CATABOLISM OF HEME

A. Introduction:

1. The average life of the red blood cells is 120 days.

2. At the end of that time, they are removed from circulation by the cells
of reticuloendothelial (RE) system present in liver, spleen and bone
marrow.
They are hemolysed and hemoglobin comes out, giving globin and heme
molecules.
a) Globin molecule hydrolyzes into free amino acids.
b) Heme gives iron and bilirubin

‫ايهاب‬/ ‫ إعداد المادة العلمية بواسطة د‬2009 - ‫محاضرات خاصة لطالب دبلومة الكيمياء الحيوية – كلية علوم دمياط‬
B. Formation of bilirubin:

1. The heme ring is catabolized by the microsomal heme oxygenase

enzymes of the RE cells.
2. In this reaction (which needs, heme oxygenase enzyme, O2 and
NADPH), iron (Fe++) is removed for re-use. The remaining of heme
ring is cleaved between pyrrole rings number I and II to form biliverdin
(green pigment) and carbon monoxide (CO)
3. Biliverdin is then reduced into bilirubin (golden yellow) in a reaction
requires biliverdin reductase enzyme.

‫ايهاب‬/ ‫ إعداد المادة العلمية بواسطة د‬2009 - ‫محاضرات خاصة لطالب دبلومة الكيمياء الحيوية – كلية علوم دمياط‬
‫محاضرات خاصة لطالب دبلومة الكيمياء الحيوية – كلية علوم دمياط ‪ 2009 -‬إعداد المادة العلمية بواسطة د ‪/‬ايهاب‬
C. Transport of bilirubin in the plasma:
1. Bilirubin is nonpolar, and is insoluble in plasma. Therefore it binds
by noncovalent bonds to plasma albumin. This form is called:
unconjugated or indirect bilirubin.

D. Uptake of bilirubin by the liver:

1. Bilirubin dissociates from the carrier albumin molecule and enters
hepatocytes.
2. Bilirubin is conjugated with one or two molecules of glucuronic
acid (the acid form of glucose) to form bilirubin monoglucuronide and
bilirubin diglucuronide. This reaction needs
UDP-glucuronyl transferase enzyme:

E. Secretion of bilirubin into bile:

Bilirubin diglucuronide is actively transported against concentration
gradient into the bile canalciuli and then into the bile.

‫ايهاب‬/ ‫ إعداد المادة العلمية بواسطة د‬2009 - ‫محاضرات خاصة لطالب دبلومة الكيمياء الحيوية – كلية علوم دمياط‬
F. Formation of urobilin in the intestine:
1. Intestinal bacteria acts on bilirubin diglucuronide leading to:
a) Removal of glucuronides (by -glucuronidases enzymes).
b) Reduction of bilirubin to colorless compounds called:
urobilinogens
2. A small fraction of urobilinogens are reabsorbed from intestine to the
liver again and re-excreted in the bile, forming the enterohepatic
urobilinogens cycle.

‫ايهاب‬/ ‫ إعداد المادة العلمية بواسطة د‬2009 - ‫محاضرات خاصة لطالب دبلومة الكيمياء الحيوية – كلية علوم دمياط‬
G. Excretion of urobilinogens in stool and urine:
1. Most of the colorless urobilinogens are oxidized to the colored
urobilin .which excreted in the stool giving its brown color.
2. Part of urobilinogens are reabsorbed to the liver, then to the blood to be
excreted by the kidney in urine and converted into urobilin.
3.Urobilin -together with urochrome-give the characteristic yellow color
of urine.

Van den Bergh reaction

1-This is a reaction between bilirubin and Ehrlich diazo reagent giving
a reddish purple compound.
2- Conjugated bilirubin reacts directly with the reagent. Thus it is
called: direct bilirubin
3- Unconjugated bilirubin does not react with the reagent directly
except after addition of methyl alcohol. Thus it may be called: indirect
bilirubin

Differences between unconjugated bilirubin and conjugated bilirubin

Unconjugated bilirubin Conjugated bilirubin
1 Present normally in plasma 1 Present normally in bile.
2 Attached non-covalently to 2 Conjugated to glucuronic acid.
albumin.
3 Has high molecular weight and 3 Has small molecular weight
cannot be filtered through the and if present in plasma can be
kidney. filtered through the kidney.
4 Nonpolar, insoluble in plasma 4 Polar, soluble In plasma and
and can cross brain barrier in can not cross brain barrier.
neonates causing brain damage.
5 Gives indirect Van den Bergh 5 Gives direct Van den Bergh
reaction.

Heme catabolism is classified into several stages:

Stage 1: Formation of bilirubin in reticuloendothelial system. In
reticuloendothelial system (RES), hemoglobin is degraded into heme
and globin. Globin is either degraded to amino acids or reused for
synthesis of hemoglohin. Heme is converted into biliverdin (green)
then to bilirubin (yellow).

One gram of hemoglobin yields 35 mg of bilirubin. The daily bilirubin

formation in adult human is about 250 mg. Bilirubin is transported to the
liver in conjunction with plasma albumin (termed indirect or

‫ايهاب‬/ ‫ إعداد المادة العلمية بواسطة د‬2009 - ‫محاضرات خاصة لطالب دبلومة الكيمياء الحيوية – كلية علوم دمياط‬
unconjugated bilirubin). Each molecule of albumin has one high
affinity site and one low affinity site for bilirubin.

Stage II: This stage occurs in the liver and can be divided into three
processes:

1-Uptake of bilirubin by liver parenchymal cells.

This is followed by removal of albumin and release of free bilirubin.

2-Synthesis of bilirubin-diglucuronide.

This is catalyzed by bilirubin-glucuronyl-transterase to form bilirubin

diglucuronide. UDP-glucuronate (UDP-GlcUA) acts as donor or the
glucuronate group to form bilirubin-monoglucuronide then bilirubin-
diglucuronide (termed cholebilirubin. direct or conjugated bilirubin)
and it is water soluble.

3-Bilirubin-diglucuronide is actively secreted into bile.

Stage III: This is produced by the action of intestinal bacteria.

Bilirubin is deconjugated from glucuronate, and then reduced to

urobilinogens (colorless). Most of urobilinogens are excreted in feces
and oxidized to the colored pigments urobilins by oxygen. This explains
darkening of feces upon standing in air. Normally small amounts of
urobilinogens are reabsorbed by portal blood and excreted by liver cells
into bile (enterohepatic circulation) and traces escape and excreted in
urine.

Plasma Bilirubin or Bile Pigments

The nonnal level of bilirubin in plasma (or serum) is less than 1.2 mg/dL
and it is present in two forms, indirect and direct.

1-Indirect reacting (bilirubin-albumin complex or unconjugated bilirubin).

It requires the addition of methanol to react with the diazo reagent of
Van den Bergh (used for its estimation). It is the main fonn present
nonnally in plasma and it ranges from 0.3 to 0.8 mg/dL. It increases in all
cases of hemolytic anemia and when liver fails to take up or conjugate
bilirubin.

‫ايهاب‬/ ‫ إعداد المادة العلمية بواسطة د‬2009 - ‫محاضرات خاصة لطالب دبلومة الكيمياء الحيوية – كلية علوم دمياط‬
2-Direct reacting (bilirubin-diglucuronide or conjugated bilirubin).
It is water-soluble and reacts directly with the diazo reagent of van den
Bergh. It represents the bilirubin-diglucuronide which escapes from
liver to systemic blood. It increases when liver fails to excrete bilirubin
after conjugation or when there is obstruction in the biliary system that
prevents its passage to the intestine. Normally its level in plasma is less
than 0.3 mg/ldL.

HYPERBILIRUBINEMIA
Hyperbilirubinemia exists when bilirubin levels exceeds 1.2 mg/dL.
Jaundice (yellow discoloration of skin and sclera of eyes) becomes
manifest usually at levels above 2 mg/dL.
Depending on the type of bilirubin present in plasma,
hyperbilirubinemias include two main types, unconjugated and
conjugated hyperbilirubinemias.

A-Unconjugated Hyperbilirubinemia

It is due to overproduction of bilirubin by reticuloendothelial system

over the capacity of the liver to remove and clear from blood. It is
characterized by high level of indirect or unconjugated bilirubin. This
type of bilirubin can cross the blood-brain barrier into the central
nervous system and cause kernicterus (encephalopathy).
Unconjugated bilirubin is water insoluble and not excreted in urine
(acholuric jaundice).

Unconjugated hyperbilirubinemia occurs in the following conditions:

I-Neonatal or Physiologic Jaundice: This is the most common cause of

jaundice in neonatal age. It results from accelerated hemolysis and
immature hepatic system for uptake, conjugation and secretion of
bilirubin. It is characterized by high plasma level of unconjugated
bilirubin, if it exceeds 20-25 mg/dL

These cases are treated by:

a-Phenobarbital which acts as inducer for UDP-glucuronyl transferase
enzyme.
b-Phototherapy (exposure to visible light) helps hepatic excretion of
bilirubin by converting bilirubin to other derivatives that are more soluble
and easily excreted in bile.

‫ايهاب‬/ ‫ إعداد المادة العلمية بواسطة د‬2009 - ‫محاضرات خاصة لطالب دبلومة الكيمياء الحيوية – كلية علوم دمياط‬
II-Hemolytic jaundice (Anemia)
Hemolytic anemia is caused by one of the following causes:
1-The presence of abnormal hemoglobins e.g. sickle cell anemia and
thalathemia.
2-Erythroblastosis fetalis: when Rh negative mother has Rh positive
fetus (due to Rh positive father).
3-Congenital spherocytosis where the red cells are abnormally fragile.
4-Favism, due to deficiency of G 6 PD.
5-Deficiency of Pyruvate kinase or other glycolytic enzymes
6-Incompatibl blood transfusion.
7-Some diseases e.g. malaria and black water fever

III-Congenital Syndromes related to uptake and conjugation of

bilirubin as follow :
1. Crigler-Najjar Syndrome
Type 1: Due to severe decrease in the activity of UDP-glucuronyl
transferase.
Type II: Due to decreased activity of UDP-glucuronyl transferase
that adds the second glucuronide group.
2. Gilbert Disease
It is mainly due to hepatic defect in the uptake of bilirubin by
liver cells.

IV-Toxic Hyperbilirubinemia
This is due to toxin-induced liver dysfunctions e.g. chloroform, carbon
tetrachloride and mushroom poisoning.

B-Conjugated Hyperbilirubinemia
Conjugated hyperbilirubinemia is due to reflux of direct or conjugated
bilirubin into blood due to biliary obstruction, conjugated bilirubin is
water soluble, so it is excreted in urine and darken its colour (Choluric
jaundice).
It occurs in the following conditions:

1-Obstructive Jaundice (Cholestatic Jaundice)

Conjugated hyperbilirubinemia results from blockage of hepatic or
common bile duct (stones and tumors), bilirubin diglucuronide is not
excreted and returns to hepatic veins and lymphatics and appears in blood
and urine.
It may be produced by micro-obstruction of intrahepatic biliary ductules
by swollen damaged hepatocytes e.g. viral hepatitis and liver cirrhosis.
Both cases are associated with marked increase of conjugated bilirubin
‫ايهاب‬/ ‫ إعداد المادة العلمية بواسطة د‬2009 - ‫محاضرات خاصة لطالب دبلومة الكيمياء الحيوية – كلية علوم دمياط‬
and slight to moderate increase of unconjugated bilirubin (mixed
hyperbilirubinemia).

2-Chronic Idiopathic Jaundice (Dubin-Johnson Syndrome)

This is a genetic disease characterized by conjugated hyperbilirubinemia
and defect in secretion of other conjugated compounds e.g. estrogens and
sulfobromophethalein.

3-Rotor's Syndrome
It is characterized by conjugated hyperbilirubinemia with normal liver

Hyperbilirubinemia or jaundice is classified clinically into three main

types as follows:
1-Prehepatic (or hemolytic) jaundice
It is characterized by the following:
1. Anemia
2. High levels of indirect bilirubin in plasma, urobilinogen in urine and
stool
3. Dark stool (contains excessive amounts of bilirubin and urobilinogen)
4. Dark urine on standing (contains excessive amounts of urobilinogen,
which gives urobilin by oxidation, so the color of urine becomes dark on
standing).

II-Hepatic jaundice
It is characterized by the following:
1. Elevated transaminases (ALT & AST) in plasma
2. Elevated levels of both direct and indirect bilirubin in plasma.
3. Dark urine and faint stool (due to decreased excretion of bile)

III-Obstructive jaundice
It is characterized by the following:
1. Marked elevation of direct bilirubin and alkaline phosphatase in
plasma.
2. Dark urine (excess bilirubin) and very faint stool (white clay
appearance).

‫ايهاب‬/ ‫ إعداد المادة العلمية بواسطة د‬2009 - ‫محاضرات خاصة لطالب دبلومة الكيمياء الحيوية – كلية علوم دمياط‬