Application of high-performance liquid chromatography to the determination of glyoxylate synthesis in chick embryo liver.
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.
D000818:Animals; D002642:Chick Embryo; D002851:Chromatography, High Pressure Liquid; D006038:Glyoxylates / Q000096:biosynthesis / Q000302:isolation & purification*; D007522:Isocitrate Lyase / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000302:isolation & purification; D008099:Liver / Q000196:embryology* / Q000378:metabolism; D010659:Phenylhydrazines; D011773:Pyruvates / Q000302:isolation & purification; D013053:Spectrophotometry
D006038:Glyoxylates; D007656:Ketoglutaric Acids; D010659:Phenylhydrazines; D011773:Pyruvates; C004787:2,4-dinitrophenylhydrazine; D007522:Isocitrate Lyase