Heme metabolism

Topics to be discussed
Structure of heme and porphyrins.
Heme biosynthesis
Defects in heme synthesis
Catabolism of heme,
Defects in Catabolism of heme.

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Learning Objectives

After the end of this session you should able to:

Understand fundamental structure and general characteristics of
porphyrins.
Discuss biosynthesis and catabolic pathways of heme and its
regulation.
Discuss the disorders of heme metabolism.

Describe metabolism of bilirubin and main types of jaundice.

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Structure of Heme

N
H
pyrrole

pyrrole rings
Heme

Heme:derivative of porphyrin.
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Porphyrins
cyclic nitrogen-containing compounds.
linkage of Tetra pyrrole rings
methyne (=HC—) bridges.
conjugated double bond.
absorb visible light, coloured compounds.

pyrrole rings (I, II, III, IV) &

methyne (=HC—) bridges :alpha, beta,

gamma and delta.

Substituent position on the ring 1 to 8. 4

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6
Substituent Side chains on Porphin Nucleus

Type I series:-symmetrical arrangement (1, 3, 5, 7 and 2, 4, 6, 8).

type III porphyrin (III series) :asymmetrical (1, 3, 5, 8, and 2, 4, 6, 7).

(in ring IV, expected order of A and P substituents reversed).

Type III :most predominant in biological systems.

• Heme & its immediate precursor, protoporphyrin IX
• type III porphyrins.

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Porphyrins
Complexes with metal ions bound to nitrogen atom of pyrrole rings.
Examples :-iron porphyrins such as heme.
• magnesium-containing porphyrin chlorophyll.
Hemoproteins widely distributed in nature.

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Structure Of Porphyrins

Porphyrinogens
bridge atom b/n pyrrole rings
reduced state (methylene).
methyl (-CH2-) .

no conjugated double bonds.

colorless and relatively less stable.
Reduced
coloured porphyrins exposure Porphyrin precursors
Colorless
Intermediates of heme
to light,(lose H). synthesis
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Biosynthesis of heme
 In most cells . But liver cells & bone marrow most
active.
• Bone marrow erythroid cells,
• Hgb(80–85% heme)
• Hepatocytes:-cytochrome, particularly cytP450.
• Mature RBCs cannot make(lack mitochondria).

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Biosynthesis of heme
four in mitochondrion &four in cytosol enzymes.

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 Primary sites: liver &bone marrow.

1)Formation of -aminolevulinic acid (ALA)

Glycine and succinyl CoA.
 Supplied all carbon and nitrogen .
 Condense to form ALA,
 by ALA synthase.
 Coenzyme : PLP

 feedback inhibition by heme/hemin. 12

Biosynthesis of heme
dehydration of two ALA molecules
• form monopyrrole, porphobilinogen.
• cytosolic ALA dehydratase (porphobilinogen synthase).
Zinc-containing enzyme
Lead inhibits its activity by displacing catalytically
important zinc.

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Biosynthesis of heme
four porphobilinogen linear tetrapyrrole.
 by loss of four ammonia

Ring closure & isomerization

⇒uroporphyrinogen III
by uroporphyrinogen synthase.

Further intermediates coproporphyrinogen III,

protoporphyrinogen IX and protoporphyrin IX.

protoporphyrin IX to heme by ferrochelatase.

first reaction & last three reactions mitochondrial.

with other four reactions in cytosol.
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Biosynthesis of heme.

• Generally four basic

processes:
Formation of pyrrole.tetrapyrrole
odification of tetrapyrrole side
chains.
⇒Oxidation of protoporphyrinogen
IX to protoporphyrin IX and
insertion of iron.
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Biosynthesis of heme
Ferrochelatase (mitochondrial inner membrane)
Inserts ferrous (Fe2+) into protoporphyrin IX.
Requires ascorbic acid, cysteine as reductants
Sensitive to Fe deprivation, heavy metals esp Pb.
Iron deficiency leads to insertion of Zn2+ important clinical indicator
of iron deficiency.
CH2 protoporphyrin IX CH2 heme
CH CH3 CH CH3

H3C CH CH2 H3C CH CH2

NH N N N
Fe++ 2H+
Fe
N HN N N
H3C CH3 H3C CH3

CH2 CH2 Ferrochelatase CH2 CH2

CH2 CH2 CH2 CH2

- - - -
COO COO COO COO
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Regulation of haem synthesis
• ALA Synthase
• short biological half-life of 1 to 3 hours.
• very unstable, low in concentration in tissues,
• rate-limiting step.

• Two forms of ALAS: ALAS1 &ALAS2.

 Regulation mediated by d/t mechanisms.
both forms require pyridoxal 5-phosphate (PLP).
 both expressed as homodimers.
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Regulation of haem synthesis
ALAS1
♣housekeeping gene & expressed in all cells.
♣key regulatory of heme syntheis in liver.
♣feedback regulated by heme.
♣induced by a variety of drugs, steroids &other chemicals
that also induce cytP450.

erythroid form of ALAS (ALAS2)

does not undergo feedback regulation by heme.
Not induced by drugs that affect ALAS1,
Not induced by factors induce liver cytP450.
Mediated by erythroid-specific transcription factors.
availability of iron as Fe/S clusters. 18
Regulation of haem synthesis
• ALA synthase In liver, control at three levels.
1. Allosteric inhibition .
by heme, hemin and hematin.
Not important control mechanism
 to high concentrations necessary(10–5 M)
2. Inhibits import of ALAS from cytosol (site of synthesis) to
mitochondria.
3. Repression of transcription of ALA synthase gene.
• by inhibiting mRNA export from nucleus or
• by targeting exported mRNA for degradation.
principal form of control (enzyme has a short half life (t1/2 = 1
hr).
most effective regulation(works at low concentration).
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Regulation of haem synthesis

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Regulation of haem synthesis
Induction of ALA synthase in liver
xenobiotics ,steroids & drugs, such as barbiturates, phenytoin,
induce synthesis of cytP450 (w/c use up heme).
Induces transcription of ALA synthase and haem synthesis.
Glucose: inhibits transcription of gene for ALA synthase.
Lack of Vit B6 /drugs like INH (isonicotinic acid hydrazide) that
decrease availability of PLP /
decrease synthesis of ALA.

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• CLINICALCORRELATES
Ferrochelatase &ALA dehydratase
highly sensitive to inhibition by lead
poisoning.
• High levels of lead combining with SH groups in
enzymes
• δ-ALA and protoporphyrin IX accumulate.
increase in ALA in circulation in absence of
increase in porphobilinogen lead poisoning.

• Heme production decreased,& anemia from a

lack of Hgb. 22
X-Linked Sideroblastic Anemia(XLSA)
• due to mutations of ALAS2 gene
• result in X-linked sideroblastic anemia.
• erythroblasts with non- heme iron organelles,
• do not make enough Hgb,
• with iron-laden mitochondria surrounding
nucleus (ring sideroblasts).
• iron-loaded erythroblasts, present in bone marrow,
• also called congenital (hereditary) sideroblastic anemia,
hereditary iron-loading anemia, X-linked hypochromic anemia.

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Porphyrias
• group of genetic or acquired rare disorders.
• defective heme synthesis.
• inherited in autosomal dominant manner,
• except:-congenital erythropoietic porphyria.
• accumulation of metabolites prior to block.
• deficiency of intermediates beyond.
 Induced by alcohol, stress, infection, starvation, hormonal changes
(e.g., menstruation),and certain drugs.
 cutaneous and non-cutaneous, (or both) or as acute and non-acute.
 Hepatic or erythropoietic (or both).

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• Acute porphyrias
 severe abdominal pain with neurological and
psychiatric symptoms.
• excess early precursors (ALA, PBG)
• toxic effects on nervous system.
• Not photosensitive (occasional photosensitivity of
skin, discoloured urine).

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Acute porphyrias
Three porphyrias known as acute cause of emergency
admissions with abdominal pain.
• also cause neuropsychiatric symptoms.
• Acute intermittent porphyria (AIC)
• most common form of acute porphyria
world-wide.
• Hereditary coproporphyria
• variegate porphyria,

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Non-acute :Cutaneous porphyrias
mainly photosensitivity syndromes.
excess later intermediates (Uro-, Copro-, Proto-)
upon exposure to sunlight mild to severe skin lesions
painful blistering of skin, scarring, loss of
fingers, corneal scarring.
Most common :porphyria cutanea tarda.

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Biochemical basis of major signs and symptoms of porphyrias.
Photosensitivity
accumulation of porphyrinogens in skin.
oxidation products, (Porphyrin), cause photosensitivity, at 400
nm.
exposed to light of this wave length, porphyrins become
“excited”
• react with molecular oxygen to form oxygen radicals.
• oxygen radicals injure lysosomes and other subcellular organelles,
releasing degradative enzymes.
• variable degrees of skin damage, including scarring.
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Biochemical basis of major signs and symptoms of porphyrias.

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• Non-cutaneous porphyrias
• acute intermittent porphyria
(AIP) &
• ALA dehydratase
deficiency porphyria.

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Porphyrias

Three most common porphyrias,

porphyria cutanea tarda
acute intermittent porphyria, &
erythropoietic protoporphyria.

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• Porphyria cutanea tarda
most common form of porphyria worldwide .
Deficiency of uroporphyrinogen III decarboxylase.
cutaneous involvement and liver abnormalities.
Inflammation, blistering, shearing of skin areas exposed to sunlight .
Hyperpigmentation.
Red-brown to deep-red urine
Symptoms exacerbated by excess hepatic iron, alcohol
consumption & induction of cytochrome P450

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• Acute Intermittent Porphyria (AIP)
• 2 nd most common overall. Most common of Acute
porphyrias,
• PBG deaminase (uroporphyrinogen -I-synthase)
deficient.
• ALA &PBG will accumulate in body tissues and
fluids .
• excretion in urine giving it a wine red colour.
• No photosensitivity.
• Hepatic porphyria, abdominal and neurological
manifestations.
• most common hepatic type porphyria .
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• Erythropoietic protoporphyria (EPP)
• 3rd overall &.
• 2nd most common cutaneous.
• most frequent childhood porphyria.
• Ferrochelatase deficiency.
• Photosensitivity: burning, itching, swelling,
and reddening of the skin.
• Liver affected only in a small
percentage of patients.

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• Treatment of porphyrias.
• avoid drugs inducing cytochrome P450.
• Ingestion of large amounts of CHOs (glucose loading) or
• administration of hematin (hydroxide of heme) or hemin
(Panhematin), .
• repress ALAS1, diminished production of harmful
heme precursors.
• Administration of β-carotene;
• to lessen production of free radicals, diminishing
photosensitivity.
• Sunscreens that filter out visible light can also
helpful to such patients.
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 Heme breakdown
In macrophages of reticulo endothelial system (liver, spleen, bone
marrow).
Most (80%) from Hgb. & 20%:other heme proteins or inefficient
erythropoiesis.
Heme oxidatively cleaved to biliverdin .
♣by heme oxygenase, substrate-inducible enzyme.
♣Heme a substrate and a cofactor for reaction.
♣iron that reaches heme oxygenase usually

its ferric form (hemin).

• rate-limiting step in catabolism of heme. 38
 Heme catabolism, bilirubin and jaundice cont…
Heme broken down into a linear molecule,
 by heme oxygenase,using NADPH and oxygen and

Generates biliverdin, plus carbon monoxide (CO) and iron

and NADP+.

Biliverdin →bilirubin
 by biliverdin reductase, using NADPH.

Birds & amphibians excrete green colored biliverdin directly.

• Bilirubin :insoluble in aqueous solution
• Bilirubin transported to Liver bound to serum albumin.
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Bilirubin metabolism

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Uptake of Bilirubin by Liver
• via facilitated transport system.
• binds to cytosolic proteins such as glutathione S-transferase,
to prevent from reentering blood.
Conjugation With Glucuronate.
♣bilirubin-specific UDP-glucosyl transferase.
♣ transfer two glucosyl moieties from UDP-glucuronate.
♣induced by several drugs, including phenobarbital.

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Secretion of Bilirubin Into the Bile
♣by active transport mechanism.
♣Need a multi specific organic anion
transporter(MOAT)
♣located in the plasma membrane of bile canaliculi.
♣inducible by same drugs that induce conjugation.
♣Most excreted type:-bilirubin diglucuronide.
♣In abnormal conjugation(eg,in obstructive jaundice),
♣mono glucuronides predominant.

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 Heme catabolism, bilirubin and jaundice
Bile duct into small intestine.
Conjugated bilirubin
excreted via bile duct to small intestine.
bacteria B-glucuronidases ,deconjugate it
to urobilinogen.
Urobilinogen excreted mostly in feces,
⇒urobilinogen⇒ stercobilinogen ⇒excreted
in stool.
Some of urobilinogen
recycled back to liver
 bloodstream & excreted in
urine(urobilin).
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Heme catabolism, bilirubin and jaundice

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Heme catabolism, bilirubin and jaundice…

Hyperbilirubinemia :imbalance b/n its production and excretion.

result from elevation of unconjugated or conjugated bilirubin levels.
Normal serum level<1.5 mg/dL (mostly unconjugated or direct).
High levels, of bilirubin is toxic :Inhibits
DNA synthesis and uncouple oxidative phosphorylation.
ATPase activity in brain mitochondria.
d/t classes of enzymes dehydrogenases, electron transport proteins,
hydrolases, & RNA synthesis, protein synthesis, and carbohydrate
metabolism.
cause jaundice,
Bilirubin encephalopathy(kernicterus) :yellow discoloration of basal
ganglia in babies with intense jaundice and is termed.

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Clinical correlates
• Jaundice : -accumulation of excessive levels of bilirubin or
bilirubin diglucuronide
• yellowish discoloration of various tissues of body
skin, nail beds, and sclerae (whites of eyes) .
Jaundice clinically obvious when plasma bilirubin
concentrations exceed 50 μ mol/L (3 mg/dL).

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Types of jaundice
Hemolytic Jaundice:
Blood unconjugated bilirubin increase.
conjugated bilirubin do not change.
Total bilirubin increases.

• Hepatic Jaundice: both conjugated and unconjugated bilirubin

levels rise,
• increase in unconjugated bilirubin is more
profound.
• Obstructive Jaundice: increased conjugated bilirubin in
blood,& increased total bilirubin.
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NORMAL BILIRUBIN METABOLISM
·Uptake of bilirubin by iver mediated by a carrier protein
(receptor)

· On smooth ER, bilirubin is conjugated with glucoronic acid

· Glucoronic acid major conjugate - catalyzed by UDP glucuronyl

tranferase

·“Conjugated” bilirubin water soluble ,secreted by hepatocytes

into biliary canaliculi.

· Converted to stercobilinogen (urobilinogen) (colorless) by

bacteria in gut

· Oxidized to stercobilin which is colored

· Excreted in feces.

· Some stercobilin may be re-adsorbed by gut and re-excreted by

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either liver or kidney.
Prehepatic (hemolytic)
jaundice
Excess production of bilirubin (beyond
livers ability to conjugate) following
hemolysis.

Excretory function of liver not impaired.

Excess RBC lysis commonly result of
autoimmune disease; hemolytic disease of
newborn ; structurally abnormal RBCs (Sickle
cell disease).

High plasma concentrations of unconjugated

(free) or ( indirect bilirubin)(normal concentration
~0.5 mg/dL).
 confirmatory test:-hematology:CBC low (Hgb) 49
Prehepatic (hemolytic) jaundice
• Plasma
• Elevation in serum free bilirubin( unconjugated
bilirubin).
• but not filtered through kidney(unconjugated
bilirubin).
• urine free from bilirubin.
• Urine
• Increased excretion of urobilinogen.
• elevated urobilinogen production, absorption from colon, and excretion in urine.
Feces
• Stools appear darker than normal color due to excessive
stercobilin. 50
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Intrahepatic jaundice
• Due to:
• Impaired uptake of bilirubin into hepatic cells.
• Disturbed intra cellular protein binding or
conjugation.
• Disturbed active secretion of bilirubin into bile
canaliculi.
Reflects a generalized liver (hepatocyte)
dysfunction .
from cirrhosis, Hepatitis virus, Viral
infections, toxins, chemicals.

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Intrahepatic jaundice
Due to liver disease conjugations of bilirubin
decreased(increased indirect bilirubin).
Bilirubin that is conjugated not efficentlly secreted into
bile but leaks to blood(increased direct bilirubin).
both serum conjugated and free bilirubin levels increased.(total
bilirubin high)
hyperbilirubinemia usually accompanied by other
abnormalities in biochemical markers of liver function.
Conformational test : ALT and AST.
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Intrahepatic jaundice…
Dark coloured urine due to excessive excretion of bilirubin and
urobilinogen.
Increased activities of alanine transaminase(SGPT) & aspartate
transaminase (SGOT) released into circulation.
pale, clay coloured stools due to absence of stercobilinogen.

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Posthepatic jaundice: obstructive jaundice

obstruction of biliary tree.

failure of biliary system to excrete
conjugated bilirubin.
blockage of hepatic or common bile
ducts,
most often due to gallstones or tumors
of pancreas head, or tumors in bile
duct.
♣Bilirubin diglucuronide that cannot
excreted
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♣ regurgitates into hepatic veins and lymphatics.
Posthepatic jaundice: obstructive jaundice
• rate of bilirubin formation is normal.
Bilirubin enters liver cells and conjugated in usual way.
Due to obstruction of bile duct
Passage of bilirubin into intestine blocked.
• conjugated bilirubin
• can not pass into small intestine
• limited urobilinogen available for reabsorption and
excretion, amount of urobilin found in urine is low.
• Urine is low/free/ from urobilinogen..
• In a complete obstruction, urobilin is absent from urine.
• Stools clay color due to absence of stercobilin.

• High amounts of soluble conjugated bilirubin

• returns back into blood, increased conjugated bilirubin in serum.
• filtered through kidney & appears in urine.
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Conjugated bilirubin appears in blood and urine.
Serum alkaline phosphatase elevated(from damaged bile
duct). ALP as conformational test.

♣dark urine and pale stools.

Dark coloured urine due to elevated excretion of
conjugated bilirubin.
pale colored stools due to
absence of stercobilinogen.
absence of fecal bilirubin or urobilin,

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Neonatal “Physiologic Jaundice“
immature hepatic system for uptake, conjugation, and
secretion of bilirubin.
Bilirubin glucosyltransferase activity and
synthesis of UDP- glucuronate, reduced.

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Neonatal “Physiologic Jaundice“

When plasma unconjugated bilirubin exceeds with

albumin (20-25 mg/dL),
bilirubin penetrate blood-brain barrier.

hyperbilirubinemic toxic encephalopathy, or

kernicterus, result in mental retardation.

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New born jaundice treatment:
Phototherapy, which converts bilirubin to other compounds that can
be excreted in urine, prevent permanent damage to nervous system
(kernicterus).

Breakdown bilirubin (IDD) &bilirubin converted into a non-toxic

isomer lumirubin. Lumirubin easily excreted by kidneys in
unconjugated form.
phenobarbital, promotes bilirubin-metabolism.

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Measurement of Bilirubin in Serum
♣Colorimetric method.
♣bilirubin reacts with diazotized sulfanilic acid and
form reddish-purple color.
♣An assay conducted in absence of added methanol
measures “direct bilirubin,”(bilirubin glucuronide).
♣only conjugated bilirubin is soluble in water,
♣ An assay conducted in presence of added methanol
measures total bilirubin.
♣Both conjugated & unconjugated :soluble in ethanol and
methanol,& participate in the reaction.
♣ D/ce b/n the two “indirect bilirubin,”(unconjugated bilirubin).
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Urinary Urobilinogen & bilirubin are Clinical Indicators
• In complete obstruction of bile duct

 bilirubin no access to intestine for conversion to urobilinogen.

 so no urobilinogen present in urine.

 Conjugated bilirubin in urine without urobilinogen suggests

intrahepatic or posthepatic obstructive jaundice.

Lack of urobilinogen in urine and feces indicates biliary obstruction;

stools whitish ("clay-colored") owing to absence of bile pigment.

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In jaundice secondary to hemolysis,

in hemolytic jaundice,

increased production of bilirubin .

Bilirubin not usually found in urine.

urobilinogen, appears in urine in large amounts.

increased urobilinogen and absence of bilirubin is suggestive of

hemolytic jaundice.

Excretion increases urinary and fecal urobilinogen.

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Causes of Hyperbilirubinemia

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