BILIRUBIN METABOLISM

&INHERITED DISORDERS
OF BILIRUBIN METABOLISM

Dr.ANAND R
 CRUNCHES….
• 80% bilirubin originates from senescent
RBCs

• 1-2 х 10*8 RBCs destroyed/hour

• 6g hb produced in body/day

• 250-300 mg bilirubin produced/day

 Pathway for RBC Scavenging
Liver, Spleen &
Bone marrow

Phagocytosis & Lysis

Hemoglobin

Globin Heme Bilirubin

Amino acids Fe2+ Through Liver

Amino acid pool Excreted

5
Bilirubin
synthesis
• Heme oxygenase (HO)- 3 isoforms
HO-1 : Inducible form
HO-2 : Constitutive form
HO-3 : Minor form

• Requires a metal and Oxygen for activation

• Iron in heme binds Oxygen –reductive

activation
Clinical significance:
• Non iron metalloporphyrins- tin and zinc
protoporphyrin , used as treatment in
hyperbilirubinemia

• Binds HO-2 with higher affinity,doesn’t

activate

• Dead end inhibitor of HO-2

CHEMISTRY OF BILIRUBIN

1’8’-Dioxo-1,3,6,7-
tetramethyl-
2,8-divinylbiladiene-a,c-
dipropionic acid
Bilirubin insolubility- a paradox
Insoluble despite of presence of 2 propionic acid
side chains,4 amino groups,2 lactam oxygens

• `ridge-tile’ configuration
• 4Z,15Z trans configuration
• Intra molecular Hydrogen bonding

SIGNIFICANCE:
• Bilirubin conjugation in body
• Basis of indirect diazo reaction
• photoisomerisation
Fog J, Jellum E. Structure of bilirubin.
Molecular model of
bilirubin structure
 BILIRUBIN TRANSPORT
Role of albumin
• Unconjugated bilirubin-sparingly soluble in water
at physiological pH.
• In circulation-bound to albumin

Purpose:
• Prevents precipitation and deposition in tissues
• Aids transport form site of production to site of
elimination-liver
• Prevents bilirubin from entering brain
Binding sites on albumin:
• 1ͦ binding site- residues 124-297
lesser extent 446-547
Gitzelmann-Cumarasamy N, Kuenzle CC, Wilson KJ. Mapping 1976

• Lysine 240-proposed to be site of high affinity

binding Jacobsen C:Biochem J 1978; 171:453.

• Unconjugated bilirubin binding –reversible

• Conjugated bilirubin can bind covalently-
delta
bilirubin
Clinical significance:
•
 HEPATIC UPTAKE
• Remains to be identified conclusively

• Enters hepatocyte across sinusoidal membrane

• Bilirubin is of enough low polarity – can cross
membranes without aid of transporters
Zucker SD, Goessling W; Biochim Biophys Acta 2000; 1463:197

• Bilirubin uptake- liver specific function

• A carrier protein thought to be involved-
OATP 2/OATP C/ SLC21A6
 STORAGE WITHIN HEPATOCYTE
• Remains bound to cytosolic proteins

• Two fractions observed- Y,Z PROTEINS

• Normal concentrations of bilirubin- bound to Y fraction-

predominant proteins

• Z fraction apparent at high concentrations of bilirubin

• Ligandin (Y Protein)-binds many other compounds

• Belong to Glutathine-S-Transferases(GST) family

 BILIRUBIN CONJUGATION
• Essential for biliary excretion of bilirubin
• Glucuronic acid – major conjugating sugar in bile
• Glucosyl,xylosyl conjugates-traces
• Catalyzed by UDP-glucuronosyltransferase(UGT)
 UGT ENZYME SYSTEM
• 2 Major families
UGT 1: Major bilirubin conjugating form in human
UGT 2: Conjugation of steroids,other endogenous and
exogenous substrates
• Multiple isoforms present
• One isoform of UGT 2 family: inducible by phenobarbital
Significance:
• UGT 1A locus abnormalities- disorders of bilirubin
conjugation
• phenobarbital used as enzyme inducer in some conditions
of hyperbilirubinemia
 UGT 1 COMPLEX
•Chromosome 2q37

•Exons 2,3,4,5- used in all isoforms expressed from this

locus,so mutations affect all isoforms

•Exons 1A1-12: variable ; each exon has different

promoter region and differently regulated,mutation
affects only the corresponding isoform
ATATATATATATATAA

ATATATA(TA)TATATATA
A
 BILIRUBIN EXCRETION
• The rate limiting step in bilirubin metabolism
• Excreted across canalicular membrane into bile
• MRP(Multidrug resistant protein) family – efflux
pumps
• MRP 2-essential for bilirubin excretion

Significance:
• Deficiency of MRP 2: DUBIN JOHNSON
SYNDROME
• MRP 2- downregulated in cholestasis
SUMMARY OF BILIRUBIN
METABOLISM IN LIVER
 BILIRUBIN IN INTESTINE

1. CB in bile is excreted into Duodenum

CB 10% diffuses in to blood CB excreted is not reabsorbed

2. Conversion of CB into uro & stercobilinogen

Urobilinogen excreted in stool Part of the UBG enters EHC

3. From gut, UBG but not CB enters EHC

Kidney excretes absorbed UBG In biliary obst. UBG absent in urine
23
BILIRUBIN HANDLING IN KIDNEY

Conjugated • Bilirubin in urine

Bilirubin is conjugated

• Not filtered or
Unconjugated secreted
Bilirubin • Nil in urine
• Normally traces
Urobilinogen •
↓ in Cholestasis
in urine • ↑ in hemolysis

24
 INHERITED DISORDERS OF
BILIRUBIN METABOLISM
UNCONJUGATED CONJUGATED
HYPERBILIRUBINEMIA HYPERBILIRUBINEMIA

• CRIGLER NAJJAR SYN. • DUBIN JOHNSON

TYPE 1 SYNDROME

• CRIGLER NAJJAR SYN. • ROTOR SYNDROME

TYPE 2 (ARIAS SYN.)

• GILBERT SYNDROME
CRIGLER NAJJAR SYNDROME
TYPE 1
• Described by Crigler and Najjar in 1952.

• Autosomal recessive inheritance

• Incidence 0.6-1.0/ million; occurs in all races

• Characterised by striking unconjugated hyperbilirubinemia

20-45 mg/dL (340-765 µmol/L)

• Jaundice Appears in neonatal period ; persists for life

 MOLECULAR MECHANISM

TYPE 1A : Defect in conjugation of various drugs and xenobiotics in

addition to bilirubin

•Constitutes majority of cases; mutations in exons 2-5

TYPE 1B : Defect limited to bilirubin conjugation

•Small subset of patients; mutations in exon A1

 CLINICAL FEATURES
• Severe unconjugated hyperbilirubinemia at birth
Prior to phototherapy:
• Kernicterus
• Death in infancy
With phototherapy:
• Child may survive infancy
• Jaundice persists throught life
• Prone to kernicterus throught life
LAB FEATURES:

• High serum bilirubin 18-30 mg, no evidence of hemolysis

• Other parameters within normal limits

• Bilirubin not present in urine; bile lacks glucuronides

• Liver biopsy shows normal histology

• Molecular diagnostics for gene sequence abnormalities for

diagnosis
 TREATMENT:
Aims at reducing bilirubin levels
Exchange tranfusion:
• in immediate neonatal period
Phototherapy:
• Main stay of treatment
• Special blue lamps used;wavelength 450-500 nm
Phototherpy for
infants
 Becomes less effective near puberty

Plasmapheresis:

•Effective in emergencies
Orthotopic liver transplantation:

•Standard treatment for CN-1

NOVEL APPROACHES:
Inhibition of heme oxygenase:
• Tried with tin-mesoporphyrin
• Showed promise in animal studies
• Effect in humans- variable

Bilirubin degradation by bilirubin oxidase:

• Obtained from myrothecium verrucaria
• In animal studies

Cytochrome p 450 induction:

• Various inducers available
• Provides alternate pathway for degradation.
Isolated liver cell tranplantation:
• Transplantation of liver cells instead of entire liver
• Results were not satisfactory

Gene therapy:
• Replacement with normal bilirubin UGT gene-
attractive therapeutic modality
• Approaches under evaluation
 CRIGLER NAJJAR SYN.2
• ARIAS Syndrome
• Milder variant
• Autosomal recessive

Differs from CN 1 by:

• Average bilirubin concentrations low
• Infrequently associated with kenicterus
• Bile deeply coloured; bilirubin monoglucuronides present
• UGT1A activity reduced;not absent totally
• Bilirubin concentration falls by >25% with phenobarbital
administration
Molecular mechanisms:

•Characterised by aminoacid substitutions – reduce

UGT1A1 activity, not abolish it

Clinical features:
•Asymptomatic in most cases

Treatment:
•Similar to CN-1
 GILBERT SYNDROME
• pronounced 'zheel-BAYR', often shortened to GS, also
called Gilbert-Meulengrachts syndrome
• Described by Gilbert in 1901
• Most common inherited disorder of bilirubin metabolism
• Males > females; Prevalence 8-10%

Presentation:
• Bilirubin levels remain < 3mg/DL; may increase during
intercurrent illness or stress
• Jaundice is the only positive finding often
• Routine lab tests are normal
• Molecular mechanism:

ATATATATATATATAA

ATATATA(TA)TATATATAA UGT 1A1*28

Promoter-reporter studies show that an

increased TATAA box length reduces UGT1A1
expression
Bosma PJ, Roy Chowdhury J, Bakker C, et al. The genetic basis
of the reduced expression of bilirubin UDP- glucuronosyltransferase 1 in Gilbert’s syndrome. N Engl J Med
1995; 333:1171.
?Defect in bilirubin uptake:
• decreased clearance may also be related to
defective uptake

Effect of fasting:

• 2-3 fold increase in s.bilirubin observed

on reducing caloric intake to 400 cal. For 48
hours.

• Limited use in diagnosis

Effect of Nicotinic Acid Administration:

• Intravenous nicotinic acid administration increases

unconjugated hyperbilirubinemia, probably by :

• increasing RBC fragility

• splenic HO activity

• splenic bilirubin formation.

• proposed as a provocative test for the diagnosis of

GS.
DIAGNOSIS:

Presumptive diagnosis made if:

• Mild unconjugated hyperbilirubinemia on several
occasions
• Serum GGT,ALP,fasting and postcibal bile acids
normal

Confirmation:
• Estimate relative concentrations of bilirubin
monoglucuronide to diglucuronide
• Genetic analysis
 BRAIN TEASER!!!!

• Handling of most xenobiotics normal in patients

with GS..

• But one drug……

• Name the drug??

 It’s IRINOTECAN

IRINOTECAN

TOXIC EFFECTS

Carboxy
esterase 2

SN-38 SN-38 G ELIMINATED

UGT 1A1
 DUBIN JOHNSON SYNDROME
• First described by DUBIN & JOHNSON in 1954
• Characterised by chronic non-hemolytic jaundice with
accumulation of conjugated bilirubin in serum
• Dark pigmented liver- dark liver jaundice
• Autosomal recessive ; both sex affected

Clinical features:

• Mild icterus
• Asymptomatic
• Hyperbilirubinemia during illness, pregnancy
 PATHOGENESIS:
Defeciency of MRP 2 protein on canalicular membrane.
• Tm reduced; storage normal
LAB FEATURES:
• S.Bilirubin levels 2-5mg/ Dl
• Other parameters: normal
• Liver grossly pigmented..
 BRAIN TEASER!!!

• Pigment accumulating in liver in

DJS??
Melanin??
No!!!

Epinephrine metabolites..
 ctd..
URINE COPROPORPHYRIN EXCRETION:

• Normally 80% of coproporphrin excreted in urine in

coproporphyrin III.

• In DJS-total coproporphyrin excreted –normal;

Over 75% is coproporphyrin I

• DIAGNOSTIC OF DJS

• Mechanism behind: not known

 ROTOR SYNDROME

• First described by ROTOR in 1948

• Rare disorder

• autosomal recessive inheritance

• Predominantly conjugated hyperbilirubinemia

 ctd..
• Shares many features with DJS.

• Liver not pigmented.

• Both Tm and storage reduced

• Abnormality: not known

Lab features:

Urine coproporphyrin excretion:

• Total coproporphyrin excretion 250-300% of normal

• 50-75% fraction is coproporphyrin I

Other parameters:
• Normal limits
• Liver histology: normal
 BILIRUBIN- FRIEND OR
FOE??
• Function as natural antioxidants in newborns

• Attenuates graft rejection in cardiac transplant models

• Inverse relation between bilirubin and coronary artery

disease

• Inverse relation between bilirubin and colorectal

cancer