1. Bilirubin metabolism and jaundice jaundice

2. 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.  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.

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

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

5. C N H M V O N H M CH 2 O OH CH 2 N H N H C O OH MM V O The linear structure of bilirubin: Two dipyrroles joined by a central methene bridge

6. C N H M V O N H M CH 2 O OH CH 2 N H N H C O OH MM V O Bilirubin contains several polar groups (shown in red): Yet, it is insoluble in water.

7. Conjugation with glucuronic acid makes bilirubin water soluble

8. N H M V O N H M CH 2 OH CH 2 N H N H C O OH MM V O CH 2 C O The internal hydrogen bonds of bilirubin are disrupted by conjugation of the propionic acid carboxyl group with glucuronic acid

9. N H M V O N H M CH 2 N H N H C O MM V O CO -GlucA GlucA- The internal hydrogen bonds of bilirubin are disrupted by conjugation of the propionic acid carboxyl group with glucuronic acid

10. Phototherapy changes the configuration of bilirubin making it transiently water soluble

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

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

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

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

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

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

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

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

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

20. B GSTs B

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

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

24. Post-hepatic jaundiceHepatic jaundicePre-hepatic jaundice  Due to obstruction of bile duct which prevents passage of bilirubin into intestine.  D.Bil will back to liver and then to circulation elevating its level in blood and urine.  Occur in:  Biliary stricture  Cancer of the pancreas or gallbladder  Gallstones  Due to liver cell damage (cancer, cirrhosis or hepatitis)  Conjugation of bilirubin decreased (ID.Bil.  ).  Blilirubin that is conjugated is not efficiently secreted into bile but leaks to blood (D.Bil.  )  Occur in :  Cirrhosis (scarring of the liver)  Hepatitis  Gilbert's disease  Due to increase in RBCs breakdown due to hemolytic anemia.  The rate of RBCs lysis and bilirubin production more than ability of liver to convert it to the conjugated form  Occur in:  Erythroblastosis fetalis  Hemolytic anemia  Transfusion reaction Causes D.Bil (High)D.Bil, ID.Bil, T.Bil all (High)ID.Bil > D.BilType of Bil. ALP ( High)ALT, AST (High) K + ( High) Hematology: CBC (low Hb) Conformational test haemolytic jaundice hepato-cellular jaundice obstructive jaundice

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

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

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

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

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

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

31. Treatment of Crigler-Najjar syndrome type 1 Routine phototherapy has extended the life expectancy. 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%.

32. Phototherapy bed

33. CN-1 syndrome-1: permanent brain damage

34. 250- 200- 150- 100- 50- 0- Effect of drugs and hormones on rat liver UGT1A1 activity Percent of basal activity Untreated Phenobarbital Clofibrate Thyroid hormone Rifampin Nuclear receptor CARCAR PPARPPAR PXRPXR TRTR

35. Inherited disorders of bilirubin metabolism causing Conjugated + Unconjugated Hyperbilirubinemia Inherited disorders of bilirubin metabolism causing Conjugated + Unconjugated Hyperbilirubinemia Dubin Johnson syndrome Dubin Johnson syndrome Rotor syndrome Rotor syndrome A disease of canalicular excretion of multiple organic anions, but not bile salts. Hepatic storage disorder

36. 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.

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

38. Inherited disorders of bilirubin metabolism causing Mixed (unconjugated and conjugated) hyperbilirubinemia Inherited disorders of bilirubin metabolism causing Mixed (unconjugated and conjugated) hyperbilirubinemia Dubin Johnson syndrome: Dubin Johnson syndrome: Rotor syndrome Rotor syndrome Excretory defect for multiple organic anions Hepatic storage disorder

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

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

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

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

43. Summary and implications Bilirubin throughput by the hepatocyte involves four steps: Process UptakeStorageConjugationExcretion Involved molecule UnidentifiedGSTsUGT1A1MRP2 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).

44. Thank you for your attention! Thank you for your attention!