2.5 Liver Function Tests, Bilirubin Metabolism, and Jaundice

Liver Function Tests, Bilirubin Metabolism, and Jaundice

The liver is examined from several directions in Clinical Chemistry: enzymes released by injured cells (covered in the enzymology section), proteins it synthesizes (covered in the protein section), and the substances it clears, above all bilirubin and ammonia. This section covers bilirubin metabolism and measurement, the laboratory classification of jaundice, inherited and neonatal hyperbilirubinemias, ammonia testing, and how the tests combine into liver injury patterns.

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1. Bilirubin Metabolism

1. Formation: About 80% of bilirubin comes from hemoglobin of aged red cells broken down in macrophages of the spleen, liver, and bone marrow. Heme oxygenase opens the heme ring to form biliverdin (green), releasing iron and carbon monoxide. Biliverdin reductase converts biliverdin to unconjugated bilirubin (orange-yellow).
2. Transport: Unconjugated bilirubin is insoluble in water, so it travels bound to albumin. It cannot be filtered by the glomerulus and does not appear in urine.
3. Hepatic uptake and conjugation: Hepatocytes take up bilirubin and conjugate it with glucuronic acid using uridine diphosphate glucuronosyltransferase (UGT1A1), forming mainly bilirubin diglucuronide. Conjugated bilirubin is water-soluble.
4. Excretion: Conjugated bilirubin is secreted into bile through the canalicular transporter MRP2. This is the rate-limiting step.
5. Intestinal conversion: Colonic bacteria reduce bilirubin to urobilinogen. Most is oxidized to stercobilin (and urobilin), which colors stool brown. About 10% to 20% of urobilinogen is reabsorbed; most returns to the liver (enterohepatic circulation), and a small amount is excreted in urine.

Delta bilirubin is conjugated bilirubin covalently bound to albumin. It forms in prolonged cholestasis, reacts as direct bilirubin, and persists with albumin's half-life (about 2 to 3 weeks), so total bilirubin can stay high for weeks after an obstruction is relieved.

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2. Measuring Bilirubin

Diazo Methods

Bilirubin reacts with diazotized sulfanilic acid to form azobilirubin. Conjugated bilirubin reacts directly in water ("direct" bilirubin); unconjugated bilirubin needs an accelerator to be released from albumin.

• Direct bilirubin measures conjugated plus delta bilirubin. Indirect (unconjugated) bilirubin = total bilirubin - direct bilirubin.
• Specimen handling: Bilirubin is destroyed by light, especially sunlight and phototherapy lamps, so specimens are protected with amber tubes or foil and tested promptly. Hemolysis interferes with diazo methods.
• Neonatal bilirubin can be measured by direct spectrophotometry (bilirubinometer), because newborn plasma has little interfering carotene. Transcutaneous bilirubin meters are used for screening.
• Reference values (adult): Total bilirubin about 0.2 to 1.0 mg/dL; direct bilirubin up to about 0.2 to 0.3 mg/dL. Multiply mg/dL by 17.1 to convert to µmol/L. Jaundice becomes visible when total bilirubin rises above about 2.5 to 3.0 mg/dL.

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3. Laboratory Classification of Jaundice

The urine findings follow directly from metabolism: only conjugated bilirubin is filtered into urine, and urobilinogen depends on bilirubin reaching the gut.

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4. Inherited and Neonatal Hyperbilirubinemias

• Physiologic jaundice of the newborn is unconjugated hyperbilirubinemia from a high red cell turnover and immature UGT1A1. It appears after the first 24 hours of life and resolves within about 1 to 2 weeks.
• Jaundice in the first 24 hours is never physiologic and suggests hemolysis (for example, HDFN or G6PD deficiency, which is common in the Philippines and included in newborn screening).
• Very high unconjugated bilirubin crosses the immature blood-brain barrier and causes kernicterus. Treatment is phototherapy, and exchange transfusion when levels are dangerous.
• Conjugated hyperbilirubinemia in an infant is always abnormal and suggests biliary atresia or neonatal hepatitis.

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5. Ammonia

• Ammonia from protein breakdown and intestinal bacteria is converted to urea by the liver's urea cycle. In severe liver failure, portosystemic shunting, Reye syndrome, and inherited urea cycle defects, ammonia rises and contributes to hepatic encephalopathy.
• Specimen: Free-flowing venous blood in a heparin or EDTA tube, placed on ice, centrifuged and analyzed within about 30 minutes. Ammonia rises in standing blood as red cells and amino acids release it. Avoid hemolysis and tobacco smoke near the specimen, and avoid ammonium heparin tubes.
• Method: The enzymatic glutamate dehydrogenase method measures the decrease in absorbance at 340 nm as NADPH (or NADH) is oxidized while ammonia and alpha-ketoglutarate form glutamate.

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6. Putting the Liver Panel Together

• Hepatocellular pattern: Large rises in ALT and AST (often more than 10 times the upper reference limit in acute viral or toxic hepatitis) with modest ALP elevation.
• Cholestatic pattern: ALP and GGT rise more than the aminotransferases, with conjugated bilirubin and urine bilirubin positive.
• Synthetic failure: A falling albumin (slow, because albumin's half-life is about 3 weeks) and a prolonged prothrombin time (fast, because factor VII's half-life is only about 6 hours) show loss of liver function rather than ongoing injury. A prolonged PT that does not correct with vitamin K points to hepatocellular failure rather than vitamin K malabsorption from cholestasis.
• Cirrhosis: Low albumin, polyclonal hypergammaglobulinemia with beta-gamma bridging on serum protein electrophoresis, a prolonged PT, low platelets from hypersplenism, and often only mildly raised aminotransferases.