A 15N-n.m.r. study of cerebral, hepatic and renal nitrogen metabolism in hyperammonaemic rats

Abstract

1. Rats were infused with 15NH4+ or L-[15N]alanine to induce hyperammonaemia, a potential cause of hepatic encephalopathy. HClO4 extracts of freeze-clamped brain, liver and kidney were analysed by 15N-n.m.r. spectroscopy in combination with biochemical assays to investigate the effects of hyperammonaemia on tissue concentrations of ammonia, glutamine, glutamate and urea. 2. 15NH4+ infusion resulted in a 36-fold increase in the concentration of blood ammonia. Cerebral glutamine concentration increased, with 15NH4+ incorporated predominantly into the gamma-nitrogen atom of glutamine. Incorporation into glutamate was very low. Cerebral ammonia concentration increased 5-10-fold. The results suggest that the capacity of glutamine synthetase for ammonia detoxification was saturated. 3. Pretreatment with the glutamine synthetase inhibitor L-methionine DL-sulphoximine resulted in 84% inhibition of [gamma-15N]glutamine synthesis, but incorporation of 15N into other metabolites was not observed. The result suggests that no major alternative pathway for ammonia detoxification, other than glutamine synthetase, exists in rat brain. 4. In the liver 15NH4+ was incorporated into urea, glutamine, glutamate and alanine. The specific activity of 15N was higher in the gamma-nitrogen atom of glutamine than in urea. A similar pattern was observed when [15N]alanine was infused. The results are discussed in terms of the near-equilibrium states of the reactions involved in glutamate and alanine formation, heterogeneous distribution in the liver lobules of the enzymes involved in ammonia removal and their different affinities for ammonia. 5. Synthesis of glutamine, glutamate and hippurate de novo was observed in kidney. Hippurate, as well as 15NH4+, was contributed by co-extracted urine. 6. The potential utility and limitations of 15N n.m.r. for studies of mammalian metabolism in vivo are discussed.

Steady-state in Vivo Glutamate Dehydrogenase Activity in Rat Brain Measured by 15N NMR

“…With the anesthetized rat still breathing, the cranium was opened by scissor dissection and the brain was removed in toto and rapidly frozen in liquid nitrogen for preparation of a perchloric acid extract, as described previously (21). This procedure takes Ͻ30 s and is as fast as similar dissection-freezing methods (22,23) that have been shown to yield brain ammonia, glutamate, and glutamine concentrations that are in good agreement with those obtained by freeze-blowing (24).…”

“…Animal Preparation and Ammonia Infusion-Male Wistar rats (250 -300 g) were anesthetized by the intraperitoneal injection of sodium pentobarbital (Nembutal; 40 mg/kg body weight), and prepared for ammonia infusion through the femoral vein (21). Two infusion protocols were used to achieve different steady-state brain ammonia concentrations for measurement of in vivo GDH activities.…”

Long-Range1H-15N Heteronuclear Shift Correlation at Natural Abundance: a Tool To Study Benzothiazole Biodegradation by TwoRhodococcusStrains

“…In our case, the delay to allow n J NH correlations was set to 80 and 140 ms in order to select assumed 3 J NH (6.2 Hz) and 4 J NH (3.5 Hz), respectively. As chemical shift is concerned, since there is not a single chemical shift referencing, we used CH 3 NO 2 as an external reference (0 ppm) and more precisely enriched urea (for practical convenience) calibrated at Ϫ306 ppm from nitromethane (13). The chemical shifts are reported negative upfield from CH 3 NO 2 resonance.…”

“…However, most of the studies rely on specific enrichment of the compounds (e.g., 2 H, 13 C, 15 N, and 19 F). To overcome the difficulty of labeled-compound synthesis and also to increase the sensitivity of detection of metabolites, in situ 1 H NMR has been recently developed that allows the study of molecules at natural abundance directly on the incubation medium without any previous purification step.…”

NH4+ assimilation in the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton, a 15N‐NMR study

“…No peak due to NH4^ was seen in spectra from intact mycelium, suggesting that on entering the mycelium •'NH4'^ was rapidly metabolized. However, the absence of "NH4^ resonance may be due in part to the relatively long T, of the NH4^ N atom (Farrow et al, 1990;Robinson et al, 1991 ;Fox et al, 1992).…”