CALCULATION OF ABSOLUTE METABOLIC FLUX AND THE ELUCIDATION OF THE PATHWAYS OF GLUTAMATE LABELING IN PERFUSED RAT-HEART BY C-13 NMR-SPECTROSCOPY AND NONLINEAR LEAST-SQUARES ANALYSIS

Absolute metabolic fluxes in isolated perfused hearts have been determ ined by a nonlinear least squares analysis of glutamate labeling kinet ics from [1-C-13]glucose, [4-C-13]beta-hydroxybutyrate, or [2-C-13]ace tate using C-13 NMR spectroscopy. With glucose as substrate, the malat e-aspartate shuttle flux was too slow to account for the reducing equi valents generated by glycolysis and to predict the observed oxygen con sumption rate. For acetate and beta-hydroxybutyrate, the malate-aspart ate shuttle had to be reversed for the network to agree with the obser ved oxygen consumption and glutamate labeling. Thus, an additional red ox shuttle was required to reoxidize the NADH produced by cytoplasmic malate dehydrogenase. Using this model there was good agreement betwee n the experimentally determined oxygen consumption and glutamate label ing and the calculated values of these parameters from the model for a ll substrates. The contribution of exogenous substrate to the overall tricarboxylic acid (TCA) cycle flux, 89.6 +/- 6.5% (mean +/- S.D.) as measured in the tissue extracts compared well with 91.4 +/- 4.2% calcu lated by the model. The ratio of TCA cycle flux to oxygen consumption for acetate, was 2.2 +/- 0.1, indicating that NADH production is princ ipally accounted for by TCA cycle flux. For glucose or beta-hydroxybut yrate, this ratio was 2.9 +/- 0.2, consistent with the existence of ot her NADH producing reactions (e.g. glycolysis, beta-hydroxybutyrate ox idation).