Bilirubin metabolism and jaundice

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

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

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

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

7. Conjugation with glucuronic acid makes bilirubin water soluble

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

9. 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 CH2 H H O CH2 M M CH2 V GlucA- C O

10. Phototherapy changes the configuration of bilirubin makingit transiently water soluble

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

12. 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. alb B

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

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

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

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

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

18. 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, oatp2 transports organic anions, such as BSP, it is not sufficient for bilirubin transport.

19. Inside the hepatocyte, bilirubin binds to cytosolic proteins termed ligandins, which are the same as glutathione-S- transferases (GSTs). GSTs GST bindinginhibits theefflux of bilirubin,thereby increasingits net uptake B B

20. GSTs B B

21. GSTs GA 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. UDP UDPGA B B UGT1A1

22. UDP-glucuronosyltransferases (UGTs) UDPGA UDP • 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. Substrate Glucuronide • UGT

23. Jaundice: • It is a medical term describes the elevation of bilirubin in blood result in yellow color of skin and sclera. • Other symptoms include nausea, vomiting, dark-colored urine andTypes of Jaundice: • fatigue. • according to the cause of jaundice it is classified to three main types: • Pre-hepatic jaundice • Hepatic jaundice • Post-hepatic (most common type)

24. haemolytic jaundice hepato-cellular jaundice obstructive jaundice

25. 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% UGT1A1 activity ~30% • Gilbert syndrome:

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

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

28. 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 bilenormal >10%) • Gilbert syndrome: Proportion of bilirubin mono-glucuronide is increased in bilenormal >10%)

29. 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

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. 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 PXR PPAR CAR TR

35. 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

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. 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.

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

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

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

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

42. HYPERBILIRUBINEMIA Normal liver enzymes Normal bile salt levels Clinical evaluation Abnormal liver enzymes • Hepatitis risk • Drugs • Alcohol • SGPT>alk. phos • Pro.-time: not corrected with vitamin K • Albumin Bilirubin: nearly all indirect-reacting 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 syndromeCrigler syndrome, types I and II • Dubin-Johnson syndrome • Rotor syndrome • Cholestatic • jaundice: • Hepatocellular • jaundice: • Viral hepatitis • Drug hepatitis • Alcoholic hepatitis • End-stage liver • disease

43. Summary and implications • Bilirubin throughput by the hepatocyte involves four discernible steps: • 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!