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Assay of Blood and Tissue Oxaloacetate and α-Ketoglutarate by Isotope Dilution Gas Chromatography-Mass Spectrometry

Abstract : 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.
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A. Laplante, Blandine Comte, C. Desrosiers. Assay of Blood and Tissue Oxaloacetate and α-Ketoglutarate by Isotope Dilution Gas Chromatography-Mass Spectrometry. Analytical Biochemistry, Elsevier Masson, 1995, 224 (2), pp.580-587. ⟨10.1006/abio.1995.1090⟩. ⟨hal-03324592⟩



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