Microdetermination of unbound bilirubin in icteric newborn sera: An enzymatic method employing peroxidase and glucose oxidase

An enzymatic assay method for the microdetermination of unbound bilirubin in newborn icteric sera is described. Unbound bilirubin is oxidized to colorless compounds by peroxidase in the presence of hydrogen peroxide derived from glucose by the mediation of glucose oxidase. In this method, the bilirubin is not significantly degraded before the addition of peroxidase, in contrast to the method using hydrogen peroxide. The oxidation rate is determined by spectrophotometry and chloroform extraction is eliminated.The unbound bilirubin concentration can be determined fromthe initial oxidation velocity of total bilirubin. The Michaelis constant, $\text{K}{}_{\mathrm{M}}$ was approximately 20 μM. The coefficient of variation for icteric serum determination was 4.4–6.5%. The concentration of unbound bilirubin was reduced after five days of storage at ?20° C.The bilirubin-albumin binding affinity was studied with purified albumin and adult serum. The dissociation constants were 2 × 10^?8 M and 5 × 10^?9 M, respectively, at bilirubin/albumin molar ratios below 1.0.Clinically, serum samples from 75 icteric newborn infants were analysed, and the sera of premature infants were found to have remarkably high levels of unbound bilirubin compared to those of fullterm infants. The sera of a Rhesus immunization infant and an ABO incompatibility infant were remarkably higher than that of the nonhemolytic icteric sera. The unbound bilirubin concentration was also affected, in an in vitro study, by the addition of hemolysate.