Nakamura H, Lee Y
Clin Chim Acta. 1977 Sep 1;79(2):411-7. doi: 10.1016/0009-8981(77)90437-5.
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 from the initial oxidation velocity of total bilirubin. The Michaelis constant, KM, was approximately 20 micrometer. 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 degrees C. The bilirubin-albumin affinity was studied with purified albumin and adult serum. The dissociation constants were 2 x 10(-8) M and 5 x 10(-9) M, respectively, at bilirubin/albuminor 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.
本文描述了一种用于微量测定新生儿黄疸血清中未结合胆红素的酶测定方法。在葡萄糖氧化酶的介导下，葡萄糖产生的过氧化氢存在时，过氧化物酶将未结合胆红素氧化为无色化合物。与使用过氧化氢的方法相比，在该方法中，在加入过氧化物酶之前胆红素不会显著降解。氧化速率通过分光光度法测定，无需氯仿萃取。未结合胆红素浓度可根据总胆红素的初始氧化速度来确定。米氏常数KM约为20微摩尔。黄疸血清测定的变异系数为4.4% - 6.5%。在-20℃储存五天后，未结合胆红素浓度降低。用纯化白蛋白和成人血清研究了胆红素 - 白蛋白亲和力。在胆红素/白蛋白摩尔比低于1.0时，解离常数分别为2×10⁻⁸M和5×10⁻⁹M。临床上，分析了75例黄疸新生儿的血清样本，发现早产儿血清中的未结合胆红素水平明显高于足月儿。恒河猴免疫婴儿和ABO血型不合婴儿的血清明显高于非溶血性黄疸血清。在体外研究中，加入溶血产物也会影响未结合胆红素浓度。
