Nuwayhid N F, Johnson G F, Feld R D
Department of Pathology, College of Medicine, University of Iowa, Iowa City 52242.
Clin Chem. 1989 Jul;35(7):1526-31.
This is a multipoint kinetic method for simultaneously determining acetoacetate (AcAc) plus beta-hydroxybutyrate and lactate plus pyruvate in a single cuvette of the Multistat III centrifugal analyzer. In the first step, AcAc and pyruvate are completely reduced, using 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) and lactate dehydrogenase (EC 1.1.1.27) in the presence of excess NADH at pH 7.5, to beta-hydroxybutyrate and lactate, respectively. After dilution, the endogenous beta-hydroxybutyrate and lactate and that resulting from reduction are simultaneously oxidized by their respective dehydrogenases in the presence of excess NAD+ at pH 9.0. Adjustment of the relative enzyme concentrations allows simultaneous estimation of AcAc plus beta-hydroxybutyrate and lactate plus pyruvate by analyzing multipoint absorbance data, collected during the oxidation reaction, with use of a two-component linear-regression model. Total run-to-run CVs were 6.4% and 6.1% at 5 mmol/L beta-hydroxybutyrate and 5 mmol/L lactate, respectively. The method was designed to be useful for identifying the cause of an increased anion gap in serum.
这是一种多点动力学方法，用于在Multistat III离心分析仪的单个比色皿中同时测定乙酰乙酸（AcAc）加β-羟基丁酸以及乳酸加丙酮酸。第一步，在pH 7.5且存在过量NADH的情况下，使用3-羟基丁酸脱氢酶（EC 1.1.1.30）和乳酸脱氢酶（EC 1.1.1.27）将AcAc和丙酮酸分别完全还原为β-羟基丁酸和乳酸。稀释后，内源性β-羟基丁酸和乳酸以及还原产生的β-羟基丁酸和乳酸在pH 9.0且存在过量NAD⁺的情况下被各自的脱氢酶同时氧化。通过调整相对酶浓度，利用在氧化反应过程中收集的多点吸光度数据，采用双组分线性回归模型，可以同时估算AcAc加β-羟基丁酸以及乳酸加丙酮酸。在5 mmol/Lβ-羟基丁酸和5 mmol/L乳酸时，每次运行的总变异系数分别为6.4%和6.1%。该方法旨在用于确定血清中阴离子间隙增加的原因。
