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

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.
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.

3. 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

4. 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)

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

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. Ridge-tile structure of bilirubin

14. Conjugation with glucuronic acid
makes bilirubin water soluble

15. 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

16. 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

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

18. 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

19. 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

20. 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

21. 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

22. 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

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

24. -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”.

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

26. 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

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

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

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

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

31. 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

32. 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.

33. 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

34. GSTs B B

35. 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

36. 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.

37. 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%

38. Unconjugated Hyperbilirubinemia
Inherited disorders of bilirubin metabolism causing
Unconjugated Hyperbilirubinemia
Crigler-Najjar syndrome
type 1:
Serum bilirubin 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.

39. 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

40. 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%)

41. 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

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

43. Genetic Lesions in UGT1A1 Deficiency Syndromes
UGT1A1 locus
1*12
1*7
1*6
1*5
1*4
1*3
1*2
1*1
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]

44. 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%.

45. Phototherapy bed

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

47. 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

48. 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

49. 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.

50. 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.

51. 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

52. 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

53. 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

54. 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

55. 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.

56. 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

57. 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

58. 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).

59. Thank you for
your attention!