[The main cause responsible for the disparity between enzymatic and diazo methods in measuring direct bilirubin from a viewpoint of bilirubin subfractionation].

An enzymatic method which measures bilirubin with bilirubin oxidase has come into use but there is often a disparity in the values of direct bilirubin measured by enzymatic and diazo methods. To determine the cause of this disparity, bilirubin subfractions were measured by HPLC. The retention time (mean +/- SD) of each subfraction was: alpha, 34.7 +/- 0.1 min; beta (biphasic), 29.3 +/- 0.1 & 28.6 +/- 0.1 min; gamma, 24.9 +/- 0.1 min; delta, 20.2 +/- 0.3 min. Samples with great difference have all four peaks (alpha + beta + gamma + delta type) while samples with little difference have large alpha and delta peaks with small beta and gamma peaks (alpha + delta type). Therefore, the beta and gamma subfractions (conjugated bilirubin) contribute substantially to the difference. Bile and synthesized direct bilirubin are comprised solely conjugated bilirubin and show a marked difference. This evidence strongly supports the findings in the present study. Next, synthetic direct bilirubin was measured by HPLC before and after the addition of bilirubin oxidase, and the absorbance at 450 nm was found to increase between 2.9 and 3.5 min. Therefore, it appears that the disparity between enzymatic and diazo methods in measuring direct bilirubin comes from the products of conjugated bilirubin by bilirubin oxidase.