Enzymatic assay for conjugated bilirubin (Bc) in serum using bilirubin oxidase (BOD)

We described an automated enzymatic assay for conjugated bilirubin (Bc) in serum using the latro D-Bil kit, with bilirubin oxidase (EC 1.3.3.5 BOD) fr om Myrothecium species. The specificity of the enzyme in the latro D-Bil ki t was examined by analyzing unconjugated bilirubin (Bu), delta bilirubin (B delta), and Bc with high-performance liquid chromatography (HPLC), before and after the enzymatic reaction using BOD. The within-assay coefficients o f variation (CV) of this method were 0.58 to 5.00% (n = 20) at 1.4 to 155.8 mu mol/L. Day-to-day CVs ranged from 1.61 to 7.14% at 1.2 to 182.1 mu mol/ L. The analytical recovery was 96 to 101%. The presence of ascorbic acid, r educed glutathione, L-cysteine, uric acid, urea, creatinine, glucose, lipem ic material, anticoagulants, hemoglobin, or human serum albumin did not aff ect this assay system. The cop-relation coefficient between values obtained with the latro D-Bil kit (y) and HPLC method as reference for conjugated f ractions (x) was; r = 0.983, y = 0.952x + 8.851 mu mol/L, Sy/x = 11.97 (n = 56). We studied serum Dc levels, not including Bu and B delta, in patients with hepatic diseases or autoimmune hemolytic anemia. Levels of Bc obtaine d by the proposed method changed more rapidly than did those of direct bili rubin (D-Bil) obtained by diazo-dye method during the course of the disease s. J. Clin. Lab. Anal. 13:219-223, 1999. (C) 1999 Wiley-Liss, Inc.