Probing the origin of acetyl-CoA and oxaloacetate entering the citric acid cycle from the C-13 labeling of citrate released by perfused rat hearts

We present a strategy for simultaneous assessment of the relative contributions of anaplerotic pyruvate carboxylation, pyruvate decarboxylation, and fatty acid oxidation to citrate formation in the perfused rat heart, This requires perfusing with a mix of C-13-substrates and determining the C-13 labeling pattern of a single metabolite, citrate, by gas chromatography-mass spectrometry, The mass isotopomer distributions of the oxaloacetate and acetyl moieties of citrate allow calculation of the flux ratios: (pyruvate carboxylation)/(pyruvate decarboxylation), (pyruvate carboxylation)/(citrate synthesis), (pyruvate decarboxylation)/(citrate synthesis) (pyruvate carboxylation)/(fatty acid oxidation), and (pyruvate decarboxylation)/(fatty acid oxidation), Calculations, based on precursor-product relationship, are independent of pool size, The utility of our method was demonstrated for hearts perfused under normoxia with [U-C-13(3)](lactate + pyruvate) and [1-C-13]octanoate under steady-state conditions, Under these conditions, effluent and tissue citrate were similarly enriched in all C-13 mass isotopomers, The use of effluent citrate instead of tissue citrate allows probing substrate fluxes through the various reactions non-invasively in the intact heart, The methodology should also be applicable to hearts perfused with other C-13-substrates, such as 1-C-13-labeled long chain fatty acid, and under various conditions, provided that assumptions on which equations are developed are valid.