Comparative Evaluation of Salivary Nitric Oxide in Caries-free and Affected Children before and after Total Oral Rehabilitation

Abstract

Background

Early childhood caries (ECC) is a multifactorial disease and a major public health concern. Saliva, a noninvasive diagnostic medium, plays a vital role in oral health. Nitric oxide (NO), beyond its physiological functions, exhibits potent antimicrobial properties and may serve as a biomarker for caries activity.

Aim

To estimate salivary NO levels in children with ECC and severe ECC (S-ECC) before and after total oral rehabilitation.

Design

Sixty-six physically and mentally healthy children aged 3–5 years were categorized into three groups: group I (caries-free), group II (ECC), and group III (S-ECC). Salivary NO and dietary nitrate were assessed at baseline. Groups II and III were reassessed at 1 week and 3 months postrehabilitation.

Results

Baseline salivary NO and dietary nitrate levels were significantly higher in the caries-free group (p < 0.05). A significant increase in NO levels was observed in ECC and S-ECC groups at 3 months postrehabilitation (p < 0.05), but not at 1 week (p > 0.05).

Conclusion

Higher NO levels in caries-free children and postrehabilitation increase in NO suggest its potential as a prognostic biomarker.

How to cite this article

Priya K, Saha S, Rai K, et al. Comparative Evaluation of Salivary Nitric Oxide in Caries-free and Affected Children before and after Total Oral Rehabilitation. Int J Clin Pediatr Dent 2026;19(2):238–243.

Introduction

Dental caries, commonly known as tooth decay, represents a pervasive microbial disease characterized by the demineralization and destruction of tooth structure, often leading to cavities.¹ Among its various forms, early childhood caries (ECC) and severe early childhood caries (S-ECC) are particularly concerning due to their impact on young children's oral health. ECC is defined as the occurrence of one or more decayed (either noncavitated or cavitated), missing (due to caries), or filled tooth surfaces in any primary tooth of a child younger than 6 years of age. S-ECC refers to the presence of any carious lesion on smooth surfaces in children under 3 years of age. For children aged 3–5, S-ECC is diagnosed when there is at least one cavitated, missing, or filled smooth surface on the primary maxillary anterior teeth, or when the number of decayed, missing, or filled teeth (dmft score) is equal to or exceeds 4 at age 3, 5 at age 4, or 6 at age 5.² The importance of ECC and S-ECC lies not only in their prevalence but also in their profound impact on children's overall health and development. Dental caries at a young age can lead to pain, infection, difficulty eating, and impaired speech development.³ Moreover, untreated ECC can result in systemic health issues and even compromise a child's academic performance due to missed school days.⁴

The development of dental caries is influenced by multiple interconnected factors,⁵ including the presence of cariogenic microorganisms—especially Streptococcus mutans—frequent intake of fermentable carbohydrates, poor oral hygiene habits, and individual host-related vulnerabilities. Other factors that contribute to the occurrence and severity of ECC and S-ECC are socioeconomic conditions, accessibility to dental services, and cultural practices.⁶

In various global contexts, the prevalence of ECC varies widely. For instance, in India, studies have reported ECC prevalence rates ranging from 44 to 49% among children aged 8–48 months, with boys being more commonly affected than girls.^6,7 This variability underscores the need for targeted interventions and research to address the specific challenges posed by ECC in different populations.

Saliva, as a diagnostic medium, offers a promising avenue for exploring biomarkers related to oral health. Salivary biomarkers, including indicators of oxidative stress, have garnered attention for their potential in assessing oral health conditions such as caries.⁸ Oral health is influenced by nitric oxide (NO), which is a key component of saliva. It acts as an antimicrobial agent by inhibiting bacterial growth and enhances the immune response in the oral cavity.^9–11 However, elevated levels of NO can also contribute to oxidative stress, highlighting its complex role in oral pathology.¹¹

The rise in oxidative stress observed in children with ECC triggers the antioxidant function of NO.¹² The antioxidant properties of NO primarily stem from its direct effects at lower concentrations. However, its efficacy as an antioxidant depends on its relative concentration compared to other potential reaction sites. Therefore, the diffusion of NO to these regions is less effective in outcompeting deleterious reactions. In such cases, NO may not effectively scavenge oxidants or play a significant antioxidant role.¹¹

Despite the importance of NO in oral health, its specific implications in ECC and S-ECC remain underexplored. Although salivary total antioxidant capacity in children with caries has been investigated earlier,^11–14 research focusing specifically on salivary NO levels in children with ECC and S-ECC before and after treatment is limited. This gap in knowledge presents an opportunity to better understand NO's role as a potential biomarker and therapeutic agent in managing ECC.

Therefore, this study aims to bridge this gap by assessing and comparing NO levels in saliva of children with ECC and S-ECC undergoing total oral rehabilitation. By examining changes in NO levels alongside clinical outcomes postrehabilitation, this research seeks to provide insights that could inform novel approaches to preventing and treating ECC effectively.

Materials and Methods

Patient Enrollment

This study was carried out from September 2022 to January 2023 and involved 66 children aged between 3 and 5 years, of both genders, who reported to the Department of Pedodontics. Detailed information regarding the treatment procedures for ECC was explained, and informed consent was obtained in the local languages. Additionally, an assent form was obtained from each child before including them in the study.

Patient Criteria

The study population included children aged 3–5 years, categorized into three categories:

Group I (control): Caries-free children.

Group II (ECC): Children diagnosed with ECC.

Group III (S-ECC): Children diagnosed with S-ECC.²

Children were excluded if they were currently on medication, had systemic or metabolic conditions, required special healthcare, exhibited uncooperative behavior, or if parental consent was not obtained.

Sample Collection

A total of 66 children (n = 22 per group) who met the inclusion criteria were enrolled. At baseline, unstimulated whole saliva was collected from all participants. For groups II and III, additional saliva samples were collected at 1 week and 3 months following complete oral rehabilitation.¹⁵

The Oral Health Assessment Form (2013) by WHO was used to evaluate caries status, and the dmft index was recorded accordingly.¹⁶ During the initial visit, a 7-day dietary recall questionnaire was used to estimate the average daily nitrate intake (in mg/kg of body weight), considering both the frequency and form (raw or cooked) of foods consumed over the week. Nitrate intake calculations were based on standard reference values from existing literature.^17,18 Saliva samples were collected with the participant seated in the coachman's position Fig. 1, centrifuged, and the resulting supernatant was stored at − 70°C for subsequent biochemical analysis.¹⁹

Fig. 1:

Procedure for salivary sample collection

Rehabilitative Intervention

Oral rehabilitation for groups II and III included (Fig. 2):

Figs 2A and B:

Pre- and postintervention

• Dietary counselling focused on nitrate-rich foods and oral hygiene instructions were given.

• Oral prophylaxis.

• Topical fluoride application using 1.23% acidulated phosphate fluoride (APF) gel via foam tray for 4 minutes.²⁰

• Placement of pit and fissure sealants on primary molars.

• Restoration of carious lesions.

• Pulp therapy where indicated.

Salivary Nitric Oxide Estimation

Through Griess reaction, salivary NO concentrations were measured. In this method, sodium nitroprusside in an aqueous solution at physiological pH releases NO, which subsequently reacts with oxygen to form nitrate. This nitrate (NO₃− ) is then enzymatically converted into nitrite (NO₂− ) by nitrate reductase in the presence of NADPH. The produced nitrite is finally quantified using the Griess reaction technique.

For the assay, 50 μL of sulphanilamide solution (1% sulphanilamide in 5% phosphoric acid) and 50 μL of N-naphthyl ethylenediamine solution (.1%) were added to each well of a 96-well ELISA plate containing the saliva sample (Fig. 3). Following incubation at room temperature for 5–10 minutes, a purple-red color developed. The optical density was then read at 540 nm using a spectrophotometer. NO concentration was determined using a standard curve, and the total salivary NO level was calculated by summing the concentrations of nitrite (NO₂− ) and nitrate (NO₃− ).

Fig. 3:

Sample preparation

Statistical Analysis

SPSS version 26.0 (SPSS Inc., Chicago, IL) was used to analyze the data, with a significance level set at p < 0.05. Descriptive statistics were used to compute mean and standard deviation. The Shapiro–Wilk test was used to assess normality.

Between-group comparisons were performed using the Kruskal–Wallis test followed by Bonferroni post hoc analysis. For within-group comparisons, the Friedman test with Bonferroni correction was employed. Chi-squared test was used to analyze the categorical data, whereas Mann–Whitney U test was used for pairwise comparisons between the groups.

Results

Caries Assessment

Caries experience showed a statistically significant difference (p = 0.0001) between ECC and S-ECC groups, indicating a greater number of teeth affected in the S-ECC group.

Dietary Nitrate Intake

Dietary nitrate intake across the three groups showed significant differences (p = 0.0001). Post hoc Bonferroni analysis showed significant pairwise differences among all groups (Table 1).

Table 1:

Dietary nitrate intake across groups

Group	Mean (mg/kg)	Standard deviation (SD)
Caries-free	5241.31	1477.39
ECC	3636.77	1072.24
S-ECC	2394.31	856.73
p-value (Kruskal–Wallis test)	0.0001*
Bonferroni post hoc test
Caries-free vs ECC	0.0001*
Caries-free vs S-ECC	0.0001*
ECC vs S-ECC	0.002*

*p < 0.05 is statistically significant (Shapiro–Wilk test, p > 0.05 for normality)

Salivary Nitric Oxide Levels

Baseline Comparison

Intergroup comparison of baseline salivary NO levels showed statistically significant differences (p = 0.0001), with the highest levels in caries-free children and the lowest in the S-ECC group.

Effect of Dental Intervention

Changes in salivary NO levels before and after dental intervention are shown in Tables 2 and 3. Significant increases were observed at 3 months posttreatment in both ECC and S-ECC groups.

Table 2:

Salivary NO levels pre- and postdental intervention

Group	Before intervention (µm/dL)	1 week after (µm/dL)	3 months after (µm/dL)
ECC	132.97 ± 75.21	237.69 ± 95.91	623.65 ± 335.46
S-ECC	111.91 ± 72.24	165.29 ± 84.65	375.74 ± 236.82
p-value (Mann–Whitney U) ECC vs S-ECC	0.93	0.01*	0.007*

*p < 0.05 is statistically significant (Shapiro–Wilk test, p > 0.05 for normality)

Table 3:

Intragroup comparison of salivary NO levels (Friedman test)

Group	Overall p-value	Post hoc comparisons	p-value
ECC	0.0001*	Baseline vs 1 week	0.21
		Baseline vs 3 months	0.0001*
		1 week vs 3 months	0.0001*
S-ECC	0.0001*	Baseline vs 1 week	0.47
		Baseline vs 3 months	0.0001*
		1 week vs 3 months	0.0001*

*p < 0.05 is statistically significant (Shapiro–Wilk test, p > 0.05 for normality)

Discussion

Early childhood caries and S-ECC are major public health issues, given their widespread occurrence and considerable effect on the well-being of young children. The development of ECC is driven by a multifactorial interaction among dietary practices, oral microbial activity, and individual host factors, leading to the breakdown of tooth structure through demineralization. A key component in this process is oxidative stress, which results from disruption of equilibrium between the reactive oxygen species (ROS) and the body's antioxidant defenses.¹⁰

Nitric oxide is a multifunctional and biologically active molecule that influences oral health by regulating oxidative stress while also supporting repair of tissues. NO is produced within the oral environment via two primary mechanisms: (1) the chemical reduction of dietary nitrates and (2) the enzymatic synthesis mediated by inducible form of nitric oxide synthase (iNOS) within the salivary glands. NO serves dual functions—on one hand, it helps counteract oxidative damage due to its antioxidant capabilities; on the other, its role as a signaling molecule influencing inflammation and promoting tissue healing is noteworthy.^10,11

The dualistic nature of NO has long been debated. While traditionally considered cytotoxic, studies have also highlighted its protective antioxidant roles.¹¹ At low concentrations, NO can directly interact with biological molecules—forming complexes with metals, reacting with metal-oxygen and oxo complexes, and neutralizing radical species. These interactions help reduce oxidative DNA damage, lipid peroxidation, and prevent metalloprotein degradation caused by peroxide oxidation, thereby serving as a protective mechanism against Fenton-type oxidation.²¹ Conversely, at higher concentrations, NO can indirectly react with oxygen or superoxide to form reactive nitrogen oxide species (RNOS), such as NO₂, N₂O₃, or peroxynitrite—a potent oxidant that contributes to cytotoxicity under certain pathological conditions.¹¹

Given these diverse roles, NO has emerged as a potential biomarker in dental caries, reflecting oxidative stress associated with ECC and S-ECC. However, compared to other biomarkers like total antioxidant capacity (TAC) or lipid peroxidation products, the assessment of NO in pediatric dental caries remains underexplored.^11–14

The present study focuses on bridging this gap by evaluating salivary NO levels in children with ECC and S-ECC, both before and after comprehensive oral rehabilitation. Understanding the modulation of NO levels in response to treatment could not only enhance insight into its role in managing caries-related oxidative stress but also inform preventive and therapeutic strategies for pediatric oral health.

The results showed that salivary NO levels increased significantly in both ECC and S-ECC groups following full oral rehabilitation. Notably, the ECC group exhibited a greater increase (623.65 ± 335.46 µm/dL) compared to the S-ECC group (375.74 ± 236.82 µm/dL) after 3 months. These values aligned closely with those observed in the caries-free group (662.20 ± 321.14 µm/dL). At the 1-week mark, salivary NO levels showed significant differences between ECC and S-ECC groups; however, no significant difference was noted at baseline (pretreatment), indicating similar levels of oxidative compromise prior to intervention.

Initial assessments revealed comparatively lower baseline NO levels in ECC and S-ECC children than their caries-free controls, indicating compromised salivary antioxidant defenses. This discrepancy may be influenced by factors beyond plaque thickness, such as plaque architecture, salivary pH, dietary cariogenicity, and the composition of the oral microflora.²² Posttreatment, the rise in NO levels—particularly in the ECC group—suggests beneficial outcomes likely attributed to dietary modifications,²² fluoride-induced iNOS activation, and the release of nitrates from restorative dental materials. Although restorative materials did not induce pathological effects, certain groups did exhibit elevated inflammatory and oxidative markers.^23,24 It is worth noting that the antioxidant efficacy of NO is concentration-dependent.²⁵

The more modest increase in NO levels observed in the S-ECC group may be attributed to the more extensive disease burden. According to Wink et al. the antioxidant activity of NO is regulated by its concentration at potential reaction sites.¹¹ In cases of severe decay, such as S-ECC, the oxidative stress burden is likely higher, thereby increasing the demand for NO and potentially overwhelming the system. Furthermore, Sueishi et al. suggest that the antioxidant system can act as an NO scavenger through redox mechanisms, possibly diminishing NO bioavailability in these cases.²⁵

Additionally, physiologic NO production via immune responses requires time—often several hours—to initiate. In disease states, particularly where stimuli are variable or extensive, this process may be further delayed.²⁶ The anti-inflammatory actions of NO are generally acute and may not be sufficient in chronic oxidative conditions such as S-ECC. The higher NO increase in the ECC group may therefore reflect better healing potential and fewer carious lesions compared to the S-ECC group.²⁷

These findings are consistent with Shahi et al. who also reported increased salivary NO levels postrehabilitation.²⁸ Dietary changes, as part of the treatment protocol, could have contributed to this rise. Salivary nitrite levels are influenced by dietary nitrate intake, nitrate-reductase activity of oral bacteria, salivary flow rate, and endogenous nitrate synthesis.²² Burleigh et al. emphasized the impact of dietary nitrates on NO production, with potential implications for oral health.²⁹ Nitrate supplementation has also been associated with increased acid resistance in saliva, thereby aiding in caries prevention.³⁰

Several studies support the association between higher salivary NO levels and caries resistance. Syed et al. and Hegde et al. observed elevated NO in caries-free individuals, suggesting a protective role in reducing caries incidence.^31,32 However, Qujeq et al. noted that poor oral hygiene might elevate NO levels without effectively preventing caries.³³ Moreover, Elyassi Gorji et al. through a meta-analysis, found no consistent correlation between salivary NO levels and caries in children, underscoring the complexity of NO's role in oral health.³⁴

Despite these complexities, oxidative stress indices remain valuable in tracking disease progression and treatment response.³⁵ The rise in NO levels posttreatment observed in this study highlights its potential as a prognostic biomarker—an assertion supported by previous studies including those by Hegde et al., Senthil Eagappan et al., and Assudani et al.^22,36,37

Nevertheless, this study has several limitations. Dietary nitrate intake, which significantly impacts NO levels, was not standardized. Variability in antioxidant consumption and physical activity—both influential to NO production—were not controlled.^38,39 Furthermore, differences in oral microbiota composition, known to influence NO synthesis and caries susceptibility, were not evaluated.⁴⁰

Future research should address these limitations by incorporating standardized dietary assessments and controlling confounding variables. Longitudinal studies could further elucidate dynamic NO level fluctuations throughout the course of caries development and treatment. Evaluating NO in conjunction with other oxidative stress biomarkers could also provide a more comprehensive understanding of its role in pediatric oral health.

Conclusion

In conclusion, the study demonstrated that salivary NO levels were significantly elevated in caries-free individuals compared to those with ECC and S-ECC. The increase in NO after 3 months of intervention was greater in the ECC group than the S-ECC group. These findings suggest that NO may serve as a potential biomarker for oxidative stress and treatment response in ECC and S-ECC, with dietary nitrate potentially playing a key role in oral health. Salivary NO may also possess antibacterial properties, making it a promising prognostic biomarker.

Orcid

Krishna Priya https://orcid.org/0009-0003-8939-2329

Swagata Saha https://orcid.org/0000-0002-2343-5977

Kavita Rai https://orcid.org/0000-0002-9677-4144

Prajna P Nayak https://orcid.org/0000-0001-8872-860X

Manju R Nair https://orcid.org/0000-0002-9391-6223

Prajna Bhandary https://orcid.org/0000-0002-3268-6743

Sharmila Kameyanda Poonacha https://orcid.org/0000-0002-1604-2120

Nishi Joshi https://orcid.org/0009-0004-0381-1586

Aishani Baksi https://orcid.org/0000-0002-7279-7827