Tabata M, Totani M
College of Medical Technology, Kyoto University, Japan.
Anal Biochem. 1995 Jul 20;229(1):133-8. doi: 10.1006/abio.1995.1389.
We describe a simple method for the highly sensitive chemiluminescence--flow injection analysis of 3-hydroxybutyrate in serum using a bioreactor column consisting of the two immobilized enzymes, 3-hydroxybutyrate dehydrogenase and NADH oxidase. The method was based on measuring the level of chemiluminescence formed by the reaction of a luminol-hexacyanoferrate mixture with hydrogen peroxide. The hydrogen peroxide was produced by the NADH oxidase reaction from NADH which was formed in the conversion of 3-hydroxybutyrate to acetoacetate by the 3-hydroxybutyrate dehydrogenase reaction. Among three immobilized enzyme columns, a coimmobilized, small 3-hydroxybutyrate dehydrogenase/NADH oxidase bioreactor alone (2 x 20 mm i.d.) readily hydrolyzed all of the injected 3-hydroxybutyrate into acetoacetate, although 3-hydroxybutyrate dehydrogenase catalyzed the reversible reaction. The present method generated linearity of the data up to 1.5 mM 3-hydroxybutyrate with satisfactory precision, reproducibility, and accurate reaction recoveries. The results from 3-hydroxybutyrate correlated satisfactorily with those obtained by other well-established methods. The coimmobilized 3-hydroxybutyrate dehydrogenase/NADH oxidase reactor unit showed good operational stability over a 5-week period, during which it was repeatedly used for 1500 analyses.
我们描述了一种使用由两种固定化酶（3-羟基丁酸脱氢酶和NADH氧化酶）组成的生物反应器柱对血清中3-羟基丁酸进行高灵敏度化学发光-流动注射分析的简单方法。该方法基于测量鲁米诺-六氰合铁酸盐混合物与过氧化氢反应产生的化学发光水平。过氧化氢是由NADH氧化酶反应从NADH产生的，而NADH是在3-羟基丁酸脱氢酶反应将3-羟基丁酸转化为乙酰乙酸的过程中形成的。在三种固定化酶柱中，单独的共固定化小型3-羟基丁酸脱氢酶/NADH氧化酶生物反应器（内径2×20mm）很容易将所有注入的3-羟基丁酸水解为乙酰乙酸，尽管3-羟基丁酸脱氢酶催化的是可逆反应。本方法在3-羟基丁酸浓度高达1.5 mM时数据呈线性，具有令人满意的精密度、重现性和准确的反应回收率。3-羟基丁酸的测定结果与其他成熟方法获得的结果具有良好的相关性。共固定化的3-羟基丁酸脱氢酶/NADH氧化酶反应器单元在5周内显示出良好的操作稳定性，在此期间它被重复用于1500次分析。
