Ammonia from Arginine

Microscopically, it may be difficult to separate O. oeni from some species Lactobacillus due to morphological similarity. In these cases, Garvie (1984) suggested that separation could be made using ammonia formation from arginine (Section 2.4.2). The described method relies on the heterofer-mentation-arginine medium (Section 13.6.3) used by Pilone et al. (1991) but without inclusion of fructose. 

Nessler s reagent contains mercury (II) iodide, which is dissolved in an aqueous solution of potassium iodide and potassium hydroxide to yield KsHgl4. The reagent is used to detect the presence of ammonia as per the following reaction  

The formation of a brick-orange color indicates the presence of ammonia. Nessler s reagent (CAS number 7783-33-7) is available from commercial sources and is not normally prepared in the laboratory. 

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PiLONE, G.J., M.G. Clayton, and RJ. van Duivenboden. 1991. Characterization of wine lactic acid bacteria Single broth culture for tests of heterofermentation. Mannitol from fructose and ammonia from arginine. Am. J. Enol Vitic. 42(2) 153-157. 

Many heterofermentative lactic acid bacteria have the ability to produce energy through the utilization of arginine in formation of ornithine, NH3, CO2, and ATP (Fig. 2.6). The ability of lactic acid bacteria to produce ammonia from arginine can be determined using the method outlined in Section 15.4.1. 

Heterocyclic enamines A -pyrroline and A -piperideine are the precursors of compounds containing the pyrrolidine or piperidine rings in the molecule. Such compounds and their N-methylated analogs are believed to originate from arginine and lysine (291) by metabolic conversion. Under cellular conditions the proper reaction with an active methylene compound proceeds via an aldehyde ammonia, which is in equilibrium with other possible tautomeric forms. It is necessary to admit the involvement of the corresponding a-ketoacid (12,292) instead of an enamine. The a-ketoacid constitutes an intermediate state in the degradation of an amino acid to an aldehyde. a-Ketoacids or suitably substituted aromatic compounds may function as components in active methylene reactions (Scheme 17). 

B12. Bessman, S. P., Shear, S., and Fitzgerald, J., Effect of arginine and glutamate on removal of ammonia from blood in normal and cirrhotic patients. New Engl. J. Med. 266, 941 (1957). 

As early as 1932, H.A. Krebs and K. Henseleit established that of the investigated amino acids, only ornithine was able to effect a real increase in the synthesis of urea from ammonia (although arginine also displayed low efficacy), (quot. 57) Thus it seemed possible to raise the turnover of ammonia in the metabolism process by using intermediates of the urea cycle. (127) (s. tab. 15.6) (s. fig. 3.10) To this end, ornithine aspartate (oral and parenteral route), arginine malic... 

The major emphasis in this chapter related to excretion is on ammonia and urea, particularly on the control of the latter as the normal excretion product. There are two major nitrogenous precursors of urea ammonia and aspartate. Before delving into the mechanism and control of urea synthesis, it would be wise to look at the different ways that these precursors can arise and the important tissues concerned with urea synthesis. In people, the predominant synthesis of urea occurs in the liver, with very small amounts of urea formed from arginine in other tissues. The complete synthesis of urea, from amino acids and other nitrogen sources, occurs via a process known as the urea cycle. 

Fig. 11.2.10. HPLC of human leucocyte interferon hydrolysate using post-column fluorescence detection with fluorescamine. Chromatographic conditions column, DC-4A (Dumim) (500 x 4.6 mm I.D.) mobile phase, 0.175 N sodium citrate, 2.5% isopropanol, pH 3.5 (Buffer A), 0.175 N sodium citrate, pH 3.45 (Buffer B), 0.2 N sodium citrate, 0.05% thiodiglycol, pH 4.1 (Buffer C), 0.52 N sodium citrate, 1 N sodium chloride, 0.05% thiodiglycol, pH 7.9 (Buffer D), elution was achieved with the following gradient Buffer A (15 min). Buffer B (17 min). Buffer C (24 min). Buffer D (41 min), flow rate, 18 ml/h temperature, 57 ° C. Peaks 1, aspartic acid 2, threonine 3, serine 4, glutamic acid 5, cysteine 6, proline 7, glycine 8, alanine 10, valine 11, methionine 12, isoleucine 13, leucine 14, norleucine 15, tyrosine 16, phenylalanine 17, histidine 18, lysine 19, ammonia 21, arginine. Reproduced from Stein and Brink (1981), with...

Pilone et al. (1991) have questioned the sensitivity of Nessler s reagent in detecting potentially low concentrations of ammonia produced from arginine initially present at concentrations described by Garvie (1967). Instead, they recommended that the concentration of the amino acid be increased from 0.3% to 0.6% (wt/vol). Using this increased concentration of L-arginine, they found that some strains of Leuc. oenos did, in fact, produce ammonia. The above procedure includes this recommendation. 

Figure 2.6. Formation of ornithine, ammonia, and carbon dioxide from arginine by some heterofermentative lactic acid bacteria.

Most of the ammonia (NH,) produced through deamination—removal of amino groups (NH,) from amino acids is converted to urea in the liver for excretion by the kidneys. To facilitate elimination, 1 mole of ammonia (NH,) combines with 1 mole of carten dioxide (CO,), another metatelic waste product. This compound is then phosphorylated to produce carbamyl phosphate. Carbamyl phosphate then combines with ornithine to form citrulline an intermediate in the urea cycle. The amino acid, aspartic acid, contributes another amino group (NH,), and citrulline is then converted to the amino acid arginine. Urea splits off from arginine forming ornithine, and the cycle is completed. Fig. U illustrates the urea cycle. 

The concept of the ornithine cycle arose from the observation that ornithine, citrulline and arginine stimulated urea production in the presence of ammonia without themselves being consumed in the process. 

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