Quantitation of metabolic compartmentation in hyperammonemic brain by natural abundance 13C-NMR detection of 13C-15N coupling patterns and isotopic shifts.
In the present study, the removal of cerebral ammonia by glutamine synthetase (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 15N enrichments of brain metabolite alpha-amino and amide positions of glutamine, glutamate, and alanine were determined by the indirect detection of 15N-labeled compounds of the 13C-15N spin coupling patterns of natural abundance 13C-NMR spectra. The 13C-NMR spectra of brain extracts were obtained from rabbits infused with 15NH4Cl with or without intraperitoneal infusion of the GS inhibitor, L-methionine DL-sulfoximine, in a reasonable acquisition time period. When 15NH4Cl was infused, [5-15N]glutamine and [2-15N]glutamine concentrations reached 5.2 mumol/100 mg protein and 3.6 mumol/100 mg protein, respectively, which indicates the relatively high activity of reductive amination of 2-oxoglutarate in the glutamate dehydrogenase reaction. The low concentration of [2-15N]glutamate, which is about 30% of that of [2-15N]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-sulfoximine, a flux of 15NH4+ via the residual activity of GS was accompanied by an apparent increase of [2-15N]glutamate and [15N]alanine concentrations (2.9 mumol/100 mg protein and 1.8 mumol/100 mg protein, respectively). These findings and those obtained from 13C-13C isotopomer analysis (Lapidot and Gopher, 1994b) suggest that astrocytic 2-oxoglutarate is partially utilized (together with an amino group donor) as a precursor for neuronal glutamate in the hyperammonemic brain when GS is inhibited. This process can partly replace GS activity in metabolizing ammonia in the hyperammonemic rabbit brain.
Lapidot
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Department of Organic Chemistry, Weizmann Institute of Science, Rehovot, Israel.
Gopher
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D000596:Amino Acids / Q000187:drug effects / Q000378:metabolism; D000641:Ammonia / Q000097:blood* / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D001923:Brain Chemistry / Q000187:drug effects; D002247:Carbon Isotopes; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D018377:Neurotransmitter Agents / Q000378:metabolism; D009587:Nitrogen Isotopes; D011817:Rabbits
D000596:Amino Acids; D002247:Carbon Isotopes; D018377:Neurotransmitter Agents; D009587:Nitrogen Isotopes; D000641:Ammonia