Mansoura University

Faculty of Science-Damietta
Chemistry Department
Branch-Biochemistry
2009

For

Biochemistry Diploma Students

Dr.Ehab
 A. Introduction
The average life of the red blood cells is 120. 1
. 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
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 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.

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C. Transport of bilirubin
in the plasma

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.

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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:

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E. Secretion of bilirubin
into bile
Bilirubin diglucuronide is actively transported
against concentration gradient into the bile

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 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.

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 G. Excretion of
urobilinogens in stool
and urine
Most of the colorless urobilinogens are . 1
oxidized to the colored urobilin .which
.excreted in the stool giving its brown color
Part of urobilinogens are reabsorbed to the . 2
liver, then to the blood to be excreted by the
.kidney in urine and converted into urobilin
Urobilin -together with urochrome-give the .3
.characteristic yellow color of urine

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 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

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Differences between unconjugated bilirubin and conjugated
bilirubin

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 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).

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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 unconjugated bilirubin).
Each molecule of albumin has one high affinity
site and one low affinity site for bilirubin.

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 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.
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 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.

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 Plasma Bilirubin or Bile
Pigments
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

Indirect reacting (bilirubin-albumin complex or -1

.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

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Direct reacting (bilirubin-diglucuronide -2
. 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.

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 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.

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A-Unconjugated
It is due to overproduction of bilirubin by
reticuloendothelial system over the
Hyperbilirubinemia
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).
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 Unconjugated
hyperbilirubinemia
occurs
I-Neonatal in the
or Physiologic following
Jaundice: This is the
most common cause of jaundice in neonatal age. It
conditions
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

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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.

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 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

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III-Congenital Syndromes related to uptake
and conjugation of bilirubin as follow :
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.
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 29
 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.

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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 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
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 Hyperbilirubinemia
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).
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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).

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