Development of Micro- and Rapid Determination Procedure of <alpha> -keto Acids by High-Performance Liquid Chromatography and Its Application for the Screening of Chronic Acidemia

高效液相色谱微量快速测定α-酮酸方法的建立及其在慢性酸血症筛查中的应用

• 批准号: 59870014
• 负责人: KOIKE Masahiko
• 金额: $ 4.86万
• 依托单位: Nagasaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research
• 财政年份: 1984
• 资助国家: 日本
• 起止时间: 1984 至 1985
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-59870014/
• 关键词: High-performance liquid chromatography; <alpha> -keto acid quinoxaline; Fluorescent Analysis; blood ＆ urine of acidemia; 先天性酸血症; 先天性代謝異常症

A rapid and microquantitative method of analysis of various <alpha> -keto acids in biological samples by high-performance liquid chromatography (HPLC) with a fluorescence detector has been urgently needed for the determination of <alpha> -keto acid in serum and urine. This is especially true for pyruvate and branched-chain <alpha> -keto acids whose concentrations are incleased in patients with congenital metabolic disorders such as the congenital pyruvated dehydrogenase or pyruvate carboxylase defect and mitochondrial myopathy associated with chronic lactic and pyruvic acidemia, and maple syrup urine disease.A. A procedure for rapid separation and microquantitative determination of various <alpha> -keto acids, i. e. <alpha> -ketoglutarate, pyruvate, <alpha> -ketobutyrate, <alpha> -ketoisovalerate , <alpha> ketoisocaproate, <alpha> -keto- <beta> -methylvalerate etc., in serum and urine has been developed. The procedure was performed by using the reverse-phase HPLC of <alpha> -keto acids … More after derivatization into strong fluorescent quinoxalines by condensation reaction with ophenylenediamine. An aqueous solution of 60% methanol was used for isocratic elution and passed through the column at a flow rate of 1 ml/min for 25 min. The column was maintained at 35゜C. The fluorescence emission was measured at 410 nm with exitation at 350 nm.B. Deproteinization of serum with tungstic acid or methanol showed good recoveries of <alpha> -keto acids and minimized the deterioration of the column. According to this procedure, each less than 0.1 ml of serum and urine are, only needed for determination of <alpha> -keto acids. Average valves of <alpha> -keto acids in serum and urine are presented. The data obtained from the samples of patients with chronic pyruvic, lactic acidemia and branched-chain <alpha> -keto aciduria.C. A slightly modification are needed for the analysis of oxaloacetate and phenylpyruvate.D. An application in screening of chronic acidosis was performed by using the filter paper method similar with Guthrie Test. From the filter paper over 90% of <alpha> -keto acids were recovered in water exaract without significant deteoriration. Less

在血清和尿液中< α > -酮酸的检测中，迫切需要一种高效液相色谱-荧光检测器快速微量分析生物样品中各种< α > -酮酸的方法。这尤其适用于丙酮酸和支链< α b> -酮酸，其浓度在先天性代谢性疾病（如先天性丙酮酸脱氢酶或丙酮酸羧化酶缺陷、慢性乳酸和丙酮酸血症相关的线粒体肌病和枫糖浆尿病）患者中升高。建立了一种快速分离和微量测定血清和尿液中各种< α > -酮酸的方法，即< α > -酮戊二酸、丙酮酸、< α > -酮丁酸、< α > -酮异戊酸、< α >酮异己酸、< α > -酮- < β > -甲基戊酸等。用反相高效液相色谱法测定了< α > -酮酸与苯二胺缩合反应衍生成强荧光喹诺啉后的产物。60%甲醇水溶液等压洗脱，以1ml /min的流速通过柱25 min。色谱柱保持在35℃。在410 nm处测定荧光发射，在350 nm. b处测定荧光发射。用钨酸或甲醇对血清进行脱蛋白处理，< α > -酮酸回收率高，使色谱柱的退化程度降至最低。根据该程序，只需要测定< α > -酮酸的血清和尿液各小于0.1 ml。血清和尿液中< α b> -酮酸的平均值。数据来自慢性丙酮酸血症、乳酸血症和支链< α > -酮酸血症患者的样本。对草酰乙酸和苯丙酮酸的分析需要稍作修改。采用类似格思里试验的滤纸法对慢性酸中毒进行了筛查。从滤纸中回收了90%以上的< α b> -酮酸，且没有明显的降解。少

项目成果

The Journal of the Japan Pediatric Society. 89-11. (1985)

日本儿科学会杂志。

Seikagaku. 55-8. (1983)

清化学。

The Journal of the Japan Pediatric Society. 89-11. (1985)Proceeding of 2nd Meeting on the Function of the Thiaminepyrophosphate Enzymes.

日本儿科学会杂志。

日本小児科学会雑誌. 89-11. (1985)

日本儿科学会杂志 89-11（1985）。

生化学. 55-8. (1983)

生物化学。55-8。