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Bilirubin Secretion,Jaundice and Evaluation

of Liver FunctionHoward J. Worman, M. D.

Evaluation of Liver Disease andHepatic Function

HistoryPhysical ExaminationLaboratory TestsSometimes Radiological/Nuclear MedicineSometimes Liver Biopsy

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Jaundice occurs as a result of excess bilirubin inthe blood. It is a hallmark of liver disease butnot always present in liver disease. Jaundiceoccurs when the liver fails to adequatelysecrete bilirubin from the blood into the bile.To understand how jaundice occurs, you mustfirst understand bilirubin synthesis,metabolism and secretion.

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BilivirdinReductase

Kikuchi et al.NatureStructuralBiology 8, 221 -225 (2001)

HemeOxygenase

Schuller et al.NatureStructuralBiology 6, 860 -867 (1999)

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Bilirubin is frequentlydepicted as a lineartetrapyrrole.

However, intramolecularhydrogen bonding fixesit in a rigid structure thatblocks exposure of itspolar groups to aqueoussolvents, making it veryinsoluble in blood.

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OATP-C?

Bilirubin in Blood is Bound to Albumin: Uptake intoHepatocyte at Basolateral (Sinusoidal) Membrane

Some bilirubinstored in cytosol boundto proteins

Bilurubin UDP-glucuronosyltransferase is localized to the endo-plasmic reticulum; it catalyzes conjugation to a diglucuronide, making it more water soluble.

A: Labeling of periphery of cell hepatocyte nucleusB: Labeling of ER with antibody to UDP-glucuronosyltransferase

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Alternative RNA splicing of different first exons ofUGT1 gives different isoforms with different substratespecificities, some for bilirubin and others to differentsubstrates, such as phenol.

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ABCC2(MRP2, cMOAT)

Bilirubin glucuronide is secreted from hepatocytes by an ATP-binding cassette protein. This is the rate limiting step in hepatocytebilirubin metabolism and disrupted in most acquired liver diseases

Bilirubin is Only Approximately 2% of Bile

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Stercobilins/Urobilins in Stool

Some urobilinogenexcreted by kidney

Diagnostic Consequences ofEnterohepatic Circulation of Bilirubin

• In hepatocyte dysfunction (hepatocellular)– May see increased urobilinogen in urine because it is

less efficiently reabsorbed by hepatocytes• In biliary obstruction

– Stools may appear white because bilirubin does not getinto intestine and therefore not converted tostercobilins/urobilins

– No urobilinogen detected in urine

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Measurement of Bilirubin in Blood

• Normally ≤17 µM (1 mg/dl)– >35 µM: can begin to detect jaundice clinically, (sclera,

mucus membranes early)– Discoloration of skin with higher concentrations

• When measured precisely (e. g. by HPLC), around96% of serum bilirubin is unconjugated

• Clinical laboratory generally “overestimates” amountof conjugated bilirubin (up to 30%) because of method– reported as “total,” “direct” (approximates conjugated) and

“indirect” (approximates unconjugated)

van den Berghand

MullerReaction (1916)

Using this method,20% to 30% serumdirect bilirubin isnormal value

R R

CONJUGATED

BILIRUBIN

UNCONJUGATED

BILIRUBIN

N+

N

SO3H

+ + ACCELERATOR

(ALCOHOL, CAFFEINE)

TOTAL – DIRECT INDIRECT=

TOTAL DIAZO REACTION OF PLASMA

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Excess conjugated bilirubin in serum may be excreted by kidneys (dark urine).Albumin-bound unconjugated bilirubin cannot be excreted by kidneys.

With longstanding elevated serum conjugated bilirubin, less is in urine because of covalent binding to albumin.

Causes ofHyperbilirubinemia and

Jaundice

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Most Common Cause of UnconjugatedHyperbilirubinemia Western Countries

Gilbert syndrome is not really a “disease” but a normal variant.

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High blood concentrations of lipid solubleunconjugated bilirubin in infants that alsohave poorly developed blood-brain barrier

can lead to kernicterus (brain damagecaused by bilirubin deposition).

Treatments include exchance transfusionand phototherapy. Heme oxygenase

inhibitors are also being studied for thisindication.

Treatment of Neonatal Jaundice by Phototherapy

Less intramolecular hydrogen bonding of E diasteriomers make them more aqueous soluble for renal excretion.

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Caused by mutation in ABCC2 encoding canalicular transporter.

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With abnormal secretion from hepatocytes, most excess bilirubin inblood is conjugated by can get “mixed picture” because of

“backup” of unconjugated bilirubin.

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Laboratory Tests inLiver Diseases

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“Liver Enzymes”

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Liver Biopsy in the Evaluation of Liver Disease

Dr. Lefkowitch Next