The majority of bilirubin (80%) is produced from the degradation of hemoglobin from erythrocytes undergoing normal (removal of aged or effete cells) or abnormal destruction (i.e. intravascular or extravascular hemolysis) within mononuclear phagocytes (principally splenic, hepatic and bone marrow macrophages). A small percentage (20%) is derived from the catabolism of various hepatic hemoproteins (myoglobin, cytochrome P450) as well as from the overproduction of heme from ineffective erythropoiesis in the bone marrow. Within macrophages, a free heme group (iron + porphyrin ring) is oxidized by microsomal heme oxygenase into biliverdin and the iron is released (the iron is then stored as ferritin or released into plasma, where it is bound to the transport protein, transferrin). Biliverdin reductase then reduces the green water-soluble biliverdin into unconjugated bilirubin. Heme oxygenase is also located in renal and hepatic parenchyma, enabling these tissues to take up heme and convert it to bilirubin. Birds lack biliverdin reductase, thus they excrete heme breakdown products as biliverdin rather than bilirubin. Unconjugated or free bilirubin is then released into plasma where it binds to albumin. Uptake of unconjugated bilirubin occurs in the liver and is carrier-mediated. The carrier-mediated uptake is shared with unconjugated bile acids and dyes such as BSP. Once within the hepatocyte, unconjugated bilirubin is transported with ligand (Y protein) or other proteins (e.g. Z protein) and the majority is conjugated to glucuronic acid by UDP-glucuronyl transferase. The remainder is conjugated to a variety of neutral glycosides (glucose, xylose). In the horse, the majority of bilirubin is conjugated to glucose. Bilirubin must be conjugated before it can be excreted into bile (conjugation makes bilirubin water soluble). Excretion into biliary canaliculi is the rate-limiting step of the entire bilirubin metabolism pathway and occurs via specific transporters, which are energy (ATP) dependent. Transfer into the canaliculi is facilitated by bile salt-dependent and bile salt-independent biliary flow (the latter of which is generated by a basolateral (sinusoidal or blood-side) Na/K ATPase pump). Bilirubin is secreted, along with bile salts (and sodium) into the intestine, where the bile salts form micelles facilitating absorption of fat. In the intestine, bacterial enzymes deconjugate bilirubin and degrade it to urobilinogen. Urobilinogen is reaborbed (about 10%) or broken down (90%) into urobilin and stercobilin (both of which are excreted in the feces). Of the resorbed urobilinogen, most is taken up by the liver (enterohepatic circulation, i.e. the urobilinogen is absorbed into the portal vein, taken up by the liver and re-excreted into bile - this re-excretion into bile is not depicted in the picture below), whilst the rest bypasses the liver and is excreted into the urine.

Conjugated bilirubin is not normally found in the urine of domestic animals, although small to 1+ amounts of conjugated bilirubin may be seen in concentrated urine from dogs (particularly males), due to the low canine renal threshold for bilirubin. In all species (but dogs, in particular), bilirubinuria may precede an increase in serum bilirubin in cholestatic disorders. Remember, only conjugated bilirubin can be excreted in urine as it is water soluble.
Circulating bilirubin exists in two main forms as determined by the Van den Bergh reaction, which differentiates bilirubin into conjugated (direct) and unconjugated (indirect) forms. There is a third form of bilirubin, called delta bilirubin (or biliprotein), which is conjugated bilirubin bound to proteins. Delta bilirubin increases in serum when hepatic excretion of conjugated bilirubin is impaired (cholestasis) and the liver retains intact conjugation mechanisms. It has a long half-life and is not excreted in the urine (as it is protein bound). Delta bilirubin may be responsible for a persistent bilirubinemia without bilirubinuria seen in some animals with cholestasis. It does react with the diazo dyes, similar to direct or conjugated bilirubin.

Although we usually think of bilirubin in terms of its diagnostic utility (i.e. to support a diagnosis of hemolytic anemia or hepatobiliary disease), bilirubin is actually an anti-oxidant, which is its main physiological function.

Causes of increased total bilirubin

Clinical icterus is observed when total bilirubin values exceed 1.5 mg/dL.

• Artifact: With some analyzers and reagents, hemolysis and lipemia (even mild) will cause artifactually high bilirubin values. The procedures used by the chemistry analyzer at Cornell University are minimally impacted by hemolysis and lipemia. The analyzer also gives an estimation of the amount of bilirubin in the sample (free from hemolysis and lipemia interference) as an icteric index. This index correlates closely (often to the nearest 1-2 mg/dL) to the total bilirubin values and can be used to confirm true increases in total bilirubin. Remember that bilirubin is unstable in light and samples stored for several days, in the presence of light, may have falsely reduced bilirubin values.
  
  
• Hemolysis: Destruction of red cells, whether through extravascular or intravascular hemolysis will increase the production of unconjugated bilirubin because of enhanced hemoglobin metabolism by mononuclear phagocytes. A healthy liver can handle substantial hemolysis without allowing an increase in total bilirubin, therefore, hyperbilirubinemia is usually due to severe, rapid hemolysis. In these cases, the bilirubin is mostly unconjugated bilirubin and the total bilirubin is usually < 2 -3 mg/dL. In some cases of hemolytic anemia (perhaps with longer standing hemolysis), secondary hepatic hypoxia/dysfunction will interfere with bilirubin excretion into the bile ducts (remember this is the rate-limiting step of bilirubin metabolism and is ATP-dependent), resulting in cholestasis. This occurs predominantly in small animal patients with immune-mediated hemolytic anemia and some foals with neonatal isoerythrolysis (NI, up to 40-60% of total bilirubin may be conjugated in foals with NI). Therefore, animals with hemolytic anemia and bilirubinemia > 2-3 mg/dL often have a cholestatic component to the icterus, i.e. there are substantial increases in both conjugated (which can dominate) and unconjugated bilirubin. This reflects both cholestasis and increased unconjugated bilirubin production from heme breakdown. Note that icterus in cattle is mostly due to hemolysis (and is usually unconjugated) and rarely due to liver disease or post-hepatic bile duct obstruction. You may find schematic illustrations of bilirubin metabolism in intravascular and extravascular hemolysis helpful.
  
  
• Liver disease: Hepatic disease may cause increases in both unconjugated and conjugated bilirubin. Increases in bilirubin in dogs often occurs after elevation of cholestatic enzymes (GGT, ALP) due to the low renal threshold for bilirubin. In acutely developing icterus, ALP and GGT levels may be normal because they require time for induction. In large animals with liver disease, increases in bilirubin are usually due to unconjugated bilirubin. Only cattle with very severe liver disease will have increased bilirubin (usually unconjugated).
  
  
• Cholestasis: This is defined as decreased bile flow and can be due to physical obstruction of bile flow or functional defects in the transporters that deliver bile salts or bilirubin into the biliary system. Obstructed bile flow can be intrahepatic (e.g. hepatocyte swelling due to hepatic lipidosis in cats) or extrahepatic (e.g. bile duct obstruction from pancreatic neoplasia, cholelithiasis, Fasciola hepatica in cattle). Changes in the character of bile (e.g. thick sludged bile in cats with dehydration) can also result in decreased bile flow. Functional defects in bile salt or bilirubin transporters occur secondary to inflammatory cytokines (e.g. endotoxemia) and drugs. Defects in these transporters also occur with physical obstructions to bile flow. Cholestasis will result in bilirubinemia, with increased direct bilirubin, and bilirubinuria (excess conjugated bilirubin in blood is excreted into the urine, because it is water soluble). Indirect bilirubin is also usually increased in cholestasis due to the toxic effect of accumulated bile salts on hepatocytes or cholestasis-induced decreases in the hepatic transporters which take up unconjugated bilirubin from blood). Cholestasis frequently (but not always) results in a higher conjugated than unconjugated bilirubin, particularly when there is a physical obstruction to bile flow (e.g. cholelithiasis, biliary mucocele). The exception is the horse, where unconjugated bilirubinemia still dominates in cholestatic conditions, i.e. direct bilirubin rarely exceeds 50% of total bilirubin in horses with cholestasis.
  
  
• Physiologic:
  1) Fasting: In horses, fasting will produce a hyperbilirubinemia due to unconjugated bilirubin. The precise mechanism for this is

  Figure reproduced with permission from: Hepatic function by Tennant B in "Clinical Biochemistry of Domestic Animals", 5th edition, Kaneko et al, eds, pg 344, 1997; Copyright Elsevier.

  unknown but it is thought to be due to either decreased uptake of bilirubin (due to competition for uptake with free fatty acids) or impaired conjugation of bilirubin (due to low glucose within hepatocytes - remember, in horses most of unconjugated bilirubin is conjugated to glucose). Increases in bilirubin are noticeable within 12 hours of fasting and may reach levels as high as 10-12 mg/dL within 2-4 days of anorexia, with clinical icterus. This occurs in the absence of significant liver disease. Note that horses have higher reference intervals for bilirubin than donkeys. Mild increases in total bilirubin (mostly unconjugated) are seen in cows that are sick and/or anorectic. The total bilirubin usually does not exceed 4 mg/dL in these cows. The precise mechanism is unknown.
  2) Neonatal: Young animals, especially foals, often have jaundice (due primarily to unconjugated bilirubin). This is due to multifactorial causes, including hemolysis of fetal red blood cells, decreased liver uptake of bilirubin, immaturity of hepatic conjugation mechanisms and poor albumin binding.
  
  
• Inherited: Inherited defects in hepatic uptake, conjugation and excretion of bilirubin occur in monkeys, sheep, and rats. Southdown sheep have an inherited defect in bilirubin uptake, resulting in a fasting hyperbilirubinemia due to unconjugated bilirubin. Corriedale sheep have Dubin-Johnson syndrome, a fasting hyperbilirubinemia due to conjugated bilirubin from defective excretion of conjugated bilirubin.
  

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