Bilirubin, a degradation product of heme catabolism, is a non-polar molecule. There are two forms of bilirubin: water-soluble (conjugated or direct) and water-insoluble (unconjugated or indirect) bilirubin. Bilirubin is produced in the endoplasmic reticulum as unconjugated bilirubin, which binds to albumin in plasma and forms albumin-bilirubin complex. This complex is transported to the liver, where it is conjugated with glucuronic acid and forms conjugated bilirubin. Bilirubin has potent antioxidant, anti-inflammatory and autoimmune properties. Bilirubin concentration in human body depends on gender, drug intake, age, etc. Low serum bilirubin is directly correlated with pathological conditions including diabetes mellitus, metabolic syndrome, and cardiovascular diseases. However, high bilirubin indicates hemolysis, jaundice, Gilbert's syndrome, hepatitis, drug toxicity, and possible blockage of bile ducts. Assay Genie's Bilirubin Assay Kit utilizes the Jendrassik-Grof principle to detect bilirubin. Total bilirubin (unconjugated + conjugated) concentration is determined in the presence of a catalyst, where bilirubin reacts with a diazo- salt to form azobilirubin, which absorbs at 600 nm. Direct bilirubin (conjugated) is determined in the absence of catalyst (550 nm).

Figure: (a) Total Bilirubin Standard Curve (0 - 4 µg). (b) Direct Bilirubin Standard Curve (0 - 4 µg). (c)Total and Direct Bilirubin concentrations in Normal Human Serum. Different volumes of human serum (10- 50 µl) were assayed following kit protocols. Reported concentrations (in mg/dl): Total Bilirubin: 4; Direct Bilirubin: 1.5. Experimental concentrations (calculated as the average of estimated bilirubin in five different human serum volumes ranging from 10 to 50 µl): Total: 3.8 mg/dl; Direct: 1.75 mg/dl