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Eighty percent of all bilirubin is derived from the metabolism of hemoglobin released from senescent red blood cells. Hemoglobin released from red blood cells is converted to unconjugated (also called indirect) bilirubin in the reticuloendothelial system. Daily production of unconjugated bilirubin is 250 to 350 mg. Because this bilirubin is water insoluble, it must be transported to the liver bound to albumin. The circulating half-life of unconjugated bilirubin is <5 minutes. In the liver, unconjugated bilirubin is transferred from albumin into hepatocytes where it is conjugated with glucuronic acid. Conjugated bilirubin (also called direct) is then excreted in the bile and transported to the intestine. Conjugated bilirubin is essentially absent from the blood of healthy individuals. In the distal ileum and colon, bacteria covert conjugated bilirubin to stercobilinogen. A small fraction is reabsorbed into the portal circulation and excreted in the urine as urobilinogen. This metabolic pathway accounts for the bilirubin detected in the plasma of healthy individuals.

Jaundice is the clinical manifestation of an elevated plasma bilirubin level. It occurs when bilirubin production exceeds the liver’s excretory capacity. This may occur because (1) too much bilirubin is being produced, (2) hepatocytes are injured and cannot metabolize or excrete bilirubin, or (3) the biliary tract is obstructed blocking the flow of conjugated bilirubin into the intestine. Examples include hemolytic anemia, acute viral hepatitis, advanced cirrhosis, and obstructive biliary tract disease.

If total bilirubin is mildly elevated (usually <6 mg/dL) and less than 20% is conjugated (direct: total ratio <0.2), the most likely diagnoses are Gilbert syndrome or hemolysis. Gilbert syndrome affects ~5% of the population and causes mild hyperbilirubinemia because of impaired UDP-glucuronyltransferase. Other possibilities include ineffective erythropoiesis, resorption of a large hematoma, pulmonary embolism with infarction, Crigler-Najjar syndrome, neonatal jaundice, and shunts. Because unconjugated bilirubin is water insoluble and bound to albumin, these patients do not have bilirubinuria. Testing of urine for bilirubin is a simple way to determine if elevated bilirubin is unconjugated or conjugated.

Increases in conjugated bilirubin are highly specific for disease of the liver or bile ducts. Hepatocellular injury or cholestasis is suspected when more than 50% of total bilirubin is conjugated bilirubin (direct: total ratio > 0.4). In common bile duct obstruction due to gallstones, total bilirubin seldom exceeds 15 mg/dL and usually remains below 6 mg/dL because obstruction is often incomplete and conjugated bilirubin is water soluble and excreted by the kidneys. Bilirubin excretion is able to keep pace with production. Total bilirubin levels >25 mg/dL usually indicate intrahepatic cholestasis.

The following table may help to differentiate the various causes of jaundice.

Lab Test



Intrahepatic cholestasis

Extrahepatic cholestasis

Total bilirubin

<6 mg/dL


Variable, may be >30 mg/dL

<30 mg/dL

Direct bilirubin







>5 fold increase

2-5 fold increase

2-3 fold increase

cholangitis higher



2-3 fold increase

3-5 fold increase

3-5 fold increase






PT corrected by vitamin K

Not Done




In adults, jaundice develops in 70% of cases of acute hepatitis A, 33 – 50% of cases of acute hepatitis B and 20 – 33% of cases of acute hepatitis C. Peak bilirubin is usually <15 mg/dL in viral hepatitis; only 10% of patients have bilirubin >15 mg/dL and only 4% have values >20 mg/dL. Bilirubin peaks a week later than ALT and AST and then gradually decreases. In adults with viral hepatitis, bilirubin remains increased for 30 +/- 20 days after peak concentrations are reached. In approximately one third of adults with hepatitis B virus infections, jaundice remains elevated more than 6 weeks. Significantly elevated bilirubin is uncommon in toxic and ischemic hepatic injury.

The presence of bilirubin in the urine reflects direct (conjugated) hyperbilirubinemia and therefore underlying hepatobiliary disease. Unconjugated bilirubin is tightly bound to albumin and is not filtered by the glomerulus. Conjugated bilirubin may be found in the urine when the total serum bilirubin concentration is normal because the renal reabsorptive capacity for conjugated bilirubin is low and the methods used can detect urinary bilirubin concentrations as low as 0.05 mg/dL. Thus, bilirubinuria may be an early sign of liver disease, while the clearance of bilirubin from the urine may be an early sign of recovery. 

In the full-term infant, serum bilirubin levels peak at 48 to 72 hours after birth. Kernicterus is a preventable lifelong neurologic syndrome caused by severe & untreated hyperbilirubinemia during the neonatal period. In full term infants, hyperbilirubinemia symptoms include severe jaundice, lethargy and poor feeding. Kernicterus may result in choreoathetoid cerebral palsy, mental retardation, sersorineural hearing loss and gaze paresis. Boys are more susceptible than girls to adverse outcomes from hyperbilirubinemia. Treating hyperbilirubinemia with phototherapy and exchange transfusions prevents kernicterus. Kernicterus virtually disappeared in full-term infants until the early 1990’s. Increases in breastfeeding and early hospital discharge after delivery have resulted in a resurgence of kernicterus.

Major risk factors for hyperbilirubinemia in full-term newborns include:

  • Jaundice within first 24 hours after birth
  • A sibling who was jaundiced as a neonate
  • ABO or Rh incompatibility
  • Nonoptimal nursing
  • G6PD deficiency
  • Infection
  • Cephalo-hematoma or bruising
  • East Asian or Mediterranean descent.

The Vitros chemistry analyzers directly measure unconjugated and conjugated bilirubin and calculate total bilirubin by adding these two fractions together. The normal range for total bilirubin is slightly lower than other chemistry methods (0 - 1.1 mg/dL vs  0.2 – 1.2 mg/dL). The major reason for this decrease in total bilirubin is that delta bilirubin is not measured. Delta bilirubin is bilirubin bound to albumin, which has a circulating half-life of 15 to 19 days. It is usually increased following a prolonged period of conjugated hyperbilirubinemia and is not clinically important. The Vitros normal range for direct bilirubin is 0 - 0.3 mg/dL, but many more patients have values of 0 or 0.1 mg/dL than with other methods.

Reference range is 0 to 1.1 mg/dL for total bilirubin and 0.0 to 0.3 mg/dL for direct bilirubin (Vitros Analyzer using Bu/Bc slide). Bilirubin levels are 33% lower beginning with the second trimester of pregnancy.

Specimen requirement is one green top (heparin tube) or SST tube.The specimen should be protected from light, because unconjugated bilirubin is unstable. One hour of light exposure can cause a 50% decrease.

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