Nitrate In Saliva Research Articles

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.

Validation of two point-of-care tests against standard lab measures of NO in saliva and in serum

Nitric oxide (NO) is an endogenous signaling molecule, which plays important roles in cardiometabolic health. A significant source of NO is dietary nitrate (NO3), which is initially metabolized by oral bacteria into nitrite (NO2−) and is subsequently converted into NO once digested in the acidic gastric environment. Inexpensive non-invasive tests for measuring nitrite from saliva have been developed as a means for individuals to monitor their NO bioavailability. However, few studies exist in the literature validating and comparing these products with standard lab assays. The objective of this study was to validate two commonly used commercial strips: Nitric Oxide Test Strips (Berkeley Test) and Nitric Oxide Indicator Strips (Neogenesis) against standard lab measures for saliva and serum nitrite/nitrate. A stratified random sample of 20 non-smoking, overweight or obese participants between 40 to 65 years of age, were selected for this study from the baseline data of the San Juan Overweight Adults Longitudinal Study (SOALS). There was a significant correlation between the measures from the two nitrite-detecting-strips after controlling for the stratification variables (metabolic syndrome, and mouthwash use) (r = 0.75). Measurements from both strips correlated significantly with salivary nitrite levels (r = 0.76 for Berkeley strips; r = 0.59 for Neogenesis). Neither of the strips had a significant correlation with the levels of saliva nitrate, serum nitrite and serum nitrate. In conclusion, commercially available Berkeley and Neogenesis strips provide a reasonable surrogate for salivary, but not for systemic nitrite levels.

Profound differences between humans and rodents in the ability to concentrate salivary nitrate: Implications for translational research

In humans dietary circulating nitrate accumulates rapidly in saliva through active transport in the salivary glands. By this mechanism resulting salivary nitrate concentrations are 10–20 times higher than in plasma. In the oral cavity nitrate is reduced by commensal bacteria to nitrite, which is subsequently swallowed and further metabolized to nitric oxide (NO) and other bioactive nitrogen oxides in blood and tissues. This entero-salivary circulation of nitrate is central in the various NO-like effects observed after ingestion of inorganic nitrate. The very same system has also been the focus of toxicologists studying potential carcinogenic effects of nitrite-dependent nitrosamine formation. Whether active transport of nitrate and accumulation in saliva occurs also in rodents is not entirely clear. Here we measured salivary and plasma levels of nitrate and nitrite in humans, rats and mice after administration of a standardized dose of nitrate.After oral (humans) or intraperitoneal (rodents) sodium nitrate administration (0.1mmol/kg), plasma nitrate levels increased markedly reaching ~300µM in all three species. In humans ingestion of nitrate was followed by a rapid increase in salivary nitrate to >6000µM, ie 20 times higher than those found in plasma. In contrast, in rats and mice salivary nitrate concentrations never exceeded the levels in plasma. Nitrite levels in saliva and plasma followed a similar pattern, ie marked increases in humans but modest elevations in rodents. In mice there was also no accumulation of nitrate in the salivary glands as measured directly in whole glands obtained after acute administration of nitrate.This study suggests that in contrast to humans, rats and mice do not actively concentrate circulating nitrate in saliva. These apparent species differences should be taken into consideration when studying the nitrate-nitrite-nitric oxide pathway in rodents, when calculating doses, exploring physiological, therapeutic and toxicological effects and comparing with human data.

A stepwise reduction in plasma and salivary nitrite with increasing strengths of mouthwash following a dietary nitrate load

Nitric Oxide (NO) bioavailability is essential for vascular health. Dietary supplementation with inorganic nitrate, which is abundant in vegetables and roots, has been identified as an effective means of increasing vascular NO bioavailability. Recent studies have shown a reduction in resting blood pressures in both normotensive and hypertensive subjects following ingestion of inorganic nitrate. Oral bacteria play a key role in this process and the use of strong antibacterial mouthwash rinses can disable this mechanism. Hence, mouthwash usage, a $1.4 billion market in the US, may potentially be detrimental to cardiovascular health. The purpose of this study was to examine the effects of different strengths of commercially available mouthwash products on salivary and plasma nitrate and nitrite concentrations following 8.4 mmol inorganic nitrate load (beetroot juice). Specifically, we examined the effects of Listerine antiseptic mouthwash, Cepacol antibacterial mouthwash, and Chlorhexidine mouthwash versus control (water).Twelve apparently healthy normotensive males (36 ± 11yrs) completed four testing visits in a randomized order, separated by one week. Testing consisted of blood pressure (BP), and saliva and venous blood collection at baseline and each hour for 4 h. Following baseline-testing participants consumed 140 ml of beet juice and then 15 min later gargled with 5 mL of assigned mouthwash. Testing and mouthwash rinse was repeated every hour for 4 h. Linear mixed effects models, followed by pairwise comparisons where appropriate, were used to determine the influence of treatment and time on plasma and saliva nitrate and nitrite, and BP.Plasma and salivary nitrate increased above baseline (time effect) for all conditions (p ≤ 0.01). There were time (p ≤ 0.01), treatment (p ≤ 0.01), and interaction (p ≤ 0.05) effects for plasma and salivary nitrite. There was a treatment effect on systolic BP (p ≤ 0.05). Further examination revealed a differentiation of plasma and salivary nitrite concentration between control/antiseptic and antibacterial/chlorhexidine treatments. When examined in this manner there was a reduction in both SBP (p ≤ 0.01) and mean arterial BP (p ≤ 0.05) from the antibacterial/chlorhexidine treatments.These results suggest a potentially differentiating effect of different commercially available mouthwash solutions on plasma and salivary nitrite concentrations and resting blood pressure responses. This raises potential public health related questions on the appropriate widespread usage of different mouthwash formulations.

Likely additive ergogenic effects of combined preexercise dietary nitrate and caffeine ingestion in trained cyclists.

Aims. To evaluate the possible additive effects of beetroot juice plus caffeine on exercise performance. Methods. In a randomized, double-blinded study design, fourteen healthy well-trained men aged 22 ± 3 years performed four trials on different occasions following preexercise ingestion of placebo (PLA), PLA plus 5 mg/kg caffeine (PLA+C), beetroot juice providing 8 mmol of nitrate (BR), and beetroot juice plus caffeine (BR+C). Participants cycled at 60% maximal oxygen uptake (max) for 30 min followed by a time to exhaustion (TTE) trial at 80% max. Saliva was collected before supplement ingestion, before exercise, and after the TTE trial for salivary nitrate, nitrite, and cortisol analysis. Results. In beetroot trials, saliva nitrate and nitrite increased >10-fold before exercise compared with preingestion (P ≤ 0.002). TTE in BR+C was 46% higher than in PLA (P = 0.096) and 18% and 27% nonsignificant TTE improvements were observed on BR+C compared with BR and PLA+C alone, respectively. Lower ratings of perceived exertion during TTE were found during 80% max on BR+C compared with PLA and PLA+C (P < 0.05 for both). Conclusions. Acute preexercise beetroot juice coingestion with caffeine likely has additive effects on exercise performance compared with either beetroot or caffeine alone.

Intragastric nitration by dietary nitrite: Implications for modulation of protein and lipid signaling

Inorganic nitrite, derived from the reduction of nitrate in saliva, has recently emerged as a protagonist in nitric oxide (•NO) biology as it can be univalently reduced to •NO, in the healthy human stomach. Important physiological implications have been attributed to nitrite-derived •NO in the gastrointestinal tract, namely modulation of host defense, blood flow, mucus formation and motility. At acidic pH, nitrite generates different nitrogen oxides depending on the local microenvironment (redox status, gastric content, pH, inflammatory conditions), including •NO, nitrogen dioxide (•NO2), dinitrogen trioxide (N2O3), and peroxynitrite. Thus, the gastric environment is a significant source of nitrating and nitrosating agents, especially in individuals consuming a nitrate/nitrite-rich diet on a daily basis. Both, the gastric lumen and mucosa contain putative targets for nitration, not only proteins and lipids from ingested aliments but also endogenous proteins secreted by the oxyntic glands. The physiological and functional consequences of nitration of gastric mediators will impact on local processes including food digestion and ulcerogenesis. Additionally, gastric nitration products (such as nitrated lipids) may be absorbed and affect systemic pathways. Thus, dietary ingestion of nitrate will have direct consequences for endogenous protein nitration, as indicated by our preliminary data.

Increase of saliva nitrate and nitrite level in patients with oral candidiasis

To observe the alterations of saliva nitrate and nitrite level in patients with oral candidiasis. Parotid saliva and whole saliva were collected from 33 patients and 34 healthy volunteers. Concentrations of nitrate and nitrite in saliva were determined by high-performance liquid chromatography. Follow-up observation was performed on 10 patients after treatment. The data were statistically analyzed with independent-samples t test or paired-samples t test at alpha = 0.05. There was significant increase of the concentrations and secretion rate of parotid saliva nitrate in patient group as compared with controls: (49.70 +/- 0.50) vs (21.51 +/- 0.60) mg/L (t = 2.692, P = 0.009) and (27.71 +/- 0.50) vs (12.55 +/- 0.60) microg/min (t = 2.554, P = 0.013), respectively. Significantly increased concentrations and secretion rate of nitrate and nitrite [nitrate: (6.46 +/- 0.94) vs (1.11 +/- 0.70) mg/L (t = 3.792, P = 0.000); nitrite: (8.48 +/- 0.58) vs (3.39 +/- 0.53) mg/L (t = 2.888, P = 0.005); nitrate secretion rate: (10.57 +/- 0.91) vs (2.10 +/- 0.74) microg/min (t = 3.464, P= 0.001); nitrite secretion rate: (13.91 +/- 0.55) vs (6.42 +/- 0.58) microg/min (t = 2.397, P = 0.020)] were revealed in whole saliva of patients group. Significantly decreased nitrate and nitrite levels were also observed in patients after treatment, especially the changes of parotid saliva nitrate secretion rate [(37.50 +/- 0.50) vs (14.34 +/- 0.64) microg/min (t = 3.142, P = 0.012)], whole saliva nitrate [(14.29 +/- 1.01) vs (2.59 +/- 1.03) mg/L (t = 3.475, P = 0.007)] and whole saliva nitrate secretion rate [(25.97 +/- 0.93) vs (4.12 +/- 1.00) microg/min (t = 3.922, P = 0.003)]. The present study revealed the significant increase of salivary nitrate and nitrite level in patients with oral candidiasis is considered to be associated with the host defense reaction.

Salivary nitrate – an ecological factor in reducing oral acidity

Human oral cavities represent a novel environment with a constant supply of concentrated nitrate. For humans, over 80% of dietary nitrate originates from fruit and vegetables. With a healthy, balanced diet, rich in fruit and vegetables, the concentration of nitrate in saliva can reach up to more than three times the European drinking water standard. The physiological function of the active excretion of salivary nitrate is unknown. Furthermore, little is known of the ecological function of oral nitrate and the effect on the oral environment during its subsequent oral microbial conversions. The objectives of the research were to investigate the effect on salivary pH coupled with oral microbial nitrate and/or nitrite reduction. Human saliva samples were incubated anaerobically in the presence of 111.0 mmol glucose (2%), with and without 1.5 mmol nitrate/nitrite, and pH and nitrate/nitrite consumption were measured during the time-course of the incubations. We found that anaerobic incubation of saliva containing a mixture of oral bacteria in the presence of nitrate/nitrite substrates and glucose resulted in a higher pH than was found in controls in the absence of nitrate/nitrite. These results suggest that the presence of these electron acceptors repressed acid fermentation, or increased alkali production, or consumed acid produced, thus reducing salivary acidity. This finding identifies salivary nitrate as a possible ecological factor in reducing oral acidity. The possibility that a symbiotic relationship between host nitrate excretion and nitrate-reducing microorganisms might help to protect against tooth decay should be explored further.

Antimicrobial effect of acidified nitrate and nitrite on six common oral pathogens in vitro

Salivary nitrate is positively correlated with plasma nitrate and its level is 9 times the plasma level after nitrate loading. Nitrate in saliva is known to be reduced to nitrite by oral bacteria. Nitrate and nitrite levels in saliva are 3 - 5 times those in serum in physiological conditions respectively in our previous study. The biological functions of high salivary nitrate and nitrite are still not well understood. The aim of this in vitro study was to investigate the antimicrobial effects of nitrate and nitrite on main oral pathogens under acidic conditions. Six common oral pathogens including Streptococcus mutans NCTC 10449, Lactobacillus acidophilus ATCC 4646, Porphyromonas gingivalis ATCC 33277, Capnocytophaga gingivalis ATCC 33624, Fusobacterium nucleatum ATCC 10953, and Candida albicans ATCC 10231 were cultured in liquid medium. Sodium nitrate or sodium nitrite was added to the medium to final concentrations of 0, 0.5, 1, 2, and 10 mmol/L. All of the microorganisms were incubated for 24 to 48 hours. The optical densities (OD) of cell suspensions were determined and the cultures were transferred to solid nutrient broth medium to observe the minimum inhibitory concentration and minimum bactericidal/fungicidal concentration for the six tested pathogens. Nitrite at concentrations of 0.5 to 10 mmol/L had an inhibitory effect on all tested organisms at low pH values. The antimicrobial effect of nitrite increased with the acidity of the medium. Streptococcus mutans NCTC 10449 was highly sensitive to nitrite at low pH values. Lactobacillus acidophilus ATCC 4646 and Candida albicans ATCC 10231 were relatively resistant to acidified nitrite. Nitrate at the given concentrations and under acidic conditions had no inhibitory effect on the growth of any of the tested pathogens. Nitrite, at a concentration equal to that in human saliva, is both cytocidal and cytostatic to six principal oral pathogens in vitro, whereas nitrate at a similar concentration has no antimicrobial effect on these organisms.

Nitrate and nitrite concentrations in rabbit saliva: Comparison with rat saliva

The purpose of this work was to investigate the interest of rabbits for studying pharmacology and toxicology of dietary nitrate. Twenty-one females were given 1, 300 and 600 mg/l nitrate in drinking water for 11 weeks. Saliva and blood were analysed for nitrate/nitrite. There is a linear relationship between the amounts of nitrate ingested and amount of nitrate in saliva, contrary to what is observed in rats. However, salivary nitrite concentrations remain low, and nitrate reductase activity in the oral cavity of the rabbit seems very weak.

Application of capillary zone electrophoresis to the analysis and to a stability study of nitrite and nitrate in saliva

The applicability of capillary zone electrophoresis for the determinations of nitrite and nitrate was studied. Using direct UV detection the limit of detection values of the analytes were 0.14 and 0.21 microg/mL, respectively. The developed method was found to be useful to directly determine nitrite, nitrate and thiocyanate in saliva. It was found that adjusting the pH of the sample to 11 and storing the saliva at 4 degrees C was adequate to make constant the nitrite/nitrate ratio in saliva samples at least 7 days.

Nitrate and nitrite concentrations in human saliva for men and women at different ages and times of the day and their consistency over time.

Salivary nitrite arises from nitrate and is the main source of gastric nitrite, a precursor of carcinogenic N-nitroso compounds. We examined nitrate and nitrite levels in unstimulated saliva from subjects consuming low-nitrate low-vitamin C diets. When saliva was collected from six men at nine times of the day (Experiment 1), night time nitrite levels were significantly higher than day time values and nitrite varied more than nitrate. When saliva was collected from 29 subjects aged 19-37 or 60-84 years at four times of the day during 1991-1993 (Experiment 2), all older subjects and older men had significantly higher nitrite levels than the corresponding younger subjects, night time nitrite levels in men were significantly raised, and nitrate and nitrite levels in the same samples were closely correlated. Saliva was collected at 6.00 a.m. on two successive days in 1997 from 16 subjects who had collected saliva in 1991-1993 (Experiment 3). Nitrate and nitrite levels on day 1 of experiment 3 were closely correlated with those on day 2. Nitrate and nitrite levels on days 1 and 2 of Experiment 3 were correlated with the corresponding parameters in Experiment 2 with P = 0.04 and 0.08 for day 1, and 0.10 and 0.28 for day 2, respectively. Hence, saliva nitrite levels rose at night and were higher in older people, especially older men, and saliva nitrate and nitrite levels varied little from day to day, but varied more after 4-6 years.

Simultaneous determination of inorganic anions in body fluids. A new method in pediatric laboratory diagnosis--results of a pilot study

The anions analysis was a methodical problem up to now. This was the reason for low interest. Biological fluids like saliva and urine which could easily receive without any stress for the children, are little investigated for its capacity on nitrite, nitrate, bromide and sulfate. In this performance there will presented an ion-chromatographic method to determine inorganic anions in the following body-fluids: serum saliva, liquor and urine. The anions chloride, nitrite, bromide, nitrate, phosphate and sulfate was determined quantitatively. The method was proved in a pilot-study on children's body-fluids serum, liquor and saliva. The objects was to get a landmark in expectation from anion concentrations. Bromide was detected as a constant part in all body fluids. The origin and importance is not clear till now. Also was found nitrate in all investigated body fluids. There seems to be a connection between diarrhea and an increase in serum levels from nitrate. We found considerable amounts of nitrate in saliva by babies and infants. The method is distinguished by little fluctuation in measurement and high specificity. Short time in analysis and simple handling will do the method for a qualified one in pediatrics.

Excretion of N-mononitrosopiperazine in urine in workers manufacturing piperazine.

Piperazine has been shown to nitrosate in vivo to N-mononitrosopiperazine (MNPZ) by oral intake. Urine from workers exposed to piperazine in a chemical plant was analysed for nitrosamines by gas chromatography-thermal energy analysis. In five out of 11 exposed cases, MNPZ excretion in urine was 0.3 to 4.7 micrograms/24 h (during and after a work shift). In four cases, MNPZ was detected in some urine samples, and in two cases MNPZ was not detected (less than 0.1 ng/ml). The individual excretion was strongly dependent on piperazine exposure, which ranged from 0.06 to 1.7 mg/m3 (time-weighted average; Spearman's rank correlation 0.78, P = 0.01). The MNPZ excretion showed no significant correlation with nitrite or nitrate in saliva (both: r = 0.50, P = 0.10).

The problem of nitrate and nitrite in human food. II. The occurrence of nitrate, nitrite, and thiocyanate in human saliva (author's transl)

When the alimentary nitrate intake is in the range of 50 mg NO3- the nitrate values in saliva of adults and children follow a Gaussian normal distribution. With high and very high nitrate intakes the individual fluctuations of nitrate in saliva are distributed randomly in a wide range with a factor of 1:4. The nitrite values in saliva fluctuate even more, so that a proper judgment should be made only in individual cases. The average content of thiocyanate in saliva of adults was found to be 134 ppm SCN-, the saliva of children contained 50 ppm SCN-. The estimated molecular ratio of thiocyanate to nitrite in saliva of adults and children may fluctuate between 8:1 and 1:1 depending on the nitrate content of food.

Studies on In Vivo Formation of Nitroso Compounds (VI)

The relationship between dietary nitrate intake and nitrite detected in human saliva was studied with a basal diet and high nitrate diet. Nitrite contents in foodstuff and in saliva were determined colorimetrically by a diazo-coupling reaction with sulfanilamide and naphthylethylenediamine, and nitrate contents were determined as nitrite after conversion to nitrite by passing a cadmium column. Determinations of nitrite and nitrate in saliva were carried out at intervals of 1-2hr except at night.When 200g of salted Chinese cabbage was taken, the average concentrations of nitrite and nitrate in saliva increased to large quantities of 71.9 and 544.5ppm, however, in the case of basal diet, decreased to 12.2 and 76.5ppm, respectively.From these results, it should be concluded that the changes of nitrite and nitrate concentrations in human saliva depended upon those contents in the diets. Furthermore, when daily secretion volume of saliva was presumed as 1, 000ml, the total amount of nitrite in saliva were 7-28 times that of the diet ingested, and it was assumed that nitrite in saliva was formed in vivo by the reduction of nitrate. We can not give a clear explanation for this reduction mechanism yet, but nitrite in saliva might be produced by the action of micro-organisms in alimentary tract, perhaps in mouth.