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1999 Fiscal Year Final Research Report Summary

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

Research Project

Project/Area Number 10672189
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Laboratory medicine
Research InstitutionFukui University of Technology

Principal Investigator

NISHINA Toshihiro  Fukui University of Technology, professor, 工学部, 教授 (20101438)

Project Period (FY) 1998 – 1999
Keywordsenzymatic cycling / lactate oxidase / lactate dehydrogenase / NADH / Thio-NAD / ketone bodies / 乳酸オキシダーゼ
Research Abstract

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.

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Published: 2001-10-23  

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