Bilirubin
Introduction
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.
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.