Laplante A, Comte B, Des Rosiers C
Department of Nutrition, University of Montreal, Quebec, Canada.
Anal Biochem. 1995 Jan 20;224(2):580-7. doi: 10.1006/abio.1995.1090.
The assay of oxaloacetate and alpha-ketoglutarate in biological samples is complicated by their chemical instability and low concentrations. We present a quantitative assay for physiological concentrations of these metabolites by isotope dilution gas chromatography-mass spectrometry. Samples are spiked with the corresponding internal standards of [U-13C4]oxaloacetate and [U-13C5] alpha-ketoglutarate prior to their treatment with hydroxylamine. After ethyl acetate extraction and evaporation of the organic phases, the oximes are converted to t-butyldimethylsilyl ethers and analyzed by selected ion monitoring gas chromatography-mass spectrometry of the [M-57]+ ion in electron impact. Although the internal standards of [U-13C4]oxaloacetate and [U-13C5] alpha-ketoglutarate are not commercially available, they can easily be synthesized in 30 min by reacting [1,2,3,6-13C4]citrate with citrate lyase, and L-[U-13C5]glutamate with pyruvate and glutamate-pyruvate transaminase, respectively. Because of their chemical instability, the internal standards are prepared on the day of the analysis. A stock solution of [1,2,3,6-13C4]citrate is prepared from L-[U-13C4]aspartate using citrate synthase and glutamate-oxaloacetate transaminase and then purified and kept frozen until required. The detection limit of the method is 0.05 nmol in a given sample. The method was applied to measurements of oxaloacetate and alpha-ketoglutarate in human blood and rat liver.
生物样品中草酰乙酸和α-酮戊二酸的测定因它们的化学不稳定性和低浓度而变得复杂。我们通过同位素稀释气相色谱-质谱法提出了一种用于这些代谢物生理浓度的定量测定方法。在用羟胺处理样品之前，将相应的[U-13C4]草酰乙酸和[U-13C5]α-酮戊二酸内标加入样品中。经过乙酸乙酯萃取和有机相蒸发后，肟转化为叔丁基二甲基甲硅烷基醚，并通过电子轰击下[M-57]+离子的选择离子监测气相色谱-质谱法进行分析。虽然[U-13C4]草酰乙酸和[U-13C5]α-酮戊二酸的内标没有商业供应，但它们可以分别通过使[1,2,3,6-13C4]柠檬酸盐与柠檬酸裂解酶反应，以及L-[U-13C5]谷氨酸盐与丙酮酸和谷氨酸-丙酮酸转氨酶反应，在30分钟内轻松合成。由于它们的化学不稳定性，内标在分析当天制备。[1,2,3,6-13C4]柠檬酸盐的储备溶液由L-[U-13C4]天冬氨酸使用柠檬酸合酶和谷氨酸-草酰乙酸转氨酶制备，然后纯化并冷冻保存直至需要。该方法在给定样品中的检测限为0.05 nmol。该方法应用于人体血液和大鼠肝脏中草酰乙酸和α-酮戊二酸的测量。
