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

1997; Elsevier BV; Volume: 272; Issue: 42 Linguagem: Inglês

10.1074/jbc.272.42.26117

ISSN

1083-351X

Autores

Blandine Comte, Geneviève Vincent, Bertrand Bouchard, Christine Des Rosiers,

Tópico(s)

Mitochondrial Function and Pathology

Resumo

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 of13C-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. 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 of13C-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. Tracing of pyruvate metabolism and of other reactions feeding into (anaplerosis) or out (cataplerosis) of the citric acid cycle (CAC) 1The abbreviations used are: CAC, citric acid cycle; αKG, α-ketoglutarate; CS, citrate synthase; GCMS, gas chromatography-mass spectrometry; MID, mass isotopomer distribution; MPE, molar percent enrichment; M i, mass isotopomers withi atoms of 13C; MF, mol fraction; OAA, oxaloacetate; PC, pyruvate carboxylation; PDC, pyruvate decarboxylation. 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 (1Magnusson I. Schumann W.C. Bartsch G.E. Chandramouli V. Kumaran K. Wahren J. Landau B.R. J. Biol. Chem. 1991; 266: 6975-6984Abstract Full Text PDF PubMed Google Scholar, 2Lee W.-N.P. J. Biol. 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Blum J.J. Biochem. J. 1989; 258: 121-140Crossref PubMed Scopus (35) Google Scholar, 9Heath D.F. Rose J.G. Biochem. J. 1985; 227: 851-867Crossref PubMed Scopus (14) Google Scholar) or the distribution of label between carbons of given molecules such as glutamate (1Magnusson I. Schumann W.C. Bartsch G.E. Chandramouli V. Kumaran K. Wahren J. Landau B.R. J. Biol. Chem. 1991; 266: 6975-6984Abstract Full Text PDF PubMed Google Scholar, 13Weiss R.G. Gloth S.T. Kalil-Filho R. Chacko V.P. Stern M.D. Gerstenblith G. Circ. Res. 1992; 70: 392-408Crossref PubMed Scopus (61) Google Scholar, 14Chance E.M. Seeholzer S.H. Kobayashi K. Williamson J.R. J. Biol. Chem. 1983; 258: 13785-13794Abstract Full Text PDF PubMed Google Scholar, 15Chatham J.C. Forder J.R. Glickson J.D. Chance E.M. J. Biol. Chem. 1995; 270: 7999-8008Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar,17Malloy C.R. Sherry A.D. Jeffrey F.M.H. Am. J. Physiol. 1990; 259: H987-H995Crossref PubMed Google Scholar, 18Di Donato L. Des Rosiers C. Montgomery J.A. David F. Garneau M. Brunengraber H. J. Biol. Chem. 1993; 268: 4170-4180Abstract Full Text PDF PubMed Google Scholar, 19Beylot M. Soloviev M.V. David F. Landau B.R. Brunengraber H. J. Biol. Chem. 1995; 270: 1509-1514Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) or citrate (4Kelleher J.K. Am. J. Physiol. 1985; 248: E252-E260Crossref PubMed Google Scholar, 6Des Rosiers C. Di Donato L. Comte B. Laplante A. Marcoux C. David F. Fernandez C.A. Brunengraber H. J. Biol. Chem. 1995; 270: 10027-10036Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The use of 13C-enriched labeled substrate(s) and measurements of 13C labeling of CAC intermediates or related metabolites by nuclear magnetic resonance (NMR) or gas chromatography-mass spectrometry (GCMS) offers several advantages over classical 14C methods. Also, these two techniques are complementary. One advantage of GCMS over NMR is its sensitivity. Thus, the 13C 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 (14Chance E.M. Seeholzer S.H. Kobayashi K. Williamson J.R. J. Biol. Chem. 1983; 258: 13785-13794Abstract Full Text PDF PubMed Google Scholar, 15Chatham J.C. Forder J.R. Glickson J.D. Chance E.M. J. Biol. Chem. 1995; 270: 7999-8008Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 17Malloy C.R. Sherry A.D. Jeffrey F.M.H. Am. J. Physiol. 1990; 259: H987-H995Crossref PubMed Google Scholar, 20Lopaschuk G.D. Saddik M. Mol. Cell. Biochem. 1992; 116: 111-116Crossref PubMed Scopus (74) Google Scholar, 21Renstrom B. Nellis S.H. Liedke A.J. Circ. Res. 1990; 66: 282-288Crossref PubMed Scopus (35) Google Scholar, 22Weiss R.G. Chacko V.P. Gerstenblith G. J. Mol. Cell. 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By replenishing CAC intermediates, anaplerotic carboxylation of pyruvate could also be a key component for restoration of cardiac function following ischemia (26Barron J.T. Kopp S.J. Tow J. Parillo J.E. Am. J. Physiol. 1994; 267: H764-H769PubMed Google Scholar, 27Russell III, R.R. Taegtmeyer H. Am. J. Physiol. 1991; 261: H1756-H1762Crossref PubMed Google Scholar, 28Peuhkurinen K.J. Nuutinen E.M. Pietiläinen E.P. Hiltunen J.K. Hassinen I.E. Biochem. J. 1982; 208: 577-581Crossref PubMed Scopus (32) Google Scholar). Substrate fluxes through pyruvate carboxylation were evaluated in hearts perfused with14C-labeled pyruvate (29Sundqvist K.E. Hiltunen J.K. Hassinen I.E. Biochem. J. 1989; 257: 913-916Crossref PubMed Scopus (22) Google Scholar, 30Peuhkurinen K.J. Hassinen I.E. Biochem. J. 1982; 202: 67-76Crossref PubMed Scopus (60) Google Scholar). However, in these studies, the partitioning of pyruvate between carboxylation and decarboxylation could not be directly quantitated. Such measurements could help clarify the relative importance of these two pathways in the cardioprotective effect of pyruvate (23McVeigh J.J. Lopaschuk G.D. Am. J. Physiol. 1990; 259: H1079-H1085PubMed Google Scholar, 26Barron J.T. Kopp S.J. Tow J. Parillo J.E. Am. J. Physiol. 1994; 267: H764-H769PubMed Google Scholar, 27Russell III, R.R. Taegtmeyer H. Am. J. Physiol. 1991; 261: H1756-H1762Crossref PubMed Google Scholar, 29Sundqvist K.E. Hiltunen J.K. Hassinen I.E. Biochem. J. 1989; 257: 913-916Crossref PubMed Scopus (22) Google Scholar, 30Peuhkurinen K.J. Hassinen I.E. Biochem. J. 1982; 202: 67-76Crossref PubMed Scopus (60) Google Scholar, 31Bünger R. Mallet R.T. Hartman D.A. Eur. J. Biochem. 1989; 180: 221-233Crossref PubMed Scopus (225) Google Scholar, 32Jessen M.E. Kovarik T.E. Jeffrey F.M.H. Sherry A.D. Storey C.J. Chao R.Y. Ring W.S. Malloy C.R. J. Clin. Invest. 1993; 92: 831-839Crossref PubMed Scopus (31) Google Scholar). In the present paper, we present a strategy for the direct and simultaneous assessment of the relative contributions of anaplerotic pyruvate carboxylation, pyruvate decarboxylation, and fatty acid oxidation to citrate formation in the intact perfused rat heart. This is accomplished using a combination of 13C-substrates and requires determination of the 13C mass isotopomer distributions (MIDs) of the acetyl and oxaloacetate (OAA) moieties of a single metabolite, effluent citrate, by GCMS. Part of this work was presented in abstract form (33Comte B. Jetté M. Cordeau S. Des Rosiers C. Circulation. 1995; 92 (abstr.): I-770Google Scholar, 34Vincent G. Comte B. Des Rosiers C. FASEB J. 1997; 11 (abstr.): 269Google Scholar, 35Comte B Des Rosiers C. FASEB J. 1997; 11 (abstr.): 268Google Scholar). The relative contributions of pathways feeding OAA and acetyl-CoA for citrate synthesis were determined in hearts perfused with a mix of13C-substrates under steady-state conditions. [U-13C3]Lactate and [U-13C3]pyruvate were supplied at physiological concentrations and in a ratio to clamp the redox state. Also a source of acetyl-CoA other than pyruvate, was provided, namely a 1-13C-labeled fatty acid, octanoate. Calculation of the various substrate flux ratios requires GCMS determination of the MIDs of (i) the acetyl and OAA moieties of citrate and of (ii) pyruvate. [1-13C]Octanoate and [U-13C3]pyruvate are metabolized to different citrate mass isotopomers. [U-13C3]Lactate, a M3 isotopomer, is converted to [U-13C3]pyruvate which is (i) decarboxylated to [1,2-13C2]acetyl-CoA (M2) by pyruvate dehydrogenase and/or (ii) carboxylated to [1,2,3-13C3]OAA (M3) by pyruvate carboxylase, or to [1,2,3-13C3]malate (M3) by NADP-linked malic enzyme. Because of the reversibility of the fumarase reaction, [2,3,4-13C3]OAA is also formed. [1-13C]Octanoate is β-oxidized to [1-13C]acetyl-CoA (M1). Through the citrate synthase reaction, [1-13C]- and [1,2-13C2]acetyl-CoA label citrate on carbon 5 (M1), and on carbons 4 + 5 (M2), respectively. Similarly, [1,2,3-13C3]- or [2,3,4-13C3]OAA label citrate on carbons 6 + 3 + 2 and 3 + 2 + 1, respectively. When there is negligible condensation between labeled acetyl-CoA and labeled OAA, citrate thus becomes enriched in M3, M2, and M1 isotopomers. Upon further metabolism in the CAC, these citrate isotopomers form a mixture of M2 and M1 positional isotopomers of OAA. Condensation between M1 and/or M2 acetyl-CoA and M3 OAA forms M4 and M5 citrate isotopomers, which upon further metabolism in the CAC form a mixture of M4, M3, M2, and M1 positional isotopomers of OAA. Theoretically, up to 64 possible citrate isotopomers can be formed, labeled in their acetyl (carbons 4 + 5) and/or OAA (carbons 1 + 2 + 3 + 6) moieties. In hearts perfused with [U-13C3]lactate, [U-13C3]pyruvate, and [1-13C]octanoate, this number is reduced to 48, because there is no formation of citrate isotopomers labeled in their acetyl moiety with only one 13C atom on carbon 4. The following notations are used in developing the equations: Metabolites: AC, acetyl-CoA; CIT, citrate; OAA, oxaloacetate; OCT, octanoate; PYRi and PYRe, intracellular and extracellular pyruvate; ACCIT, the acetyl moiety of citrate, equivalent to carbons 4 and 5 of citrate (C-4 + 5); OAACIT, the OAA moiety of citrate equivalent to C-1 + 2 + 3 + 6. Isotopomer specifications: OAAMi, mass isotopomer of a given metabolite, for example OAA, labeled with i atoms of 13C; MF, mol fraction in a given mass isotopomer (Mi) of a metabolite, calculated as MF (M i ) =A M i /(ΣA M i ) where A represents the peak area of each fragmentogram, determined by computer integration and corrected for naturally occurring heavy isotopes (6Des Rosiers C. Di Donato L. Comte B. Laplante A. Marcoux C. David F. Fernandez C.A. Brunengraber H. J. Biol. Chem. 1995; 270: 10027-10036Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 7Fernandez C.A. Des Rosiers C. J. Biol. Chem. 1995; 270: 10037-10042Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 18Di Donato L. Des Rosiers C. Montgomery J.A. David F. Garneau M. Brunengraber H. J. Biol. Chem. 1993; 268: 4170-4180Abstract Full Text PDF PubMed Google Scholar, 36Des Rosiers C. Montgomery J.A. Desrochers S. Garneau M. David F. Mamer O.A. Brunengraber H. Anal. Biochem. 1988; 173: 96-105Crossref PubMed Scopus (49) Google Scholar); MPE, molar percent enrichment in a given mass isotopomer of a metabolite, equivalent to MF × 100. Flux rates: FCPYRi→OAA, fractional contribution (FC) of one metabolite to the total flux of the other, e.g.intracellular pyruvate to OAA. For a given reaction, the sum of the different FCs equals 1. The principle of the calculation of relative input fluxes from measured MID of given metabolites was described in previous publications for liver and heart perfusions (6Des Rosiers C. Di Donato L. Comte B. Laplante A. Marcoux C. David F. Fernandez C.A. Brunengraber H. J. Biol. Chem. 1995; 270: 10027-10036Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 7Fernandez C.A. Des Rosiers C. J. Biol. Chem. 1995; 270: 10037-10042Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 37Des Rosiers C. Fernandez C.A. David F. Brunengraber H. J. Biol. Chem. 1994; 269: 27179-27182Abstract Full Text PDF PubMed Google Scholar, 38Laplante A. Vincent G. Poirier M. Des Rosiers C. Am. J. Physiol. 1997; 272: E74-E82PubMed Google Scholar). Briefly, we first assume, for simplicity, that carboxylation of M3 pyruvate is the only pathway forming M3 OAA. Then, we consider the possible formation of M3 OAA through the metabolism in the CAC of some citrate isotopomers. The balance of M3 isotopomers of OAA and adjacent metabolites yields Equation 1, (FCPYRi→OAA)=(OAAM3)/(PYRiM3)Equation 1 where FCPYRi→OAA is the fractional contribution of pyruvate to OAA via pyruvate carboxylation, and OAAM3 and PYRiM3 are the MF in M3 of intracellular OAA and pyruvate, respectively. Since for a given reaction, the sum of all FC terms equals 1, then (1 − FCPYRi→OAA) represents the OAA molecules coming from all other sources, namely from the CAC and from aspartate transamination. Note that FCPYRi→OAA represents the carboxylation of pyruvate derived from both exogenous and endogenous sources. The fractional contribution of extracellular pyruvate and/or lactate to tissue pyruvate (FCPYRe→PYRi) is obtained from Equation 2, (FCPYRe→PYRi)=(PYRiM3)/(PYReM3)Equation 2 where PYReM3 is the MF in M3 of the incoming pyruvate tracer. The contribution of unlabeled pyruvate arising from glucose or glycogen through glycolysis is (1 − FCPYRe→PYRi). Since M3 OAA is the only source of M3 citrate labeled in its OAA moiety (OAACIT), the balance of M3 isotopomers of OAA moiety of citrate and adjacent metabolites yields Equation 3, (FCOAA→CIT)=(OAAM3CIT)/(OAAM3)Equation 3 where FCOAA→CIT is the fractional contribution of OAA to citrate via citrate synthase, and OAAM3CIT is the MF in M3 of the OAA moiety of citrate. Multiplying Equation 1 by Equation 3 yields the fractional contribution of pyruvate to citrate via the carboxylation of pyruvate (FCPYRi→CIT(OAA)). The latter expression is equivalent to the flux ratio (pyruvate carboxylation)/(citrate synthesis), also named factor "y" (2Lee W.-N.P. J. Biol. Chem. 1989; 264: 13002-13004Google Scholar, 3Lee W.-N.P. J. Biol. Chem. 1993; 268: 25522-25526Abstract Full Text PDF PubMed Google Scholar, 6Des Rosiers C. Di Donato L. Comte B. Laplante A. Marcoux C. David F. Fernandez C.A. Brunengraber H. J. Biol. Chem. 1995; 270: 10027-10036Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 39Katz J. Lee W.-N.P. Wals P.A. Bergner E.A. J. Biol. Chem. 1989; 264: 12994-13001Abstract Full Text PDF PubMed Google Scholar, 40Cline G.W. Shulman G.I. J. Biol. Chem. 1995; 270: 28062-28067Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 41Malloy C.R. Sherry A.D. Jeffrey F.M.H. Am. J. Physiol. 1990; 259: H987-H995Crossref PubMed Google Scholar), when flux through citrate synthase is set equal to 1 (see Equation 4). FCPYRi→OAA(CIT)=(FCPYRi→OAA)×(FCOAA→CIT)=OAAM3CIT/PYRiM3Equation 4 Similar reasoning yields the flux ratios (pyruvate decarboxylation)/(citrate synthesis) or factor "x" (2Lee W.-N.P. J. Biol. Chem. 1989; 264: 13002-13004Google Scholar, 3Lee W.-N.P. J. Biol. Chem. 1993; 268: 25522-25526Abstract Full Text PDF PubMed Google Scholar, 6Des Rosiers C. Di Donato L. Comte B. Laplante A. Marcoux C. David F. Fernandez C.A. Brunengraber H. J. Biol. Chem. 1995; 270: 10027-10036Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 39Katz J. Lee W.-N.P. Wals P.A. Bergner E.A. J. Biol. Chem. 1989; 264: 12994-13001Abstract Full Text PDF PubMed Google Scholar) and (octanoate oxidation)/(citrate synthesis) using Equations 5 and 6, respectively, FCPYRi→AC(CIT)=(FCPYRi→AC)×(FCAC→CIT)=ACM2CIT/PYRiM3Equation 5 FCOCT→AC(CIT)=(FCOCT→AC)×(FCAC→CIT)=4×ACM1CIT/OCTiM1Equation 6 where (i) FCPYRi→AC and FCOCT→AC are the fractional contributions of pyruvate decarboxylation and of octanoate oxidation to acetyl-CoA, respectively; (ii) FCAC→CIT is the fractional contribution of acetyl-CoA to citrate via citrate synthase; and (iii) OCTiM1 is the MF in M1 of intracellular octanoate, which we assumed equal to that of extracellular octanoate, since octanoate is not a physiological fatty acid. The factor 4 in Equation 6 takes into account that octanoate is oxidized to 4 acetyl-CoA; only one of which is labeled. The sum of Equations 5 and 6 (FCPYRi→CIT + FCOCT→CIT) reflects the relative combined contributions of pyruvate decarboxylation and octanoate oxidation to the acetyl moiety of citrate; the contribution of other substrates (FCOS→AC(CIT)), such as endogenous fatty acids and/or leucine, is given by Equation 7. FCOS→AC(CIT)=[1−(FCPYRi→AC(CIT)+FCOCT→AC(CIT))]Equation 7 In developing Equations Equation 1, Equation 2, Equation 3, Equation 4, Equation 5, Equation 6, we assumed that carboxylation of M3 pyruvate was the only reaction forming M3 OAA molecules. Such assumptions are likely to prevail in hearts perfused with a low enrichment of [U-13C3]lactate and [U-13C3]pyruvate. However, in hearts perfused simultaneously with a mix of 13C-substrates, the probability of condensation between labeled acetyl-CoA and labeled OAA is increased. Some citrate molecules labeled in both their acetyl and OAA moieties could be metabolized to M3 OAA in the CAC. Therefore, to assess the validity of our initial assumption, we evaluated the proportion of the M3 OAA molecules formed through the recycling of some citrate isotopomers in the CAC and subsequently corrected the measured MF M3 of the OAA moiety of citrate (OAAM3CIT). This was done using Equation8 and requires estimating the (i) MPE of citrate isotopomer precursor of M3 OAA (OAAM3PR) and (ii) the13C dilution in the CAC (DF). Values for (i) MPE OAAM3PR and (ii) DF, obtained using Equations 9 and 10 (see below), are approximations. More precise estimation of these values would require extensive modeling of the metabolism of our mix of 13C-substrates in the CAC, OAAM3−CIT*=OAAM3CIT−(OAAM3PR×DF)Equation 8 where (i) OAAM3PR is the enrichment of citrate isotopomer precursor of M3 OAA and (ii) DF is the correction factor for the 13C dilution in the CAC. The enrichment of citrate isotopomer precursor of M3 OAA was estimated as follows. We identified which of the 48 possible citrate isotopomers are precursors (PR) of M3 OAA (OAAM3PR), assuming steady-state conditions. For example, there are four citrate isotopomers formed from the condensation of M2 acetyl-CoA and M1 OAA molecules: (A) [1,4,5-13C3]citrate; (B) [2,4,5-13C3]citrate; (C) [3,4,5-13C3]citrate; and (D) [4,5,6-13C3]citrate. Upon metabolism in the CAC, citrate isotopomers B and C form M3 OAA, whereas isotopomers A and D form M2 OAA. For simplicity, it is assumed that citrate isotopomers A to D are similarly enriched, although this is an approximation of the actual situation (see "Discussion" for details). Assessing the validity of this assumption requires more extensive modeling of the metabolism of our mix of 13C-substrates in the CAC. Then, the enrichment of citrate isotopomers B and C is estimated from ((ACM2CIT) × (OAAM1CIT) × 0.5) where ACM2CIT and OAAM1CIT are experimentally determined enrichments of the acetyl and OAA moieties of citrate, and the number 0.5 takes into account that only half of citrate isotopomers formed through the condensation of M2 acetyl-CoA and M1 OAA are metabolized to M3 OAA. This constitutes the first term of Equation 9. Note that for clarity, the superscript CIT was omitted. A similar reasoning was applied for each term of this equation. OAAM3PR=[1/2×(ACM2×OAAM1)]+[2/3×(ACM2×OAAM2)] +[1/2×(ACM2×OAAM3)]+[1/2×(ACM1×OAAM3)]Equation 9 +[(ACM1×OAAM4)]The 13C dilution in the CAC due to entry of unlabeled molecules was estimated from the total enrichment in tissue citrate (CITΣM i ) and succinate (SUCΣM i ) using Equation 10, DF=[CITΣMi−(Σfi×OAAM1CIT)]/[SUCΣMi]Equation 10 where the factor f takes into account that a fraction of all citrate molecules enriched with one 13C in any one carbon of its OAA moiety are converted to unlabeled succinate. Note that the magnitude of f depends on the nature of the13C-substrate. For example, f equals 1 for13C-substrates forming [5-13C] citrate, such as 1-13C-labeled fatty acid or acetate. However,f equals 0.5 for 13C-substrates forming [4,5-13C2]citrate, such as [U-13C3]pyruvate or [U-13C2]acetate. With a mix of13C-substrates, the relative contribution of each substrate to the M1 enrichment of the OAA moiety of citrate needs to be considered. 2Under our perfusion conditions, the percentage contributions of [1-13C]octanoate and [U-13C3](lactate + pyruvate) to M1 OAA enrichment are evaluated to be 78.9 ± 0.6 and 21.2 ± 0.4%, respectively, from experiments where hearts were perfused with a single13C-substrate (MPE M1 OAA (citrate); Table II; Conditions A and B). Therefore, Σf i equals ((0.79 × 1) + (0.21 × 0.5)) or 0.895. Alternatively, the relative contributions of these 13C-substrates can be estimated from the M1 and M2 enrichments of the acetyl moiety of citrate, 88.0 ± 0.3 and 12.0 ± 0.7%, respectively (Table IIIC). Chemicals, organic solvents, enzymes, and coenzymes were purchased from Boehringer Mannheim (Laval, Quebec), Fisher (Montreal, Quebec), Sigma, and Anachemia (Dorval, Quebec). [U-13C3]Lactate, [U-13C3]pyruvate, [1-13C]octanoate, [U13C2]acetate, and [U-13C5]glutamate (all 99%) were obtained from Isotec (Miamisburg, OH) and Cambridge Isotope Laboratories (Woburn, MA). The derivatization agentN-methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide was supplied by Regis Chemical (Morton Glove, IL). All aqueous solutions were made with water purified by the "Milli-Q" system (Millipore, St-Laurent, Quebec). Fed male Sprague-Dawley rats (Charles River, Quebec) weighing 120–220 g were anesthetized by intraperitoneal injection of sodium pentobarbital (65 mg/kg). After opening the chest and insertion of a cannula into the aorta, hearts were excised and transferred to a Langendorff set up as described previously (38Laplante A. Vincent G. Poirier M. Des Rosiers C. Am. J. Physiol. 1997; 272: E74-E82PubMed Google Scholar, 42Brunet J. Boily M.J. Cordeau S. Des Rosiers C. Free Radical Biol. & Med. 1995; 19: 627-638Crossref PubMed Scopus (65) Google Scholar). Briefly, hearts (wet weight 1.1–1.3 g) were perfused for up to 60 min retrogradely through the aorta at a constant pressure of 80 mmHg with a non-recirculating Krebs-Ringer bicarbonate buffer containing 1.3 mm calcium, 11 mm glucose, 1 or 0.5 mm lactate, 0.2 or 0.05 mm pyruvate, 0.2 mm octanoate, with or without 0.5 mm glutamate or 0.1 mm acetate. The buffer was gassed with 95% O2:5% CO2 (pH 7.4) at 38 °C. The following parameters were continuously monitored through instruments linked to a microcomputer: (i) coronary flow, using an electromagnetic flow probe (model FM501, Carolina Medical Electronics, King, NC) installed above the aortic cannula; (ii) temperature using a thermocouple (Yellow Springs Instrument, Yellow Springs, OH) attached to the surface of the heart; and (iii) contractile function using a latex balloon filled with fluid inserted into the left ventricular cavity and connected to a pressure transducer (Digi-Med Heart Performance Analyzer, Micro-Med, Louisville, KY). Hearts that did not show an increase in coronary flow on release of a 20–25-s period of in-flow occlusion were discarded. Under our conditions, hearts were beating spontaneously at 280 ± 19 beats/min and maintained a contractile function (dP/dT) of 2099 ± 152 mmHg/s and a coronary flow rate of 9.3 ± 0.6 ml/min. After a 15–20-min equilibration period, one or more unlabeled substrate(s) were replaced by the corresponding labeled substrates either (i) [1-13C]octanoate, (ii) [U-13C3]lactate + [U-13C3]pyruvate, (iii) [1-13C]octanoate + [U-13C3]lactate + [U-13C3]pyruvate, (iv) [U-13C5]glutamate, or (v) [U-13C2]acetate. Starting 10 min before the labeling period, samples of effluent perfusate (20 ml) were collected every 5 min and processed as follows: (i) 7 ml was immediately made 10 mm hydroxylamine-hydrochloride and sonicated for 1 min to convert α-ketoglutarate (αKG) to its oxime derivative (43Laplante A. Comte B. Des Rosiers C. Anal. Biochem. 1995; 224: 580-587Crossref Scopus (19) Google Scholar); (ii) 10 ml was made 1% sulfosalicylic acid; and (iii) 1 ml was left untreated. Samples were stored at −20 °C until analysis. After perfusing for 40 min with 13C-substrate(s), hearts were freeze-clamped and stored in liquid nitrogen. The MID of tissue and effluent perfusate citrate and other CAC metabolites (αKG, OAA, succinate, fumarate, malate) was determined by published GCMS methods (6Des Rosiers C. Di Donato L. Comte B. Laplante A. Marcoux C. David F. Fernandez C.A. Brunengraber H. J. Biol. Chem. 1995; 270: 10027-10036Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 18Di Donato L. Des Rosiers C. Montgomery J.A. David F. Garneau M. Brunengraber H. J. Biol. Chem. 1993; 268: 4170-4180Abstract Full Text PDF PubMed Google Scholar, 37Des Rosiers C. Fernandez C.A. David F. Brunengraber H. J. Biol. Che

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