Sources of production in the rat

Early Bilirubin Late Bilirubin 15% 65% Bilirubin and 0-3 Days 40-80 Days

Increased Erythropoiesis

Red Cell Non- Senescence Hemoglobin Sources (liver)

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

Pathways of Bilirubin Synthesis and Catabolism NADPH NADP Bone Reticuloendothelial Hemin + Marrow RBCs System (RES) N N Hgb Hgb Fe Fe OH NADPH O2 Globin Precursors NADP NN Myoglobin Globin N N + Non-Hgb Heme Fe Heme COO COO Proteins NN Bilirubin O2 CO heme-oxygenase complex Fe Fe (II) + COO COO Porphogens Shunt OH HO Free Bilirubin – Albumin Complex Pathway hydroxyheme-oxygenase complex N N

OH HO NADPH H Conjugated NADP NH N Urine Bilirubin 68mg 2mg N N Urobilinogen in H Biliverd OOC COO N N reductase 70mg C biliverdin HH Fecal OOC COO Urobilinogen Pigments bilirubin

Labeling of RBC hemoglobin and fecal stercobilin

0.3 N of haemin N N of stercobilin of N 15 15 B 0.1 0.2 X X 0.08 X X 0.06 X 0.1 X 0.04 A X X X X X X 0.02 X X X B: Atomexcess % A: Atomexcess % 20 40 60 80 100 120 140 160 180 Times ( days)

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

1 BILIRUBIN CONJUGATION IN MICROSOMES INVOLVES TWO STEPS, MEDIATED BY ONE BILIRUBIN-UDPGA TRANSFERASE

BILIRUBINS: C O H UDPGA H O H UNCONJUGATED C N N O (ligandin) (UCB) O N N H O H H 1 O C C O GA UDPGA O UDP MONO- H H TRANSFERASE C N N O UDPGA MDR2 GLUCURONIDE O N N OATP H O H H (BMG) O C

2 C O GA O H H C N N O UDP DIGLUCURONIDE O N N H H (BDG) O Conjugation prevents GA O C internal H-bonding by the -COOH groups of Bilirubin

A.

OATP MDR2

B. (=cMOAT)

CH2 CH Me UROBILINOGENS ARE FORMED BY β CO (excreted via lungs) Me A B CH2 CH2 CO2H DECONJUGATION AND REDUCTION OF NH N α Fe γ Fe (reutilized) N NH BILIRUBINS BY INTESTINAL BACTERIA C CH CH CO H H2C CH D 2 2 2 CO H CO H δ 2 2 Me Me CONJUGATED BILIRUBIN CH2 CH2 CH2 CH2 A UROBILINOGEN Me CH Me CH CH Me 2 2 Me CH COOGA COOGA CH BCDCH COOH COOH A. A CH2 O N N N N O H H H H M M BILIRUBIN – IX α (planar structure) M M N UNCONJUGATED N N H H N BILIRUBIN H H D H H2C CH A N H H H H 2 B. B N N H N V M COOH COOH C E M O O O M V O BILIRUBIN – IX α (convoluted structure) M M M E

COOH COOH N N O O Deconju- H H C. OH O O OO HO H Reduction HO C C OH gation N H OH HO CH2 CH CH N CH2 2 2 Me Me CH CH2 CH2 Me Me CH V M CH CH M = Methyl CH2 O O + 8H O N N N N O 2 GA H H H H E = Ethyl M V BILIRUBIN - IX α DIGLUCURONIDE V = Vinyl

2 Bilirubinuria in Jaundice

UNCONJUGATED PLASMA BILIRUBIN (B) CONJUGATED

P L A S M A

(A=ALBUMIN)

U R I NO BILIRUBIN N BILIRUBIN ++ E

FRACTIONAL DIAZO REACTION OF PLASMA

RR SO H 3 Fast Direct + reaction N+

N CONJUGATED BILIRUBIN

SO3H DIAZO REAGENT Slow Indirect + reaction N+

N UNCONJUGATED BILIRUBIN

TOTAL DIAZO REACTION OF PLASMA

RR

CONJUGATED SO3H BILIRUBIN

+ + ACCELERATOR

N+ (ALCOHOL, CAFFEINE)

N UNCONJUGATED BILIRUBIN

TOTAL – DIRECT = INDIRECT

3 Phototherapy for h

p Sn-PP Liver Co-PP Liver

g m

/ 2.5 16

n

i

b

u

r

i l

i 2.0

b

l 12

o

m n

( 1.5

e

s 8

a n

e 1.0

g

y

x O

4

e 0.5

m

e H

0 8 16 24 0 2 4 6 8 10 (hours) (days) Time

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

Bile-Duct RAT PBC Treatment for Neonatal Jaundice Sn-PP 10.0 Ligation (50 ) 18 Prevention of kernicterus Control 7.5 16

14 5.0 Sn-PP 12

Plasma Bilirubin mg/dl Bilirubin Plasma 2.5 10

-40 2 4 012 34567 Time (days)

Effect of Sn-protoporphyrin (Sn-PP) (100 μmol/kg body wt) administered at 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).

COOH COOH UGT1 gene CH2 CH2 CH2 CH2 5’ 3’ Me CH Me CH2 CH2 Me Me CH CH BCDCH A CH2 O N N N N O H H H H 1J 1I 1H 1G 1F 1E 1D 1C 1B 1A 234 5

mRNA Bilirubin IX α B-UGT1

COOH COOH CH Phenol 2 CH2 CH2 CH2 Me CH2 Me CH2 Me CH Me CH UGTs CH CH CH2 O N N N N O H H H H Bilirubin IX β NH2 COOH

substrate binding site UDPGA membrane binding site spanning Structure of the α and β isomers of bilirubin IX. In the IXβ isomer (and in the enzyme region γ and δ isomers, not shown) the propionic acid groups are moved to positions 285 amino acids 246 amino acids other than those indicated on the central pyrrole rings B and C of bilirubin IXα.

4 Unconjugated Hyperbilirubinemia

Characteristics Crigler-Najjar Type I Gilbert Syndrome

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

Plasma BSP retention at 45 Normal Usually normal; elevated min in a minority of cases

Hepatic bilirubin-UDPGT Undetectable 30-50% of normal activity

Effect of Phenobarbital on No effect Reduction plasma bilirubin

Pigments in bile Small amounts of unconjugated Increased proportion of bilirubin monoglucuronide

Prevalence Rare 2-7% of population

Prognosis Kernicterus Benign

Animal Model Gunn rat Bolivian Squirrel Monkey

Mutation UDPGT null UDPGT promoter

A(TA)6TAA

Chronic conjugated hyperbilirubinemias

Characteristic Dubin-Johnson Syndrome Rotor Syndrome Jaundice Appearance of liver Grossly black Normal Histology of liver Dark pigment; predominantly in Normal centrilobular areas; otherwise normal Serum bilirubin Elevated, usually between 2 and 5 mg%, Elevated, usually between 2 Conjugated bilirubin occasionally as high as 20 mg%; and 5 mg%, occasionally as predominantly direct-reacting high as 20 mg%; Unconjugated bilirubin predominantly direct- reacting Routine liver function Normal except for bilirubin Normal except for bilirubin tests 45-min plasma BSP Normal or elevated; secondary rise at 90 min Elevated; no secondary rise at Cholestatic Defect in Hepatocellular retention 90 min Conjugation Urinary Normal total >80% as coproporphyrin I Elevated total; elevated coproporphyrin proportion of coproporphyrin I but <80% Mode of inheritance Autosomal recessive Autosomal recessive Intrahepatic Extrahepatic Prevalence Uncommon (1:1300 in Persian Jews) Rare Prognosis Benign Benign Mutation MRP2

Jaundice

Conjugated bilirubin Unconjugated bilirubin

Cholestatic Hemolysis Defect in Hepatocellular Conjugation

Intrahepatic Extrahepatic

5 Liver Function Tests

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

Imaging • Ultrasound •CT scan Bilirubin • Liver-spleen scan Metabolism • Radionuclide biliary scan •Blood • ERCP (endoscopic retrograde •Conjugated & cholangiopancreatography) Conjugated • Transhepatic cholangiography •Urine – Urobilinogen •Stool – Stercobilin

plus 3 O minus Fe Jaundice ↑ BR V minus CO α M

Conjugated bilirubin M D A V COOH COOH N N CH CH δ CH2 2 2 CH2 Unconjugated bilirubin Fe β Me CH Me CH2 CH2 Me Me CH N N CH CH A BCCH D ↑ ALT ↑ AP M C B M 2 O N N N N O H H H H γ CH2 CH2 Hemolysis Defect in Cholestatic CH2 CH2 Hepatocellular Bilirubin IX α Conjugation COOH COOH Nl BD Dilated BD Ferroprotoporphyrin IX (Protoheme) Intrahepatic Extrahepatic 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.

6 BILIRUBIN IS PRODUCED BY OXIDATION OF HEME AND REDUCTION OF THE RESULTANT BILIVERDIN

HEME BILIRUBIN IXα M = Methyl Pr Pr H2 E = Ethyl C M M γ M Pr = Propionyl M N N N N H H β Fe δ H N H N N N BILIVERDIN IXα V α M V M Pr Pr O O C M M H V V M M Biliverdin O & Heme 2 N N Reductase + NADPH Oxygenase H NADP H N H N V M NADP+ Fe3+ O O NADPH CO M V

cMOAT MDR2

7 8