Multiplet structure of 13C NMR signal from glutamate and direct detection of tricarboxylic acid (TCA) cycle intermediates

Publisher Website
Google Scholar
Abstract
For the first time, 13C NMR signals are shown from 13C-en-riched, low-level tricarboxylic acid (TCA) cycle intermediates from extracts of normal cardiac tissue. As the low tissue content of the key intermediates α-ketoglutarate (α-KG) and succinate (SUC) in normal, well perfused tissues has until now precluded direct NMR detection from intact tissues and tissue extracts, 13C NMR signal from glutamate has generally been used to infer the isotopomer patterns of intermediates that are in chemical exchange with glutamate. However, the required assumptions regarding intracellular compartmentation for such indirect analysis have not been previously tested, as glutamate is largely cytosolic while the TCA cycle enzymes are located in the mitochondria. Chromatographic isolation of α-KG and SUC from heart tissue extracts allowed isotopomer analysis to be performed for comparison with that of glutamate. At steady state, a direct relationship between glutamate and α-ketoglutarate isotopomers was found, but succinate isotopomers matched those of glutamate only in hearts that displayed negligible contributions from the oxidation of unla-beled endogenous carbon sources.