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Summary Background Considerable controversy exists regarding the association between nitrate intake and risk of colorectal cancer. Therefore, we performed a dose–response meta-analysis of observational studies. Methods We identified relevant studies by searching PubMed, Scopus and ISI Web of Knowledge until April 2020 and references of retrieved relevant articles. The random-effects model was used to calculate pooled effect size (ESs) and 95% confidence intervals (CIs). Results Fifteen prospective cohort and case–control papers were included in this systematic review and meta-analysis. In total, 2,573,524 participants with an age range between 20 and 85 years were included. The total number of colorectal cancer cases was 38,848. Intake of nitrate from diet was associated with a risk of colorectal cancer (Pooled HR: 1.13; 95% CI: 1.04–1.23, I2 = 38%; P = 0.08). Nitrite in diet was not significantly associated with risk of colorectal cancer (pooled HR: 1.07; 95% CI: 0.95–1.21, I2 = 61.6%; P = 0.005). Nitrate in water did not show an association with risk of colorectal cancer (pooled HR: 1.04; 95% CI: 0.92–1.19, I2 = 64.7%; P = 0.002). Non-linear dose–response analysis revealed no significant association of dietary nitrite and also nitrate of drinking water with risk of colorectal cancer. However, dietary nitrate was marginally associated with a greater risk of colorectal cancer. Linear dose–response analysis of nitrate from diet was not associated with colorectal cancer risk by an additional 50 mg per day. Such a non-significant association was also seen for colorectal cancer risk by an additional 1 mg per day and 1 mg/l from dietary nitrite and water nitrate respectively. Conclusions Dietary nitrate was related to a higher risk of colorectal cancer risk. However, intake of nitrite from diet and nitrate from the drinking water was not associated with colorectal cancer risk.

Determination of the modulation of nitrite and nitrate levels in biological samples usually poses a major challenge, owing to their high background concentrations. To effectively investigate the variation of nitrite/nitrate in vivo, in this study, we developed a15N-labelled nitrite/nitrate tracer analysis using LC-MS/MS following the derivatization with 2,3-diaminonaphthalene. This method allows for the determination of 15N-labelled nitrite/nitrate as 15N-2,3-naphthotriazole (15N-NAT) that can efficiently differentiate newly introduced nitrite/nitrate from the background nitrite/nitrate in biological matrices. We also investigated the contribution of background 14N-NAT isotopomers to 15N-NAT, which has long been overlooked in the literature. Our results indicated that the contribution of background 14N-NAT isotopomers to 15N-NAT is significant. Such contribution is constant (~2.2% under positive ion mode and 1.1% under negative ion mode), and does not depend upon the concentration of 14N-NAT or the sample matrix measured. An equation has been therefore developed, for the first time, to correct the contribution of background 14N-NAT isotopomers to 15N-NAT. With the proposed 15N-labelled nitrite/nitrate tracer analysis, the amount and percentage distribution of 15NO2- and 15NO3-, both in urine and feces, after oral administration of 15N-labelled nitrite/nitrate are clearly demonstrated. The excretions of 15NO2- and 15NO3- were significantly increased with the increasing dose implying that the dietary nitrite/nitrate intake is an important source in urine/feces. The present method allows for the simple, reliable and accurate quantification of 15NO2- and 15NO3-, and it should also be useful to trace the biotransformation of nitrite and nitrate in vivo.

Streptococcus mutans resides in the oral polymicrobial biofilm and is a major contributor to the development of dental caries. Interestingly, high salivary nitrite concentrations have been associated with a decreased prevalence of dental caries. Moreover, the combination of hydrogen peroxide–producing oral commensal streptococci and nitrite has been shown to mediate the generation of reactive nitrogen species, which have antimicrobial activity. The goal of this study was to examine whether nitrite affects S. mutans virulence during polymicrobial infections with the commensal Streptococcus parasanguinis. Here, we report that the combination of S. parasanguinis and nitrite inhibited S. mutans growth and biofilm formation in vitro. Glucan production, which is critical for S. mutans biofilm formation, was also inhibited in 2-species biofilms with S. parasanguinis containing nitrite as compared with biofilms that contained no nitrite. In the in vivo caries model, enamel and dentin carious lesions were significantly reduced in rats that were colonized with S. parasanguinis prior to infection with S. mutans and received nitrite in the drinking water, as compared with animals that had a single S. mutans infection or were co-colonized with both bacteria and received no nitrite. Last, we report that S. mutans LiaS, a sensor kinase of the LiaFSR 3-component system, mediates resistance to nitrosative stress. In summary, our data demonstrate that commensal streptococci and nitrite provide protection against S. mutans pathogenesis. Modulating nitrite concentrations in the oral cavity could be a useful strategy to combat the prevalence of dental caries.

Aim Endothelial dysfunction accompanied by an increase in oxidative stress is a key event leading to hypertension. As dietary nitrite has been reported to exert antihypertensive effect, the present study investigated whether chronic oral administration of sodium nitrite improves vascular function in conduit and resistance arteries of hypertensive animals with elevated oxidative stress. Methods Sodium nitrite (50 mg/L) was given to angiotensin II-infused hypertensive C57BL/6J (eight to ten weeks old) mice for two weeks in the drinking water. Arterial systolic blood pressure was measured using the tail-cuff method. Vascular responsiveness of isolated aortae and renal arteries was studied in wire myographs. The level of nitrite in the plasma and the cyclic guanosine monophosphate (cGMP) content in the arterial wall were determined using commercially available kits. The production of reactive oxygen species (ROS) and the presence of proteins (nitrotyrosine, NOx-2 and NOx-4) involved in ROS generation were evaluated with dihydroethidium (DHE) fluorescence and by Western blotting, respectively. Results Chronic administration of sodium nitrite for two weeks to mice with angiotensin II-induced hypertension decreased systolic arterial blood pressure, reversed endothelial dysfunction, increased plasma nitrite level as well as vascular cGMP content. In addition, sodium nitrite treatment also decreased the elevated nitrotyrosine and NOx-4 protein level in angiotensin II-infused hypertensive mice. Conclusions The present study demonstrates that chronic treatment of hypertensive mice with sodium nitrite improves impaired endothelium function in conduit and resistance vessels in addition to its antihypertensive effect, partly through inhibition of ROS production.

Nitric oxide (NO) is synthesized not only from L-arginine by NO synthases (NOSs), but also from its inert metabolites, nitrite and nitrate. Green leafy vegetables are abundant in nitrate, however whether or not a deficiency in dietary nitrite/nitrate spontaneously causes disease remains to be clarified. In this study, we tested our hypothesis that long-term dietary nitrite/nitrate deficiency induces metabolic syndrome (MetS) in mice. To this end, we prepared a low nitrite/nitrate diet (LND) consisting of an amino acid-based low nitrite/nitrate chow in which the contents of L-arginine, fat, carbohydrates, protein, and energy were identical with a regular chow, and potable ultrapure water. Nitrite and nitrate were undetectable in both the chow and the water. Intriguingly, in comparison with a regular diet, 3 months of the LND significantly elicited visceral adiposity, dyslipidaemia, and glucose intolerance; 18 months of the LND significantly provoked increased body weight, hypertension, insulin resistance, and impaired endothelium-dependent relaxations to acetylcholine; and 22 months of the LND significantly led to death due to cardiovascular disease, including acute myocardial infarction. These abnormalities were reversed by simultaneous treatment with sodium nitrate, and were significantly associated with endothelial NOS down-regulation, adiponectin insufficiency, and gut microbiota dysbiosis. These results provide the first evidence that long-term dietary nitrite/nitrate deficiency gives rise to MetS, endothelial dysfunction, and cardiovascular death in mice, indicating a novel pathogenetic role of the exogenous NO production system in MetS and its vascular complications.

Nitrite represents an endocrine reserve of bioavailable nitric oxide (NO) that mediates a number of physiological responses including conferral of cytoprotection after ischemia/reperfusion (I/R). It has long been known that nitrite can react with non-heme iron to form dinitrosyliron complexes (DNIC). However, it remains unclear how quickly nitrite-dependent DNIC form in vivo, whether formation kinetics differ from that of NO-dependent DNIC, and whether DNIC play a role in the cytoprotective effects of nitrite. Here we demonstrate that chronic but not acute nitrite supplementation increases DNIC concentration in the liver and kidney of mice. Although DNIC have been purported to have antioxidant properties, we show that the accumulation of DNIC in vivo is not associated with nitrite-dependent cytoprotection after hepatic I/R. Further, our data in an isolated mitochondrial model of anoxia/reoxygenation show that while NO and nitrite demonstrate similar S-nitrosothiol formation kinetics, DNIC formation is significantly greater with NO and associated with mitochondrial dysfunction as well as inhibition of aconitase activity. These data are the first to directly compare mitochondrial DNIC formation by NO and nitrite. This study suggests that nitrite-dependent DNIC formation is a physiological consequence of dietary nitrite. The data presented herein implicate mitochondrial DNIC formation as a potential mechanism underlying the differential cytoprotective effects of nitrite and NO after I/R, and suggest that DNIC formation is potentially responsible for the cytotoxic effects observed at high NO concentrations., Graphical abstract fx1, Highlights • Dietary nitrite results in DNIC formation in many tissues, most notably the liver. • Nitrite-dependent DNIC accumulate within the mitochondrion. • NO generates greater DNIC formation in the mitochondrion than nitrite. • At high concentrations of NO DNIC formation is associated with mitochondrial injury.

Nitrate and nitrite are precursors of N-nitroso compounds (NOC), probable human carcinogens that cause pancreatic tumors in animals. Disinfection by-products (DBP) exposures have also been linked with digestive system cancers, but few studies have evaluated relationships with pancreatic cancer. We investigated the association of pancreatic cancer with these drinking water contaminants and dietary nitrate/nitrite in a cohort of postmenopausal women in Iowa (1986-2011). We used historical monitoring and treatment data to estimate levels of long-term average nitrate and total trihalomethanes (TTHM; the sum of the most prevalent DBP class) and the duration exceeding one-half the maximum contaminant level (>½ MCL; 5 mg/L nitrate-nitrogen, 40 µg/L TTHM) among participants on public water supplies (PWS) >10 years. We estimated dietary nitrate and nitrite intakes using a food frequency questionnaire. We computed hazard ratios (HR) and 95% confidence intervals (CI) using Cox regression and evaluated nitrate interactions with smoking and vitamin C intake. We identified 313 cases among 34,242 women, including 152 with >10 years PWS use (N = 15,710). Multivariable models of average nitrate showed no association with pancreatic cancer (HRp95vs. Q1 = 1.16, 95% CI: 0.51-2.64). Associations with average TTHM levels were also null (HRQ4vs. Q1 = 0.70, 95% CI:0.42-1.18). We observed no trend with increasing years of exposure to either contaminant at levels >½ MCL. Positive associations were suggested in the highest dietary nitrite intake from processed meat (HRp95vs. Q1 = 1.66, 95% CI 1.00-2.75;ptrend = 0.05). We found no interactions of nitrate with known modifiers of endogenous NOC formation. Our results suggest that nitrite intake from processed meat may be a risk factor for pancreatic cancer.

Loss of nitric oxide (NO) bioavailability underlies the development of hypertensive heart disease. We investigated the effects of dietary nitrite on NG-nitro-l-arginine methyl ester (l-NAME)-induced hypertension. Sprague-Dawley rats were divided into five groups: an untreated control group, an l-NAME-treated group, and three other l-NAME-treated groups supplemented with 10 mg/L or 100 mg/L of nitrite or 100 mg/L of captopril in drinking water. After the 8-week experimental period, mean arterial blood pressure was measured, followed by sampling of blood and heart tissue for assessment of nitrite/nitrate levels in the plasma and heart, the plasma level of angiotensin II (AT II), and the heart transcriptional levels of AT II type 1 receptor (AT1R), transforming growth factor-β1 (TGF-β1), and connective tissue proteins such as type 1 collagen and fibronectin. Heart tissue was analyzed by histopathological morphometry, including assessments of ventricular and coronary vascular hypertrophy and fibrosis, as well as immunohistochemistry analyses of myocardial expression of AT1R. l-NAME treatment reduced the plasma nitrate level and led to the development of hypertension, with increased plasma levels of AT II and increased heart transcriptional levels of AT1R and TGF-β1-mediated connective tissue proteins, showing myocardial and coronary arteriolar hypertrophy and fibrosis. However, dietary nitrite supplementation inhibited TGF-β1-mediated cardiac remodeling by suppressing AT II and AT1R. These results suggest that dietary nitrite levels achievable via a daily high-vegetable diet could improve hypertensive heart disease by inhibiting AT II-AT1R-mediated cardiac remodeling.

Menopausal women are at greater risk of developing metabolic syndrome with reduced endothelial nitric oxide synthase (eNOS) activity. Hormone replacement therapy increases eNOS activity and normalizes some characteristics of metabolic syndrome. We hypothesized that nitric oxide (NO) supplementation should have a therapeutic effect on this syndrome. We examined the effect of dietary nitrite in a mouse model with postmenopausal metabolic syndrome induced by ovariectomy (OVX) and a high fat diet (HF). C57BL/6 female mice were divided into five groups, sham+normal fat diet (NF), sham+ HF, OVX+HF with or without sodium nitrite (50 mg and 150 mg/l) in the drinking water. Daily food intake and weekly body weight were monitored for 18 wk. OVX and HF significantly reduced plasma levels of nitrate/nitrite (NOx), and mice developed obesity with visceral hypertrophic adipocytes and increased transcriptional levels of monocyte chemoattractant protein-1, TNF-α, and IL-6 in visceral fat tissues. The proinflammatory state in the adipocytes provoked severe hepatosteatosis and insulin resistance in OVX+HF group compared with sham+NF group. However, dietary nitrite significantly suppressed adipocyte hypertrophy and transcriptions of proinflammatory cytokines in visceral fat in a dose-dependent manner. The improvement of visceral inflammatory state consequently reversed the hepatosteatosis and insulin resistance observed in OVX+HF mice. These results suggest that an endogenous NO defect might underlie postmenopausal metabolic syndrome and that dietary nitrite provides an alternative source of NO, subsequently compensating for metabolic impairments of this syndrome.

Nitric oxide (NO) is synthesised not only from l-arginine by NO synthases (NOSs), but also from its inert metabolites, nitrite and nitrate. Green leafy vegetables are abundant in nitrate, but whether or not a deficiency in dietary nitrite/nitrate spontaneously causes disease remains to be clarified. In this study, we tested our hypothesis that long-term dietary nitrite/nitrate deficiency would induce the metabolic syndrome in mice. To this end, we prepared a low-nitrite/nitrate diet (LND) consisting of an amino acid-based low-nitrite/nitrate chow, in which the contents of l-arginine, fat, carbohydrates, protein and energy were identical with a regular chow, and potable ultrapure water. Nitrite and nitrate were undetectable in both the chow and the water. Three months of the LND did not affect food or water intake in wild-type C57BL/6J mice compared with a regular diet (RD). However, in comparison with the RD, 3 months of the LND significantly elicited visceral adiposity, dyslipidaemia and glucose intolerance. Eighteen months of the LND significantly provoked increased body weight, hypertension, insulin resistance and impaired endothelium-dependent relaxations to acetylcholine, while 22 months of the LND significantly led to death mainly due to cardiovascular disease, including acute myocardial infarction. These abnormalities were reversed by simultaneous treatment with sodium nitrate, and were significantly associated with endothelial NOS downregulation, adiponectin insufficiency and dysbiosis of the gut microbiota. These results provide the first evidence that long-term dietary nitrite/nitrate deficiency gives rise to the metabolic syndrome, endothelial dysfunction and cardiovascular death in mice, indicating a novel pathogenetic role of the exogenous NO production system in the metabolic syndrome and its vascular complications.

Nitrate and nitrite are precursors in the endogenous formation of N-nitroso compounds (NOC) which are potent animal carcinogens for the organs of the digestive system. We evaluated dietary intakes of nitrate and nitrite, as well as nitrate ingestion from drinking water (public drinking water supplies (PWS)), in relation to the incidence (1986–2014) of cancers of the esophagus (n = 36), stomach (n = 84), small intestine (n = 32), liver (n = 31), gallbladder (n = 66), and bile duct (n = 58) in the Iowa Women’s Health Study (42,000 women aged from 50 to 75 in 1986). Dietary nitrate and nitrite were estimated using a food frequency questionnaire and a database of nitrate and nitrite levels in foods. Historical nitrate measurements from PWS were linked to the enrollment address by duration. We used Cox regression to compute hazard ratios (HR) and 95% confidence intervals (CI) for exposure quartiles (Q), tertiles (T), or medians, depending on the number of cancer cases. In adjusted models, nitrite intake from processed meats was associated with an increased risk of stomach cancer (HRQ4vsQ1 = 2.2, CI: 1.2–4.3). A high intake of total dietary nitrite was inversely associated with gallbladder cancer (HRQ4vsQ1 = 0.3, CI: 0.1–0.96), driven by an inverse association with plant sources of nitrite (HRQ4vsQ1 = 0.3, CI: 0.1–0.9). Additionally, small intestine cancer was inversely associated with a high intake of animal nitrite (HRT3vsT1 = 0.2, CI: 0.1–0.7). There were no other dietary associations. Nitrate concentrations in PWS (average, years ≥ 1/2 the maximum contaminant level) were not associated with cancer incidence. Our findings for stomach cancer are consistent with prior dietary studies, and we are the first to evaluate nitrate and nitrite ingestion for certain gastrointestinal cancers.

Background There is no epidemiological study on the association between dietary nitrate (NO 3 ) and nitrite (NO 2 ) and intakes and the risk of type 2 diabetes (T2D). Objective The aim of this study was therefore to examine the potential effect of dietary NO 3 and NO 2 on the occurrence of T2D. Design This longitudinal study was conducted within the framework of the Tehran Lipid and Glucose Study (TLGS) on 2139 T2D-free adults, aged 20–70 years, followed for a median of 5.8 y. Dietary intakes of NO 3 and NO 2 were estimated using a 168-food items validate semi-quantitative food frequency questionnaire, at baseline. Multivariate Hazard Ratios (HR) and 95% confidence intervals (CI), adjusted for diabetes risk score (DRS), and dietary intakes of fat, fiber and vitamin C, were calculated for residual energy-adjusted NO 3 and NO 2 intakes. Since significant interaction ( P = 0.024) was found between NO 2 and vitamin C intakes in the multivariable model, stratified analyses were done for Results Median (inter quartile range; IQR) daily intake of NO 3 and NO 2 were 410 mg/d (343–499) and 8.77 mg/d (7.53–10.2). An increased risk of T2D was observed among participants who had higher intake of total and animal-based NO 2 in participants who had low vitamin C intake (HR = 2.43, 95% CI = 1.45–4.05, HR = 1.88, 95% CI = 1.12–3.15, respectively). We found no significant association between NO 3 in overall, and plant- and animal sources as well, with the risk of T2D. Plant-derived NO 2 was also unrelated to incidence of T2D. Conclusion Our findings indicated that higher intakes of total and animal-based NO 2 may be an independent dietary risk factor for development of T2D in subjects with lower vitamin C intakes.

Background: There are now indisputable health benefits of nitrite when administered in a clinical setting for specific diseases. Most of the published reports identify the production of nitric oxide (NO) as the mechanism of action for nitrite. Basic science as well as clinical studies demonstrates nitrite and/or nitrate can restore NO homeostasis as an endothelium independent source of NO that may be a redundant system for endogenous NO production. Nitrate must first be reduced to nitrite by oral commensal bacteria and then nitrite further reduced to NO along the physiological oxygen gradient. Despite decades of rigorous research on its safety and efficacy as a curing agent, sodium nitrite is still regarded by many as a toxic undesirable food additive. However, research within the biomedical science community has revealed enormous therapeutic benefits of nitrite that is currently being developed as novel therapies for conditions associated with nitric oxide insufficiency. This review will highlight the fundamental biochemistry of nitrite and nitrate in human physiology and provide evidence that nitrite and nitrate be considered essential nutrients. Foods or diets enriched with nitrite can have profound positive health benefits. Keywords: nitrite, nitrate, nitric oxide, curing, nutrition, epidemiology, cardiovascular, cancer, diet, nitrosamines, antioxidants

Dietary nitrate is now recognized as an alternative substrate for nitric oxide (•NO) production in the gut. This novel pathway implies the sequential reduction of nitrate to nitrite, •NO and other bioactive nitrogen oxides but the physiological relevance of these oxidants has remained elusive. We have previously shown that dietary nitrite fuels an hitherto unrecognized nitrating pathway at acidic gastric pH, through which pepsinogen is nitrated in the gastric mucosa, yielding a less active form of pepsin in vitro. Here, we demonstrate that pepsin is nitrated in vivo and explore the functional impact of protein nitration by means of peptic ulcer development. Upon administration of pentagastrin and human nitrite-rich saliva or sodium nitrite to rats, nitrated pepsin was detected in the animal's stomach by immunoprecipitation. •NO was measured in the gastric headspace before and after nitrite instillation by chemiluminescence. At the end of each procedure, the stomach's lesions, ranging from gastric erosions to haemorrhagic ulcers, were scored. Nitrite increased gastric •NO by 200-fold (p, Graphical abstract fx1, Highlights • The nitrate-nitrite-nitric oxide pathway promotes gastric protein nitration. • Pepsin is nitrated by dietary nitrite through the production of ∙NO2. • Nitrated pepsin prevents the development of acute peptic ulcers.

Vascular stiffness is a major cause of cardiovascular disease during normal aging and in Hutchinson–Gilford progeria syndrome (HGPS), a rare genetic disorder caused by ubiquitous progerin expression. This mutant form of lamin A causes premature aging associated with cardiovascular alterations that lead to death at an average age of 14.6 years. We investigated the mechanisms underlying vessel stiffness in LmnaG609G/G609G mice with ubiquitous progerin expression, and tested the effect of treatment with nitrites. We also bred LmnaLCS/LCSTie2Cre+/tgand LmnaLCS/LCSSM22αCre+/tg mice, which express progerin specifically in endothelial cells (ECs) and in vascular smooth muscle cells (VSMCs), respectively, to determine the specific contribution of each cell type to vascular pathology. We found vessel stiffness and inward remodeling in arteries of LmnaG609G/G609G and LmnaLCS/LCSSM22αCre+/tg, but not in those from LmnaLCS/LCSTie2Cre+/tgmice. Structural alterations in aortas of progeroid mice were associated with decreased smooth muscle tissue content, increased collagen deposition, and decreased transverse waving of elastin layers in the media. Functional studies identified collagen (unlike elastin and the cytoskeleton) as an underlying cause of aortic stiffness in progeroid mice. Consistent with this, we found increased deposition of collagens III, IV, V, and XII in the media of progeroid aortas. Vessel stiffness and inward remodeling in progeroid mice were prevented by adding sodium nitrite in drinking water. In conclusion, LmnaG609G/G609G arteries exhibit stiffness and inward remodeling, mainly due to progerin-induced damage to VSMCs, which causes increased deposition of medial collagen and a secondary alteration in elastin structure. Treatment with nitrites prevents vascular stiffness in progeria, This study was supported by the Spanish Ministerio de Ciencia, Innovación y Universidades (MCIU, grants SAF2016‐79490‐R and SAF2016‐80305‐P), with co‐funding from the European Regional Development Fund (ERDF, “Una manera de hacer Europa”). RvK and RKA are supported by NIH grants AG047373, T32‐GM008076, F31HL142160 and NSF grant CMMI 1548571. L.d.C. was supported by a Jordi Soler postdoctoral grant from the Red de Investigación Cardiovascular (RETIC Program, Instituto de Salud Carlos III), and A.S.‐L. was supported by a Severo Ochoa predoctoral grant from the MCIU (SVP‐2014‐068334) and by a grant from Asociación Apadrina la Ciencia‐Ford España‐Ford Motor Company Fund. The CNIC is supported by the MCIU and the Pro‐CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV‐2015‐0505)

Nitrite compounds are very important in many industrials that may cause some significant problems, but in more or less cases it has useful effect. Therefore, the present study was carried out to shed the light on the effect of nitrite on streptozotocin (STZ) inducing diabetic rats. In this study, thirty-two male Wister rats were distributed into four groups (n= 8 per group): Group (1) control group; Group (2) normal rats received NaNO2 30 mg/kg body weight; Group (3) diabetic group rats received 45 mg/kg STZ intraperitoneal (IP); Group (4) diabetic rats treated daily with NaNO2 for three months. Serum from each group was collected from rat's blood samples to measure aminotransferase (AST), alanine aminotransferase (ALT), Urea, and Creatinine. A self-assembled nanostructure titania (TiO2) based surface similar to honey cells was synthesized by a simple, fast, low-cost procedure using the 2D electrochemical cell at sequence potential from 5 V to 30 V for 2 h followed by annealed method at 350°C and 750°C for 3 h. A linear sweep voltammetry electrochemical method was used to deposit the Ni-Cu nanoparticles on titania surface providing a highly sensing platform of Ni-Cu/nanotitania array. Both the degree of sensing (46 µA mM−1) for the electrode were calculated through low detection range from 0.00 to 0.35 mM of glucose via cyclic voltammetry and amperometery techniques. By increasing the glucose concentration through the detection range from 0.4 to 2.5 mM the sensitivity increased to 248 µA mM−1. This significant change indicates that the sensitivity of the electrode is suitable for diabetes measurements. We reported for the first electrochemical sensor fabricated from Ni-Cu/nanotitania to detect glucose level in real urine samples and we used this level as a marker for the effect of nitrite present in the drinking water. The results showed that the presence of low concentration of sodium nitrite can enhance the liver and kidney functions in diabetic rats and also lead to the decrease of the glucose concentration in urine. Additionally, Ni-Cu/nanotitania sensor also has stability, profitable good reproducibility and applicability that guarantee its value in the medical applications as in the environmental applications that has been proven previously.

Pulmonary emphysema (PE) is a major pathological feature of chronic obstructive pulmonary disease (COPD) and is characterized by proteolytic destruction of the alveolar structure and subsequent inflammation of the respiratory tract. We hypothesized that nitrite attenuates the development of PE via anti-inflammatory actions. PE was induced by intratracheal instillation of porcine pancreas elastase (PPE) in mice. Dietary nitrite dose-dependently (50 and 150 mg/L in drinking water) attenuated emphysematous development and macrophage accumulation in the alveolar parenchyma 21 d after PPE treatment. The present study shows that dietary nitrite might be a possible nutritional strategy in preventing the development of PE in mice.

The goal of this study was to find the relationship between dietary nitrite and risk of esophageal cancer, and determine the amount of nitrite intake to establish the oral highest daily intake to prevent the occurrence of esophageal cancer. Duplicate portions of three-consecutive-day diets were collected from 100 patients with esophageal precancerous lesions and 100 controls. The average nitrite daily intakes for esophageal precancerous lesions and normal people were 15.72 mg/d and 11.11 mg/d. The median nitrite daily intakes for cases and controls were 8.76 mg/d and 5.33 mg/d. Positive association was observed between the risk of esophageal precancerous lesions and dietary nitrite intake (p = 0.035). An increased risk of esophageal precancerous lesions was observed for cases or controls in the highest intake quartile of nitrite (highest vs. lowest quartile odds ratio (OR) = 2.256, 95% confidence interval (CI): 1.012-5.026). These results suggest that dietary nitrite intake may influence the risk of esophageal cancer; populations with high incidence of esophageal cancer should take control of nitrite intake as one of the measures to prevent esophageal cancer.

Endogenous and dietary nitrite produces reactive nitrogen species (RNS) that react with DNA causing mutations. The nitrosation of 2′-deoxyguanosine (dGuo) and DNA with nitrite was studied under different conditions, and the reaction and degradation products identified and analysed by HPLC-DAD-MS. Nitrosative deamination of dGuo produced xanthine along with 2′-deoxyxanthosine whereas DNA afforded xanthine. Formation of xanthine increased with nitrite concentration and in low pH such as that of stomach. Xanthine was measured as a marker of nitrosation of dGuo and DNA, and it was subsequently used to study the antinitrosating activity of β-carboline alkaloids, and selected antioxidants. Food-occurring tetrahydro-β-carbolines (THβCs) decreased nitrosative deamination of dGuo and DNA under conditions simulating the stomach. Antinitrosating activity was also evidenced for flavonoids (catechin, quercetin) and indole (melatonin) antioxidants. Among THβCs the most active antinitrosating compounds were 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acids (THβC-3-COOHs) that reacted with nitrite to give N-nitroso derivatives as main products along with 3,4-dihydro-β-carboline-3-carboxylic acids and aromatic β-carbolines (norharman and harman). Antinitrosating activity of THβCs correlated well with the formation of N-nitroso-THβC-3-COOHs. These N-nitroso derivatives were stable at pH 7 but degraded in acid conditions affording nitrosating species., The authors thank MINECO-FEDER (projects SAF2015-66690-R and SAF2015-68580-C2-2-R) and CSIC (project 200470E658) for supporting this work.

The Panel on Food Additives and Nutrient Sources added to Food (ANS) provided a scientific opinion re-evaluating the safety of potassium nitrite (E 249) and sodium nitrite (E 250) when used as food additives. The ADIs established by the SCF (1997) and by JECFA (2002) for nitrite were 0-0.06 and 0-0.07 mg/kg bw per day, respectively. The available information did not indicate in vivo genotoxic potential for sodium and potassium nitrite. Overall, an ADI for nitrite per se could be derived from the available repeated dose toxicity studies in animals, also considering the negative carcinogenicity results. The Panel concluded that an increased methaemoglobin level, observed in human and animals, was a relevant effect for the derivation of the ADI. The Panel, using a BMD approach, derived an ADI of 0.07 mg nitrite ion/kg bw per day. The exposure to nitrite resulting from its use as food additive did not exceed this ADI for the general population, except for a slight exceedance in children at the highest percentile. The Panel assessed the endogenous formation of nitrosamines from nitrites based on the theoretical calculation of the NDMA produced upon ingestion of nitrites at the ADI and estimated a MoE > 10,000. The Panel estimated the MoE to exogenous nitrosamines in meat products to be

Nitrate and nitrite are precursors in the endogenous formation of N-nitroso compounds (NOC), potential human carcinogens. We evaluated the association of nitrate and nitrite ingestion with postmenopausal ovarian cancer risk in the Iowa Women's Health Study. Among 28,555 postmenopausal women, we identified 315 incident epithelial ovarian cancers from 1986 to 2010. Dietary nitrate and nitrite intakes were assessed at baseline using food frequency questionnaire data. Drinking water source at home was obtained in a 1989 follow-up survey. Nitrate-nitrogen (NO3 -N) and total trihalomethane (TTHM) levels for Iowa public water utilities were linked to residences and average levels were computed based on each woman's duration at the residence. We computed multivariable-adjusted hazard ratios (HR) and 95% confidence intervals (CI) using Cox proportional hazards regression. We tested interactions of nitrate with TTHMs and dietary factors known to influence NOC formation. Ovarian cancer risk was 2.03 times higher (CI = 1.22-3.38, ptrend = 0.003) in the highest quartile (≥2.98 mg/L) compared with the lowest quartile (≤0.47 mg/L; reference) of NO3 -N in public water, regardless of TTHM levels. Risk among private well users was also elevated (HR = 1.53, CI = 0.93-2.54) compared with the same reference group. Associations were stronger when vitamin C intake was

Diabetes mellitus type 2 is a syndrome of disordered metabolism with inappropriate hyperglycemia owing to a reduction in the biological effectiveness of insulin. Type 2 diabetes is associated with an impaired nitric oxide (NO) pathway that probably serves as the key link between metabolic disorders and cardiovascular disease. Insulin-mediated translocation of GLUT4 involves the PI3K/Akt kinase signal cascade that results in activation of endothelial NO synthase (eNOS). eNOS is dysfunctional during diabetes. We hypothesize that loss of eNOS-derived NO terminates the signaling cascade and therefore cannot activate GLUT4 translocation and that dietary nitrite may repair this pathway. In this study, we administered 50mg/L sodium nitrite to db/db diabetic mice for 4 weeks. After 4 weeks treatment, the db/db mice experienced less weight gain, improved fasting glucose levels, and reduced insulin levels. Cell culture experiments using CHO-HIRc-myc-GLUT4eGFP cell lines stably expressing insulin receptor and myc-GLUT4eGFP protein, as well as L6 skeletal muscle cells stably expressing rat GLUT4 with a Myc epitope (L6-GLUT4myc), showed that NO, nitrite, and GSNO stimulate GLUT4 translocation independent of insulin, which is inhibited by NEM. Collectively our data suggest that nitrite improves insulin signaling through restoration of NO-dependent nitrosation of GLUT4 signaling translocation. These data suggest that NO-mediated nitrosation of GLUT4 by nitrite or other nitrosating agents is necessary and sufficient for GLUT4 translocation in target tissue. Description of this pathway may justify a high-nitrate/nitrite diet along with the glycemic index to provide a safe and nutritional regimen for the management and treatment of diabetes.

Dietary nitrate and nitrite are sources of gastric NO, which modulates blood flow, mucus production, and microbial flora. However, the intake and importance of these anions in infants is largely unknown. Nitrate and nitrite levels were measured in breast milk of mothers of preterm and term infants, infant formulas, and parenteral nutrition. Nitrite metabolism in breast milk was measured after freeze-thawing, at different temperatures, varying oxygen tensions, and after inhibition of potential nitrite-metabolizing enzymes. Nitrite concentrations averaged 0.07 ± 0.01 μM in milk of mothers of preterm infants, less than that of term infants (0.13 ± 0.02 μM) (P < .01). Nitrate concentrations averaged 13.6 ± 3.7 μM and 12.7 ± 4.9 μM, respectively. Nitrite and nitrate concentrations in infant formulas varied from undetectable to many-fold more than breast milk. Concentrations in parenteral nutrition were equivalent to or lower than those of breast milk. Freeze-thawing decreased nitrite concentration ∼64%, falling with a half-life of 32 minutes at 37°C. The disappearance of nitrite was oxygen-dependent and prevented by ferricyanide and 3 inhibitors of lactoperoxidase. Nitrite concentrations in breast milk decrease with storage and freeze-thawing, a decline likely mediated by lactoperoxidase. Compared to adults, infants ingest relatively little nitrite and nitrate, which may be of importance in the modulation of blood flow and the bacterial flora of the infant GI tract, especially given the protective effects of swallowed nitrite.

The potential toxic effects of nitrate-nitrite-nitrosamine on pancreatic β cell have remained a controversial issue over the past two decades. In this study, we reviewed epidemiological studies investigated the associations between nitrate-nitrite-nitrosamines exposure, from both diet and drinking water to ascertain whether these compounds may contribute to development of type 1 diabetes. To identify relevant studies, a systematic search strategy of PubMed, Scopus, and Science Direct was conducted using queries including the key words “nitrate”, “nitrite”, “nitrosamine” with “type 1 diabetes” or “insulin dependent diabetes mellitus”. All searches were limited to studies published in English. Ecologic surveys, case-control and cohort studies have indicated conflicting results in relation to nitrate-nitrite exposure from drinking water and the risk of type 1 diabetes. A null, sometimes even negative association has been mainly reported in regions with a mean nitrate levels < 25 mg/L in drinking water, while increased risk of type 1 diabetes was observed in those with a maximum nitrate levels > 40-80 mg/L. Limited data are available regarding the potential diabetogenic effect of nitrite from drinking water, although there is evidence indicating dietary nitrite could be a risk factor for development of type 1 diabetes, an effect however that seems to be significant in a higher range of acceptable limit for nitrate/nitrite. Current data regarding dietary exposure of nitrosamine and development of type 1 diabetes is also inconsistent. Considering to an increasing trend of type 1 diabetes mellitus (T1DM) along with an elevated nitrate-nitrite exposure, additional research is critical to clarify potential harmful effects of nitrate-nitrite-nitrosamine exposure on β-cell autoimmunity and the risk of T1DM.

BACKGROUND Nitrosatable drugs can react with nitrite in the stomach to form N-nitroso compounds, and results from animal studies suggest that N-nitroso compounds are teratogens. With data from the National Birth Defects Prevention Study, the relation between prenatal exposure to nitrosatable drugs and limb deficiencies, oral cleft, and heart malformations in offspring was examined. METHODS Maternal reports of drugs taken during the first trimester of pregnancy were classified with respect to nitrosatability for mothers of 741 babies with limb deficiencies, 2774 with oral cleft malformations, 8091 with congenital heart malformations, and 6807 without major congenital malformations. Nitrite intake was estimated from maternal responses to a food frequency questionnaire. RESULTS Isolated transverse limb deficiencies and atrioventricular septal defects were associated with secondary amine drug exposures (adjusted odds ratios [aORs], 1.51; 95% confidence limit [CI], 1.11–2.06 and aOR, 1.97; 95% CI, 1.19–3.26, respectively). Tertiary amines were associated with hypoplastic left heart syndrome (aOR, 1.50; 95% CI, 1.10–2.04) and single ventricle (aOR, 1.61; 95% CI, 1.06–2.45). These two malformations were also significantly associated with amide drugs. For several malformations, the strongest associations with nitrosatable drug use occurred among mothers with the highest estimated dietary nitrite intake, especially for secondary amines and atrioventricular septal defects (highest tertile of nitrite, aOR, 3.30; 95% CI, 1.44–7.58). CONCLUSION Prenatal exposure to nitrosatable drugs may be associated with several congenital malformations, especially with higher nitrite intake. The possible interaction between nitrosatable drugs and dietary nitrite on risk of congenital malformations warrants further attention. Birth Defects Research (Part A) 94:701–713, 2012. © 2012 Wiley Periodicals, Inc.

Mice lacking all three nitric oxide synthase (NOS) genes remain viable even though deletion of the major downstream target of NO, soluble guanylyl cyclase, is associated with a dramatically shortened life expectancy. Moreover, findings of relatively normal flow responses in eNOS knockouts are generally attributed to compensatory mechanisms including upregulation of remaining NOS isoforms, but the alternative possibility that dietary nitrite/nitrate (NOx) may contribute to basal levels of NO signaling has never been investigated. Aim: The aim of the present study was to examine how NO signaling products (nitrosated and nitrosylated proteins) and NO metabolites (nitrite, nitrate) are affected by single NOS deletions and whether dietary NOx plays a compensatory role in any deficiency. Specifically, we sought to ascertain whether profound alterations of these products arise upon genetic deletion of either NOS isoform, inhibition of all NOS activity, NOx restriction, or all of the above. Results: Our results indicate that while some significant changes do indeed occur, they are surprisingly moderate and compartmentalized to specific tissues. Unexpectedly, even after pharmacological inhibition of all NOSs and restriction of dietary NOx intake in eNOS knockout mice significant levels of NO-related products remain. Innovation/Conclusion: These findings suggest that a yet unidentified source of NO, unrelated to NOSs or dietary NOx, may be sustaining basal NO signaling in tissues. Given the significance of NO for redox regulation in health and disease, it would seem to be important to identify the nature of this additional source of NO products as it may offer new therapeutic avenues for correcting NO deficiencies. Antioxid. Redox Signal. 17, 422–432.

Background— Pulmonary hypertension (PH) is a multifactorial disease characterized by increased pulmonary vascular resistance and right ventricular failure; morbidity and mortality remain unacceptably high. Loss of nitric oxide (NO) bioactivity is thought to contribute to the pathogenesis of PH, and agents that augment pulmonary NO signaling are clinically effective in the disease. Inorganic nitrate (NO 3 − ) and nitrite (NO 2 − ) elicit a reduction in systemic blood pressure in healthy individuals; this effect is underpinned by endogenous and sequential reduction to NO. Herein, we determined whether dietary nitrate and nitrite might be preferentially reduced to NO by the hypoxia associated with PH, and thereby offer a convenient, inexpensive method of supplementing NO functionality to reduce disease severity. Methods and Results— Dietary nitrate reduced the right ventricular pressure and hypertrophy, and pulmonary vascular remodeling in wild-type mice exposed to 3 weeks of hypoxia; this beneficial activity was mirrored largely by dietary nitrite. The cytoprotective effects of dietary nitrate were associated with increased plasma and lung concentrations of nitrite and cGMP. The beneficial effects of dietary nitrate and nitrite were reduced in mice lacking endothelial NO synthase or treated with the xanthine oxidoreductase inhibitor allopurinol. Conclusions— These data demonstrate that dietary nitrate, and to a lesser extent dietary nitrite, elicit pulmonary dilatation, prevent pulmonary vascular remodeling, and reduce the right ventricular hypertrophy characteristic of PH. This favorable pharmacodynamic profile depends on endothelial NO synthase and xanthine oxidoreductase -catalyzed reduction of nitrite to NO. Exploitation of this mechanism (ie, dietary nitrate/nitrite supplementation) represents a viable, orally active therapy for PH.

Nitrate and nitrite are precursors in the formation of N-nitroso compounds. We recently found a 40% increased risk of NHL with higher dietary nitrite intake and significant increases in risk for follicular and T-cell lymphoma. It is possible that these compounds also affect NHL prognosis by enhancing cancer progression in addition to development by further impairing immune system function. To test the hypothesis that nitrate and nitrite intake affects NHL survival, we evaluated the association in study participants that have been followed post-disease diagnosis in a population-based case-control study among women in Connecticut. We did not observe a significant increasing trend of mortality for NHL overall or by subtype for nitrate or nitrite intake for deaths from NHL or death from any cause, although a borderline significant protective trend was observed for follicular lymphoma with increasing nitrate intake. We did not identify a difference in overall survival for nitrate (P = 0.39) or for nitrite (P = 0.66) or for NHL specific survival for nitrate (P = 0.96) or nitrite (P = 0.17). Thus, our null findings do not confer support for the possibility that dietary nitrate and nitrite intake impacts NHL survival by promoting immune unresponsiveness.

Biomedical interest in the nitrite anion (NO2−) was once limited primarily to its toxic effects following ingestion of contaminated food or well water. The discovery that nitric oxide (NO) is an endogenously-produced signaling molecule, and that nitrite is a relatively stable product of NO-metabolism, brought increased focus on nitrite concentrations as an index of endogenous NO production. More recently, however, it has become apparent that nitrite is more than just a byproduct of NO, but that it can also be converted back into NO by a number of different biochemical pathways (see recent review by Lundberg and Weitzberg [1]). As a result, nitrite is now widely regarded as a bioactive molecule with potentially wide-ranging physiological function and therapeutic application. A majority of circulating nitrite is derived from the oxidation of NO produced by endothelial NO synthase [2]. A significant portion of circulating nitrite is also derived from plasma nitrate (NO3−) which is concentrated ~10-fold from the plasma into the saliva where oral bacterial nitrate reductases efficiently reduce it to nitrite that is then swallowed and absorbed in the gastrointestinal tract [1]. Nitrite is metabolized in a number of different organs as well as in the blood, or excreted by the kidneys. When O2 is present, nitrite can be oxidized to produce nitrate. In contrast, under hypoxic conditions nitrite can be reduced to NO that can then produce an array of nitrogen oxide species including nitrosothiols, N-nitrosamines, and iron-nitrosyls. Nearly all of these products can be converted back into nitrite again under physiological conditions, raising the possibility of bidirectional flux between nitrite and these other nitrogen oxide species (Figure 1). The recently described reduction of nitrate (NO3−) to nitrite (NO2−) in the mouth, and nitrite to NO in the tissues constitutes a nitrate→nitrite→NO axis that provides for NO production independent of the traditional NO synthase pathways. While the half-life of NO in whole blood (~2 ms) is so short that the effects of free NO are limited to a few hundred microns from its site of production, the elimination half-lives of nitrite (15 to 30 min) and nitrate (4 to 6 h) are long enough for these species to serve as circulating reservoirs of NO bioactivity. Thus the widely different biologic half lives provide responses by NO in seconds, nitrite as a substrate for NO production changing over minutes, and nitrate as a large-capacity reservoir changing over hours. Figure 1 Circulating concentrations and half-lives of nitric oxide, nitrite, and nitrate. Nitric oxide is oxidized to nitrite, which is further oxidized to nitrate. Nitrate is reduced back into nitrite by commensal bacteria in the mouth, and nitrite is reduced ... Most of the bioactivity of nitrite requires it first be reduced to NO. This redox reaction can occur with a number of different proteins that carry transition metals such as hemoglobin, myoglobin, cytoglobin, and neuroglobin, other heme-containing proteins such as cytochrome c oxidase, and the molybdenum-containing proteins xanthine oxidase and aldehyde oxidase (see recent review by Kim-Shapiro and Gladwin [3]). Importantly, when O2 binds to the transition metals of these proteins the reduction of nitrite to NO is blocked, and thus nitrite reduction to NO occurs only under hypoxic conditions when there is need for vasodilation and increased O2 delivery. This negative feedback system maintains O2 delivery without requiring neural or endocrine control. Nitrite can also be reduced to NO under acidic conditions in which it is protonated to form nitrous acid (HNO2, pKa = 3.2) that then decomposes into NO in seconds. As a result, significant amounts of NO can be derived from nitrite in the stomach and in severely ischemic/acidotic tissues, with the possibility of clinical relevance in myocardial ischemia, for example. The NO-bioactivity of nitrite opens the door to a wide array of potential clinical applications. In the lung, nitrite is being considered for the treatment of pulmonary hypertension. Inhaled nitrite aerosol is effective at reducing pulmonary hypertension caused by hypoxia or hemolysis in newborn lambs and adult humans [4–6]. Although the original mechanism proposed for this effect was the reduction of nitrite to NO by deoxyhemoglobin flowing into the lung [4], more recent studies indicate that nitrite may be converted to NO within the airway itself to effect the vasodilatory response [5]. In the current issue of this journal, Rix et al. present the first human data for safety and pharmacokinetics of inhaled nitrite aerosol [7]. In healthy volunteers, they find inhaled nitrite to be well tolerated within the expected therapeutic dose range. Systemic hypotension was the dose-limiting adverse effect. These results are consistent with the NO-bioactivity of nitrite, in accord with earlier animal studies, and provide a rational basis for a trial of aerosolized nitrite in treatment of pulmonary hypertension. Given the hypoxia-sensitive nature of NO production from nitrite, it is surprising that systemic hypoxia did not potentiate the hypotensive effects of nitrite, but this may have been due to a counteracting sympathetic response to the hypoxic stimulus. However, it is unknown in what form nitrite crosses the bronchial/alveolar membranes. Other questions that remain include whether the transport is carrier-mediated or pH dependent, what level of the vascular tree smooth muscle cells are relaxed, and whether this reduced smooth muscle tone is distributed throughout the lungs such that blood flow is more evenly matched with ventilation. In addition to pulmonary hypertension, other potential uses of nitrite are being explored. Because the conversion of nitrite to NO is accelerated when O2 levels are low, much research has focused on nitrite’s role in responses to ischemia/reperfusion stress. Modest increases in circulating nitrite concentrations prior to an ischemic insult confer marked protection against injury in many organ systems (Lundberg & Weitzberg review [1]). The mechanism(s) for these protective effects appears to involve reduction of nitrite to NO since the addition of NO scavengers is found to eliminate protection. Putative pathways for protection include the suppression of O2 consumption and lessened superoxide production during the ischemic insult due to NO competing with O2 for binding to the mitochondrial complex IV. In addition, the reduction of nitrite may result in S-nitrosation of a number of enzymes, ion channels, and transcription factors resulting in functional changes that confer protection against ischemia and/or reperfusion injury [8]. Although the exact mechanism(s) of protection remain to determined, supplemental nitrite is currently being studied in a number of indications including myocardial infarction, subarachnoid hemorrhage, cardiac arrest, organ transplant, and coronary bypass surgery. Research over the past two decades has alerted us to the important contribution of nitrite to NO signaling. However, although the beneficial effects of dietary nitrite on exercise performance are already widely accepted [1] and the pharmaceutical use of nitrite is under investigation for a number of different indications, there is still much to learn regarding how such broad effect is achieved by this anion we once thought to be physiologically irrelevant.

Until recently, nitrite has been considered a stable oxidation inert metabolite of nitric oxide ((∙)NO) metabolism. This view is now changing as it has been shown that nitrite can be reduced back to (∙)NO and thus one may consider a reversible interaction regarding (∙)NO:nitrite couple. Not only physiological regulatory actions have been assigned to nitrite but also may represent, in addition to nitrate, the largest (∙)NO reservoir in the body. This notion has obvious importance when considering that (∙)NO is a ubiquitous regulator of cell functions, ranging from neuromodulation to the regulation of vascular tone. Particularly in the stomach, following ingestion of nitrate and food or beverages-containing polyphenols, a rich chemistry occurs in which (∙)NO, (∙)NO-derived species and nitroso or nitrated derivatives may be formed. Most of these molecules may play an important role in vivo. For instance, it has been shown that polyphenol-catalyzed nitrite reduction to (∙)NO may induce local vasodilation and that ethanol (from wine) reacts with (∙)NO-derived species yielding nitroso derivatives endowed with (∙)NO-donating properties. Thus, this review reveals new pathways for the biological effects of dietary nitrite encompassing its interaction with dietary components (polyphenols, red wine, lipids), yielding products with impact on human physiology and pathology, namely cardiovascular, urinary and gastrointestinal systems. Novel therapeutic strategies are therefore expected to follow the elucidation of the mechanisms of nitrite biology.

Dietary nitrite and nitrate are important sources of nitric oxide (NO). However, the use of nitrite as an antihypertensive drug may be limited by increased oxidative stress associated with hypertension. We evaluated the antihypertensive effects of sodium nitrite given in drinking water for 4weeks in two-kidney one-clip (2K1C) hypertensive rats and the effects induced by nitrite on NO bioavailability and oxidative stress. We found that, even under the increased oxidative stress conditions present in 2K1C hypertension, nitrite reduced systolic blood pressure in a dose-dependent manner. Whereas treatment with nitrite did not significantly change plasma nitrite concentrations in 2K1C rats, it increased plasma nitrate levels significantly. Surprisingly, nitrite treatment exerted antioxidant effects in both hypertensive and sham-normotensive control rats. A series of in vitro experiments was carried out to show that the antioxidant effects induced by nitrite do not involve direct antioxidant effects or xanthine oxidase activity inhibition. Conversely, nitrite decreased vascular NADPH oxidase activity. Taken together, our results show for the first time that nitrite has antihypertensive effects in 2K1C hypertensive rats, which may be due to its antioxidant properties resulting from vascular NADPH oxidase activity inhibition.

It has been estimated that 65,980 individuals were diagnosed with non-Hodgkin lymphoma (NHL) and 19,500 died from NHL in the United States in 2009. Although established risk factors such as immunodeficiency and viral infections may be responsible for a portion of the cases, the majority of NHL cases remain unexplained. Dietary nitrate and nitrite intake are exposures of particular interest for NHL risk as they are precursors in the endogenous formation of N-nitroso compounds, which cause lymphomas in animal studies. We investigated NHL risk overall and by histologic type in relation to dietary nitrate and nitrite intake in a population-based case-control study of 1,304 women in Connecticut. Nitrate and nitrite intake were assessed using a 120-item food frequency questionnaire. We found no association between risk of NHL overall and dietary nitrate and a slightly increased risk of NHL with higher dietary nitrite intake (highest vs. lowest intake quartile OR = 1.4; 95% CI: 0.9-2.2). When we evaluated intake by subtype, a significant positive trend was observed for follicular lymphoma and nitrate (p-trend = 0.04) and nitrite (p-trend < 0.01) with an over twofold risk in the highest nitrite intake quartile (OR = 2.3; 95% CI: 1.1-4.9). An increased risk in the highest quartile of nitrite intake was also observed for T-cell lymphoma (OR = 3.4; 95% CI: 1.0-11.9). Animal products containing nitrite were more strongly associated with risk of follicular lymphoma; whereas, both animal and plant sources of nitrite were associated with elevated ORs for T-cell lymphoma. Our results confirm a previous finding for nitrite intake and NHL risk and highlight the importance of evaluating histologic type. We conclude that these results should be replicated in a larger study with data on drinking water as well as dietary sources of nitrate intake.

The discovery of the nitric oxide (NO) pathway in the 1980s represented a critical advance in understanding cardiovascular disease, and today a number of human diseases are characterized by NO insufficiency. In the interim, recent biomedical research has demonstrated that NO can be modulated by the diet independent of its enzymatic synthesis from l-arginine, e.g., the consumption of nitrite- and nitrate-rich foods such as fruits, leafy vegetables, and cured meats along with antioxidants. Regular intake of nitrate-containing food such as green leafy vegetables may ensure that blood and tissue levels of nitrite and NO pools are maintained at a level sufficient to compensate for any disturbances in endogenous NO synthesis. However, some in the public perceive that dietary sources of nitrite and nitrate are harmful, and some epidemiological studies reveal a weak association between foods that contain nitrite and nitrate, namely cured and processed meats, and cancer. This paradigm needs revisiting in the face of undisputed health benefits of nitrite- and nitrate-enriched diets. This review will address and interpret the epidemiological data and discuss the risk-benefit evaluation of dietary nitrite and nitrate in the context of nitric oxide biology. The weak and inconclusive data on the cancer risk of nitrite, nitrate and processed meats are far outweighed by the health benefits of restoring NO homeostasis via dietary nitrite and nitrate. This risk/benefit balance should be a strong consideration before there are any suggestions for new regulatory or public health guidelines for dietary nitrite and nitrate exposures.

High dietary nitrate and nitrite intake may increase the risk of gastro-intestinal cancers due to the in vivo formation of carcinogenic chemicals known as N -nitroso compounds. Water and leafy vegetables are natural sources of dietary nitrate, whereas cured meats are the major sources of dietary nitrite. This paper describes a simple and fast analytical method for determining nitrate and nitrite contents in vegetables and meat, using reversed-phase HPLC-UV. The linearity R 2 value was >0.998 for the anions. The limits of quantification for nitrite and nitrate were 5.0 and 2.5 mg/kg, respectively. This method is applicable for both leafy vegetable and meat samples. A range of vegetables was tested, which contained

The presence of nitrates and nitrites in food is associated with an increased risk of gastrointestinal cancer and, in infants, methemoglobinemia. Despite the physiologic roles for nitrate and nitrite in vascular and immune function, consideration of food sources of nitrates and nitrites as healthful dietary components has received little attention. Approximately 80% of dietary nitrates are derived from vegetable consumption; sources of nitrites include vegetables, fruit, and processed meats. Nitrites are produced endogenously through the oxidation of nitric oxide and through a reduction of nitrate by commensal bacteria in the mouth and gastrointestinal tract. As such, the dietary provision of nitrates and nitrites from vegetables and fruit may contribute to the blood pressure–lowering effects of the Dietary Approaches to Stop Hypertension (DASH) diet. We quantified nitrate and nitrite concentrations by HPLC in a convenience sample of foods. Incorporating these values into 2 hypothetical dietary patterns that emphasize high-nitrate or low-nitrate vegetable and fruit choices based onthe DASH diet,we foundthat nitrate concentrations in these 2 patterns vary from 174 to 1222 mg. The hypothetical high-nitrate DASH diet pattern exceeds the World Health Organization’s Acceptable Daily Intake for nitrate by 550% for a 60-kg adult. These data call into question the rationale for recommendations to limit nitrate and nitrite consumption from plant foods; a comprehensive reevaluation of the health effects of food sources of nitrates and nitrites is appropriate. The strength of the evidence linking the consumption of nitrate- and nitrite-containing plant foods to beneficial health effects supports the consideration of these compounds as nutrients. Am J Clin Nutr 2009;90:1–10.

Enteric neuropathy is emerging as a central theme in a range of digestive tract pathologies. Aberrant nitric ox- ide (NO) signaling by cellular components of the enteric nervous system or by inappropriate processing of dietary nitrite represents potential disease factors in delayed gastric emptying, diabetic gastroparesis, hypertrophic pyloric stenosis, and in inflammatory barrier dysfunction of the gut. Exploiting the neuronal nitric oxide synthase (nNOS) system to generate NO as a form of treatment is considered an option in these clinical disorders. Here we review the transcriptional regula- tion of nNOS activity and its pathophysiological role in the digestive tract. We also highlight potentially novel therapeutic strategies that exploit nNOS-derived S-nitrosothiols (SNO) and advanced glycation end products (AGEs).

To evaluate the associations between diet and non-Hodgkin lymphoma (NHL) according to t(14;18) status, one of the most common chromosomal abnormalities in NHL, as t(14;18)-positive NHL represents a genetically more homogeneous group than NHL overall. We determined the presence of the t(14;18)(q32;q21) by fluorescence in situ hybridization in 172 of 175 tumor blocks from a population-based, case–control study conducted in Nebraska during 1983–1986. Information on the frequency of consumption as an adult of 30 food items was derived from the parent case–control study. Dietary factors in 60 t(14;18)-positive and 87 t(14;18)-negative cases were compared with 1,075 controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using polytomous logistic regression. The risk of t(14;18)-positive NHL for the highest versus the lowest approximate tertile of intake was elevated for milk (OR = 2.2; 1.0–5.0) and dietary nitrite (OR = 2.8; 1.3–6.1), whereas coffee consumption was inversely associated with risk (OR = 0.4; 0.2–0.7). We also found inverse associations between the intake of fish (OR = 0.5; 0.3–1.0) and carotene (OR = 0.5; 0.2–0.9) and risk of t(14;18)-negative NHL. There was no association between the intake of meats, vegetables, protein, or vitamin C and risk of either t(14;18)-positive or t(14;18)-negative NHL. We observed differences in associations between diet and t(14;18)-defined subgroups of NHL. These findings should be interpreted cautiously because of the small sample.

Prenatal exposure to nitrosatable drugs, including secondary or tertiary amines, has been associated with preterm birth. Associations may be accentuated by higher intakes of dietary nitrites because of the increased formation of N-nitroso compounds. Using data from mothers of babies without major birth defects (controls) from the National Birth Defects Prevention Study, we examined the relationship between nitrosatable drug exposure in conjunction with dietary nitrite intake and preterm birth among 496 mothers of preterm infants and 5,398 mothers with full-term deliveries in 1997-2005. A protective association was observed with a high intake of plant nitrites (adjusted hazard ratio (AHR) = 0.72, 95% confidence interval (CI): 0.53, 0.97). Secondary amines in conjunction with high nitrite intake were associated with preterm birth during the first (AHR = 1.84, 95% CI: 1.14, 2.98), second (AHR = 1.89, 95% CI: 1.17, 3.07), and third (AHR = 2.00, 95% CI: 1.22, 3.29) trimesters. The adjusted hazard ratios for tertiary amine use in the third trimester by increasing tertiles of nitrite intake were 0.67 (95% CI: 0.35, 1.31), 1.25 (95% CI: 0.71, 2.19), and 2.02 (95% CI: 1.17, 3.49). Prenatal exposure to nitrosatable drugs, particularly secondary and tertiary amines, in conjunction with higher levels of dietary nitrite intake may increase the risk of preterm birth.

Objective: Dietary nitrite has been associated with increased glioma risk; however, drinking water nitrate has not been extensively evaluated. Methods: We conducted a population-based case-control study of adult glioma in Nebraska. Water utility nitrate measurements were linked to residential water source histories. We computed average nitrate exposure over a 20-year period. A food frequency questionnaire was used to assess dietary nitrate and nitrite. Results: Increasing quartiles of the average nitrate level in drinking water were not significantly associated with risk (adjusted odd ratios: 1.4, 1.2, 1.3). Risk was similar among those with both higher and lower intakes of vitamin C, an inhibitor of N-nitroso compound formation. Dietary nitrite intake was not associated with risk. Conclusions: Our study does not support a role for drinking water and dietary sources of nitrate and nitrite in risk of adult glioma.

NO generated from l -arginine by NO synthases (NOSs) in the endothelium and in other cells plays a central role in several aspects of vascular biology. The biological activity of NO is acutely terminated by oxidation to nitrite and nitrate, and these compounds have long been considered only as inert end-products of NO. However, this dogma is now being challenged because recent research convincingly has shown that the nitrite ion can be recycled back to bioactive NO again in blood and tissues. Nitrite reduction to NO can occur via several routes involving enzymes, proteins, vitamins, or even simple protons. This pathway may serve as a backup system for NO generation in conditions such as hypoxia, in which the NOS/ l -arginine system is compromised, but detrimental effects can also be foreseen. With this new knowledge, nitrate and nitrite should probably be viewed as storage pools for NO rather than inert waste products. Here we discuss novel aspects of nitrite-dependent NO generation in vivo and its role in vascular control.

Nitrite represents a circulating and tissue storage form of NO whose bioactivation is mediated by the enzymatic action of xanthine oxidoreductase, nonenzymatic disproportionation, and reduction by deoxyhemoglobin, myoglobin, and tissue heme proteins. Because the rate of NO generation from nitrite is linearly dependent on reductions in oxygen and pH levels, we hypothesized that nitrite would be reduced to NO in ischemic tissue and exert NO-dependent protective effects. Solutions of sodium nitrite were administered in the setting of hepatic and cardiac ischemia-reperfusion (I/R) injury in mice. In hepatic I/R, nitrite exerted profound dose-dependent protective effects on cellular necrosis and apoptosis, with highly significant protective effects observed at near-physiological nitrite concentrations. In myocardial I/R injury, nitrite reduced cardiac infarct size by 67%. Consistent with hypoxia-dependent nitrite bioactivation, nitrite was reduced to NO, S-nitrosothiols, N-nitros-amines, and iron-nitrosylated heme proteins within 1–30 minutes of reperfusion. Nitrite-mediated protection of both the liver and the heart was dependent on NO generation and independent of eNOS and heme oxygenase-1 enzyme activities. These results suggest that nitrite is a biological storage reserve of NO subserving a critical function in tissue protection from ischemic injury. These studies reveal an unexpected and novel therapy for diseases such as myocardial infarction, organ preservation and transplantation, and shock states.

Nitric oxide (NO • ) is thought to protect against the damaging effects of myocardial ischemia–reperfusion injury, whereas xanthine oxidoreductase (XOR) normally causes damage through the generation of reactive oxygen species. In the heart, inorganic nitrite \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}({\mathrm{NO}}_{2}^{-})\end{equation*}\end{document} has the potential to act as an endogenous store of NO • , liberated specifically during ischemia. Using a detection method that we developed, we report that under ischemic conditions both rat and human homogenized myocardium and the isolated perfused rat heart (Langendorff preparation) generate NO • from \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{NO}}_{2}^{-}\end{equation*}\end{document} in a reaction that depends on XOR activity. Functional studies of rat hearts in the Langendorff apparatus showed that nitrite (10 and 100 μM) reduced infarct size from 47.3 ± 2.8% (mean percent of control ± SEM) to 17.9 ± 4.2% and 17.4 ± 1.0%, respectively ( P < 0.001), and was associated with comparable improvements in recovery of left ventricular function. This protective effect was completely blocked by the NO • scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO). In summary, the generation of NO • from \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{NO}}_{2}^{-}\end{equation*}\end{document} , by XOR, protects the myocardium from ischemia–reperfusion injury. Hence, if XOR is presented with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{NO}}_{2}^{-}\end{equation*}\end{document} as an alternative substrate, the resultant effects of its activity may be protective, by means of its production of NO • , rather than damaging.