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 About 5ml of the aliquots pounds interested us to study further con- were withdrawn from the reaction mixture cerning the effect of other organic acids and the at definite time intervals and poured onto structures of nitrosatable compounds or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.