tRNA)