13C isotopomer analysis of glutamate by heteronuclear multiple quantum coherence total correlation spectroscopy (HMQC-TOCSY)

C-13 has become an important tracer isotope for studies of intermediary metabolism, Information about relative flux through pathways is encoded by the distribution of C-13 isotopomers in an intermediate pool such as glutamate, This information is commonly decoded either by mass spectrometry or by measuring relative multiplet areas in a C-13 NMR spectrum, We demonstrate here that groups of glutamate C-13 isotopomers may be quantified by indirect detection of protons in a 2D HMQC-TOCSY NMR spectrum and that fitting of these data to a metabolic model provides an identical measure of the C-13 fractional enrichment of acetyl-CoA and relative anaplerotic flux to that given by direct C-13 NMR analysis. The sensitivity gain provided by HMQC-TOCSY spectroscopy will allow an extension of C-13 isotopomer analysis to tissue samples not amenable to direct C-13 detection(similar to 10 mg soleus muscle) and to tissue metabolites other than glutamate that are typically present at lower concentrations. (C) 1998 Federation of European Biochemical Societies.