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' 11111111111111 11111.11.» 1'1 , n. - J—u... l "‘ .1 ‘ '1 I . . o.‘ -. .‘.-~ A I C ." 1 .i f: {11131.41 21"}51‘1' ' 1"..'""".|1'£) "1'." 11"1"1JJI'J "HJ" JJ11111‘ THESIS LIBRARY indickfigfiun1533gg University This is to certify that the dissertation entitled N—NITROSAMIDES IN FOOD: FORMATION AND TOXICOLOGY presented by Mohamad Hassan Fooladi has been accepted towards fulfillment of the requirements for Ph. D. Food Science degree in fl 74 WW ajor professor Date December 15, 1981 MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771 mmmmmmgwnmlmllmm 31293 109 25 OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to move charge from circulation records N-NITROSAMIDES IN FOOD: FORMATION AND TOXICOLOGY BY Mohamad Hassan Fooladi A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1981 ABSTRACT N-NITROSAMIDES IN FOOD: FORMATION AND TOXICOLOGY BY Mohamad Hassan Fooladi Formation of N—substituted amides was investigated using both a model system and bacon. Fatty acids were shown to react readily with selected -amino acids in model systems at 200°C to give N-substituted amides. This reac- tion involves decarboxylation of the amino acids and dis- placement of the alcohol moiety of the fatty esters by the amine that was formed. However, formation of primary amines via the decarboxylation of -amino acids appears to be un- likely at temperatures normally encountered in pan frying of bacon due to insufficient energy for the decarboxylation step. Under these conditions only amines would react readi- ly with fatty acids to yield secondary amides. N-Substituted amides were shown to be nitrosated readi- ly under acid conditions in a model system. It was demon- strated that the normal pH of foods militates against the formation of N-nitrosamides in foods, even when their precursors are present. It was also demonstrated that Mohamad Hassan Fooladi N-nitrosamides are very unstable under conditions commonly encountered in cooking of bacon. The major conclusion from this investigation was that N-nitrosamides are unlikely to be present in heat-processed foods. In the event that N-substituted amides are formed during processing and cooking of foods, however, they could be nitrosated in yigg. Therefore, the carcinogenicity of these compounds was investigated in a feeding trial using Swiss Mice. None of the N-substituted amides alone or in combination with nitrite or their corresponding N-nitro- samides caused development of tumors in mice within a seven month feeding trial. Only the mice which were fed N-nitroso- methylurea (positive control) had lesions which were localized in the lungs. Since the result of mutagenicity assays showed that N-nitrosopentylpalmitamide and N-nitroso- methylurea are strong' mutagens and that N-nitrosomethyl— propionamide is a weak mutagen the possibility that these compounds are carcinogenic cannot be ruled out. Therefore, the remaining mice are being held on the same diet and will continue for two years before sacrificing and. examining their tissues for tumor development. ACKNOWLEDGEMENTS I would like to express sincere gratitude to Drs. A.M. Pearson and J.l. Gray for their guidance, encouragement and the velues they have instilled in me over the past several yeras. It would also like to express my appreciation to the guidance committee, Drs. L.R. Dugan, L.E. Dawson, R.W. Leucke, and P. Markakis, for their critical review of this thesis. Appreciation is also expressed to D.J. Skrypec and A.K. Mandagere and Dr. D.S. Sleight for conducting the Ames tests, mass spectrometric analyses and autopsies on mice, respectively. ii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . Chemistry of Formation . . . . . . Kinetics of Nitrosation Reactions . In Vivo Formation of N- -Nitroso Compounds . . . . . . . . . . Stability of N- -Nitroso Compounds . . Precursors of N- Nitroso Compounds . . Amines in Foods . . . . . . . Formation of N-substituted Amides . Nitrate and Nitrite . . . . . . . . Toxicology of N-Nitrosamides . . . . . Carcinogenicity . . . . . . . . . . Mutagenic Action of N-Nitroso Compounds . . . . . . . . . . . . MATERIALS AND METHODS O O O C I I O O O 0 Reagents . . . . . . . . . . . Preparation of Amides in Model Systems (1) Amino Acids plus Saturated Fatty Acids . . . . . . . . . (ii) Amino Acids plus Unsaturated Fatty Acids . . . . . . . . (iii) Amino Acids plus Lard or Pork Belly Adipose Tissue . . . . . Nitrosation of N-Substituted Amides . Preparation of N-Nitroso- N-pentylpalmitamide . . . . . . . Preparation of N-Nitroso- N-methylpropionamide (NOMP) . . . iii Page vi 10 12 13 15 18 22 22 26 31 31 31 31 32 33 33 33 34 Analysis and Identification of Amides and N-Nitrosamides . . . . . . . . . . Formation of N-substituted Amides in Pork Belly Slices . . . . . . . . . Processing of Pork Bellies . . . . . . . Frying of the Bacon Slices . . . . Analysis of N- substituted Amides in Fried Pork Belly Slices and Cooked-out Fat . Formation of N-Nitrosamides in Pork Belly Slices . . . . . . . . . . . . Thermal Stability of N-Nitrosamides under Frying Conditions of Bacon . . . Determination of Fatty Acid Composition of Lard and Pork Belly Adipose Tissue . . Preparation of Methyl Esters for GLC Analysiss . . . . . . . . . . . . Effects of N—substituted Amides and N-Nitrosamides on Tumor Development in Mice . . . . . . . . . . . . . . . Ames Test . . . . . . . . . . . . . . . RESULTS AND DISCUSSION 0 O O O O C C O C 0 O 0 Preparation of N-Substituted Amides . . . Fatty Acid Composition of Lard and Pork Belly Adipose Tissue . . . . Formation of Amides in Pork Belly Slices Treated with Norleucine and/or Pentylamine . . . . . . . . . Nitrosation of N- Substituted Amides . . Formation of N- ~Nitrosamides in Bacon . . Thermal Decomposition of N-Nitrosamides Mutagenicity of N-Substituted Amides and Their Corresponding N-Nitrosamides . Carcinogenicity of N-Nitrosamides . . . . SUMMARY AND CONCLUSIONS . . . . . . . . . . . BIBLIOGRAPHY O O O O O O O O O O O O O O O O 0 APPENDIX C O O O O O O O O O O O O O O O O O 0 iv Page 35 36 37 38 39 39 40 40 41 43 45 45 50 54 59 65 69 72 77 80 89 LIST OF TABLES Table l. Nitrate and nitrite usage recommended by USDA Expert Panel . . . . . . . . . . . 2. Carcinogenicity of N-nitrosamides . . . . . 3. Additives that has been added to purified diet for different groups of mice in feeding trial . . . . . . . . . . . . . . 4. Fatty acid composition of pork bellies and N-substituted amides which formed upon heating pork bellies containing pentYIamine O O O O O O O O O O O O O O O 5. Mutagenic potenatial of N-substituted amides and their corresponding N-nitrosamides . . 6. Effect of additives on feed and water consumption of mice . . . . . . . . . . . 7. Data on gross examination of the mice for tumors and other abnormalities upon autopsy O I O O O O O O O O O O O O O Page 20 24 42 56 71 73 75 LI ST OF FIGURES Figure Page 1. Gas chromatogram of N-pentylstearamide . . . . . . 46 2. Mass spectrum of N-pentylstearamide prepared from the interaction between norleucine and stearic acid . . . . . . . . . . . 47 3. Gas chromatogram of standard N-pentyl- myristamide (NPM), N-pentylpalmitamide (NPP), N-pentyllinoleamide (NPL), N-pentyl oleamide (NOP) and N-pentylstearamide (NPS) formed from reaction of pentylamine and fatty acids . . . . . . . . . . . . . . . . . . . 51 4. Gas chromatogram of standard mixture containing N-isobutylmyristamide, N-isobutylpalmitamide, N-isobutylstearamide prepared from reactions of valine and corresponding fatty acids . . . . . . . . . . . . 52 5. Gas chromatogram showing fatty acid composition of pork belly adipose tissue . . . . . 53 6. Gas chromatogram of N-substituted amides (N-pentylmyristamide, N-pentylpalmitamide, N-pentyllinoleamide, N-pentyloleamide and N—pentylstearamide) present in cook-out fat from rehydrated freeze dried pork belly slices containing pentylamine, heated for 16 minutes at 175 C . . . . . . . . . SS 7. Gas chromatogram of N-pentylmyristamide (MPM), N-pentylpalmitamide (NPP), N-pentyloleamide (NPO), N-pentyllinoleamide (NPL) and N-pentylstearamide (NPS) present in pork belly adipose tissue wiSh added norleucine heated 1 hour at 200 C . . . . . . . . 57 8. Gas chromatogram of N-nitroso— pentylstearamide . . . . . . . . . . . . . . . . 60 vi Figure Page 9. Mass spectrum of pentyl stearate, a breakdown product of N-nitroso- pentylstearamide . . . . . . . . . . . . . . . . 61 10. Gas chromatogram of standard mixture containing N-nitrosopentylmyristamide (NOPM), N-nitrosopentylpalmitamide (NOPP), N-nitrosopentyloleamide (NOPO), N—nitroso- pentyllinoleamide (NOPL), and N-nitro- pentylstearamide (NOPS) derived from nitrosation of their corresponding N-substituted amides . . . . . . . . . . . . . . 64 11. Gas chromatogram of recovery of N-nitroso pentylpalmitamide from raw bacon . . . . . . . . 66 12. Gas chromatogram of N-nitrosopentyl- palmitamide residue in cook-out fat . . . . . . 67 13. Gas chromatogram of N—nitrosopentyl- palmitamide residue in fried bacon . . . . . . . 68 vii LIST OF APPENDICES Appendix Page 1 Mass spectrum of N-pentyllauramide prepared from reaction of norleucine and lauric acid at 200 C . . . . . . . . . . 89 2 Mass spectrum of N-pentylmyristamide prepared from reaction 05 norleucine and myristic acid at 200 C . . . . . . . . . 91 3 Mass spectrum of N-pentylpalmitamide prepared from reaction 08 norleucine and palmitic acid at 200 C . . . . . . . . . 93 4 Mass spectrum of N-isobutylmyristamide prepared from reaction of valine and myristic aCid O O O O O I O O O O O O O O O 95 5 Mass spectrum of N-isobutylpalmitamide prepared from reacti8n of valine and palmitic acid at 200 C . . . . . . . . . . . 97 6 Mass spectrum of N—3-methy1thiopropyl- myristamide prepared from reaction 8f methionine and myristic acid at 200 C . . . 99 7 Mass spectrum of pentyl myristate a breakdwon product of N-nitrosopentyl- myristamide . . . . . . . . . . . . . . . . . 101 8 Mass spectrum of pentyl palmitate a breakdwon product of N-nitrosopentyl- palmitamide O O O O O O O O O O O O O O O O O 103 9 Mass spectrum of isobutyl myristate a breakdwon product of N-nitrosoisobutyl- myriStamide O O O O O O O O O O O O O O O O O 105 10 Mass spectrum of 3-methylthiopropyl myristate a breakdwon product of N-nitroso- 3-methylthi0propylmyristamide . . . . . . . . 107 11 Composition of purified diet . . . . . . . . 109 viii INTRODUCTION Since the first report of experimental induction of liver cancer in rats by feeding low levels (50 u/Kg) of N- nitrosodimethylamine (NDMA) (Magee and Barnes, 1956), N- nitroso compounds have been intensively studied as carcino- gens. These compounds have been found to produce a variety of tumors at different organ sites, the extent depending on their structure and dosage rate, route of administration and the test species (Preussmann, 1973). N-Nitroso compounds can be devided into two groups according to their chemical reactivity: (l) N-nitrosamines, and (2) N-nitrosamides. They have the following general for- mula: N-Nitrosamines are formed principally from the reac- tion between secondary' amines and nitrous acid. 131 this reaction, R1 is an alkyl group while R may be an alkyl, aryl or a wide variety of other functional groups. R R >NH+HN02 ------- > >N-N=O+H20 R1 R1 2 N-Nitrosamines can also be formed from tertiary amines, quarternary ammonium compounds and primary polyamines. Many of these N-nitroso compounds have been identified in vari- ous food systems (Gray and Randall, 1979). N-Nitrosamines are largely systemic agents and can be converted to reac- tive netabolites, probably alkylating agents, by enzymatic processes in the mammalian organism (Preussmann, 1973). On the other hand, N-nitrosamides which arise from the reac- tion of secondary amides with nitrite (Mirvish, 1977), usually have one alkyl residue (R1) and an acyl residue (R2). They are chemically reactive compounds and are relatively easily hydrolyzed to alkylating diazoalkanes (Preussmann, 1972). They are considered to exert both local and systemic activity in the carcinogenesis of experimental animals (Preussmann, et al., 1972). Although their powerful carcinogenic responses are well known, there have been only a limited. number of studies on the occurrence of non- volatile N-Nitroso compounds in food systems, due in part to their instability under neutral and alkaline conditions (Mirvish, 1971). However, the precursors of N-nitrosamides have been reported in certain foods. Such compounds include uridine and ureas which have been isolated from fish by Mirvish (1975). High concentrations of agmatine, a decar- boxylation products of arginine, have been reported in fresh abalone (Kawabata et al., 1978), and citrulline has been reported by Wada (1930) in watermelon. 3 Recently, Sims and Fioriti (1975) reported that heat- ing fatty acids (or esters) and triglycerides and -amino acids at temperature above 150°C resulted in substantial yields of N-substituted amides. These results were confirm— ed by Kakuda and Gray (1980a) using a model system contain- ing amino acids or free amines and fatty acids. They report- ed that presence of a secndary amino group in these com- pounds makes them susceptible to KFhitrosation. These com- pounds may represent another source of nitrosatable com- pounds available for reaction with nitrite. The present study was undertaken to establish whether N-substituted. amides can be formed under conditions en- countered in processing and cooking of foods, and thus, be potential precursors of N-nitrosamides. Specific objectives of the present study were: (1) to study the formation of N- substituted amides from reactions between fatty acids and/or triglycerides with -amino acids and amines in both model and bacon systems: (2) to investigate the nitrosation of N-substituted amides by sodium nitrite in both model system and bacon systems: (3) to study the thermal stabili- ty of N—nitrosamides during the cooking of food: (4) to determine the mutagenicity of N-substituted amides and their corresponding N-nitrosamides using the Salmonella/ microsome mutagenicity test; and (5) to estimate the carci— nogenicity of N-substitutes amides and N-nitrosamides in a feeding trial using Swiss mice. LITERATURE REVIEW Chemistry of Formation Kinetics of Nitrosation Reactions N-Nitrosamines are formed principally from the reac- tion between secondary amines and nitrous acid (Mirvish, 1975). N-Nitrosamines can also be formed from tertiary amines, quarternary ammoniunl compounds and primary' poly- amines (Gray and Randall, 1979). In addition, the formation of relatively non-volatile N-nitroso compounds have been suggested by model system studies (Mirvish, 1971; Kakuda and Gray, 1980b). Mirvish (1975) stated, that for nitrosation to occur, nitrite must first be converted to nitrous acid (HNO2 - PKa 3.36), indicating that the reaction is catalyzed by acid. Nitrous acid is then converted to an active nitrosat- ing species. The actual nitrosating species can be one of the following, depending on the reaction conditions: ni- trous anhydride (N203), nitrous acidium ion (H2N02+), free nitrosonium ion (NO+) and nitrosyl halide (NOX). The following reactions have been reported by Mirvish (1970) and show the equilibrium equations for the nitrite 5 ion in aqueous solution: H+ H+ - —-=== ====== + ===== O-N—O<—- >HN02< >H2N02< >H20 + NO+ FAST Nitrite Nitrous Nitrous Nitrosonium Ion Acid Acidium Ion Ion :N/ x" Or "CNS H 3O+ + O=N- O- -N=O H20 + O=N-X Or O=N-CNS Nitrous Nitrosyl Nitrosyl Anhydride Halide Thiocynate The kinetics of N-nitrosamines formation from seconda- ry amines and nitrous acid has been studied in detail by Mirvish (1972; 1975). Mirvish (1975) reported that the nitrosation reaction for most secondary amines proceeds via the active nitrosating species, nitrous anhydride. He fur- ther proposed that the reaction proceeds according to the overall third order rate equation as shown below: Rate of N-nitrosamine = k [total amine] [nitrite]2 formation where k is the rate constant. The equation shows that the reaction rate is propor— tional to the concentration of the free amine and to the square of the concentration of nitrite. Mirvish (1975) stated that since it is the free amine and not the pro- tonated amine that is nitrosated, both pH and amine basici- ty may influence N-nitrosamine formation. Sander’ et a1. 6 (1972) found that there is an inverse relationship between the basicity' of amines and the ease of nitrosation. In other words, the lower the basicity of a secondary amine, the easier it is to achieve nitrosation in acid solution, Sander et a1. (1972) pointed out that at increasing acid concentrations, a higher proportion of the nitrite is con- verted to nitrous anhydride, which is the nitrosating agent. At the same time, however, salt formation by the amines is emhanced. Thus, there is an optimal pH value for the nitrosation of secondary amines which is about pH 3. Sander et a1. (1972) reported that nitrosation of alkylamides follows the same principle as that for seconda- ry amines. Because of resonance and inductive effects, however, the ability to nitrosate depends on the structure of the amides. Mirvish (1977) proposed the following equa- tions for nitrosation of alkylamides: + + HN02 + H <-— —————— > H2NO2 (I) RNH CORl + HZNOZ+ <========> RN(NO).COR1 + HZO (II) rate of N-nitrosamide = k1 [RNH CORl] [HNOZ] [H+] (III) formation rate of N-nitrosamide = k2 [amide] [nitrite] [H+] (IV) formation He showed that the nitrosation rate is proportional to the amide and nitrous acidium ion concentrations. Nitrous acidium ion concentration in turn is proportional to H+ and HNO2 concentration as shown in equation I. In equa- tion III, kl depends on nitrite ionization, and hence on 7 pH but does not depend on ionization of the amides since amides are usually not ionized above pH2. Mirvish (1975) suggested that the main nitrosating agent for amides is probably nitrous acidium ion (H2N02+), the protonated nitrous acid. Therefore, nitrosation of secondary amides is pH dependent. In con- trast to nitrosation of secondary amines, there is no pH for nitrosation because alkylamide have low basicity. How- ever, the extent of nitrosation increases as the pH of the reaction medium is lowered. Similar results have been re- ported by Kakuda and Gray (1980b) for the nitrosation of secondary amides. In their model system, they found no apparent pH maximum for the reaction, N-nitrosamide forma- tion increased with increasing hydrogen ion concentration. The rate of nitrosation decreased rapidly as the pH increas- ed, with littly reaction occurring above pH3. They noted that a drop in pH from 2 to 1 increased nitrosation by 5-8 times. The rate constant remained relatively stable over a pH range of 1:8.5, thus supporting the nitrous acidium ion mechanism. Mirvish (1975) studied the nitrosation of 21 amides and reported that all of them followed the nitrous acidium mechanism. The rate constant values varied. some 300,000 times between different amides. They were lowest for simple alkyl- and arylamides and for guanidines, and highest for ethyleneurea. There is no simple rule relating ease of 8 nitrosation to other properties of the amides as is true for the amines. Kakuda and Gray (1980b) pointed out that the nitrous acidium ion cannot be considered an important N-nitrosating species in food systems, since low pH is not normally en- countered. Thus, the pH of foods would militate against the occurrence of N-nitrosamides, even if their precursors are present. Mirvish (1971), however, showed that in 2139 N-nitrosation of alkylamides is possible. In Vivo Formation of N-Nitroso Compounds 1 vivo formation of N-nitroso compounds was first indicated by Sander and Burkle (1969). Although they did not perform chemical analyses for N-nitroso compounds in the stomach, they noted esophageal and hepatic tumors in rats fed nitrite together with morpholine and N-methylbenza— mine. They also observed tumors characteristic of the cor- responding N-nitroso compound in rats upon feeding nitrite plus methylurea, ethylurea and 1,3-dimethylurea. They pro- posed that nitrosation probably occurs in the stomach and is catalyzed by hydrochloric acid. Mirvish et a1. (1978) measured N-nitrosomethylurea (NMU) formation in the stomach contents of rats fed 3H methylurea and sodium nitrite. The rats were killed 3 hours later and the NMU yield was calculated to be 0.46% of the methylurea. If sodium ascorbate was added to the feed in equimolar amounts, NMU production was completely inhibited. 9 Between 45-90 minutes after gavage of urea and nitrite to starved rats, Mirvish and Chu (1973) obtained yields of 27% for lufll and 9% for N-nitrosoethylurea (NEU). Gavage of sodium bicarbonate before the methylurea plus nitrite treat- ment neutralized the stomach content and prevented NMU for- mation. Mirvish et a1. (1974) showed that starved rats given feed containing sodium nitrite still had 57 and 8% of the nitrite in the stomach after 1 and 5 hours, respectively. The mean nitrite concentration in the acidic glandular part of the stomach was 50% less than that of the non glandular portion. They reported that factors other than emptying of the stomach accounted for about 40% of the nitrite loss, which occurred especially rapidly in the glandular portion of the stomach. They suggested that these factors probably include absorption of nitrite from. the stomach, decomposi- tion of nitrite and its reaction with food components. All these processes probably require conversion of nitrite to HNO2 by the action of gastric HCl. They reported that the transit time in the stomach was 1.2 hours, the pH was 3.6 and the nitrite concentration comprised 11% of that in the food. They concluded that conditions in the stomach would permit significant nitrosation to occur in rodents if the compounds were readily nitrosatable. Varghese et a1. (1978) also presented evidence sup- porting 1 vivo formation of N-nitroso compounds. They 10 detected a bacterial mutagen in ether extracts of freeze dried feces of humans on western diets. They suggested that the mutagens may be N—nitroso compounds, which were not detected in the diet but were formed _i_n_ Mg and elimi- nated in the feces. Sato et a1. (1959, 1961) found a corre- lation between gastric cancer and high intake of salted fish and vegetables in Japan. They suggested that 12 21319 nitrosation of ureas and methylguanidine, which may be present in fish included in the diet, could be responsi- ble for the high incidence of gastric cancer. In light of above findings, lg 3132 nitrosation might present a hazard, especially with the more readily nitrosatable compounds that are present in drugs, agricul- tural chemicals or as food components (Mirvish, 1977). Stability of N-Nitrosamides Mirvish (1971) reported that decomposition of N-nitro- samides occurs under either neutral or alkaline conditions. Chow (1979) studied the thermal and photolytic decomposi- tion of N-nitrosamides and reported that at temperatures from .wmbient to 100°C. N-nitrosamides undergo irreversi- ble thermal rearrangements to form diazo esters. He pointed out that diazo esters decompose rapidly to give carboxylic esters or acids and olefins as shown here. 11 o R' 0 N A I g \N g R C ——+ II ./'\ \N/ \R.__-—:_? \fiv/ \R IN-O R I h N f 0%, \w l R'-OCCR RCO?ll Olefins The stability of N-nitrosamides and the final products from diazo esters are strongly dependent on the nature of the R1 group (primary, secondary, tertiary’ alkyls, phenyl or benzyl groups, etc), but less so on the R group (Chow, 1979). Photolytic decomposition of N-nitrosamides in polar and non-polar solvents yield amidyl and nitric oxide radi- cals, which further undergo typical free radical reacitons as shown by Chow (1979) below: 0 N0 0 H I H R-C-N-R' ----------- > R-C-N-R' + ON. 1... o H H I R-C- N-R' Chow (1979) reported that the first step in photolysis of N-nitrosamides is the homolytic scission of the N-N bond. The second step is usually abstraction of hydrogen from the alcholic, hydrocarbon or olefinic solvent, which 12 forms an amide. In the third step, the nitroso radical re- acts with the new carbon radical to give ketones from alco- holic solvents or nitrosodimers and nitrite, from hydro- carbons or olefinic solvents (Thomas, 1972). N-Nitrosamides also decompose readily in neutral and alkaline solutions. In contrast to N-nitrosamines, Mirvish (1971) reported that N-nitrosamide are unstable in neutral and alkaline solutions, and decompose readily to yield a1- kyldiazo hydroxide as illustrated below: 0 NO Druckery (1975) proposed that alkyldiazo hydroxides are proximate alkylating carcinogens. These compounds have been widely used in chemistry as alkylating substances. There have been only a limited number of reports on the occurrence of N-nitrosamides in food systems. Thus, one may suspect that lack of detection in environmental samples may be due to the instability of the N-nitrosamide linkage. Precursors of N—Nitroso Compounds Precursors of N-nitroso compounds constitute nitrite and various amines and amides, many of which are natural constituents of foodstuffs or drugs (Sander and Schweinsberg, 1972). Each of these reactants and then 13 possible sources in foods will be discussed herein. Amines in Foods Amines in foods are formed by both biological and chemical pathways (Maga, 1978). These include: (a) amino acid decarboxylation, which is responsible for the forma- tion of spermidine from methionine (Lakritz, et al., 1975), putrescine from ornithine (Tabor et al., 1958), cadaverine from lysine (Tabor et al., 1958) tyramine from tryosine (Kristoffersen, 1963) and histamine from histidine (Dierick et al., 1974); (b) trimethylamine oxide conversion, such as the enzymatic conversion of trimethylamine oxide to tri- nethylamine (Tarr, 1940); (c) aldehyde amination as in the amination and transamination of aldehydes, which is the po- tential pathway for formation of most monoamines associated with foods (Hartmann, 1967; Maier, 1970); (d) phospholipid decomposition, such as the formation of ethanolamine from the splitting of cephalin (Herdlicka and Janicek, 1964); and (e) thermal amino decomposition, which accounts for the appearance of a wide variety of amines, such as ethanol- amine, methylamine, propylamine and either iso-or pentyl- amines, which are formed during heating of cysteine (Mulders, 1973). Velisek and Davidek (1974) also postulated that amines in foods could easily be formed during the non- enzymatic browning process. A wide range of simple aliphatic amines and mono- amines, such as tyramine, histamine and tryptamine has been l4 detected in cheeses (Golovnya and Zhuravleva, 1970; Gray et al., 1979). Spinelli et a1. (1974) reported that pyrolysis of proteins on cooking of foods may produce free amino acids and rdtrosatable secondary amines. In their analysis of amines in fresh processed pork, they demonstrated the presence of spermidine, spermine» putrescine, cadaverine, tryptamine, tyramine, histamine and ethanolamine. Lijinsky and Epstein (1970) have pointed out that putrescine, which is a decomposition product of arginine, may undergo cycliza- tion to form pyrrolidine during the cooking of fish and meat. Pyrrolidine can undergo nitrosation to form N-nitro- sopyrrolidine (NPYR) as reported by Bills et al., (1973). The aliphatic polyamines, spermidine and spermine are widely distributed in biological material, including viruses, bacteria, plant and animal tissues (Tabor and Tbor, 1964). Lakritz et a1. (1975) reported maximum values of 125 mg and 1013 mg of spermidine per 100 g of tissue for fresh pork and putrified pork, respectively. Spermine values were 55.7 mg and 2769 mg per 100 g of fresh and putrified pork, respectively. Formation of NPYR from reac- tions of spermidine with sodium nitrite upon heating has been reported by Ferguson et a1. (1973). Polyamines and free amino acids can also produce the precursors of N-nitroso compounds. Lien and Nawar (1974a) reported that free amino acids and polyamines are converted U3 N-nitrosatable secondary amines by thermal degradation. Simmonds et a1. (1972) found decarboxylation to be the 15 major thermal decomposition pathway for production of free amines from -amino acids. The free amino acid content of meat increases upon aging due to the actions of the naturally occurring cathe- psins (McCain et al., 1968). Bharucha et a1. (1979) demon- strated that raw bacon contained 2 to 3 times more free amino acids than was found in fresh pork bellies. Gray and Collins (1977) reported that the free proline concentration in fresh pork bellies increased with storage time. Kakuda and Gray (1980a), however, concluded that formation of free amine via decarboxylation of amino acids under conditions normally encountered in cooking and processing of food seems unlikely, since there is insufficient energy for decarboxylation. They demonstrated that high temperatures (minimum 150°C for 45 min) are required for decarboxyla- tion of norleucine. A high activation energy for decaroxyla- tion of amino acids was previously reported by Sims and Fioriti (1975). Formation of N-substituted Amides Beckwith (1970) stated that amides can be formed by the acylation of ammonia or amines. This is in agreement with the observations of Lien and Nawar (1974b), who report- ed formation of amides upon pyrolysis of a mixture of amino acids and triglycerides. Lien and Nawar (1974a) also report- ed that ammonia and amines are produced by thermal decompo- sition of amino acids, whereas, acylating agents (i.e., 16 carboxylic acid and acid anhydride) can be formed from pyrolysis of triglycerides. They proposed the following reaction pathway for formation of amides: - HYDROCARBONS - CARBONYL COMPOUNDS AMINO ACID AMMONIA ------- 1 u - NITRILE 1 PRIMARY . -------------- - ACID : AMIDE I — PRIMARY AMINE - . L AMMONIA I ; P NITRILE I ' r ACID 1 SECONDARY I AMIDE — OLEFIN I I I ' L ; AMMONIA ACID or : I FACID ANHYDRIDE-;j TRIGLYCERIDE--- LHYDROCARBONS Sims and Fioriti (1975) have investigated reactions which occur between fatty esters and. amino acids under thermal stress. They reported that fatty esters react rapidly with many -amino acids at temperatures as low as 150°C to give N-substituted amides as the major reaction 17 products. The reaction involves decarboxylation of the amino acid and displacement of the alcohol moiety of the fatty ester by the amine, which is formed. They reported yields of N-substituted amides as high as 50% of theoretic- al can be obtained. They proposed the following reaction: O RCHZCOZRl + R2 ea COZH 150-200 C ; ONH n 2 RCHZ-C-I'N-CHZ-Rz'l-COZ'FRIOH H Sims and Fioriti (1975) reported that on heating methionine in mineral oil at 200°C for prolonged periods, no CO2 was evolved, and the methionine could be recovered quantitatively. When fatty acids were present in the mix- ture however, the corresponding N-substituted amides were formed. They suggested that high temperature reactions of fatty acids and amino acids are consistent with a concerted mechanism. It is possible that two succesive steps are in- volved, i.e., decarboxylation, which is the rate determin- ing step, and amidation. Lien and Nawar (1974b) reported formation of caproic amide and isobutylcapric amide upon heating of valine and 18 tricaproin at 270°C. Breitbart (1977) reported the radio- lytic interaction of some amino acids and triglycerides. He identified caproic amides and caproic nitrile from an irra- diated lysine and tributyrin mixture. Kakuda and Gray (1980a) studied the formation of N- substituted amides in a model system containing fatty esters or triglycerides and free amines. They reported that fatty acid ester and free amines reacted readily to form N- substituted amides under conditions involving thermal stress. They showed that free fatty acids are not as reac- tive as their respective triglycerides, but at high tempera- ture, both reacted readily with amines. They theorized that formation of primary amines via decarboxylation of -amino acids is unlikely under normal cooking conditions, because there is insufficient energy for decarboxylation. They su- ggested that under conditions encountered in the processing and cooking of foods, only amines would react with fatty acids and/or esters to yield substantial quantities of secondary amides. Furthermore, they reported that the presence of a secondary amino group in the amide compounds would make them susceptible to nitrosation. Thus, they may provide another precursor for N-nitroso compounds. Nitrate and Nitrite Nitrate and nitrite have been used in the curing of meat for many years. Nitrite serves several purposes during meat curing, including production of the characteristic l9 cured meat color (Brooks et al., 1940), contributing to cured meat flavor (Bailey and Swain, 1973), elimination of warmed-over flavor (Bailey and Swain, 1973; Fooladi et al., 1979) and retardation of botulinal toxin formation (Christiansen et al., 1973). During the past decade however, nitrite has become the center of widespread controversy as a result of its interac- tion with the naturally occurring amino compounds in meat to produce N-nitroso compounds (Gray, 1976). N-Nitrosamines are formed in cured meat products under certain conditions and have been found sporadically in hams, wieners, bologna, and similar products (Gray, 1976). The effect of various amounts of nitrite on the forma- tion of NPYR during cooking of bacon has been extensively studied by Sen et a1. (1974). They demonstrated a gradual increase in formation of NPYR in fried bacon with increas- ing nitrite concentrations. The amount of NPYR correlated closely with the initial concentration of nitrite, but not with the residual nitrite level found in raw bacon. Gray and Dugan (1974) showed that the concentration of nitrite and the nitrite to amine ratio are important on formation of nitrosamines. They indicated that minimum or perhaps no N-nitrosamine formation may be expected when nitrite levels are low. They reported maximum formation occurred at or above a nitrite to amine ratio of 2:1. The rate of N-nitrosation of secondary amines was re- ported by Mirvish (1970) to be directly proportional to the 20 square of the nitrite concentration. Thus, the amount of nitrite permitted in curing of meat has received considera- ble attention. The current levels of nitrate and nitrite used in various meat products as recommended by the USDA Expert Panel (1978) are given in Table 1. Table 1. Nitrate and nitrite usage recommended by USDA Expert Panel (1978) Levels of nitrate Levels of nitrite Product (mg/kg) mg/kg Cooked sausages 0 100-156 Fermented sausages 0 60-156 Dry cured cuts 300 100 Pickle cured products 0 110-200 Commercially-sterile products 0 50 Perishable-canned products 0 80-200 Shelf-stable products 0 156 Bacona o 120 a Level established by regulation on May 16, 1978. The public health aspects of N-nitroso compounds have so far focused mainly on whether or not nitrite should be used to preserve meat products. However, nitrite also occurs in other foods and human saliva. Tannenbaum et a1. (1974) reported that nitrite is a normal constituent of human saliva, resulting from the reduction of nitrate by a 21 variety of microorganisms that inhabit the mouth. They reported that the level of salivary nitrite in healthy individuals is fairly constant and average about 6-10 mg/liter. According to White (1975) the estimated average daily ingestion of nitrate per capita in the U.S.A. is 80 mg, coming principally from an average daily intake of 306 grams of vegetables. He concluded that the amount varies enormously depending to the type of vegetable and its ni- trate content. Nitrate also occurs in water, especially in well water from some rural areas (Comly, 1945; Burden, 1961). The concentration of nitrite in vegetables and water, on the other hand, is usually very low, although fairly high levels have been detected on storage of tempera- ture abused spinach and beets (Heisler et al., 1974). Tannenbaum et a1. (1978) reported that nitrite and nitrate are formed d§| 9932 in the human intestine, possibly by heterotrophic nitrification of ammonia or organic nitrogen compounds. They suggested that this reac- tion takes place in the upper aerobic portion of the intes- tine. As a result, they concluded that human exposure to nitrite may be much greater than previously recognized. In light of the above findings on the precursors of N-nitroso compounds the presence of amines, amides, and nitrite in human diets is therefore, unavoidable, even without the consumption of cured meat items. 22 Toxicology of N-Nitroso Compounds Carcinogenicity Lijinsky (1977) reported that N-nitroso compounds can be divided into two classes according to their action as carcinogens: 1) N-nitrosamines 2) N-nitrosamides. N-Nitrosamines are considered indirect acting carcino- gens and require metabolic activation (Lijinsky, 1977). These compounds have been studied extensively as potential environmental carcinogens. Only a few have been found to be non-carcinogenic (Magee and Barnes, 1956). Some of these di- alkylnitrosamines have been shown to be carcinogenic to a variety Of animal species, including subhuman primates, hamsters, mice, pigs, dogs, parakeets and monkeys (Preussmann 25 al., 1976; Wishnok, 1979). N-Nitrosamides on the other hand are direct acting carcinogens and considered not to need metabolic activation (Narisaw et al., 1971). Druckrey (1975) reported that N- nitrosamides, in contrast to dialkyl nitrosamines, are un- stable in alkaline solutions and decompose readily to yield alkyldiazo hydroxide which is the proximate alkylating carcinogen. Accordingly they are to be considered as direct acting carcinogens. Druckrey (1972) stated that N-nitrosamides unlike N- nitrosamines often cause cancer at the site of application in animals. Their biological efficacy corresponds closely to the chemical reactivity of the compound. It is highest 23 with adkylnitrosocarboxylamides, and decreases in the following order: carbamic esters (urethans), ureas, biurets, and nitroguanidines. Druckrey (1975) also reported that within individual groups of N-nitrosamides, the smaller the molecule the less carcinogenic it is. The carcinogenicity of N-nitrosamides generally decreases with increasing number of C atoms in both the acyl and alkyl groups. Table 2 lists some of the N-nitrosamides and some of the sites where they induce tumors (Preussmann, 1973). The data in Table 2 demonstrate that tumors can be pro- duced in a great many tissues. The organ specificity of action depends mainly on the chemical structure of the com- pound, and to a minor degree on the animal species, the route Of application and the dosage rate (Preussmann, 1973). Mirvish (1977) reported that N-nitrosamides and relat- ed compounds like 1-methyl-1-nitroso-3-nitroguanidine, l- methyl-l-nitroso-3-acety1urea and. methylnitrosourea are among the few compounds that induce glandular stomach tu- mors in rodents. Since N-nitrosamides are rather unstable and decompose readily under the influence of light, tempera- ture and alkaline pH (ChOW' 1979; Mirvish, 1971), their formation _i__q v_i\_r_g may be more important than their occurrence in foods. Mirvish (1971) stated that if N-nitro- samides are formed 1 vivo in the stomach, they may induce tumors. Sato gt_ 21. (1959) found a significant 24 m momzuou ouoo / Hmcfiam .Emummm .O.a +++ Zuzuo monoahcumEMOI moo>umc .cfioun \\ mmo Nmznoo 595 .3 / nooEoumumuOm .O.Q +++ znzuo monoaxnumel m .\\ mo memoonoo mesa .>fi // nooEOumnmuOm .O.m +++ 2: no woosumuoawnumEIZI mmo\\\ mmuuoo CODEODMIOHOM .O.m ++ ./IZIzuo mofleouwomamnuwfil m8\ comuo :Owuom OMOHOACIZ uwwumu own: COMDDOAHQQ¢ oficmmocfioumu mHoEuom OCMEomouuwz .mmoweomOHOACIz mo hawOwcwmocflOuou .N OHDMB 25 wsocm>ouucfl u .>fi canoe an n .O.Q mmuooumzuoo nooEOum .// owns Hoaoocoam .O.m +++ zazuo lazumOMI.ZIH>:umEI m \ mu NxmmoCZIoo ouou Hocflmm .>w //. mw>umc .nmwumm .O.m +++ \\\2| no mounamzumeuul mmo comuo :Owuom Omouuwcuz ammuou Cam: cofiuoOmem< vasomocfioumu waosuom moflEmmOuuwz Ao.ucoov ~ manna 26 positive correlation between the high incidence of gastric cancer and the high intake of salted fish and vegetables in Japan. They suggested that gastric cancer couLd be due to N-nitrosamides formed i_n 1132 from N-nitrosation of ureas and methylguanidine which are present in fish includ- ed in the diet (Mirvish, 1971). Mirvish (1975) failed to induce tumors in rats by feeding nitrite plus the amides, N-methylacetamide, N-methylurethan, N-ethylurethan, phenylurea and l-methyl-B- acetylurea. However, the corresponding N-nitroso deriva- tives are known carcinogens. They concluded that of the many amines and amides that may be found in the human en- vironment, in food, and in drugs, only a few are easily nitrosated under the conditions prevailing in the human organism, especially in the stomach. Mutagenic Action of N-Nitroso Compounds Magee (1972) pointed out that an identical molecular mechanism may account for both the carcinogenic and muta- genic activity of N-nitroso compounds since much of the evi- dence favors alkylation as the biochemical mechanism. N-Nitrosamides, but not N-nitrosamines, are mutagenic in bacterial systems 1 vivo. The N-nitrosamines appear to require metabolic activation by the mammalian enzyme sys- tem before they can exert a mutagenic effect. This was demonstrated with NDMA in the host-mediated microbial assay 27 of Gabridge and Legator (1969) and by the liver microsomal- activated microbial system of Ames et a1. (1973). N-Nitrosoguanidine and other N-nitrosamides have been reported as being among the most powerful mutagens (Magee and Barnes, 1967; Zimmermann, 1971; Mandell and Greenberg, 1960). Although the N-nitrosamides are mutagenically active in bacteria, yeasts, Neurospora, plants and drosophila, N- nitrosamines have generally been reported to be inactive in all the above organisms except drosophila (Magee, 1972). Magee and Barnes (1967) reported that the relatively un- stable N-nitrosamines which are believed to require enzyma- tic decomposition before becoming active carcinogens, are mutagenic only in drosophila and inactive in microorgani— sms, such as Escherichia coli, Neurospora and Sac- charomyces. Magee and Barnes (1967) state that this maybe related to the presence of enzymes capable of -oxidation of the N-nitrosamines in drosophila and their absence in the microorganisms. Zimmerman 21.; a1. (1965) observed that N-methyl- nitrosamides are mutagenic in saccharomyces at pH 2, and that the compounds decomposed to yield nitrous acid. They further demonstrated that deamination of adenine occurred when the base was exposed to some N-nitrosamides at pH 2. These results led them to conclude that N-nitrosamides may exert their mutagenic action via deamination by nitrous acid at low pH and by alkylation at high pH. 28 Microorganisms, i.e., certain species of bacteria, are most commonly used for testing for mutagenic effects. The chemical in question is introduced into the grwoth medium of these organisms. The changes if any, both in metabolism and in the genetic material of the cell, are examined for mutagens. Magee and Barnes (1967) stated that the validity of such tests is questioned if the results are compared to humans, because most chemicals ingested and absorbed through the samall intestine in human pass through the liver first. The liver has many mechanisms for detoxifying chemicals, rendering them harmeless to the body. They are then excreated in the urine. Bacteria do not have this pro- perty. The mutagenic test receiving the widest attention at the present time is the reverse mutation test developed by Ames et 11. (1975). This test uses a mutant strain of Salmonella typhimurium that lacks coding for the enzyme phosphoribosyl ATP synthetase, which is required for histi- dine synthesis, thus, this strain is unable to grow in a histidine-deficient medium unless a reverse mutation has occrred. Since many chemicals are not mutagenic or carcino- genic unless they are biotransformed to a toxic product by the endoplasmic reticulum (microsomes), rat liver micro- somes are usually added to the medium containing the mutant strain and the reverse mutation is then quantitated by the growth of the strain on a histidine-deficient medium. The principle of the test is based on the use of mutants with 29 an unique type of DNA damage. Ames (1975) introduced a base substitution strain TA 1535 and two different kinds of frameshift mutations, TA 1537, TA 1538, for detecting muta- tions by the sensitive and convenient back mutation test. The sensitivity of the test strains has been increased by adding to them an additional deletion mutation, designated as UVrB. This effectively eliminates any DNA repair system that normally would protect salmonella from mutation by ultraviolet light. The effect of choosing strains carring the UVrB defect has been shown to increase the sensitivity of the strains to most chemical mutagens. Still another mutation, rfa, was also incorporated into the test which changed the nature of the lipopolysaccharide bacteria cell wall. It became more permeable to chemicals like the poly- cyclic and heterocyclic hydrocarbons and thus more sensi- tive to the mutagenic activity of such compounds. Two new test strains (TA 100 and TA 98) have recently been intro- duced by McCann gt al. (1975b). They were developed by transferring a resistance transfer factor, PKM 101, to both standard TA 1535 and TA 1538. This made new test strains much more sensitive to reversion with a variety of potent carcinogens, such as aflatoxin B1. Ames get 31. (1975) stated that the sensitivity of the bacterial mammalian-microsomal system makes it useful as a tool for rapidly obtaining information on the muta- genic and potential carcinogenic activity of uncharacteriz- ed compounds in complex mixtures. This test commonly known 30 as the Ames test, can be used as an assay to identify the mutagenic components of complex mixtures. The test is high- ly selective for the detection of carcinogens. The Ames sal- monella tester strains have been used by .McCann '§t_'§1. (1976) to screen large numbers of carcinogens for mutagenic activity. The results showed a positive correlation of 90% between mutagenicity and carcinogenicity. Ames and McCann (1976) reported that out of 106 run: carcinogens tested about 85% were non-mutagenic. Sugimura gt a1. (1976) determined the correlation between mutagenicity and carcino- genicity of various substances. Using carcinogenicity data from the published literature and data on mutagenicity ob- tained in their own laboratory, they concluded that many mutagenic compounds are also carcinogenic while many non- mutagenic compounds are not carcinogenic. They found that number of mutagenic compounds that are noncarcinogenic, and conversely, the number of nonmutagenic compounds that are carcinogenic are very small. Their work demonstrated that most carcinogenic compounds give positive tests by the Ames procedure, whereas, very few noncarcinogens give positive results. This indicates the validity of the Ames mutagenici- ty test as a.rapid screening procedure for determining en- vironmental carcinogens. MATERIALS AND METHODS Reagents All chemicals and solvents employed were of analytical grade and used without further purification. A11 fatty acids and their methyl esters were purchased from Fisher Scientific CO. (Fair Lawn, N.J.). Pentylamine, norleucine, valine, methionine and N-methylpropionamide were purchased from Eastman Kodak Co. (Rochester, N.Y.). Column packing materials were obtained from Supelco, Inc., Bellefonte, PA. Lard was purchased from a local retail market. Pork bellies were purchased from a local supplier soon after slaughter and stored in a cooler at 2°C until used. Preparation of Amides in Model Systems (1) Amino Acids plus Saturated Fatty Acids Palmitic acid (1 mole) and norleucine (1.5 moles) were heated together for 1 hour at 200°C in a 150-ml round bottom flask fitted with a water-cooled condenser. After cooling, the reaction mixture was slurried in warm (30°C) diethyl ether, and any unreacted amino acid was removed by filtration. The solvent was evaporated in a Buchii rotary 31 32 evaporator. The residue was redissolved in a minimum of warm (40°C) petroleum ether and then left at rOom tempera- ture to crystallize. The crude amide mixture was dissolved in 100 m1 of methanol and then made alkaline with 1 N methanolic KOH. The solution was stirred for 1 hour before evaporating to dryness under vacuum. The dried residue was extracted with diethyl ether and filtered under suction. The ether extract was washed twice with aqueous 0.1 N KOH, followed by two washings with water. A series of amides was prepared in a similar manner by reacting norleucine with lauric, myristic and stearic acids, and by reacting lauric acid with valine and methionine. (ii) Amino Acids plus unsaturated Fatty Acids A 4 g aliquot of oleic (or linoleic) acid was heated with 1.6 g of norleucine at 200°C for 1 hour as described previously. Removal of the unreacted amino acid and evapora- tion of the diethyl ether left an oily liquid. This crude product was purified by column chromatography using Supel- cosil (ATF 061) as described by Sims and Fioriti (1975). A 3 9 sample of the crude product was dissolved in a 4 m1 of petroleum ether and applied on the column. The material on the column was eluted with 200 m1 of petroleum ether, which was discarded. A 250 ml aliquot of petroleum ether/diethyl ether (90:10, v/v) was then used to elute a second fraction which was collected and evaporated down until only an oily liquid comprising the amide remained. 33 (iii) Amino Acids jlus Lard or Pork Bellies Adipose Tissue Lard or pork belly adipose tissue (4 g) and 1.6 of norleucine were heated for 1 hour at 200°C. The reaction mixture was slurried in warm diethyl ether and filtered. The filtrate was evaporated to a paste and purified by thin-layer chromatography (Sims and Fioriti, 1975). The TLC plates were developed in a solvent system of petroleum ether/diethyl ether/acetic acid (40:60:1, v/v) dried, and sprayed with water to visualize the bands. The amide- containing bands were scraped off the plates, dried and the amides eluted from the absorbent with diethyl ether. Nitrosation of N-Substituted Amides Preparation of N-Nitroso-N-pentylpalmitamide. The nitrosation procedure was based on the method described by White (1955) with a few modifications as out- lined by Kakuda and Gray (1980b). A 3.25 g aliquot of recrystallized N-pentylpalmitamide (prepared as previously described) was dissolved in a solvent mixture containing glacial acetic acid (50 ml), acetic anhydride (50 ml) and chloroform (95 ml). The mixture was cooled in an ice bath and 15 g of sodium nitrite were slowly added with stirring over a 4-5 hour period. After reacting overnight at 4°C, the mixture was carefully poured into ice water. The chloro- form phase was collected and the water phase was extracted 34 with another 100 ml aliquot Of chloroform. The pooled chloroform extracts were washed with 'water, 5% K CO 2 3 solution and again with water before evaporating to dryness under vaccum. The crude N-nitrosamide preparation remaining was partially purified by precipitating the unreacted amide in cold petroleum ether (4°C) followed by vacuum filtra- tion of the cold mixture. The clear yellow filtrate was placed on a Supelcosil AFT 061 column and eluted with 60 ml of petroleum ether. All the N-substituted amides, which were prepared previously from the reactions of norleucine, valine and methionine with capric, lauric, myristic, stearic, oleic and linoleic acids were nitrosated in the same manner . Preparation of N-Nitroso-N-methylprgpionamide (NOMP) A 25-9 aliquot of N-methylpropionamide (Eastman Kodak CO., Rochester, N.Y.) was dissolved in a mixture containing 120 m1 of glacial acetic acid and 138 mL of acetic anhy- dride and cooled to 0°C. Sodium nitrite (61 g) was added slowly to the mixture over a 4 hour period. After allowing the mixture to react overnight at 4°C, the N-nitrosamide was extracted with chloroform. The chloroform extract was washed successively with water, 5% K CO 2 3' with water. The solvent was removed by vacuum evaporation. and again The remaining N-nitroso-N-methylpropionamide was purified by vacuum distillation, which was repeated a second time. The distillations were conducted at 40°C (15.0 mm Hg) and 35 distillates were collected in a receiving falsk packed in ice. The first and final 5-10 mL portions of the distillate were discarded during each distillation. Analysis and Identification of Amides and N-Nitrosamides The purity of the N-substituted amides and N-nitro- samides was determined by gas liquid chromatography (GLC) while their identities were confirmed by gas liquid chroma- tography-mass spectometry (GLC-MS). A Hewlett Packard gas chromatograph (Model 5830A) equipped with a flame ionization detector (FID) and a Hewlett Packard 18850A GC therminal was used for analysis of the amides and N-nitrosamides. A glass column (2m x 2mm, i.d.) was packed with 3% OV-lOl on 80/100 Supelcoport (Supelco Inc., Bellfonte, PA). The chromatograph was operat- ed under the following conditions: Initial temperature (Tl) . 240°C (30°C for N-methyl- propionamide and NOMP) Time at T1 (t1) : 1 min Final temperature (T2) : 260°C Time at T2 : 10 min Injection port temperature : 250°C Flame ionization detector 0 temperature : 350 C Chart speed 1 cm per min Attenuation variable 36 Slope sensitivity : 0.5 Carrier gas : Nitrogen Carrier gas flow rate : 30 ml per min Hydrogen flow rate 30 ml per min Air flow rate 200 ml per min The samples were injected using a 10 ul Hamilton Syringe (Hamilton CO., Reno, Nevada). The volumes of sample injected varied from 0.5 to 1.5 ul. The GC/MS system was a Hewlett Pakard 5985A gas chroma- tograph/Hewlett Packard mass spectrometer (Hewlett Packard Corp., Arondale, PA). The column was the same as that used for GC analysis. Helium was the carrier gas with a flow rate of 25 ml/min. The analysis were carried out using a temperature program from 240 to 260°C! at SOC/min, with a one minutes hold time at 240°C. The ion source and analyzer temperature of the mass spectrometer were maintained at 200°C. The electron multi- plier voltage was 2000 V and the ionization potential was 70 eV. Formation of N-substituted Amides in Pork Belly Slices Processing of Pork Bellies A fresh pork belly weighing approximately 8 lbs was sliced to 1/8 inch in thickness. The slices were divided 37 into two groups. One of the groups was sprayed on the sur- face wtih pentylamine, while the other group of slices was freeze dried and then rehydrated with water containing pentylamine, followed by equilibration for 48 hours. Two pork bellies approximately the same size were selected and stitch pumped with water containing pentyl- amine and/or norleucine. All treated bellies were smoked for 4 hours at 58°C (dry bulb) and 3 hours at 52°C (dry bulb) at ambient relative humidity in a laboratory smoke house (Drying System Inc., Chicago, IL). Smoke was applied throughout cooking with a midget size Mepaco Smoke genera- tor (Meat Packers Equipment CO., Oakland, CA) utilizing mixed hard wood sawdust. The smoked bellies were transferr- ed Ix> a tempering cooler (-2°C) where they were held over- night prior to slicing. Frying of the Bacon Slices The treated slices were fried in a Sunbeam (Sears and Roebuck , Chicago , IL) electric fry pan . Each group of slices was fried such that half of them were held at 180°C for 4 minutes on each side and the other half for an additional 4 minutes on each side. Cook-out fat was taken for analysis after each cooking interval. 38 Analysis of N-substituted Amides in Fried Pork Belly Slices and Cook-out Fat. Twenty grams of fried pork belly slices were homogeniz- ed with 10 ml distilled water in a Waring Blender and extracted three times with 50 m1 of methylene chloride. The extract and tissue residue were then transferred to a medium grade sintered glass funnel and filtered under vacuum. The homogenizer and the residue in the funnel were washed with an additional volume of methylene chloride and filtered. The extract was quantitatively transferred to a 500 m1 separating funnel and distilled water (10% by volume) was added and throughly mixed. The mixture was allowed to se- parate into two phases until the interface was Clear. The upper phase was transferred to a 500 m1 volumetric flask and evaporated to dryness in a vacuum Rotavapor-R (Buchi, Switzerland) at 30-400C. The crude product was purified by column chromatography (Supelcosil-ATP 061). The crude dried extract was dissolved in 5 ml of petroleum ether and applied on the column. The sample was eluted with 200 ml of petroleum ether, followed by 250 m1 of petroleum ether/di- ethyl ether (90:10, v/v). The first fraction was discarded and the second fraction was collected and evaporated to dryness. The purified amide was dissolved in 5 ml diethyl ether for GLC analysis. 39 Formation of N-Nitrosamides in Pork Belly Slices A solution of N-pentylpalmitamide in diethyl ether (100 mg in 3 ml) and sodium nitrite in water (0.5 g in 3 ml) was injected into 50 g of pork belly slices with a Hamilton syringe and stored 24 hours at 4°C. The slices were fried as previously described. The fried pork belly slices and cooked-out fat were extracted with chloroform. The pooled chloroform extracts were washed with water and evaporated to dryness. The crude product was partially puri- fied by precipitating the unreacted amide in cold petroleum ether (4°C) and vacuum filtering. The filtrate was placed on a Supelcosil ATP-061 column eluted with 100 m1 of petro- leum ether. The petroleum ether was evaporated and the purified compound was dissolved in 5 m1 of diethyl ether for GLC analysis. Thermal Stability of N-Nitrosamides under Frying Condi- tions of Bacon. 100 mg of N-nitrosopentylpalmitamide were dissolved in 3 ml of diethyl ether and injected into 100 g of pork belly slices with a Hamilton microsyringe. 50 g of pork belly slices were fried as before. The fried pork belly slices and cooked-out fat were extracted and analyzed for N-nitro- sopentylpalmitamide. The remaining 50 g of raw pork belly were extracted and analyzed in a similar manner and used as the control. 40 Determination of Fatty Acid Composition of Lard and Pork Belly Adipose Tissue. The fatty acid composition of lard and pork belly adipose tissue was determined by gas chromatographic analy- sis of their fatty acid methyl esters. Preparation of Methyl Esters for GLC Analysis Esterification of the fatty acides was carried. out according to the procedure of Morrison and Smith (1964). An aliquot of the lipid was placed in a test tube fitted with a teflon-lined screw cap. Boron trifluoride-methanol rea- gent was added under nitrogen in the proportions of 1 m1 of reagent per 4-16 mg of lipid and the tube was closed with the screw cap. The tube was then heated in a boiling water bath for 30 minutes, cooled and opened. The esters were extracted by adding 2 volumes of pentane, then 1 volume of water, shaking briefly and centrifuging until both layers were clear. The top organic layer was removed using a micro- pipette and placed in a clean vial. The solution was concen- trated by slowly removing part of the solvent in stream of nitrogen. A Hewlett Packard gas chromatograph (Model 5830A) equipped with a flame ionization detector (FID) and Hewlett Packard 18850A GC therminal was used for the analy- sis of the fatty acid methyl esteres. The glass column (2m x 2mm. i.d.) was packed with 15% diethylene glycol succin- ate (DEGS) on Chromosorb W 60/80 mesh. The instrument was 41 operated under the following conditions: Initial temperature (Tl) : 190°C Time at Tl(t1) : 20 min Final temperature (T2) : 260°C Time at T2 : 10 min Ingection port temperature : 210°C FID temperature : 350°C Chart speed : 1 cm per min Attenuation : 8 Nitrogen carrier gas : 30 ml per min Hydrogen flow rate 30 ml per min Air flow rate : 200 ml per min Standard fatty acid methyl esters were prepared under iden- tical conditions and used for identification and quantitat- ing the fatty acids in the samples. Effects of N-substituted Amides and N-Nitrosamides on Tumor Development in Mice 160 female mice were allotted into 9 groups of 20 mice each, except for groups 8 and 9 had only 10 mice. The mice were idenfified by numbering from 1 though 10 by clipping their toes. The animals were watered and fed purified diet (Appendix 11) containing one of the following additives as indicated in Table 3. 42 Table 3. Additives that has been added to purified diet for different groups of mice in feeding trial. Group Compound Group 1: Control Group 2: (300 mg/kg) N-Methylpropionamide r3 Group 3: (300 mg/kg) N-Methylpropionamide + E; (300 mg/kg NaNOZ) 9 Group 4: (300 mg/kg) N-Pentylpalmitamide Group 5: (300 mg/kg) N-Pentylpalmitamide + (I4 300 mg/kg NaNO2 Group 6: (300 mg/kg) Nitrosomethylpropionamide Group 7: (300 mg/kg) Nitrosopentylpalmitamide Group 8: (300 mg/kg) Nitrosomethylurea (positive control) Group 9: (300 mg/kg) Nitrosopentyldecanamide 43 Food and water were given ad libitum and the intake per group of 10 mice was recorded. After 7 months on the diet, 10 mice from each group were sacrificed by etherizing (ex- cept groups 8 and 9 in which 4 mice were sacrificed). Dr. Stuart D. Sleight of the department of Pathology grossly examined the internal organs and glands for tumors and other abnormalities. Samples of liver, lungs, spleen, heart and stomach were then placed in 10% neutral formalin. Fixed tissues were processed routinely, embedded in paraffin and cut into 6-um sections-tissue. Sections were stained with hematoxylin and eosin and examined by light microscopy, and the histologically Observed lesions were characterized as benign or malignant. The remaining mice were kept on the same diet which will continue for a 2 year period. Ames TBSt The Ames test was performed according to the procedure described by Ames at at. (1975). The compounds were tested for mutagenecity using the Salmonella strain G46 (mutation in TA 1535) with and without S-9 mix. Instead of the standard plate test, a liquid suspension test was used. This involves pre-incubatian of chemicals with S-9 mix and bacteria in liquid suspension for approximately 20 minutes at 37°C before plating. The S-9 mix was prepared following the procedure Of Ames gt gt. (1975). Male rats (300 g) were injected 44 interaperitoneal (ip) with Aroclor 1254 (500 mg/kg). Five days before exsanguination, the 9000x g supernatant was col- lected and frozen until needed. RESULTS AND DISCUSSION PreEaration of N-Substituted Amides Long chain and short chain N-substituted amides were prepared in gram quantities and purified by crystallization in petroleum ether, or by column chromatography. The forma- tion of these N-substituted amides was confirmed by GLC-MS analysis. Figure 1 shows a gas chromatogram of N-pentylsteara- mide (NPS), prepared by reaction of norleucine and stearic acid at 200°C for two hours. The reaction involves decar- boxylation of norleucine and replacement of the alcohol moiety of the fatty acid with the amine group which is form- ed (Sims and Fioriti, 1975). The mass spectrum of NPS is Shown in Figure 2. and exhibits major ion peaks at m/e 129, 297, 353 (M+), 267, 57, and 30. The most intense peak in the mass spectrum of NPS is a rearrangement ion at m/e 129, which is correlated with Cleavage of the C-C bond beta to the carbonyl group, and is aceompanied by rearrangement of a hydrogen atom (Gilpin, 1959). The breakdown of NPS to form the m/e 129 peak is 45 zo'ommxn mm Figure 1. 46 —NPS ] 2 3 4 5 6 7 3. THE (f'eIHUTES) Gas chromatogram of N-pentylstearamide. 47 Figure 2. Mass spectrum of N-pentylstearamide prepared from the interaction between norleucine and stearic acid. a/m 48 RELATIVE INTENSITY _a ._a N b 05 m C) N 4:- 03 0’) O O O O O O O O O C) O O C) 1 I 1 l L j I l l 1 N 31 3.. L...) O 5‘; W: .I C) O p A) _ (J... N_ m = \‘ Ch Dc: 0 I‘d i- Ch )--— V I— ‘4 1* w) M m 00.] Cal 0 U.) N __a O \O O \I o" w d — E N N. crfi c: # -‘ Q 1 o w ._a 4:... #4 O o ._a ‘ b F N w — (A) w ._a on. C: CH c: (a; c: v .—I w m oo— o" O N b O O O O Ke'h.‘ l l Ll- 49 shown below: 0 H H l CH3 -(CH2)13 -CH2 -CH2 -CH2 -C -N -CH2 -(CH2)3 -CH3 m/e 353 HO H I l 3 -(CH2)13 -CH =CH + CH2 =C -N -CH2 -(CH2)3 -CH3 m/e 224 m/e 129 CH The second most intense peak (m/e 30) resulted from cleavage of the C-C bond beta to the nitrogen atom. It was accompanied by a hydrogen rearrangement on the nitrogen- containing fragment (Budzikiewicz gt. ‘gl., 1967) as illustrated below: CH -(CH ) -C -NH -CH -(CH ) -CH 3 2 16 N 2 2 3 3 O +. + CH3 -(CH2)16 -fi -NH =CH2 + CH3 -CH2 -CH2 -CH2 0 (m/e 297) (m/e 57) + + CH3 -(CH2)16 -C0 + HZN =CH2 (m/e 267) (m/e 30) Other important peaks in the spectrum and their structures are shown below: - (CH ) - CO+ (m/e 267) CH3 2 16 - (CH ) + (m/e 239) CH3 2 l6 50 The mass spectrum of N-pentylpalmitamide exhibited major ions at m/e 129, 296, 325 (M), 239, 268, and 30. A similar fragmentation pattern has been reported by Kakuda and Gray (1980a) for NPP. The mass spectrum of the other amides are presented in Appendices (1-6) and confirmed their formation from amino acids (valine and norleucine) and fatty acids (lauric, myristic, oleic and linoleic). A mixture of purified amides was prepared and used as standards for identificaiton on the basis of retention times of the amides formed in pork belly slices. Figure 3 shows the GLC chromatogram of the standard mixture of N- pentylmyristamide, 'N-pentylpalmitamiden 'N-pentylstearamide, N-pentyloleamide and N-pentyllinoleamide. Figure 4 shows the GLC chromatogram of N-isobutymyristamide, N-isobutyl- palmitamide and N-isobutylstearamide and were derived from reaction of valine with the corresponding fatty acids. Fatty Acid Composition of Lard and Pork Belly Adipose Tissue. The fatty acid composition of lard. and. pork belly adipose tissue was determined by gas chromatographic analy- sis Of their fatty acid methyl esters. Figure 5 shows that pork belly adipose tissue contains myristic, palmitic, stearic, oleic, and linoleic acids. The analysis revealed that lard contained 1.0, 19.5, 17.0, 51.0 and 10.6% of myristic, palmitic, stearic, oleic and linoleic acids, res- pectively. Figure 3. 51 ,NPO n1cn 22c: Taco n1 :0 NPL NPS NPP TII’E (IIIIUTES) Gas chromatogram of standard N-pentylmyristamide (NPM), N-pentylpalmitamide (NPP), N-pentyllinole- amide (NPL), N-pentyloleamide (NOP) and N-pentyl- stearamide (NPS) formed from reaction of pentyl- amine and fatty acids. 52 Q) “U '1- . E '. '5 f +4 u- _L E w v '- 'U (U '0— . D. E 2, E I ' H (U 3 Q) I .o .u l O m I (I) l— . .'_ >1 ' 44 Z 3 : , .D . O - U1 ' I Z N-isobutylmyristamide I rn ”’33 CD'U U7le :0 I ‘ I ......._--....___ .. 1 ... ... _.—_—\\;h—— . , I} THE (MINUTES) Figure 4. Gas chromatogram of standard mixture containing N-isobutylmyristamide, N-isobutylpalmitamide, N- isobutylstearamide prepared from reactions of valine and corresponding fatty acids. 53 T WMZO'UMITIIU C18:1 123459F3‘910I‘II2‘T Time (Minutes) Figure 5. Gas chromatogram showing fatty acid composition of pork belly adipose tissue. 54 Formation of Amides in Pork Belly Slices Treated with Norleucine and/or Pentylamine Freeze dried pork belly slices were rehydrated in water containing 1000 mg/kg of pentylamine. After equili- bration for 24 hours, the treated slices were fried 4 minutes on each side at 175°C and analyzed for amide for- mation. The analysis revealed that there was neither amide formation in the cook-out fat nor in the fried bacon after heating. After heating of the fried bacon and cook-out fat for an additional 8 minutes, five N-substituted amides were identified in the cook-out fat and cooked residue (Figure 6), indicating that the time of frying is the limiting factor in their formation. It was noted that the relative percentages of the various amides formed closely approximat- ed the fatty acid composition of pork belly adipose tissue as indicated in Table 4. Similar results were obtained when pentylamine was sprayed on the surface of pork belly slices, followed by frying. N-Substituted amides were fOrmed from the reaction of added pentylamine and the fatty acids naturally present in the pork belly slices. The results are in agreement with the findings of Kakuda and Gray (1980a), who reported that free amines react readily upon heating with fatty acids and fatty esters. They repoted that a temperature of 100°C for 15 minutes was sufficient for amide formation from the reaction of pentylamine with tripalmitin or palmitic acid. Figure 6. 'U (DFT‘I 73 I '—~ _. 4.- (H (r) " ' 55 .. ._.____.._.____..__._......______.....‘3 : ,et NPO __ . ..--.. - - NPP NPM TNE:(HU1HES) Gas chromatogram of N-substituted amides (N- pentylmyristamide, N-pentylpalmitamide, N-pentyl- linoleamide, N-pentyloleamide and N-pentylstear- amide) present in cook-out fat from rehydrated freeze dried pork belly slices containing pentyl- amine, heated for 16 minutes at 175°C. 56 Table 4. Fatty acid composition of pork bellies and N-subs- tituted amides which formed upon heating pork bellies containing pentylamine Fatty Acid Percent % Amide N-Substituted Amide C14 1.3 0.4 Pentylmyristamide C16 16.5 16.3 Pentylapalmitamide C18 11.1 14. Pentylstearamide C18:l 49.8 43. Pentyloleamide C18:2 15.4 15.5 Pentyllioleamide When the study‘ was repeated. with norleucine, amide formation did not take place in the cook-out fat or fried bacon, even after an additional heating period of 8 minutes at 175°C. However, on heating norleucine and pork belly adipose tissue or lard for one hour at 200°C, five N- substituted amides were identified (Figure 7). These re- sults indicate that time and temperature of frying was not sufficient for decarboxylation of norleucine. Sims and Fioriti (1975) reported that decarboxylation of an amino acid in the presence of a fatty acid ester is zero order and is much slower than the aminolysis reaction. When pork bellies were stitch pumped with pentylamine or norleucine and trilaurine, followed by smoking, slicing and frying, amide formation was not evident in either the smoked belly, the smoked and fried belly, or in the cook- out fat. This was true even though previous experiments had Figure 7. :2 CD 13 oo n1 mm 57 NPO + NPL NPP NPM l , 1‘ I n I I." \' m} \l. 1]]! I i iii: ,4 i 2 3 4 5 o 7 8. THE (HIWTES) Gas chromatogram of N-pentylmyristamide (MPM), N-pentylpalmitamide (NPP), N-pentyl- oleamide (NPO), N-pentyllinoleamide (NPL) and N-pentylstearamide (NPS) present in pork belly adipose tissue with added norleu- cine heated 1 hour at 200 C. 1.1—. 73 58 confirmed the formation of amides in pork belly slices treated with pentylamine under identical frying conditions. Patterson and Mottram (1974) reported that the concentra- tion of volatile amines in pork carcass meat decreased dur- ing the curing and processing. Therefore, these results suggest that low temperature and long periods of smoking resulted in volatilization, and, loss of ;penty1amine. The smoking conditions and frying temperature were not suf- ficient to decarboxylate the norleucine, since, smoked pork belly slices which contained norleucine, when heated 1 hour at 200°C formed five N-substitutes amides corresponding to the fatty acid content of pork belly adipose tissue. These results indicate that formation of primary amines via the decarboxylation of amino acids appear to be unlikely under norml cooking conditions. There does not appear to be sufficient energy for the decarboxylation reac- tion. Kakuda and Gray (1980a) reported high temperatures (minimum 150°C for 45 minutes) were required for the decarboxylation of norleucine. However, the amount of free amines in foods is not limited to those formed by thermal decarboxylation. Many enzymatic and bacterial decarboxyla- tion reactions are known to occur in many foodstuffs (Maga, 1978), and these reactions may serve as sources of free amines. The presence of amines, fatty acids, and high temperatures during cooking and processing may lead to for- mation of secondary amides in foods. 59 Nitrosation of N-Substituted Amides Gram quantities of different N-nitrosamides were pre- pared and purified by column chromatography or vacuum dis- tillation. The purity of the N-nitrosamides was determined by GLC and their identity was confirmed by GLC-MS analysis. All compounds had purities in excess of 95%. Figure 8 shows the chromatogranl of N-nitrosopentyl- stearamide (NOPS) which was prepared by nitrosation of recrystallized N-pentylstearamide. N-nitrosopentylsteara- mide contained small amounts of N-pentylstearamide (NPS). Figure 9 shows the mass spectrum of NOPS and represents only the corresponding ester produced by thermal elimina- tion of nitrogen. White (1955b) repoted that formation of esters and olefins occur according to the following scheme: N=O O l‘ 1 III R - N - C - R ------ > N2 + R O C R (esters) H l 0 N2 + olefins The compound detected by GLC-MS analysis of NOPS correspond- ed to pentylstearate as shown below: r— 267 ) o - ,q (CH) - CH (M+ 354) CH3 (CH2 4 ’ C ‘ 2 16 3 i + 40H c ’\ OH R m/e 285 60 , I '\ r. . C I v I I :' I l m o. f o I z I I I: M II . ' , \ I : C. »«-~————./ -———— I W - I l 2 3 ‘1 5 6 7' 8 '9'!- “II! “51‘. (iIlII'JTES) Figure 8. Gas chromatogram of N-nitrosopentylstearamide. 61 Figure 9. Mass spectrum of pentyl stearate, a breakdown product of N-nitrosopentylstearamide. am 62 RELATIVE INTENSITY ..n _a N D 03 m C N A C‘ CO 0 O O O O O O O O O O O Q j 1 1 J L J o J l W R N cf cf N b #- _— (A) I I_ U.) I— m N- °‘ 0‘ O I_ '8 I— \J \I O N m 84 O— b N J 03 01 w _a c: ‘3. O" Q g —l ch c: w _a h ad O o =- I” w (A) ....I ON 0') O‘- O b O 3 + V ._a w m ar- cf' 0 N «D o O o O 63 The major ions present were m/e 70, 43, 267, 354 (M+) and 285. The mass spectrum of N-nitrosopentylpalmitamide (NOPP) exhibited major inos at m/e 70, 43, 326 (M+) and 239. A similar fragmentation pattern was reported by Kakuda and Gray (1980b) for NOPP. The mass spectra of the other N-nitrosamide confirmed their formation by nitrosating their corresponding N-substi- tuted amides. A standard mixture of purified N-nitrosopen- tylmyristamide, N-nitrosopentylpalmitamide, N-nitrosopenty- lstearamide, N-nitrosopentyloleamide and N-nitrosopentyl- linoleamide was prepared (Figure 10) and used as standards for the identification Of the N-nitrosamides in bacon. Formation of N-Nitrosamides in Bacon Nitrosation of secondary amides under acid conditions in the model system was confirmed by GLC-MS analysis. In order to determine whether nitrosation of amides can occur in bacon, a solution of N-pen-tylpalmitamide and sodium ni- trite were injected separately into the pork belly slices and stored 24 hours at 4°C. The bacon was then analyzed for the presence of 'N-nitrosopentypalmitamide before and after frying. Results showed that N-nitrosamide formation did not occur in the raw or cooked bacon or in the cook-out fat. This is not surprising since the main nitrosating agent for amides is the nitrous acidium ion (Mirvish, 1975a). Thus, nitrosation of secondary amides requires low pH. The extent of nitrosation of secondary amides decreases "I “U ()3 I71 f- I I ~.._.. -.__. ‘.- FT] (0 ~—_-.—o—.,~_ 64 ..-.W- 1 .- _. ...---- , c * a l ; O I2 z ‘ ..J ’I g, I 0- r.i g l(‘,‘(: I In, : + '33 i O ‘f If I a. 3' w In a Z w I) g] h. C) I & II'I' "'I'. Iri Z O «I w Z - , .— o I 2 3 £3 5 <6 } 53. T If}? (MINUTES) Figure 10. Gas chromatogram of standard mixture contain- ing N-nitrosopentylmyristamide (NOPM), N-ni- trosopentylpalmitamide (NOPP), N-nitrosopen- tyloleamide (NOPO), N-nitrosopentyllinole- amide (NOPL), and N-nitrosopentylstearamide (NOPS) derived from nitrosation of their cor- responding N-substituted amides. 65 as the pH of the environment increases and little reaction occurs above pH 3 (Kakuda and Gray 1980b). Thus, the pH of foods would militate aginst the occurrence of nitrosamides, even if their precursors are present. However, 1 vivo nitrosation of alkylamides has been reported by Mirvish (1971). The nitrosation of amides require acid conditions, which occur in gastric juices plus sufficient quantities of nitrite. The eating of vegetables and vegetable juices results in increases in salivary nitrite, 3 levels of hun- dreds of parts per million being reported by Tannenbaum, gt a_]._., 1976. Also, _d_e_ m synthesis of nitrate and nitrite in the upper aerobic portion of the intestine has been reported by Tannenbaum gt al., (1978). Thus, 1 vivo nitrosation of N-substituted amides in the di- gestive tract is possible. Thermal Decomposition of N-Nitrosamides The stability of NOPP under frying conditions similar to those used in the cooking of bacon was also studied. An aliquot (1000 mg/kg) of NOPP solution in corn oil was injected into pork belly slices and stored overnight at 4°C, and then fried. A 95% recovery of the NOPP was obtained from the raw pork belly slices (Figure 11). Analysis revealed that only 8 and 5% of the original NOPP was found in cook-out fat (Figure 12) and fried bacon (Figure 13), respectively. This indicates that 87% of the 66 rm 0):: cp‘raoo n1 :3 5 6 7 8: THE (MINUTES) Figure 11. Gas chromatogram of recovery pentylpalmitamide from raw bacon. of N—nitroso— 67 ZO'Umm :3 mm l ‘. a ‘f“" W ‘E\:;PP (j o 1 13 3 4 5 6 i 33: TM": (HUM-HES) Figure 12. Gas chromatogram of N-nitrosopentylpalmitamide residue in cook-out fat. 68 rm 0):: CD‘U our“ :3 o I 2 :3 4 5 6 L, 3,. THE (MINUTES) Figure 13. Gas chromatogram of N-nitrosopentylpalmitamide residue in fried bacon. ori re: re W 69 original amount of NOPP was lost during heating. These results support the finding of Kakuda and Gray (1980b) who reported N-nitrosamides are much less stable than volatile N-nitrosamines. In their thermal decomposition studies on N-nitrosamides utilizing heating conditions commonly encountered in the pan frying of bacon or in the oven roasting of pork, Kakuda and Gray (1980b) found that NOPP was degraded to the extent of 74-97% compared to 3-14% for NPYR and NDMA. Chow (1979) reported that at temperatures ranging from ambient to 100°C, N-nitrosamides undergo irreversible thermal rearrangements to form diazo esters. Diazo esters, in turn, decompose rapidly to give carboxylic esters or acids and olefins. The instability of N-nitrosa- mides under alkaline and neutral pH and their rapid thermal decomposition leads to the conclusion that the occurrence of N-nitrosamides in food systems is unlikely. The major contribution of Ehsubstituted amides, if present in foods, may be as precursors of N—nitroso compounds formed by _i_n_ vivo nitrosation reactions. Mutagenicity of N-Substituted Amides and Their Correspond- ing N-Nitrosamides The mutagenicity of some short chain and long chain N— substituted amides and their corresponding N-nitrosamides was tested by using Salmonella strain G 46 (mutation in TA 1535) with and without S-9 mix. The results of the test are 70 presented in Table 5. The numbers shown in Table 5 repre- sent the frequency of histidine-positive revertants per plate after substraction of the spontaneous mutation rate (background). The starred values (*) deserve consideration as possi- ble mutagenic compounds. None of the N-substituted amides which were tested showed.:mutagenic potential. N-Nitroso- pentylpalmitamide was the only N-nitrosamide which gave a strong mutagenic response without S-9 activation. N-Nitroso- methylpropionamide showed. weak. mutagenic activity at ‘the concentration tested upon addition of S-9 mix. N-Nitroso- methylurea was not mutagenic in the Ames test but micro- somes from induced rat liver (S-9 mix) can convert it to derivatives, which are mutagenic. These results are in con- trast to the findings of Magee (1972), who reported that N-nitrosamides are mutagenic without metabolic activation. McCann 25. al. (1975a) reported that N-nitrosomethylurea is mutagnic in Salmonella strain TA 100 and TA 1535 without the addition of S-9 mix. Brundrett g gl. (1979) studied the mutagenecity of seven N-nitrosoureas and nitro- samides and reported that mutagenecity of none of the com- pounds was enhanced by the addition of S-9 extract to the standard Ames test. In fact, mutagenic activity was often decreased slightly by S-9 addition. Furthermore, these authors showed a decrease in mutagenecity of N-nitroso- acetamide and nitrosoureas with an increase in N-alkyl chain length. 71 Table 5. Mutagenicity potential of N-substituted amides and their corresponding N-nitrosamides. His+ Revertants/plate Concn. Compound (+) S-9 (-) S-9 1 mM N-pentylpalmitamide ND ND 1 mM N-nitrosopentylpalmitamide ND 2350* 1 mM N-methylpropionamide ND ND F 1 mM N-nitrosomethylpropionamide 280* ND 1 mM N-nitrosomethylurea 1086* 43 1 mM N-(3-methylbutyl) myristamide ND ND ' E N-(3-methylbutyl) stearamide ND ND 1 mM N-nitroso—(3-methylbutyl) stearamide ND ND ND=non detectable 72 The present observations on mutagenecity of N-nitrosamides which can be considered only as preliminary tests are incon- sistent with the literature. The contradictory nature of the results could be due to the instability of the N-nitro- samides and rapid decomposition via photolysis, alkaline pH or temperature (ambient to 100°C). Therefore, The observ- ed mutagenicity reponse could be due to the breakdown pro- ducts. Further studies on mutagenicity of these compounds with different strains of Salmonella are required before any positive conclusions can be drawn. Carcinogenicity of N-Nitrosamides The effect of N-substituted amides and N-nitrosamides on tumor development was investigated in feeding trials using Swiss mice. Table 6 presents the data on feed and water consumption per mouse per day in each group. The data on Table 6 indicate that the feed and water consumption of the mice in group 6 was greatly reduced, being only half as much as for the other groups. within two weeks on the diet, two mice from group 6 died and after 3 weeks, the remaining mice began sneezing, chattering and showing signs of respiratory distress. They refused to consume their feed and water and started to lose the hair from their backs. Two mice from this group were sent to the Animal Health Diagnostic Laboratory at Michigan State University. The laboratory examination report indicated that a light brownish red discoloration was observed in the 73 Table 6. Effect of additives on feed and water consumption of mice Group Additivesa 9 feed/ mL H20/ mg additives/ mouse/day mouse/day mouse/7 months Control 1 none 4.2 7.98 0 2 NMP 4.2 7.61 264.6 3 NMP+NaNO2 4.00 7.93 252 4 NPP 3.82 7.52 239.4 5 NPP+NaNO2 4.90 8.10 245.7 6 NOMP 2.1* 4.23* 163.8 7 NOPP 3.85 7.87 242.5 8 NMU 3.93 8.02 241.9 9 NOPD 3.84 7.89 247.6 a. NMP = N-methylpropionamide NPP = N-pentylpalmitamide NOMP = N-nitrosomethylpropionamide NOPP = N-nitrosopentylpalmitamide NMU = N-nitrosomethylurea NOPD = N-nitrosopentyldecanamide 74 carnial lung lobes of these mice. The problem was suspected to be a viral disease triggered by the stress induced by NOMP. However, the laboratory examination was unable to identify a specific disease in these mice. 18 mice out of 20 were dead within 7 months while mice in other groups appeared quite normal. After seven months on the experiment, 10 mice from each group were sacrificied except for groups 8 and 9 with 4 mice each and group 6 with 2 mice. Table 7 presents the results of the gross examination of the mice for tumors and other abnormalities. Microscopic examination of liver, lungs, spleen, heart and stomach tissues showed that the only mice that had any consistent number of lesions were in group 8, which was fed the purified diet plus N-nitrosomethylurea. Two of four mice in this group had adenocarcinomas in the lungs and the other two had adenomas. These lesions were seen grossly in three of four mice. The carcinogenicity of N-nitrosomethylurea has been demonstrated in several species for a variety of tissues depending on animal species, the route of application and the dosage rate. Preussmann (1973) reported that NMU deve- loped tumors of the forestomach in mice when the compound was administered by mouth, and tumors of the lungs by intravenous (i.v.) route of application. Denlinger gt 31. (1978) reported that metastasis occurred to liver, spleen, lymph nodes, lungs and adrenal of purebred Boxer 75 Table 7. Data on gross examination of the mice for tumors and other abnormalities upon autopsy. Group Abnormalities 1 Normal 2 Mouse #1 had enlarged uterus, filled with olive green watery fluid 3 All mice normal 4 Mouse #2 had enlarged spleen - 1 l/2 times normal size 5 Mice #3, 8 and 9 had enlarged uterus filled with olive green liquid 6 Only 2 mice survived. The hair was sparse on both 7 All normal 8 Small modules in lungs found in four mice that were examined 9 All normal 76 dogs when given weekly i.v. injections (5 mg/kg) of NMU for 36 weeks and observed for approximately 3 years. Although the hair was sparse, skin sections from the surviving 2 mice in group 6 had no histological lesions. Sections of spleen, kidney, stomach and intestine were normal. N-Nitrosopentylpalmitamide and N-nitrosopentyl— decanamide also did not develop any tumors in the above tissues. These compounds are high molecular weight with long chain alkyl and acyl groups, therefore, the possibili- ty that these compounds are not carcinogenic exists. Druckrey (1975) reported that the carcinogenic potential of N-nitrosamides corresponds closely to the chemical reactive of the compound. and, generally’ decreases with increasing number of carbon atoms in the acyl and alkyl groups. The only conclusion from these results would be that N-substituted amides with and without the addition of nitrite, and their corresponding N-nitrosamides did not cause tumor development in mice given the dosages and time periods used in this study. However, some of these com- pounds (NOPP and NOMP) were found to be mutagenic by the Ames test. Since there was no evidence of tumors at this time, the remaining mice are being kept on the same diet and will continue to be held for two years at which time will sacrifice and examined for tumor. SUMMARY AND CONCLUSION A study was conducted to establish the formation of N- substituted amides from the reaction of amino acid or free amines and fatty acids. GLC-MS analysis confirmed their for- mation at 200°C. Formation of N-substituted amides under frying condition of bacon was investigated by rehydrating freeze dried pork belly slices with water contianing pentyl— amine. After equilibrating for 24 hours at 4°C, the slices were fried for 4 minutes on each side at 175°C in a preheated pan. Analysis revealed no amide formation in either the fried slices or cook-out fat. However, upon heat- ing for an additional 6 minutes, N-substituted amides were formed in proportion to the fatty acid composition of pork belly adipose tissue. When the study was repeated with norleucine, no amide formation took place in the cook-out fat or in fried slices, even after the additional heating period. However, heating pork belly adipose tissue and norleucine together at 200°C for 1 hour resulted in forma- tion of N-substituted amides proportional to the fatty acid composition of the pork belly adipose tissue. Furthermore, when pork bellies were stitch-pumped with pentylamine or norleucine and trilaurin, smoked, sliced and fried, amide 77 78 formation was not evident in either the bacon or in cook- out fat. It is concluded that under the conditions en- countered in the frying of bacon, only amines would react with fatty acids to yield secondary amides. It was further concluded that the temperature of frying is not sufficient for decarboxylation of the amino acids and formation of N- substituted amides. Nitrosation of N-substituted amides was also carried out under acid conditions. Formation of N-nitrosamides was confirmed by GLC-MS. However, the normal pH of bacon would militate against the formation of N-nitrosamides, even if their precursors were present. The stability of N-nitrosopentylpalmitamide under fry- ing conditions used for bacon was also studied. The overall decomposition was approximately 87%. The major conclusion from this investigation was that N-nitrosamides are unlike- ly to be present in heat processed foods. In the event that N-substituted amides are formed during processing and cooking of foods, they could be nitro- sated 1 vivo. Therefore, the carcinogenicity of these compounds was investigated in a feeding trial using Swiss mice. 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Pharmacol. 20:985. APPENDICES 89 Appendix 1: Mass spectrum of N-pentyllauramide prepared from reaction of norleucine and lauric acid at 200 C. a/m 90 RELATIVE INTENSITY —l —l N b 05 (D O N J:- C\ m 0 O O O O O O O O O D O D 1 1 4‘ L 1 J J 1 1 If N —l N N [\‘T N O" O“ll N .5; hu— 0 0 fi A — w (A) h.)_ 01 C" D" O .—— [\3 \j i 3 90 $34 A 8 c: '1 ‘ v (A) ...; o O..____ O" O -—J —l L0 ...1 4; r0 | 0" €34 c> —l N k0 w —-l «b __1 EH 0" -?= l\ (.4) .—| 05- O“ O O o —J to 00—1 G)- O O N 45 D O O O 91 Appendix 2: Mass spectrum of N-pentylmyristamide prepared from rgaction of norleucine and myristic acid at 200 C. a/m 92 RELATIVE INTENSITY —.a .._a N 4:5 03 a) O N b O} CI) 0 O O O O O O O O O O O O l l 1 J L l __l I I _l N 8* 84 h (A) N 0‘ -—J 03 O. O .L— N ' m ‘— a> '-- \: (A; N oo 8‘: O- N 8 .5 8 d _J O o- q ‘ O "’+ v —J _a (A) __I b N N— D-I o r— __l A— b O o" _l A N g —a .— C‘- CD 0 0 w —I GD G)- " D D g '8 O O 93 Appendix 3: Mass spectrum of N-pentylpalmitamide prepared from rceaction of norleucine and palmitic acid at 200 C. a/w 94 RELATIVE INTENSITY —l —l N .b on oo o m 4: Ch 0:) o O O o o o o o o o o o o l l 1 l L J J l I _L N N N (.0 o N \3 DJ 9,) b- O (0 O ..__ 4:- co N_ as 0‘ Dc: 0 N C» CD a: s c>‘4 ‘ (A) —J o O... O“ 0 w —l N Na CH o —l :3 Iv on no (A) A E -‘>-- " ..J c: 7% C3 V (A) —.l as- on O O I —l co en ar- cf‘ o N A O O O O 95 Appendix 4: Mass spectrum of N-isobutylmyristamide prepar- ed from reaction of valine and myristic acid. 96 RELATIVE INTENSITY Loo mo mo so No Loo mo mo so No ..m Emm aw ms my As , mm mm .mm ..mo Ema Ewe 1mg Mme wa Az+v mmd was Nwo was wow wmm “Aml wmo use Boo a\m 97 Appendix 5: Mass spectrum of N-isobutylpalmitamide prepar- ed from reaction of valine and palmitic acid 0 at 200 C. a/w 98 RELATIVE INTENSITY —l u—J N h m 00 O N A 0‘ CO CD O O O O O O O O O C CD 0 1 1 A l L j J l l 1 R; N c> cf' N N O o O b 00 N‘ N on E E a-J 0'1 0‘ On CD 03 N 00 84 04-: DJ __0 o O...— O“ 0 w P -—1 d o w A -' —‘ N '7 M —l '1. o 01 v N CD w -—l A: 9.; O 0 w —l 05.. ON— O O I —I co oo oo- o“ O N J3 O O O O 99 Appendix 6: Mass spectrum of N-3-methylthiopropylmyrist- amide prepared from<§eaction of methionine and myristic acid at 200 C. a /u1 RELATIVE INTENSITY 100 —l —l N b OS 00 O N .5 0‘ m C) C) O O O O O ' O O O O O D 1 1 1 1 l J I I J 1 N F— N ——J N ‘5‘ 8" .5 w ‘ m N o. \l 0“ O. O N ‘_—._..._._ m \I N O) m w 00 O- 0 LA) ..a O O O" 0 w ‘ DO __a N —" Nd CH 0‘ o 3+ w —l -§—1 .54 O O u—l 4: \l w —l 03.. Oh O O 0 ...-J u (I) oo— o" O N J:- O O O O 101 Appendix 7: Mass spectrum of pentyl myristate a breakdwon product of N-nitrosopentylmyristamide. a/Lu RELATIVE 102 INTENSITY _o ._a N .p 0‘ co 0 N «b 0‘ 0‘) O O C) O O O O D O O O O O 1 I 1_ l l j I 1 I 1 T — N —J N __a an e 1 N . N \O N “-1 O O p b.) __.— U1 0'! N 03 03" o q o - . __ \I O N on g... o. 00 I _ \l N w .—l o ‘0 O CH a> ca‘ T-E + v w ...: N N—J o—J o w —4 115. DJ O 0 OJ —0 m- CH O O 8 —J u oo- 8‘ O N b D O O O 103 Appendix 8: Mass spectrum of pentyl palmitate a breakdwon product of N-nitrosopentylpalmitamide. a/m 104 RELATIVE INTENSITY __l —J N 4:: 0‘ 00 O N b 0‘ CO C O O O O o O O C) O O O l I l l l J I l I 1 N N N‘ OTI D N N g-J w 'o ‘0 b (A) N ‘— a: U" N 0‘ 03—4 \l D a O \l " o N 0° C?“ 0‘ 0 w —J O O o" o" w and N [\H C,“ o Q) d N- N 0‘ w 3+ —-I 4';- v A. O 0 w n-l cs... 0W 0 O 8 and w oo oo— C3"I O N b O o O O 105 Appendix 9: Mass spectrum of isobutyl myristate a break- dwon product of N—nitrosoisobutylmyristamide. a /u1 106 RELATIVE INTENSITY _‘ —J N h m G) O N b m CO O O C) C) O O O O O O C C O I J 1 I l J I I I l N ...—I I—J R? m cf CV N N LO 3) A- .4 b (A) 3 N of“ 8.. U“- 0 \l N on Good 0‘ N C) (A) A z .— 0.) + ...; go o v 8.. \l % —I Nd o—J o p—I ‘_ N LO w —l J; b.- O 0 w c—l as- O‘- O O I —-l w 00 oo— 0" O T” —l 00 m N .9 c: O O C) 107 Appendix 10: Mass spectrum of 3-methylthiopropyl myristate a breakdwon product of N-nitroso-3-methy1thio- propylmyristamide. a /u1 RELATIVE 108 INTENSITY u—l —‘ N 4:- C\ m C) N -D C‘. CO C 0 C3 0 O O O C3 C) C) O C) O I I L I I J I I I l N g. g. — N N (D N O O __— J} (A) U" _ (A) N _1 Ch 01 0‘ o‘ — o \l N 0‘ m m C O- O _ _ 0‘ s a; Lu) T— p—I w can! 0" N Nd y ’\ o '1- w ...-J N b—J O O w —-l CL; 05-1 ..— O O 3 —J w m 00-- o" O N b O O O C) 109 Appendix 11: Composition of purified diet 110 Ingredient D-Dextrose Vitamine Free Casein Corn oil Salt mix CaCO3 CaHPO4.2H20 NaCl K HPO 2 4 K3C6HSO7.H20 MgCO3 FeC6H507.3H20 MnSO4.H20 ZnCO3 CuSO4 KI .5820 Vitamin mix Vitamin A as retinyl palmitate Vitamin D as ergocalciferol Menadione (Vitamin K) biotin Vitamin 812 Calcium pantothenate folic acid niacin pyridoxine HCl riboflavin choline chloride selenium Amount (g) 649 200 100 6.54 14.7 4.3 3.09 9.46 1.64 0.64 0.055 0.018 0.007 0.0018 25,000 I U 2,000 I U 1 mg 0.1 mg 0.1 mg 10 mg 1 mg 25 mg 5.0 mg 10 mg 59 .04 mg "IIII'IIIIIIII’IIIT