Hemoglobin Metabolism

 Heme is an iron containing porphyrin.

 Heme is the prosthetic group of several proteins and

enzymes including hemoglobin, myoglobin,
cytochrome, cytochrome P450, enzymes like catalase,
certain peroxidase, and tryptophan pyrrolase.

 Heme is synthesized from porphyrin and iron

 Porphyrin ring is coordinated with an atom of iron to

form heme
Heme molecule showing a pyrrole molecule and an atom of iron
(Fe 2+) in the center of the porphyrin ring,
which is tetrapyrrole structure.
(M: methyl; P: propionyl; V: vinyl)
 Biosynthesis Of Heme

 Heme synthesis takes place in all cells (except in mature

erythrocytes), but occurs to the greatest extent in the
bone marrow and liver.

 Biosynthesis of heme may be divided into three stages:

 Stages of Heme Synthesis

1. Biosynthesis of δ-aminolevulinic acid (ALA) from

precursor glycine and succinyl-CoA

2. Formation of porphobilinogen (PBG) from δ-

aminolevulinic acid

3. Formation of porphyrins and heme from

porphobilinogen.
Regulation of Heme Synthesis

 δ-aminolevulinic acid synthase (ALAS), a

mitochondrial allosteric enzyme that catalyzes first

step of heme biosynthetic pathway, is a regulatory

enzyme. It is feedback inhibited by heme.

 Regulation also occurs at the level of enzyme synthesis.

• Increased level of heme represses the synthesis of

δ-aminolevulinic acid synthase

• Decreased level of heme induces the synthesis of δ-

aminolevulinic acid synthase.

 The iron atom itself activates heme synthesis.

 Several drugs induce the synthesis of hepatic ALAS,

e.g.steroid hormone métabolites, éthanol, barbiturates, etc.

Disorders of heme biosynthesis

Disorders of heme biosynthesis may be genetic or

acquired.

1. The most common acquired defect is lead poisoning.

Lead can inactivate d-ALA dehydratase and ferrochelatase

by combing with essential thiol groups of these enzymes.

Signs of lead poisoning include

 Elevated levels of protoporphyrin in erythrocytes

 Elevated urinary levels of ALA and coproporphyrin III.

 Production of heme is decreased.

 Anemia results from lack of hemoglobin and

 Energy production decreases due to lack of cytochromes

for the electron transport chain.
2. X-linked sideroblastic anemia (XLSA):

 It is a congenital disorder due to a defect in δ-

aminolevulinate synthase (ALAS), which is involved
in the first step of heme synthesis.

 Sideroblastic anemia is a form of anemia in which the

bone marrow produces ringed sideroblasts rather than
healthy red blood cells.
 Sideroblasts are nucleated erythroblasts (precursors to
mature red blood cells) with granules of iron
accumulated in the mitochondria surrounding the
nucleus.

 In sideroblastic anemia, iron is available but cannot

incorporate into hemoglobin.

 Symptoms of sideroblastic anemia include skin

paleness, fatigue, dizziness, and enlarged spleen and
liver.
3. Porphyrias are a group of genetic diseases that result

from defect in enzymes of the biosynthetic pathway

from glycine to porphyrins. In porphyrias, specific

porphyrin precursors accumulate in erythrocytes,

body fluids, and in the liver.

 The Porphyrias

 Porphyria is named from the ancient Greek word

porphura, meaning purple. Purple color is caused by
pigment-like porphyrins in the urine of some patients with
defects in heme synthesis.

 The porphyrias are rare group of inherited disorders

resulting from deficiencies of enzymes in the biosynthetic
pathway for heme, and lead to accumulation and
increased excretion of porphyrins or porphyrin precursors
(ALA and PBG).
 With the exception of two, congenital erythropoietic
porphyria and aminolevulinic acid dehydratase
deficiency porphyria (ALAD Porphyria), which are
genetically autosomal recessive diseases, all the
porphyrias are inherited as autosomal dominant
disorders.
 Aminolevulinic acid dehydratase deficiency porphyria
(ALAD Porphyria) is a hepatic and extremely rare
congenital condition.
 Most individuals inheriting this condition are
heterozygotes and are usually asymptomatic, because
the single copy of the normal gene provides a sufficient
level of enzyme function.

 However, certain nutritional and environmental factors

can cause a formation of δ-aminolevulinate and
porphobilinogen, leading to attacks of acute
abdominal pain and neurological dysfunction.
Classification of porphyria

The hereditary porphyrias are classified into two categories

on the basis of the organs or cell that are mostly affected.
Thus, porphyria are classified into:

1. Erythropoietic porphyria : Enzyme deficiency occur in

erythropoietic cells of the bone marrow.

2. Hepatic porphyria : Enzyme deficiency occur in

hepatic cell.
Porphyrias caused by mutations of enzymes.
 Biochemical Basis of the Major Signs and Symptoms of

Porphyrias

 Where the enzyme lesion occurs early in the pathway

prior to the formation of porphyrinogens, ALA and PBG
will accumulate in the body tissues and fluids.

 These compounds can impair the function of abdominal

nerve and central nervous system, resulting in abdominal
pain and neuropsychiatric symptoms.
 This toxic effect of ALA and PBG may be due to
inhibition of ATPase in nervous tissue by ALA. ALA
may be taken up by brain which causes conduction
paralysis.

 On the other hand, enzyme blocks later in the pathway,

result in the accumulation of the porphyrinogens which
on exposure to light auto-oxidized to corresponding
porphyrin derivatives, and causes photosensitivity to
visible light and skin injuries.
 The injuries occur in the exposed areas where sunlight
activates porphyrins, which reacts with molecular
oxygen to form oxygen radicals in the skin.

 These oxygen radicals injure lysosomes and other

organelles. Damaged lysosomes release their degradative
enzymes, causing skin damage.
Biochemical basis of the major signs and
symptoms of porphyrias.
 In congenital erythropoietic porphyria the urine of

these patients is red because of excretion of large

amounts of uroporphyrinogen I and their teeth

exhibit a strong red fluorescence under ultraviolet

light because of the deposition of porphyrins.

 Furthermore, their skin is usually very sensitive to

light because of photosensitive porphyrins.

Fluorescent teeth of a patient with
porphyria cutanea tarda.
Skin lesion in a patient with porphyria
cutanea tarda.
Treatment of Porphyria

• Present treatment of porphyria is basically symptomatic.

 The symptoms of most porphyrias are now readily

controlled with dietary changes or administration of heme
or heme derivatives.

 Avoid drugs that cause production of cytochrome P450.

 Patients exhibiting photosensitivity may benefit from

administration of β-carotene.
 β-carotene decreases the production of free radicals and
helps to diminish photosensitivity.

 Use of sunscreens that filter out visible light can also be

helpful to decrease the photosensitivity.

 Ingestion of large amounts of carbohydrate and

administration of hematin to repress synthesis of ALA
synthase.

 ALA synthase diminish production of harmful heme

precursors .
Laboratory Test for the Detection of Porphyrins

and their Precursors

 Spectrophotometry is used to detect porphyrins and their

precursors.

 Coproporphyrins and uroporphyrins are excreted in urine

increased amounts in the porphyrias.

 When present in urine or feces, they can be separated by

extraction with appropriate solvents then identified and
quantified using spectrophotometric methods.
CATABOLISM OF HEME TO BILIRUBIN
 After approximately 120 days, the heme group of
hemoglobin released from dying erythrocytes in the
spleen is degraded to yield free Fe 2+ and ultimately
bilirubin.

 First hemoglobin is dissociated into heme and globin.

 Globin is degraded to its constituent amino acids, which

are reused.
Formation of Bilirubin

 The catabolism of heme is carried out in the

microsomes by a complex enzyme system called heme
oxygenase in the presence of NADPH and O2.

 Ferric ion and carbon monoxide (CO) are released with

production of the green pigment biliverdin.

 Biliverdin is reduced to red orange colored bilirubin

 Bilirubin and its derivatives are collectively termed as

bile pigments
Degradation of hemoglobin to bilirubin.
 FATE OF BILIRUBIN

Metabolism and excretion of bilirubin occurs in the

liver and intestine. It can be divided into four processes:

1. Uptake of bilirubin by liver

2. Conjugation of bilirubin in the liver

3. Secretion of conjugated bilirubin into the bile

4. Excretion of bilirubin.
Five major processes involved in the metabolism of
hemoglobin.
Conjugation of bilirubin with glucuronic acid in the liver
Disorders Of Bilirubin Metabolism
 Serum Bilirubin

The normal concentration of serum bilirubin is:

1. Total bilirubin = 0.1 to 1.0 mg/dL

2. Conjugated (direct) bilirubin = 0.1 to 0.4 mg/dL.

3. Unconjugated (indirect) bilirubin = 0.2 to 0.7 mg/dL.

Diseases or conditions that interfere with bilirubin

metabolism may cause a rise in its serum concentration.

When bilirubin in the blood (unconjugated or conjugated)

exceeds 1 mg/dL, hyperbilirubinemia exists.

 Hyperbilirubinemia may be due to:

• Increased bilirubin production

• Decreased hepatic uptake

• Decreased hepatic conjugation

• Decreased excretion of bilirubin into bile.

 When bilirubin in blood reaches a certain concentration
approximately 2.2 to 5 mg/dL it diffuses into the
tissues.

 The skin and sclera appear yellowish due to deposition

of bilirubin in the tissues.

 This clinical condition is called jaundice or icterus

(French : jaune = yellow).

Hyperbilirubinemia may be acquired or inherited

Acquired hyperbilirubinemia are:

• Hemolytic jaundice (Prehepatic jaundice)

• Hepatic jaundice (Hepatocellular jaundice)

• Obstructive jaundice (Posthepatic jaundice)

• Neonatal jaundice (Physiological jaundice).

Inherited hyperbilirubinemia are:

• Gilbert’s syndrome

• Crigler-Najjar syndrome

• Dubin-Johnson syndrome

• Rotor syndrome.
 Pre-hepatic or Hemolytic jaundice

 Increased breakdown of hemoglobin to bilirubin at a rate

in excess of the ability of the liver cell to conjugate and
excrete it.

 Excess hemolysis may be due to:

 Sickle hemoglobin

 Deficiency of enzyme glucose-6-phosphate

dehydrogenase

 Incompatible blood transfusion.

Biochemical features

 Increased plasma concentration of unconjugated

bilirubin

 Increased amount of urobilinogen in urine and feces

 Absence of bilirubin in the urine. Since the excess

bilirubin is unconjugated, it is not excretable in the
urine
Hepatocellular or hepatic jaundice

 Hepatic parenchymal cell damage impairs uptake and

conjugation of bilirubin and results in unconjugated
hyperbilirubinemia.

 Liver damage is usually caused by:

 Infections (viral hepatitis)

 Toxic chemicals (such as alcohol, chloroform, CCl4 etc.)

 Drugs

 Cirrhosis.
 Hepatocellular damage commonly have obstruction of

the liver biliary tree that leads to increased plasma level

of conjugated bilirubin also.

Biochemical features

 Increased plasma concentration of conjugated and

unconjugated bilirubin

 Decreased amount of urobilinogen in urine and feces

 Presence of bilirubin in the urine

 Raised level of alanine transaminase (ALT) and

aspartate transaminase (AST) enzymes.
 Post-hepatic or obstructive jaundice

 Obstruction in the common bile duct that prevents the

passage of conjugated bilirubin from the liver cells to the

intestine.
 The obstruction may be due to:

 Blockage of the common bile duct by gallstones

 Carcinoma of the head of the pancreas

 Carcinoma of the duct itself.

Biochemical manifestations

 Increased plasma concentration of conjugated bilirubin

 Absence of urobilinogen in feces and urine

 The presence of bilirubin and bile salts in the urine

 Raised level of plasma alkaline phosphatase (ALP).

 Neonatal or Physiologic Jaundice

 Cause: Increased hemolysis

Immature liver UDP- glucuronyl transferase

enzyme system necessary for conjugation.

 When unconjugated bilirubin concentration in plasma

exceeds 20 to 25 mg/dL. This results in a
hyperbilirubinemic toxic encephalopathy or kernicterus,
which can cause mental retardation.
 INHERITED HYPERBILIRUBINEMIAS

Gilbert’s syndrome

Inherited disease characterized by mild unconjugated

hyperbilirubinemia due to:

 Impaired hepatic uptake of bilirubin

 Partial conjugation defect due to reduced activity of

UDP-glucuronyl transferase
 Crigler-Najjar syndrome

 Inherited disease due to deficiency of hepatic glucuronyl

transferase enzyme.

 There are two forms :

1. Type-I is characterized by complete absence of the

conjugating enzyme glucuronyl transferase and therefore no
conjugated bilirubin is formed. It causes severe jaundice with
kernicterus and early death.

2. Type-II is a less severe form in which the enzyme

deficiency is partial and is compatible with more prolonged survival.
 Dubin-Johnson syndrome

 This is harmless autosomal recessive disorder and is due

to defective hepatic secretion of conjugated bilirubin into
the bile and is characterized by slightly raised plasma
conjugated bilirubin level.

 It is characterized by abnormal black pigment in the

hepatocytes, imparting a dark brown to black color to the
liver
Laboratory Tests done to Differentiate Jaundice

1. Serum bilirubin:

Van den Bergh test is a chemical method to estimate

bilirubin in serum.

 Bilirubin + Ehrlich’s Diazo reagent (diazotized

sulfanilic acid).

 Radish purple azo pigment is formed.

They are analyzed by photometry at 540 nm.

The two types of reactions of Van den Bergh test are:
Direct and Indirect.

 For conjugated bilirubin direct Van den Bergh test is

positive, so conjugated bilirubin is called direct
bilirubin.

 For unconjugated bilirubin indirect Van den Bergh

test is positive, so unconjugated bilirubin is called
indirect bilirubin.
2. Test in urine

Urine is tested for presence of bilirubin, urobilinogen,

and bile salts.

 Urine bilirubin is tested by modified Fouchet’s test.

 Urine and fecal urobilinogen is tested by Ehrlich’s test.

 Urine bile salts are tested by Hay’s test and Pettenkofer

test.
3. Liver enzyme assay

 Transaminases (AST and ALT) elevated in hepatitis.

 Alkaline phosphatase elevated in obstructive jaundice.

 5’ Nucleotidase elevated in obstructive jaundice.