December 1977 509

Studies on the Formation of Nitrosamines (VI)

The Effects of Organic Acids on the Rate of Nitrosation of Ureido Compounds

(Received May 17, 1977)

Miyako YAMAMOTO, Takashi YAMADA and Akio TANIMURA

(Department of Food Additives, National Institute of Hygienic Sciences: 18-1, Kamiyoga 1-chome, Setagaya-ku, Tokyo)

The effect of various organic acids, such as citric acid, tartaric acid, malic acid, tricarballylic acid and so forth, on the rates of nitrosation of hydantoic acid (HA) and methylurea (MU), which were ureido compounds, was studied. These organic acids accel- erated the rate of nitrosation of the ureido compounds. Extents of accelerating effect of the organic acids were greater in the compounds which have more carboxyl groups or hydroxy groups. The rate of nitrososarcosine formation from creative, one of guanido compounds, and nitrite was accelerated by citric acid, tartaric acid and sodium thiocyanate. The reaction of HA and sodium nitrite in presence of citric acid was first order with respect to each substance, and the following equation was set up: rate=k1[HA] [nitrite] +k2[citrate] [HA] [nitrite]. The rate of carboxymethyl nitrosourea (CMNU) formation from HA and nitrite was increased with lowering pH, and this pH dependence was not remarkably affected in the presence of citric acid.

1. Introduction 2. Methods In the previous paper,1) we reported that Measurements of the rate of nitrosation citrate and tartrate accelerated the rate of 2.1 The reaction of hydantoic acid (HA) nitrosation of ureas or carbamates, but did or methylurea (MU) with nitrite not affect the rate of nitrosation of secondary The rate of nitrosation was determined in amines. In contrast, thiocyanate accelerated the same manner as described in the previous only the rate of nitrosation of secondary paper.1) Aqueous solution of sodium nitrite amines. was added to aqueous solution of HA or MU. Citrate and tartrate are present in foods, The mixed solution was immediately adjusted and there are some ureido compounds in vivo. with aqueous solution of hydrochloric acid or The fact that citrate and tartrate accelerated sodium hydroxide to the desired pH and incu- only the rates of nitrosation of ureido com- bated at 37•Ž. About 5ml of the aliquots pounds interested us to study further con- were withdrawn from the reaction mixture cerning the effect of other organic acids and the at definite time intervals and poured onto structures of nitrosatable compounds or organic crystalline sulfamic acid, more than five equiv- acids which might take parts in accelerating alent to sodium nitrite, to stop the reaction. mechanism. The absorbance of the reaction mixture was The kinetic orders with respect to each immediately measured at 394nm for nitrosa- substance in nitrosation in the presence of tion of HA and at 392nm for that of MU. organic acids were also studied, because the When the effects of organic acids were de- order with respect to nitrite concentration termined, aqueous solutions of the acids were might depend on the reaction form of nitrite previousy mixed with aqueous solutions of HA in nitrosation.2•`7) or MU. Other procedures were the same as 510 J . Food Hyg. Soc. Vol.18, No.6

described above. Only in the case of nitro- 2.3 Effects of ethylenediaminetetraacetic sation of HA in the presence of citrate at pH acid (EDTA) or metal ions 3.5 and 4.0, citrate concentration and pH were Twenty millimoles of HA was reacted with adjusted with citric acid and sodium citrate, 50mM of sodium nitrite at pH 2.5 and 37•Ž because the addition of 50mM of citric acid in the presence or absence of EDTA, cupric lowered the pH of reaction mixture below chloride, calcium chloride, magnesium chloride 3.5. or citric acid. The rates of nitrosation were In each experiment, the reaction mixture measured in the same manner as described in omitting HA or MU was incubated and the •˜ 2.1. Deionized and distilled water was used absorbance of the solution was measured as a for the experiments except for the experiment blank value. Each blank value was subtracted with EDTA. from the corresponding experimental value. 3. Results The initial rates were calculated from the 3.1 Identification of the reaction products slopes of the time courses of the reaction. The CMNU was identified as described in the e values at wavelengths used for measure- previous paper.1) The identification of MNU ments were as follows: carboxymethylnitroso- was performed as follows. Five millimolars of urea (=nitrosohydantoic acid) (CMNU), ƒÃ384nm MU and 5mM of sodium nitrite were reacted =88;8) nitrosomethylurea (MNU), ƒÃ392nm=93.3) at pH 2.5 and 37•Ž for 10min, and sulf amic 2.2 The reaction of creative with nitrite acid was added. The absorption spectrum at Aqueous solution of sodium nitrite was 310•`420nm used for the reaction mixture was added to aqueous solution of creatine, and the same as that of the authentic MNU, and Amax the mixed solution was adjusted to pH 3.0 (392nm) was coincident with that of authentic and incubated at 37•Ž for 2hr. The amount one.3) After extraction of reaction mixture of nitrososarcosine (NS) formed from the re- with dichloromethane, aqueous layer exhibited action of creatine and sodium nitrite was no absorption above 350nm, and the absorp- determined in the same manner as described tion spectrum of dichloromethane layer showed previously.9) Thinlayer chromatography was three characteristic peaks which were coinci- performed using Silicagel HF254 (Merck) and dent with those of authentic MNU (379, 393, developing solvent, methyl acetate-iso-pro- 411nm3)). On a thin-layer chromatogram of panol-concentrated ammonium hydroxide (9: the extract (Silicagel HF254+388 (Merck), devel- 7: 4). The amount of NS was calculated from oping solvent: ethyl acetate-dichloromethane the absorbance of sample solution at 260nm (1:9)), only one spot which was coincident and ƒÃ260nm of NS (1500). with the authentic MNU was detected with The reaction mixture containing thiocyanate ultra-violet lamp (365.0nm). was colorless before extraction with ethyl It was confirmed that the nitrosation of HA acetate. But after extraction, ethyl acetate and MU could be followed by measuring the layer became yellow and the yellow crystal was absorbance at Amax of CMNU (394nm) and separated out by concentration of ethyl acetate. MNU (392nm), respectively, in the presence The yellow substance was also formed in the and absence of organic acids in the same man- ethyl actate extract from reaction mixture of ner as described in the previous paper.1) nitrite and thiocyanate. Thin-layer chromato- 3.2 Effects of various acids on the rate graphy was performed after filtration of the of nitrosation of HA or MU. yellow powder. The initial rate of CMNU formation from In each experiment, blank value was deter- 20mM of HA and 50mM of sodium nitrite mined as described in •˜2.1. at pH 2.5 and 37•Ž, in the presence of various The final concentration of each substance concentrations of citric acid, tartaric acid, ma- in the reaction mixture was 25mM for creatine, lic acid, lactic acid, tricarballylic acid, succinic 150mM for sodium nitrite and 100mM for acid, glutaric acid, gluconic acid and acetic acid citric acid, tartaric acid or sodium thiocyanate. were shown in Fig. 1. The rates of CMNU formation increased linearly with the concen- trations of organic acids. The accelerating December 1977 Studies on the Formation of Nitrosamines (VI) 511

effects were in a order of citric acid>tartaric The initial rates of MNU formation from acid>malic acid-tricarballylic acid>lactic acid 5mM of MU and 5mM of sodium nitrite at succinic acid. Acetic acid scarcely accelerated pH 2.5 and 37•Ž, in the presence of various the reaction. concentrations of citric acid, tartaric acid, malic acid, tricarballylic acid and sodium thio- cyanate were shown in Fig. 3. Thiocyanate had no effects on the rates of MNU formation. 3.3 The additive effect of citric acid and tartaric acid on the rate of nitrosa- tion of HA As shown in Table 1, initial rate of CMNU formation from 20mM of HA and 50mM of

Fig. 3. Effects of various organic acids and

Fig. 1. Effects of various organic acid on CMNU thiocyanate on MNU formation from MU

formation from HA and nitrite and nitrite The reactions of 20mM of HA and 50mM of The reactions 5mM of MU and 5mM of sodium

sodium nitrite in the presence of various con- nitrite in the presence of various concentrations

centrations of organic acids were carried out at of organic acids or thiocyanate were carried out

pH 2.5 and 37•Ž. at pH 2.5 and 37•Ž.

Fig. 2. The structures of organic acids 512 J. Food Hyg. Soc. Vol.18, No.6

Table 1. The Additive Effect of Citric Acid and Tartaric Acid on the Rate of CMNU Forma- tion from HA and Sodium Nitrite Twenty millimolars of HA and 50mM of sodium nitrite were reacted at pH 2.5 and 37•Ž in the presence and absence of organic acids.

sodium nitrite at pH 2.5 and 37•ŽC was 10.48 M/min when organic acid was not added. The rate of increment by the addition of 50mM of citric acid and 50mM of tartaric acid were 8.16M/min and 5.77M/min, respectively. The Fig. 4. Effect of pH on the rates of CMNU sum of these values agreed with the rate formation from HA and sodium nitrite in of increment (13.72M/min) by the addition of the presence and absence of citric acid 50mM of citric acid and 50mM of tartaric acid. 3.4 The rates of nitrosation of HA at various pH Changes of the rate of CMNU formation from 20mM of HA and 50mM of sodium nitrite at various pH in the presence and absence of 50mM of citric acid were shown in Fig. 4. The rate of CMNU formation increased with lowering pH in the presence and absence of citric acid. 3.5 Stoichiometric reaction of HA, sodium nitrite and citric acid Changes of the rate of CMNU formation from 5•`40mM of HA and 5•`40mM of sodium nitrite at pH 2.5 and 37•Ž in the presence of 0•`500mM of citric acid were shown in Fig. 5. When the concentration of citric acid was constant, the rates of CMNU formation were proportional to [HA]•~[NaNO2] as shown in Fig. 5. 3.6 Effects of acids on the reaction of creatine and sodium nitrite

The amounts of NS formed from 25mM of Fig. 5. Second-order rate plots for UMN U for- creatine and 150mM of sodium nitrite at pH mation in the presence or absence of citric 3.0 and 37•Ž for 2hr in the presence of 100 acid mM of citric acid, tartaric acid and sodium Reactions were carried out at pH 2.5 and 37•Ž; -•›- thiocyanate were shown in Table 2. These , citric acid 0mM; -• -, citric acid acids accelerated NS formation from creatine 200mM; -•¢-, citric acid 500mM. December 1977 Studies on the Formation of Nitrosamines (VI) 513

Table 2. Effects of Citric Acid, Tartaric Acid addition of sulf amic acid, because pH of the and Sodium Thiocyanate on Nitrososarcosine reaction mixture became less than 0.5. How- Formation from Creatine and Sodium Nitrite ever, they were quite stable in the reaction The reactions were carried out at pH 3.0 and mixture at pH 2.5 and 37•Ž during the ex- 37•Ž for 2hr. The concentrations of the com- periments. Therefore, it is improbable that ponents were 25mM for creatine, 150mM for citric acid increased the rates of CMNU for- sodium nitrite and 100mM for each anion. mation by retardation of CMNU decomposition in the reaction mixture. The experiments in •˜3.7 were performed to make clear the possibility of participation of citric acid as a chelating agent. If citric acid

played a role in acceleration of nitrosoureas formation as a chelating agent, there might be two possibilities, 1) Metal ions present in Table 3. Effect of Metal Ions on the Rates of the water used inhibited the formation of CMNU Formation from HA and Sodium nitrosoureas, but the inhibitory effect was Nitrite lost by chelating with citric acid. 2) Chelate The reactions were carried out at pH 2.5 and compounds which were formed from citric acid 37•Ž. The concentration of HA was 20mM and and metal ions present in the water used ac- that of sodium nitrite was 50mM. celerated nitrosation. However, no changes of the rates of CMNU formation were observed by addition of EDTA to the reaction mixture of HA and sodium nitrite. When Ca2+, Mg2+ or Cu2+ was added to the reaction mixture, no changes of the rates of CMNU formation were also observed. Therefore, metal ions had no effects on the rates of CMNU formation. Further, when both metal ions and citric acid were added, the rates of nitrosation were the same as those when only citric acid was added. Therefore, chelate compounds formed from metal ions and citric acid also had no effects and sodium nitrite. on nitrosation. 3.7 Effect of EDTA or metal ions on the According to Mirvish,3•`7) the main nitrosat- reaction of HA and sodium nitrite ing agent in the reaction of secondary amines When 1 or 10mM of EDTA was added to with nitrite under mild acidic condition is the reaction mixture containing 20mM of HA nitrous anhydride (N2O3), and the rates of and 50mM of sodium nitrite at pH 2.5 and nitrosation are proportional to the square of 37•Ž, no changes of the rates of CMNU for- nitrite concentration. On the other hand, the mation were observed. main nitrosating agent in the reaction of The rates of CMNU formation from 20mM ureas or urethanes with nitrite is nitrous of HA and 50mM of sodium nitrite at pH 2.5 acidium ion (H2NO2+), and the rates are pro- and 37•Ž in the presence of 10mM of metal portional to the nitrite concentration. It has ions or 100mM of citric acid were shown in been reported that thiocyanate forms nitrosyl Table 3. When metal ions were added to the thiocyanate (NOSCN)T and accelerates the reaction mixture with and without citric acid, nitrosation of secondary amines.10•`13) In the there were no effects of metal ions on the present study, as well as in the previous rate of CMNU formation compared with the paper,1) we observed that thiocyanate had no controls. effects on the nitrosation of ureido compounds, 4. Discussion but citrate and tartrate remarkably accelerated CMNU and MNU were unstable after the it. The different effects of citrate and tartrate 514 J. Food Hyg. Soc. Vol.18, No.6

from thiocyanate on nitrosation might depend tion as shown in Fig. 1. Consequently, the on the difference of reaction mechanism. reaction was first order with respect to the To compare the effects of acids, which have concentrations of HA, sodium nitrite and similar structures to citric acid or tartaric citric acid, respectively, and the following acid, on nitrosation of ureido compounds, equation can be set up.: rate=k1[HA] [nitrite] hydroxycarboxylic acids and carboxylic acids +k2[HA] [nitrite] [citrate]. shown in Fig. 2 were added to the reaction As for the effect of pH on nitrosation of mixture of HA and sodium nitrite. Most of HA, the rates of CMNU formation were in- them accelerated CMNU formation, and the creased with lowering pH in the range of 1.5 extents of acceleration were greater in the and 4, in the presence and absence of citric compounds which have more carboxyl groups acid, but remarkable difference of pH depend- or hydroxy groups (Fig. 2). ence was not observed by the addition of Similar results were obtained in the case of citric acid. Therefore, the dissociation of nitrosation of MU, but the extents of accele- citric acid or protonation of nitrite might ration by acids were smaller than those in not play an important role in the acceleration nitrosation of HA (Fig. 3). Thiocyanate had mechanism. no effects on the rates of nitrosation of MU, As shown in Table 1, the accelerating effects as well as HA.1) by organic acids can be added each other. The Generally, nitrosations of amides other than content of one kind of organic acid in vivo alkylureas or alkylurethanes proceed very or in foods might not be so high, but many slowly. We found that acetylglycine was kinds of organic acids might have action scarcely nitrosated at pH 2.5 and 37•Ž for together. 2•`5hr, but nitrosated in the presence of This work was supported by Cancer Research citrate or tartrate*. Consequently, nitrosation Fund from the Ministry of Health and Welfare of the compounds which have amide groups (1976 and 1977) entitled •gStudies on chemical seemed to be accelerated by organic acids. carcinogens in the life environment.•h Though it is not clear that organic acids have interations with amide compounds or nitrite, References or both, hydrogen bond might play some 1) Yamamoto, M., Yamada, T., Tanimura, A.: roles in acceleration of nitrosation of amide J. Food Hyg. Soc. Japan, 17, 363 (1976). compounds by organic acids. 2) Turney, T.A., Wright, G.A.: Chem. Rev., Creatine and glycocyamine, which are gua- 59, 497 (1959). nido compounds, form NS9,14) and CMNU,8,15) 3) Mirvish, S.S.: J. Nat. Cancer Inst., 46, 1183 respectively, via some steps in the reaction (1971). with nitrite. These reactions were accelerated 4) Mirvish, S.S.: ibid., 44, 633 (1970). by citrate, tartrate and thiocyanate. But 5) Mirvish, S.S., Sams, J., Fan, T.Y., Tannen- baum, S.R.: ibid., 51, 1833 (1973). citrate and tartrate might have accelerating 6) Mirvish, S.S., Garcia, H.: Z. Krebsforsch., effects on the different steps from those on 79, 304 (1973). which thiocyanate has the effects, because 7) Mirvish, S.S.: Toxicol. Appl. Pharmacol., 31, thiocyanate has no effects on the rates of 325 (1975). nitrosation of amide compounds.1) 8) Yamamoto, M., Yamada, T., Tanimura, A.: The order of nitrite concentration which J. Food Hyg. Soc. Japan, 17, 176 (1976). participates to nitrosation depends on reaction 9) Yamamoto, M., Yamada, T., Tanimura, A.: form of nitrite2•`7). The rates of CMNU for- ibid., 15, 461 (1974). mation from HA and sodium nitrite in the 10) Boyland, E., Nice. E., Williams, K.: Fd. Cosmet. Toxicol., 9, 639 (1971). presence or absense of citric acid were pro- 11) Fan, T.Y., Tannenbaum, S.R.: J. Agr. Food portional to the concentrations of HA and ni- Chem., 21, 237 (1973). trite (Fig. 5). And when the concentrations of 12) Boyland, E., Walker, S.A.: Nature, 248, 601 HA and sodium nitrite were constant, the rates (1974). increased linearly with citric acid concentra- 13) Yamada, T., Yamamoto, M., Tanimura, A.: * Yamamoto , M., unpublished data J. Food Hyg. Soc. Japan, 15, 201 (1974). December 1977 Studies on the Formation of Nitrosamines (VI) 515

14) Archer, M.C., Clark, S.D., Thilly, J.E., Tan- 15) Yamada, T., Yamamoto, M., Tanimura, A.: nenbaum, S.R.: Science, 174, 1341 (1971) . J. Food Hyg. Soc. Japan, 17, 182 (1976).