Qureshi A A, Elson C E, Lebeck L A
J Chromatogr. 1982 Nov 19;249(2):333-45. 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.
已描述了18日龄鸡胚肝脏中异柠檬酸裂解酶反应的三种主要α-酮终产物（乙醛酸、丙酮酸、α-酮戊二酸）的分离与鉴定。这是通过高效液相色谱法（HPLC）将这些α-酮酸分离为它们的2,4-二硝基苯腙（DNPHs）来实现的。α-酮酸的DNPHs用含有5 mM磷酸四丁铵的甲醇-水-乙酸（60:38.5:1.5）等度溶剂系统从反相超球C18（IP）柱和径向压缩C18柱上洗脱。与传统反相柱需要30 - 40分钟相比，在径向压缩柱上15 - 20分钟内即可完成分离。对测定样品和参考化合物的保留时间和峰面积进行积分。通过与标准品比较计算峰中α-酮酸的相对含量。每个峰的鉴定基于保留时间匹配、与真实化合物的共色谱以及在240至440 nm之间的停流紫外-可见扫描。乙醛酸占异柠檬酸裂解酶反应总产物的5%。第18天与胚胎肝脏糖原生成的高峰期平行，此时期原始卵中的葡萄糖储备已耗尽。
