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 13 C NMR spectroscopy was used to non-invasively observe label turnover of [4- 13 In summary, It is possible to detect 13 C labeling of glutamate and glutamine in liver via non-invasive 13 C NMR. Additionally, the in vivo 13 C labeling kinetics of glutamate and glutamine in liver and glutamine in blood may be used to calculate the liver tricarboxylic acid cycle flux.In liver, the tricarboxylic acid cycle is not only coupled to mitochondrial energy production but also provides substrates for gluconeogenesis and other metabolic pathways. Therefore, in order to quantify the tricarboxylic acid cycle flux from the labeling kinetics of glutamate and glutamine in vivo, the influx/ efflux of labeled substrates coupled to the tricarboxylic acid cycle must be included in the mathematical analysis.Tricarboxylic acid cycle flux measurements in hepatocyte preparations have previously been made using a variety of substrates (1), and more recently measurements of tricarboxylic acid cycle activity in perfused liver have been made using an isotopomer steady state analysis (2, 3). Magnusson et al. (4) have measured relative metabolic fluxes including the tricarboxylic acid cycle flux in human liver via a steady state isotopomer analysis of glutamine which conjugates to phenylacetate in liver and is excreted in the urine. Although these in vivo methods provide relative flux information on a number of metabolic pathways in liver, they lack the ability to calculate absolute fluxes unless the absolute flux of one pathway is known. This is possible in perfused systems but may be difficult in the whole body.In vivo kinetic measurements of tricarboxylic acid cycle activity have been made non-invasively in brain (5-7) and in perfused heart (8 -11) and liver (12) by measuring turnover of 13 C label in glutamate by NMR, and absolute tricarboxylic acid cycle fluxes have been calculated from these measurements in the brain (5, 13) and heart (8 -11) using a variety of mathematical models. In kinetic experiments of glutamate isotope labeling in brain, label dilution of the brain glutamate/glutamine pool via exchange with the blood glutamate/glutamine pool is limited by the blood-brain barrier (14). Therefore, the tricarboxylic acid cycle flux has been calculated from the brain [4-13 C]glutamate/glutamine turnover data using a linear metabolic pathway analysis (5, 13) in which to a first approximation the exchange with blood metabolite pools is neglected. However in liver, glutamine is in active exchange with blood causing label dilution of the liver glutamate and glutamine pool (15). Thus in order to calculate the tricarboxylic acid cycle flux in the liver using in vivo NMR measurements of 13 C label turnover in glutamate and glutamine, the mathematical analysis must incorporate labeled liver glutamine exchange with blood. [2-13 C]Ethanol was used as...

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