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

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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.