Selective Enzymatic Methods for Measuring Bilirubin Fractions in Serum

Abstract

Selective enzymatic methods for measuring total and conjugated bilirubins in serum have been developed by using bilirubin oxidase (BOD). BOD can oxidize all bilirubin fractions to non-colored substances at pH 7.8 in the presence of anionic surfactant. On the other hand, BOD can oxidize only the conjugated bilirubin fraction at pH 5.5 in the presence of reagent components such as sodium fluoride and N-acetylcysteine, which can decrease the enzymatic reactivity to unconjugated bilirubin. The resulting decrease in absorbance at 450nm is linearly related to the concentration of bilirubin fractions in serum. These methods were found to have the satisfactory results with respect to the range of the measurement, the reproducibility, absence of interference by coexisting substances in serum and the stability of the reagents in practical use. The value obtained by the method for measuring the total bilirubin correlated well with those obtained by the conventional methods. The method for measuring the conjugated bilirubins was characterized to react only with conjugated bilirubins, but not with unconjugated and delta bilirubins, by its reactivity to the unconjugated bilirubin and the HPLC analysis. The measured value for the conjugated bilirubins by this method correlated well with that by the HPLC analysis. These methods were applicable to the fractional determination of bilirubin fractions in the neonatal serum. These findings suggest the usefulness of these new methods for the accurate measurement of bilirubin fractions present in serum.