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

Sources of bilirubin Erythroid Non-erythroid (80%) (20%) Normal: (80%) (20%) Normal: Senescent erythrocytes Free heme Abnormal: Hemolysis: Extravascular Intravascular Ineffective erythropoiesis Cytochromes Catalase Peroxidase Tryptophane pyrrolase Myoglobin

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

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

The linear structure of bilirubin: Two dipyrroles joined by a central methene bridge N H M V O CH2 OH OH O C C CH2 V CH2 M M N N CH2 H H O

Bilirubin contains several polar groups (shown in red): Yet, it is insoluble in water. O O OH OH C C CH2 CH2 V V M M CH2 CH2 M M N N N N O H H CH2 H H O

Water insolubility of bilirubin is explained by internal hydrogen bonding. N H M V O CH2 OH C H H O N N CH2 CH2 M M V CH2 C OH O

This is explained by internal hydrogen bonding. OH C CH2 V M M CH2 H H O N N N N O H H CH2 CH2 M M V CH2 C OH O

This is explained by internal hydrogen bonding. CH2 C V M M OH CH2 O H H O N N N N O H H CH2 OH O CH2 M M CH2 V C

This is explained by internal hydrogen bonding. CH2 C V M M OH CH2 O H H O N N N N O H H CH2 OH O CH2 M M CH2 V C

As a consequence of hydrogen bonding, all polar groups are engaged. The central methene bridge becomes buried. CH2 C V M M OH CH2 O C H H O N N N N O H H CH2 C OH O CH2 M M CH2 V C

Ridge-tile structure of bilirubin

Conjugation with glucuronic acid makes bilirubin water soluble

The internal hydrogen bonds of bilirubin are disrupted by conjugation of the propionic acid carboxyl group with glucuronic acid CH2 C V M M OH CH2 O H H O N N N N O H H CH2 OH O CH2 M M CH2 V C

The internal hydrogen bonds of bilirubin are disrupted by conjugation of the propionic acid carboxyl group with glucuronic acid CH2 -GlucA C O V M M CH2 H H O N N N N O H H CH2 CH2 M M CH2 V GlucA- O C

Phototherapy changes the configuration of bilirubin making it transiently water soluble

The dipyrrole carbon bridges switch direction. Internal hydrogen bonds are disrupted transiently upon exposure of bilirubin to light. The dipyrrole carbon bridges switch direction. CH2 C V M M OH CH2 O C H H O N N N N O H H CH2 C OH O CH2 M M CH2 V C

The dipyrrole carbon bridges switch direction. Thus a carbon atom comes in the way of the hydrogen bonds. N H M V O CH2 OH C

The dipyrrole carbon bridges switch direction. Thus a carbon atom comes in the way of the hydrogen bonds. N H M V O CH2 OH C C C

The bulky carbon atom disrupts the hydrogen bonds by steric hindrence. V M M OH CH2 O C H H O N N N N O H H CH2 C OH O CH2 M M CH2 V C

The bulky carbon atom disrupts the hydrogen bonds by steric hindrence. V M M OH CH2 O C H H O N N N N O H H CH2 C OH O CH2 M M CH2 V C

Exposure to diazo reagents result in “direct” and “indirect” van den Burgh reaction, roughly corresponding to conjugated and unconjugated fractions of bilirubin.

-GlucA GlucA- N H M V O CH2 C In conjugated bilirubin, the central carbon bridge is accessible. Therefore, the van den Burgh reaction is “direct”. N H M V O CH2 OH C In unconjugated bilirubin, the central carbon bridge is buried by hydrogen bonds. Therefore, the van den Burgh reaction is “indirect”.

Bilirubin throughput: schema of a hepatocyte Tight junction Liver sinusoid Fenestrated endothelium Sinusoidal surface Canalicular surface

B alb Bilirubin circulates bound to serum albumin. Albumin- binding: Keeps bilirubin soluble Prevents tissue deposi- tion. Prevents renal excretion Drugs that displace bilirubin from albumin may precipitate kernicterus: Sulfonamides Coumadin, etc. B alb

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

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

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

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

Bilirubin enters through the sinusoidal surface, probably by facilitated diffusion. Uptake is energy independent and bidirectional. Bilirubin uptake is reduced: In neonates In cirrhosis From drug effect: novobiocin In some cases of Gilbert syndrome B B

What is the mechanism of facilitated diffusion of bilirubin? Zucker has proposed that no transporter protein is needed. In a recent report, organic anion transport protein 2 (oatp2) has been implicated in bilirubin uptake. However, our recent studies show that although oatp2 transports organic anions, such as BSP, it is not sufficient for bilirubin transport.

Inside the hepatocyte, bilirubin binds to cytosolic proteins termed ligandins, which are the same as glutathione-S- transferases (GSTs). GST binding inhibits the efflux of bilirubin, thereby increasing its net uptake GSTs B B

GSTs B B

Conjugation of bilirubin with glucuronic acid is catalyzed by UGT1A1, which transfers glucuronic acid from UDP-glucuronic acid to bilirubin Conjugation with glucuronic acid makes bilirubin water-soluble and non-toxic. Glucuronidation is essential for biliary excretion of bilirubin. GSTs UDP UDPGA B B B GA UGT1A1

UDP-glucuronosyltransferases (UGTs) UDPGA UDP Substrate Glucuronide UGT UGTs are ER proteins that convert many internal and exogenous toxins to non-toxic metabolites. UGT’s are a family of enzymes concentrated in the liver. One UGT isoform, UGT1A1, conjugates bilirubin and is essential for its excretion. Inherited UGT1A1 deficiency causes jaundice.

Unconjugated Hyperbilirubinemia Inherited disorders of bilirubin metabolism causing Unconjugated Hyperbilirubinemia Crigler-Najjar syndrome type 1: Virtually no UGT1A1 activity Crigler-Najjar syndrome type 2: UGT1A1 activity below 10% Gilbert syndrome: UGT1A1 activity ~30%

Unconjugated Hyperbilirubinemia Inherited disorders of bilirubin metabolism causing Unconjugated Hyperbilirubinemia Crigler-Najjar syndrome type 1: Serum bilirubin 18-40 mg/dl: Kernicterus, unless treated vigorously Crigler-Najjar syndrome type 2: Serum bilirubin 8-18 mg/dl: Kernicterus is rare Gilbert syndrome: Serum bilirubin normal to 5 mg mg/dl (increases during fasting, intercurrent illness, etc. No cerebral toxicity.

Unconjugated Hyperbilirubinemia Inherited disorders of bilirubin metabolism causing Unconjugated Hyperbilirubinemia Crigler-Najjar syndrome type 1: Rare autosomal recessive Crigler-Najjar syndrome type 2: Rare autosomal recessive Gilbert syndrome: Very common, autosomal recessive. 9% of population homozygous. ~4% exhibit clinical jaundice intermittently

Unconjugated Hyperbilirubinemia Inherited disorders of bilirubin metabolism causing Unconjugated Hyperbilirubinemia Crigler-Najjar syndrome type 1: Bilirubin conjugates are almost absent in bile Crigler-Najjar syndrome type 2: Proportion of bilirubin mono- glucuronide is increased in bile normal >10%) Gilbert syndrome: Proportion of bilirubin mono- glucuronide is increased in bile normal >10%)

Unconjugated Hyperbilirubinemia Inherited disorders of bilirubin metabolism causing Unconjugated Hyperbilirubinemia Crigler-Najjar syndrome type 1: Phenobarbital treatment: little or no effect. Crigler-Najjar syndrome type 2: Phenobarbital reduces serum bilirubin is by >25% Gilbert syndrome: Serum bilirubin is normalized

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

Genetic Lesions in UGT1A1 Deficiency Syndromes UGT1A1 locus 1*12 1*7 1*6 1*5 1*4 1*3 1*2 1*1 2 3 4 5 CN-I Stop codon or frame-shift Signal peptide CN-II Substitution Splice-site mutation Gilbert A(TA)7 TAA [Normal: A(TA)6 TAA]

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 damage

Effect of drugs and hormones on rat liver UGT1A1 activity 250- 200- 150- 100- 50- 0- Percent of basal activity Thyroid hormone Clofibrate Untreated Rifampin Phenobarbital Nuclear receptor CAR PPAR PXR TR

Conjugated + Unconjugated Hyperbilirubinemia Inherited disorders of bilirubin metabolism causing Conjugated + Unconjugated Hyperbilirubinemia Dubin Johnson syndrome A disease of canalicular excretion of multiple organic anions, but not bile salts. Rotor syndrome Hepatic 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) Inherited disorders of bilirubin metabolism causing Mixed (unconjugated and conjugated) hyperbilirubinemia Dubin Johnson syndrome: Excretory defect for multiple organic anions Rotor syndrome Hepatic storage disorder

Mixed (unconjugated and conjugated) Inherited disorders of bilirubin metabolism causing Mixed (unconjugated and conjugated) hyperbilirubinemia Dubin Johnson syndrome: Benign, rare autosomal recessive disorder. 1:1300 in Sephardic Jews Rotor syndrome Benign, rare, autosomal recessive disorder

Mixed (unconjugated and conjugated) Inherited disorders of bilirubin metabolism causing Mixed (unconjugated and conjugated) hyperbilirubinemia Dubin Johnson syndrome: Accumulation of pigments Rotor syndrome No pigmentation

Mixed (unconjugated and conjugated) Inherited disorders of bilirubin metabolism causing Mixed (unconjugated and conjugated) hyperbilirubinemia Dubin Johnson syndrome: Injected BSP is taken up, conjugated and regurgitated back to plasma (“double hump” curve) Rotor syndrome BSP clearance is slower, but the double hump curve is not seen

Mixed (unconjugated and conjugated) Inherited disorders of bilirubin metabolism causing Mixed (unconjugated and conjugated) hyperbilirubinemia Dubin Johnson syndrome: Highly characteristic urinary porphyrin excretion pattern. Rotor syndrome Urinary porphyrin excretion pattern is similar to that in many cholestatic diseaess.

Urinary coproporphyrin excretion pattern in Dubin-Johnson and Rotor syndromes 500- 400- 300- 200- 100- 0- Coproporphyrin I: Hatched bar Coproporphyrin III: open bar mg of coproporphyrin per g creatinine D-J Rotor Normal D-J hetero Rotor hetero

HYPERBILIRUBINEMIA Clinical evaluation Normal liver enzymes Normal bile salt levels Clinical evaluation Abnormal liver enzymes Bilirubin: nearly all indirect-reacting Hepatitis risk Drugs Alcohol SGPT>alk. phos Pro.-time: not corrected with vitamin K Albumin Large direct-reacting component History suggests obstruction SGPT<alk. phos Pro.-time: corrected with vitamin K Cholesterol Hemolysis? Splenomegaly, anemia, high LDH, high retic. count, low haptoglobin Drugs? Rifampin, radiographic contrast Inherited disorders of bilirubin conjugation: Gilbert syndrome Crigler syndrome, types I and II Dubin-Johnson syndrome Rotor syndrome Hepatocellular jaundice: Viral hepatitis Drug hepatitis Alcoholic hepatitis End-stage liver disease Cholestatic jaundice: Extrahepatic Vs. Intrahepatic

Summary and implications Bilirubin throughput by the hepatocyte involves four discernible steps: Process Uptake Storage Conjugation Excretion Involved molecule 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).

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