Studies on micromethod for the determination of blood ingredents by enzymatic cycling

利用乳酸脱氢酶(LD)和乳酸氧化酶(LOX)的酶促循环反应，建立了一种高灵敏度测定乳酸和丙酮酸的新方法。该方法采用了一种新的NADH、LD和LOX的酶循环技术，测量了在37℃时氧化的NADH在340 nm处的吸光度下降。本方法的试剂设计用于自动化临床分析。与10 mmol/L标准溶液的反应速率进行比较，计算乳酸和丙酮酸的浓度。线性范围为1~20 mm ol/L，每次测定的血量或血液量相当于0.16min的L，1μ/L标准品的吸光度为0.0045/m in。批内精密度(n=10)CV值分别为2.3%(2.51 mmol/L)和1.4%(7.28 mmol/L)，不受胆红素(300 mg/L)、血红蛋白(5g/L)和抗坏血酸(1g/L)的影响。与LOX-POD-TOOS法的相关系数为0.987(n=93)。建立了简便、高灵敏的乳酸和丙酮酸的动力学测定方法，受干扰因素影响较小。另一方面，通过3-羟基丁酸脱氢酶(3-HBAD)和硫代-NAD的酶促环化反应，建立了测定酮体(乙酰乙酸酯和3-羟基丁酸)的高灵敏方法。该方法测量了在37℃时扣除的硫代-NADH在405 nm处的吸光度增加。

We developed a new and highly sensitive method for determination of lactate and pyruvate by the enzymatic cycling reaction with lactate dehydrogenase (LD) and lactate oxidase (LOX). The method involves a new enzymatic cycling technique with NADH, LD and LOX, and measures decrease of absorbance at 340nm of NADH oxidized at 37℃ during the reaction. The reagents for present method were designed to be used automated clinical analyzers. Lactate and pyruvate concentration was calculated by comparison with the reaction rate obtained with 10mmol/L of standard solution. The linearity ranged from 1 to 20mmol/L. The blood or the serum quantity per test are equivalent to 0.16μL, and absorbance for 1mmol/L standard was 0.0045/min. The CV values in within-run precision (n=10) were 2.3% (2.51mmol/L) and 1.4% (7.28mmol\L).This method was not affected by bilirubin (300 mg/L), hemoglobin (5g/L) and ascorbate (1g/L). The coefficient of correlation with LOX-POD-TOOS method was 0.987 (n=93). Thus the simple and highly sensitive kinetic assay method of lactate and pyruvate, less affected by interferents, was established.On the other hand, we developed a highly sensitive method for determination of ketone bodies (acetoacetate and 3-hydroxybutylate) by the enzymatic cycling reaction with 3-hydroxy butyrate dehydrogenase (3-HBAD) and Thio-NAD. The method measures increase of absorbance at 405nm of Thio-NADH deducted at 37℃ during the reaction.