Bilirubin metabolism and jaundice jaundice Jayanta Roy Chowdhury Professor of Medicine and Molecular Genetics Albert Einstein College of Medicine.

BilirubinBilirubin metabolism metabolism andand

jaundicejaundice

BilirubinBilirubin metabolism metabolism andand

jaundicejaundice

Jayanta Roy ChowdhuryJayanta Roy Chowdhury

Professor of Medicine and Molecular GeneticsProfessor of Medicine and Molecular GeneticsAlbert Einstein College of MedicineAlbert Einstein College of Medicine

Pathophysiological importance of bilirubin metabolism

It is the end product of heme degradation.

Serum bilirubin level is an important clinical marker of hepatobiliary excretory function.

Bilirubin is an endogenous model for plasma carriage and hepatic throughput of organic anions.

Hepatic uptake, storage, conjugation and excretion of bilirubin are finely balanced. Therefore, enhancement of bilirubin throughput requires coordinated induction of multiple genes, which may be mediated by nuclear receptors.

Erythroid Non-erythroidErythroid Non-erythroid

Normal:

Senescent erythrocytes Free heme

Abnormal:Abnormal:

• Hemolysis: Extravascular Intravascular

• Ineffective erythropoiesis

Normal:

Senescent erythrocytes Free heme

Abnormal:Abnormal:

• Hemolysis: Extravascular Intravascular

• Ineffective erythropoiesis

(80%) (20%)(80%) (20%)(80%) (20%)(80%) (20%)

• CytochromesCytochromes• CatalaseCatalase• PeroxidasePeroxidase• Tryptophane pyrrolaseTryptophane pyrrolase

• MyoglobinMyoglobin

• CytochromesCytochromes• CatalaseCatalase• PeroxidasePeroxidase• Tryptophane pyrrolaseTryptophane pyrrolase

• MyoglobinMyoglobin

Sources of bilirubinSources of bilirubinSources of bilirubinSources of bilirubin

Early bilirubin(15-20%)0- 3 days

Late bilirubin(65%)

40- 80 days

Non-Hbsources (liver)

Increased erythropoiesis

Erythrocytesources

Early and late labeled peaks of radioisotope incorporation into bilirubinAfter injection of labeled porphyrin precursor (14C-glycine)

Opening of the heme ring and Enzyme-catalyzed formation of bilirubin

C

NH

MV

ONH

M CH2

CH2

O OH

CH2

NH

NH

CH2

CH2

C

OOH

M MV

O

The linear structure of bilirubin:Two dipyrroles joined by a central methene bridge

C

NH

MV

ONH

M CH2

CH2

O OH

CH2

NH

NH

CH2

CH2

C

OOH

M MV

O

Bilirubin contains several polar groups (shown in red):Yet, it is insoluble in water.

C

NH

MV

ONH

M CH2

CH2

O OH

CH2

NH

NH

CH2

CH2

C

OOH

M MV

O

Water insolubility of bilirubin is explained by internal hydrogen bonding.

C

NH

MV

ONH

M CH2

CH2

O OH

CH2

NH

NH

CH2

CH2

C

OOH

M MV

O

This is explained by internal hydrogen bonding.

NH

MV

ONH

M CH2OH

CH2

NH

NH

CH2

CH2

C

OOH M MV

O

CH2 C

O


NH

MV

ONH

M CH2OH

CH2

NH

NH

CH2

CH2

C

OOH M MV

O

CH2 C

O


NH

MV

ONH

M CH2OH

CH2

NH

NH

CH2

CH2

C

OOH M MV

O

CH2 C

O

As a consequence of hydrogen bonding, all polar groups are engaged.

The central methene bridge becomes buried.

C

C

Ridge-tile structure of bilirubin

Conjugation with glucuronic acid makes bilirubin water soluble

NH

MV

ONH

M CH2OH

CH2

NH

NH

CH2

CH2

C

OOH M MV

O

CH2 C

O

The internal hydrogen bonds of bilirubin are The internal hydrogen bonds of bilirubin are disrupted by conjugation of the propionic aciddisrupted by conjugation of the propionic acidcarboxyl group with glucuronic acidcarboxyl group with glucuronic acid

NH

MV

ONH

M CH2

CH2

NH

NH

CH2

CH2

C

O

M MV

O

CH2 CO-GlucA

GlucA-

The internal hydrogen bonds of bilirubin are The internal hydrogen bonds of bilirubin are disrupted by conjugation of the propionic aciddisrupted by conjugation of the propionic acidcarboxyl group with glucuronic acidcarboxyl group with glucuronic acid

Phototherapy changes the configuration of bilirubin makingit transiently water soluble

NH

MV

ONH

M CH2OH

CH2

NH

NH

CH2

CH2

C

OOH M MV

O

CH2 C

O

Internal hydrogen bonds are disrupted transiently upon exposure of bilirubin to light.

C

C

The dipyrrole carbon bridges switch direction.

NH

MV

ONH

M CH2OH

CH2

NH

NH

CH2

CH2

C

OOH M MV

O

CH2 C

O

Thus a carbon atom comes in the way of the hydrogen bonds.


NH

MV

ONH

M CH2OH

CH2

NH

NH

CH2

CH2

C

OOH M MV

O

CH2 C

O

Thus a carbon atom comes in the way of the hydrogen bonds.


C

C

NH

MV

ONH

M CH2OH

CH2

NH

NH

CH2

CH2

C

OOH M MV

O

CH2 C

O

C

C

The bulky carbon atom disrupts the hydrogen bonds by steric hindrence.

NH

MV

ONH

M CH2OH

CH2

NH

NH

CH2

CH2

C

OOH M MV

O

CH2 C

O

C

C

The bulky carbon atom disrupts the hydrogen bonds by steric hindrence.

Exposure to diazo reagents resultin “direct” and “indirect” van den Burgh reaction, roughlycorresponding to conjugated andunconjugated fractions of bilirubin.

NH

MV

ONH

M CH2OH

CH2

NH

NH

CH2

CH2

C

OOH M MV

O

CH2 C

O

C

C

In unconjugatedbilirubin, the central carbonbridge is buried byhydrogen bonds. Therefore, the van den Burgh reaction

is “indirect”.

NH

MV

ONH

M CH2

CH2

NH

NH

CH2

CH2

CO

M MV

O

CH2 CO-GlucA

GlucA-

In conjugatedbilirubin, the central carbonbridge is accessible. Therefore, the van den Burgh reaction

is “direct”.

Bilirubin throughput: schema of a hepatocyteBilirubin throughput: schema of a hepatocyte

Sinusoidalsurface

Canalicularsurface

Tight junction

Liversinusoid

Fenestratedendothelium

Bilirubin circulates bound to serum albumin.

BBalb

Albumin- binding: Keeps bilirubin soluble

Prevents tissue deposi- tion.

Prevents renal excretion

Drugs that displace bilirubin from albumin may precipitate kernicterus: Sulfonamides Coumadin, etc.

Bilirubin circulates bound to serum albumin. At the sinusoidal surface of hepatocytes, it dissociates from albumin.

BBalb


BBalb


BB

alb


BB

alb

Bilirubin enters through the sinusoidal surface, probably by facilitated diffusion. Uptake is energy independent and bidirectional.

BB

Bilirubin uptakeis reduced: In neonates

In cirrhosis

From drug effect: novobiocin

In some cases of Gilbert syndrome

What is the mechanism of What is the mechanism of facilitated diffusion of bilirubin?facilitated diffusion of bilirubin?

• Zucker has proposed that no transporter protein Zucker has proposed that no transporter protein is needed. is needed.

• In a recent report, organic anion transport In a recent report, organic anion transport protein 2 (oatp2) has been implicated in bilirubin protein 2 (oatp2) has been implicated in bilirubin uptake. uptake.

• However, our recent studies show that althoughHowever, our recent studies show that although oatp2 transports organic anions, such as BSP, oatp2 transports organic anions, such as BSP, it is not sufficient for bilirubin transport. it is not sufficient for bilirubin transport.

B

Inside the hepatocyte, bilirubin binds to cytosolic proteins termed ligandins, which are the same as glutathione-S- transferases (GSTs).

GSTs

B

GST bindinginhibits theefflux of bilirubin,thereby increasingits net uptake

B

GSTs

B

B

Conjugation of bilirubin with glucuronic acid is catalyzed by UGT1A1, which transfers glucuronic acid from UDP-glucuronic acid to bilirubin

GSTsUDPUDPGAGA UDPUDP

BBGAGAUGT1A1

B

Conjugation with glucuronic acid makes bilirubin water-soluble and non-toxic.

Glucuronidation is essential for biliary excretion of bilirubin.

UDP-glucuronosyltransferases (UGTs)UDP-glucuronosyltransferases (UGTs)UDP-glucuronosyltransferases (UGTs)UDP-glucuronosyltransferases (UGTs)

• UGTs are ER proteins that convert many internal and UGTs are ER proteins that convert many internal and exogenous toxins to non-toxic metabolites.exogenous toxins to non-toxic metabolites.

• UGT’s are a family of enzymes concentrated in the liver.UGT’s are a family of enzymes concentrated in the liver.

• One UGT isoform, UGT1A1, conjugates bilirubin and is One UGT isoform, UGT1A1, conjugates bilirubin and is essential for its excretion. essential for its excretion.

• Inherited UGT1A1 deficiency causes jaundice.Inherited UGT1A1 deficiency causes jaundice.

Substrate Substrate

UDPGAUDPGAUDPGAUDPGA

•UGTUGT•UGTUGT GlucuronideGlucuronideGlucuronideGlucuronide

UDPUDPUDPUDP

Inherited disorders of bilirubin metabolism causing Inherited disorders of bilirubin metabolism causing

Unconjugated HyperbilirubinemiaUnconjugated HyperbilirubinemiaInherited disorders of bilirubin metabolism causing Inherited disorders of bilirubin metabolism causing

Unconjugated HyperbilirubinemiaUnconjugated Hyperbilirubinemia

• Crigler-Najjar syndrome Crigler-Najjar syndrome type 1:type 1:


• Gilbert syndrome:Gilbert syndrome:

Virtually no UGT1A1 activityVirtually no UGT1A1 activity

UGT1A1 activity below 10% UGT1A1 activity below 10%

UGT1A1 activity ~30% UGT1A1 activity ~30%







Serum bilirubin 18-40 mg/dl:Serum bilirubin 18-40 mg/dl:Kernicterus, unless treated Kernicterus, unless treated vigorouslyvigorously

Serum bilirubin 8-18 mg/dl:Serum bilirubin 8-18 mg/dl:Kernicterus is rare Kernicterus is rare

Serum bilirubin normal to Serum bilirubin normal to 5 mg mg/dl 5 mg mg/dl (increases during fasting, (increases during fasting, intercurrent illness, etc.intercurrent illness, etc.No cerebral toxicity.No cerebral toxicity.







RareRareautosomal recessiveautosomal recessive

RareRareautosomal recessive autosomal recessive

Very common, Very common, autosomal recessive.autosomal recessive.

9% of population homozygous.9% of population homozygous.~4% exhibit clinical jaundice ~4% exhibit clinical jaundice intermittentlyintermittently







Bilirubin conjugates are almost Bilirubin conjugates are almost absent in bileabsent in bile

Proportion of bilirubin mono-Proportion of bilirubin mono-glucuronide is increased in bileglucuronide is increased in bilenormal >10%) normal >10%)

Proportion of bilirubin mono-Proportion of bilirubin mono-glucuronide is increased in bileglucuronide is increased in bilenormal >10%)normal >10%)







Phenobarbital treatment: Phenobarbital treatment: little or no effect.little or no effect.

Phenobarbital reduces serum Phenobarbital reduces serum bilirubin is by >25%bilirubin is by >25%

Serum bilirubin is normalizedSerum bilirubin is normalized

In 1953, Crigler and Najjar described “a mysterious illness that caused jaundice and severe neurological damage”

Genetic Lesions in UGT1A1 Deficiency Genetic Lesions in UGT1A1 Deficiency SyndromesSyndromes

Genetic Lesions in UGT1A1 Deficiency Genetic Lesions in UGT1A1 Deficiency SyndromesSyndromes

1*11*12 1*7 1*6 1*5 1*4 1*3 1*2 2 3 4 5

Stop codonor frame-shift

Substitution

Splice-sitemutation

CN-I

CN-II

Signal peptide

Gilbert

A(TA)7 TAAA(TA)7 TAA[Normal: A(TA)6 TAA][Normal: A(TA)6 TAA]

UGT1A1 locus

Treatment of Crigler-Najjar syndrome type 1

• Routine phototherapy has extended the life expectancy to adolescence and beyond.

• During emergency, bilirubin may be removed by plasmapheresis.

• Tin mesoporphyrin can be used for transient reduction of serum bilirubin levels

• At puberty, phototherapy becomes progressively ineffective.

• Liver transplantation is the only curative therapy.

• In one patient, liver cell transplantation reduced serum bilirubin level by 50%.

Phototherapy bed

CN-1 syndrome-1: permanent brain damageCN-1 syndrome-1: permanent brain damage

250-

200-

150-

100-

50-

0-

Effect of drugs and hormones on rat liver UGT1A1 activity

Per

cen

t of

bas

al a

ctiv

ity

Untreat

ed

Phenob

arbita

l

Clofib

rate

Thyroid

hor

mon

e

Rifam

pin

Nuclear receptor CARCARCARCAR PPARPPARPPARPPAR PXRPXRPXRPXR TRTRTRTR


Conjugated + Unconjugated HyperbilirubinemiaConjugated + Unconjugated HyperbilirubinemiaInherited disorders of bilirubin metabolism causing Inherited disorders of bilirubin metabolism causing

Conjugated + Unconjugated HyperbilirubinemiaConjugated + Unconjugated Hyperbilirubinemia

• Dubin Johnson syndromeDubin Johnson syndrome

• Rotor syndromeRotor syndrome

A disease of canalicular A disease of canalicular excretion of multiple organic excretion of multiple organic anions, but not bile salts.anions, but not bile salts.

Hepatic storage disorderHepatic storage disorder

• Inherited deficiency or abnormality of MRP2 causes Dubin-Johnson syndrome

• Biliary excretion of many organic anions, but not most bile acids, is deficient in Dubin-Johnson syndrome. Abnormality of biliary excretion causes the retention of a pigment in the liver.

• Inherited deficiency or abnormality of MRP2 causes Dubin-Johnson syndrome

• Biliary excretion of many organic anions, but not most bile acids, is deficient in Dubin-Johnson syndrome. Abnormality of biliary excretion causes the retention of a pigment in the liver.

• However, serum bilirubin is only mildly elevated (3-5 mg/dl), suggesting the existence of alternative pathways for excretion of bilirubin glucuronides.


Mixed (unconjugated and conjugated) Mixed (unconjugated and conjugated) hyperbilirubinemiahyperbilirubinemia



• Dubin Johnson syndrome:Dubin Johnson syndrome:


Excretory defect for Excretory defect for multiple organic anionsmultiple organic anions

Hepatic storage disorderHepatic storage disorder







Benign, rare autosomal Benign, rare autosomal recessive disorder.recessive disorder.1:1300 in Sephardic Jews1:1300 in Sephardic Jews

Benign, rare, autosomal Benign, rare, autosomal recessive disorderrecessive disorder







Accumulation of pigmentsAccumulation of pigments

No pigmentationNo pigmentation







Injected BSP is taken up, Injected BSP is taken up, conjugated and regurgitated conjugated and regurgitated back to plasma (“double hump” back to plasma (“double hump” curve)curve)

BSP clearance is slower, BSP clearance is slower, but the double hump curve but the double hump curve is not seenis not seen







Highly characteristic Highly characteristic urinary porphyrin urinary porphyrin excretion pattern.excretion pattern.

Urinary porphyrin excretion Urinary porphyrin excretion pattern is similar to that in pattern is similar to that in many cholestatic diseaess.many cholestatic diseaess.

500-

400-

300-

200-

100-

0-

Normal

Normal

Normal

Normal

D-J

D-J hetero

RotorRotorRotorRotor

Rotor hete

ro

Rotor hete

ro

Rotor hete

ro

Rotor hete

ro

g o

f co

pro

por

ph

yrin

per

g c

reat

inin

e

Urinary coproporphyrin excretion pattern in Urinary coproporphyrin excretion pattern in Dubin-Johnson and Rotor syndromesDubin-Johnson and Rotor syndromes

Coproporphyrin I: Hatched barCoproporphyrin III: open bar

HYPERBILIRUBINEMIAHYPERBILIRUBINEMIAHYPERBILIRUBINEMIAHYPERBILIRUBINEMIA

Clinical evaluationClinical evaluationClinical evaluationClinical evaluationNormal liver enzymesNormal liver enzymesNormal bile salt levelsNormal bile salt levelsNormal liver enzymesNormal liver enzymesNormal bile salt levelsNormal bile salt levels Abnormal liver enzymesAbnormal liver enzymes

Bilirubin: nearly allBilirubin: nearly all indirect-reactingindirect-reacting

Large direct-reactingLarge direct-reacting componentcomponent

•Hepatitis riskHepatitis risk•DrugsDrugs•AlcoholAlcohol•SGPT>alk. phosSGPT>alk. phos• Pro.-time: Pro.-time: not corrected not corrected with vitamin K with vitamin K• AlbuminAlbumin

• History suggestsHistory suggests obstruction obstruction• SGPT<alk. phosSGPT<alk. phos• Pro.-time: Pro.-time: corrected with corrected with vitamin K vitamin K• CholesterolCholesterol

• Dubin-JohnsonDubin-Johnson syndrome syndrome• Rotor syndromeRotor syndrome

• Hemolysis? Hemolysis? Splenomegaly, Splenomegaly, anemia, high LDH, anemia, high LDH, high retic. count, high retic. count, low haptoglobin low haptoglobin

• Drugs?Drugs? Rifampin, Rifampin, radiographic contrast radiographic contrast

• Inherited disorders ofInherited disorders of bilirubin conjugation: bilirubin conjugation: Gilbert syndrome Gilbert syndrome Crigler syndrome, Crigler syndrome, types I and II types I and II

HepatocellularHepatocellularjaundice:jaundice:

• Viral hepatitisViral hepatitis• Drug hepatitisDrug hepatitis• Alcoholic hepatitisAlcoholic hepatitis• End-stage liver End-stage liver diseasedisease

Cholestatic Cholestatic jaundice:jaundice:

• Extrahepatic Extrahepatic Vs.Vs.• IntrahepaticIntrahepatic

Summary and implicationsSummary and implications• Bilirubin throughput by the hepatocyte involves four discernible steps:

Process Uptake Storage Conjugation Excretion

Involvedmolecule

Unidentified GSTs UGT1A1 MRP2

• The four steps are finely balanced. Therefore,

Reduction at any step may cause hyperbilirubinemia.

Enhancement of the throughput requires induction of multiple genes, probably coordinated by nuclear receptors, such as the constitutive androstene receptor (CAR).

Thank you for your attention!Thank you for your attention!

Bilirubin metabolism and jaundice jaundice Jayanta Roy Chowdhury Professor of Medicine and Molecular Genetics Albert Einstein College of Medicine.

Documents

exposure of bilirubin

excretion of bilirubin

configuration of bilirubin

dayslate bilirubin

internal hydrogen bonding

serum bilirubin level

cglycineearly bilirubin

ccthe dipyrrole carbon