Novel assay for measuring serum conjugated bilirubin and its clinical relevance

We have developed a new enzymatic assay for the determination of conjugated bilirubin (Bc) using stable liquid reagents. In this assay, only Bc is selectively oxidized by bilirubin oxidase at pH 5. 0 in the presence of nitrilotris (methylenephosphonic acid) trisodium salt, ethylenediaminetetraacetic acid disodium manganese (II) salt, and 4-hydroxy-2,2,6,6,-tetramethylpiperidine 1-oxyl. Bc is quantitatively determined from a decrease in the absorbance at 450 nm caused by Bc oxidization. The reagent solutions of the assay were developed so that they could be stably stored for one year together with bilirubin oxidase, in order to eliminate the need to prepare working solutions every time they are required. The assay has good reactivity, differentiability, measurability, and precision. Neither ascorbic acid nor hemoglobin interfered with the measurement. Bc values determined by the assay reflected more clearly the pathophysiological condition of hepatobiliary disease patients with jaundice than the values of total bilirubin or direct bilirubin determined by conventional methods. From these observations, we concluded that this Bc assay is valuable for the evaluation of jaundice.