A NEW ENZYMATIC ASSAY FOR SELECTIVELY MEASURING CONJUGATED BILIRUBIN CONCENTRATION IN SERUM WITH USE OF BILIRUBIN OXIDASE

A new enzymatic assay for selectively measuring conjugated bilirubin c oncentration in serum with use of bilirubin oxidase (BOD) has been dev eloped. At pH 5.5 BOD can oxidize only conjugated bilirubin in the pre sence of reagents such as sodium fluoride and N-acetylcysteine which c an decrease BOD reactivity to unconjugated bilirubin and bilirubin cov alently bound to albumin (delta bilirubin). The resulting decrease in absorbance al 450 nm is linearly related to the concentration of conju gated bilirubin in serum. The BOD in this new assay was confirmed to o xidize conjugated bilirubin, and neither unconjugated nor delta biliru bin, based on both its reactivity to unconjugated bilirubin and HPLC r esults. This assay was found to give satisfactory results, such as in terms of the range of measurement, the reproducibility of the results, the lack of interference with coexisting substances in serum and the stability of the reagent solutions, in practical applications. The ser um conjugated bilirubin concentrations determined using this assay cor relate well with those determined by the HPLC analysis. This assay can be used for accurate monitoring of changes in the conjugated bilirubi n concentration in patient sera. These findings suggest that the conju gated bilirubin assay is useful for fractional determination of biliru bin in icteric sera. (C) 1998 Elsevier Science B.V.