Transformation of ethyl alcohol to ethyl nitrite in acidified saliva: possibility of its occurrence in the stomach

After taking alcoholic beverages, the ethanol is mixed with saliva and then gastric juice. As pH of gastric juice is around 2, the ethanol might be transformed to ethyl nitrite in the stomach by reacting with salivary nitrite. In this study, reactions between ethanol and nitrite in acidified saliva were investigated. The result indicates that nitrite in acidified saliva reacted with ethanol producing ethyl nitrite. It is discussed that ethyl nitrite might be formed in the stomach after drinking alcoholic beverages and that the ethyl nitrite might function as a donor of NO in intestinal and gastric tissues.     

Enhanced plasma ghrelin levels in rats with streptozotocin-induced diabetes

Human saliva, which contains nitrite, is normally mixed with gastric juice, which contains ascorbic acid (AA). When saliva was mixed with an acidic buffer in the presence of 0.1 mM AA, rapid nitric oxide formation and oxygen uptake were observed. The oxygen uptake was due to the oxidation of nitric oxide, which was formed by AA-dependent reduction of nitrite under acidic conditions, by molecular oxygen. A salivary component SCN⁻ enhanced the nitric oxide formation and oxygen uptake by the AA/nitrite system. The oxygen uptake by the AA/nitrite/SCN⁻ system was also observed in an acidic buffer solution. These results suggest that oxygen is normally taken up in the stomach when saliva and gastric juice are mixed.     

Dietary nitrate inhibits stress-induced gastric mucosal injury in the rat

Dietary nitrate is reduced to nitrite by some oral bacteria and the resulting nitrite is converted to nitric oxide (NO) in acidic gastric juice. The aim of this study is to elucidate the pathophysiological role of dietary nitrate in the stomach. Intragastric administration of nitrate rapidly increased nitrate and NO in plasma and the gastric headspace, respectively. Water-immersion-restraint stress (WIRS) increased myeloperoxidase (MPO) activity in gastric mucosa and induced hemorrhagic erosions by a nitrate-inhibitable mechanism. In animals that had received either cardiac ligation or oral treatment with povidone-iodine, a potent bactericidal agent, administration of nitrate failed to increase gastric levels of NO and to inhibit WIRS-induced mucosal injury. WIRS decreased gastric mucosal blood flow by a mechanism which was inhibited by administration of nitrate. These data suggested that the enterosalivary cycle of nitrate and related metabolites consisted of gastrointestinal absorption and salivary secretion of nitrate, its conversion to nitrite by oral bacteria and then to NO in the stomach might play important roles in the protection of gastric mucosa from hazardous stress.     

Apples increase nitric oxide production by human saliva at the acidic pH of the stomach: a new biological function for polyphenols with a catechol group?

Dietary inorganic nitrate is secreted in saliva and reduced to nitrite by bacterial flora. At the acidic pH of the stomach nitrite is present as nitrous acid in equilibrium with nitric oxide (*NO), and other nitrogen oxides with nitrating and nitrosating activity. *NO in the stomach exerts several beneficial effects, but nitrosating/nitrating species have been implicated as a possible cause of epithelial neoplasia at the gastroesophageal junction. We investigated the effects of apple extracts on *NO release by human saliva at pH 2. A water extract obtained from apple homogenate increased *NO release caused by acidification of saliva. Data show that polyphenols were responsible for this activity, with chlorogenic acid and (+)-catechin the most active and concentrated species. However, ferulic acid, a hydroxycinnamic acid with only one aromatic hydroxyl group, did not increase *NO release. Fructose, the most representative sugar in apples, was also inactive. Interestingly, ascorbic acid in saliva induced a SCN(-)-enhanced burst of *NO but, unlike apple, the release was transient. The simultaneous addition of ascorbic acid and apple extract caused a burst of *NO followed by the increased steady-state level characteristic of saliva containing apple extract. Chlorogenic acid and (+)-catechin, but not ferulic acid, formed o-semiquinone radicals and nitrated polyphenols, suggesting the scavenging of *NO(2) by o-semiquinones. Our results propose that some apple polyphenols not only inhibit nitrosation/nitration but also promote *NO bio-availabilty at the gastric level, a previously unappreciated function.     

The bactericidal effect and chemical reactions of acidified nitrite under conditions simulating the stomach

AIMS: To examine the hypothesis of non-immune defence mechanisms based on nitrite. METHODS AND RESULTS: The acidified media (nutrient broth or citrate-phosphate buffer) under aerobic conditions with additions of physiological levels of nitrite, L-ascorbic acid, iodide and thiocyanate were used to simulate gastric juice. The bactericidal effects of acidified nitrite on Escherichia coli and lactobacilli were investigated using bacterial plate counts. Conversion of acidified nitrite to nitric oxide, nitrogen dioxide and nitrate was also studied. Nitrite significantly increased the bactericidal effects on E. coli and lactobacilli. The bactericidal effects were enhanced by thiocyanate but not by L-ascorbic acid and iodide. L-Ascorbic acid and thiocyanate, but not iodide, enhanced the decomposition of acidified nitrite in nutrient broth. Acidified nitrite was converted to both nitric oxide and nitrate, but a portion of the acidified nitrite in nutrient broth may have been converted to other unidentified nitrogen compounds. Nitrogen dioxide was not detected in any of the samples. CONCLUSION: The bactericidal effects of nitrite appeared to be primarily related to nitrous acid, and possibly to other unidentified nitrogenous metabolites, but not to nitric oxide and nitrogen dioxide. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential role of nitrite as an antimicrobial substance in the stomach may be of some importance in the ecology of the gastrointestinal tract and in host physiology.     

Diet-induced endogenous formation of nitroso compounds in the GI tract

Red or processed meat, but not white meat or fish, is associated with colorectal cancer. The endogenous formation of nitroso compounds is a possible explanation, as red or processed meat--but not white meat or fish--causes a dose-dependent increase in fecal apparent total N-nitroso compounds (ATNC) and the formation of nitroso-compound-specific DNA adducts. Red meat is particularly rich in heme and heme has also been found to promote the formation of ATNC. To investigate the underlying mechanism of ATNC formation, fecal and ileal samples of volunteers fed a high red meat or a vegetarian diet were analyzed for nitrosyl iron, nitrosothiols, and heme. To simulate the processes in the stomach, food homogenates and hemoglobin were incubated under simulated gastric conditions. Nitrosyl iron and nitrosothiols were significantly (p < 0.0001) increased in ileal and fecal samples after a high red meat diet compared with a vegetarian diet; significantly more nitrosyl iron than nitrosothiols was detectable in ileal (p < 0.0001) and fecal (p < 0.001) samples. The strong correlation between fecal nitrosyl iron and heme (0.776; p < 0.0001) suggested that nitrosyl heme is the main source of nitrosyl iron, and ESR confirmed the presence of nitrosyl heme in fecal samples after a high red meat diet. Under simulated gastric conditions, mainly nitrosothiols were formed, suggesting that acid-catalyzed thionitrosation is the initial step in the endogenous formation of nitroso compounds. Nitrosyl heme and other nitroso compounds can then form under the alkaline and reductive conditions of the small and large bowel.     

Formation of an adduct by clenbuterol, a beta-adrenoceptor agonist drug, and serum albumin in human saliva at the acidic pH of the stomach: evidence for an aryl radical-based process

Clenbuterol (CLB) is an antiasthmatic drug used also illegally as a lean muscle mass enhancer in both humans and animals. CLB and amine-related drugs in general are nitrosatable, thus raising concerns regarding possible genotoxic/carcinogenic activity. Oral administration of CLB raises the issue of its possible transformation by salivary nitrite at the acidic pH of gastric juice. In acidic human saliva CLB was rapidly transformed to the CLB arenediazonium ion. This suggests a reaction of CLB with salivary nitrite, as confirmed in aerobic HNO(2) solution by a drastic decrease in nitric oxide, nitrite, and nitrate. In human saliva, both glutathione and ascorbic acid were able to inhibit CLB arenediazonium formation and to react with preformed CLB arenediazonium. The effect of ascorbic acid is particularly pertinent because this vitamin is actively concentrated within the gastric juice. EPR spin trapping experiments showed that preformed CLB arenediazonium ion was reduced to the aryl radical by ascorbic acid, glutathione, and serum albumin, the major protein of saliva. As demonstrated by anti-CLB antibodies and MS, the CLB-albumin interaction leads to the formation of a covalent drug-protein adduct, with a preference for Tyr-rich regions. This study highlights the possible hazards associated with the use/abuse of this drug.     

NO Synthesis in Human Saliva

Human saliva contains nitrate that is converted into nitrite by the activity of facultative, anaerobic bacteria of the oral cavity. Nitrite can be reduced to NO in the acidic gastric milieu; some NO may also form in the mouth at acidic pH values. In this paper, we show that bacteria ( S. salivarius, S. mitis and S. bovis ) isolated from saliva, may contribute to NO production in human saliva. NO formation by bacteria occurs at neutral pH values and may contribute to the antibacterial activity of saliva.     

NO synthesis in human saliva

Human saliva contains nitrate that is converted into nitrite by the activity of facultative, anaerobic bacteria of the oral cavity. Nitrite can be reduced to NO in the acidic gastric milieu; some NO may also form in the mouth at acidic pH values. In this paper, we show that bacteria ( S. salivarius , S. mitis and S. bovis ) isolated from saliva, may contribute to NO production in human saliva. NO formation by bacteria occurs at neutral pH values and may contribute to the antibacterial activity of saliva.     

Nitrite, a naturally occurring precursor of nitric oxide that acts like a 'prodrug'

There are enzymatic and non-enzymatic mechanisms that generate NO* from nitrite in blood, stomach, saliva, urine and skin. In blood vessels, nitrite-derived NO* can provide protection via compensatory vasodilation during hypoxia, and in various body fluids it may have antibacterial activity. In the skin, nitrite-derived NO* may contribute to skin tanning, as well as to protection against UV-induced cell damage. Current knowledge on nitrite acting like an NO* 'prodrug' is presented, emphasizing the role of nitrite in skin.     

Saliva plays a dual role in oxidation process in stomach medium

The aim of this study was to evaluate the role of saliva in the oxidation process under the acidic condition of the stomach. Saliva specimens played varied roles in the lipid peroxidation process of heated muscle tissue in simulated gastric fluid: pro-oxidant effects, no effects, and antioxidant effects. To elucidate these differences, selected saliva components were examined. The pseudoperoxidase activity of lactoperoxidase increased lipid peroxidation, while thiocyanate and nitrite-reduced lipid peroxidation. The effect of a saliva specimen on lipid peroxidation was correlated with the concentration of nitrite in the specimen, but not with that of other saliva components. The inhibitory effect of nitrite may be due to its conversion to NO. Elucidation of the antioxidant effect of saliva on co-oxidation of d-alpha-tocopherol in gastric fluid, demonstrated that saliva alone cannot protect d-alpha-tocopherol from co-oxidation, although it partially protected against lipid peroxidation. The presence of red wine polyphenols in stomach medium totally inhibits food lipid peroxidation and d-alpha-tocopherol co-oxidation.     

Features of the metabolism of nitric oxide in normal state and inflammation

Brief analysis of the metabolism of nitric oxide in living cells in normal state and pathology and also the analysis of the causes that hampered the progress of these studies were carried out. It was established that most of physiological fluids, including blood, normally contain nitrite and non-thiolate nitroso compounds in concentration less than 100 nM. Literature data from different researchers on the normal range of nitrite concentration in plasma of healthy people from several hundreds of nM to several μM is probably the result of low selectivity of the methods used. But nitrite and non-thiolate nitroso compounds concentration in blood is dramatically increased in case of inflammatory diseases. It is proposed that the main mechanism for the production of these substances in blood is the nitrosyl iron complexes transformation by active oxygen species but not the activation of NO production as it was considered previously.     

Effects of pH, Nitrite, and Ascorbic Acid on Nonenzymatic Nitric Oxide Generation and Bacterial Growth in Urine

Nitrite may be generated by bacteria in urine during urinary tract infections. Acidification of nitrite results in the formation of nitric oxide (NO) and other reactive nitrogen oxides, which are toxic to a variety of microorganisms. We have studied NO formation and bacterial growth in mildly acidified human urine containing nitrite and the reducing agent vitamin C. Urine collected from healthy subjects was incubated in closed syringes at different pH values with varying amounts of nitrite and/or ascorbic acid added. NO generation was measured in headspace gas using a chemiluminescence technique. A similar setup was also used to study the growth of three strains of bacteria in urine. Mildly acidified nitrite-containing urine generated large amounts of NO and this production was greatly potentiated by ascorbic acid. The growth of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus saprophyticus was markedly reduced by the addition of nitrite to acidified urine. This inhibition was enhanced by ascorbic acid. In conclusion, we show that the growth of three common urinary pathogens is markedly inhibited in mildly acidified urine when nitrite is present. The bacteriostatic effect of acidified nitrite is likely related to the release of NO and other toxic reactive nitrogen intermediates. These results may help to explain the well-known beneficial effects of urinary acidification with, e.g., vitamin C in treatment and prevention of urinary tract infection.     

Salivary thiocyanate/nitrite inhibits hydroxylation of 2-hydroxybenzoic acid induced by hydrogen peroxide/Fe(II) systems under acidic conditions: possibility of thiocyanate/nitrite-dependent scavenging of hydroxyl radical in the stomach

Formation of OH radicals in the stomach is possible by Fenton-type reactions, as gastric juice contains ascorbic acid (AA), iron ions and H2O2. An objective of the present study is to elucidate the effects of salivary SCN- and NO2- on the hydroxylation of salicylic acid which was induced by H2O2/Fe(II) and AA/H2O2/Fe(II) systems. Thiocyanate ion inhibited the hydroxylation of salicylic acid by the above systems in acidic buffer solutions and in acidified saliva. The inhibition by SCN- was deduced to be due to SCN- -dependent scavenging of OH radicals. Nitrite ion could enhance the SCN- -dependent inhibition of the hydroxylation induced by AA/H2O2/Fe(II) systems. The enhancement was suggested to be due to scavenging of OH radicals by NO which was formed by the reactions among AA, HNO2 and SCN- contained in the reaction mixture. The concentrations of SCN- and NO2-, which were effective for the inhibition, were in ranges of their normal salivary concentrations. These results suggest that salivary SCN- can cooperate with NO2- to protect stomach from OH radicals formed by AA/H2O2/Fe(II) systems under acidic conditions.     

The presence of 4-hydroxyphenylacetic acid in human saliva and the possibility of its nitration by salivary nitrite in the stomach

Human saliva contained 4-hydroxyphenylacetic acid (HPA) (2-10 microM) and nitrite (60-300 microM). HPA was nitrated to 4-hydroxy-3-nitrophenylacetic acid (NO2HPA) when HPA and sodium nitrite were mixed at pH 1.0. NO2HPA was also formed when saliva was incubated under acidic conditions. These results suggest that salivary HPA is nitrated to NO2HPA when saliva is swallowed into the stomach.     

Oxidative Modulation of the Glutathione-redox Couple Enhances Lipopolysaccharide-induced Interleukin 12 P40 Production by a Mouse Macrophage Cell Line,J774A.1

Abstract

Reactions of salivary nitrite with components of wine were studied using an acidic mixture of saliva and wine. The formation of nitric oxide (NO) in the stomach after drinking wine was observed. The formation of NO was also observed in the mixture (pH 3.6) of saliva and wine, which was prepared by washing the oral cavity with wine. A part of the NO formation in the stomach and the oral cavity was due to the reduction of salivary nitrite by caffeic and ferulic acids present in wine. Ethyl nitrite produced by the reaction of salivary nitrite and ethyl alcohol in wine also contributed to the formation of NO. In addition to the above reactions, caffeic acid in wine could be transformed to the oxathiolone derivative, which might have pharmacological functions. The results obtained in this study may help in understanding the effects of drinking wine on human health.     

The pathway of nitrogen and reductive enzymes of denitrification

Some recent studies on the pathway of nitrogen and the reductases of denitrification are reviewed. The available evidence suggests that while the intermediates of denitrification can remain enzyme-bound (presumably to nitrite reductase) prior to formation of N₂O, NO and nitroxyl (HNO) can be released in part by certain bacteria. Release of NO is recognized by a nitrite/NO?¹⁵N exchange reaction and isotopic scrambling in product N₂O; release of nitroxyl by Pseudomonas stutzeri is recognized by isotopic scrambling of nitrite and NO in product N₂O in absence of exchange and affords evidence that the first N?N bond forms in denitrification at the N¹⁺ redox level. The recent purification and partial characterization of nitrous oxide reductase are described. The ability of the dissimilatory nitrite reductase to activate nitrite for nitrosyl transfer affords a new chemical probe into the mechanism of action of this central enzyme. It would appear that reduction of nitrite is subject to electrophilic catalysis. ¹⁸O studies show that dissociation of nitrite from nitrite reductase can be slow relative to competing reduction or nitrosyl transfer.     

Proteomic analysis of human gastric juice: a shotgun approach

Gastric juice is the most proximal fluid surrounding the stomach tissue. The analysis of gastric juice protein contents will thus be able to accurately reflect the pathophysiology of the stomach. This biological fluid is also a potential reservoir of secreted biomarkers in higher concentration as compared to the serum. Unlike the rest of the gastrointestinal fluids, there were very few studies reported on gastric juice proteome. To date, the proteins that routinely populate this biofluid are largely unknown. This is partly due to the technical difficulties in processing a sample that contains a collection of other gastrointestinal fluids, especially saliva. In this study, we attempt to profile the protein components of the gastric fluids from chronic gastritis patients using a direct shotgun proteomics approach. These data represent the first report of the proteome of human gastric juice with gastritis background.     

Dietary nitrite induces occludin nitration in the stomach

The clinical implications of the nitrate–nitrite–nitric oxide pathway have been extensively studied in recent years. However, the physiological impact of bioactive nitrogen oxides produced from dietary nitrate has remained largely elusive. Here, we report a hitherto unrecognized nitrite-dependent nitrating pathway that targets tight junction proteins in the stomach. Inorganic nitrate, nitrite or saliva obtained after the consumption of lettuce were administered by oral gavage to Wistar rats. The enterosalivary circulation of nitrate was allowed to occur for 4?h after which the animals were euthanized and the stomach collected. Nitrated occludin was detected by immunoprecipitation in the gastric epithelium upon inorganic nitrite administration (p??NO production rates from inorganic and salivary nitrite under simulated gastric conditions, suggests that competing reactions at acidic pH determine the production of nitrating agents (^?NO₂) or other, more stable, oxides. Accordingly, it is shown in vitro that salivary nitrite yields higher steady state concentrations of ^?NO (0.37?±?0.01?μM) than sodium nitrite (0.12?±?0.03?μM). Dietary-dependent reactions involving the production of nitrogen oxides should be further investigated as, in the context of occludin nitration, the consumption of green leafy vegetables (with high nitrate content), if able to modulate gut barrier function, may have important implications in the context of leaky gut disorders.     

Increase in gastric pH reduces hypotensive effect of oral sodium nitrite in rats

The new pathway nitrate-nitrite-nitric oxide (NO) has emerged as a physiological alternative to the classical enzymatic pathway for NO formation from l-arginine. Nitrate is converted to nitrite by commensal bacteria in the oral cavity and the nitrite formed is then swallowed and reduced to NO under the acidic conditions of the stomach. In this study, we tested the hypothesis that increases in gastric pH caused by omeprazole could decrease the hypotensive effect of oral sodium nitrite. We assessed the effects of omeprazole treatment on the acute hypotensive effects produced by sodium nitrite in normotensive and L-NAME-hypertensive free-moving rats. In addition, we assessed the changes in gastric pH and plasma levels of nitrite, NO(x) (nitrate+nitrite), and S-nitrosothiols caused by treatments. We found that the increases in gastric pH induced by omeprazole significantly reduced the hypotensive effects of sodium nitrite in both normotensive and L-NAME-hypertensive rats. This effect of omeprazole was associated with no significant differences in plasma nitrite, NO(x), or S-nitrosothiol levels. Our results suggest that part of the hypotensive effects of oral sodium nitrite may be due to its conversion to NO in the acidified environment of the stomach. The increase in gastric pH induced by treatment with omeprazole blunts part of the beneficial cardiovascular effects of dietary nitrate and nitrite.