PROBING THE ORIGIN OF ACETYL-COA AND OXALOACETATE ENTERING THE CITRIC-ACID CYCLE FROM THE C-13 LABELING OF CITRATE RELEASED BY PERFUSED RATHEARTS

We present a strategy for simultaneous assessment of the relative cont ributions of anaplerotic pyruvate carboxylation, pyruvate decarboxylat ion, and fatty acid oxidation to citrate formation in the perfused rat heart, This requires perfusing with a mix of C-13-substrates and dete rmining the C-13 labeling pattern of a single metabolite, citrate, by gas chromatography-mass spectrometry, The mass isotopomer distribution s of the oxaloacetate and acetyl moieties of citrate allow calculation of the flux ratios: (pyruvate carboxylation)/(pyruvate decarboxylatio n), (pyruvate carboxylation)/(citrate synthesis), (pyruvate decarboxyl ation)/(citrate synthesis) (pyruvate carboxylation)/(fatty acid oxidat ion), and (pyruvate decarboxylation)/(fatty acid oxidation), Calculati ons, based on precursor-product relationship, are independent of pool size, The utility of our method was demonstrated for hearts perfused u nder normoxia with [U-C-13(3)](lactate + pyruvate) and [1-C-13]octanoa te 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 s ubstrate fluxes through the various reactions non-invasively in the in tact heart, The methodology should also be applicable to hearts perfus ed with other C-13-substrates, such as 1-C-13-labeled long chain fatty acid, and under various conditions, provided that assumptions on whic h equations are developed are valid.