Abstract
We developed a new and highly sensitive method for determining 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 the decrease of absorbance at 340nm of NADH oxidized at 37° during the reaction. The reagents for the present method were designed for use on automated clinical analyzers. The lactate and pyruvate concentration was calculated by comparison with the reaction rate obtained with 1 Ommol/l of standard solution. The linearity ranged from 1 to 20 mmol/l. The blood or the serum quantity per test was equivalent to 0.16μl, and absorbance for a lmmol/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 (300mg/l), hemoglobin (5g/l) or ascorbate (1g/l). The coefficient of the correlation with the 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.
