S-3-羟基异丁酸的分光光度酶法测定

Spectrophotometric enzymatic assay for S-3-hydroxyisobutyrate.

作者信息

Rougraff P M, Paxton R, Goodwin G W, Gibson R G, Harris R A

机构信息

Department of Biochemistry, Indiana University School of Medicine, Indianapolis 46202-5122.

出版信息

Anal Biochem. 1990 Feb 1;184(2):317-20. doi: 10.1016/0003-2697(90)90687-5.

DOI:10.1016/0003-2697(90)90687-5

PMID:2183647

Abstract

An enzymatic spectrophotometric end-point assay has been developed for determination of S-3-hydroxyisobutyrate in biological fluids. The assay measures NADH production at 340 nm after initiation of the reaction with rabbit liver 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31). The assay is not affected by R-3-hydroxyisobutyrate, lactate, malate, 3-hydroxybutyrate, 2-methyl-3-hydroxybutyrate, 3-hydroxyisovalerate, 3-hydroxy-n-valerate, 2-methyl-3-hydroxy-valerate, and 3-hydroxypropionate. The assay does measure 2-ethyl-3-hydroxypropionate, a minor metabolite produced by catabolism of alloisoleucine. Application of the method to measure S-3-hydroxyisobutyrate in plasma obtained from normal, 48-h starved, and mildly and severely diabetic rats gave levels of 28, 42, 112, and 155 microM, respectively.

摘要

已开发出一种酶促分光光度终点分析法，用于测定生物体液中的S-3-羟基异丁酸。该分析法在与兔肝3-羟基异丁酸脱氢酶（EC 1.1.1.31）启动反应后，测量340 nm处的NADH生成量。该分析法不受R-3-羟基异丁酸、乳酸、苹果酸、3-羟基丁酸、2-甲基-3-羟基丁酸、3-羟基异戊酸、3-羟基-n-戊酸、2-甲基-3-羟基戊酸和3-羟基丙酸的影响。该分析法确实能检测2-乙基-3-羟基丙酸，这是别异亮氨酸分解代谢产生的一种次要代谢产物。将该方法应用于测量正常、饥饿48小时、轻度和重度糖尿病大鼠血浆中的S-3-羟基异丁酸，其水平分别为28、42、112和155 microM。