Determination of the sum of bilirubin sugar conjugates in plasma by bilirubin oxidase

Background: A reliable indicator of cholestasis is the presence of abnormal concentrations of bilirubin mono- and diglucuronide [conjugated bilirubin (CB)] in blood. A routine assay of CB is available only to those who posses s a certain type of clinical analyzer. We describe a two-point manual metho d for CB that could be adapted as a rate assay to automated clinical analyz ers. Methods: The measurement of CB is based on its oxidation to biliverdin by b ilirubin oxidase. The resulting decrease in absorbance at 460 nm is proport ional to the CB concentration. The assay is calibrated with solutions of di taurobilirubin in human serum. Results: Under the conditions of the assay (0.1 mol/L glycine buffer, pH 10 .0; reaction time, 2 min), only 5% of unconjugated bilirubin is oxidized an d delta-bilirubin is not oxidized at all. Results obtained with the bilirub in oxidase method agreed well with those obtained by HPLC. The long-term CV s at CB concentrations of 6 and 63.4 mg/L were 20% and 2.6%, respectively. The reference values, established by analyzing 51 plasma specimens from hea lthy adults, were 0.0-1.2 mg/L, with a mean value of 0.2 mg/L. Conclusions: The proposed method for CB has good analytical specificity and obviates the requirement for HPLC or a dry chemistry analyzer. The measure ment of CB in blood is superior to the measurement of direct bilirubin beca use an abnormal concentration of direct bilirubin does not necessarily indi cate the presence of cholestasis. (C) 1999 American Association for Clinica l Chemistry.