1

Bilirubin and Jaundice

Pathways of Bilirubin Synthesis and Catabolism

Porphogens

Fe

Heme

Globin

Hgb

+

+

Urobilinogen StercobilinFecalPigments

UrineUrobilinogen

Heme PrecursorsMyoglobinNon-Hgb HemeProteins

ShuntPathwayFree Bilirubin – Albumin Complex

ConjugatedBilirubin

Hgb

Biliverdin

Bilirubin

Fe

Globin

RBCsBone

MarrowReticuloendothelial

System (RES)

2mg

70mg

68mg

2

Labeling of RBC hemoglobin and fecal stercobilin

0.3

0.2

0.1

0.1

0.08

0.06

0.04

0.02

20 40 60 80 100 120 140 160 180

A:

Ato

m %

exc

ess

15N

of

ster

cob

ilin

B:

Ato

m %

exc

ess

15N

of

hae

min

B

A

Times ( days)

Labeling of red cell hemoglobin and fecal stercobilinin a normal human given 15N orally

X

X X

X X X

XX

X

X X

X

X

X

X

Sources of bilirubin production in the rat

Early Bilirubin15%

0-3 Days

Late Bilirubin65%

40-80 Days

Non-HemoglobinSources (liver)

Red CellSenescence

IncreasedErythropoiesis

Sources of bilirubin production in the rat, as adduced from studies of thelabeling of plasma bilirubin in Gunn rats and bile bilirubin in normal rats.

3

NN

N NFe

COO COO

C

NN

N N

OOC COO

OH HO

H H

H

NN

NH N

OOC COO

OH HO

H

NADPH NADP

O2NADPHNADP

Fe (II)O2 CO

Biliverdin

reductase

NADPHNADP

Hemin +

heme-oxygenase complex

hydroxyheme-oxygenase complex

biliverdin

bilirubin

NN

N NFe

COO COO

OH

4

OATP MDR2

(ligandin)

A.

B.

5

N NH

NNHFe

Me

CH

CHCH2

Me

CH2 CH2 CO2H

MeMe

H2C CH2 CH2 CO2H

A B

CD

γ

β

α

δ

NH

NH

NH

CH CH2CH

NH

O

MeCH2MeCHMe Me CH

O

CH2

C

CH2

CH2

CCH2

OO O OO O

OH

OHHO

COOH COOH

OHHO

HOCH2

NH

NH

NH

CH CH2CH

NH

O

MeCH2MeMe Me CH

OA B C D

CHCH2 CH2

CO2H

CH2

CH2

CO2HCH2

A B

C

D

BILIRUBIN - IX α DIGLUCURONIDE

BILIRUBIN – IX α (convoluted structure)

BILIRUBIN – IX α (planar structure)

A.

B.

C.

CO (excreted via lungs)

Fe (reutilized)

HN

HN

NH

NH

C OO

C

C

OO

OO

H

H

HN

HN

NH

NH

C OO

C

C

OO

OO

H

GA

HN

HN

NH

NH

C OO

C

C

OO

O

GA

OGAConjugation prevents

internal H-bonding by the-COOH groups of Bilirubin

BILIRUBINS:

UNCONJUGATED (UCB)

MONO-GLUCURONIDE

(BMG)

DIGLUCURONIDE(BDG)

2

1

BILIRUBIN CONJUGATION IN MICROSOMESINVOLVES TWO STEPS, MEDIATED BY

ONE BILIRUBIN-UDPGA TRANSFERASE

UDPGA

UDP

UDPGA

UDP

UDPGATRANSFERASE

6

OATP MDR2

(=cMOAT)

UROBILINOGENS ARE FORMED BY DECONJUGATION AND REDUCTION OF BILIRUBINS BY INTESTINAL BACTERIA

CONJUGATED BILIRUBIN

UNCONJUGATEDBILIRUBIN

Deconju-gation

A UROBILINOGEN

Reduction

NHN

HH

N HN

COOGA COOGA

M

MV

M VO O

M

NHN

HH

N HN

COOH COOH

M

MV

M VO O

M

NHN

HH

N HN

H2C CH2

COOH COOH

M

ME

M EO O

M

H H

2 GA + 8HM = MethylE = EthylV = Vinyl

7

Slow

Fast

FRACTIONAL DIAZO REACTION OF PLASMA

R R

Directreaction

Indirectreaction

CONJUGATEDBILIRUBIN

UNCONJUGATEDBILIRUBIN

N+

N

SO3H

+

N+

N

SO3H

+

DIA

ZO

RE

AG

EN

T

8

R R

CONJUGATEDBILIRUBIN

UNCONJUGATEDBILIRUBIN

N+

N

SO3H

+ + ACCELERATOR

(ALCOHOL, CAFFEINE)

TOTAL – DIRECT INDIRECT=

TOTAL DIAZO REACTION OF PLASMA

Bilirubinuria in Jaundice

UNCONJUGATED PLASMA BILIRUBIN (B) CONJUGATED

NO BILIRUBIN BILIRUBIN ++

URINE

PLASMA

(A=ALBUMIN)

9

10

Phototherapy for neonatal jaundice

Treatment for Neonatal JaundicePrevention of kernicterus

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NH

NH

NH

CH CH2CH

NH

O

MeCH2MeMe Me CH

OA B C D

CHCH2 CH2

COOH

CH2

CH2

COOHCH2

NH

NH

NH

CH CH2CH

NH

O

CH2MeMe Me CH

O

CH2

COOHCH2

CH2

CH2

COOH

Me CHCH2

Structure of the α and β isomers of bilirubin IX. In the IXβ isomer (and in the γ and δ isomers, not shown) the propionic acid groups are moved to positions other than those indicated on the central pyrrole rings B and C of bilirubin IXα.

Bilirubin IX α

Bilirubin IX β

2.5

2.0

1.5

1.0

0.5

16

12

8

4

0 8 16 24 0 2 4 6 8 10

LiverCo-PPLiverSn-PP

Hem

e O

xyge

nase

(nm

ol b

ilirub

in/m

g ph

Effect of Sn-protoporphyrin (Sn-PP) and CO-protoporphyrin (Co-PP) when administeredonce at a dose of 50 μmol/kg body wt on hepatic heme oxygenase activity in the rat.

Time(hours) (days)

12

18

16

14

10

-4 0 2 4 0 1 2 3 4 5 6 7

12

2.5

5.0

7.5

10.0

Effect of Sn-protoporphyrin (Sn-PP) (100 μmol/kg body wt) administeredat 4d before and on the day of surgery on hyperbilirubinemia in the

bile-duct-ligated rat. Effect of Sn-PP (2 x 0.25 μmol/kg body wt) on the levels of serum bilirubin in a patient with primary biliary cirrhosis (PBC).

Time (days)

Bile-DuctLigation

RAT PBC(50 )

Sn-PP

Pla

sma

Bilir

ubin

mg/

dl Control

Sn-PP

UGT1 gene

1J 1I 1H 1G 1F 1E 1D 1C 1B 1A 2 3 4 5

5’ 3’

mRNA B-UGT1

Phenol

UGTs

NH2 COOH

enzymesubstrate binding site UDPGA

binding sitemembranespanningregion

285 amino acids 246 amino acids

13

UDPGT promoter A(TA)6TAA

UDPGT nullMutation

Bolivian Squirrel MonkeyGunn ratAnimal Model

BenignKernicterusPrognosis

2-7% of populationRarePrevalence

Increased proportion of monoglucuronide

Small amounts of unconjugatedbilirubin

Pigments in bile

ReductionNo effectEffect of Phenobarbital on plasma bilirubin

30-50% of normalUndetectableHepatic bilirubin-UDPGT activity

Usually normal; elevated in a minority of cases

NormalPlasma BSP retention at 45 min

50-85 μM (fluctuates)340-860 μMPlasma bilirubin

Gilbert SyndromeCrigler-Najjar Type ICharacteristics

Unconjugated Hyperbilirubinemia

MRP2Mutation

BenignBenignPrognosis

RareUncommon (1:1300 in Persian Jews)Prevalence

Autosomal recessiveAutosomal recessiveMode of inheritance

Elevated total; elevated proportion of coproporphyrin I but <80%

Normal total >80% as coproporphyrin IUrinary coproporphyrin

Elevated; no secondary rise at 90 min

Normal or elevated; secondary rise at 90 min45-min plasma BSP retention

Normal except for bilirubinNormal except for bilirubinRoutine liver function tests

Elevated, usually between 2 and 5 mg%, occasionally as high as 20 mg%; predominantly direct-reacting

Elevated, usually between 2 and 5 mg%, occasionally as high as 20 mg%; predominantly direct-reacting

Serum bilirubin

NormalDark pigment; predominantly in centrilobular areas; otherwise normal

Histology of liver

NormalGrossly blackAppearance of liver

Rotor SyndromeDubin-Johnson SyndromeCharacteristic

Chronic conjugated hyperbilirubinemias

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Hemolysis Hepatocellular

Conjugated bilirubin

Jaundice

Defect in Conjugation

Unconjugated bilirubin

Intrahepatic Extrahepatic

Cholestatic

15

Hemolysis Hepatocellular

Conjugated bilirubin

Jaundice

Defect in Conjugation

Unconjugated bilirubin

Intrahepatic Extrahepatic

Cholestatic

16

Liver Function Tests

• Bilirubin• PT (Prothrombin time) • Glucose• Cholesterol• ALT (alanine aminotransferase)• AST (aspartate aminotransferase)• Alkaline phosphatase• GGT (γ-glutamyltranspeptidase)

Imaging

• Ultrasound• CT scan• Liver-spleen scan• Radionuclide biliary scan• ERCP (endoscopic retrograde

cholangiopancreatography)• Transhepatic cholangiography

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Hemolysis Hepatocellular

Conjugated bilirubin

Jaundice

Defect in Conjugation

Unconjugated bilirubin

Intrahepatic Extrahepatic

Cholestatic

↑ BR

Nl BD Dilated BD

↑ AP↑ ALT

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Bilirubin Metabolism

•Blood•Conjugated & Conjugated

•Urine – Urobilinogen•Stool – Stercobilin

NH

NH

NH

CH CH2CH

NH

O

MeCH2MeMe Me CH

OA B C D

CHCH2 CH2

COOH

CH2

CH2

COOHCH2

N

N N

N

Fe

MV

V

M

CH2

CH2

COOH

M

M

CH2

CH2

COOH

α

β

γ

δ

plus 3 O minus Fe

minus CO

A

B

D

C

Ferroprotoporphyrin IX(Protoheme)

Bilirubin IX α

Conversion of protoheme (ferroprotoporphyrin IX) to bilirubin IXα. Cleavage of the protoporphyrin ring occurs selectively at the α-methene bridge. The bridge carbon atom is oxidized to carbon monoxide.

19

BiliverdinReductase

M = MethylE = EthylPr = Propionyl

NN

N N

M

M

M V

M

Pr Pr

V

Fe

CH

α

β

γ

δ

NNH

HN H

N

M

M

M VO O

M

Pr Pr

V

NHN

HH

N HN

H2C

M

M

M

VO O

M

V

HemeOxygenase

BILIVERDIN IXα

BILIRUBIN IXαHEME

BILIRUBIN IS PRODUCED BYOXIDATION OF HEME AND

REDUCTION OF THE RESULTANT BILIVERDIN

O2 &NADPH

NADP+

CO Fe3+ NADPH

NADP+

20

cMOAT MDR2

21

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