Research on the development of highly sensetive methods for trace metals in biological fluids

生物体液中痕量金属高灵敏检测方法开发研究

• 批准号: 05671923
• 负责人: TABATA Masayoshi
• 金额: $ 1.34万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05671923/
• 关键词: Enzymatic analysis; Biological fluids; Calcium; Zinc; Phospholipase D; Choline oxidase; Alkaline phosphatase; Chemiluminescence; 亜鉛; アルコール脱水素酵素

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.

对生物体液中Ca^2+的酶法测定进行了研究.其原理是利用胆碱氧化酶和过氧化物酶测定Ca^<2+>激活的磷脂酶D的活性。我发现，虽然来自色褐链霉菌的磷脂酶D在非常窄的Ca浓度范围内被激活，但当向反应混合物中加入除Ca^2+之外的二价金属离子时，激活磷脂酶D的Ca^2+浓度范围扩大了。在二价以上的金属离子中，Mn^<2+>表现出最好的效率，在含Mn^<2+>的反应混合物中，磷脂酶D的反应速率与20 mM Ca^<2 +>的浓度呈线性增加。Ca测定不受其它离子如Mg^<2+>的影响。本法测定血清钙的结果与原子吸收光谱法测定结果一致。采用磷脂酶D和胆碱氧化酶组成的生物反应器，用化学发光法对钙进行了高灵敏度的分析。用乙醇脱氢酶（ALDH）和碱性磷酸酶（ALP）等锌酶作酶法测定锌。虽然将Zn溶液添加到去除Zn的apo-ALDH中，但其不再显示活性。螯合剂去除Zn后的apo-ALP活性随Zn浓度的增加而增加。嗜热菌蛋白酶分子中含有1摩尔Zn，其Zn容易被螯合剂除去。但由于嗜热菌蛋白酶是一种蛋白酶，其活性测定比较困难。

项目成果

Masayoshi, Tabata: "High molecular reagents for diagnosis." Polymer Preprints, Japan. 43. 2546-2547 (1994)

Masayoshi, Tabata：“用于诊断的高分子试剂。”

Masayoshi, Tabata: "Chemiluminometry and its application to the bioreactor for diagnosis." Japanese Journal of Clinical Chemistry. 22. 71a-75a (1993)

Masayoshi，Tabata：“化学发光法及其在生物反应器诊断中的应用。”

Masayoshi, Tabata: "Highly sensitive flow injection analysis for serum 3-hydroxy-butyrate using the immobilized enzyme and chemiluminescence." The Japanese Journal of Clinical Pathology. Supplement, 42. 156 (1994)

Masayoshi，Tabata：“使用固定化酶和化学发光对血清 3-羟基丁酸盐进行高灵敏度流动注射分析。”

Masayoshi Tabata: "Use of a bioreactor consisting of an immobilized NADH oxidase column and a hydrogen peroxide electrode for the determination of serum lactate dehydrogenase activity" Analytica Chimica Acta. 298. 113-119 (1994)

Masayoshi Tabata：“使用由固定化 NADH 氧化酶柱和过氧化氢电极组成的生物反应器测定血清乳酸脱氢酶活性”Analytica Chimica Acta。

Masayoshi, Tabata: "Use of a bioreactor consisting of an immobilized NADH oxidase column and a hydrogen peroxide electrode for the determination of serum lactate dehydrogenase activity." Analytica Chimica Acta. 298. 113-119 (1994)

Masayoshi, Tabata：“使用由固定化 NADH 氧化酶柱和过氧化氢电极组成的生物反应器来测定血清乳酸脱氢酶活性。”