A comparison between NMR and GCMS 13C-isotopomer analysis in cardiac metabolism. Export

@article{citeulike:693709, 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-(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.}, address = {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}, author = {Chatham, J. C. and Bouchard, B. and Des Rosiers, C.}, citeulike-article-id = {693709}, citeulike-linkout-0 = {http://view.ncbi.nlm.nih.gov/pubmed/12956405}, citeulike-linkout-1 = {http://www.hubmed.org/display.cgi?uids=12956405}, issn = {0300-8177}, journal = {Mol Cell Biochem}, keywords = {13c, isotopomer, nmr}, month = {July}, number = {1-2}, pages = {105--112}, posted-at = {2006-06-12 14:13:45}, priority = {3}, title = {A comparison between NMR and GCMS 13C-isotopomer analysis in cardiac metabolism.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/12956405}, volume = {249}, year = {2003} }

TY - JOUR ID - citeulike:693709 L3 - citeulike-article-id:693709 N2 - 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. IS - 1-2 JF - Mol Cell Biochem SN - 0300-8177 AD - 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 EP - 112 TI - A comparison between NMR and GCMS 13C-isotopomer analysis in cardiac metabolism. VL - 249 SP - 105 KW - 13c KW - isotopomer KW - nmr AU - Chatham, JC AU - Bouchard, B AU - Des Rosiers, C PY - 2003/07// UR - http://view.ncbi.nlm.nih.gov/pubmed/12956405 ER -