Bilirubin

Introduction

Bilirubin is formed from the metabolism of heme groups in the liver, spleen and bone marrow and is taken up and conjugated by hepatocytes before excretion in the bile. Increased levels are associated with accelerated haemolysis, hepatobiliary disease and cholestatic disorders. Bilirubin is a fairly crude marker of hepatic function. Refrences: NationWide Laboratories

Image of a jaundiced cat, note the discoloured pinnae
(Image source Sabar 2007, Wikimedia Commons)

Red blood cells either undergo phagocytosis in the case of ageing cells or haemolysis in haemolytic crises. Haemoglobin is freed from the red cells and is further broken down in the reticulo-endothelial system to haeme and globulin. Haeme is a mixture of iron and porphyrin. The enzymatic conversion occurs within cells of the monocyte-phagocyte system (MPS) when haemoglobin is released by the degradation of red blood cells.

Both the iron and globulin are recycled for further use in erythropoiesis. The porphyrin from haemoglobin breakdown is converted to biliverdin, a green pigment, which may contribute to the greenish appearance seen in local bruising. Biliverdin is subsequently changed into bilirubin. This unconjugated bilirubin is not water soluble and is thus bound to albumin to be transported in the blood to the liver. In the hepatocyte, bilirubin is released from the albumin and conjugated with glucuronic acid, forming water soluble conjugated bilirubin. This is secreted into bile which then moves into the small intestine.

Unconjugated bilirubin is sometimes referred to as indirect, in contrast to conjugated which can be referred to as direct bilirubin.

The conjugated bilirubin is degraded to urobilinogen by gastro-intestinal bacteria and a small proportion of this product is reabsorbed and excreted in the urine. The remaining urobilinogen is further degraded to stercobilin, a brown pigment which contributes to the colour of faeces. Therefore, in animals with complete biliary obstruction, urobilinogen is absent from the urine and the faeces have a white/grey 'acholic' colour due to the absence of stercobilin. The latter alteration in faecal colour also results from steatorrhoea.

Small quantities of conjugated bilirubin are found in the urine of normal dogs because it has a low renal threshold.

The technical name for Jaundice is Icterus, which refers to the staining of tissues by bilirubin pigment or bilirubin complexes, a phenomenon that is clinically evident on physical examination of the sclera and mucous membranes. It can also been seen on a separated blood sample - the serum will appear yellow/orange in colour.

Hyperbilirubinaemia

Bilirubin increases when the production of bilirubin exceeds the ability of the liver to recover unconjugated bilirubin from the bloodstream and process it, or when clearance of the conjugated form is altered by impaired hepatocyte processes or obstruction to the discharge of bilirubin into the intestines. Clinically, this means that hyperbilirubinaemia can be caused by heamolysis or choleostasis - the next step diagnostically is to establish if the inciting cause is prehepatic, hepatic or posthepatic.

Small animals

Causes of hyperbilirubinaemia

• Biliary obstruction and cholestasis (intra or posthepatic)
• Reduced hepatocellular function (loss of >70% of functional hepatocellular mass)
• Haemolysis (intravascular or marked extravascular)

Complementary tests

The differentiation of total serum bilirubin into direct (conjugated) and indirect (unconjugated) bilirubin may be performed but is of strictly limited diagnostic value. As a rule of thumb, >90% of bilirubin must be either direct acting or indirect acting before it is of diagnostic value (direct acting indicating biliary tract obstruction and indirect acting indicating haemolysis). A haemogram should be used to rule out haemolysis. Bile acids will be elevated due to loss of hepatocellular mass or biliary obstruction but not due to haemolysis. Marked increases in ALP and GGT will be seen with posthepatic cholestasis.

Small animals and complementary tests references: NationWide Laboratories

Bilirubin in horses

Horses have a different biliary excretion system to other animals and also readily develop jaundice in response to anorexia or intestinal disruption, so hyperbilirubinaemia can be difficult to interpret in the horse and elevated unconjugated bilirubin levels in the absence of any clinical signs might be of little consequence.

Causes of hyperbilirubinaemia

• Anorexia
• Biliary obstruction and cholestasis (intra or posthepatic)
• Reduced hepatocellular function (particularly due to acute hepatic failure)
• Haemolysis (intravascular or marked extravascular)
• Intestinal obstruction and a variety of other serious systemic diseases

Extrahepatic causes for example, anorexia, other systemic disease may see an increase in bilirubin of up to 150umol/l

Complementary tests

In contrast to small animals, only modest increases in direct acting bilirubin are seen in either liver failure or in cholestasis. Where direct acting bilirubin forms >25-50% of the total bilirubin, this indicates significant cholestasis, which may be intra- or post hepatic.

Causes of hyperbilirubinaemia and complementary test references: NationWide Laboratories

Identification

Bilirubin stains brown with H&E, like both haemosiderin and lipofuscin. They must be distinguished from each other by special stains. Bilirubin stains bright green with a Fouchet stain.

Distinguishing Conjugated from Unconjugated Bilirubin

The Van de Berg test can be used to distinguish conjugated from unconjugated bilirubin. Plasma from an icteric animal is treated with an aqueous solution of the reagent diazotised sulphanilic acid and this produces a red-purple colour reaction. The intensity of this colour is directly proportional to the amount of water soluble (conjugated ) bilirubin in the sample. Further addition of alcohol intensifies the colour if there is non-water soluble (unconjugated) bilirubin also present. The intensified colour is directly proportional to the total amount of bilirubin present in the sample and the difference between the two readings gives the amount of unconjugated bilirubin in the sample.

References

Text referenced 'Nationwide Laboratories': NationWide Laboratories