Bilirubin is another blood test that can reflect injury to hepatocytes and/or the biliary system. That is, hyperbilirubinemia can result from both hepatocellular and cholestatic liver injury.

Bilirubin is a major breakdown product of hemoglobin and is derived from red blood cells that have died and removed by the spleen. Heme is converted to bilirubin through a series of enzymatic reactions. This bilirubin is initially water-insoluble bilirubin (unconjugated bilirubin) and is transported in blood bound to albumin. It is then taken up by hepatocytes where it becomes water-soluble through its conjugation with glucuronic acid. Conjugated bilirubin is then excreted into the bile canaliculus and is eventually secreted into the duodenum.

Hepatocytes transform bilirubin into a water soluble product by a process known as conjugation. Conjugated bilirubin can then be excreted into bile. Hyperbilirubinemia can result from either the conjugated or unconjugated fractions.

The conjugated fraction reacts directly with Ehrlich's diazo reagent and is thus called direct bilirubin. The unconjugated bilirubin requires the addition of alcohol to develop color reaction. It is also known as indirect bilirubin. Total bilirubin is the summation of the two fractions.

Similar to other common liver tests, abnormal bilirubin levels may or may not be caused by liver disease process. For example, hemolysis leads to indirect hyperbilirubinemia the liver is normal.