189

HEME SYNTHESIS AND BREAKDOWN

HEMOGLOBIN
Questions:
1.
Explain the structural organization of haemoglobin. Explain how the
structure of hemoglobin is carrying out its functions? Pon May 2012
2.
Abnormal hemoglobins Nov 1991; March 2002
3.
Structure of hemoglobin - Aug 2004
4.
What does the shift of oxy-dissociation curve of hemoglobin to the right
mean? Pon May 2012
5.
Hemoglobinopathies - March 2002
6.
Sickle cell anemia Pon May 2013
7.
A 3 year old girl with complaints of lethargy, weakness and frequent
episodes of abdomen and bon pain was brought to the hospital. On
examination sclera was yellow and spleen enlarged. The blood smear
showed crescent shaped RBcs and the indirect van den Berg test was
positive. What is the probable diagnosis? How would you confirm your
diagnosis? Pon Nov 2011
8.
Sickle cell anemia is due to an abnormal globin chain in Hb. Discuss ots
molecular basis. Pon Nov 2006
9.
What is the molecular defect in HBS and HbM? Pon May 2004
10.
How does HbS and HbS differ in their primary structure?
11.
What is carboxy Hb? In what conditions does it increase in blood? Pon
May 2007
12.
Write a note on HBA1c and its significance. Pon Nov 2011
Structure of hemoglobin:
1. Hemoglobin is found exclusively in red blood cells (RBCs), where its main function is
to transport oxygen (O2 ) from the lungs to the capillaries of the tissues. Normal level
of Hemoglobin (Hb) in blood in males is 14-16 g/dl and in females, 13-15 g / dl.
2. Hb is globular in shape. Hemoglobin has a mol. wt. 64 450. Hb is a conjugated
protein, containing globin and the heme.
3. Globin:
a. The globular protein units of Hb is made up of four (two identical pairs)
polypeptide chains
a. Two identical alpha () chains containing 141 amino acids and
b. Two identical non- chains (beta(),gamma(),delta() or epsilon () chains.
c. In adult humans the non- chains are beta (), containing 146 amino acids.
d. The pairing of one alpha chain and one non-alpha chain produces a
hemoglobin dimer (two chains).
e. Two dimers combine to form a hemoglobin tetramer, which is the functional
form of hemoglobin.
4. Heme:
f. Heme contains a porphyrin molecule namely protoporphyrin lX, with iron at its
center. Protoporphyrin lX consists of four pyrrole rings to which four methyl,
two propionyl and two vinyl groups are attached.
g. Porphyrins are cyclic compounds formed by fusion of 4 pyrrole rings linked by
methenyl (=CH) bridges
h. The pyrrole rings are named as I, II, III, IV and the bridges as alpha, beta,
gamma and delta.
i. Heme is a prosthetic group and is present in :
i. cytochrome
ii. Catalase

190
iii. Tryptophan pyrolase
iv. Chloropyll

5.
a.

b.

c.

d.

e.

Transport Of Oxygen By
Hemoglobin:
Hemoglobin structure has all the
requirements of an ideal respiratory pigment:
i. It can transport large quantities of oxygen
ii. It has great solubility
iii. It can take up and release oxygen at appropriate Partial pressures
iv. It is a powerful buffer.
v. The properties of individual hemoglobins are consequences of their
quaternary as well as of their secondary and tertiary structures. The
quaternary structure of hemoglobin confers striking properties, absent
from monomeric myoglobin, which adapts it to its unique biologic roles.
vi. The subunit composition of hemoglobin tetramers undergoes complex
changes during development. The human fetus initially has Hb F (high
affinity for oxygen) and
after birth the Hb A
predominates.
The binding ability of hemoglohin
with 02 at different partial
pressures of oxygen (pO2) can be
measured by a graphic
representation known as 02
dissociation curve.
The oxygen dissociation curve for
hemoglobin is sigmoidal in shape.
A shift of the curve to right means
that Hb has reduced affinity and
gives up O2 more to tissues.
The ability of hemoglobin to bind
oxygen is affected by the following, which are collectively called allosteric
(other site) effectors.
i. The po2 (heme-heme interactions),
ii. The ph of the environment,
iii. The partial pressure of carbon dioxide
iv. The availability of 2,3-bisphosphoglycerate.
Heme-heme interaction:
i. One molecule of hemoglobin (four hemes) can bind with four molecules
of 02. The binding of oxygen to one heme increases the binding of
oxygen to other hemes. Thus the affinity of Hb for the last 02 is about
100 times greater than the binding of the first 02 to Hb. This

191
phenomenon is referred to as cooperative binding of 02 to Hb or
heme-heme interaction. The release of 02 from one heme facilitates
the release of 02 from others. There is a communication among heme
groups in the hemoglobin function.
ii. In the lungs, where the concentration of 02 is high, the hemoglobin
gets fully saturated with 02. At the tissue level, where the 02
concentration is low, the oxyhemoglobin releases its 02 for cellular
respiration.
f. T & R form: The four subunits (a2b2) of hemoglobin are held together by weak
forces. The deoxy Hb exists in a T or taut (tense) form. T-form of Hb has low
oxygen affinity. Oxy Hb exists in R (relaxed) form and has more affinity for
oxygen.
g. Bohr effect:
i. The binding of oxygen to hemoglobin decreases with increasing H+
concentration (lower pH) or when the hemoglobin is exposed to
increased
partial pressure of CO2 (pCO2). This phenomenon is
known as Bohr effect. lt is due to a change in the binding affinity of
oxygen to hemoglobin. Bohr effect causes a shift in the oxygen
dissociation curve to the right.
ii. Mechanism of Bohr effect:
1. Most of the CO2 produced in metabolism is hydrated and
transported as bicarbonate ion. Some CO2 is carried as
carbamate bound to the N-terminal aminogroups of hemoglobin
(forming carbaminohemoglobin)
2. When the pCO2 is high in tissues, CO2 diffuses into the red
blood cells. The carbonic anhydrase in the red cells favors the
formation of carbonic acid (H2CO3). In RBC, the intracellular pH
falls. This reduces the affinity of Hb for O2 and O2 is released
to the tissues.
Carbonic anhydrase
CO2 + H2O ----------------- H2CO3 H+ + HCO3
3. The binding of CO2 stabilizes the T (taut) or deoxy form of
hemoglobin, resulting in a decrease in its affinity for oxygen and
a right shift in the oxygen dissociation.
4. In the lungs, CO2 dissociates from the hemoglobin, and is
released in the breath.
h. Chloride shift:
i. Chloride (Cl-) is bound more tightly to deoxyhemoglobin than to
oxyhemoglobin. This facilitates the release of 02
ii. From tissues, CO2 is taken up by RBC and the HCO3 concentration
within the cell increases in RBC. This would diffuse out into the plasma.
Simultaneously, chloride ions from the plasma would enter in the cell
to establish electrical neutrality. This is called chloride shift or
Hamburger effect.
iii. In the lung, CO2 gets out of RBC and HCO3 enters RBC while Cl- gets
out.
i. Effect of 2,3.bisphospho glycerate on O2 affinity of Hb:
i. 2,3-BPC is produced in the erythrocytes from glycolysis through a short
pathway, referred to as Rapaport-Leubering cycle. lt specifically binds
to deoxyhemoglobin (and not to oxyhemoglobin) and decreases the 02
affinity to Hb by stabilizing the deoxygenated hemoglobin to T-form.

192
The binding of 2,3-BPC with hemoglobin is associated with the release
of 02 to the tissues. The concentration of 2,3-BPC in erythrocytes is
elevated in chronic hypoxia and severe anemia.
ii. Storage of blood in acid citrate-dextrose medium results in the

decreased
concentration of
2,3-BPC. Such blood when transfused fails to supply 02 to the tissues
immediately.
iii. The binding of 2,3-BPC to fetal hemoglobin is very weak' Therefore, HbF has higher
affinity for 02 compared to adult hemoglobin (HbA).
6. Binding of CO: Carbon monoxide (CO) binds tightly (but reversibly) to the hemoglobin
iron, forming carbon monoxy hemoglobin (or carboxyhemoglobin). When CO binds to
one or more of the four heme sites, hemoglobin shifts to the relaxed conformation,
causing the remaining heme sites to bind oxygen with high affinity. This shifts the
oxygen dissociation curve to the left, and changes the normal sigmoidal shape
toward a hyperbola. As a result, the affected hemoglobin is unable to release oxygen
to the tissues.
7. Types of normal Hb:
a. Hb A: This is denoted as a2b2 and termed hemoglobin A.
b. Hb F: The combination of two alpha chains and two gamma chains form fetal
hemoglobin, termed hemoglobin F.
c. Hb A2The product of the delta globin gene is called hemoglobin A 2.
Hemoglobin

Globin Chain
Composition

Adult
Concentration

HbA

a2b2

96-98%

HbA2

a2d2

2.3-3.5%

HbF

a2r2

<2%

HbAlc

a2b2- glucose

< 5%

8. Hb derivatives: Hemoglobin (specifically heme) combines with different ligands and

forms hemoglobin derivatives. The normal blood contains oxyHb and deoxyHb.
Besides these, methemoglohin (metHb) and carboxyhemoglobin are the other
important Hb derivatives' The Hb derivatives have characteristic colour and they can
be detected by absorption spectra.

193
Methemoglobin
1. When the ferrous (Fe++) iron is oxidized to ferric (Fe+++) state, met-Hb is formed.
Small quantities of met-Hb formed in the RBCs are readily reduced back to the
ferrous state by met-Hb reductase enzyme systems.
2. About 75% of the reducing activity is due to enzyme system using NADH and
cytochrome b5. Another 20% of the reducing activity is due to NADPH dependent
system. Glutathione dependent Met-Hb-reductase accounts for the rest 5% activity.
3. Normal blood has only less than 1% of methemoglobin. It has markedly decreased
capacity for oxygen binding and transport. An increase in methemoglobin in blood,
(methemoglobinemia) is manifested as cyanosis. Causes may be congenital or
acquired.
4. Congenital mHb:
a. Presence of Hb variants like HbM can cause congenital methemoglobinemia.
Cytochrome b5 reductase deficiency is characterized by cyanosis from birth.
10-15% of hemoglobin may exist as methemoglobin.
b. Oral administration of methylene blue, 100-300 mg/day or ascorbic acid 200500 mg/day decreases met-Hb level to 5-10% and reverses the cyanosis.
5. Acquired or Toxic Methemoglobinemia:
a. Met-hemoglobinemia may develop by intake of water containing nitrates or
due to absorption of aniline dyes.
b. Drugs which produce met-hemoglobinemia are: acetaminophen, phenacetin,
sulphanilamide, amyl nitrite, and sodium nitroprusside.
c. Glucose-6-phosphate dehydrogenase deficiency methemoglobinemia may be
manifested even with small doses of drugs.
d. Intravenous leukomethylene blue 2 mg/kg is effective, which will substitute for
the NADPH.
Carboxyhemoglobin {COHb}
1. Carbon monoxide (CO) is a toxic compound (an industrial pollutant) that can bind with
Hb in the same manner as 02 binds. However, CO has about 200 times more affinity than
02 for binding with Hb.
2. Clinical manifestations of CO toxicity are observed when the COHb concentration
exceeds 20%. The symptoms include headache, nausea, breathlessness, vomiting and
irritability.
3. Administration of 02 through oxygen masks will help to reverse the manifestations of CO
toxicity'
HEMOGLOBINOPATHIES
1. Abnormal hemoglobins are the resultant of mutations in the genes that code for a or
b chains of globin. As many as 400 mutant hemoglobins are known. About 95% of
them are due to alterationi n a singlea mino acid of globin.
2. Molecular defects:
a. Qualitative defects: Abnormalities in the primary sequence of globin chains
lead to hemoglobinopathies, e.g. HbS.
b. Quantitative defect: Abnormalities in the rate of synthesis would result in
thalassemias.
c. In humans, alpha chain genes are located on chromosome No. 16 and beta
type genes are clustered in chromosome No. 11. Mutations in the genes that
encode the a or b chains of hemoglobin produce yemoglobinopathies.
d. Sickle cell anemia (Hb S), hemoglobin C disease (Hb C), hemoglobin SC
disease (Hb S + Hb C), and the thalassemia syndromes are representative

194
hemoglobinopathies that can have severe clinical consequences.
e. The first three conditions result from production of hemoglobin with an altered
amino acid sequence (qualitative hemoglobinopathy), whereas the
thalassemias are caused by decreased production of normal hemoglobin
(quantitative hemoglobinopathy).
3. Sickle-cell anemia (HbS)
a. Mostc ommon form of abnormal hemoglobins; largely confined to tropical
areas of the world.
b. The hemoglobin (HbS) has two normal a-globin chains and two abnormal
(mutant) p-globin chains. This is due to a difference in a single amino acid. In
HbS, glutamate at sixth position of p-chain is replaced by valine.
c. Sickle cell anemia is said to be homozygous, if caused by inheritance of two
mutant genes (one from each parent) that code for b-chains. In case of
heterozygous HbS, only one gene (of b-chain) is affected while the other is
normal.
d. Sickle-cell anemia is characterized by the following abnormalities:
1. Life-long hemolytic anemia
2. Tissue damage and pain: The sickled cells block the capillaries
resulting in poor blood supply to tissues. This leads to extensive
damage and inflammation of certain tissues causing pain.
3. Increased susceptibility to infection: Hemolysis and tissue damage
are accompanied by increased susceptibility to infection and
diseases.
4. Premature death: Homozygous individuals of sickle-cell anemia die
before they reach adulthood (< 20 years).
5. Mechanism of sickling in sickle-cell anemia: Glutamate is a polar
amino acid and it is replaced by a non-polar valine in sickle-cell
hemoglobin. This causes a marked decrease in the solubility of HbS
in deoxygenated form (T form). Sickling is due to polymerization of
deoxy HbS.
6. Sickle-cell trait (heterozygous state with about 40% HbS) provides
resistance to malaria which is a major cause of death in tropical
areas. lncreased lysis of sickled cells (shorter life span of
erythrocytes) interrupts the parasite cycle.
e. Lab tests:
i. Sickling test : This is a simple microscopic examination of blood smear
prepared by adding reducing agents such as sodium dithionite. Sickled
erythrocytes can be detected under the mrcroscope.
ii. Electrophoresis : When subjected to electrophoresis in alkaline medium
(pH 8.6), sickle-cell hemoglobin (HbS) moves slowly towards anode
than does adult hemoglobin (HbA). The slow mobility of HbS is due to
less negative charge, caused by the absence of glutamate residues
that carry negative charge. In case of sickle-cell trait, the fast moving
HbA and slow moving HbS are observed. The electrophoresis of
hemoglobin obtained from lysed erythrocytes can be routinely used for
the diagnosis of sickle cell anemia and sickle-cell trait.
f. Treatment:
i. In patients with severe anemia, repeated blood transfusion is required.
This may result in iron overload and cirrhosis of liver.
ii. Hydroxyurea, an antitumor drug, is therapeutically useful because it
increases circulating levels of Hb F, which decreases RBC sickling.
iii. Gene-replacement therapy is being tried.

195
4. Hemoglobin C disease:
a. Cooley's hemoglobinemia (HbC) is characterized by substitution of glutamate
by lysine in the sixth position of b-chain. Due to the presence of lysine, Hb C
moves more slowly on electrophoresis compared to HbA and HbS, HbC disease
occurs only in blacks.
b. Both homozygous and heterozygousin dividuals of Hb C disease are known.
This disease is characterized by mild hemolytic anemia. No specific therapy is
recommended.
5. Hemoglobin D:
a. This is caused by the substitution of glutamine in place of glutamate in the
121st position of B-chain.
b. Several variants of HbD are identified from different places indicated by the
suffix. For instance, HbD (Punjab), HbD (Los Angeles). Hb D, on
electrophoresis moves along with Hb S.
6. Hemoglobin E:
a. This is the most common abnormal hemoglobin after HbS. lt is estimated that
about 1O% of the population in South-East Asia (Bangladesh, Thailand,
Myanmar) suffer from HbE disease. In India, it is prevalent in West Bengal.
b. HbE is characterized by replacement of glutamate by lysine at 26th position of
B-chain. The individuals of HbE (either homozygous or heterozygous) have no
clinical manifestations.
THALASSEMIAS
1. Thalassemias are characterized by a defect in the production of a-or b-globin chain.
There is however, no abnormal ity in the amino acids of the individual chains.
Thalassemias occur due to a variety of molecular defects:
1. Gene deletion or substitution,
2. Underproduction or instability of mRNA,
3. Defect in the initiation of chain synthesis,
4. Premature chain termination
2. Reduction in alpha chain synthesis is called alpha thalassemia, while deficient beta chain
synthesis is the beta thalassemia. Other types like delta-beta thalassemia, Hb Lepore,
hereditary persistence of HbF (HPF) are related conditions.
3. Beta thalassemia is more common than alpha variety. Beta type is characterized by a
decrease or absence of synthesis of beta chains. As a compensation, gamma or delta
chain synthesis is increased. It can exist in homozygous(major) and heterozygous(minor)
and intermediate forms. Alpha thalassemias are caused by a decreased synthesis or total
absence of a-globin chain of Hb. There are four copies of a-globin gene, two on each one
of the chromosome 16. Four types of a-thalassemias occur which depend on the number
of missing a-globin genes:
a. Silent carrier state is due to loss of one of the four a-globin genes with no physical
manifestations.
b. a-Thalassemia trait caused by loss of two genes. Minor anemia is observed.
c. Hemoglobin H disease, due to missing of three genes, is associated with
moderate anemia.
d. Hydrops fetalis is the most severe form of a-thalassemias due to lack of all the
four genes. The fetus usually survives until birth and then dies.
e. Homozygous beta thalassemia is characterized by severe anemia, hypersplenism
and hepatosplenomegaly. The marrow in the skull bones expand producing the
hair-on-end
f. appearance described in X-ray.

196
g. Repeated transfusion is the only available treatment. This may lead to iron
overload.
h. Splenectomy may also lessen the anemia.
i. Marrow transplantation has been successfully tried in a few cases.

HEME
1. Name the rate limiting enzyme of heme synthesis. What is its coenzyme? Pon
2009
2. Heme degradation - Aug 2005;
3. Heme catabolism - April 2001; Pon 2014
Heme Biosynthesis :
1. lt is primarily synthesized in the liver and the erythrocyte-producing cells of bone
marrow (erythroid cells). Heme synthesis also occurs to some extent in other tissues.
2. Step 1: ALA synthesis: The synthesis starts with the condensation of succinyl CoA
and glycine in the presence of pyridoxal phosphate to form delta amino levulinic acid
(ALA). The enzyme ALA synthase is located in the mitochondria and is the ratelimiting enzyme of the pathway. This reaction requires pyridoxal phosphate (PLP) as
a coenzyme & anemia may be manifested in pyridoxal deficiency.
3. Step 2: Two molecules of ALA are condensed to form porphobilinogen (PBG). The
condensation involves removal of 2 molecules of water and the enzyme is ALA
dehydratase . Porphobilinogen is a monopyrrole. The enzyme contains zinc and is
inhibited by lead.
4. Step 3: Condensation of 4 molecules of the PBG, results in the formation of
uroporphyrinogen (UPG). Uroporphyrinogen I is converted to uroporphyrinogen III by
the enzyme, uroporphyrinogen III synthase.
5. Step 4: The UPG-III is next converted to coproporphyrinogen (CPG-III) by
decarboxylation. Four molecules of CO2 are eliminated by uroporphyrinogen
decarboxylase. The acetate groups (CH2COOH) are decarboxylated to methyl (CH3)
groups.
6. Step 5: Further metabolism takes place in the mitochondria. CPG is oxidized to
protoporphyrinogen (PPG-III) by coproporphyrinogen oxidase.
7. Step 6: The Protoporphyrinogen-III is oxidized by the enzyme protoporphyrinogen
oxidase to protoporphyrin-III (PP-III) in the mitochondria. The oxidation requires
molecular oxygen. The methylene bridges (CH2) are oxidised to methenyl bridges (
CH=) and colored porphyrins are formed. Protoporphyrin- 9 is thus formed.
8. Step 7: The last step in the formation of heme is the attachment of ferrous iron to the
protoporphyrin. The enzyme is heme synthase or ferrochelatase which is also located
in mitochondria.
Regulation of Heme Synthesis
4.
Heme inhibits the synthesis of ALA synthase by acting as a co-repressor. The
enzyme ALA synthase is located in the mitochondria and is the rate-limiting
enzyme of the pathway. ALA synthase (ALAS) have both erythroid and nonerythroid (hepatic) forms. Erythroid form is called ALAS2; it is not induced by the
drugs that affect ALAS1. Erythroid form is not subject to feedback inhibition by
heme.
5.
ALA synthase is also allosterically inhibited by hematin. When there is excess of
free heme, the Fe++ is oxidized to Fe+++ (ferric), thus forming hematin.
6.
Drugs like barbiturates induce heme synthesis. Barbiturates require the heme

197
containing cytochrome p450 for their metabolism. Out of the total heme
synthesized, two thirds are used for cytochrome p450 production.
7.
The steps catalyzed by ferrochelatase and ALA dehydratase are inhibited by lead.
8.
INH (Isonicotinic acid hydrazide) that decreases the availability of pyridoxal
phosphate may also affect heme synthesis.
9.
High cellular concentration of glucose prevents induction of ALA synthase. This is
the basis of administration of glucose to relieve the acute attack of porphyrias.
Catabolism of Heme: Degradation of Heme
In RE System:
1. Erythrocytes have a life span of 120 days. A t the end of this period, they are taken
up and degraded by the macrophages of the reticuloendothelia (RE) system in the
spleen and liver. The hemoglobin is cleaved to the protein part globin and non-protein
heme. About 6 g of hemoglobin per day is broken down, and resynthesized in an
adult man
2. The globin may be reutilized as such for the formation of hemoglobin or degraded to
the individual amino acids.
3. 80% of the heme comes from the erythrocytes and the rest (2O%) comes from
immature RBC, myoglobin and cytochromes
4. Heme oxygenase and NADPH and O2 complex cleaves the methyl bridges of pyrrole
rings to form biliverdin and Carbon monoxide. Heme oxygenase utilizes NADPH and
02 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).
5. Biliverdin is reduced by biliverdin reductase to Bilirubin (yellow pigment). One gram
of hemoglobin on degradation finally yields about 35 mg biliruhin. The term bile
pigments is used to collectivelyrepresent bilirubin and its derivatives
In Plasma:
6. Bilirubin is bound to albumin and transported to liver. One molecule of albumin can
bind 2 molecules of bilirubin. Certain drugs like sulfonamides & salicylates can
displace bilirubin from albumin. Due to this, bilirubin can enter the central nervous
system and cause damage to neurons.
In Liver:
7. As the albumin-bilirubin complex enters the liver , bilirubin dissociates and is taken
up by sinusoidal surface of the hepatocytes by a carrier mediated active transport.
Inside the hepatocytes, bilirubin binds to a specific intracellular protein namely
ligandin.
8. In liver Bilirubin is released and conjugated with two molecules of glucuronate
supplied by UDP-glucuronate catalysed by glucuronyl transferase to form water
soluble Bilirubin diglucuronide. The enzyme bilirubin glucuronyl transferacsaenbe
induced by a number of drugs (e.g. Phenobarbital). Drugs like primaquine,
novobiocin, chloramphenicol, androgens and pregnanediol may interfere in this
conjugation process and may cause jaundice.

198

curonide

Bilirubin diglucuronide
In Biliary System:
9. Conjugated Bilirubin is excreted to bile and enters intestine through gall bladder and
bile duct. The transport of hilirubin diglucuronide is an active, energy-dependent and
rate limiting process. Excretion of conjugated bilirubin into bile is mediated by an ATP
binding cassette protein which is called Multispecific organic anion transporter
(MOAT), located in the plasma membrane of the biliary canaliculi.This step is
susceptible to any impairment in liver function. Bile containing bilirubin is collected
by gall bladder and transported to 1st part of duodenuam by bileduct.
In Intestine: Enterohepatic circulation:
10. In intestine conj.bilirubin is hydrolysed by bacterial b-glucuronidase to form free
Bilirubin .
11. Free bilirubin is again converted to urobilinogen in intestines; part of Urobilinogen is
reabsorbed and excreted in urine as urobilin. And major part is converted to
stercobilin by bacteria and excreted in feces.
12. Enterohepatic circulation: 20% urobilinogen is reabsorbed from the intestine and
returned to liver. It is re excreted in to intestine through bile. This is called
enterohepatic circulation. Small portion is excreted in urine. Both urobilin and
stercobilin are present in urine and feces.
13. If intestinal flora is decreased by prolonged administration of antibiotics, bilirubin is
not reduced to bilinogens, and in the large gut, it is re-oxidized by O2 to form
biliverdin. Then green tinged feces is seen, especially in children.

199
PORPHYRINS
a. Porphyrias - April 2000
b. Acute intermittan porphyria. Pon may 2013
a. Porphyrins are cyclic compounds composed of 4 pyrrole rings held together by methenyl
(=CH-) bridges. Heme is an iron-containing porphyrin . The structure of porphyrins has
four pyrrole rings namely l, II, lll and lV. Forphyrins in cancer therapy
b. 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.

Porphyrias:
1. Porphyrias are rare, inherited (or occasionally acquired) defects in heme synthesis,
resulting in the accumulation and increased excretion of porphyrins or porphyrin
precursors.
2. Porphyrias are inherited mostly as autosomal dominant disorders. Porphyrias may be broadly
grouped into 3 types:
i. Erythropoietic porphyrias:Enzyme deficiency occurs in the erythrocytes.
ii. Hepatic : Enzyme defect lies in the liver
iii. Porphyrias with both erythropoietic and hepatic abnormalities

3. Acute
Intermittent Porphyria (AIP)
a. This disorder occurs due to the deficiency of the enzyme uroporphyrinogen I
synthase. Acute intermittent porphyria is characterized by increased excretion of
porphobilinogen and 6-aminolevulinate. The urine gets darkened on exposure to
air due to the conversion of porphobilinogen to porphobilin and porphyrin.

200

4.

5.

6.

7.

b. It is inherited as an autosomal dominant trait. lt is usually expressed after puberty.

The symptoms include abdominal pain, vomiting and cardiovascular abnormalities.
The neuropsychiatric distrubances observed in these patients are believed to be
due to reduced activity of tryptophan pyrrolase resulting in the accumulation of
tryptophan and 5-hydroxytryptamine.
c. The symptoms are more severe after administration of drugs (e.g.barbiturates)
that induce the synthesis of cytochrome P450. This is due to the increased activityo
f ALA synthase causing accumulation of PBG and ALA. These patients are not
photosensitive since the enzyme defect occurs prior to the formation of
uroporphyrinogen.
d. Acute intermittent porphyria is treated by administration of hematin which inhibits
the enzyme-ALA synthase and the accumulation of porphobilinogen.
Congenital erythropoietic porphyria:
a. This disorder is due to a defect in the enzyme uroporphyrinogen III cosynthase. lt
is a rare congenital disorder caused by autosomal recessive mode of inheritance
mostly confined to erythropoietic tissues.
b. The individuals excrete uroporphyrinogen I and coproporphyrinogen I which
oxidized respectively to uroporphyrin I and coproporphyrin | (red pigments).
c. The patients are photosensitive (itching and burning of skin when exposed to
visible light) due to the abnormal prophyrins that accumulate. Increased
hemolysis is also observed in the individuals affected by this disorder.
d. Porphyria cutanea tarda
e. This is also known as cutaneous hepatic porphyria and is the most common
porphyria, usually associated with liver damage caused by alcohol
overconsumption and iron overload.
f. The partial deficiency of the enzyme uroporphyrinogen decarboxylase is
responsible for the defect.
g. There is increased excretion of uroporphyrins (l and lll) and rarely
porphobilinogen.
h. Cutaneous photosensitivity is the most important clinical manifestation of these
patients. Liver exhibits fluorescence due to high concentration of accumulated
porphyrins.
Hereditary coproporphyria
a. This disorder is due to a defect in the enzvme coproporphyrinogen oxidase. As a
result of this, coproporphyrinogen lll and other intermediates (ALA and PBG) of
heme synthesis prior to the blockade are excreted in urine and feces.
b. The victims of hereditary coproporphyria are photosensitive. They exhibit the
clinical manifestations observed in the patients of acute intermittent porphyria.
Infusion of hematin is used to control this disorder. Hematin inhibits ALA synthase
and thus reduces the accumulation of various intermediates.
Variegate porphyria
a. The enzyme protoporphyrinogen oxidase is defective in this disorder. Due to this
blockade, protoporphyrin lX required for the ultimate synthesis of heme is not
produced. Almost all the intermediates (porphobilinogen, coproporphyrin,
uroporphyrin, protoporphyrin etc.) of heme synthesisa ccumulate in the body and
are excreted in urine and feces. The urine of these patients is coloured and they
exhibit photosensitivity.
Protoporphyria
a. This disorder, also known as erythropoietic protoporphyria, is caused by a
deficiency of the enzyme ferrochelatase. Protoporphyrin lX accumulates in the
tissues and is excreted into urine and feces. Reticulocytes (young RBC) and skin

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biopsy exhibit red flourescence.
b. Acquired {toxic} porphyrias
c. The porphyrias, though not inherited, may be acquired due to the toxicity of
several compounds. Exposure of the body to heavy metals (e.g. lead), toxic
compounds (e.g hexachlorobenzene) and drugs (e.g.griseofulvin) inhibits many
enzymes in heme sylthesis. These include ALA dehydratase, urporphyrin I
synthase and ferrochelatase

1.
2.
3.
4.
5.
6.
7.
8.
9.

BILIRUBIN (Jaundice)
How is bilirubin formed and excreted? Pon Nov 11
Discuss the use of Vandenberg test in differential diagnosis of jaundice.
Pon Nov 2007
Synthesis and conjugation of Bilirubin.
Vandenberg test - march 2002
Excretion of Bilirubin and clinical importance of Bilirubin estimation Sep 2002
Unconjugated hyperbilirubinemia - Oct 2000
How is bilirubin estimated? The level of which type of bilirubin is altered
in different types of jaundice. Pon Nov 2010
Discuss the formation and fate of bilirubin in the body. On May 2010

1. Bilirubin consists of an open chain of four pyrrole-like rings (tetrapyrrole).

Unconjugated bilirubin is toxic to neonatal brain. Cojugated brain merely indicates
obstruction in biliary patway.
2. Plasma Bilirubin: Normal values:
Total:
0.2 to .8 mg /dl
Unconjugated:
0.2 to .6 mg /dl
Conjugated:
< 0.2 mg /dl
3.
If the plasma bilirubin level exceeds 1 mg/dl, the condition is called
hyperbilirubinemia. Levels between 1 and 2 mg/dl are indicative of latent jaundice.
When the bilirubin level exceeds 2 mg/dl, it diffuses into tissues producing yellowish
discoloration of sclera, conjunctiva, skin and mucous membrane resulting in jaundice.
Icterus is the Greek term for jaundice.
4.
Van Den Berg Reaction:
a. The test identifies increase in serum bilirubin and is negative in normal level.
b. Basis: The regent is a mixture of equal volumes of sulfanilic acid and sodium
nitrite. The principle is that diazotized sulfanilic acid reacts with Bilirubin to
form purple colored azobilirubin.
c. Direct Reaction: Van den berg reagent gives purple color in 30 seconds with
conjugated Bilirubin. This is called direct positive reaction.
d. Indirect reaction: In Unconjugated Bilirubin the reagent produce purple color
in 30 seconds after addition of methanol.
e. If both Bilirubin are present initial pink color is deepened after addition of
methanol and is known as biphasic reaction.
f. Interpretation of results:
i. Indirect positive:
Hemolytic jaundice
ii. Direct positive:
Obstructive jaundice
iii. Biphasic:
Hepatic jaundice.
5.
Classification of Hyperbilirubinemia or Jaundice
a. Hemolytic jaundice: Elevated serum unconjugated bilirubin, and increased
urinary excretion of urobilinogen.
b. Obstructive jaundice : Elevated serum conjugated bilirubin and increased

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activities of alkaline phosphatase (ALP), alaninet ransaminase (ALT) and
aspartate transaminase (AST)
c. Hepatic jaundice: Elevated serum unconjugated and conjugated bilirubin, and
increased activities of ALT and AST.
6. Hemolytic jaundice:
a. This condition is associated with increased hemolysis of erythrocytes. This
results in the overproduction of bilirubin beyond the ability of the Iiver to
conjugate and excrete the same.
b. Cuases:
i. Congenital Hemolytic jaundice occurs due to:
1. Hemoglobin defect: Thalassemias; sickele cell disease
2. Enzyme defect:
G6PD & pyruvate kinase deficiency
3. Membrane defect:
Cong. Sphaerocytosis
ii. Immune hemolytic jaundice:
1. Rh incompatibility
2. ABO incompatibility
iii. Acquired causes:
1. Incompatible blood transfusion,
2. Malaria,
c. Hemolytic jaundice is characterized by:
i. Elevation in the serum unconjugated bilirubin.
ii. Increased excretion of urobilinogen in urine.
iii. Dark brown colour of feces due to high content of stercobilinogen
7.
Hepatic (hepatocellular) jaundice:
a. This type of jaundice is caused by dysfunction of the Iiver due to damage to
the parenchymal cells. This may be attributed to viral infection (viral hepatitis,
poisons and toxins (chloroform, carbon tetrachloride, phosphorus etc.)
cirrhosis of liver, cardiac failure etc. Among these, viral hepatitis is the most
common.
b. Damage to the liver adversely affects the bilirubin uptake and its conjugation
by liver cells. Hepatic jaundice is characterized by:
i. Increased levels of conjugated and unconjugatedbilirubin in the serum.
ii. Dark coloured urine due to the excessive excretion of bilirubin and
urobilinogen.
iii. lncreased activities of alanine transaminase (SGPT) and aspartate
transaminase (SCOT) released into circulation due to damage to
hepatocytes.
iv. The patients pass pale, clay coloured stools due to the absence of
stercobilinogen.
v. The affected individuals experience nausea and anorexia (loss of
appetite).
8.
Obstructive (regurgitation) jaundice:
a. 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.
b. Due to the blockage in bile duct, the conjugated bilirubin from the liver enters
the circulation.
c. Obstructive jaundice:
i. Congenital: Biliary atresia
ii. Acquired: Viral hepatitis.
d. Obstructive jaundice is characterized by:
i. Increased concentration of conjugated bilirubin in serum.
ii. Serum alkaline phosphatase is elevated as it is released from the cells
of the damaged bile duct.

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iii. 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).
iv. The patients experience nausea and gastrointestinal pain.
9.
Neonatal - physiologic jaundice:
a. lt is caused by increased hemolysis coupled with immature hepatic system for
the uptake, conjugation and secretion of bilirubin.
b. The activity of the enzyme UDP-glucuronyltransferase is low in the newborn.
Further, there is a limitation in the availability of the substrate UDP-glucuronic
acid for conjugation. The net effect is that in some infants the serum
uncojugated bilirubin is highly elevated from day 2 of life but does not go
beyond toxic leveles that produce kernicterus.
10.
Jaundice due to genetic defects: Congenital Non hemolytic Hyperbilirubinemias. They
result from abnormal uptake, conjugation or excretion of bilirubin due to inherited
defects.
a. Crigler-Najjar Syndrome:
i. Here the defect is in conjugation.
1. In Type 1, there is severe deficiency of UDP glucuronyl
transferase. The disease is often fatal and the children die
before the age of 2. Jaundice usually appears within the first 24
hours of life. Unconjugated bilirubin level increases to more
than 20 mg/dl, and hence kernicterus results.
2. The Type 2 disease is a milder form; only the second stage of
conjugation is deficient. When barbiturates are given, some
response is seen and jaundice improves. Bilirubin level in blood
exceeds 20 mg/dl in Crigler-Najjar syndrome Type 1 and does
not exceed 20 mg/dl in Crigler-Najjar syndrome Type 2.
b. Gilbert's Disease
i. It is inherited as an autosomal dominant trait. The defect is in the
uptake of bilirubin by the liver and an impairnrent in conjugation due to
reduced activity of UDP-glucuronyltransferase.
ii. Bilirubin level is usually around 3 mg/dl, and patient is asymptomatic,
except for the presence of mild jaundice.
c. Dubin-Johnson Syndrome
i. It is an autosomal recessive trait leading to defective excretion of
conjugated bilirubin; so conjugated bilirubin in blood is increased.
ii. The disease results from the defective ATPdependent organic anion
transport in bile canaliculi. There is a mutation in the MRP-2 protein
which is responsible for transport of conjugated bilirubin into bile. The
bilirubin gets deposited in the liver and the liver appears black,
referred to as Black liver jaundice.
iii. Rotor Syndrome: It is a similar condition, but the exact defect is not
identified. Bilirubin excretion is defective, but there is no staining of the
liver. It is an autosomal recessive condition.
11. Breast milk jaundice:
a. Early onset: This type indirect hyperbilirubinemia is due to lack of adequate
breast milk and increased entero hepatic circulation.
b. Late onset: In some breast-fed infants, prolongation of the jaundice has been
attributed to high level of an estrogen derivative (pregnandiol) in maternal
blood, which is excreted through the milk. This would inhibit the glucuronyl
transferase system.

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c. These are mostly benign and self-limiting conditions.
12. Immune hemolytic jaundice:
a. Rh Incompatibility:
i. This condition results from incompatibility between maternal and fetal
blood groups. Rh +ve fetus may produce antibodies in Rh ve mother.
In Rh incompatibility, the first child often escapes. But in the second
pregnancy, the Rh antibodies will pass from mother to the fetus. They
would start destroying the fetal red cells even before birth.
ii. Sometimes the child is born with severe hemolytic disease, often
referred to as erythroblastosis fetalis. When blood level is more than 20
mg/dl, the capacity of albumin to bind bilirubin is exceeded. In young
children before the age of 1 year, the blood-brain barrier is not fully
matured, and therefore free bilirubin enters the brain (Kernicterus). It is
deposited in brain, leading to mental retardation, fits, toxic encephalitis
and spasticity.
iii. If the child develops hemolytic disease, child may be given exchange
transfusion along with phototherapy and barbiturates. Phototherapy
with blue light (440 nm wave length) isomerizes the insoluble bilirubin
to more soluble isomers. These can be excreted through urine without
conjugation.
b. ABO incompatibility: This is due to mostly O + mother and A or B group fetus.
The disease is milder than Rh type. But even first pregnancy is affected.
13. Obstructive Jaundice:
a. Obstruction can occur due to intrhepatic cause like hepatitis
b. Bile duct obstruction may be congenital or due to calculus & cancer head of
pancreas.
14. Tests for Bile Pigments
a. Bilinogens (UBG and SBG) react with Ehrlich's aldehyde reagent (para
dimethyl amino benzaldehyde) to form red color.
b. In Fouchet's test, urine is heated with barium sulphate which adsorbs bilirubin.
Ferric chloride oxidizes bilirubin to produce a green color.
c. In Gmelin's test, nitric acid is used as the oxidizing agent.
d. The bilinogens (UB and SB) form complexes with zinc ions which exhibit
brilliant green fluorescence. This is the basis for Schlesinger's reaction. It is
negative in normal urine.
15. Van Den Berg Reaction:
a. The test identifies increase in serum Bilirubin and is negative in normal level.
It identifies conjugated and unconjugated bilirubins.
b. Basis: The regent is a mixture of equal volumes of sulfanilic acid and sodium
nitrite. The principle is that diazotized sulfanilic acid reacts with Bilirubin to
form purple colored azobilirubin.
c. Direct Reaction: Van den berg reagent gives purple color in 30 seconds with
conjugated Bilirubin. This is called direct positive reaction.
d. Indirect reaction: In Unconjugated Bilirubin the reagent produce purple color
in 30 seconds after addition of methanol.
e. If both Bilirubin are present initial pink color is deepened after addition of
methanol and is known as biphasic reaction.
f. Interpretation of results:
Indirect positive:
Hemolytic jaundice
Direct positive:
Obstructive jaundice
Biphasic:
Hepatic jaundice.

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MYOGLOBIN (Mb)
1. It is seen in muscles. Myoglobin content of skeletal muscle is 2.5 g/l00 g; of cardiac
muscle is 1.4 g% and of smooth muscles 0.3g%.
2. Mb is a single polypeptide chain. Human Mb contains 152 amino acids with a
molecular weight of 17,500 Daltons.
3. One molecule of Mb can combine with 1 molecule of oxygen. The Hb carries oxygen
from lungs to tissue capillaries, from where oxygen diffuses into tissues. In the
muscles, the oxygen is taken up by Mb for the sake of tissue respiration.
4. Mb has higher affinity for oxygen than that of Hb.
5. In severe physical exercise, pO2 in muscles lowers to 5 mm Hg, when myoglobin
releases all the bound oxygen.
6. Mb has a high oxygen affinity while Bohr effect, co-operative effect and 2,3-BPG
effect are
absent.
7. Myoglobin in Urine and Blood: Severe crush injury causes release of myoglobin from
the damaged muscles. Being a small molecular weight protein, Mb is excreted
through urine (myoglobinuria). Urine color becomes dark red.
8. Myoglobin will be released from myocardium during myocardial infarction (MI), and is
seen in
serum. Serum myoglobin estimation is useful in early detection of myocardial
infarction

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