Nitrosation Rate Research Articles

Role of glucosinolates in the formation of N-nitroso compounds

Since thiocyanate ion is able to catalyse the nitrosation reaction, the breakdown products of glucosinolates were further tested for their ability to catalyse the nitrosation rate of proline. Because the low pH of the stomach may result in the chemical breakdown of glucosinolates, the hydrolysis of glucosinolates (sinigrin, gluconapin, progoitrin, glucotropaeolin, sinalbin gluconasturtiin, glucobrassicin, and 4-hydroxyglucobrassicin) were also examined.

Nitrosation of aspartic acid, aspartame, and glycine ethylester. Alkylation of 4-( p-nitrobenzyl)pyridine (NBP) in vitro and binding to DNA in the rat

In a colorimetric assay using 4-( p-nitrobenzyl)pyridine (NBP) as a nucleophilic scavenger of alkylating agents, the nitrosation and alkylation reactions were investigated for a number of amino acids and derivatives. The alkylating activity increased with the square of the nitrite concentration. The nitrosation rate constants for aspartic acid, aspartame, and glycine ethylester (= precursors C) were 0.08, 1.4 and ⩽0.2, respectively, expressed in terms of the pH-dependent k 2 rate constant of the equation dNOC/dt = k 2 · [C] · [nitrite] 2. The rates correlated inversely with the basicity of the amino group. The stability of the alkylating activity was astonishingly high, both in acid and at neutral pH. Half-lives of 500, 200, and 30 min were determined for aspartic acid (pH 3.5), aspartame (pH 2.5), and glycine ethylester (pH 2.5). Values of 60, 15, and 2 min, respectively, were found at pH 7. It is concluded that rearrangement of the primary N-nitroso product to the ultimate alkylating agent could be rate-limiting. The potential of nitrosated α-amino acids to bind to DNA in vivo was investigated by oral gavage of radiolabelled glycine ethylester to rats, followed immediately by sodium nitrite. DNA was isolated from stomach and liver and analysed for radioactivity and modified nucleotides. No indication of DNA adduct formation was obtained. Based on an estimation of the dose fraction converted from glycine ethylester to the nitroso product under the given experimental conditions, the maximum possible DNA-binding potency of nitroso glycine ethylester is about one order of magnitude below the methylating potency of N-nitrosomethylurea in rat stomach. The apparent discrepancy to the in vitro data could be due to efficient detoxification processes in mammalian cells.

The rapid nitrosation of 2,3-diaminonaphthalene by gastric isolates of Neisseria subflava

Three gastric isolates of Neisseria subflava nitrosated 2,3-diaminonaphthalene to a fluorescent triazole product. The bacterial nitrosation rate was optimal at pH 7.5 with glucose as the electron donor for the reduction of nitrite to a nitrosating species. There was a delay period of 6 rnin with nitrite concentrations lower than 20 mmol/l before nitrosation was detected.

The stability of the nitrosated products of indole, indole-3-acetonitrile, indole-3-carbinol and 4-chloroindole

The nitrosation rates of indole-3-acetonitrile, indole-3-carbinol, indole and 4-chloroindole and the stability of their nitrosated products were investigated. Each of the nitrosated indole compounds was directly mutagenic to Salmonella typhimurium TA 100 in the following order of potency: 4-chloroindole ⪢ indole-3-carbinol ⩾ indole > indole-3-acetonitrile. Total N-nitroso determinations, carried out according to a modified method of Walters et al. ( Analyst, Lond. 1978, 103, 1127), and Ames test results revealed that each of the indole compounds immediately formed mutagenic N-nitroso products upon nitrite treatment under acidic conditions. However, the nitrosation rates of indole and 4-chloroindole were higher than those of indole-3-acetonitrile and indole-3-carbinol. For indole-3-carbinol, indole-3-acetonitrile and indole, no change in the amount of nitrosated products was observed at increasing incubation times from about 15 up to 60 min. For 4-chloroindole the amount of nitrosated products decreased with increasing incubation times. In all cases the responses in the Ames test paralleled the amounts of nitrosated products. The stabilities of the nitrosated products of the indole compounds were investigated at pH 2 and 8. Both mutagenicity data and measurements by high-performance liquid chromatography using a photohydrolysis detector indicated that the nitrosation products of indole-3-acetonitrile, indole-3-carbinol and indole were more stable at pH 8 than at pH 2. Conversely, nitrosated 4-chloroindole was stable at pH 2 but not at pH 8. The pH 8 chromatograms showed a large nitrite peak. From this we hypothesized that the presence of free nitrite might be responsible for the stability of nitrosated indole-3-acetonitrile, indole-3-carbinol and indole at pH 8. Experiments confirmed the existence of an equilibrium between the nitrosated indole compound and the free indole compound plus nitrite.

Diazotisations in Highly Concentrated Mineral Acids: The nitrosation mechanism of anilinium and hydroxylammonium ions through proton loss from the ammonio group

AbstractIt is shown that the acidity dependence of the rate of nitrosation of aromatic amines and of hydroxylamine in strongly acidic aqueous solutions does not necessarily involve the rearrangement of a charge transfer complex (consisting of the NO+ ion and the substrate with an NH group) in concert with a proton loss at the NH group. More likely, proton loss of the charge complex preceeds the π → N rearrangement of the NO+ ion.

ChemInform Abstract: Nitrosation by Alkyl Nitrites. Part 2. Kinetics of Reactions in Aqueous Acid Solution with Isopropyl and tert.‐Butyl Nitrites.

AbstractThe rates of nitrosation of sulfamic acid, HN3, HS‐CH2‐COOH, Cys, Ph‐NH‐Me, and H2N‐CS‐NH2 by isopropyl nitrite are markedly reduced when isopropanol is added.

Endogenous nitrosation in relation to nitrate exposure from drinking water and diet in a Danish rural population

Increasing levels of nitrate in drinking water is of concern due to the possibility of an associated increase in long-term exposure to endogenously formed carcinogenic N-nitroso compounds. Excretion of N-nitrosoproline in 12-h overnight urine after intake of 500-mg L-proline was used to quantify the rate of endogenous nitrosation in 285 individuals in an area in northern Denmark with large variation in nitrate concentration of the drinking water. Nitrate intake was measured in a 24-h duplicate diet sample. The crude association between nitrate concentration in drinking water and rate of endogenous nitrosation in individuals is only weakly positive and not quite statistically significant (P = 0.08). The risk of having detectable nitrosation increases significantly with total nitrate intake and tobacco smoking. In nonsmokers, nitrosation is determined by nitrate intake. Smokers have increased nitrosation which does not depend on nitrate intake. Effect modification through dietary factors was evaluated and indicated a protective effect of tea consumption, while the effect of eating vegetables was not clear-cut. The experimental design (12-h urine sample; proline dose taken in the evening) is likely to underestimate the effect of nitrate in drinking water relatively to nitrate in the diet.

Nitrosation of mefenorex in the presence of cyclodextrins

β- and γ-Cyclodextrin (CD) and heptakis-2,6-di-O-methyl-β-cyclodextrin (DIMEB) form soluble inclusion compounds with mefenorex (MEF); with α-CD a partial inclusion occurs. No solid inclusion compound could be obtained with the four CDs. β-, γ-CD and DIMEB, but not α-CD, enhance the nitrosation rate of MEF if the nitrosation assay procedure (NAP test) is applied. During this reaction with β- and γ-CD, solid inclusion compounds of the CDs and nitrosomefenorex (NMEF) precipitate.

In vitro nitrosation of ephedrine in the presence of cyclodextrins

β-, and γ-cyclodextrin and heptakis-2,6-di-O-methyl-β-cyclodextrin enhance the nitrosation rate of l-ephedrine if the nitrosation assay procedure (NAP test) is applied. During this reaction with β-cyclodextrin a solid inclusion compound of β-cyclodextrin andN-nitrosoephedrine precipitates. Solubilities and stabilities of inclusion compounds of the cyclodextrins with ephedrine and nitrosoephedrine, respectively, explain especially the catalytic effects of some cyclodextrins on ephedrine.

Possible mycological etiology of oral mucosal cancer: catalytic potential of infecting Candida aibicans and other yeasts in production of N-nitrosobenzylmethylamine

Yeasts were isolated from 12 cases of oral precancerous lesions (leukoplakia and erythroleukoplakia) by sampling the lesion as well as normal mucosa of each patient, yielding 21 strains of Candida albicans belonging to 15 biotypes, two strains of C. tropicalis, one strain of C. parapsilosis and two strains of Torulopsis glabrata. Biopsies were obtained from the lesions for histologic examination. The catalytic potential of the yeast strains to form N-nitrosobenzylmethylamine (NBMA) from the precursors N-benzylmethylamine and nitrite was assessed at pH 6.8. The NBMA produced was identified and quantitated by h.p.l.c. and confirmed by g.c.-m.s. Nitrosation rates were calculated as total nitrosamine subtracted the chemically produced nitrosamine, and related to number of yeast cells. The yeast strains differed in nitrosation potential (P less than 0.001), ranking from 0 to 1.2 micrograms NBMA/10(6) cells. Candida albicans strains, belonging to the biotypes 051, 147, 151, 153, 157 and 353, which constitute more rarely occurring biotypes, exhibited the highest nitrosation potential. Candida tropicalis, C. parapsilosis and T. glabrata were ranked lower. Strains with high nitrosation potential were generally isolated from lesions with more advanced precancerous changes. The yeast cells were present in the superficial part of the epithelium of the lesions as branching mycelium, and in some cases extending from the mucosal surface to the deeper epithelial cell layers. This might represent a fungal transportation system which could channel precursors in the saliva at the mucosal surface to the deeper part of the epithelium where the produced nitrosamine could be deposited. Thus, further evidence is provided supporting the hypothesis that certain strains of C. albicans and of other yeasts play a causal role in the development of oral cancer, by means of endogenous nitrosamine production.

Experimental studies on the N-nitrosamine formation in bacon during cooking

The present study aimed to clarify the mechanism and conditions for carcinogenic N-nitrosopyrrolidine (NPYR) formation in bacon during the cooking process, analyzing the precursors of N-nitrosamines in various materials, including raw pork and spices. Results obtained are summarized as follows:1. When bacon samples were brought to 100°-200°C within 10 minutes, the amounts of NPYR formed from free proline via pyrrolidine (Pyr) were almost the same levels as those formed via N-nitrosoproline (NPRO). In contrast, when samples were heated at 225°C, the yield of NPYR via Pyr was apparently greater than that formed via NPRO.2. Concerning the precursors of N-nitrosamines in fresh pork samples, spermine, a polyamine, was found to range from 50-100mg/kg, while levels of other polyamines were less than 1/10 of the spermine level.3. With regard to the precursors of nitrosamines in spices used for preparing commercial bacon, it was found that only pepper contained such compounds among the various spices so far tested, viz., pyrroperine and Pyr were detected, and both compounds are precursors of NPYR. In addition, piperine and piperidine which form N-nitrosopiperidine after a nitrosation reaction were isolated. The rates of nitrosation of the aforementioned compounds under acidic conditions at 37°C and 100°C were also studied.

Kinetics and mechanism of the nitrosation of N-acetyltryptophan and of the denitrosation of N-acetyl-N1-nitrosotryptophan

Both the formation and the denitrosation of N-acetyl-N1-nitrosotryptophan have been studied kinetically in aqueous solution at 25 °C at acidities between 1 M-HClO4 and pH 4. A value of 850 l mol–1 has been obtained for the equilibrium constant for the formation of N-acetyl-N1-nitrosotryptophan. At acidities ([H+]) greater than 0.1 M the rate constants for both nitrosation and denitrosation increase linearly with the concentration of acid, and the reaction rates are unaffected by the addition of nucleophiles (Br– and SCN–). The results are consistent with a mechanism for nitrosation where the rate-limiting step is the proton transfer from the protonated N-nitroso species to the medium. For denitrosation the corresponding protonation of the nitrosamine is rate-limiting. These conclusions are confirmed by the results obtained when the reactions are carried out in heavy water. However, in the pH range 1–4 the rates of both nitrosation and denitrosation are independent of the acidity of the medium and are again unaffected by the presence of nucleophiles or buffers. It is suggested that in this region nitrosation occurs at C-3. This is followed by deprotonation and an internal NO migration from C to N which is rate-limiting. This mechanism also accounts for earlier results on the denitrosation reaction at even lower acidities (pH 4–7), where acid catalysis and nucleophilic catalysis are found. Results of experiments in heavy water are compatible with this mechanism.

Screening of microorganisms for nitrosation catalysis at pH 7 and kinetic studies on nitrosamine formation from secondary amines by E. coli strains.

Thirty-eight strains of microorganisms isolated from infected human trachea, urine, blood and faeces were examined for their ability to form N-nitrosomorpholine from morpholine and nitrite at pH 7.25. Twenty-five bacterial strains exhibited nitrosation activity, including 18 out of 19 strains of Escherichia coli and three out of nine Pseudomonas aeruginosa, Proteus morganii, Proteus mirabilis, Klebsiella pneumoniae and Neisseria strains; E. coli A10 strain showed the highest activity. Formation of N-nitrosomorpholine was proportional to the incubation time up to 2 h and to the number of resting E. coli A10 cells; the reaction followed Michaelis-Menten kinetics. Nitrosation rate appeared to be dependent on the pKa value of several amines studied. As the nitrosation catalysis was heat-labile, our data suggest that N-nitrosation is catalysed by a bacterial enzyme(s). This reaction may lead to enhanced endogenous nitrosation in subjects suffering from an achlorhydric stomach or from chronic urinary tract infections.

N-Nitrosation of Proline in Smokers and Nonsmokers<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

Endogenous nitrosation of proline was investigated in smokers and nonsmokers. Volunteers consumed a volume of beet juice equivalent to 325 mg nitrate, and 1 hour later they consumed 500 mg proline. In separate experiments volunteers ingested proline alone. Twenty-four-hour urine samples were collected and analyzed for N-nitrosoproline. When proline alone was ingested, there was no significant difference in urinary nitrosoproline excretion between smokers and nonsmokers. When beet juice and proline were consumed, however, smokers produced approximately 2.5 times as much N-nitrosoproline as nonsmokers. Salivary thiocyanate levels were approximately 3.2 times higher in smokers compared to those in nonsmokers. Salivary nitrite levels of smokers and nonsmokers, either before or after beet juice consumption, were not different. Salivary nitrate concentrations, however, were higher in nonsmokers than in smokers after beet juice consumption but not before. Our results suggest that the higher level of salivary thiocyanate in smokers is responsible for the increased rate of endogenous nitrosation of proline in this group compared to the rate in nonsmokers. Nitrosating agents in cigarette smoke do not appear to play a significant role.

A kinetic study of the influence of alcohols on the nitrosation of morpholine in acid media. Equilibrium constants for the formation of alkyl nitrites

The effect of the aliphatic alcohols methanol, ethanol, propanol, isopropanol, 2-butanol, isobutanol, and tert-butanol on the rate of nitrosation of morpholine at pH 3 and 25 °C has been studied. The inhibition observed is attributed to the formation of alkyl nitrites, which are poor nitrosating agents in acid media. The equilibrium constants for the formation of alkyl nitrites from nitrous acid and alcohols have been calculated both spectrophotometrically and from kinetic data for the inhibitory effect, and there is agreement between the two sets of results. The molar absorptivities at 265 nm of the alkyl nitrites have also been determined. When the concentration of alkyl nitrite is very low, a slight catalytic effect considered to be an effect of the medium has been observed.

Kinetic study of phenol nitrosation in protic and aprotic solvents

The specific constants of the phenol nitrosation reaction were determined over a temperature range of 0°C to 40°C in the following solvents: water, ethanol, dimethylsulfoxide, acetonitrile, chloroform, benzene and cyclohexane. The influence of several strong electrolytes on the nitrosation rate in aqueous media was studied in the presence of a strong acid, 0.1 N, HCl, and without it. The magnitude of the primary isotopic effect which is produced in protic and aprotic solvents was also investigated. On the basis of an analysis of the yields and from the reaction kinetics found in this work we propose a nitrosation reaction mechanism in aprotic solvents. The nitrosonium ion plays no role in the mechanism.

Monitoring N-nitrosamino acids excreted in the urine and feces of rats as an index for endogenous nitrosation.

A simple and sensitive method for the quantitative estimation of endogenous N-nitrosation in rats has been developed. This approach is based on the findings that N-nitrosamino acids (e.g., nitrosoproline (NPRO), nitrosohydroxyproline (NHPRO) and nitrososarcosine (NSAR)) when administered orally to rats, are excreted unchanged almost quantitatively (88-96% of the dose) in the urine and feces. After sequential administration of a nitrosatable amino acid and sodium nitrite the nitrosamino acid excreted in the urine and feces was analyzed. The amount of NPRO excreted in the urine of rats was proportional to the dose of proline and to the square of the nitrite dose. Co-administration of ascorbic acid and alpha-tocopherol together with the precursors decreased the urinary NPRO whereas thiocyanate increased the yield. After feeding an amino acid precursor and nitrite, the yield of nitrosamino acids formed in vivo and excreted in the urine increased in the order: NPRO less than NSAR less than NHPRO. The same order was seen when the nitrosation rates of the amino acids in vitro were compared. Thus N-nitrosation in vivo in rats occurs via a similar mechanism as observed in vitro. Monitoring of N-nitrosamino acids excreted in the urine and feces thus appears to provide a valuable index for endogenous N-nitrosation.

Nitrite stability and nitrosation potential in human gastric juice

Human gastric juice samples were investigated from high- and low-risk areas (Colombia and Boston) for factors which influence the stability of nitrite and its potential for nitrosamine formation. The samples from individuals with chronic atrophic gastritis and intestinal metaplasia were not reactive to nitrite and supported a rate of nitrosation largely compatible with the nitrite and thiocyanate concentration. The samples from other individuals contained factors which destroyed nitrite and inhibited nitrosation. There were several samples from both groups which had an elevated rate of nitrosation catalyzed by unknown factors. It is suggested that continued hypochlorhydria might deplete gastric juice of its natural protective factors and lead to an elevated risk of gastric cancer in agreement with our earlier etiological model [1].

純硫酸中シクロヘキサンカルボン酸のニトロシル硫酸による三酸化硫黄触媒ニトロソ化反応の速度論的研究

The rates of nitrosation of cyclohexanecarboxylic acid (CCA) with nitrosyl hydrogensulfate (NHS) in 7.29-14.28% oleum forming ε-caprolactam have been followed by the volumetric determination of evolved carbon dioxide. The reaction dose never occur without sulfur trioxide. The rate of the reaction is expressed by Rate={k2cs-k3cnsCNHS}CCCACSO3-{k2cc-k3ccnCNHS}C2CCA A probable mechanism which involves pentamethyleneketene (PMK) formed by the elimination of sulfuric acid and proton from the protonated hexahydrobenzoyl hydrogensulfate (2) and proton, and the cyclohexanecarboxylic anhydride (CCAN) formed by the elimination of proton, sulfur trioxide and water from on adduct of 2 and CCA, was discussed.

The exposure to humans to nitrite.

Although nitrate is more abundant than nitrite in food and the environment in general, it requires reduction by, for instance, bacterial or plant enzymes before it is involved in the nitrosation of amines or amides. Part of the exposure of humans to nitrite arises from its use as a food additive where it performs a very useful function in protecting the consumer from pathogenic micro-organisms such as Clostridium botulinum. Some untreated foodstuffs, such as potatoes, tomatoes and beets, also contain low levels of nitrite. Nevertheless, the main source of human contact is that produced in vivo from nitrate ingested in foods in general and in vegetables in particular. Nitrate also occurs widely in drinking water supplies and this source can also contribute in some measure to human exposure. As yet, it is not possible to compute with accuracy the contribution from any endogenous synthesis within the gastrointestinal tract. Since the rate of nitrosation of an amine is dependent on the nitrite concentration to a power of greater than unity, it is probable that nitrite ingested in one application over a short period will be more active in the synthesis of N-nitroso compounds than a continuous supply at lower concentrations over long periods.