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J Biol Chem, Vol. 273, Issue 20, 12187-12194, May 15, 1998

In Vivo ¹³C NMR Measurements of Hepatocellular Tricarboxylic Acid Cycle Flux

Beat M. Jucker^§, Joon Y. Lee, and Robert G. Shulman

From the Departments of  Chemistry and ^§ Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510 and the  Yale University School of Medicine, New Haven, Connecticut 06520

A combined isotopic steady state and in vivo isotopic non-steady state analysis was used to calculate tricarboxylic acid cycle flux in livers of anesthetized rats infused with ethanol. In vivo ¹³C NMR spectroscopy was used to non-invasively observe label turnover of [4-¹³C]glutamate, [4-¹³C]glutamine, and [2-¹³C]glutamate/glutamine in liver following a bolus intravenous infusion of [2-¹³C]ethanol. The isotopic steady state analysis of [2-¹³C], [3-¹³C], and [4-¹³C]glutamate isotopomers (Malloy, C. R., Sherry, A. D., and Jeffrey, F. M. H. (1988) J. Biol. Chem. 263, 6964-6971) in liver extracts was used to indirectly calculate the anaplerotic flux (0.90 ± 0.07 × citrate synthase flux) and [2-¹³C]acetyl-CoA fractional enrichment (51.4 ± 3.4%). The [4-¹³C]glutamate, [4-¹³C]glutamine, and [2-¹³C]glutamate fractional enrichments determined in liver extracts were 23.0 ± 1.1, 17.2 ± 1.5, and 7.7 ± 0.5%, respectively. These data in addition to blood [2-¹³C]acetate and [4-¹³C]glutamine enrichment time course data were used in conjunction with a metabolic steady state mathematical analysis designed to account for liver glutamate and glutamine label dilution as a consequence of glutamine exchange with blood to calculate the tricarboxylic acid (tca) cycle flux (V_tca = 0.33 ± 0.09 µmol/g wet weight/min) in liver.

In summary, It is possible to detect ¹³C labeling of glutamate and glutamine in liver via non-invasive ¹³C NMR. Additionally, the in vivo ¹³C labeling kinetics of glutamate and glutamine in liver and glutamine in blood may be used to calculate the liver tricarboxylic acid cycle flux.

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