ENZYMES - it is the most specific pancreatic marker

- these are proteins produced by living cells that - major source: pancreas
hastens chemical reactions in organic matter. IV. Lactate dehydrogenase
- They are measured in terms of their activity - catalyzes the conversion of lactic and pyruvic
- They are large molecules and they are normally acids.
confined within cells - is a zinc-containing enzyme that is a part of the
- Abnormal large amounts of enzymes in serum are glycolytic pathway
used clinically as evidence of organ damage. - in plasma, majority comes from the breakdown of
erythrocytes and platelets
FACTORS AFFECTING ENZYMATIC LD-1 and LD-2 – heart, RBCs, kidneys
REACTIONS LD-3 – lungs, pancreas, spleen
Enzyme as a protein, composed of a specific amino LD-4 and LD-5 – skeletal muscles, liver, intestine
acid forming polypeptide chains which then folds IV. Creatine Kinase
and results in structural cavities - catalyzes the transfer of a phosphate group
between creatine phosphate and adenosine
MAJOR CLINICAL ENZYMES diphosphate
I. Phosphatases - found in high concentrations only in muscle and
A. Alkaline phosphate (ALP) brain
- a nonspecific enzyme capable of reacting with Major tissue sources: brain tissue, smooth and
many different substrates. skeletal muscles and cardiac muscles
- It is to liberate inorganic phosphate from an IV. Aldolase
organic phosphate ester with the concominant - is a glycolytic enzyme that splits fructose-1-6-
production of an alcohol diphosphate into two triose phosphate molecules in
Major Isoenzymes the metabolism of glucose.
Liver ALP, Bone ALP, Placental ALP, Intestinal Increased: Skeletal muscle disease, leukemia,
ALP hemolytic anemia and hepatic cancer
B. Acid Phosphatase (ACP)
- It catalyzes the same reaction made by ALP, CARBOHYDRATES: TESTS BASED ON THE
except that it is FORMATION OF FURFURAL AND ITS
active at pH 5.0 DERIVATIVES
- for detection of prostatic adenocarcinoma INTRODUCTION
- useful for forensic (in the investigation of rape • A carbohydrate is a polyhydroxy aldehyde, a
cases) polyhydroxy ketone, or a compound that yields
II. Transferases polyhydroxy aldehydes or polyhydroxy ketones
A. Aspartate Aminotransferase (AST) upon hydrolysis.
- is involved in the transfer of an amino group
between
aspartate and alpha-keto acids with the formation
of oxaloacetate and glutamate.
- major sources: cardiac tissue, liver and skeletal
muscle
B. Alanine Aminotrasnferase (ALT)
- it has enzymatic activity similar to AST.
- It catalyzes the transfer of an amino group from
alanine to alpha-ketoglutarate with the formation of
glutamate and pyruvate. CLASSIFICATION OF CARBOHYDRATES
- ALT is more liver specific • Monosaccharides – “simple sugars”, are highly
- Major tissue source: liver soluble in water, less soluble in ethanol,
III. Amylase and insoluble in ether.
- it catalyzes the breakdown of starch and glycogen • They are either aldoses or ketoses
- it is the smallest enzyme in size • They may also be classified into tetroses,
- it is the earliest pancreatic marker pentoses, or hexoses
- Major tissue source: salivary glands, pancreas • Free monosaccharides are all reducing sugars.
IV. Lipase • They also exhibit mutarotation
- it hydrolyzes the ester linkages of fats to produce • Disaccharides – are formed by two molecules of
alcohol and fatty acid monosaccharides. Examples are maltose,
which are abundant in germinating barley; sucrose,
also known as cane sugar or beet sugar; and
lactose or milk sugar, which does not taste very PROCEDURE
sweet and is not fermented by yeast. 1. Mix 4 ml of distilled water and two drops of the
• Polysaccharides – found in nature function either Molisch’s reagent in a test tube. This tube will serve
as structural units (e.g. cellulose), or as the control.
for storage such as starch, dextrin, glycogen, and 2. Place 4 ml of 3% solution of glucose in a second
inulin test tube. Add two dropsof the Molisch’s reagent
and mix the contents by gently shaking the
testtube.
3. Incline the test tube and cautiously add about 5
ml of concentratedsulfuric acid, allowing the acid to
run down the side of the tube. Sulfuric acidis
denser than water and will form a lower layer. Note
the color of the ring formed at the junction if the two
liquids.
4. In the same manner of adding acid, add sulfuric
acid to the controltube.
5. Repeat the above test with 3% sample solutions
of glucose, xylose, lactose,
and starch.
GENERAL TESTS FOR CARBOHYDRATES 6. Record all results.
BIAL’S ORCINOL TEST
• used to determine the presence of pentoses and
nucleotides that contain pentose sugars.
• When pentoses are treated with orcinol, furfurals
are formed and they will yield a blue-green
compound in the presence of ferric ions.
• The reaction is not specific for pentoses because
other compounds like trioses, uronic acids, and
certain heptoses will also give blue or green
products. Hydroxymethyl furfural is formed from
hexoses to give yellow-brown condensation
products
MOLISCH TEST PROCEDURE
• is the general test for carbohydrates. 1. Place 1 ml of each 3% solution of xylose,
• It is 5% solution of a-naphthol in alcohol glucose, and starch in separately labeled test tubes
• The sugars are mixed with a-naphthol. 2. Add 3 ml of Bial’s reagent to each test tube
The test tube is inclined, and concentrated H2SO4 3. Carefully heat each tube over a Bunsen flasme
is added along the side of the tube, causing the until the solution begins to boil. Add one to two
formation of a lower layer of acid. The concentrated drops of 10% FeCl3 solution.
H2SO4 will dehydrate the sugar allowing it to react 4. Note the color of the product formed
with the alcohol forming furfural or hydroxymethyl- 5. Record your results in the table.
furfural. SELIWANOFF’S TEST
• This test is used to differentiate ketohexoses from
aldohexoses. Ketohexoses react faster with the
solution containing hydrochloric acid and resorcinol
than aldohexoses.
• The dehydrated ketohexoses form a bright cherry
red condensation product, while the aldohexose
yields only a pale pink coloration, a
negative result. In this test, prolonged heating of
samples should be avoided.
• It contains Resorcinol and
concentrated HCl.
 Benedict’s solution are reduced to Copper (I) ions,
which causes the color change.

Sample: Urine
• Aim: To detect the presence of reducing sugar in
urine
• PROCEDURE:
1. Using a pipette, accurately take 5 mL of
PROCEDURE
Benedict’s reagent and slowly transfer it to the test
1. Place 1 ml each of 3% xylose, glucose, fructose,
tube.
and sucrose in separately labeled test tubes.
2. Take 5 mL of freshly collected urine by pipette
2. Add 4 ml of Seliwanoff’s reagent to each test
and add it to the test tube with Benedict’s reagent.
tube
3. Place the test tube in boiling water bath for 2-3
3. Place the test tubes in a water bath filled with
minutes
boiling water and allow them to stay there for
4. Observe the color of the solution and note
exactly 30seconds to 1 minute
whether a precipitate was formed
4. Note the changes and record which test tube
5. Avoid prolonged heating
gives a positive result in the shortest time.
6. Record your results
5. Continue heating and observe the color changes
at 1 minute intervals
6. Record the time required for a positive test for
each sample

TESTS BASED ON REDUCING PROPERTY OF

SUGARS

REDUCING SUGARS
It must have a free aldehyde or ketone group

Common dietary monosaccharides: glucose,

galactose and fructose
BARFOED’S TEST
1. Benedict’s Test • Barfoed’s reagent – contains cupric acetate in
2. Barfoed’s Test dilute acetic acid
3. Tollen’s Test • It oxidizes monosaccharides but not
oligosaccharides
BENEDICT’S TEST • Disaccharides are less oxidized but are oxidized if
• It is a very sensitive test done under mildly they undergo prolonged heating → causing
alkaline conditions hydrolysis of the disaccharides into
• Benedict’s Reagent: Composed of CuSo4, monosaccharides → produces a positive result
Na2CO3, sodium citrate • It is used to distinguish between monosaccharide,
• Positive result: Change of color of Benedict’s disaccharides, and oligosaccharides
reagent • Positive result: Brick red precipitate
• Used to test for the presence of glucose in urine • Unlike Benedict’s test, this test is carried out
PRINCIPLE: under acidic rather than basic medium
• When Benedict’s solution and simple
carbohydrates are heated, the solution
changes to orange red/ brick red. This reaction is
caused by the reducing property of simple
carbohydrates. The copper (II) ions in the
 TOLLEN’S TEST (SILVER MIRROR TEST)
• Sugars with aldehyde groups are capable
reducing Tollen’s reagent
• Tollen’s reagent: Ammoniacal solution of silver
• Positive result: Gray to black precipitate (silver
mirror)
BARFOED’S TEST

TOLLEN’S TEST
• PROCEDURE:
1. Place 5 drops of 3% solutions of glucose,
xylose, and sucrose in separate test tubes
2. Add 2 mL of Tollen’s reagent into each tube
Preparation: Add 1 drop of NaOH solution to 6
mL of 5% AgNO3. Add dilute NH4O4 (1 mL
concentrated NH4OH + 5 mL water) until the
brown precipitate of silver oxide that forms just
dissolves
This reagent must be prepared fresh and not
stored since it decomposed when left standing
and yields an explosive decomposition product.
Discard all leftover materials.
3. Boil for about 5 minutes. Note and record your
observations.