Bilirubin Metabolism

DR RAGHUVEER CHOUDHARY ASSISTANT PROFESSOR DEPARTMENT OF PHYSIOLOGY DR S.N.MEDICAL COLLEGE JODHPUR

Introduction
y Bilirubin is the orange-yellow pigment derived from

senescent red blood cells.
y It is a toxic waste product in the body. y It is extracted and biotransformed mainly in the liver, and

excreted in bile and urine.
y It is a bile pigment y Elevations in serum and urine bilirubin levels are normally

associated with Jaundice.

Erythrocytes become ³old´ as they lose their flexibility and become pikilocytes (spherical), 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:SPLEEN: The Graveyard of Red Blood Cells ‡ Secondary site of synthesis:LIVER & BONE MARROW 

An average

person produces about 4 mg/kg of bilirubin per day. bilirubin production from all sources in man averages from 250 to 300 mg.

TOTAL BILIRUBIN

85%
HEMOGLOBIN FROM SENESCENT RBC¶S DESTROYED IN RETICULOENDOTHELIAL CELLS OF LIVER, SPLEEN & BONE MARROW

15%
RBC PRECURSORS DESTROYED IN THE BONE MARROW 

The daily

CATABOLISM OF HEME-CONTAINING PROTEINS (MYOGLOBIN, CYTOCHROMES & PEROXIDASES)

Extravascular Pathway for RBC Destruction
(Liver, Bone marrow, & Spleen)
Phagocytosis & Lysis

Hemoglobin

Globin

Heme

Bilirubin

Amino acids

Fe2+

Amino acid pool

Recycled

Excreted

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 . Fe 2+ is released from the ring in this process. I

Oxidation Heme Oxygenase

II I

IV

II

Heme Oxygenase

I I V
Fe2+

C
II

NADPH

II I

O2

O2

IV

III

II

I

Biliverdin

H NADPH Bilirubin

I
y In the next reaction, a second

II I

IV

II

bridging methylene (between rings III and IV) is reduced by biliverdin reductase, producing bilirubin. Reductio n
Biliverdin Reductase

I

II I

IV

II

y biliverdin causing a change in the color of the molecule from

blue-green (biliverdin) to yellow-red (bilirubin).
y 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.
y Peripherally arising bilirubin is transported to the liver in

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

Excretion of Bilirubin
In Blood
y The bilirubin synthesized in spleen, liver & bone marrow is

unconjugated bilirubin.
y It is hydrophobic in nature so it is transported to the liver as

a complex with the plasma protein, albumin.
y Bilirubin-albumin complex diffuses between endothelial

cells in the liver sinusoids into the space of dissé.

Unconjugated bilirubin 
   

Lipid soluble : limits excretion 1 gm albumin binds 8.5 mg bilirubin Fatty acids & drugs can displace bilirubin 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 (-) charge Ligandin (-) charge

Ligandin Prevents bilirubin from going back to plasma

In Liver
y After uptake by hepatocytes, bilirubin is reversibly bound

to soluble proteins called ³Y´ Proteins or ³Ligandins´.
y Ligandins are cytosolic proteins & constitute 5% of total

protein of liver cytosol.
y Ligandins play an important role in the processing of

bilirubin by limiting the passive reflux of bilirubin back into the plasma.
y It also promotes the transfer of bilirubin through the

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

In Endoplasmic Reticulum
y 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.
y The microsomal enzyme bilirubin uridine diphosphate (UDP)-

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.
y The sugar acid, Glucuronic acid is first activated by enzymatic

formation of UDP-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
y Hepatic Uptake

‡ Conjugation ‡ Excretion

-Unconjugated 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
y Hepatic Uptake

‡ Conjugation ‡ Excretion

-Bilirubin which is now water soluble in the Unconjugated bilirubin (water insoluble) is is presented can now be excreted from the liver cell to(water converted to bilirubin diglucoronide the liver cell biliary system. soluble) -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

BILIRUBIN DIGLUCORONIDE

Excretion of Bilirubin

In Biliary Tract
y The excretion of bilirubin into bile is against a marked

concentration gradient is thought to be an energydependent, active-transport process.
y 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.
y 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
y

In the small intestine, conjugated bilirubins are poorly reabsorbed, but are partly hydrolyzed back to unconjugated bilirubin by catalytic action of bacterial ß-glucuronidases. In the distal ileum and colon, anaerobic flora mediate further catabolism of bile pigments:
a)

y

hydrolysis of conjugated bilirubin to unconjugated bilirubin by bacterial -glucuronidases; multistep hydrogenation (reduction) of unconjugated bilirubin to form colorless urobilinogens; and oxidation of unconjugated bilirubin to brown colored mesobilifuscins.

b)

c)

y Urobilinogens is a collective

term for a group of 3 tetrapyrroles;
± ± ±

Stercobilinogen (6H) Mesobilinogen (8H)&, Urobilinogen (12H)

y Upto 20 % of urobilinogen

produced daily is reabsorbed from the intestine & enters the entero-hepatic circulation.
Urobilinogen Structure

y Most of the reabsorbed urobilinogen is taken up by the liver

& is re-excreted in the bile.
y A small fraction (2 % - 5 %) enters the general circulation &

appears in the urine.
y In the lower intestinal tract, the 3 urobilinogens

spontaneously oxidize to produce the corresponding bile pigments; 
 

Stercobilin Mesobilin & Urobilin;

which are orange-brown in color and are the major pigments of stool.

JAUNDICE

Clinical Significance
Hyperbilirubinemia & Types of Jaundice
y Hyperbilirubinemia : Increased plasma concentrations

of bilirubin (> 3 mg/dl) occurs when there is an imbalance between its production and excretion.
y Recognized clinically as jaundice. y Also known as icterus, a yellow discoloration of the skin,

sclerae and mucous membrane.

y Jaundice becomes clinically evident when the serum

bilirubin level exceeds 2.5mg/dL.
y Several types of Jaundice:  Hemolytic  Hepatocellular  Obstructive y Symptoms:  Yellow discoloration of the skin, sclerae and mucous membranes  Itching (pruritus) due to deposits of bile salts on the skin  Stool becomes light in color  Urine becomes deep orange and foamy

Different Causes of Jaundice

y Excessive Production of Bilirubin y Reduced Hepatocyte Uptake y Impaired Bilirubin conjugation y Impaired Bile Flow

Jaundice

Classification

Pre-hepatic

Hepatic

Post-Hepatic

Prehepatic (hemolytic) jaundice
y Results from excess production of

bilirubin (beyond the livers ability to conjugate it) following hemolysis
y 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
y High plasma concentrations of

unconjugated bilirubin (normal concentration ~0.5 mg/dL)

Hepatic jaundice
y Impaired uptake, conjugation,

or secretion of bilirubin
y Reflects a generalized liver

(hepatocyte) dysfunction
y In this case,

hyperbilirubinemia is usually accompanied by other abnormalities in biochemical markers of liver function

Posthepatic jaundice
y Caused by an obstruction of the

biliary tree.
y Plasma bilirubin is conjugated,

and other biliary metabolites, such as bile acids accumulate in the plasma.
y Characterized by pale colored

stools (absence of fecal bilirubin or urobilin), and dark urine (increased conjugated bilirubin).
y In a complete obstruction,

urobilin is absent from the urine.

Pre-hepatic Jaundice

Hepatic Jaundice

Post-hepatic Jaundice

Total bilirubin Normal / Increased Increased Conjugated bilirubin Unconjugated bilirubin Urobilinogen Urine Colour Stool colour Normal Increased Increased Normal Normal Normal / Decreased

Increased Increased

Normal / Increased Normal Normal / Increased Dark Normal normal Decreased / Negative Dark Pale increased

Alkaline normal phosphate levels Alanine transferase n normal Aspartate transferase levels

increased

normal

Diagnoses of Jaundice

Neonatal Jaundice
y Common, particularly in premature infants. y Transient (resolves in the first 10 days). y Due to immaturity of the enzymes involved in bilirubin

conjugation.
y 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
y If bilirubin levels are judged to be too high, then

phototherapy with UV light is used to convert it to a water soluble, non-toxic form.

y If necessary, exchange blood transfusion is used to remove

excess bilirubin
y Phenobarbital is oftentimes administered to Mom prior to

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

D U R I N G P H O T O T H E R A P Y, T H E T R E AT M E N T O F CHOICE FOR JAUNDICE, BABIES ARE PLACED UNDER BLUE LIGHTS ( B I L I L I G H T S ) T H AT CONVERT THE BILIRUBIN INTO C O M P O U N D S T H AT C A N B E E L I M I N AT E D F R O M T H E B O D Y.
‡

y Phototherapy is the use of visible light for the treatment of

hyperbilirubinemia, or jaundice, in the newborn.

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

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

y Phototherapy is usually not needed unless the bilirubin levels rise

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

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

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

The mechanism of phototherapy
y When bilirubin molecules absorb light, 2 main photochemical reactions

occur:

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

photobilirubin) and to lumirubin.

y Unlike 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. 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. 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

y Lumirubin is not reversible. Although much less lumirubin than

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

reversible irreversibl e

Exchange Transfusion
y 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). removal of the patient's blood and replacement with fresh donor blood or plasma. Description

y The procedure involves the incremental

y 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.

y The exchange transfusion proceeds in cycles, each generally of a few

minutes duration.

y 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. patient¶s size and the severity of illness procedure needs to be repeated. post the procedure.

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

y After the exchange transfusion, catheters may be left in place in case the

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

y So exchange therapy is generally never used until after intensive

phototherapy has been attempted & exhausted.

Bilirubin Toxicity - Kernicterus
y Kernicterus or brain encephalopathy refers to the yellow

staining of the deep nuclei (i.e., the kernel) of the brain namely, the basal ganglia. jaundice.

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

y 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. jaundice due to: 


y This condition develops in newborns with prolonged

Polycythemia Rh incompatibility between mother & fetus

y 3 major features: 
 

Movement disorder Gaze abnormality, esp. upward gaze limitation Auditory abnormalities

y 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
y Gilbert¶s Syndrome y Crigler-Najjar (Type I) y Crigler-Najjar (Type II) y Lucey-Driscoll y Dubin-Johnson y Rotor¶s Syndrome

Algorithm for differentiating the familial causes of Hyperbilirubinemia

Isolated increased serum bilirubin

Ruling out of hemolysis, subsequent fractionation of the bilirubin

Conjugated Possibility of the following syndromes: ‡ Dublin-Johnson ‡ Rotor

Unconjugate d Possibility of following syndromes based on the bilirubin concentration: ‡ Gilbert¶s - <3 mg/dl ‡ Crigler-Najjar (Type I) - >25 mg/dl ‡ Crigler-Najjar (Type II) - 5 to 20 mg/dl ‡ Lucey-Driscoll - Transiently ~ 5 mg/dl

Gilbert¶s Syndrome
y Gilbert¶s syndrome is also called as familial non-hemolytic

non-obstructive jaundice.
y It is a benign condition manifested by mild unconjugated

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

as chronic Hepatitis.
y The concentration of Bilirubin in serum fluctuates between 1.5

& 3 mg/dl.
y In this condition the activity of hepatic glucuronyltransferase

is low as a result of mutation in the bilirubin-UDPglucuronyltransferase gene(UGT1A1).

y It is easily distinguished from chronic hepatitis by the

absence of anemia & bilirubin in urine, & also by normal liver function tests
y Special diagnostic tests are occasionally necessary &

include demonstrating a rise in bilirubin on fasting and a fall in bilirubin on taking Phenobarbital.
y Gilbert¶s syndrome is inherited as an autosomal recessive

trait.
y Males are more frequently affected then females. Onset of

symptoms is seen in teens, early 20¶s or 30¶s.
y 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)
y Crigler-Najjar Syndrome (Type I) is a rare genetic disorder

caused by complete absence of UDP-glucuronyltransferase and manifested by very high levels of unconjugated bilirubin.
y It is inherited as an autosomal recessive trait. y Most patients die of severe brain damage caused by

kernicterus within the first year of life.
y Early liver transplantation is the only effective therapy.

Crigler-Najjar Syndrome (Type II)
y This is a rare autosomal dominant disorder. y It is characterized by partial deficiency of UDP-

glucuronyltransferase.
y Unconjugated bilirubin is usually 5 ± 20 mg/dl. y Unlike Crigler-Najjar Type I, Type II responds

dramatically to Phenobarbital & a normal life can be expected.

Dubin-Johnson Syndrome
y It is a benign, autosomal recessive

condition characterized by jaundice with predominantly elevated conjugated bilirubin and a minor elevation of unconjugated bilirubin. anions and bilirubin into bile is impaired, reflecting the underlying defect in canalicular excretion. black appearance and liver biopsy reveals a dark brown melanin-like pigment in hepatocytes and kupffer cells.

y Excretion of various conjugated

y The Liver has a characteristic greenish

Rotor¶s Syndrome

y It is another form of conjugated hyperbilirubinemia. y It is similar to dubin-johnson syndrome but without

pigmentation in liver.

Clinical Conditions Related to Increased Conjugated Hyperbilirubinemia
Dubin-Johnson Rotor Syndrome Syndrome Defect (hepatocytes) Presence of Pigmentation Metabolism Secretory Yes Abnormal Porphyrin Metabolism Transport No None

Lucey-Driscoll Syndrome
y Lucey-Driscoll Syndrome is a familial form of

unconjugated hyperbilirubinemia caused by a circulating inhibitor of bilirubin conjugation.
y The hyperbilirubinemia is mild and lasts for the

first 2 to 3 weeks of life.