A sensitive determination of α-keto acids by gas-liquid chromatography and its application to the assay of l-glutamate dehydrogenase and aminotransferases

✍ Scribed by Shinji Ohmori; Yukari Tanaka; Mikiko Ikeda; Kazuhiro Hirota

• Publisher: Elsevier Science
• Year: 1981
• Tongue: English
• Weight: 687 KB
• Volume: 112
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(81)90282-7

✦ Synopsis

A sensitive and selective determination of a-keto acids was established by the use of a gas chromatograph equipped with an electron capture detector. a-Keto acids (pyruvic, oxaloacetic, a-ketobutyric, and a-ketoglutaric acids) were reacted with pentafluorophenylhydrazine, and the derivatives were extracted with ethyl ether, reacted with diazomethane, and were subjected to gas-liquid chromatography with an electron capture detector. In the course of the reaction, oxaloacetic acid was decarboxylated, and yielded pyruvic acid. In the case of pyruvic (oxaloacetic) and a-ketobutyric acids two peaks corresponding to the syn and anti forms of the hydrazone appeared, and in the case of a-ketoglutaric acid, two peaks corresponding to the hydrazone and the cyclization compound produced from the hydrazone. The sum of the two peaks was taken for the determination. The present method was applicable to the assay of L-glutamate dehydrogenase, aspartate : 2-oxoglutarate, and L-alanine : 2-oxoglutarate aminotransferases.

CHEMICALS AND INSTRUMENTS

PFPH

was purchased from Aldrich Chemical Company.

GLDH, GOT, and GPT were obtained from Boehringer-Mannheim GmbH. PA, OAA, a-KBA, CX-KGA, and other reagents (all analytical grade) were purchased from Wako Pure Chemicals Ind. Ltd. (Osaka). 3-Methyl-lphenyl-5-pyrazolone and ethyl 5-0x0-1phenyl-2-pyrazoline-3-carboxylate were