N-nitrosamino Acids Research Articles

Interaction of total N-nitroso compounds in environment and in vivo on risk of esophageal cancer in the coastal area, China

Shantou, China, is one of the endemic regions of esophageal cancer (EC) in the coastal areas, but the relationship between the multiple exposures of total N-nitroso compounds (TNOCs) and the high risk of EC to date is unclear. This study is to estimate the potential integrative role of multiple exposures of environmental and endogenous TNOC on the EC risk. We collected randomly the samples of 72 h diet and 24 h urine from 256 healthy male (aged 30–65 years) in the high- and low-risk area for EC, and determined the contents of TNOC and 4 N-nitrosamino acids (NAAs) by a method of chemical fission-Thermal Energy Analyzer (TEA) and gas chromatography — TEA, respectively. We used TNOC and NAAs index to evaluate the integrative effect of multiple TNOC exposures on the EC risk by a logistic model. TNOC intake and urinary TNOC content were higher in the high-risk subjects than that in the low-risk subjects by 1.60 μmol/day and 1.45 μmol/L, respectively. TNOC intake, urinary TNOC content and its interaction were significant factors involved in the higher risk of EC ( R 2 = 0.685, p = 0.000) with predicted Correct Class of 85.6%. The risk of the occurrence of a larger SMR of EC may increase by 4.841-fold for every 1 μmol/day increase in TNOC intake (95% CI 2.370–9.888, p = 0.000) and by 4.113-fold for every 1 μmol/L increase in urinary TNOC content (95% CI 2.108–8.023, p = 0.000), respectively. The integrally role of multiple exposures of TNOC in environment and in vivo may increase the risk of EC in the coastal areas in eastern Guangdong, China.

Five Leading U.S. Commercial Brands of Moist Snuff in 1994: Assessment of Carcinogenic N-Nitrosamines

Moist snuff is the only tobacco product in the United States with increasing sales (an increase of 38.4% between 1981 and 1993) and with increased consumption, primarily by male adolescents aged 12-18 years old and young adults aged 19 years old or older. It is known from previous studies that levels of nicotine and the proportion of unprotonated (free) nicotine, as well as the pH, which affects nicotine delivery, vary considerably among the leading snuff brands. Whether concentrations of major carcinogens, such as the nicotine-derived tobacco-specific N-nitrosamines (TSNAs), like N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), also vary among these brands has not been determined previously. Our purpose was threefold: 1) to determine the concentrations of major carcinogenic nicotine-derived N-nitrosamines in each of the five most popular moist snuff brands; 2) to analyze the quantitative differences in the various snuff components (e.g., NNN) between two major brand categories: a category comprising the brands known to have high levels of unprotonated nicotine (Copenhagen, Skoal fine cut, and Kodiak) versus a category comprising the brands known to have low levels (Hawken and Skoal Bandits); and 3) to compare the differences in the concentrations of nicotine (previously determined), NNN, NNK, and total TSNAs between these two major brand categories. Three boxes of each of the five leading U.S. moist snuff brands were bought in July 1994 from retailers in six areas and transferred immediately to the analytical laboratory. After extraction, N-nitrosamino acids and TSNAs were determined on a gas chromatograph interfaced with a thermal energy analyzer (GC-TEA) and integrator. Each 5-g sample of ground, freeze-dried tobacco was extracted twice, and each extract was analyzed twice by GC-TEA. All P values reported are two sided. Copenhagen, Skoal fine cut, and Kodiak as a group had statistically significant higher levels of nicotine (P = .0017), NNN (P < .0001), NNK (P = .0119), and total TSNAs (P < .0001) than the Hawken and Skoal Bandits group. Concentrations (means +/- SD) of nicotine, NNN, NNK, and total TSNAs comparing the two major brand categories are as follows: nicotine--11.6 +/- 1.01 mg/g versus 6.96 +/- 3.62 mg/g (P = .0017), NNN--7.74 +/- 1.70 micrograms/g versus 4.17 +/- 1.35 micrograms/g (P < .0001), NNK--1.23 +/- 0.68 micrograms/g versus 0.61 +/- 0.41 micrograms/g (P = .0119), and total TSNAs--14.3 +/- 3.82 micrograms/g versus 6.3 +/- 2.56 micrograms/g (P < .0001). The three leading U.S. snuff brands (Copenhagen, Skoal fine cut, and Kodiak; making up 92% of the U.S. market) showed not only high levels of pH, nicotine, and unprotonated (free) nicotine, but also high concentrations of the strongly carcinogenic TSNAs in comparison with the fourth and fifth best selling moist snuff brands, Hawken and Skoal Bandits (3% of the U.S. market).

Effects of storage conditions on levels of tobacco-specific N-nitrosamines and N-nitrosamino acids in U.S. moist snuff

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEffects of storage conditions on levels of tobacco-specific N-nitrosamines and N-nitrosamino acids in U.S. moist snuffMirjana V. Djordjevic, Jingrun. Fan, Lowell P. Bush, Klaus D. Brunnemann, and Dietrich. HoffannCite this: J. Agric. Food Chem. 1993, 41, 10, 1790–1794Publication Date (Print):October 1, 1993Publication History Published online1 May 2002Published inissue 1 October 1993https://doi.org/10.1021/jf00034a051RIGHTS & PERMISSIONSArticle Views290Altmetric-Citations18LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (598 KB) Get e-Alerts Get e-Alerts

Geographic association between urinary excretion of N‐nitroso compounds and oesophageal cancer mortality in china

Overnight urine samples were collected from approximately 60 male adults in each of 69 counties of China in 1989. Two specimens were collected from each subject--one after a loading dose of proline and ascorbic acid and another after a loading dose of proline only. Levels of N-nitrosamino acids and nitrate were measured in urine samples and correlated with cumulative mortality rates for subjects aged between 0 and 64 years in the 1970s. Oesophageal cancer mortality rates were positively and significantly associated with (i) urinary levels of excreted N-nitrosoproline (NPRO) (after proline and ascorbic acid loading or proline loading only), (ii) N-nitrososarcosine levels, and (iii) nitrosation potential (the decrease in the amount of urinary NPRO after adding ascorbic acid to the proline load). There were also positive correlations between the urinary level of NPRO or other N-nitrosamino acids and that of nitrate. The urinary excretion of nitrate was associated with consumption of various nitrate-rich vegetables. The results suggest that N-nitroso compounds (NOC) or other nitrite-derived carcinogens are implicated in the aetiology of oesophageal cancer in China.

Cross-Sectional Study with Multiple Measurements of Biological Markers for Assessing Stomach Cancer Risks at the Population Level

A cross-sectional study to determine correlations between measurable biologic markers and mortality from stomach cancer was performed in various areas of Japan. Blood and urine were collected from randomly selected 40- to 49-year-old men and their spouses in four areas with different rates of mortality from stomach cancer. The samples were analyzed for levels of the micronutrients vitamins A, C, and E, beta-carotene, and lycopene in plasma and for levels of NaCl, nitrate, and N-nitrosamino acids (N-nitrosoproline, N-nitrosothioproline [NTPRO] and N-nitrosomethylthioproline [NMTPRO]) in 24-hr urine. A significant, strong correlation was found between the amount of salt excreted in urine and stomach cancer mortality in both men and women. Although the amounts of nitrate and of the three N-nitrosamino acids in 24-hr urine were not correlated with stomach cancer rates, the low excretion levels of NTPRO and NMTPRO in the lowest risk area for stomach cancer were noteworthy, regardless of the high level of nitrate excretion in the same area. This suggests a lesser degree of endogenous nitrosation in the body. No protective effect of micronutrients was observed in this correlation study; there was, however, a negative correlation between plasma lycopene level and stomach cancer mortality. Salt intake was thus confirmed to play an important role in the development of stomach cancer and is likely to be a rate-regulating factor in Japanese populations. N-Nitrosamino acids and lycopene may also be related to stomach cancer mortality.

Cross-sectional study with multiple measurements of biological markers for assessing stomach cancer risks at the population level.

A cross-sectional study to determine correlations between measurable biologic markers and mortality from stomach cancer was performed in various areas of Japan. Blood and urine were collected from randomly selected 40- to 49-year-old men and their spouses in four areas with different rates of mortality from stomach cancer. The samples were analyzed for levels of the micronutrients vitamins A, C, and E, beta-carotene, and lycopene in plasma and for levels of NaCl, nitrate, and N-nitrosamino acids (N-nitrosoproline, N-nitrosothioproline [NTPRO] and N-nitrosomethylthioproline [NMTPRO]) in 24-hr urine. A significant, strong correlation was found between the amount of salt excreted in urine and stomach cancer mortality in both men and women. Although the amounts of nitrate and of the three N-nitrosamino acids in 24-hr urine were not correlated with stomach cancer rates, the low excretion levels of NTPRO and NMTPRO in the lowest risk area for stomach cancer were noteworthy, regardless of the high level of nitrate excretion in the same area. This suggests a lesser degree of endogenous nitrosation in the body. No protective effect of micronutrients was observed in this correlation study; there was, however, a negative correlation between plasma lycopene level and stomach cancer mortality. Salt intake was thus confirmed to play an important role in the development of stomach cancer and is likely to be a rate-regulating factor in Japanese populations. N-Nitrosamino acids and lycopene may also be related to stomach cancer mortality.

Levels of direct-acting mutagens, total N-Nitroso compounds in nitrosated fermented fish products, consumed in a high-risk area for gastric cancer in southern China

A high gastric cancer mortality in Fujian province (Peoples Republic of China) has been associated with the consumption of certain salted fermented fish products such as fish sauce (FS). We have investigated the levels and nature of N-nitroso compounds (NOC) and genotoxins present, before and after nitrosation, in 49 FS samples collected from villages in this high-risk area, pooled into six samples. The concentrations of total NOC before nitrosation ranged from 0.2 to 16 μmoles/1, and after nitrosation at pH 2 and pH 7, they rose by up to 4800- and 100-fold, respectively. In nitrosated samples, 40–50% of total NOC was not extractable into organic solvents; volatile N nitrosamines accounted for 1–2% and N-nitrosamino acids for 8–16% of total NOC. None of the FS samples exhibited genotoxic activity, but after nitrosation all were weakly active in the SOS chromotest. The highest SOS-inducing potency was observed with nitrosated ethyl acetate extracts of most samples. The formation of methylating agents was measured by incubation of nitrosated FS with DNA and subsequent analysis of 7-methylguanine adduct. 2 of the 6 nitrosated FS samples caused a slight increase in DNA methylation. 1 pooled home-made FS sample (the only one tested) contained tumour promoter-like substances, as measured by expression of certain EBV genes in Raji cells. HPLC fractionation of ethyl acetate extracts of FS samples allowed identification of three UV-absorbing peaks that, upon nitrosation, produced direct-acting genotoxins. This genotoxicity was partly ascribed to the formation of nitrite-derived arene diazonium cations that were characterized by a coupling reaction with N-ethyl-1-naphthylamine and thin-layer chromatography. The presence of nitrite-dependent genotoxins and tumour promoter-like substances supports the hypothesis that FS consumption is a risk factor for gastric cancer in certain areas of China.

Volatile, non‐volatile and totalN‐nitroso compounds in bacon

Twenty-five smoked and unsmoked fried bacon samples have been analysed by a group selective procedure to measure the concentration of apparent total N-nitroso compounds (ATNC). The levels of a range of individual N-nitroso compounds, including simple volatile N-nitrosamines, N-nitrosothiazolidines, N-nitrosamino acids and N-nitrosothiazolidine carboxylic acids have also been examined. Concentrations of ATNC varied from 430 to 6800 micrograms(N-NO)/kg with a mean of 2700 micrograms(N-NO)/kg. Protein-bound N-nitrosoproline was the most abundant compound detected in unsmoked bacon, mean 260 micrograms/kg, and on average accounted for 4% of the ATNC concentration. For smoked bacon, bound N-nitrosoproline was detected in levels of up to 890 micrograms/kg and contributed 5% to the ATNC total. The most abundant compound present in smoked bacon was N-nitrosothiazolidine-4-carboxylic acid, mean 660 micrograms/kg, and this accounted for 6% of the ATNC. N-Nitrosothiazolidine, mean 340 micrograms/kg, and 2-(hydroxymethyl)-3-nitrosothiazolidine-4-carboxylic acid, mean 180 micrograms/kg, were the next most prominent compounds detected in smoked bacon. The combined sum of all the individual N-nitroso compounds measured accounted for, on average, 16% of the total ATNC. The identities of the N-nitroso compounds comprising the majority of the ATNC in bacon remain unknown.

Urinary excretion of N-nitrosamino acids and nitrate by inhabitants of high- and low-risk areas for stomach cancer in Poland.

Urine samples were collected from 96 inhabitants of a high-risk rural area and a low-risk urban area for stomach cancer in Poland, according to the following protocol: (1) when they were undosed; (2) after ingestion of proline 3 times a day; and (3) after ingestion of proline together with vitamin C 3 times a day. The samples were analyzed for N-nitrosamino acids and nitrates, as indices of exposure to preformed and endogenously formed N-nitrosamines. The median values of N-nitrosoproline (NPRO) and N-nitrosothiazolidine 4-carboxylic acid (NTCA) excreted in the urine of undosed subjects were not different between the two areas; but N-nitrososarcosine and 3-(N-nitroso-N-methylamino)propionic acid levels were 3- to 4-fold higher in subjects of the high-risk area. After intake of proline, the NPRO level increased (p less than 0.02) only in subjects in the high-risk area; intake of vitamin C tended to inhibit this increase in NPRO and lowered the levels of other nitrosamino acids. The urinary level of nitrates was 1.4-fold, but significantly higher among subjects in the high-risk area than among those in the low-risk area; nitrate levels were not correlated with the amounts of cured meat or types of vegetables consumed. Urinary nitrate levels and excretion of NPRO, NTCA and the sum of all nitrosamino acids analyzed showed positive, though modest, correlations. These results indicate a higher potential for endogenous nitrosamine formation, possibly by intragastric nitrosation among subjects in the high-risk rural area.

Identification in rats of N-nitrosonipecotic acid as a major urinary metabolite of the areca-nut alkaloid-derived nitrosamines, N-nitrosoguvacoline and N-nitrosoguvacine

N-Nitrosamines derived from areca-nut alkaloids have been implicated in cancer of the oral cavity and esophagus caused by betel quid chewing in India and other Asian countries. A major urinary metabolite of N-nitrosoguvacoline and N-nitrosoguvacine, both present in saliva of betel quid chewers of ppb levels, was isolated from rat urine and identified as N-nitrosonipecotic acid by comparison with the authentic compound. When a dose of 50 or 500 μg/rat of either compound was administered orally to BDIV rats, 66–85% of the dose was excreted in the urine as N-nitrosonipecotic acid and 2–8% as N-nitrosoguvacine. These N-nitrosamino acids could be analysed in the urine of betel quid chewers as a marker of exposure to areca-nut specific nitrosamines.

Macrophage-mediated N-nitrosation of thioproline and proline

Thioproline (Thiazolidine-4-carboxylic acid) and proline were nitrosated by stimulated mouse macrophages in vitro . A macrophage cell line (J774.1, 1.0 × 10 6/well, 1 ml) was incubated with Escherichia coli lipopolysaccharide, interferon-γ and thioproline (5 mM) or proline (5 mM). After 72 hr incubation at 37°C, 4 μM N-nitrosothioproline was produced. The amount of N-nitrosoproline was much lower than that of N-nitrosothioproline. Thioproline and proline inhibited the formation of carcinogenic N-nitrosomorpholine. N-nitrosothioproline and N-nitrosoproline are found as major N-nitroso compounds in human urine. Macrophage mediated N-nitrosation may contribute to the formation of these N-nitrosamino acids in the human body.

Levels of mutagens in the urine of smokers of black and blond tobacco correlate with their risk of bladder cancer.

Levels of urinary mutagens, thioethers, N-nitrosamino acids, nitrate, nicotine, cotinine and creatinine were measured in 21 non-smokers, 26 smokers of blond tobacco, 9 smokers of black tobacco and 5 smokers of both types of tobacco, all eating a similar diet. Results were expressed either per 24 h urine or per mmol creatinine. The sum of urinary nicotine and cotinine levels (N + C) was used as a measure of exposure to the number of cigarettes smoked. Statistically significant positive dose-effect relationships were obtained between the urinary N + C levels and (i) the number of revertants (Salmonella typhimurium TA98, with a metabolic activation system); (ii) the concentration of thioethers; (iii) the levels of N-nitrosoproline or the sum of all nitrosamino acids excreted and (iv) the amount of urinary nitrate. No such correlation was found between N + C levels and induction potency in the SOS chromotest. A linear dose-effect relationship between urinary mutagenicity (i.e. log revertants of S. typhimurium TA98) and N + C levels or number of cigarettes per day was established for smokers of blond tobacco. After adjustment for N + C levels, the urine of smokers of black tobacco contained twice as much mutagenic material as did the urine of blond tobacco smokers (P = 0.02). For other exposure markers, no statistically significant difference was found between the two types of smokers. Epidemiological studies have shown that the risk of urinary bladder cancer is 2.5 times higher in smokers of black tobacco than in blond tobacco. Therefore, our findings on urinary mutagenicity provide experimental evidence that the type of tobacco is the factor responsible for the observed difference in risk and that smoking of black as compared to blond tobacco results in a higher exposure of the urinary bladder to genotoxic hence potentially carcinogenic substances.

Identification and analysis of a nicotine-derived N-nitrosamino acid and other nitrosamino acids in tobacco

Research on carcinogenic, tobacco-specific N-nitrosamines (TSNA) led to the identification and analysis of 4-(methylnitrosamino)-4-(3-pyridyl)butyric acid (iso-NNAC) in tobacco and tobacco smoke. In order to isolate iso-NNAC, an aqueous tobacco extract at pH 4 was partitioned with ethyl acetate after the other N-nitrosamino acids and TSNA were removed at pH 2 and pH 9 respectively. The structure of iso-NNAC was confirmed by GC-MS after enrichment of the methylated pH 4 fraction by chromatography on an alumina column. Iso-NNAC, 3-(methylnitrosamino)propionic acid, 4-(methylnitrosamino)butyric acid, N-nitrosoproline and TSNA were determined by GC-TEA in various smokeless tobaccos as well as in reference cigarettes. The levels of iso-NNAC in tobacco products ranged from 0.01 p.p.m. in chewing tobacco to 0.95 p.p.m. in dry snuff. The transfer rate of unchanged iso-NNAC into the mainstream smoke of a non-filter cigarette amounted to 0.85%. Iso-NNAC does not induce DNA repair in primary rat hepatocytes and is inactive as a tumorigenic agent in strain A mice.

Group-selective determination of total N-nitroso compounds in nitrate-containing human urine samples.

A group-selective method for the determination of total N-nitroso compounds (NOC) has been adapted for analysing human urine samples. Nitrate was first removed from urine by an anion-exchange procedure that prevented the significant loss of various added reference NOC and unidentified urinary NOC. The total NOC were then determined by injecting the urine sample (nitrate content less than 1 mmol l-1) or anion-exchange eluate into refluxing ethyl acetate containing either acetic acid for determining heat and acetic acid labile thermal energy analyser responsive compounds (TAC) or into hydrogen bromide for the determination of TAC and NOC. The nitrogen monoxide levels released were measured using thermal energy analysis with chemiluminescence detection, and the differnce between the two determinations represented the concentrations of NOC. The optimum conditions for preventing artefactual nitrosation in urine samples by the addition of sodium hydroxide or sulphamic acid without decomposition of NOC were determined. The influence of time and storage conditions on NOC stability was investigated. Fifteen urine samples collected from volunteers dosed with proline were analysed for total NOC and N-nitrosamino acids revealing a preponderance of unknown NOC. The determination of total NOC in human urine using this group-selective method offers a new approach to the estimation of human exposure to NOC and to isolate hitherto unknown NOC and their metabolites.

Urinary excretion of nitrate, nitrite and N-nitroso compounds in Schistosomiasis and bilharzia bladder cancer patients.

Saliva and 24-h urine samples were collected from male Schistosomiasis (bilharzia) patients with S. haematobium infection and possible concurrent S. mansoni infection without diagnosed bladder cancer (n = 27), bilharzia patients with diagnosed bladder cancer (n = 23) as well as a comparative control group (n = 27) of healthy Egyptian volunteers with no current bilharzia infection and/or bacterial urinary tract infections from the Nile Delta area of Egypt. Saliva samples were analysed for the presence of nitrate and nitrite; urine samples were analysed for the presence of nitrate, nitrite, volatile and non-volatile N-nitroso compounds. Bilharzia patients prior to, and after, diagnosed bladder cancer regularly excreted free nitrite as well as volatile nitrosamines (N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosopiperidine and N-nitrosopyrrolidine) in addition to which elevated concentrations of non-volatile N-nitrosamino acids (N-nitrosoproline, N-nitrososarcosine, N-nitrosothiazolidine-4-carboxylic acid and its 2-methyl derivative) were also present. Total urinary excretion of volatile N-nitroso compounds (0.32 +/- 0.64 micrograms/day; mean +/- SD) and non-volatile N-nitroso compounds (31.20 +/- 22.07 micrograms/day) was observed in the Egyptian control group. Significantly higher concentrations were found in bilharzia patients: 3.47 +/- 6.42 (P less than 0.05) and 62.91 +/- 21.96 (P less than 0.05); as well as in bilharzia patients with diagnosed bladder cancer: 1.71 +/- 1.96 (P less than 0.02) and 44.94 +/- 7.31 respectively. Free nitrite was found in the urine of two volunteers in the Egyptian control group (1.7 and 3.0 micrograms/day), urinary nitrite was significantly increased in bilharzia patients (5.18 +/- 9.11 micrograms/day, P less than 0.02) and in bladder cancer patients (1.75 +/- 2.81 micrograms/day, P less than 0.05). Nitrate concentrations were elevated from 139.3 +/- 82.2 in the control group to 143.6 +/- 136.3 and 175 +/- 190 in the bilharzia and bladder cancer groups respectively. These results indicate that significant in vivo formation of nitrite and volatile N-nitroso compounds occurs in the urinary bladder of bilharzia patients and this may be an oetiological factor in the induction of bilharzial bladder cancer associated with S. haematobium infection.

High-performance liquid chromatography separations of nitrosamines : III. Conformers of N-nitrosamino acids

The separation of a selected group of N-nitrosamino acids and their syn and anti conformers by high-performance liquid chromatography using an α-cyclodextrin bonded silica gel column and a mobile phase of acetonitrile—triethylammonium acetate was achieved. The effects of mobile phase pH and concentration of acetonitrile on resolution and elution times are also reported.

Contribution of wood smoke to in vivo formation of N-nitrosothiazolidine-4-carboxylic acid: Initial studies

The effect of administering liquid smoke or smoked food products to rats on in vivo formation of N-nitrosamino acids was investigated. Rats treated by gavage with either cysteine, formaldehyde or nitrite excreted urine containing no detectable levels of N-nitrosothiazolidine-4-carboxylic acid (NTCA). All three precursor compounds were required for NTCA formation. Two liquid smokes, when administered to rats in combination with cysteine and nitrite also produced measurable quantities of NTCA. Ascorbate inhibited in vivo formation of NTCA by approximately 90% when given to rats simultaneously with cysteine, formaldehyde, and nitrite. α-Tocopherol was much less effective than ascorbate in blocking NTCA formation. When smoked bacon, smoked Swiss cheese, and chicken barbecued with a sauce containing smoke flavouring were fed to rats along with nitrate, NTCA was again detected in the urine.

Review of methodologies for the determination of nonvolatile N-nitroso compounds in foods.

An attempt has been made to briefly review methods available for the determination of total N-nitroso compounds, N-nitrosamides, N-nitrosamino acids, and miscellaneous other nonvolatile N-nitroso compounds in foods and beverages, giving special emphasis to the progress made during the past five years. It appears that a wide variety of acceptable methods are available for N-nitrosamino acids and simple hydroxylated N-nitrosamines, but none of them has yet been adequately validated. Only limited progress has been made for the analysis of N-nitrosamides, N-nitroso sugar amino acids, and other N-nitroso compounds. Various mass spectrometric methods available for the determination and confirmation of these compounds are also discussed.

Influence of cysteine and nitrate on the endogenous formation of N-nitrosamino acids

The excretion of N-nitrosamino acids and nitrate in 24-h urine was studied over a continuous 45-day period for a single volunteer on supplemented diet regime. Days 1–10 (normal diet), 11–20 (normal diet + 600 mg nitrate/day), 21–25 (normal diet), 26–30 (normal diet + 200 mg L-cysteine/day), 31–35 (normal diet), 36–40 (normal diet + 600 mg nitrate + 200 mg l-cysteine/day) and days 41–45 (normal diet). The presence of N-nitrosohydroxyproline (NHPRO) in human urine is reported for the first time as well as the influence of l-cysteine on the formation of N-nitrosothiazolidine-4-carboxylic acid (NTCA) and N-nitroso-2-methylthiazolidine-4-carboxylic acid (NMTCA) under a normal nitrate burden (75 mg/day) and a high nitrate burden (675 mg/day). After estimation of the amount of dietary nitrite consumed and the extent of nitrite formation by bacterial reduction of nitrate in the oral cavity, the total daily exposure to nitrite was calculated. From a total daily exposure of 7.2 mg nitrite/day from a typical European diet, 0.08% is required for the endogenous formation of N-nitrosamino acids found in urine. Under an increased nitrate burden (675 mg/day), the total exposure to nitrite was increased to 38 mg/day of which 0.14% was required for the endogenous formation on N-nitrosamino acids.

Urinary N-nitrosamino acids as indices of endogenous formation of N-nitroso compounds.

Exposure to their precursors (e.g., amines, nitrate/nitrite, NOx) can lead to formation in the human body of N-nitroso compounds (NOC), a class of potent animal carcinogens, which are also suspected of being carcinogenic in man. A non-invasive method, the 'N-nitrosoproline (NPRO) test', for estimating endogenous nitrosation in man was developed in our laboratory. This test, which monitors 24-hr-excretion of urinary N-nitrosamino acids, is now applied in clinical and field studies, with the aim of measuring nitrosamine exposure and of identifying dietary, life-style, and host factors, or disease states, that affect nitrosation in man. Results from such studies are used to identify populations/individuals at high risk for cancers of the stomach, oesophagus, and oral cavity possibly caused by endogenous nitrosamines, and to indicate preventive measures by which the body burden of endogenous nitroso carcinogens can be lowered efficiently.