Labelled ammonia

H. H. Janzen and Y. Bruinsma, Methodology for the quantification of root and rhizosphere nitrogen dynamics by exposure of shoots to N labelled ammonia. Soil Biology and Biochemistry 2/ 189 (1989). 

The first example of a dynamic flux analysis was a study performed in the 1960s [269]. In the yeast Candida utilis, the authors determined metabolic fluxes via the amino acid synthesis network by applying a pulse with 15N-labeled ammonia and chasing the label with unlabeled ammonia. Differential equations were then used to calculate the isotope abundance of intermediates in these pathways, with unknown rate values fitted to experimental data. In this way, the authors could show that only glutamic acid and glutamine-amide receive their nitrogen atoms directly from ammonia, to then pass it on to the other amino acids. 

Investigation of the deethylation with N-labeled ammonia showed that in 18 the label is incorporated for about 100% in the pyrimidine ring. Evidently the Sn(ANRORC) mechanism is operative in the replacement of the quaternary ring nitrogen by the amino nitrogen. The initial step in this deethylation reaction will probably be adduct formation on C-6, i.e., 17. Addition at C-2 is less likely because of the presence of the phenyl group at that position (Scheme III.12). 

Similar reactions were also observed with 1-nitropurine derivatives. Treatment of 2, 3, 5 -tri-0-acetyl-l-nitroinosine (51) with N-labeled ammonia gave [l- N]-2, 3, 5 -tri-0-acetylinosine (52), which after deprotection yielded [1- V]inosine (53) (Scheme III.29) (95JA3665). 

The reduction of these compounds would give mole of labelled aniline, H moles of ordinary aniline, i mole of labelled ammonia and mole of ordinary ammonia, which is incompatible with the experimental results actually obtained. 

Interestingly, hydroboration-amination as an isotope incorporation methodology has led to the synthesis of isomerically pure 13N- and 15N-labeled primary amines from labeled ammonia [23-25] (Scheme 8). These labeled amines are valuable intermediates in pharmacology. 

Co-condensation of silicon atoms, generated from a resistively heated, doped silicon rod (1350 -1380 °C) with very diluted ammonia/argon gas mixtures (1 1000) at 10 K leads to the formation of a primary Si,NH3 n-adduct 1, which is stable under these conditions[2]. This compound can be identified by three prominent IR absorptions ( >013 3387.3, 1599.1, and 1185.6 cm ). The silicon-nitrogen stretching band is too low in intensity to be observed. These bands show the expected shifts when the co-deposition is repeated with isotopically labeled ammonia ( ND3 2525.4,1171.5, and 912.5 cm 3 3378.8, 1596.1, and 1180.0 cm ). 

Although the liver is the principal organ for the conversion of ammonia to urea, it has been demonstrated by Sporn et al. (S15) that this process can occur also in the brain, although the activity of the cycle is small. The urea cycle enzymes were later demonstrated in cerebral tissue by Tomlinson and Westall (T6), but the activities were very small, less than 1%, compared with the liver (R5). In vivo experiments in cats showed that N-labeled ammonia as ammonium acetate injected in the brain was found largely in glutamine. That injected in the body was found mostly as urea and free NH3 in the liver (B3). This suggests that glutamine is more important for the removal of ammonia in the brain whereas in the liver the urea cycle is more important. 

Cooper AJL, McDonald JM, Gelbard AS, Gledhill RF, Duffy TE (1979) The metabolic fate of N-labeled ammonia in rat brain. J Biol Chem 254 4982-4992. 

Nitrogen-13-labeled ammonia (ty2 = 10 min) is produced by reduction of 13N-labeled nitrates and nitrites that are produced by proton irradiation of water in a cyclotron. The reduction is carried out with titanium chloride in alkaline medium. 13N-NH3 is then distilled and finally trapped in acidic saline solution. Wester et al (1991) has used a pressurized target of aqueous ethanol, in which ethanol acts as a hydroxyl free radical scavenger to improve the... 

Tn recent years there has been a great deal of interest in understanding the mechanisms involved in ammonia metabolism in brain. The reasons for this interest are twofold ammonia is thought to be a major toxin contributing to the symptoms of encephalopathy associated with liver disease (1,2) and of Reye s disease (3) and earlier experiments with N-labeled ammonia suggested the existence of at least two distinct metabolic pools in brain (4). 

N-Labeled ammonia has been generated by the bombardment of methane gas (26) or metal carbides (27) with high-energy deuterons. The method that is now used at the Memorial Sloan-Kettering Cancer Center is to bombard water with protons. The chemical form of the generated... 

The avid accumulation of N-radioactivity within the myocardium following intravenous administration of N-labeled ammonia has been well documented (10, II, 67,68). From the known complexity of ammo-... 

Figure 6. Anion exchange radiochromatographic analysis of in rabbit septum effluent collected from 9.5 to 10 5 min after addition of labeled ammonia to perfusate...

As a final point, it should be noted that amino acids themselves exhibit some myocardial specificity (71, 72,73) and, thus, the metabolic-ally generated labeled amino acids contribute to total heart uptake and further confuse the issue. One can readily see that this use of N-labeled ammonia as a tracer for myocardial metabolism presents a very complex situation in which determination of even relative metabolic rates is di Scult at best and absolute measurements become virtually impossible. 

Tn this volume, with several chapters devoted to the assimilation and metabolism of N-labeled ammonia and nitrate into amino acids, purines, and pyrimidines that are the building units of cellular macromolecules, it is perhaps worthwhile to consider the larger context of basic inorganic biochemistry, or how cells view and utilize the elements of the periodic table (1,2,3),... 

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