Bilirubin Metabolism

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 Introduction

þ Bilirubin is the orange-yellow pigment derived from

senescent red blood cells.

þ It is a toxic waste product in the body.

þ It is extracted and biotransformed mainly in the liver, and

excreted in bile and urine.

þ It is a bile pigment

þ Elevations in serum and urine bilirubin levels are normally

associated with Jaundice.
Erythrocytes become ³old´ as they lose their flexibility
and become _

_   increasingly rigid
and fragile. Once the cell become fragile, they easily
destruct during passage through tight circulation
spots, especially in spleen, where the intra-capillary
space is about 3 micron as compared to 8 micron of
cell size
RBCs useful life span is 100 to 120 days,After which
they become trapped and fragment in smaller circulatory
channels, particularly in those of the spleen. For this
reason, the spleen is sometimes called the ³red blood cell
graveyard.´
Dying erythrocytes are engulfed and destroyed by
macrophages.
 Formation of Bilirubin

‡ Primary site of synthesis:-

: The Graveyard
of Red Blood Cells
‡ Secondary site of synthesis:-


 
 
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 Pathophysiology
RBCs
Breakdown

Hemoglobin Produces
& Breakdown

Heme
Heme
Oxygenase
Biliverdin
Biliverdin
Reductase
Bilirubin
‡ The globin is recycled or converted into amino acids, which
in turn are recycled or catabolized as required.
‡ Heme is oxidized, with the heme porphyrin ring being
opened by the endoplasmic reticulum enzyme, heme
oxygenase.
‡ The oxidation occurs on a specific carbon producing
equimolar amounts of the linear tetrapyrrole biliverdin,
iron , and carbon monoxide (CO). This is the only reaction
in the body that is known to produce CO.
‡ Most of the CO is excreted through the lungs, with the
result that the CO content of expired air is a direct measure
of the activity of heme oxygenase in an individual.
In the first reaction, a
bridging m eth yl en e
group is cleaved by
h em e o x y gen a se to
form Linear Biliverdin
from Cyclic Heme
m o l e c u l e .
Oxidation Heme Oxygenase
Fe 2+ is released from
the ring in this process.

I II IV II
I
 Heme Oxygenase

I
C NADPH
I /3 II
V
O2 O2
II
I
IV III II I

Biliverdin
 H

Bilirubin
NADPH
 I II IV II
þ In the next reaction, a second I
bridging methylene (between
rings III and IV) is reduced by
biliverdin reductase,
producing $+,+"$+.
Reductio Biliverdin Reductase
n

I II IV II
I
þ biliverdin causing a change in the color of the molecule from
blue-green (biliverdin) to yellow-red (bilirubin).

þ The latter catabolic changes in the structure of tetrapyrroles

are responsible for the progressive changes in color of a
hematoma, or bruise, in which the damaged tissue changes
its color from an initial dark blue to a red-yellow and finally
to a yellow color before all the pigment is transported out of
the affected tissue.

þ Peripherally arising bilirubin is transported to the liver in

association with albumin, where the remaining catabolic
reactions take place.
 Excretion of Bilirubin

In Blood

þ The bilirubin synthesized in spleen, liver & bone marrow is

unconjugated bilirubin.

þ It is hydrophobic in nature so it is transported to the liver as

a complex with the plasma protein, albumin.

þ Bilirubin-albumin complex diffuses between endothelial

cells in the liver sinusoids into the space of dissé.
„nconjugated bilirubin

w Lipid soluble
w : limits excretion
w 1 gm albumin binds 8.5 mg bilirubin
w Fatty acids & drugs can displace bilirubin
w Indirect positive reaction in van den Bergh test
Role of Blood Proteins in the Metabolism
of Bilirubin

1. Albumin

Dissolved in Blood
Blood

Liver
Ligandin Ligandin

(-) charge (-) charge

Ligandin Prevents bilirubin

from going back to plasma
In Liver

þ After uptake by hepatocytes, bilirubin is reversibly bound

to soluble proteins called ³Y´ Proteins or ³Ligandins´.

þ Ligandins are cytosolic proteins & constitute 5% of total

protein of liver cytosol.

þ Ligandins play an important role in the processing of

bilirubin by limiting the passive reflux of bilirubin back
into the plasma.

þ It also promotes the transfer of bilirubin through the

cytosol to the smooth endoplasmic reticulum where it is
further processed.
In Endoplasmic Reticulum

þ In the microsomes of the endoplasmic reticulum,

unconjugated bilirubin is converted to water soluble mono- or
di- conjugates by sequential covalent coupling with glucuronic
acid.

þ The microsomal enzyme bilirubin uridine diphosphate („DP)-

glucuronyltransferase catalyzes the formation of bilirubin mono
glucuronide.It is not yet clear whether the same enzyme also
catalyzes the conversion of bilirubin mono glucuronide to
bilirubin di-glucuronide.

þ The sugar acid, Glucuronic acid is first activated by enzymatic

formation of „DP-glucuronic acid and then transferred to
bilirubin by the enzyme glucuronyltransferase
Bilirubin is conjugated in
a two step process to
form bilirubin mono- &
di- glucuronide
 3 Steps of Bilirubin Metabolism

þ Hepatic „ptake ‡ Conjugation

‡ Excretion
-„nconjugated bilirubin is presented in the
liver cell
-The albumin associated with it is dissociated
-Ligandin is delivered to prevent efflux of
bilirubin back to plasma
 3 Steps of Biliverdin Metabolism
þ Hepatic „ptake ‡ Conjugation
‡ Excretion
-Bilirubin whichbilirubin
„nconjugated is now water
(water
is presented
soluble
insoluble)
in
cantheis
now
converted
liver be
cellexcreted
to bilirubin
from the
diglucoronide
liver cell to(water
the
biliary
soluble)system.
-The albumin associated with it is dissociated
-Takes place in the smooth endoplasmic
-Ligandin is delivered to prevent efflux of
reticulum of the liver
bilirubin back to plasma
-Catalyzed by glucoronyl transferase
Conjugation with Glucoronates

BILIR„BIN
DIGL„CORONIDE
Excretion of Bilirubin
In Biliary Tract

þ The excretion of bilirubin into bile is against a marked

concentration gradient is thought to be an energy-
dependent, active-transport process.

þ Bilirubin conjugates pass with the bile successively from

the canaliculi, through the bile ductules and intrahepatic
ducts of progressively increasing caliber, to the
extrahepatic bile ducts.

þ Between meals, bile bilirubins are temporarily stored in

the gallbladder, from which they are emptied into the
duodenum during feeding.
The Biliary System
In the Intestine

þ In the small intestine, conjugated bilirubins are poorly

reabsorbed, but are partly hydrolyzed back to unconjugated
bilirubin by catalytic action of bacterial ß-glucuronidases.

þ In the distal ileum and colon, anaerobic flora mediate further

catabolism of bile pigments:

a) hydrolysis of conjugated bilirubin to unconjugated bilirubin by

bacterial ǃ-glucuronidases;

b) multistep hydrogenation (reduction) of unconjugated bilirubin

to form colorless urobilinogens; and

c) oxidation of unconjugated bilirubin to brown colored

mesobilifuscins.
þ „robilinogens is a collective
term for a group of 3
tetrapyrroles;
± Stercobilinogen (6H)
± Mesobilinogen (8H)&,
± „robilinogen (12H)

þ „pto 20 % of urobilinogen
produced daily is reabsorbed
from the intestine & enters the
entero-hepatic circulation.
„robilinogen Structure
þ Most of the reabsorbed urobilinogen is taken up by the liver
& is re-excreted in the bile.

þ A small fraction (2 % - 5 %) enters the general circulation &

appears in the urine.

þ In the lower intestinal tract, the 3 urobilinogens

spontaneously oxidize to produce the corresponding bile
pigments;
w Stercobilin
w Mesobilin &
w „robilin;
which are orange-brown in color and are the major
pigments of stool.
i

 Clinical Significance

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þ Hyperbilirubinemia : Increased plasma concentrations

of bilirubin (> 3 mg/dl) occurs when there is an
imbalance between its production and excretion.

þ Recognized clinically as jaundice.

þ Also known as icterus, a yellow discoloration of the skin,

sclerae and mucous membrane.
þ Jaundice becomes clinically evident when the serum
bilirubin level exceeds 2.5mg/dL.

þ Several types of Jaundice:

w Hemolytic
w Hepatocellular
w Obstructive

þ Symptoms:
w Yellow discoloration of the skin, sclerae and mucous membranes
w Itching (pruritus) due to deposits of bile salts on the skin
w Stool becomes light in color
w „rine becomes deep orange and foamy
Different Causes of Jaundice

þ Excessive Production of Bilirubin

þ Reduced Hepatocyte „ptake
þ Impaired Bilirubin conjugation
þ Impaired Bile Flow
 Jaundice

Classification

Pre-hepatic Hepatic Post-Hepatic

Prehepatic (hemolytic) jaundice
þ Results from excess production of
bilirubin (beyond the livers ability
to conjugate it) following
hemolysis

þ Excess RBC lysis is commonly the

result of autoimmune disease;
hemolytic disease of the newborn
(Rh- or ABO- incompatibility);
structurally abnormal RBCs
(Sickle cell disease); or
breakdown of extravasated blood

þ High plasma concentrations of

unconjugated bilirubin (normal
concentration ~0.5 mg/dL)
Hepatic jaundice
þ Impaired uptake, conjugation,
or secretion of bilirubin

þ Reflects a generalized liver

(hepatocyte) dysfunction

þ In this case,
hyperbilirubinemia is usually
accompanied by other
abnormalities in biochemical
markers of liver function
Posthepatic jaundice
þ Caused by an obstruction of the
biliary tree.

þ Plasma bilirubin is conjugated,

and other biliary metabolites,
such as bile acids accumulate in
the plasma.

þ Characterized by pale colored

stools (absence of fecal
bilirubin or urobilin), and dark
urine (increased conjugated
bilirubin).

þ In a complete obstruction,
urobilin is absent from the
urine.
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Diagnoses of Jaundice
 Neonatal Jaundice
þ Common, particularly in premature infants.

þ Transient (resolves in the first 10 days).

þ Due to immaturity of the enzymes involved in bilirubin

conjugation.

þ High levels of unconjugated bilirubin are toxic to the

newborn ± due to its hydrophobicity it can cross the
blood-brain barrier and cause a type of mental
retardation known as kernicterus

þ If bilirubin levels are judged to be too high, then

phototherapy with „V light is used to convert it to a
water soluble, non-toxic form.
þ If necessary, exchange blood transfusion is used to remove
excess bilirubin

þ Phenobarbital is oftentimes administered to Mom prior to

an induced labor of a premature infant ± crosses the
placenta and induces the synthesis of „DP glucuronyl
transferase

þ Jaundice within the first 24 hrs of life or which takes longer

then 10 days to resolve is usually pathological and needs to
be further investigated
 Phototherapy

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þ Phototherapy is the use of visible light for the treatment of
hyperbilirubinemia, or jaundice, in the newborn.

þ It is one of the most common non-routine therapy applied in the

newborn population & is a treatment for all types of jaundice.

þ Phototherapy is usually not needed unless the bilirubin levels rise

very quickly or go above 16-20 mg/dl in healthy, full term babies.

þ Phototherapy is used at much lower levels of jaundice in premature

or sick infants.

þ Phototherapy does not have to be continuous to be useful.

 The mechanism of phototherapy

þ When bilirubin molecules absorb light, 2 main photochemical reactions

occur:

þ Native 4Z,15Z-bilirubin converts to 4Z,15E bilirubin (also known as

photobilirubin) and to lumirubin.

þ „nlike 4Z,15Z bilirubin, photobilirubin can be excreted via the liver without
conjugation, but its clearance is very slow, and its conversion is reversible. In
the bowel (away from the light), photobilirubin is converted back to native
bilirubin.

þ Lumirubin is not reversible. Although much less lumirubin than

photobilirubin is formed, lumirubin is cleared from the serum much more
rapidly, and it is likely that lumirubin formation is primarily responsible for
the decline in serum bilirubin that results from phototherapy.

þ Small amounts of native bilirubin are also oxidized to monopyrroles and

dipyrroles that can be excreted in the urine. This is a slow process and only a
minor contributor to the elimination of bilirubin during phototherapy
reversible

irreversibl
e
 Exchange Transfusion
þ Exchange transfusion is a potentially life-
saving procedure performed to counteract
the effects of serious jaundice or changes
in the blood (from, for example, sickle cell
anemia).

þ The procedure involves the incremental

removal of the patient's blood and
replacement with fresh donor blood or
plasma.

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þ In order to perform an exchange

transfusion, it is essential to have the
ability to both remove and replace blood.
In most cases, this involves the insertion
of more than one intravenous (or arterial)
catheter.
þ The exchange transfusion proceeds in cycles, each generally of a few
minutes duration.

þ The patient¶s blood is slowly withdrawn, and an equal amount of fresh, pre-
warmed blood or plasma is transfused. This cycle is repeated until a
predetermined volume of blood has been replaced.

þ The blood is withdrawn usually in increments of 5 to 20 ml depending on the

patient¶s size and the severity of illness

þ After the exchange transfusion, catheters may be left in place in case the
procedure needs to be repeated.

þ The exchange transfusion process is riddled with complications in process &

post the procedure.

þ So exchange therapy is generally never used until after intensive

phototherapy has been attempted & exhausted.
 Bilirubin Toxicity - Kernicterus
þ Kernicterus or brain encephalopathy refers to the yellow
staining of the deep nuclei (i.e., the kernel) of the brain
namely, the basal ganglia.

þ It is a form of permanent brain damage caused by excessive

jaundice.

þ The concentration of bilirubin in serum is so high that it can

move out of the blood into brain tissue by crossing the fetal
blood-brain barrier.

þ This condition develops in newborns with prolonged

jaundice due to:
w Polycythemia
w Rh incompatibility between mother & fetus
þ 3 major features:
w Movement disorder
w Gaze abnormality, esp. upward gaze limitation
w Auditory abnormalities

þ Concentrations of bilirubin in the blood serum of affected

infants with hemolytic disease should be monitored so that
treatment can begin before dangerous concentrations are
reached. Treatment is with blood transfusion, and can be
administered before birth.
Inherited Disorders of Bilirubin
Metabolism

þ Gilbert¶s Syndrome
þ Crigler-Najjar (Type I)
þ Crigler-Najjar (Type II)
þ Lucey-Driscoll
þ Dubin-Johnson
þ Rotor¶s Syndrome
 Algorithm for differentiating the familial causes
of Hyperbilirubinemia

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Ruling out of hemolysis, subsequent fractionation of the bilirubin

Conjugated „nconjugate
d
Possibility of following syndromes
Possibility of the
based on the bilirubin concentration:
following syndromes:
‡ Gilbert¶s - <3 mg/dl
‡ Dublin-Johnson
‡ Crigler-Najjar (Type I) - >25 mg/dl
‡ Rotor
‡ Crigler-Najjar (Type II) - 5 to 20
mg/dl
‡ Lucey-Driscoll - Transiently ~ 5
mg/dl
 Gilbert¶s Syndrome
þ Gilbert¶s syndrome is also called as familial non-hemolytic
non-obstructive jaundice.

þ It is a benign condition manifested by mild unconjugated

Hyperbilirubinemia.

þ It affects 3% ± 5% of the population. It is often misdiagnosed

as chronic Hepatitis.

þ The concentration of Bilirubin in serum fluctuates between 1.5

& 3 mg/dl.

þ In this condition the activity of hepatic glucuronyltransferase

is low as a result of mutation in the bilirubin-„DP-
glucuronyltransferase gene(„GT1A1).
þ It is easily distinguished from chronic hepatitis by the
absence of anemia & bilirubin in urine, & also by normal
liver function tests

þ Special diagnostic tests are occasionally necessary &

include demonstrating a rise in bilirubin on fasting and a
fall in bilirubin on taking Phenobarbital.

þ Gilbert¶s syndrome is inherited as an autosomal recessive

trait.

þ Males are more frequently affected then females. Onset of

symptoms is seen in teens, early 20¶s or 30¶s.

þ Gilbert¶s syndrome does not require any treatment as it

does not interfere with the normal lifestyle of a person.
 Crigler-Najjar Syndrome (Type I)
þ Crigler-Najjar Syndrome (Type I) is a rare genetic disorder
caused by complete absence of „DP-glucuronyltransferase
and manifested by very high levels of unconjugated bilirubin.

þ It is inherited as an autosomal recessive trait.

þ Most patients die of severe brain damage caused by

kernicterus within the first year of life.

þ Early liver transplantation is the only effective therapy.

 Crigler-Najjar Syndrome (Type II)

þ This is a rare autosomal dominant disorder.

þ It is characterized by partial deficiency of „DP-

glucuronyltransferase.

þ „nconjugated bilirubin is usually 5 ± 20 mg/dl.

þ „nlike Crigler-Najjar Type I, Type II responds

dramatically to Phenobarbital & a normal life can be
expected.
 Dubin-Johnson Syndrome
þ It is a benign, autosomal recessive
condition characterized by jaundice
with predominantly elevated
conjugated bilirubin and a minor
elevation of unconjugated bilirubin.

þ Excretion of various conjugated

anions and bilirubin into bile is
impaired, reflecting the underlying
defect in canalicular excretion.

þ The Liver has a characteristic greenish

black appearance and liver biopsy
reveals a dark brown melanin-like
pigment in hepatocytes and kupffer
cells.
 Rotor¶s Syndrome

þ It is another form of conjugated hyperbilirubinemia.

þ It is similar to dubin-johnson syndrome but without

pigmentation in liver.
Clinical Conditions Related to Increased
Conjugated Hyperbilirubinemia

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 Lucey-Driscoll Syndrome

þ Lucey-Driscoll Syndrome is a familial form of

unconjugated hyperbilirubinemia caused by a
circulating inhibitor of bilirubin conjugation.

þ The hyperbilirubinemia is mild and lasts for the

first 2 to 3 weeks of life.