QUANTITATION OF METABOLIC COMPARTMENTATION IN HYPERAMMONEMIC BRAIN BYNATURAL-ABUNDANCE C-13-NMR IR DETECTION OF C-13-N-15 COUPLING PATTERNS AND ISOTOPIC SHIFTS

In the present study, the removal of cerebral ammonia by glutamine syn thetase (GS) and by reductive amination of 2-oxoglutarate by glutamate dehydrogenase in the presence of an amino donor group, was determined in hyperammonemic rabbit brains. The N-15 enrichments of brain metabo lite alpha-amino and amide positions of glutamine, glutamate, and alan ine were determined by the indirect detection of N-15-labeled compound s of the C-13-N-15 spin coupling patterns of natural abundance C-13-NM R spectra. The C-13-NMR spectra of brain extracts were obtained from r abbits infused with (NH4Cl)-N-15 with or without intraperitoneal infus ion of the GS inhibitor, L-methionine DL-sulfoximine, in a reasonable acquisition time period. When (NH4Cl)-N-15 was infused, [5-N-15]glutam ine and [2-N-15]glutamine concentrations reached 5.2 mu mol/100 mg pro tein and 3.6 mu mol/100 mg protein, respectively, which indicates the relatively high activity of reductive amination of 2-oxoglutarate in t he glutamate dehydrogenase reaction. The low concentration of [2-N-15] glutamate, which is about 30% of that of [2-N-15]glutamine obtained in this study, suggests that very little glutamine serves as a precursor of neuronal glutamate. When GS was inhibited by L-methionine DL-sulfo ximine, a flux of (NH4+)-N-15 via the residual activity of GS was acco mpanied by an apparent increase of [2-N-15]glutamate and [N-15]alanine concentrations (2.9 mu mol/100 mg protein and 1.8 mu mol/100 mg prote in, respectively). These findings and those obtained from C-13-C-13 is otopomer analysis (Lapidot and Gopher, 1994b) suggest that astrocytic 2-oxoglutarate is partially utilized (together with an amino group don or) as a precursor for neuronal glutamate in the hyperammonemic brain when GS is inhibited. This process can partly replace GS activity in m etabolizing ammonia in the hyperammonemic rabbit brain.