Salivary Nitrate Research Articles

Dietary nitrate maintains intestinal epithelia homeostasis in aged mice.

The intestinal tract, which is the primary site of digestion and absorption of nutrients, is one of the most vulnerable organs during aging. Dietary nitrate, which is mainly derived from the diet and absorbed in the intestinal tract, is a key messenger that connecting oral and general health. However, whether dietary nitrate regulates intestinal tract homeostasis remains unclear. Our data revealed that the serum and salivary nitrate levels decreased during mice aging. The functional proteins of the epithelial barrier (E-cadherin, Claudin-1 and Zonula Occludens-1) in the colon tissues decreased during the aging process. Long-term nitrate supplement in drinking water restored the serum and salivary nitrate levels and increased the functional proteins expression of the colon epithelial barrier. Dietary nitrates increase the relative abundance of some intestinal probiotics, particularly those associated with the production of short-chain fatty acids, such as Blautia, Alloprevotella, Butyricicoccus, and Ruminococcaceae, while promoting the butyric acid production in the colon. Moreover, the expression of Sialin (encoded by Slc17a5), which is a nitrate transporter, increased in the colon epithelial cells by nitrate supplementation. The epithelial cell-conditional Slc17a5-knockout mutant mice (K14-cre; Slc17a5fl/fl) revealed that the functional proteins expression of the colon epithelial barrier and the proliferation of PCNA-positive intestinal epithelial cells in the colon crypts was significantly decreased compared with those of the K14-cre; Slc17a5fl/+ mice. Taken together, our findings suggested that nitrate supplementations were associated with the increased expression of colonic epithelial barriers-related proteins and the increased Sialin expression. Nitrate may serve as a potential therapeutic approach in maintaining aged colonic homeostasis.

The Probiotic Streptococcus salivarius M18 Increases Plasma Nitrite but Does Not Alter Blood Pressure: A Pilot Randomised Controlled Trial

Some species of oral bacteria can reduce dietary nitrate to nitrite, which can later be converted to nitric oxide in the nitrate—nitrite—nitic oxide pathway. Increasing nitric oxide availability can reduce blood pressure (BP) and improve exercise performance. Streptococcus salivarius M18 (Streptococcus salivarius M18) is a bacteriocin-producing probiotic that is known to improve oral health by inhibiting pathogenic oral bacteria. However, it is presently unclear whether probiotic-induced alterations to the oral microbiome will influence circulating levels of nitric oxide metabolites and BP. Purpose: To determine the effects of Streptococcus salivarius M18 supplementation on plasma and salivary nitrate and nitrite levels and BP. Methods: Ten healthy males (age 32 ± 8 y, body mass 88.2 ± 15.1 kg) completed 2 × 14-day supplementation phases in a randomized order at least 14 days apart. In one phase, participants consumed Streptococcus salivarius M18 probiotic lozenges (2.5 billion colony-forming units/dose) once per day, and in the other, they ingested water (placebo). The abundance of bacteria on the tongue was assessed via Illumina 16S rRNA gene sequencing, unstimulated saliva, and venous blood samples were collected, and BP was measured pre and post each phase. Saliva and plasma were analysed for nitrate and nitrite using chemiluminescence, and pH was measured in saliva. The change in each outcome from pre- to post-supplementation was compared between phases using repeated measures ANOVA. Results: Plasma nitrite increased from baseline following probiotic supplementation (from 173 ± 39 to 223 ± 63 nM, p = 0.003, 95% CI 192–250 nM). In comparison, there was no change in the placebo phase or between baselines (all p > 0.05). The abundance of nitrite-producing bacteria was not altered, salivary nitric oxide metabolites and pH did not change, and the increase in plasma nitrite did not result in reductions in BP (all p > 0.05). Conclusions: Supplementation with Streptococcus salivarius M18 increased plasma nitrite, a key marker of NO availability. Despite this, Streptococcus salivarius M18 did not lower BP in these healthy normotensive participants. Additionally, the increase in plasma nitrite was not associated with abundance changes in bacteria thought important to NO generation. Further research is required to determine the mechanism behind the increase in plasma nitrite and the potential therapeutic and ergogenic benefits of Streptococcus salivarius M18 supplementation.

Caloric Restriction (CR) Plus High-Nitrate Beetroot Juice Does Not Amplify CR-Induced Metabolic Adaptation and Improves Vascular and Cognitive Functions in Overweight Adults: A 14-Day Pilot Randomised Trial

Caloric restriction (CR) and dietary nitrate supplementation are nutritional interventions with pleiotropic physiological functions. This pilot study investigates the combined effects of CR and nitrate-rich beetroot juice (BRJ) on metabolic, vascular, and cognitive functions in overweight and obese middle-aged and older adults. This was a two-arm, parallel randomized clinical trial including 29 participants allocated to CR + BRJ (n = 15) or CR alone (n = 14) for 14 days. Body composition, resting energy expenditure (REE), and hand-grip strength were measured. Resting blood pressure (BP) and microvascular endothelial function were measured, and Trail-Making Test A and B were used to assess cognitive function. Salivary nitrate and nitrite, and urinary nitrate and 8-isoprostane concentrations were measured. Changes in body composition, REE, and systolic and diastolic BP were similar between the two interventions (p > 0.05). The CR + BRJ intervention produced greater changes in average microvascular flux (p = 0.03), NO-dependent endothelial activity (p = 0.02), and TMT-B cognitive scores (p = 0.012) compared to CR alone. Changes in urinary 8-isoprostane were greater in the CR + BRJ group (p = 0.02), and they were inversely associated with changes in average microvascular flux (r = -0.53, p = 0.003). These preliminary findings suggest that greater effects on vascular and cognitive functions could be achieved by combining CR with dietary nitrate supplementation.

Estimation of salivary nitrate and nitrite levels in different types of tobacco users - A comparative study

Estimation of salivary nitrate and nitrite levels in different types of tobacco users - A comparative study

Dietary nitrate supplementation for preventing and reducing the severity of winter infections, including COVID-19, in care homes (BEET-Winter): a randomised placebo-controlled feasibility trial

Key summary pointsAimTo assess the feasibility of supplementing dietary nitrate (NO substrate) intake in care home residents.FindingsExpressions of interest by care homes were not realised during the alpha COVID-19 wave of infections. Background dietary nitrate was < 30% of acceptable daily intake; most residents received a majority of their nitrate supplementation and supplementation increased urinary and salivary nitrate concentrations.Message Recruiting UK care homes during the COVID-19 pandemic was partially successful and supplemented dietary nitrate was tolerated and elevated urinary nitrate concentrations.

Double-blind controlled dietary cross-over intervention with differentially fertilised intact lettuce leaves shows acute reduction in blood pressure in young adults, associated with faster uptake of nitrate than of phenolics

PurposeTo compare acute effects on blood pressure (BP) of ingestion of visually similar lettuce with controlled high and low content of either nitrate or phenolic compounds.MethodsIn a randomised cross-over design, 19 healthy participants (22–31 years) received 50 g of lettuce containing either 530 mg (8.4 mmol) nitrate + 11 mg (0.03 mmol) phenolic compounds (HNLP); or 3 mg nitrate (0.05 mmol) + 77 mg (0.2 mmol) phenolic compounds (LNHP), obtained by differential fertilisation. Ambulatory BP was recorded along with plasma, salivary and urinary nitrate and nitrite and plasma concentrations of cyclic guanosine monophosphate (cGMP), phenolic metabolites, Trolox equivalent antioxidant capacity (TEAC) and ferric reducing antioxidant power (FRAP).ResultsCompared with LNHP, 3 h post ingestion of HNLP, plasma nitrate increased 0.31 ± (95%CI) 0.12 mM (+ 240%), and salivary nitrate 5.5 ± 1.4 mM (+ 910%); accumulated urinary nitrate excretion increased 188 ± 72 mg (+ 296%) (all P < 0.001). Systolic BP was reduced 4.9 ± 4.2 mmHg (P = 0.031) between 3 and 6 h after ingestion of HNLP compared with LNHP; systolic BP differences were negatively correlated (P = 0.004) with differences in saliva nitrate concentrations. LNHP increased plasma phenolics at 6 h, predominantly 3ʹ-methoxycinnamic acid-4ʹ-glucuronide (ferulic acid-4ʹ-glucuronide), 116%, 204 ± 138 nM more than HNLP (P = 0.001); increased cGMP 14% (P = 0.019); and reduced FRAP 3.1% (P = 0.009).ConclusionThe acute BP difference within 6 h of consumption matched the plasma/saliva nitrate peak, not the slower changes of plasma phenolics. This is the first double-blind controlled dietary intervention demonstrating differential effects on human physiology by consumption of an intact plant food, where compositional differences were obtained by controlling growing conditions, indicating potential opportunities for health claims relating to precision/vertical farming.Clinical trial registrationThe trial was retrospectively registered on ClinicalTrials.gov, with identifier NCT02701959, on March 8, 2016.

Reduced nitric oxide synthesis in winter: A potential contributing factor to increased cardiovascular risk

BackgroundNitric oxide is a key signalling molecule that elicits a range of biological functions to maintain vascular homeostasis. A reduced availability of nitric oxide is implicated in the progression of cardiovascular diseases and increases the risk of pathogenic events. AimsTo compare the concentration of nitric oxide metabolites in healthy adults between winter and summer months. DesignAn observational study of healthy adults (age 32 ± 9 years) living in central Scotland. MethodsThirty-four healthy adults (13 females) were monitored for 7 days in summer and winter to record sunlight exposure (ultraviolet-A (UV-A) radiation), diet, and physical activity. At the end of each phase, blood pressure was measured, and samples of blood and saliva collected. The samples were analysed to determine the concentrations of plasma and salivary nitrate and nitrite and serum 25-hydroxyvitamin D (25(OH)D). ResultsThe participants maintained similar diets in each measurement phase but were exposed to more UV-A radiation (550%) and undertook more moderate-vigorous physical activity (23%) in the summer than in winter. Plasma nitrite (46%) and serum 25(OH)D (59%) were higher and blood pressure was lower in the summer compared to winter months. Plasma nitrite concentration was negatively associated with systolic, diastolic, and mean arterial blood pressure. ConclusionsPlasma nitrite, an established marker of nitric oxide synthesis, is higher in healthy adults during the summer than in winter. This may be mediated by a greater exposure to UV-A which stimulates the release of nitric oxide metabolites from skin stores. While it is possible that seasonal variation in nitric oxide availability may contribute to an increased blood pressure in the winter months, the overall impact on cardiovascular health remains to be determined.

Pharmacokinetic Profile of Incremental Oral Doses of Dietary Nitrate in Young and Older Adults: A Crossover Randomized Clinical Trial

BackgroundDietary nitrate consumption can increase concentrations of nitrate and nitrite in blood, saliva, and urine. Whether the change in concentrations is influenced by age is currently unknown. ObjectivesWe aimed to measure changes in nitrate and nitrite concentrations in plasma, urine, and saliva and exhaled NO concentrations after single incremental doses of dietary nitrate in young and older healthy adults. MethodsTwelve young (18–35 y old) and 12 older (60–75 y old) healthy, nonsmoking participants consumed single doses of 100 g, 200 g, 300 g whole beetroot (BR) and 1000 mg potassium nitrate (positive control) ≥7 d apart in a crossover, randomized clinical trial. Plasma nitrate and nitrite concentrations and exhaled NO concentrations were measured over a 5-h period. Salivary nitrate and nitrite concentrations were measured over a 12-h period and urinary nitrate over a 24-h period. Time, intervention, age, and interaction effects were measured with repeated-measures ANOVAs. ResultsDose-dependent increases were seen in plasma, salivary, and urinary nitrate after BR ingestion (all P ≤ 0.002) but there were no differences between age groups at baseline (all P ≥ 0.56) or postintervention (all P ≥ 0.12). Plasma nitrite concentrations were higher in young than older participants at baseline (P = 0.04) and after consumption of 200 g (P = 0.04; +25.7 nmol/L; 95% CI: 0.97, 50.3 nmol/L) and 300 g BR (P = 0.02; +50.3 nmol/L; 95% CI: 8.57, 92.1 nmol/L). Baseline fractional exhaled NO (FeNO) concentrations were higher in the younger group [P = 0.03; +8.60 parts per billion (ppb); 95% CI: 0.80, 16.3 ppb], and rose significantly over the 5-h period, peaking 5 h after KNO3 consumption (39.4 ± 4.5 ppb; P < 0.001); however, changes in FeNO were not influenced by age (P = 0.276). ConclusionsBR is a source of bioavailable dietary nitrate in both young and older adults and can effectively raise nitrite and nitrate concentrations. Lower plasma nitrite and FeNO concentrations were found in older subjects, confirming the impact of ageing on NO bioavailability across different systems.This trial was registered at www.isrctn.com as ISRCTN86706442.

Inorganic nitrate attenuates endothelial dysfunction consequent to systemic inflammation

Abstract Introduction Chronic cardiovascular diseases are characterised by low-grade systemic inflammation and attenuated nitric oxide (NO) bioavailability resulting in endothelial dysfunction. Inorganic nitrate augments NO bioavailability and improves markers of vascular dysfunction in patients with cardiovascular risk factors. However, the exact mechanism of this effect is uncertain. Purpose To determine whether inorganic nitrate supplementation alters systemic inflammation-induced endothelial dysfunction. Methods 62 healthy male volunteers were randomised 1:1 to receive ∼8–10 mmol of dietary inorganic nitrate in beetroot juice or nitrate-free beetroot juice (placebo) once daily for 6 days. Measures of brachial artery flow-mediated dilatation (FMD), brachial blood pressure (BP), pulse wave analysis and carotid-femoral pulse wave velocity (PWV) by Vicorder were taken prior to and at 8 hours after a typhoid vaccine (to induce mild systemic inflammation). Plasma, urine and saliva samples were also collected. Clinicaltrials.gov: NCT02715635. Results Baseline characteristics were similar between the two groups. Inorganic nitrate significantly elevated plasma nitrite (placebo = Δ0.02±0.5 μM, inorganic nitrate = Δ0.63±1.2 μM; p=0.01) and nitrate levels (p&amp;lt;0.0001) compared to placebo. There were significant increases in urine nitrite (p&amp;lt;0.0001) and nitrate (p&amp;lt;0.0001) in addition to salivary nitrite (p&amp;lt;0.0001) and nitrate (p&amp;lt;0.0001) compared to placebo. After 8 hours, typhoid vaccine induced an increase in circulating white cells (placebo = Δ3.34±3.37x109/L, inorganic nitrate = Δ2.9±2.78x109/L; p=0.58) that was similar in in both arms. However, there was a significant reduction in the FMD response in the placebo group at 8-hours post vaccine; an effect that was absent in volunteers treated with inorganic nitrate (placebo = Δ−1.33±1.53%, inorganic nitrate = Δ−0.07±1.84%, p=0.005). Importantly, there were no statistically significant differences in baseline vessel diameter (p=0.78), time to peak diameter in response to flow (p=0.87) and peak shear rate (p=0.57) between the groups. When comparing change from baseline to 8 hours after the vaccine, there were no significant differences in brachial systolic BP (p=0.12), central systolic BP (p=0.12) and PWV (p=0.60) between groups, but a significant reduction in brachial diastolic BP in the inorganic nitrate group (p=0.048). Conclusions Inflammation-induced endothelial dysfunction was prevented in those receiving dietary inorganic nitrate suggesting that elevating circulating nitrite and delivering NO to the blood vessel wall, through dietary approaches may offer potential therapeutic benefit in those cardiovascular diseases which typically exhibit low grade inflammation and deficiencies in bioavailable NO. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): British Heart Foundation

Nitrite in paraffin-stimulated saliva correlates with blood nitrite

Nitrite anion (NO2−) is a circulating nitric oxide (NO) metabolite considered an endothelial function marker. Nitrite can be produced from nitrate (NO3−) secreted from plasma into saliva. The nitrate reductase of oral bacteria converts salivary nitrate to nitrite, which is swallowed and absorbed into circulation. In this study, we aimed to examine the relevance between these species' salivary and blood levels. We collected three whole saliva samples (unstimulated, paraffin-stimulated, and post-chlorhexidine mouthwash stimulated saliva) and blood from 75 healthy volunteers. We measured the nitrite and nitrate by the chemiluminescence method. The nitrite levels in stimulated saliva and post-mouthwash stimulated saliva exhibited weak correlations with blood nitrite. There was no correlation between nitrite in unstimulated saliva with blood nitrite. The baseline platelet activity, determined as P-selectin expression, negatively correlated with nitrite in plasma and post-mouthwash stimulated saliva. The salivary nitrate in all saliva samples showed correlations with its plasma levels. We conclude that nitrite in stimulated saliva correlates with blood nitrite.

Point-of-Care Detection of Salivary Nitrite Based on the Surface Plasmon-Assisted Catalytic Coupling Reaction of Aromatic Amines.

Rapid quantification of nitrite (NO2−) in food, drink and body fluids is of significant importance for both food safety and point-of-care (POA) applications. However, conventional nitrite analytical methods are complicated, constrained to sample content, and time-consuming. Inspired by a nitrite-triggered surface plasmon-assisted catalysis (SPAC) reaction, a rapid point-of-care detection salivary nitrate was developed in this work. NO2− ions can trigger the rapid conversion of p-aminothiophenol (PATP) to p,p′-dimercaptozaobenzene (DMAB) on gold nanoparticles (GNPs) under light illumination, and the emerged new bands at ca. 1140, 1390, 1432 cm−1 originating from DMAB can be used to the quantification of nitrite. Meanwhile, to make the method entirely suitable for on-site fast screen or point-of-care application, the technique is needed to be further optimized. The calibration graph for nitrates was linear in the range of 1–100 µM with a correlation coefficient of 0.9579. The limit of detection was 1 µM. The facile method could lead to a further understanding of the progression and treatment of periodontitis and to guide professionals in planning on-site campaigns to effectively control periodontal diseases.

THE VASCULAR ENDOTHELIUM FUNCTION INDICATORS IN ORAL LIQUID OF IQOS SMOKING ADOLESCENTS

Heated tobacco products (HTP) were introduced to the world market as an alternative to conventional tobacco cigarettes. Active advertising, readily available to purchase and the public's perception that HTP are a safer alternative to conventional tobacco cigarettes lead to increasing their consumption by young age group in Ukraine. Despite the claims by the tobacco corporations about reduction of adverse effects the HTP on the activity of cardiovascular system, the published studies of potential vascular endothelial consequences were not found. Therefore, the aim of our study is to assess the HTP effect on vascular endothelial cells by determination of the nitrate, nitrite, S-nitrosothiols, and endothelin-1 levels in saliva of smoking adolescents. The object and research methods. Thirty adolescents aged 14-17 years old were divided to three groups. Group I was introduced by ten healthy non-smoking adolescents (control group). Group II consists of ten smoking adolescents have smoked the IQOS for two years. Group III is formed by ten smoking adolescents who have smoked the IQOS for three years. The salivary nitrate and nitrite levels were measured using the Griess assay such as an indicator of NO production. The salivary S-nitrosothiols were measured spectrophotometrically. The salivary endothelin-1 level was measured with ELISA kit. Statistical analysis of the data was carried out using the STATISTICA 7.0 program. The data were presented as Median values (Me), lower and upper quartile, i.e. Me [Lq; Uq]. Statistical significance was accepted if p &lt; 0.05. Results. The salivary nitrate concentrations in groups II (Me = 14.11 [13.98; 14.52] μmol/l) and III (Me = 12.06 [11.44; 12.31] μmol/l) were significantly decreased compared to group I (Me = 15.89 [14.98; 16.89] μmol/l, KW test: H = 23.59, p=0.000. The values of nitrite in groups II and III were the following: Me = 2.32 [1.47; 2.48] μmol/l and 1.37 [1.25; 1.44] μmol/l, respectively. The indices were lower in 1.41 and 2.36 times than in group I: Me = 3. 24 [3.05; 3.78] μmol/l, KW test: Н = 22.92, p = 0.000. The levels of salivary S-nitrosothiols in group II are: Me = 1.02 [0.84; 1.27] μmol/l and group III Me = 0.36 [0.25; 0.62] μmol/l were reduced in 1.75 and 4.97 times compared to group I: Me = 1.79 [1.67; 1.97] μmol/l, KW test: Н = 24.46, р = 0.000. We assume that the reduction of salivary nitrate and nitrite concentrations is associated with a low level of S-nitrosothiols which serve as deposited and released nitric oxide (NO). Probably, the decrease of nitrates and nitrites is associated with the inhibition of endothelial NO synthase. The salivary endothelin-1 level was significantly higher in group II: Ме=0.58 [0.53; 0.73] pmol/ml and in group III: Ме = 0.82 [0.79; 0.93] pmol/ml than in group I: Ме = 0.17 [0.14; 0.26] pmol/ml, KW test: Н = 23.94, р = 0.000. It should be noted, the significant differences between data of nitrate, nitrite, S-nitrosothiol and endothelin-1 content in groups II and III were found (Mann-Whitney U test, nitrates - Z = 3.62, p = 0.000; nitrites - Z = 2.91 , p = 0.003; S-nitrosothiols - Z = 3.74, p = 0.00; endothelin-1 - Z = 3.36, p = 0.000). These results showed interrelation between adverse IQOS effect on vascular endothelium of adolescents duration of IQOS smoke. Conclusions. The adverse effect of heated tobacco product (IQOS) on the biochemical indicators of the vascular endothelium function of adolescents was found in our experimental study. The salivary nitrate, nitrite and S-nitrosothiols levels were significantly decreased in IQOS smoking adolescents. The salivary endothelin-1 content was significantly elevated in IQOS smoking adolescents. The revealed changes indicate the endothelial dysfunction in young smokers. The development of pathological processes in the vascular endothelium depends on the smoking duration.

Effects of Chewing Gum on Nitric Oxide Metabolism, Markers of Cardiovascular Health and Neurocognitive Performance after a Nitrate-Rich Meal

Objectives Cardiovascular and neurocognitive responses to chewing gum have been reported, but the mechanisms are not well understood. Chewing gum after a nitrate-rich meal may upregulate the reduction of oral nitrate to nitrite and increase nitric oxide (NO), a molecule important to cardiovascular and neurocognitive health. We aimed to explore effects of chewing gum after a nitrate-rich meal on nitrate metabolism (through the enterosalivary nitrate-nitrite-NO pathway), endothelial function, blood pressure (BP), neurocognitive performance, mood and anxiety. Methods Twenty healthy men (n = 6) and women (n = 14) with a mean age of 48 years (range: 23–69) were recruited to a randomized controlled cross-over trial. After consumption of a nitrate-rich meal (180 mg of nitrate), we assessed the acute effects of chewing gum, compared to no gum chewing, on (i) salivary nitrate, nitrite and the nitrate reductase ratio (100 x [nitrite]/([nitrate] + [nitrite]); (ii) plasma nitrite, S-nitrosothiols and other nitroso species (RXNO); (iii) endothelial function (measured by flow mediated dilatation); (iv) BP; (v) neurocognitive performance; (vi) mood; and (vii) anxiety. Results Consumption of the nitrate-rich meal resulted in a significant increase in markers of nitrate metabolism. A significantly higher peak flow mediated dilatation was observed with chewing compared to no chewing (baseline adjusted mean difference: 1.10%, 95% CI: 0.06, 2.14; p = 0.038) after the nitrate-rich meal. A significant small increase in systolic BP, diastolic BP and heart rate were observed with chewing compared to no chewing after the nitrate-rich meal. The study did not observe increased oral reduction of nitrate to nitrite and NO, or improvements in neurocognitive performance, mood or anxiety with chewing compared to no chewing. Conclusion Chewing gum after a nitrate-rich meal resulted in an acute improvement in endothelial function and a small increase in BP but did not result in acute effects on neurocognitive function, mood or anxiety.

Abundance of Veillonella spp. does not Reflect Salivary Nitrite Production after Nitrate Ingestion

Veillonella spp. have been reported to be the most prevalent nitrate-reducing bacterial species in the oral cavity. The purpose of this study was to examine the relationship between the abundance of Veillonella spp. and nitrite production after nitrate ingestion. Bacterial samples were obtained from the tongue surfaces of 50 university students. The predominant Veillonella spp., V. atypica, V. dispar, and V. rogosae were identified and enumerated using real-time polymerase chain reaction (qPCR). Salivary nitrate and nitrite were measured before and 30, 60, and 90 min after ingestion of 100 ml of beetroot juice. Increased nitrite concentrations were observed in all participants, with a mean increase of 0.61 (0.42-1.10) mM expressed as the median (interquartile range). Veillonella atypica was detected in 40 subjects (80%), V. dispar in 48 (96%), and V. rogosae in 48 (96%), at quantities ranging from 1.3 × 102 to 2.8 × 107 CFU/ml per subject. The strengths of the correlations of the log colony forming unit (CFU) values of V. atypica, V. dispar, V. rogosae, and the log CFU value of the three species together with the increase in nitrite levels were 0.091, 0.114, -0.228, and 0.060, respectively, none of which were significant (p > 0.05). Our results indicate that the abundance of Veillonella spp. is not related to salivary nitrite production after nitrate ingestion.

Potential role for age as a modulator of oral nitrate reductase activity

Reduction of salivary nitrate to nitrite by oral nitrate reductase (NR) expressing bacteria has emerged as an integral pathway in regulating nitric oxide (NO) homeostasis and signaling. The oral microbiome is critical for this pathway. Variations in this pathway may underlie variable responses in the magnitude by which dietary or therapeutic nitrate modulates NO-signaling. The relationships between oral microbes and NR activity, and the factors that affect this relationship remain unclear however. Using a cross-sectional study design, the objective of this study was to determine the relationships between oral microbes and oral NR activity using a protocol that directly measures initial NR activity. Tongue swabs were collected from 28 subjects ranging in age from 21 to 73y. Initial NR activity showed a bell-shaped dependence with age, with activity peaking at ~40-50y and being lower but similar between younger (20-30y) and older (51–73) individuals. Microbiome relative abundance and diversity analyses, using 16s sequencing, demonstrated differences across age and identified both NR expressing and non-expressing bacteria in modulating initial NR activity. Finally, initial NR activity was measured in 3mo and 13mo old C57BL/6J mice. No differences in bacterial number were observed. However initial NR activity was significantly (80%) lower in 13mo old mice. Collectively, these data suggest that age is a variable in NR activity and may modulate responsiveness to dietary nitrate.

Isolation and Characterization of Nitrate-Reducing Bacteria as Potential Probiotics for Oral and Systemic Health.

Recent evidence indicates that the reduction of salivary nitrate by oral bacteria can contribute to prevent oral diseases, as well as increase systemic nitric oxide levels that can improve conditions such as hypertension and diabetes. The objective of the current manuscript was to isolate nitrate-reducing bacteria from the oral cavity of healthy donors and test their in vitro probiotic potential to increase the nitrate-reduction capacity (NRC) of oral communities. Sixty-two isolates were obtained from five different donors of which 53 were confirmed to be nitrate-reducers. Ten isolates were selected based on high NRC as well as high growth rates and low acidogenicity, all being Rothia species. The genomes of these ten isolates confirmed the presence of nitrate- and nitrite reductase genes, as well as lactate utilization genes, and the absence of antimicrobial resistance, mobile genetic elements and virulence genes. The pH at which most nitrate was reduced differed between strains. However, acidic pH 6 always stimulated the reduction of nitrite compared to neutral pH 7 or slightly alkaline pH 7.5 (p < 0.01). We tested the effect of six out of 10 isolates on in vitro oral biofilm development in the presence or absence of 6.5 mM nitrate. The integration of the isolates into in vitro communities was confirmed by Illumina sequencing. The NRC of the bacterial communities increased when adding the isolates compared to controls without isolates (p < 0.05). When adding nitrate (prebiotic treatment) or isolates in combination with nitrate (symbiotic treatment), a smaller decrease in pH derived from sugar metabolism was observed (p < 0.05), which for some symbiotic combinations appeared to be due to lactate consumption. Interestingly, there was a strong correlation between the NRC of oral communities and ammonia production even in the absence of nitrate (R = 0.814, p < 0.01), which indicates that bacteria involved in these processes are related. As observed in our study, individuals differ in their NRC. Thus, some may have direct benefits from nitrate as a prebiotic as their microbiota naturally reduces significant amounts, while others may benefit more from a symbiotic combination (nitrate + nitrate-reducing probiotic). Future clinical studies should test the effects of these treatments on oral and systemic health.

Different Pharmacokinetic Responses to an Acute Dose of Inorganic Nitrate in Patients with Type 2 Diabetes.

In this study, we aimed to compare the pharmacokinetics of nitrate (NO3) in patients with type 2 diabetes mellitus (T2DM) and healthy adults. Potential effects of salivary nitrate reductase (NR) activity on cardiometabolic responses to an acute dose of NO3 was also assessed. Nine healthy adults and nine T2DM patients were recruited to consume a NO3-rich breakfast (~410 mg NO3). Pharmacokinetics of NO3 were examined using repeated measurements of NOx (nitrate+ nitrite) concentrations of serum and saliva over 8 hours and NO3 concentrations of spot and 24-h urine samples. Cardiometabolic parameters, including serum levels of glucose, insulin, and triglycerides as well as blood pressure were also measured. Compared to patients with T2DM, serum NOx concentration (Δ1= 16.7 vs. 4.4 μmol/L, P=0.057) of healthy subjects sharply increased within 1 hour after NO3 loading. Healthy subjects had a higher NR activity index, and higher peak salivary NO3 concentration with a lower time to peak. Diabetic patients with high- compared to low-NR values had a higher whole-body NOx exposure (103±31.4 vs. 58.9±22.1 μmol.h/L); they also showed a better glycemic response and more reduction of blood pressure following ingestion of a NO3-rich meal. T2DM may be associated with a different pattern of NOx pharmacokinetics (especially salivary NOx metabolism). Salivary NR activity may have a critical role in postprandial metabolism of NO3, and diabetic patients with higher NR activity may take more advantages from NO3 supplementation.

Nitrate as a potential prebiotic for the oral microbiome

The salivary glands actively concentrate plasma nitrate, leading to high salivary nitrate concentrations (5–8 mM) after a nitrate-rich vegetable meal. Nitrate is an ecological factor that can induce rapid changes in structure and function of polymicrobial communities, but the effects on the oral microbiota have not been clarified. To test this, saliva of 12 healthy donors was collected to grow in vitro biofilms with and without 6.5 mM nitrate. Samples were taken at 5 h (most nitrate reduced) and 9 h (all nitrate reduced) of biofilm formation for ammonium, lactate and pH measurements, as well as 16S rRNA gene Illumina sequencing. Nitrate did not affect biofilm growth significantly, but reduced lactate production, while increasing the observed ammonium production and pH (all p < 0.01). Significantly higher levels of the oral health-associated nitrate-reducing genera Neisseria (3.1 ×) and Rothia (2.9 ×) were detected in the nitrate condition already after 5 h (both p < 0.01), while several caries-associated genera (Streptococcus, Veillonella and Oribacterium) and halitosis- and periodontitis-associated genera (Porphyromonas, Fusobacterium, Leptotrichia, Prevotella, and Alloprevotella) were significantly reduced (p < 0.05 at 5 h and/or 9 h). In conclusion, the addition of nitrate to oral communities led to rapid modulation of microbiome composition and activity that could be beneficial for the host (i.e., increasing eubiosis or decreasing dysbiosis). Nitrate should thus be investigated as a potential prebiotic for oral health.

Nitrate: a pioneer from the mouth to the systemic health and diseases

Dietary nitrate which mainly comes from green leafy vegetables, is absorbed into blood circulation by the intestinal mucosa. Parotid gland is an important organ for transporting nitrate. Nitrate in blood is taken up by sialin, a nitrate transporter and concentrated in salivary glands and secreted into saliva. The salivary nitrate is partially reduced to nitrite and nitric oxide by oral bacteria, and then salivary nitrate and nitrite return into blood circulation with swallowing and intestinal mucosal absorption. As a non-classic source of nitric oxide, nitrate-nitrite-nitric oxide pathway plays an important role on physiological and pathological conditions, especially on the condition of hypoxia and ischemia. These functions include body protection, such as gastrointestinal tract, cardiovascular system, anti-inflammation, regulation of glucose/lipid metabolism, improvement of sport ability, maintaining gut microbiome hemostasis, and alleviating senility. The traditional view on nitrate as a harmful substance to human body has been proved to be lack of scientific evidence. With further research and application, as a pioneer from the mouth to the whole body, nitrate is expected to play a crucial part in human health, and prevention and treatment of systemic diseases.

Effects of age, sex and diet on salivary nitrate and nitrite in infants

The inorganic anions nitrate and nitrite are oxidation products from endogenous nitric oxide (NO) generation and constituents in our diet. A nitrate-nitrite-NO pathway exists in which nitrate can be serially reduced to bioactive NO. The first step of this pathway occurs in the oral cavity where oral bacteria convert salivary nitrate to nitrite, whereafter nitrite is reduced to NO systemically by several enzymatic and non-enzymatic pathways. Data are scarce regarding salivary levels and oral conversion capacity of these anions in infants. We measured salivary nitrate and nitrate in infants at 4 and 12 months of age and related values to age, sex, dietary pattern and oral microbiome.Saliva was collected from a total of 188 infants at 4 and 12 months of age. Salivary nitrate, nitrite and nitrite/nitrate ratio as a measure of oral nitrate-reducing capacity were analyzed by HPLC and related to age, sex, type of diet (breast milk or formula) and oral microbiome. There was no difference in salivary nitrate, nitrite or nitrite/nitrate ratio between boys and girls at any age. At 4 months levels of these parameters were lower than what has been described in adults but they had all increased significantly at 12 months of age. At 4 months of age salivary nitrite/nitrate ratio was lower in breast-fed compared to formula-fed infants, but these differences disappeared at 12 months. Several bacterial species were associated with oral nitrate reducing capacity including Prevotella, Veillonella, Alloprevotella and Leptotrichia.We conclude that in infants there is an increase in salivary nitrate and nitrite as well as in oral nitrate-reductase capacity during the first year of life. Differences observed at 4 months of age between breast-fed and formula-fed infants disappear at one year of age.