MULTIPLET STRUCTURE OF (13)G NMR SIGNAL FROM GLUTAMATE AND DIRECT-DETECTION OF TRICARBOXYLIC-ACID (TCA) CYCLE INTERMEDIATES

For the first time,C-13 NMR signals are shown from C-13-enriched, low- level tricarboxylic acid (TCA) cycle intermediates from extracts of no rmal cardiac tissue, As the low tissue content of the key intermediate s alpha-ketoglutarate (alpha-KG) and succinate (SUC) in normal, well p erfused tissues has until now precluded direct NMR detection from inta ct tissues and tissue extracts, C-13 NMR Signal from glutamate has gen erally been used to infer the isotopomer patterns of intermediates tha t are in chemical exchange with glutamate, However, the required assum ptions regarding intracellular compartmentation for such indirect anal ysis have not been previously tested, as glutamate is largely cytosoli c while the TCA cycle enzymes are located in the mitochondria. Chromat ographic isolation of alpha-KG and SUC from heart tissue extracts allo wed isotopomer analysis to be performed for comparison with that of gl utamate, At steady state, a direct relationship between glutamate and alpha-ketoglutarate isotopomers was found, but succinate isotopomers m atched those of glutamate only in hearts that displayed negligible con tributions from the oxidation of unlabeled endogenous carbon sources.