C-13 ISOTOPOMER ANALYSES IN INTACT TISSUE USING (C-13)HOMONUCLEAR DECOUPLING

Entry of C-13-enriched acetyl-CoA into the citric acid cycle results i n scrambling of C-13 into the various carbon positions of all intermed iate pools. The eventual result is that the C-13 resonances of all det ectable intermediates or molecules exchanging with those intermediates appear as multiplets due to nearest neighbor spin-spin couplings. We have previously shown that an isotopomer analysis of the glutamate C-1 3 multiplets provides a history of C-13 flow through the cycle pools a nd that relative substrate utilization and relative anaplerotic flux c an be quantitated (C.R. Malloy, A.D. Sherry, and F.M.H. Jeffrey, Am. J . Physiol. 259, H987-H995 (1990)). A major limitation of the method fo r in vivo applications is spectral resolution of multiline resonances required for a complete isotopomer analysis. We now show that {C-13}ho monuclear decoupling of the glutamate C3 resonance collapses nine-line C4 and C2 resonances into three-line multiplets. We demonstrate that these three-line C-13 multiplets are well resolved in isolated, perfus ed rat hearts and present steady-state equations that allow an isotopo mer analysis from data obtained in intact tissue. This advancement off ers for the first time the possibility of extending C-13 isotopomer me thods to complex metabolic conditions in vivo.