Effects of Nitrate and Nitrite on Plaque pH Decrease and Nitrite-Producing and -Degrading Activities of Plaque in vitro

Nitrate is found in food, especially in vegetables such as green leafy vegetables [1]. Ingested nitrate is rapidly absorbed from the upper gastrointestinal tract, circulates through tissues, about 70% of which is excreted through urine, and about 25% is concentrated about 10-fold in the salivary glands, mainly parotid, and secreted again in saliva in the oral cavity [2‒5].

Nitrate in food taken into the oral cavity and nitrate re-secreted in saliva in the oral cavity are reduced to nitrite by a wide range of oral commensal bacteria, including Veillonella, Actinomyces, Rothia, Neisseria, Schaalia, and Streptococcus spp. [6‒8]. When the nitrite produced in the oral cavity is swallowed with the remaining nitrate and exposed to the acidic environment of the stomach, some is reduced non-enzymatically to nitric oxide [9]. These nitrogen oxides (nitrate, nitrite, and nitric oxide) are absorbed by the intestinal tract, circulate through tissues, are gradually re-oxidized to the chemically more stable nitrate, and secreted back into the saliva, as described above. Thus, the entero-salivary circulation of nitrate occurs [4, 5].

Nitrite has been reported to inhibit the growth [10] and acid production [11] of the caries-associated bacterium Streptococcus mutans as well as plaque acid production [12]. It has also been shown to inhibit the growth of periodontal disease-associated bacteria such as Porphyromonas gingivalis and Fusobacterium nucleatum [13, 14].

Individual differences in oral nitrite production have also been reported [7, 15]. As an example of the influence of these individual differences on oral disease, it was reported that children with high salivary nitrate concentrations and high nitrite productivity had less dental caries [16]. Furthermore, our previous study showed that nitrite inhibited the acid-producing capacity of dental plaque in children and that this effect was greater in plaque with a higher acid-producing capacity [12]. These studies suggest that in actual dental plaque, food/saliva-derived nitrate is converted to nitrite by plaque bacteria, and that this nitrite inhibits the acid-producing capacity of sugar-metabolizing bacteria. In addition, the higher the acid-producing capacity of plaque, the more susceptible it is to inhibition by nitrite [12], suggesting that nitrite production by plaque may function that inhibits excessive acid production by plaque. Furthermore, plaque bacteria have been reported to not only produce nitrite, but also to further degrade the nitrite produced [8, 17, 18], indicating that bacterial nitrate metabolism is more complex.

However, the effects of nitrate on the pH-lowering and nitrite-producing and -degrading activities of human plaque are unknown. In this study, therefore, we investigated the dynamics and functions of nitrate/nitrite in human plaque by measuring the nitrite-producing and -degrading activities and clarifying the relationship between these activities and the pH-lowering activity. In addition, we examined the inter-subject differences in these activities.

Patients who visited a general practitioner were included in the study. They were checked to ensure that they had not taken any antibiotics within at least 2 weeks and had not eaten or drank within 2 h prior to plaque sampling. This study was conducted in accordance with the approval of the Research Ethics Committee of Tohoku University Graduate School of Dentistry (Approval No. 2018-3-17: Research Ethics Committee of Tohoku University Graduate School of Dentistry). Written informed consent was obtained from the subjects. One dentist collected samples from the buccal aspect of the maxillary molar with a dental spoon excavator (#2, YDM, Saitama) under no-shadow lighting. The following experiments were conducted in air.

We previously reported that nitrite inhibits acid production in plaque. In this experiment (Experiment 1 in Fig. 1), we aimed to determine how nitrite produced in plaque from nitrate affects endogenous (before addition of glucose to plaque) and exogenous (after addition of glucose to plaque) acid production. In addition, the effect of glucose metabolism on nitrite production from nitrate was also investigated.

Plaque samples from 18 subjects (8 females, 10 males; mean: 49.6 ± 21.1 years; DMFT = 15.2 ± 10.2) were collected and plaque suspensions were prepared at a final concentration of 0.1 mg/μL of 8.3 mm potassium phosphate buffer (pH 7.0) containing 9.2 mm MgCl₂. After measuring the initial pH of the plaque suspension (E1-pH0), potassium nitrate (final concentration: 4.95 mm) or purified water was added to the plaque suspension, and the nitrite concentration (E1-N1) and pH (E1-pH1) were measured after incubation at 36°C for 7 min. Then, glucose (final concentration: 55.6 mm) was added, it was incubated at 36°C for 7 min, and the nitrite concentration (E1-N2) and pH (E1-pH2) were measured. The nitrate concentration was determined based on the concentration in saliva [19].

The objective of this experiment (Experiment 2 in Fig. 1) was to confirm the effect of nitrite on endogenous (before addition of glucose to plaque) and exogenous (after addition of glucose to plaque) acid production, and to further investigate the effect of glucose metabolism on nitrite-degrading activity. Plaque samples from 15 subjects (8 females, 7 males; mean: 37.9 ± 20.6 years; DMFT = 10.5 ± 8.4) were collected and plaque suspensions were prepared at a final concentration of 0.1 mg/μL of 8.3 mm potassium phosphate buffer (pH 7.0) containing 9.2 mm MgCl₂. After measuring the initial pH of the plaque suspension (E2-pH0), potassium nitrite (final concentration: 0.78 mm) or purified water was added to the plaque suspension and it was incubated at 36°C for 7 min (E2-pH1), and then glucose (final concentration: 39.7 mm) was added and it was incubated for 7 min (E2-pH2).

Potassium nitrite (final concentration: 0.67 mm) was added to the plaque suspension, it was incubated for 7 min, and the nitrite concentration (E2-N1) was measured. In addition, 20 μL of these reaction solutions were collected in new centrifuge tubes, 10 μL of purified water and glucose (final concentration 92.7 mm) were added to each, and the nitrite level (E2-N2) was measured after incubation at 36°C for 7 min. The decrease in nitrite was defined as nitrite-degrading activity. The nitrite concentration was determined based on the concentration in saliva [19].

The purpose of this experiment (Experiment 3 in Fig. 1) was to measure the nitrite-producing and -degrading activities of plaque and determine if there is a correlation between the two. Plaque from 18 subjects (9 females, 9 males; mean: 41.9 ± 25.8 years; DMFT = 12.9 ± 9.3) was collected and plaque suspensions were prepared at a final concentration of 0.1 mg/μL of 8.3 mm potassium phosphate buffer (pH 7.0) containing 9.2 mm MgCl₂. Nitrite concentrations (E3-N0, E3-N1) were measured before and after incubation of the plaque suspensions with potassium nitrate (final concentration: 3.30 mm) or potassium nitrite (final concentration: 0.78 mm) for 10 min at 36°C. An increase in nitrite in the nitrate-added samples was considered nitrite-producing activity, while a decrease in nitrite in the nitrite-added samples was considered nitrite-degrading activity.

For statistical analysis, measurements were analyzed by the paired t test, and p < 0.05 was considered significant. Correlations were determined by single regression analysis.

The addition of nitrate significantly inhibited the pH decrease due to the endogenous acid production of plaque (before addition of glucose to plaque) (p < 0.0001) (Table 1). Furthermore, the pH decrease due to the exogenous acid production of plaque (after addition of glucose to plaque) was also significantly suppressed (p < 0.02) (Table 1). The addition of glucose significantly enhanced nitrite production in the plaque by approximately 3.3-fold (p < 0.002) (Table 1). No correlation was found between nitrite-producing activity and the ability to inhibit acid production of plaque (Fig. 2a1, a2).

The addition of nitrite significantly inhibited the pH decrease due to endogenous acid production (before addition of glucose) (p < 0.0002) but did not inhibit the pH decrease due to exogenous acid production (after addition of glucose) (Table 2). Glucose addition significantly enhanced nitrite-degrading activity (p < 0.05), but the enhancement was only about 1.1-fold, being much lower than the enhancement of nitrite-producing activity (Table 2). No correlation was found between nitrite-degrading activity and the ability to inhibit acid production of plaque (Fig. 2b1, b2).

All plaque samples showed nitrite-producing activity as well as nitrite-degrading activity. Individual differences in nitrite-producing activity were large (0.073–1.496 mm for 7 min), whereas such differences in nitrite-degrading activity were smaller (0.053–0.526 mm for 7 min), and there was no correlation between the two (Fig. 3a). Furthermore, nitrite-producing activity was correlated with age (Fig. 3b: r = 0.75, p < 0.002), whereas nitrite-degrading activity was not (Fig. 3c). No correlation was found with other parameters, including gender.

We previously showed that nitrite inhibits the acid-producing activity of plaque in children [12], and in this study, we found that nitrate also has an inhibitory effect on plaque pH-lowering (Table 1), which is due to nitrite production by nitrate reduction. Furthermore, the enhancement of nitrite production by glucose is considered to efficiently suppress the glucose-induced pH decrease. Nitrite production from nitrate linked to glucose metabolism may function as a mechanism to maintain plaque pH, suppressing its excessive acidification and contributing to plaque environmental homeostasis. Moreover, the metabolic reaction of nitrite production from nitrate is a reduction in which hydrogen ions are consumed (nitrate + 2H⁺ + 2e⁻ → nitrite + H₂O), resulting in increased acid-buffering capacity to counter plaque acidification. Degradation of nitrite can also be involved in reduction and may contribute to acid-buffering, but it is unclear which of the multiple degradation pathways functioned [8] in the present study.

Unlike our previous study [12] using plaque samples from children, in which the pH decrease was suppressed by nitrite after glucose addition as well as before, the pH decrease was not significantly suppressed by nitrite after glucose addition (Table 2). Although nitrite degradation activity was not measured in the previous study, it is possible that nitrite degradation activity was low in children’s plaque and that sufficient nitrite concentration was maintained to suppress the pH decrease after glucose addition. On the other hand, in the present study of plaque, the overall nitrite-degrading activity of these plaque samples was high, indicating that most of the nitrite may have been consumed before glucose addition (Table 2). As a result, the nitrite concentration may have been too low after glucose addition, resulting in little or no inhibition of the pH decrease (Table 2). This suggests that for nitrite to exert its inhibitory function effectively in the oral cavity, it must be continuously produced, and the continuous supply of nitrate to the oral cavity as a component of saliva through the entero-salivary circulation is a reasonable mechanism to make this possible.

Recently, diverse bacteria have been reported to exhibit nitrite-degrading activity and their metabolic processes are also diverse [8, 17]. However, the metabolic system of dental plaque has been reported to involve denitrification [18], where nitrite⁻ is converted to nitrogen via nitric oxide and nitrous oxide (N₂O).

In our current study, the nitrite-producing activity of plaque was observed in all samples, although there were large individual differences (Table 1). The activity was also present without glucose addition and was significantly increased by approximately 3.3-fold with glucose addition. Since bacteria in plaque can store endogenous metabolic substrates such as intracellular polysaccharides [20‒22], it is considered that in the absence of glucose addition, these endogenous substrates are metabolized to acquire reducing power and used to reduce nitrate to nitrite. When glucose is added, the reducing power obtained by efficiently metabolizing glucose in glycolysis can be used for the reduction of nitrate to nitrite. In addition, lactate produced from glucose may also increase nitrite production from nitrate [8, 23], although lactate production was not measured in the present study. Lactate can provide the reducing power to reduce nitrate to nitrate, linked to the oxidation of lactate [23].

On the other hand, nitrite-degrading activity was also observed in all samples, but it was lower than nitrite-producing activity, and the individual differences were also smaller (Table 2). Furthermore, this activity was hardly enhanced by glucose addition (approximately 1.1-fold). Similar to nitrite-producing activity, nitrite-degrading activity is considered to utilize the reducing power derived from endogenous metabolic substrates. However, the activity did not increase with glucose addition, suggesting that the reducing power obtained by glycolysis is not linked to nitrite-degrading activity.

The lack of a correlation between nitrite-producing and -degrading activities (Fig. 3a) suggests that different bacterial groups may be responsible for each activity, or that the same bacteria may have different mechanisms of production and degradation. In fact, previous studies identified Veillonella, Actinomyces, Neisseria, and Rothia spp. as the main nitrite-producing bacteria [6, 7, 22, 24, 25], whereas nitrite-degrading bacteria have not been clearly identified and require further study.

The nitrite-producing activity of plaque was found to be positively correlated with the age of the subjects (Fig. 3b). The reason why nitrite productivity correlates with age is unknown. Previous studies also reported that nitrite productivity is low in saliva of infants [26] and that nitrite productivity of the tongue is related to age, with a bell-shaped peak at 40–50 years of age [27], while no significant differences between children and adults in nitrite production in tongue and dental plaque were reported [7]; thus, there is no consistent trend between age and nitrite productivity. Further research is needed to assess the relationship between age and dietary habits, e.g., daily intake of nitrate, and the resulting differences in the composition of the oral microbiome, especially in the nitrite productivity of nitrite-producing bacteria. On the other hand, the nitrite-degrading activity of plaque did not correlate with age (Fig. 3c). This again suggests that the bacterial groups responsible for nitrate reduction are different from those responsible for nitrite degradation, or that the metabolic systems for nitrate reduction and nitrite degradation are different.

The present study demonstrated that nitrate is converted to nitrite by plaque bacteria, which may contribute to caries prevention by suppressing the plaque pH decrease caused by food-derived carbohydrates in the oral cavity (Table 1). The plaque pH decrease before the addition of glucose was also suppressed (Table 1), suggesting that salivary nitrate can be continuously converted to nitrite and contribute to caries prevention by suppressing the pH decrease derived from bacterial intracellular polysaccharides during the time when no food is consumed. Furthermore, the production of nitrite from nitrate was enhanced by the addition of glucose (Table 1), indicating that nitrite production inhibits acid production from glucose as a self-regulatory mechanism (resilience) and contributes to plaque pH homeostasis by countering excessive acidification [28]. In addition, nitrite has been reported to inhibit the growth of caries-associated and periodontal disease-associated bacteria [10, 11, 13, 14] and is likely to contribute to the maintenance and promotion of oral health.

On the other hand, nitrate has been the subject of concern regarding its toxicity to the human body. Nitrite, which is formed from nitrate, has been implicated in methemoglobinemia in early childhood [29] and in the formation of carcinogenic N-nitrosamines by reaction with amines [29] and so is subject to regulation in food and drinking water [30]. However, the prevention of methemoglobinemia in early childhood (blue baby syndrome) is now possible, as it has been shown to be caused by the rapid growth of nitrite-producing bacteria in stomachs that are not sufficiently acidic [30]. In addition, many epidemiological studies have been conducted on the carcinogenicity of nitrates, but no evidence has been found to support a correlation between nitrates and cancer incidence [30, 31]; furthermore, the incidence rate of gastric and intestinal cancer was reduced in groups with a high vegetable-based nitrate intake [32], suggesting that nitrates are not carcinogenic, which is becoming the main view.

In recent years, nitrate supplied to the oral cavity has been found to be beneficial to health via the entero-salivary circulation of nitrogen oxide, in which nitrate is partially reduced to nitrite by nitrite-producing bacteria in the oral cavity, absorbed into the blood, gradually returned to nitrate, and secreted back into the oral cavity as saliva (Fig. 4). Nitrite has been found to exhibit antimicrobial activity not only against oral bacteria [10, 11, 13, 14] but also organisms causing gastrointestinal diseases [33]. The antimicrobial effect of nitrite is thought to be due in part to the formation of nitric oxide (Fig. 4), inactivation of Fe-S containing proteins, and inhibition of bacterial respiration [34, 35]. Nitrite also reacts with hydrogen peroxide produced by microorganisms to synthesize reactive nitrogen species such as peroxynitrite (ONOO⁻) [36], which has a stronger antimicrobial effect. Furthermore, absorbed nitrite has been shown to become nitric oxide in blood vessels and exhibit hypotensive effects due to vasodilation [37‒40] (Fig. 4). More recently, other beneficial therapeutic or preventive effects of nitrate have also been reported for hypertension, cardiovascular disease, and exercise performance [5, 41, 42].

This nitrate-derived nitric oxide is produced by the nitrate-nitrite-nitric oxide pathway, in which nitrite produced by oral bacteria is converted to nitric oxide non-enzymatically, by xanthine oxidase, by reaction with deoxyhemoglobin or deoxymyoglobin, or by other enzymes [43‒45] (Fig. 4). This nitrate-nitrite-nitric oxide pathway is different from the well-known nitric oxide synthase that produces nitric oxide from arginine [5]. For this pathway to function and contribute to oral and systemic health, as described above, it is essential to form symbiosis with oral nitrite-producing bacteria as well as obtain nitrate from food. It should be noted, however, that while there are limits to the amount of nitrate salts added to foods, we obtain most nitrate from vegetables. Vegetables, as well as tea leaves, contain antioxidants and polyphenols that may promote nitric oxide production while preventing the potential formation of N-nitroso compounds [38, 46‒48]. Low doses of nitrate to stimulate oral and general health could be safe, but high doses of nitrate could lead to N-nitroso compound formation in the absence of antioxidants. In addition, blue baby syndrome is not common in developed countries because of water quality regulations; however, water contaminated with nitrate remains a hazard for unweaned infants. Consuming adequate amounts of vegetables and living in harmony with nitrite-producing bacteria may be a cost-effective way to maintain and promote oral and systemic health.

The present study was conducted in vitro and in air using human dental plaque. Environmental factors such as buffering capacity and oxygen concentration may differ from those in the oral cavity since the former depends on salivary secretion and its reachability while the latter fluctuates between aerobic and anaerobic. To fully account for these factors, in vivo plaque pH measurements [49] are necessary, although standardization of human plaque is not easy. In the future, the antimicrobial effect of nitrate/nitrite on plaque acidification should be confirmed in vivo and combine with the present in vitro study to fully understand and verify the nitrate/nitrite effects in the oral cavity.

This study used human plaque and was conducted in accordance with the approval of the Research Ethics Committee of Tohoku University Graduate School of Dentistry (Approval No.: 2018-3-17). Written informed consent was obtained from the subjects.

The authors declare no conflict of interest.

This study was supported in part by Grants-in-Aid for Scientific Research B (21H03151) and Grants-in-Aid for Scientific Research C (20K10241) from the Japan Society for the Promotion of Science, and AMED under Grant Number (JP 23zf0127001h).

All authors contributed to the present study. Yuji Yamamoto contributed to design, acquisition, and analysis, and drafted the manuscript. Jumpei Washio contributed to design, acquisition, and analysis, and critically reviewed the manuscript. Koichi Shimizu contributed to acquisition and analysis. Nobuhiro Takahashi contributed to conception, design, analysis, and interpretation, drafted the manuscript, and critically reviewed it.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.