Application of high-performance liquid chromatography to the determination of glyoxylate synthesis in chick embryo liver

• Published in: Journal of chromatography Vol. 249; no. 2; pp. 333 - 345
• Main Authors: Qureshi, A.A, Elson, C.E, Lebeck, L.A
• Format: Journal Article
• Language: English
• Published: Netherlands 01.01.1982
• Subjects: animal physiology; Animals; Chick Embryo; Chromatography, High Pressure Liquid; Glyoxylates - biosynthesis; Glyoxylates - isolation & purification; Isocitrate Lyase - metabolism; Ketoglutaric Acids - isolation & purification; Liver - embryology; Liver - metabolism; Phenylhydrazines; Pyruvates - isolation & purification; Spectrophotometry
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(00)86343-8

The isolation and identification of three major alpha-keto end products (glyoxylate, pyruvate, alpha-ketoglutarate) of the isocitrate lyase reaction in 18-day chick embryo liver have been described. This was accomplished by the separation of these alpha-keto acids as their 2,4-dinitrophenylhydrazones (DNPHs) by high-performance liquid chromatography (HPLC). The DNPHs of alpha-keto acids were eluted with an isocratic solvent system of methanol-water-acetic acid (60:38.5:1.5) containing 5 mM tetrabutylammonium phosphate from a reversed-phase ultrasphere C18 (IP) and from a radial compression C18 column. The separation can be completed on the radial compression column within 15-20 min as compared to 30-40 min with a conventional reversed-phase column. Retention times and peak areas were integrated for both the assay samples and reference compounds. A relative measure of alpha-keto acid in the peak was calculated by comparison with the standard. The identification of each peak was done on the basis of retention time matching, co-chromatography with authentic compounds, and stopped flow UV-VIS scanning between 240 and 440 nm. Glyoxylate represented 5% of the total product of the isocitrate lyase reaction. Day 18 parallels the peak period of embryonic hepatic glycogenesis which occurs at a time when the original egg glucose reserve has been depleted.