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: • Cytochromes
Senescent erythrocytes • Catalase
Free heme • Peroxidase
Abnormal:
• Tryptophane pyrrolase
• Hemolysis: • Myoglobin
Extravascular
Intravascular
• Ineffective erythropoiesis
Early and late labeled peaks of radioisotope incorporation into bilirubin
After injection of labeled porphyrin precursor (14C-glycine)

Early bilirubin Late bilirubin

(15-20%) (65%)
0- 3 days 40- 80 days

Increased
erythropoiesis

Non-Hb Erythrocyte
sources (liver) sources
 Opening of the heme ring and
Enzyme-catalyzed formation of bilirubin
The linear structure of bilirubin:
Two dipyrroles joined by a central methene bridge

O OH OH O

C C
CH2 CH2
V V
M M CH2 CH2 MM

N N N N
O H H CH2 H H O
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 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.

O 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.

O 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 CH2 O OH
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 CH2 O OH
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 CH2 O OH

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 CH2 O OH
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
CO-GlucA
V
M M CH2
H H O
N N
N N
O H H CH2
CH2 M M
CH2 V
GlucA- C
O
Phototherapy changes the
configuration of bilirubin making
it transiently water soluble
 Internal hydrogen bonds are disrupted
transiently upon exposure of bilirubin to light.
The dipyrrole carbon bridges switch direction.

CH2
C
V
M M CH2 O OH

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

CH2
C
V
In unconjugated M M CH2 O OH
bilirubin, the
central carbon C H
N
H O
N
bridge is buried by N N
hydrogen bonds. O H H CH2
Therefore, the van C
OH O CH2 M M
den Burgh reaction CH2
is “indirect”.
V
C
 Bilirubin throughput: schema of a hepatocyte
Tight
junction
Liver
sinusoid

Sinusoidal
surface
Fenestrated
endothelium

Canalicular
surface
  Bilirubin circulates bound to serum albumin.

Albumin-
binding:
 Keeps bilirubin
soluble
 Prevents
tissue deposi-
tion.
 Prevents alb B
renal excretion
 Drugs that
displace
bilirubin from
albumin may
precipitate
kernicterus:
Sulfonamides
Coumadin, etc.
 Bilirubin circulates bound to serum albumin.
 At the sinusoidal surface of hepatocytes, it dissociates
from albumin.

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

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

alb
B
 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 B B
effect:
novobiocin
 In some cases
of Gilbert
syndrome
 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 GSTs
its net uptake

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.
GSTs
UDPGA UDP
 Glucuronidation
is essential for B B B GA
biliary excretion UGT1A1
of bilirubin.
 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.
 Inherited disorders of bilirubin metabolism causing
Unconjugated Hyperbilirubinemia

• Crigler-Najjar syndrome Virtually no UGT1A1 activity

type 1:

• Crigler-Najjar syndrome UGT1A1 activity below 10%

type 2:

• Gilbert syndrome: UGT1A1 activity ~30%

 Inherited disorders of bilirubin metabolism causing
Unconjugated Hyperbilirubinemia

• Crigler-Najjar syndrome Serum bilirubin 18-40 mg/dl:

type 1: Kernicterus, unless treated
vigorously

• Crigler-Najjar syndrome Serum bilirubin 8-18 mg/dl:

type 2: Kernicterus is rare

• Gilbert syndrome: Serum bilirubin normal to

5 mg mg/dl
(increases during fasting,
intercurrent illness, etc.
No cerebral toxicity.
 Inherited disorders of bilirubin metabolism causing
Unconjugated Hyperbilirubinemia

• Crigler-Najjar syndrome Rare

type 1: autosomal recessive

• Crigler-Najjar syndrome Rare

type 2: autosomal recessive

• Gilbert syndrome: Very common,

autosomal recessive.
9% of population homozygous.
~4% exhibit clinical jaundice
intermittently
 Inherited disorders of bilirubin metabolism causing
Unconjugated Hyperbilirubinemia

• Crigler-Najjar syndrome Bilirubin conjugates are almost

type 1: absent in bile

• Crigler-Najjar syndrome Proportion of bilirubin mono-

type 2: glucuronide is increased in bile
normal >10%)

• Gilbert syndrome: Proportion of bilirubin mono-

glucuronide is increased in bile
normal >10%)
 Inherited disorders of bilirubin metabolism causing
Unconjugated Hyperbilirubinemia

• Crigler-Najjar syndrome Phenobarbital treatment:

type 1: little or no effect.

• Crigler-Najjar syndrome Phenobarbital reduces serum

type 2: 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

Substitution CN-II
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-

Percent of basal activity 200-

150-

100-

50-

0-

Nuclear CAR PPAR PXR TR

receptor
 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.
 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.
Urinary coproporphyrin excretion pattern in
Dubin-Johnson and Rotor syndromes

mg of coproporphyrin per g creatinine 500- Coproporphyrin I: Hatched bar

Coproporphyrin III: open bar
400-

300-

200-

100-

0-
 HYPERBILIRUBINEMIA
Normal liver enzymes
Clinical evaluation Abnormal liver enzymes
Normal bile salt levels

Bilirubin: nearly all Large direct-reacting •Hepatitis risk • History suggests

indirect-reacting component •Drugs obstruction
•Alcohol • SGPT<alk. phos
• Hemolysis? • Dubin-Johnson •SGPT>alk. phos • Pro.-time:
Splenomegaly, syndrome • Pro.-time: corrected with
anemia, high LDH, not corrected vitamin K
high retic. count, • Rotor syndrome with vitamin K
• Cholesterol
low haptoglobin • Albumin
• Drugs?
Rifampin, Hepatocellular Cholestatic
radiographic contrast jaundice: jaundice:
• Inherited disorders of
bilirubin conjugation: • Viral hepatitis • Extrahepatic Vs.
Gilbert syndrome • Drug hepatitis • Intrahepatic
Crigler syndrome, • Alcoholic hepatitis
types I and II • End-stage liver
disease
 Summary and
implications
• Bilirubin throughput by the hepatocyte involves
four discernible steps:

Process Uptake Storage Conjugation Excretion

Involved Unidentified GSTs UGT1A1 MRP2
molecule

• 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).
Thank you for
your attention