Quantitation of Metabolic Compartmentation in Hyperammonemic Brain by Natural Abundance ¹³C‐NMR Detection of ¹³C‐¹⁵N Coupling Patterns and Isotopic Shifts

Abstract

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 "N enrichments of brain metabolite a-amino and amide positions of glutamine, glutamate, and alanine were determined by the indirect detection of ' Wlabeled compounds of the lT-''N spin coupling patterns of natural abundance IT-NMR spectra. The 'IC-NMR spectra of brain extracts were obtained from rabbits infused with "NH,CI with or without intraperitoneal infusion of the GS inhibitor, L-methionine DL-sulfoximine, in a reasonable acquisition time period. When "NH,CI was infused, ]alanine concentrations (2.9 pmoM00 mg protein and 1.8 pmol/l00 mg protein, respectively). These findings and those obtained from 'T-''C 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.

Multinuclear NMR Spectroscopy Studies on NH₄Cl-Induced Metabolic Alterations and Detoxification Processes in Primary Astrocytes and Glioma Cells

“…The present data show that treatment of glial cells with pathologically high ammonium concentrations for 3 h increases the intracellular glutamine concentration, and are in good accord with results from previous 1 H-NMR studies of acute hyperammonemia in human and rat brain [12][13][14][15]. After an ammonia load, the glutamate concentration transiently decreases in brain during hyperammonemia (see Cooper and Plum [9] for review), as it is consumed by glutamine synthesis, but recovers after 3 h [42,43]. This is in agreement with our cell studies, in which the portion of relatively newly synthesized 13 C-labelled glutamate is increased in ammonium-treated cells.…”

“…Nonetheless, the glutamate dehydrogenase (GDH, EC 1.4.1.2) is far less efficient at removing abundant ammonia than is GS. These results correspond to 13 C-15 N-NMR studies in rabbit brain by Lapidot et al [42] and studies of [ 15 N]glutamate metabolism in astrocytes by Yudkoff et al. [44], who also observed a minor active flux via GDH.…”

“…The increased anaplerotic metabolism is confirmed by the large increase in 13 C enrichment in positions C2 and C3 of glutamine and glutamate compared to C4 during hyperammonemia (table 3, 4). Lapidot and Gopher [42,49] also found an increase in PC activity in rabbit brain under hyperammonemic conditions, which corresponds well with increased glutamine synthesis without any reduction in glutamate concentration during hyperammonemia.…”

Non-Invasive Methods for Studying Brain Energy Metabolism: What They Show and What It Means

“…Thus, glutamine synthase flux reflects largely the glutamate/glutamine shuttle [46][47][48], but is also a means of ammonia detoxification, coupled to anaplerosis [48,54,55]. 'Mitochondrial exchange' between (mitochondrial) 2-oxoglutarate and (cytosolic) glutamate is due to aminotransferases [46,56] and also glutamate dehydrogenase [57,58]. Pyruvate recycling occurs in vivo [43], mainly in astrocytes [59].…”

In vivo Investigation of Glutamate–Glutamine Metabolism in Hyperammonemic Monkey Brain Using ¹³C-Magnetic Resonance Spectroscopy

“…It is believed that the excess ammonia in the brain is detoxified by the process of glutamine synthesis, which is catalysed by glutamine synthetase localized in astroglia. The rate of turnover of ammonia in the rat brain has been estimated using the 15 N isotope [3][4][5][6]. Noninvasive in vivo investigation by 13 C magnetic resonance spectroscopy (MRS) of cerebral glutamate and glutamine metabolism, which play a central role in ammonia metabolism in the brain, should provide valuable information about the biochemical significance of amino acid metabolism in the brain under physiological and pathological conditions.…”