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

Abstract

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 13 C-substrates and determining the 13 C 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-13 C 3 ](lactate ؉ pyruvate) and [1-13 C]octanoate under steady-state conditions. Under these conditions, effluent and tissue citrate were similarly enriched in all 13 C 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 13 C-substrates, such as 1-13 C-labeled long chain fatty acid, and under various conditions, provided that assumptions on which equations are developed are valid.Tracing of pyruvate metabolism and of other reactions feeding into (anaplerosis) or out (cataplerosis) of the citric acid cycle (CAC) 1 with radioactive or stable isotopes is complicated by label recycling and exchange reactions between CAC intermediates and other metabolites such as aspartate and glutamate. Mathematical models of increasing complexity were developed for the study of the CAC in various organs or tissues (1-12), including the heart (13-17). Solving for flux parameters in equations derived from these models requires measuring the incorporation of 14 C-or 13 C-labeled substrate(s) into various CAC metabolites (8, 9) or the distribution of label between carbons of given molecules such as glutamate (1,(13)(14)(15)(17)(18)(19) or citrate (4, 6). The use of 13 C-enriched labeled substrate(s) and measurements of 13 C labeling of CAC intermediates or related metabolites by nuclear magnetic resonance (NMR) or gas chromatography-mass spectrometry (GCMS) offers several advantages over classical 14 C methods. Also, these two techniques are complementary. One advantage of GCMS over NMR is its sensitivity. Thus, the 13 C labeling of the actual CAC intermediates can be determined.Investigations on the cardioprotective effects of pyruvate have emphasized the concerted regulation of pyruvate decarboxylation and fatty acid oxidation (14 -15, 17, 20 -23). For this purpose, elegant 13 C NMR techniques were developed to quantitate from the 13 C labeling of carbons of glutamate, the relative contributions of pyruvate, fatty acid, and ketone ...

A 13C Mass Isotopomer Study of Anaplerotic Pyruvate Carboxylation in Perfused Rat Hearts

“…When this process is taken into account, the 13 C dilution attributed to entry of unlabeled ␣KG and/or succinate was estimated (from Eq. 10, Comte et al (67)) to be 1.13 Ϯ 0.04 (p Ͻ 0.05, n ϭ 8) and was similarly increased by NE and DCA (1.29 Ϯ 0.03, n ϭ 9; p Ͻ 0.05 unpaired t test).…”

“…Calculations-Equations to calculate the relative contributions of pyruvate and octanoate to citrate formation from the 13 C labeling pattern of citrate released from hearts perfused with [U- 13 C 3 ]lactate, [U- 13 C 3 ]pyruvate, and [1-13 C]octanoate are presented in the accompanying report (67). Note that in the present report, we will refer to these equations using the same numbering.…”

“…GCMS Assays-Procedures for determination of the 13 C-mass isotopomer distribution (MID) of tissue and effluent metabolites (citrate, OAA, and acetyl moieties of citrate, ␣KG, succinate, malate, fumarate, and pyruvate) are described in the accompanying report (67). MID data are expressed as mol fraction (MF) or molar percent enrichment (MPE).…”

“…6 of Ref. 67, not shown). The contribution of pyruvate decarboxylation to acetyl-CoA formation was less than 5% (PDC/CS, Table IIA) and was not significantly modified by 1 mM DCA or 1 M NE or by increasing the octanoate concentration to 1 mM.…”

“…1) Also a minimal estimate of the CAC flux was calculated as the sum of acetyl-CoA production from pyruvate decarboxylation and octanoate oxidation (Equations 5 and 6, Comte et al (67)). Absolute rates of pyruvate carboxylation were similar in all conditions, on average 0.14 Ϯ 0.02 mol/min (n ϭ 14).…”