In vivo 13C NMR measurements of hepatocellular tricarboxylic acid cycle flux

Abstract

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 MR spectroscopy was used to non-invasively observe label turnover of [4-13C]glutamate, [413C]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 x citrate synthase flux) and [2-13C]acetylCoA fractional enrichment (51.4 ± 3.4%). The [4-1SC]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(tcm) = 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 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.