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Volume 272, Number 42, Issue of October 17, 1997 pp. 26117-26124
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Probing the Origin of Acetyl-CoA and Oxaloacetate Entering the Citric Acid Cycle from the ¹³C Labeling of Citrate Released by Perfused Rat Hearts

(Received for publication, February 13, 1997, and in revised form, August 8, 1997)

Blandine Comte , Geneviève Vincent ^§ , Bertrand Bouchard and Christine Des Rosiers ^§

From the Departments of  Nutrition and ^§ Biochemistry, University of Montréal, Montréal, Québec H3C 3J7, Canada

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 ¹³C-substrates and determining the ¹³C 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-¹³C₃](lactate + pyruvate) and [1-¹³C]octanoate under steady-state conditions. Under these conditions, effluent and tissue citrate were similarly enriched in all ¹³C 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 ¹³C-substrates, such as 1-¹³C-labeled long chain fatty acid, and under various conditions, provided that assumptions on which equations are developed are valid.

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