Bilirubin metabolism and jaundice Jayanta Roy Chowdhury Professor
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%) Normal: Senescent erythrocytes Free heme Abnormal: • Hemolysis: • Ineffective erythropoiesis Extravascular Intravascular (20%) • • Cytochromes Catalase Peroxidase Tryptophane pyrrolase • Myoglobin
Early and late labeled peaks of radioisotope incorporation into bilirubin After injection of labeled porphyrin precursor (14 C-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 OH OH O C V M O N H C CH 2 M CH 2 N H O CH 2 V MM CH 2 N H O
Bilirubin contains several polar groups (shown in red): Yet, it is insoluble in water. OH OH O C V M O N H C CH 2 M CH 2 N H O CH 2 V M M CH 2 N H O
Water insolubility of bilirubin is explained by internal hydrogen bonding. OH O C CH 2 M M V CH 2 C O OH N H O N H M O V CH 2
This is explained by internal hydrogen bonding. OH O C CH 2 M M V CH 2 C O OH N H O N H M O V CH 2
This is explained by internal hydrogen bonding. CH 2 N H CH 2 V CH 2 O OH O N H CH 2 M M M N H M O V C OH O C
This is explained by internal hydrogen bonding. CH 2 N H CH 2 V CH 2 O OH O N H CH 2 M M M N H M O V C OH O C
ØAs a consequence of hydrogen bonding, all polar groups are engaged. ØThe central methene bridge becomes buried. CH 2 C N H CH 2 O N H OH O C V CH 2 M M M N H M O V C OH O 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 CH 2 N H CH 2 V CH 2 O OH O N H CH 2 M M M N H M O V C OH O C
The internal hydrogen bonds of bilirubin are disrupted by conjugation of the propionic acid carboxyl group with glucuronic acid CH 2 N H CH 2 M M V CH 2 Gluc. A- C CH 2 N H O N H M O V CO-Gluc. A O
Phototherapy changes the configuration of bilirubin making it transiently water soluble
Ø Internal hydrogen bonds are disrupted transiently upon exposure of bilirubin to light. ØThe dipyrrole carbon bridges switch direction. CH 2 C N H CH 2 O N H OH O C V CH 2 M M M N H M O V C OH O C
ØThe dipyrrole carbon bridges switch direction. ØThus a carbon atom comes in the way of the hydrogen bonds. CH 2 N H CH 2 V CH 2 O OH O N H CH 2 M M M N H M O V C OH O C
ØThe dipyrrole carbon bridges switch direction. ØThus a carbon atom comes in the way of the hydrogen bonds. CH 2 N H OH C CH 2 V CH 2 C O M M O CH 2 N H M O V C OH O C
ØThe bulky carbon atom disrupts the hydrogen bonds by steric hindrence. CH 2 N H OH C CH 2 V CH 2 C O M M O CH 2 N H M O V C OH O C
ØThe bulky carbon atom disrupts the hydrogen bonds by steric hindrence. CH 2 N H OH C CH 2 V CH 2 C O M M O CH 2 N H M O V C OH O C
Exposure to diazo reagents result in “direct” and “indirect” van den Burgh reaction, roughly corresponding to conjugated and unconjugated fractions of bilirubin.
CH 2 M N H CH 2 M M CH 2 V C N H CH 2 N H OH O C V CH 2 M M M C N H CH 2 OH O C In unconjugated M bilirubin, the central carbon bridge is buried by hydrogen bonds. O Therefore, the van den Burgh reaction is “indirect”. V N H O CH 2 O Gluc. A- C N H CH 2 O M In conjugated bilirubin, the central carbon bridge is accessible. Therefore, the van O den Burgh reaction is “direct”. V CO-Gluc. A
Bilirubin throughput: schema of a hepatocyte Tight junction Liver sinusoid Sinusoidal surface Fenestrated endothelium Canalicular surface
Ø Bilirubin circulates bound to serum albumin. Albuminbinding: § Keeps bilirubin soluble § Prevents tissue deposition. § Prevents renal excretion § Drugs that displace bilirubin from albumin may precipitate kernicterus: Sulfonamides Coumadin, etc. alb B
Ø Bilirubin circulates bound to serum albumin. Ø At the sinusoidal surface of hepatocytes, it dissociates from albumin. alb B
Ø Bilirubin circulates bound to serum albumin. Ø At the sinusoidal surface of hepatocytes, it dissociates from albumin. alb B
Ø Bilirubin circulates bound to serum albumin. Ø At the sinusoidal surface of hepatocytes, it dissociates from albumin. alb B
Ø Bilirubin circulates bound to serum albumin. Ø At the sinusoidal surface of hepatocytes, it dissociates from albumin. alb B
Ø Bilirubin enters through the sinusoidal surface, probably by facilitated diffusion. Ø 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 (oatp 2) has been implicated in bilirubin uptake. • However, our recent studies show that although oatp 2 transports organic anions, such as BSP, it is not sufficient for bilirubin transport.
Ø Inside the hepatocyte, bilirubin binds to cytosolic proteins termed ligandins, which are the same as glutathione-Stransferases (GSTs). GST binding inhibits the efflux of bilirubin, thereby increasing its net uptake GSTs B B
GSTs B B
Ø Conjugation of bilirubin with glucuronic acid is catalyzed by UGT 1 A 1, 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 UDPGA UDP B B B GA UGT 1 A 1
UDP-glucuronosyltransferases (UGTs) UDPGA Substrate • UGT UDP Glucuronide • 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. • Inherited UGT 1 A 1 deficiency causes jaundice. One UGT isoform, UGT 1 A 1, conjugates bilirubin and is essential for its excretion.
Inherited disorders of bilirubin metabolism causing Unconjugated Hyperbilirubinemia • Crigler-Najjar syndrome Virtually no UGT 1 A 1 activity • Crigler-Najjar syndrome UGT 1 A 1 activity below 10% • Gilbert syndrome: UGT 1 A 1 activity ~30% type 1: type 2:
Inherited disorders of bilirubin metabolism causing Unconjugated Hyperbilirubinemia • Crigler-Najjar syndrome Serum bilirubin 18 -40 mg/dl: Kernicterus, unless treated vigorously • Crigler-Najjar syndrome 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. type 1: type 2:
Inherited disorders of bilirubin metabolism causing Unconjugated Hyperbilirubinemia • Crigler-Najjar syndrome Rare autosomal recessive • Gilbert syndrome: Very common, autosomal recessive. type 1: type 2: 9% of population homozygous. ~4% exhibit clinical jaundice intermittently
Inherited disorders of bilirubin metabolism causing Unconjugated Hyperbilirubinemia • Crigler-Najjar syndrome Bilirubin conjugates are almost absent in bile • Crigler-Najjar syndrome Proportion of bilirubin monoglucuronide is increased in bile normal >10%) • Gilbert syndrome: Proportion of bilirubin monoglucuronide is increased in bile normal >10%) type 1: type 2:
Inherited disorders of bilirubin metabolism causing Unconjugated Hyperbilirubinemia • Crigler-Najjar syndrome type 1: • Crigler-Najjar syndrome type 2: • Gilbert syndrome: Phenobarbital treatment: little or no effect. Phenobarbital reduces serum bilirubin is by >25% Serum bilirubin is normalized
In 1953, Crigler and Najjar described “a mysterious illness that caused jaundice and severe neurological damage”
Genetic Lesions in UGT 1 A 1 Deficiency Syndromes UGT 1 A 1 locus 1*12 1*7 1*6 1*5 1*4 1*3 1*2 CN-I Stop codon or frame-shift Signal peptide Substitution CN-II Splice-site mutation Gilbert A(TA)7 TAA [Normal: A(TA)6 TAA] 1*1 2 3 4 5
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
Percent of basal activity Effect of drugs and hormones on rat liver UGT 1 A 1 activity 25020015010050 Th y ho roi rm d on e n pi m fib ifa R l C ar ob Ph en lo bi ta ed at nt re U ra te 0 - Nuclear receptor CAR PPAR PXR TR
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 MRP 2 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 MRP 2 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.
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
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
Inherited disorders of bilirubin metabolism causing Mixed (unconjugated and conjugated) hyperbilirubinemia • Dubin Johnson syndrome: Accumulation of pigments • Rotor syndrome No pigmentation
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
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.
mg of coproporphyrin per g creatinine Urinary coproporphyrin excretion pattern in Dubin-Johnson and Rotor syndromes 500 - Coproporphyrin I: Hatched bar Coproporphyrin III: open bar 4003002001000 - l a m r No J tero D he J D o r r o e t t o R r he to o R
HYPERBILIRUBINEMIA Normal liver enzymes Normal bile salt levels Bilirubin: nearly all indirect-reacting Clinical evaluation Large direct-reacting component • Hemolysis? • Dubin-Johnson Splenomegaly, syndrome anemia, high LDH, • Rotor syndrome high retic. count, low haptoglobin • Drugs? Rifampin, radiographic contrast • Inherited disorders of bilirubin conjugation: Gilbert syndrome Crigler syndrome, types I and II Abnormal liver enzymes • Hepatitis risk • Drugs • Alcohol • SGPT>alk. phos • Pro. -time: not corrected with vitamin K • Albumin Hepatocellular jaundice: • Viral hepatitis • Drug hepatitis • Alcoholic hepatitis • End-stage liver disease • History suggests obstruction • SGPT<alk. phos • Pro. -time: corrected with vitamin K • Cholesterol Cholestatic jaundice: • Extrahepatic Vs. • Intrahepatic
Summary and implications • Bilirubin throughput by the hepatocyte involves four discernible steps: Process Involved molecule Uptake Unidentified Storage Conjugation GSTs UGT 1 A 1 Excretion MRP 2 • The four steps are finely balanced. Therefore, Ø Reduction at any step may cause hyperbilirubinemia. Ø 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!
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