METABOLISM OF [U-C-13]GLUTAMATE IN ASTROCYTES STUDIED BY C-13 NMR-SPECTROSCOPY - INCORPORATION OF MORE LABEL INTO LACTATE THAN INTO GLUTAMINE DEMONSTRATES THE IMPORTANCE OF THE TRICARBOXYLIC-ACID CYCLE

Primary cultures of cerebral cortical astrocytes were incubated with [U-C-13]glutamate (0.5 mM) in modified Dulbecco's medium for 2 h. Perchloric acid (PCA) extracts of the cells as well as redissolved lyophilized media were subjected to NMR spectroscopy to identify C-13-labeled metabolites. NMR spectra of the PCA extracts exhibited distinct multiplets for glutamate, aspartate, glutamine, and malate. The culture medium showed peaks for a multitude of compounds released from the astrocytes, among which lactate, glutamine, alanine, and citrate were readily identifiable. For the first time incorporation of label into lactate from glutamate was clearly demonstrated by doublet formation in the C-3 position and two doublets in the C-2 position of lactate. This labeling pattern can only occur by incorporation from glutamate, because natural abundance will only produce singlets in proton-decoupled C-13 spectra. Glutamine, released into the medium, was labeled uniformly to a large extent, but the C-3 position not only showed the expected apparent triplet but also a doublet due to C-13 incorporation into the C-4 position of glutamine. The doublet accounted for 11% of the total label in the glutamine synthesized and released within the incubation period. The corresponding labeling pattern of [C-13]glutamate in the PCA extracts showed that 19% of the glutamate contained C-12. Labeling of lactate, citrate, malate, and aspartate as well as incorporation of C-12 into uniformly labeled glutamate and glutamine could only arise via the tricarboxylic acid cycle. The relative amount of glutamate metabolized via this route is at least 70% as calculated from the areas of the C-3 resonances of these compounds. Only a maximum of 30% was converted to glutamine directly. Key