J. Sci. Fd Agric. 1975, 26, 1785-1791

Formation of C- and S-Nitroso Compounds and their Further Reactions“

John Gilbert, Michael E. Knowles and David J. McWeeny

Ministry of Agriculture, Fisheries and Food, Food Science Division, Colney Lane, Novwich NR4 7UA

(Manuscript received 14 May 1975 and accepted I Aiigusf 1975)

The possible involvement of nitrite in C- and S-nitrosation reactions with either food components or food additives is reviewed. C-nitrosation of phenols has been shown to occur in smoked bacons with the formation of trace amounts of nitrophenols, and protein-nitrite interactions have been studied under simulated stomach conditions where three products were identified. Also some theoretically possible reactions of nitrite with the activated methylene groups of creatinine and ketones are considered. The implication of reaction with sulphydryl groups of meat proteins with loss of nitrite in meat products and of the potential formation ot S-nitrosothiols is discussed with reference to current literature.

1. Introduction

Nitrite is a reactive species and in a food matrix can be lost by a variety of routes through interaction with food components. Interest in these reactions naturally centres on their relationships to quality and safety of foods and studies on nitrite reactivity have concentrated on flavour, colour, nitrosamine formation and micro- biology. After allowing for nitrite consumed by these major routes, during the course of curing and storage of meat products, a significant proportion of nitrite (estimated at about 20731 is unaccountably ‘‘lost’’ in terms of a total balance. Although a pro- portion of this nitrite may have been lost in the form of gaseous products (nitric oxide, nitrous oxide and nitrogen,ZJg there is stillaneed to consider the nature of other possible reactions. The aim of this paper is to draw attention to some other theoretically possible reactions which may account for the “missing” nitrite, and to mention some of our own work in identifying certain nitrite-derived products both in model systems and in a foodstuff.

2. General reactions of nitrite

A variety of reactions in which nitrite might participate is outlined in Figure 1. The first is C-nitrosation of an activated aromatic nucleus, such as a phenol, where a

a This paper is the text from which verbal presentation of more limited context was derived for the SCIjIFST Symposium “The Ro!e of Nitrate/Nitrite in Food”, London, March 1975.

1785

1786 J. Gilbert et a1

facile substitution reaction yields the corresponding nitrosophenol. Nitrosation may occur ortho and/or para to the hydroxyl group and the initial product, the nitroso compound, will be readily susceptible to oxidation to the corresponding nitro com- pound. The phenolic substrate could be present as a food additive, e.g. as a component of smoke flavour or naturally present as a protein amino acid such as tyrosine; reaction with the tyrosine moiety will, of course, modify the protein itself. C-nitro- sation reactions are pH independent from pH 1-5,4 and kinetically they are generally

R'- co -cH, - R" --+ R'- co - CH - R" ==== R' - co -C - R" I1 NOH

I NO

OH OH Me \ Me I Me I

"aN

R - SH + HONO ==== R - S -NO + H,O

Figure 1. General reactions of nitrite. ( I ) C-nitrosation of phenol; (2) C-nitrosation of a ketone to form the oxime; (3) C-nitrosation of creatinine; (4) S-nitrosation of a sulphydryl compound.

likely to be more favoured than N-nitrosation. A comparison of pseudo first order rate coefficients at pH 1.5 and 25°C for the interaction of dimethylamine (to form N-nitrosodimethylamine) and phenol (to form p-nitrosophenol) with 3 x l o - 3 ~

sodium nitrite shows phenol to react about lo4 times more rapidly than dimethyl- a m i ~ ~ e . ~ The second and third C-nitrosation reactions shown in Figure 1 occur with an activated methylene group in an organic substrate. Here the product will be the corresponding oxime following irreversible prototropic rearrangement. The substrate

Formation of C- and S-nitroso compounds 1787

may be present as a ketone from fat oxidation (in reaction (2)) or naturally occurring as in the example of creatinine (shown in reaction (3)).

Tn the laboratory preparation of a-oximino ketones the nitrosating agent is generated from an alkyl nitrite in the presence of a strong acid,6 but lower yields can be obtained simply by the action of nitrous acid on the ketone-as would be the situation in a cured meat. Recently the in vitro reactions of creatinine with nitrous acid have been reported7 the products being identified as creatinine-5-oxime and 1 -methylhydantoin- 5-oxime. Our own measurements (see Table 1) of both creatine and creatinine have confirmed the relatively large amounts of each which occur in muscle tissue, but after typical Wiltshire-style curing and when allowances are made for leaching losses of creatine and creatinine during the immersion period in the brine, the total figures for

Table 1. Creatine and creatinine levels in pork and bacons (% wt. wt. averaged values)

Sample Creatine Creatinine

Salt pork 0.35 0.035 Bacon 0.36 0.035 Smoked bacon 0 .38 0.041

creatine and creatinine are the same for salt pork and bacon. Whilst this does not preclude the occurrence of the reaction, it does seem that only trace amounts of these products can be present in cured meats. The last reaction of nitrite shown in Figure 1 is its interaction with sulphydryl compounds-an example of S- rather than C- nitrosation. Meat proteins could provide free -SH groups as substrates for such reactions. Indeed the amino acid cysteine has been shown to react in solution with an equimolar amount of nitrite to yield a red crystalline products identified as S- nitrosocysteine. Studies of such reactions showed them to proceed at considerably slower rates in weakly acid solutions (e.g. at a pH of 5.0, approximating meat condi- tions) than under stronger acid conditions (pH 2.3). However despite this reduction in the rate of reaction, in the competitive situation at pH 5.0 it was found that reaction of nitrite with -SH occurred much more rapidly than with -NHz groups.*

3. C-Nitrosation reactions 3.1. Nitrite interaction with phenols in smoked bacongJO Nitrite interaction with phenols in smoked bacon represents an example of a C- nitrosation reaction involving a food additive.

The phenols in smoked bacon can be present at levels ranging up to a few hundred parts/106 and are mainly responsible for its smoked flavour.gJ1 The principal phenols involved in smoked bacon are phenol itself, ortho, meta and para cresol, guaiacol, syringol and their para-substituted derivatives. When a liquid smoke-which is a convenient way of experimentally handling wood-smoke, is nitrosated on its own under fairly drastic conditions, then the phenols behave in the manner shown in

1788 J. Gilbert et at

Figure 1. A mixture of ortho and para nitro phenols are formed after undergoing oxidation via the intermediate nitroso compounds. Of the mixture the products are however largely ortho-nitro because in the majority of cases the para position is blocked by an alkyl substituent. This model nitrosation of a liquid smokelo enabled us to identify the kinetically and sterically most favoured products formed under these reaction conditions, and characterise these compounds unequivocally in terms of mass spectra, etc. The next stage was to examine both traditionally smoked bacons and bacons “smoked” with liquid smokes. They were analysed in the raw state, after frying and the volatiles collected from frying. The phenols were extracted after precipitation of the meat protein and fractionated according to acidity, the final concentrate being examined by gas chromatography and mass spectrometry.

Of the many nitrogen containing components extracted from the bacons which appeared to correspond with compounds in the nitrosated smoke, three were clearly identified as ortho-nitro-m-cresol, ortho-nitroguaiacol and ortho-nitro-4-methyl- guaiacol. These three compounds were found in both types of fried bacon and in the volatiles from the liquid smoke prepared bacon. Only these three compounds were unequivocally identified (on the basis of g.c. retention data and low resolution mass spectra) but the evidence suggests that many more are present and many tentative identifications have been made. It remains for these other compounds to be identified, for measurements to be made of the amounts present, and for their significance to be assessed. Extraction problems made quantitative analysis difficult but present

I CH

+NH, /“”\ coo- +N$, ‘COO- /CH\

+NH, coo-

CH

+NL, ‘coo- /CH,

+NH, coo- Figure 2. Reactions of nitrite with tyrosine. (I) Tyrosine; (11) 3-nitrosotyrosine; (111) 3-nitro-

tyrosine; (IV) tyrosine-3-diazonium nitrate; (V) 3,4-dihydroxyphenylalanine.

Formation of C- and S-nitroso compounds 1789

indications are that quantities present may be expressed in terms of parts/106 rather than parts/lOg (as in the case of nitrosamines).

3.2. Nitrite interaction with tyrosine To investigate potential C-nitrosation reactions involving proteins the interaction of albumin with nitrite has been studied under conditions of pH and temperature normally found in the human stomach.12 In an unbuffered system at an initial pH of 2.5, reaction between bovine serum albumin and sodium nitrite was carried out at 37°C for 2 h. The yellow product after dialysis was hydrolysed by conventional acid and by enzymic methods and then the amino acids were analysed by ion-exchange chromatography. Together with the expected protein amino acids four “abnormal” peaks were detected. Three of these four products of nitrite interaction were identified on the basis of their elution times compared with authentic compounds. These were shown to be 6-hydroxynorleucine, 3-nitrotyrosine and 3,4-dihydroxyphenylalanine (DOPA). The 6-hydroxynorleucine is believed to arise by nitrous acid deamination of lysine and the other two products are formed by the route shown in Figure 2. Tyrosine (I) initially undergoes C-nitrosation, ortho to the phenolic hydroxyl group forming 3-nitrosotyrosine (11). The nitrosotyrosine can undergo nitric oxide or aerial oxidation to 3-nitrotyrosine (111), or with nitric oxide can form the intermediate 3-diazonium nitrate (IV), which readily hydrolyses to the corresponding hydroxyl compound-3,4-dihydroxyphenylalanine (V).

After showing that albumin could interact with nitrite under stomach conditions the next step was to subject bacon to the same treatment. In this case there was no evidence for the presence of 3-nitrotyrosine although there were some indications of a peak in the position of 3,4-dihydroxyphenylalanine. This identification however has only been tentative because of problems of analysis.

4. S-Nitrosation reactions

There are many reports in the literature which implicate the sulphydryl groups of meat protein either in the mechanism of formation of typical cured meat colours13J4 (as reductants) or in nitrite depletion by “nitrite binding”. Hence there have been recently made a number of studies of both the amount and changes in -SH content of meats, and of the chemical properties and stabilities of S-nitroso thiols (the pro- ducts of S-nitrosation reactions). The stability of simple nitroso thiols (i.e. those of cysteine and glutathione) has been found to be low and indeed 50% decomposition in solution occurred in a matter of hourss. In dilute solution decomposition occurs with liberation of nitrous acid and reformation of the parent sulphydryl compound.

R-SNO + H 2 0 S R S H + HN02

Nevertheless, despite this instability it still remains possible that higher molecular weight SH compounds occurring in meat could form nitrosothiols which would remain stable even under weakly acid conditions.

Other workers have examined the role of -SH groups in nitrite depletion through protein-nitrite binding by measuring the rate of nitrite depletion both before and

1790 J. Gilbert et al.

after blocking of the -SH g r o ~ p s . l ~ ? ~ When the SH group was blocked by 4-vinyl- pyridine3 there was some depression in the rate of nitrite depletion, but it did not occur to such an extent as to suggest that the -SH groups played averyimportant role in the mechanism. On the other hand when cysteine (30 mM) was incubated with nitrite (4 mM) for 5 h at 60°C in a meat slurry the nitrite loss was greater by 30-50 % of that in a “control” with no added cysteine.16 In other work reaction between nitrite and the sulphydryl groups of myosin (in equimolar ratios) at 100°C and pH 5.0 for 1 h only showed a 10 % loss in sulphydryls and free nitrite.17 Half of this nitrite lost was bound to myosin as nitrosothiols, the nitrosothiol group being easily split during prolonged heat treatment.17 Measurements on a large number of differing types of cured meat products have indicated a correlation between the level of nitrite and the number of free -SH groups, viz. the higher the addition of nitrite the lower the SH content.l*>s On prolonged storage of certain of these products the slow continuous decrease in -SH groups is said to indicate progressive reaction with nitrite.18

Clearly from these reports in the literature there is some evidence to suggest -SH involvement with nitrite. However the mechanism of reaction and the nature of the products need elucidation and this is an area of work which might reward more detailed investigation.

5. Conclusion

During the last few years there have been a number of major investigations of the factors involved in the formation of traces of nitrosamines in some cured meats; at the same time there have been a lesser number of studies on the other reactions of nitrites in food. Whilst the information on these other reactions is still fragmentary it is growing steadily and already certain features are becoming recognised as important. Kinetic studies in simple solution systems show some of the C-nitrosation reactions to be very much faster than N-nitrosation. This is confirmed by the detection of products of C-nitrosation of smoke phenols in bacon in relatively large amounts. The occurrence of reactions of nitrite with sulphur compounds, particularly thiols in meat has been demonstrated and their role is being investigated from a number of standpoints. Finally the failure to detect nitrosation products of tyrosine, creatine and creatinine in meat when they can be formed fairly readily in solution emphasises the major difference between reactions in a meat matrix and in simple solutions. Whilst the outcome of these investigations cannot be anticipated one might hope that they will help in assessing the role of C- and S-nitrosations in moderating the amount of N-nitrosation which occurs in meat and may also help to improve the chemical understanding of some of the practical effects, such as flavour change which are associated with the use of nitrite in meat.

References 1. Knowles, M. E. Unpublished results. 2. Walters, C. L. Proc. 17th Europ. Meeting of Meat Kes. Workers, Bristol 1971, p. 182. 3. Olsman, W. J . Proc. Znt. Symp. Nitrite Meat Prod. 1973, p. 129.

Formation of C- and S-nitroso compounds 1791

4. Challis, B. C.; Lawson, A. J. J. chem. SOC. B, 1970, 770. 5. Challis, B. C. Nature, Lond. 1973, 244, 466. 6. Smith, P. A. S. Open Chain Nitrogen Compounds 1966, Vol. 2, Benjamin Inc. 7. Archer, M. C.; Clarke, S. D.; Thilly, J. E.; Tannenbaum, S . R. Science 1971, 174, 1341. 8. Mirna, A.; Hofmann, K. Fleischwirtschaft 1969, 49, 1361. 9. Knowles, M. E.; Gilbert, J.; McWeeny, D. J. J . Sci. Fd Agric. 1975, 26, 189.

10. Knowles, M. E.; Gilbert, J.; McWeeny, D. J. J. Sci. Fd Agric. 1975, 26, 267. 11. Gilbert, J.; Knowles, M. E. J. Fd Technol. 1975, 10, 245. 12. Knowles, M. E.; McWeeny, D. J.; Couchman, L.; Thorogood, M. Nature, Lond. 1974,247,288. 13. Watts, B. M.; Erdman, A. M.; Wentworth, J. J. agric. Fd Chem. 1955, 3, 147. 14. Fox, J. B.; Ackerman, S. A. J. Fd Sci. 1968, 33, 364. 15. Olsman, W. J.; Krol, B. Proc. 18th Europ. Meeting of Meat Res. Workers, Ontario, Canada

1972, p. 409. 16. Fox, J. B.; Nicholas, R. A. J. agric. Fd Chem. 1974, 22, 302. 17. Kubberad, K.; Cassens, R. G.; Greaser, M. L. J. Fd Sci. 1974, 39, 1228. 18. Susic, M.; Hofmann, K.; Manojlovic, D.; Nickolic, G . Fleischwirtschaft 1974, 54, 1081. 19. Woolfold, G.; Casselden, R. J.; Walters. C. L. Biochem J. 1972, 130, 82P.

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