ASSAY OF BLOOD AND TISSUE OXALOACETATE AND ALPHA-KETOGLUTARATE BY ISOTOPE-DILUTION GAS-CHROMATOGRAPHY MASS-SPECTROMETRY

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-C-13(4)]oxaloacetate and [U-C-13(5)]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-C-13(4)]oxaloacetate and [U-C-13(5)]alpha-ketoglutarate are not commercially available, they can easily be synthesized in 30 min by reacting [1,2,3,6-C-13(4)]citrate with citrate lyase, and L-[U-C-13(5)]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-C-13(4)]citrate is prepared from L-[U-C-13(4)]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. (C) 1995 Academic Press, Inc.