1994 Fiscal Year Final Research Report Summary
Research on the development of highly sensetive methods for trace metals in biological fluids
| Project/Area Number |
05671923
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Laboratory medicine
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
TABATA Masayoshi Kyoto University, College of Medical Technology, 医療技術短期大学部, 助教授 (70115880)
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| Project Period (FY) |
1993 – 1994
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| Keywords | Enzymatic analysis / Biological fluids / Calcium / Zinc / Phospholipase D / Choline oxidase / Alkaline phosphatase / Chemiluminescence |
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| Research Abstract |
The development of the enzymatic method for determining Ca^<2+> in biological fluids was trid. The assay principle was based on determining the activity of phospholipase D activated by Ca^<2+> using choline oxidase and peroxidase. I found that though phospholipase D from Streptomyces chromofuscus was activated in the very narrow range of the Ca concentration, the range of the Ca^<2+> concentration activating phospholipase D expanded, when a divalent metal ion except Ca^<2+> was added to the reaction mixture. Mn^<2+> among the metal ions above divalent showed the best efficiency, and the reaction rate of phospholipase D increased linearly with the concentration of 20 mM Ca^<2+> in the reaction mixture containing Mn^<2+>. The Ca assay was not influenced with other ions, such as Mg^<2+>. The results of serum Ca by the present method correlated with those by the atomic absorption spectrometry. The highly sensitive analysis of Ca was tried using chemiluminescence and the bioreactor consisting of phospholipase D and choline oxidase. It detected 10 pmol H_2O_2 per muL sample.
The enzymatic method for determining Zn wa tried using the Zn-enzyme such as alcohol dehydrogenase (ALDH) and alkaline phosphatase (ALP) . Though a Zn solution was added to the apo-ALDH removed Zn, it did not show the activity again. The activity of the apo-ALP removed Zn by a chelate agent increased with the concentration of Zn added. Thermolysin had 1 mol Zn in the molecule, and its Zn was easily removed by a chelate agent. But because thermolysin was a kind of protease, the determination of its activity was difficult.
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