Measurement of direct bilirubin by use of bilirubin oxidase

Abstract

We developed an enzymatic method for measuring direct-reacting bilirubin (DBIL) in serum. At pH 4.5, bilirubin oxidase (BOX) oxidizes mono-conjugated bilirubin, di-conjugated bilirubin, and most of the δ-bilirubin to biliverdin. The resulting decrease in absorbance at 460 nm is linearly related to the concentration of DBIL in serum. Mean DBIL values in the 51 patients' sera examined by the BOX method and a diazo procedure (Clin Chem 1982;28:2305) were 45.4 and 42.8 mg/L, respectively. For the same samples, mean values for DBIL and conjugated bilirubin by the Kodak 'Ektachem' methods were 50.2 and 24.8 mg/L, respectively. Hemoglobin, up to 1.5 g/L, does not interfere. Unconjugated bilirubin reacts negligibly. Day-to-day CVs were 2.2% and 2.4% at DBIL concentrations of 37 and 74 mg/L, respectively.