Heme metabolism and associated diseases

BY: MENGISTU .W (B.phrm .Msc in med. Bchm )

Assi.. Professor

October, 2023 1
At the end of lecture, students should be able to understand:
• Fundamental structure and general characteristics of
porphyrins
• Metabolic pathway enzymes and intermediates
• regulation of heme biosynthesis
• biochemical defects in different type of porphyrias
• Pathway of heme catabolism that produces bilirubin;
intermediates as well as the enzymes catalyzing various steps
of bilirubin metabolism and the associated pathology.
• jaundice & cause of jaundice
• jaundice is classification

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Biological Significance of Porphyrins
• In nature, the metalloporphyrins (heme) are linked with
proteins to form conjugated proteins termed metallo-
porphyrino-proteins.
• Most important of these are
– in oxygen transport (hemoglobin)
– oxygen storage (myoglobin)
– electron transport (cytochromes)
– hydrogen peroxide inactivation (catalase), hydroxylation

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 Porphyrins
• Porphyrins are cyclic compounds composed of 4 pyrrole
rings held together by;
– methenyl ( = CH-bridges)
• The metal ions can bind with nitrogen atoms of pyrrole
rings to form complexes
• Heme is an iron-containing porphyrin while
– chlorophyll is a magnesium-containing porphyrin
• Thus both are classical examples of metalloporphyrins

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 Nomenclature of porphyrins
 Hans Fischer, the father of
porphyrin chemistry, proposed a
model for presentation of porphyrin
structures. Fig.1
1. The structure of porphyrins
(C20H14N4) has four pyrrole rings
namely I, II, III and IV
Are joined through methenyl bridges
• Naturally occurring porphyrins contain
substituent (Side chains) groups w/c
replacing
– the 8 hydrogen atoms of porphyrin nucleus
Fig. 1: Structures of porphyrin [I-IV are
pyrrole rings; 1-8 are substituent
positions; α,β,γ, & δ are methylene
( =CH- ) bridges.]
 Nomenclature

2. porphyrins vary in the nature and position of the side

chains attached to these sites
• The three most important porphyrins in humans are
uroporphyrin(URO), coproporphyrin(COPRO), and
protoporphyrin (PROTO)
– URO has four propionate and four acetate side chains
Uroporphyrins contains acetate(-CH2-COO-) and propionate
(-CH2-CH2-COO-) side chains
– COPRO has four propionate and four methyl groups.
– PROTO has four methyl, two vinyl and two propionate groups

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Nomenclature of porphyrins

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 Nomenclature of porphyrins
3) These side chains are either;
symmetrically or asymetrically ordered on pyrrole rings
e.g. Type I uroporphyrins-I, arranged as A acetate (A) alternates with
P(propionate) around the tetra pyrrole ring
Type-III porphyrines (e.g. uroporphyrin III) which contain an
asymmetric substitution on ring D
 are more common in the biological system ,meaning that the
expected arrangement of side chains is reversed on ring IV
 Originally, Fischer placed them as IX series hence they are
more popularly known as type IX porphyrins.

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 Nomenclature of porphyrins…
In case of PROTO, it is possible to arrange the two propionate,
two vinyl and four methyl groups in 15 different configurations.
Only the type IX isomer (Fig.) is produced in the human body

Fig. . Structure of protoporphyrin IX

Heme is a cyclic tetrapyrrole with a hexavalent iron (Fe2) atom at the centre that is
coordinate with four pyrrole nitrogens.
Haem is the final product of the porphyrin synthetic pathway

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Nomenclature of porphyrins
• The Fischer’s models
of important
porphyrins
(uroporphyrin I and III;
coproporphyrin I and
III; protoporphyrin IX
and heme) are depicted
in Fig.1

Fig. 1 :Fischer’s shorthand models of physiologically important

porphyrins [A–Acetate( CH3COO–); P–Propionyl CH2CH2COO–);M–
Methyl ( CH3 ); V–Vinyl ( CH CH2)] 10
 Nomenclature of porphyrins…
• The two major sites of the heme biosynthetic pathway
are
– bone marrow (erythroid cells), which synthesize
• approximately 85% of the body’s heme
– the liver, second important sites for the synthesis of heme,
though some other tissues also synthesize heme in small
amounts
• It requires participation of eight enzymes to synthesize heme
of these enzymes, four (first and the last three) are
mitochondrial and the rest are cytosolic (Fig.)
• The reactions are all irreversible, occurring in 3 stages:

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 Biosynthesis of heme

• Heme is the most important porphyrin containing compound

• It is primarily synthesized
– in the liver and the erythrocyte
• Heme synthesis occurs in all cells due to the requirement for heme
as
– a prosthetic group on enzymes ,Tryptophan pyrrolase
etc…
– electron transport chain proteins with d/f Cytochrome
– the formation of hemoproteins e.g. cytochrome P450
involved in detoxification , while
– in the erythroid cells, it is necessary for the synthesis of
hemoglobin, Myoglobin

• Mature red blood cells lack mitochondria and are unable to

synthesize heme 13
 Porphyrins in cancer therapy

• The photodynamic properties of porphyrins can be

used in the treatment of certain cancers.
• This is carried out by a technique called cancer
phototherapy.
• Tumors are capable of taking up more porphyrins
than normal tissues
• The cancer phototherapy/ activated by light
– is carried out by administering hematoporphyrin (or
other related compounds) to the cancer patient.
• When the tumor is exposed to an argon laser, the
porphyrins get excited and produce cytotoxic
effects on tumor cells 14
 Biosynthesis of heme ….
The rxn occur partly mitochondrial and partly cytosolic:
The pathway is initiated in the mitochondria, and the first
intermediate, ALA, diffuses into the cytosol where next
few reactions, up to formation of coproporphyrinogen III
takes place.
The latter compound diffuses into the mitochondrion where
the last three reactions occur

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 Biosynthesis of heme occurs in the following stages
1. Formation of δ-aminolevulinate :
• Glycine, a non-essential amino acid
and succinyl CoA, an intermediate
in the citric acid cycle, are
– the starting materials for porphyrin
synthesis
• Glycine combines with succinyl
CoA to form δ-aminolevulinate
(ALA).
• The reaction catalyzed by a
pyridoxal phosphate dependent δ
amino levulinate synthase occurs in
Figure 2. Pathway of porphyrin
the mitochondria synthesis: ALAS = δ-
• ALA is a rate limiting step in the aminolevulinic acid synthase;
pathway of heme biosynthesis CoA = coenzyme A; PLP =
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pyridoxal phosphate.
 Biosynthesis of heme …

• Many drugs (e.g. antifungal, anticonvulsants) increase ALA

synthase activities
• Because these drugs are metabolized in liver by Cyt. P450,
a heme containing enzyme
• This results in increase synthesis of Cyt. P450, leading to
consumption of heme
In erythroid cells
– heme synthesis is under the control of erythropoietin hormone
and the availability of intracellular iron
– heme is synthesized for incorporation into hemoglobin
– Accumulation of heme in erythroid cells is desired :
as it stimulate more globin chain synthesis

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 Biosynthesis of heme…

2.Synthesis of porphobilinogen:
Two molecules of ALA condenses
to form porphobilinogens by ALA
dehydratase, a Zn-containing
enzyme
• It is sensitive to inhibition by heavy
metals such as lead by combining
with SH groups
• Hemin negatively regulates hepatic
ALA synthase enzyme &
decreasing synthesis of hepatic
ALA
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 Biosynthesis of heme…
3) Formation of uroporphyrinogen
(In cytosol)
• The condensation of four molecules
of porphobillinogens results in the
formation of tetrapyrrrole,
hydroxymethyl bilane, a reaction
catalyzed by Hydroxymethyl
bilanesynthase
• Defects of heme synthesis after
formation of hydroxymethylbilane
leads to photosensitivity of patients
• Isomerization and cyclization by
uroporphyinogen III synthase
 leads to formation of
Uroporphyrinogen III 19
 Biosynthesis of heme…

4. Conversion of uroporphyrinogen III to protoporphyrin IX:

This is catalysed by a series of reactions

(a) Uropprphyrinogen III undergoes decarboxylation at its acetate

groups, to metyl groups & generating coproporphyrinogen III, a
reaction carried out by uroporphyrinogen decarboxylase

(b) Coproporphyrinogen oxidase converts (oxidative

decarboxylation) two of the propionate side chains (P) to vinyl
groups (V) & results in formation of protoporphyrinogen IX

c) Protoporphyrinogen oxidase, oxidizes methylene

groups( CH2-) interconnecting pyrrole rings to methenyl groups
This leads to the synthesis of protoporphyrin IX
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5.Synthesis of heme from
protoporphyrin IX heme (In
mitochondria):
• The incorporation of ferrous iron
(Fe2+) into protoporphyrin IX is
catalysed by the enzyme
ferrochelatase or heme
synthetase.
• This enzyme can be inhibited by
Lead poisoning
• If *Ferrochelatase and ALA DH
are inhibited
• Protoporphyrin and ALA
accumulate in urine 21
Fig. 3 Biosynthesis of heme [A–Acetyl ( CH2 COO– ); P–Propionyl (CH2 CH2 COO–);M–Methyl ( CH3); V–Vinyl
(- CH CH2)].
 Biosynthesis of heme…

Two different mechanisms exist for the regulation of heme

biosynthesis in the liver and the erythroid cells
Regulation in the liver :
• The first committed step in heme biosynthesis, catalyzed by
δ-aminolevulinate (ALA) synthase, is regulatory.
• Heme or its oxidized product hemin (Fe3+) controls this
enzyme activity by three mechanisms
1. Feedback inhibition
2. Repression of ALA synthatase
3. Inhibition of transport of ALA synthase from outside
cytosol to mitochondria (the site of action)

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• Acute intermittent porphyria: This occurs due to a
mutation in hydroxymethylbilane synthase, which
– leads to an accumulation of ALA and porphobilinogen
• Defects of heme synthesis after formation of
hydroxymethylbilane leads to photosensitivity of patients

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 Biosynthesis of heme…

Effect of drugs on ALA synthase activity :

e.g. phenobarbital etc.
• these compounds, are mostly metabolized by a heme
containing protein, cytochrome P450
• On administration of drugs, cellular levels of heme are
depleted
– due to its increased incorporation into cytochrome P450
• The reduced heme concentration
– increases synthesis of ALA synthase to meet the cellular
demands
 Biosynthesis of heme…

Regulation in the erythroid cells :

• The enzyme ALA synthase does not appear to control the
heme synthesis in the erythroid cells
• Uroporphyrinogen synthase and ferrochelatse mostly
regulate heme formation in these cells
• Further, the cellular uptake of iron also influences heme
synthesis
• It is observed that heme stimulates globin synthesis

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 PORPHYRIAS

• Porphyrias are the metabolic disorders of heme synthesis,

characterized by ;
– the increased excretion of porphyrins or
– porphyrin precursors
• Each porphyria results in the accumulation of a unique
pattern of intermediates caused by the deficiency of an
enzyme in the heme synthetic pathway
• Porphyrias are either inherited or acquired
are classified as
– erythropoeitic (enzyme deficiency is in the erythropoitic cell)
or
– Hepatic (enzyme deficiency is in the liver)
– In the liver Porphyrias as acute and chronic 27
 Porphyrias …

• All the known porphyrias are inherited as autosomal

dominant disorders. Photosensitivity
• One common feature of porphyria is decrease synthesis of
heme, and causing increase in ALA synthatase activity
• Porphyrin accumulation leads to cutaneous symptoms and
urine that is red to brown in natural light and pink to red
in fluorescent light Fig. 4:

Fig. 4:Porphyrin accumulation

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The following points about this biosynthetic p.way are noteworthy

1. Localization: Major sites of biosynthesis include

– liver, which synthesizes a number of heme-proteins ( prominently, the
cytochrome P-450), and
– erythrocyte-producing stem cells, which are active in hemoglobin synthesis.
2. Polarity of intermediates: The initial intermediates of this pathway
are polar.
Uroporphyrinogen is strongly polar since all its side chains (four
acetate and four propionate) are predominantly polar.
• The subsequent intermediates become more and more non-polar .
3. Porphyrinogens (reduced) vs Porphyrins:
• They are not coloured because their pyrrole rings are connected by methylene (–
CH2-) bridges ,and the double bonds are not conjugated over the whole system.
• Porphyrinogens can be converted to the corresponding coloured porphyrins on
exposure to light, when they lose hydrogen atoms

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 Features of Porphyrias

4. The route of excretion of the accumulated porphyrins ,

urine or faeces-depends on their polarity.
For example:
– Uroporphyrinogen with its eight carboxyl groups is most
water-soluble and is excreted almost entirely in urine.
– Coproporphyrin has four carboxylic groups and is excreted by
either route
– Protoporphyrin, with only two carboxyl groups is excreted
exclusively in faeces

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 Features of Porphyrias

4. Level of metabolic block determines clinical features:

(a)Early block: If the metabolic block lies early in the pathway,
so that synthesis of even the first tetrapyrrole ring does not
occur, the defect may present as an acute pain crisis.
This is because the accumulated metabolites (ALA and PBG)
cause excitation of visceral pain fibres.
(b)Later block: When the metabolic block lies beyond the
formation of the first tetrapyrrole ring, cutaneous manifestations
are the predominant features.

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The different types of porphyrias are described (Table)

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Fig .Summary of heme synthesis and porphyrias
 Heme Breakdown

• End products of heme breakdown are bile

pigments: bilirubin and biliverdin.
About 80-85% of the heme degraded each day is
provided by
old erythrocytes; and
 the rest of the 15-20 % comes from
the immature erythrocytes and other heme containing
proteins,myoglobin and cytochromes

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 DEGRADATION OF HEME TO BILE PIGMENTS

• Erythrocytes have a life span of 120 days.

• At the end of period, they are removed from the
circulation
• Erythrocytes are taken up and degraded by:
– the macrophages of the reticuloendothelial (RE) system in
the spleen and liver
• The hemoglobin is cleaved to ;
– the protein part, globin and non-protein part heme
• About 6 g of hemoglobin per day is broken down, and
resynthesized in an adult man (70 kg)

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 Degradation of heme …
• Microsomal heme oxygenase :

A
• An enzyme utilizes NADPH and
O2 and cleaves the methenyl
bridges between the two pyrrole
rings (A and B) to form biliverdin
• Simultaneously, ferrous
iron(Fe2+) is oxidized to ferric
form (Fe3+) and released.
• The products of heme oxygenase
reaction are
– biliverdin (a green pigment), Fe3+
and carbon monoxide (CO) Fig : 5. Degradation of heme
Fig. 6:Degradation of heme to bile pigments (Note :Colours used in structures represent change in the
specific reaction only).
 Degradation of heme …

• Transport of bilirubin to liver : Bilirubin is lipophilic (because

of polar nature of albumin) which enables it to move freely in
plasma.
• Certain drugs and antibiotics (e.g sulfonamides, salicylates)
can displace bilirubin from albumin
• thus increasing the unbound-bilirubin concentration in
plasma
• This may have hazardous consequences in newborn infants
because , unbound-bilirubin can cross the blood-brain barrier
and enter the central nervous system, to cause neuronal tissue
damage: the condition is known as bilirubin encephalopathy.

Fig. Breakdown of hemoglobin in reticulo-endothelial cells of liver, spleen and bone marrow.
(Total amount of hemoglobin in adult body is about 750 g and daily turnover rate is around 6 g)
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 Degradation of heme …

Unconjugated bilirubin:
• Bilirubin that are not conjugated with gluconic acid ,
also called hemo-bilirubin, indirected bilirubin
• Uncojugated bilirubin is normally not excreted
conjugated bilirubin:
• Bilirubin that are conjugated with gluconic acid, also
called hepatic bilirubin, directed bilirubin

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 Degradation of heme …
• On coming in contact with the hepatocyte
surface, unconjugated bilirubin is
preferentially metabolized
• The metabolism involved 3 steps:
1. Hepatic uptake :is an active(energy
requiring) process, which requires mediation
of the carrier protein.
• Within the hepatocyte, two proteins: ligand
in Y & Z protein, bind the bilirubin
2. Conjugation : occur in endoplasmic
reticulum UDP-glucuronyl transferase,
– Product: mono or diglucuronides.
– The process of conjugation can be induced
by drugs phenobarbital
3. Secretion in bile 40
Major differences between unconjugated and conjugated bilirubin

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 Formation of urobilins in the intestine
• in the intestine bilirubin diglucuronide is
– hydrolyzed and
– reduced by bacteria in the gut to yield urobilinogen, a colorless
compound
• Most of the urobilinogens in intestine are oxidized by
intestinal bacteria to stercobilin, which gives stools their
characteristic brown color.
• Some urobilinogen is reabsorbed from the gut into the portal
blood and transported to the kidney, where it is converted to
the yellow urobilin and excreted, giving urine its characteristic
color.
– Other urobilinogen back via enterohepatic circulation

Bilirubin diglucuronide urobilinogen 42

 JAUNDICE

• The normal serum total bilirubin concentration is in the range

of 0.2 to 1.0 mg/dl.
• Jaundice (French : Jaune-yellow) is a clinical condition
characterized by yellow colour of the white of the eyes
(sclerae) and skin
• It is caused by the deposition of bilirubin in the blood/serum
& term as hyperbilirubinemia

Fig.7: Jaundice : ( also called icterus considered as a symptom rather than a disease
) refers to the yellow color of the eyes & skin and scleare caused by deposition of bilirubin,
secondary to increased bilirubin levels in the blood. 43
 Classification of jaundice

• jaundice is classified into three major types

– Pre /hepatic or hemolytic
– hepatic and
– Post hepatic (obstructive jaundice).
1. Hemolytic jaundice :This condition is associated with
increased hemolysis of erythrocytes
e.g. incompatible blood transfusion, malaria, sickle-cell
anemia & thalassemia
• This results in the overproduction of bilirubin, beyond
– the ability of the liver to conjugate and excrete.
– A portion of the bilirubin remains unconjugated, which results
in rise in serum concentration, hyperbilirubinaemia

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 Classification of jaundice

• More bilirubin is excreted into the

bile,
• the amount of the urobilinogen
entering the enterohepatic
circulation is increased, and urinary
urobilinogen is increased.
– Unconjugated bilirubin is elevated in
blood.
• Hemolytic jaundice is X-’ized by
– Increased excretion of urobilinogen
in urine
– Dark brown colour of feces due to
high content of stercobilinogen
Fig :8 Summary of bilirubin
metabolism (UDP-GlcUA—UDP-
glucuronic acid) 45
 Jaundice…
2. Intra Hepatic (hepatocellular) jaundice :
This type of jaundice is caused by dysfunction of the liver
cells , due to damage to the parenchymal cells
• viral hepatitis is the most common
• toxic liver damage
• Damage to the liver cells adversely affects
– the bilirubin uptake and
– its conjugation by liver cells
Hepatic jaundice is characterized by
• the serum levels of both the conjugated and the unconjugated
bilirubin rises in this type of jaundice ,referred to as a “biphasic
rise”
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 Jaundice…

• The reasons for “biphasic rise”

– The damaged hepatocytes have impaired capacity to
take up the circulating bilirubin and to conjugate
– The inflammatory edematous cells produces
intracellular biliary obstruction element
Consequently,
– the conjugated bilirubin formed in the hepatocytes may
leak out of the damaged cells into the blood circulation
In Hepatocellular jaundice, Since there is less conjugated of
bilirubin ,the amount that enters the gut is also less, leading to
decreased formation of urobilinogen and stercobilinogen
• As a result, urinary and fecal excretion of urobilin and
stercobilin falls 47
 Jaundice…
– Increased activities of alanine transaminase (SGPT) and
aspartate transaminase(SGOT) released into circulation
due to damage to hepatocytes.
– The patients pass pale, clay colure stools due to the
decrease of stercobilinogen.
3.Obstructive jaundice :
– This is due to an obstruction in the bile duct that prevents
the passage of bile into the intestine.
– The obstruction may be caused by gall stones, tumors etc.
– Due to the blockage in bile duct, the conjugated bilirubin
from the liver enters the circulation

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 Jaundice…
Obstructive jaundice is characterized by
– Increased concentration of conjugated bilirubin in serum.
– Serum alkaline phosphatase is elevated as it is released from
the cells of the damaged bile duct.
– Dark coloured urine due to elevated excretion of bilirubin
and
– clay coloured feces due to absence of stercobilinogen.
– Feces contain excess fat indicating
• impairment in fat digestion and absorption in the absence of
bile (specifically bile salts)

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 JAUNDICE DUE TO GENETIC DEFECTS

Neonatal jaundice
• Physiological jaundice is not truly a genetic defect
• It is caused by increased hemolysis coupled with immature
hepatic system for the uptake, conjugation and secretion of
bilirubin.
• The activity of the enzyme UDP-glucuronyltransferase is low
in the newborn
• in some infants the serum uncojugated bilirubin is highly
elevated (may go beyond 25mg/dl), which can cross the blood
brain barrier.
– kernicterus that causes mental retardation

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 Neonatal jaundice…

• Phototherapy : Bilirubin can absorb blue light (420–470

nm) maximally.
• Phototherapy deals with the exposure of the jaundiced
neonates to blue light.
• By a process called photoisomerization, the toxic native
unconjugated bilirubin gets converted into a non-toxic
isomer namely lumirubin.
• Lumirubin can be easily excreted by the kidneys in the
unconjugated form (in contrast to bilirubin which cannot be
excreted)

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 JAUNDICE …
Drug phenobarbital
• is used in the treatment of neonatal jaundice, as it can
induce bilirubin metabolizing enzymes in liver.
• is indicated when the bilirubin level stays dangerously
high despite phototherapy.
• Blood exchange transfusion is required in extreme cases,
when sustained increase in serum bilirubin above 20
mg/dL occurs

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• Conjugated hyperbilirubinaemia is typical for biliary
obstruction (cholestasis);
• unconjugated hyperbilirubinaemia occurs in haemolytic
conditions and in otherwise normal newborns; and
• a mixed hyperbilirubinaemia occurs in hepatocellular damage,
e.g. in viral hepatitis and toxic liver damage.

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 Genetic Causes of Jaundice

• There are a number of genetic disorders, referred to as

inherited hyperbilirubinaemia, that impair bilirubin
conjugation or secretion.
• Gilbert’s syndrome, Crigler–Najjar syndrome, Dubin
Johnson’s syndrome and Rotor syndrome are some
commonly described disorders.
• All except Gilbert’s syndrome are extremely rare
disorders; the latter affects 5% of the population

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