Abstract
NMR spectroscopy and gas chromatography-mass spectrometry (GCMS) have both been used to study cardiac metabolism using substrates labeled with the stable isotope carbon-13. 13C-NMR studies of substrate oxidation are based on the assumption that the 13C-enrichment of glutamate reflects that of 2-ketoglutarate (2-KG). This assumption appears reasonable; however, it has not been thoroughly validated. The higher sensitivity of GCMS enables the direct determination of 13C-enrichment of 2-KG and other tricarboxylic acid (TCA) cycle intermediates. Therefore, using extracts from normal and diabetic hearts perfused with physiological concentrations of unlabeled glucose and 13C-labeled substrates, [3-13C](lactate + pyruvate) and [U-13C]palmitate, we compared the mass isotopomer distribution (MID) of citrate, 2-KG, succinate and malate measured directly by GCMS with that extrapolated from 13C-NMR glutamate isotopomer analysis. A significant correlation between the absolute molar percent enrichments (MPE) of the various mass isotopomers of glutamate determined by 13C-NMR and 2-KG determined by GCMS was observed for all sixteen-heart samples. This correlation was improved if the contribution from unlabeled 2-KG was removed (i.e. relative MPE) indicating that 13C-NMR under estimated the unlabeled fraction. We attribute this discrepancy in the measurement of unlabeled 2-KG to the fact that GCMS measures MO directly, while the NMR analysis calculates it by difference, since unlabeled glutamate is not detected by 13C-NMR spectroscopy. Despite the differences between the two methods, 13C-MID of glutamate determined by NMR provides a simple and reliable indicator of fluxes of 13C-enriched substrates through the TCA cycle. It is also clear that MID analysis of TCA cycle intermediates by GCMS is a sensitive and direct approach to assess substrate selection for citrate synthesis as well as a potential indicator of sites and extent of anaplerosis and/or compartmentation. This study demonstrates that the alliance of NMR and GCMS represents a powerful approach for investigating the control and regulation of cardiac carbon metabolism. (Mol Cell Biochem 249: 105–112, 2003)
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References
Langendorff O: Untersuchungen am überlebenden Säugethierherzen. Pflügers Arch Gesente Physiol 61: 291–332, 1895
Taegtmeyer H: One hundred years ago: Oscar Langendorff and the birth of cardiac metabolism. Can J Cardiol 11: 1030–1035, 1995
Opie LH: Cardiac metabolism—emergence, decline, and resurgence. Part I. Cardiovasc Res 26: 721–733, 1992
Stanley WC, Lopaschuk GD, Hall JL, McCormack JG: Regulation of myocardial carbohydrate metabolism under normal and ischaemic conditions. Potential for pharmacological interventions. Cardiovasc Res 33: 243–257, 1997
Lopaschuk GD, Rebeyka IM, Allard MF: Metabolic modulation: A means to mend a broken heart. Circulation 105: 140–142, 2002
Taegtmeyer H, Goodwin GW, Doenst T, Frazier OH: Substrate metabolism as a determinant for postischemic functional recovery of the heart. Am J Cardiol 80: 3A–10A, 1997
Taegtmeyer H: Six blind men explore an elephant: Aspects of fuel metabolism and the control of tricarboxylic acid cycle activity in heart muscle. Basic Res Cardiol 79: 322–336, 1984
Randle PJ, Garland PB, Hales CN, Newsholme EA, Denton RM, Pogson CI: Interactions of metabolism and the physiological role of insulin. Recent Prog Horm Res 22: 1–48, 1966
Neely JR, Morgan HE: Substrate and energy metabolism of the heart. Ann Rev Physiol 36: 413–459, 1974
Hall JL, Stanley WC, Lopaschuk GD, Wisneski JA, Pizzurro RD, Hamilton CD, McCormack JG: Impaired pyruvate oxidation but normal glucose uptake in diabetic pig heart during dobutamine-induced work. Am J Physiol Heart Circ Physiol 271: H2320–H2329, 1996
Goodwin GW, Taylor CS, Taegtmeyer H: Regulation of energy metabolism of the heart during acute increase in heart work. J Biol Chem 273: 29530–29539, 1998
Belke DD, Larsen TS, Lopaschuk GD, Severson DL: Glucose and fatty acid metabolism in the isolated working mouse heart. Am J Physiol Regulatory Integrative Comp Physiol 277: R1210–R1217, 1999
Barr RL, Lopaschuk GD: Methodology for measuring in vitro/ex vivo cardiac energy metabolism. J Pharmacol Toxicol Meth 43: 141–152, 2000
Magnusson I, Schumann WC, Bartsch GE, Chandramouli V, Kumaran K, Wahren J, Landau BR: Non-invasive tracing of Krebs cycle metabolism in liver. J Biol Chem 266: 6975–6984, 1991
Neurohr KJ, Barrett EJ, Shulman RG: In vivo carbon-13 nuclear magnetic resonance studies of heart metabolism. Proc Natl Acad Sci USA 80: 1603–1607, 1983
Malloy CR, Sherry AD, Jeffrey FMH: Carbon flux through citric acid cycle pathways in perfused heart by 13C NMR spectroscopy. FEBS Lett 212: 58–62, 1987
Weiss RG, Chacko VP, Gerstenblith G: Fatty acid regulation of glucose metabolism in the intact beating rat heart assessed by carbon-13 NMR spectroscopy: The critical role of pyruvate dehydrogenase. J Mol Cell Cardiol 21: 469–478, 1989
Lewandowski ED: Metabolic heterogeneity of carbon substrate utilization in mammalian heart: NMR determination of mitochondrial vs. cytosolic compartmentation. Biochemistry 31: 8916–8923, 1992
Laughlin MR, Taylor J, DeGroot M, Balaban RS: Pyruvate and lactate metabolism in the in vivo dog heart. Am J Physiol Heart Circ Physiol 264: H2068–H2079, 1993
Chatham JC, Forder JR, Glickson JD, Chance EM: Calculation of absolute metabolic flux and the elucidation of the pathways of glutamate labeling in perfused rat heart by 13C NMR spectroscopy and nonlinear squares analysis. J Biol Chem 270: 7999–8008, 1995
Chatham JC, Gao Z-P, Forder JR: The impact of 1 week of diabetes on the regulation of myocardial carbohydrate and fatty acid oxidation. Am J Physiol Endocrin Metab 277: E342–E351, 1999
Comte B, Jetté M, Bouchard B, Cordeau S, Des Rosiers C: A 13C-mass isotopomer study of anaplerotic pyruvate carboxylation in perfused rat hearts. J Biol Chem 272: 26125–26131, 1997
Comte B, Vincent G, Bouchard B, Des Rosiers C: Probing the origin of acetyl-CoA and oxaloacetate entering the citric acid cycle from the 13C-labeling of citrate released by perfused rat hearts. J Biol Chem 272: 26117–26124, 1997
Laplante A, Vincent C, Poirier M, Des Rosiers C: Effects and metabolism of fumarate in the perfused rat heart. A13C mass isotopomer study. Am J Physiol Endocrin Met 272: E74–E82, 1997
Vincent G, Comte B, Poirier M, Des Rosiers C: Citrate release by perfused rat hearts: A window on mitochondrial cataplerosis. Am J Physiol Endocrinol Metab 278: E846–E856, 2000
Panchal AR, Comte B, Huang H, Kerwin T, Darvish A, Des Rosiers C, Brunengraber H, Stanley WC: Partitioning of pyruvate between oxidation and anaplerosis in swine hearts. Am J Physiol Heart Circ Physiol 279: H2390–H2398, 2000
Panchal AR, Comte B, Huang H, Dudar B, Roth B, Chandler M, Des Rosiers C, Brunengraber H, Stanley WC: Acute hibernation decreases myocardial pyruvate carboxylation and citrate release. Am J Physiol Heart Circ Physiol 281: H1613–H1620, 2001
Jeffrey FMH, Roach JS, Storey CJ, Sherry AD, Malloy CR: 13C Isotopomer analysis of glutamate by tandem mass spectrometry. Anal Biochem 300: 192–205, 2002
Chatham JC, Gao ZP, Bonen A, Forder JR: Preferential inhibition of lactate oxidation relative to glucose oxidation in the rat heart following diabetes. Cardiovasc Res 43: 96–106, 1999
Malloy CR, Sherry AD, Jeffrey FMH: Analysis of tricarboxylic acid cycle of the heart using 13C isotope isomers. Am J Physiol Heart Circ Physiol 259: H987–H995, 1990
Weiss RG, Gloth ST, Kalil-Filho R, Chacko VP, Stern MD, Gerstenblith G: Indexing tricarboxylic acid cycle flux in intact hearts by carbon-13 nuclear magnetic resonance. Circ Res 70: 392–408, 1992
Lewandowski ED, Doumen C, White LT, LaNoue KF, Damico LA, Yu X: Multiplet structure of 13C NMR signal from glutamate and direct detection of tricarboxylic acid (TCA) cycle intermediates. Magn Reson Med 35: 149–154, 1996
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Chatham, J.C., Bouchard, B., Des Rosiers, C. (2003). A comparison between NMR and GCMS 13C-isotopomer analysis in cardiac metabolism. In: Gilchrist, J.S.C., Tappia, P.S., Netticadan, T. (eds) Biochemistry of Diabetes and Atherosclerosis. Developments in Molecular and Cellular Biochemistry, vol 42. Springer, Boston, MA. https://doi.org/10.1007/978-1-4419-9236-9_14
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DOI: https://doi.org/10.1007/978-1-4419-9236-9_14
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