Delineation of substrate selection and anaplerosis in tricarboxylic acid cycle of the heart by 13C NMR spectroscopy and mass spectrometry

Abstract

(13)C NMR and mass spectrometry (MS) provide complementary information regarding the (13)C labeling of intermediary metabolites. Currently, these two techniques are rarely used together because of the complexity of modeling the distribution of both positional and mass isotopomers. In this study, we developed a matrix-based model for the assessment of (13)C label distribution in the tricarboxylic acid cycle and related metabolites. The model was applied to the analysis of NMR- and MS-measured (13)C isotopomers for quantification of substrate utilization and anaplerotic fluxes in isolated perfused rat hearts. NMR and MS data were acquired from two groups of rat hearts perfused with substrates in complementary labeling patterns, i.e. the (13)C-PAL + GLC group (0.6 mM [(13) C(16) ]palmitate + 5.5 mM glucose) and the PAL + (13)C-GLC group (0.6 mM palmitate + 5.5 mM [(13)C(6) ]glucose). Relative flux parameters were obtained by fitting the model to the NMR data, MS data and their combination, respectively. Our results suggest that, although both NMR and MS can provide accurate quantification of substrate selection in oxidative metabolism, the accuracy of estimation of anaplerotic fluxes relies on the combination of these two experimental methods.