A comparison between NMR and GCMS 13C-isotopomer analysis in cardiac metabolism.
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-(13)C](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 M0 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.
Chatham
John C
JC
Center for NMR Research and Development, Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294-4470, USA. jchatham@uab.edu
Bouchard
Bertrand
B
Des Rosiers
Christine
C
D000818:Animals; D002247:Carbon Isotopes; D019343:Citric Acid / Q000378:metabolism; D002952:Citric Acid Cycle; D004734:Energy Metabolism; D008401:Gas Chromatography-Mass Spectrometry; D005971:Glutamates / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008293:Malates / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D010477:Perfusion; D051381:Rats; D019802:Succinic Acid / Q000378:metabolism
D002247:Carbon Isotopes; D005971:Glutamates; D007656:Ketoglutaric Acids; D008293:Malates; D019343:Citric Acid; C030298:malic acid; D019802:Succinic Acid