Nitrites, ascorbic acid reaction

Cytochrome c is a heme containing protein which occurs in muscle at lower concentrations than does myoglobin. It was demonstrated some time ago (18) that oxidized cytochrome c reacts with gaseous nitrite oxide to produce a nltrosyl compound. Recent work (19, 20, 21) has examined the reactions of cytochrome c with nitrite and the contribution of the product formed to cured meat color in considerably more detail. The general conclusion is that even at the pH normally encountered in meat, the reaction can take place in the presence of ascorbic acid but probably does not affect meat color because of the unstable nature of the reaction product and the low concentration. 

Mirvish (53,54) discovered that vitamin C could inhibit ni-trosation reactions. The purely chemical interaction of ascorbic acid with nitrite has been studied for theoretical reasons and because of its importance in the preservation of foods. This interaction has received increased attention for minimizing the presence of nitrosamines and nitrosamides in the environment, and especially in foods. We have studied the relationship in gastric carcinogenesis between high levels of nitrite, including pickling, and of vitamin C as a protective and inhibiting element. 

Inhibitors of nitrosation generally function by competing with the amine for the nitrosating agent. An inhibitor would thus react with nitrite at a faster rate than with amines. The inhibition reaction has recently been reviewed ( 35). The ability of ascorbate to act as a potent inhibitor of nitrosamine formation has resulted in the use of the vitamin in nitrite-preserved foods and pharmaceuticals. Furthermore, the effectiveness of ascorbate in inhibiting nitrosamine formation is dependent on (1) the concentration of ascorbate (an excess is required) (2) pH (ascorbate is nitrosated 240 times more rapidly than ascorbic acid) (3) the reactivity of the amine toward nitrosation and (4) the extent of oxygenation of the system. 

Principally the same, but chemically simpler, sequence was used to prepare arylnitro anion-radicals from arylamines, in high yields. For instance, aqueous sodium nitrite solution was added to a mixture of ascorbic acid and sodium 3,5-dibromo-4-aminobenzenesulfonate in water. After addition of aqueous sodium hydroxide solution, the cation-radical of sodium 3,5-dibromo-4-nitro-benzenesulfonate was formed in the solution. The latter was completely characterized by its ESR spectrum. Double functions of the nitrite and ascorbic acid in the reaction should be underlined. Nitrite takes part in diazotization of the starting amine and trapping of the phenyl a-radical formed after one-electron reduction of the intermediary diazo compound. Ascorbic acid produces acidity to the reaction solution (needed for diazotization) and plays the role of a reductant when the medium becomes alkaline. The method described was proposed for ESR analytical determination of nitrite ions in water solutions (Lagercrantz 1998). 

The reaction of nitrous acid with ascorbic acid (41) also involves 0-nitrosa-tion. The final product is dehydroascorbic acid which arises from the nitrite by a series of rapid reactions involving homolytic fission and forming nitric oxide (Dahn et al., 1960). 

Many samples have redox potentials such fiiat fiiey can be oxidized by iodine. Therefore, file iodine in file titrant may be consumed by readily oxidizable samples fiiat will give a false high value for file water content. Some common substances fiiat can be oxidized by iodine are ascorbic acid, arsenite (As02 ), arsenate (As04 ), boric acid, tetraborate (3407 ), carbonate (COs ), disulfite (8205 ), iron(ll) salts, hydrazine derivatives, hydroxides (OH ), bicarbonates (HCOs"), copper(l) salts, mercaptans (RSH), nitrite (N02 ), some metal oxides, peroxides, selenite (SeOs "), silanols (RsSiOH), sulfite (SOs ), tellurite (TeOs ), fiiiosulfate (8203 ), and tin(ll) salts. For situations such as fiiese where file material under analysis reacts wifii iodine, an oven can be used to liberate fiie moisture from file sample, which is fiieii carried into file reaction vessel and titrated wifiiout interference. 

Ascorbic acid is known to react rapidly with nitrite as well as with other nitrosating agents (16). This, along with its low toxicity and known nutritional importance, has naturally led to an evaluation of its usefulness as an inhibitor of nitrosation reactions (17). We have begun both experimental and theoretical studies of the interactions of ascorbic acid with amines, amides, and nitrosating agents. Our approaches and results are described in the following sections. 

In principle, then, ascorbic acid appears to have considerable importance as an inhibitor of in vivo nitrosation of ingested or biosynthesized amines via endogenous nitrite. However, this potential is far from realized. The reaction conditions actually encountered in living organisms are complex and varied and—perhaps more importantly—the in vitro interactions among ascorbic acid, nitrite, and amines are not straightforward. 

In addition to these aspects of the amine nitrosation reaction, the reactions of ascorbic acid with various components of the nitrite equilibria involve transformations that are also aflFected by the presence or absence of oxygen (1,23), Some of these are shown schematically in Scheme 3. If attention is then focussed on the reactions of ascorbic acid/ascorbate rather than on the nitrosation of amines, it can be seen that the amount of ascorbic acid or ascorbate available for inhibition of nitrosation can be diminished by the presence of oxygen. 

The observations above, taken together, suggest that the reactions among amines, nitrite, and ascorbic acid under physiological conditions would be expected to be extremely complex and that kinetic studies might be experimentally intractible even in vitro. A summary of the equilibria and reactions that might occur in an anaerobic model system is shown in Scheme 4. The question "How does ascorbic acid affect... 

Figure 2. Reaction of ascorbic acid with nitrite and air at pH 4 (25°C), (Reproduced, with permission, from Ref. 24.)...

Figure 7. Time course of nitrosomorpholine formation. The reaction system was kept under N2 unless stated otherwise, 10 mM morpholine, 10 mM nitrite, pH 4, 0°C, varying oxygen a, without ascorbic acid b, [Asc] = I mM c,d, [Asc] = 3 mM, degassing of the solution was done by bubbling N2 through pasteur pipet for 5 min d,f, exposed to air 30 min after the reaction started e,f, [Asc] = 3 mM, degassing of the solution was done by bubbling N2 through gas dispersion tube for 1 h.

N-Nitrosamines are formed in foods by the reaction of secondary and tertiary amines with a nitrosating agent, usually nitrous anhydride, which forms from nitrite in acidic, aqueous solution. NDMA is the most common volatile amine found in food. Food constituents and the physical make-up of the food can affect the extent of nitrosamine formation. Ascorbic acid and sulfur dioxide have been used to inhibit the formation of nitrosamines. 

When the reaction was buffered with solid CaC03, the pH of the aqueous phase after sparking was 7.1 and amino acid yields were increased 2-15 fold. Moreover, it was found that direct acid hydrolysis of the reaction mixtures gave dramatically lower amino acid yields than when an oxidation inhibitor was added prior to hydrolysis. When hydrolysis is carried out in the presence of ascorbic acid, which is typically added to natural water samples to inhibit oxidation by nitrate and nitrite (/2), recoveries are increased a factor of 10-100 fold (Table I). Thus, the yield of amino acids from the neutral gas mixture with the addition of ascorbic acid prior to acid hydrolysis is comparable to that obtained from a reducing gas mixture (Table I). 

To show that the source of the amino acids in our experiments was not the result of the reaction of the various nitrogen species produced in the reaction with ascorbic acid, we reacted ascorbate individually and in combination with ammonia, hydrazine, nitrite, and nitrate. Very low traces of amino acids were produced in these reactions, indicating that the amino acids detected are in fact produced from the electric discharge reaction. While ascorbic acid is not likely to have been an abundant prebiotic species, oxidation could have been inhibited by other available chemical species such as sulfides and reduced metal ions. 

Ascorbic acid has been shown to exert a protective effect against carcinogenesis by nitrite reacting with aminopyrine (123), morpholine (174), piperazine (Ref. 174, p. 175), and raw fish (204) as well as to reduce the acute hepatotoxicity resulting from feeding nitrite and dimethylamine (61) or nitrite and aminopyrine (Refs. 145, 168, and 169 Ref. 174, p. 160). Studies with preformed nitrosamines have shown that ascorbic acid exerts little or no protective effect (Ref. 174, p. 175). Ascorbic acid does not react with amines, nor does it increase the rate of nitrosamine decomposition it exerts its protective effect largely by reaction with nitrite... 

Phosphate is mostly determined photometrically through the reaction with ammonium heptamolyb-date in acidic medium in the presence of potassium antimonyl tartrate as a catalyst. 12-Molybdophos-phoric acid is formed that upon reduction with, for example, ascorbic acid has a blue color. The lower limit of detection is lOpgl. Chromium(VI), nitrite, and high concentrations of chloride and iron interfere. The lower recoveries caused by iron are eliminated by addition of hydrogensulfite. 

Miura, Y. Hatakeyama, M. Hosino, T. Haddad, P.R. Rapid ion chromatography of L-ascorbic acid, nitrite, sulfite, oxalate, iodide and thiosulfate by isocratic elution utilizing a postcolunm reaction with cerium(IV) and fluorescence detection. J. Chromatogr. A, 2002, 956,11-8A. 

Nitrosamines are produced in foodstuffs from reactions of nitrites (added as preservatives to bacon and other processed meat products) with amines in (or derived from) the foodstuff, particularly during cooking, e.g., frying anti-oxidants such as ascorbic acid (vitamin C) are added to such foodstuffs to inhibit formation of nitrosamines. A source of nitrosamines that may be of interest to some analytical chemists is beer, in this case attributed (Sen 1983) to reaction of nitrogen oxides with alkaloids (usually present in germinated malt) during the drying process. NMDA can also be formed inadvertently in a number of industrial processes. 

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