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-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.
Laplante
A
A
Department of Nutrition, University of Montreal, Quebec, Canada.
Comte
B
B
Des Rosiers
C
C
D000818:Animals; D001224:Aspartic Acid; D002247:Carbon Isotopes; D002845:Chromatography; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D018698:Glutamic Acid; D007553:Isotope Labeling; D007656:Ketoglutaric Acids / Q000032:analysis* / Q000097:blood; D008099:Liver / Q000737:chemistry; D008297:Male; D010071:Oxaloacetates / Q000032:analysis* / Q000097:blood; D051381:Rats; D017207:Rats, Sprague-Dawley; D012015:Reference Standards
D002247:Carbon Isotopes; D007656:Ketoglutaric Acids; D010071:Oxaloacetates; D001224:Aspartic Acid; D018698:Glutamic Acid