| Budget Amount *help |
¥7,600,000 (Direct Cost: ¥7,600,000)
Fiscal Year 1985: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1984: ¥4,900,000 (Direct Cost: ¥4,900,000)
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| Research Abstract |
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
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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
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