| Project/Area Number |
10672189
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Laboratory medicine
|
|---|
| Research Institution | Fukui University of Technology |
|---|
Principal Investigator |
NISHINA Toshihiro Fukui University of Technology, professor, 工学部, 教授 (20101438)
|
|---|
| Project Period (FY) |
1998 – 1999
|
| Project Status |
Completed (Fiscal Year 1999)
|
| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1999: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1998: ¥2,600,000 (Direct Cost: ¥2,600,000)
|
|---|
| Keywords | enzymatic cycling / lactate oxidase / lactate dehydrogenase / NADH / Thio-NAD / ketone bodies / 乳酸オキシダーゼ / 酵素サイクハング法 / 血牛乳酸 / 血中アセトン体 |
|---|
| 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.
|
