|Year : 2016 | Volume
| Issue : 1 | Page : 17-22
Correlation of plaque nitric oxide levels with plaque Streptococcus mutans, plaque pH and decayed, missing and filled teeth index of children of different age groups
Sawinderjit Saini, Hina Noorani, PK Shivaprakash
Department of Pedodontics, PMNM Dental College and Hospital, Bagalkot, Karnataka, India
|Date of Web Publication||2-Feb-2016|
Department of Pedodontics, PMNM Dental College and Hospital, Bagalkot, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: Dental plaque is considered one of the most important etiological factors for dental caries. Nitrate and Nitrite levels in saliva are considered protective against oral and gastrointestinal disease. Plaque nitric oxide (NO) levels and its role in dental caries has not be explored in the literature. Aim: Determine the correlation of the plaque nitric oxide (NO) with dental caries in vivo. Materials and Method: 75 healthy children between ages of 3-15 years were selected. The age, state of dentition and the level of caries in all individuals were determined by the same examiner, using DMFT/deft index. The plaque samples collected were subjected to nitric oxide estimation, streptococcus mutans count and pH for correlation. Statistical Analysis Used: SPSS computer based software was used for descriptive data analysis. ANOVA one way analysis and Karl pearson's correlation was carried out. Results and Conclusion: Statistically significant relationship was observed between plaque nitric oxide and dental caries in all age groups. Similarly, significant correlation of nitric oxide was found in relation to plaque streptococcus mutans and plaque pH. Thus concluded, plaque nitric oxide can be considered as a risk assessment tool for prediction of dental caries.
Keywords: Dental caries, plaque nitric oxide, plaque Streptococcus mutans
Key message: Plaque nitric oxide results in the activation of the host defense mechanism for protection. Thus can be considered as a risk assessment tool for prediction of dental caries.
|How to cite this article:|
Saini S, Noorani H, Shivaprakash P K. Correlation of plaque nitric oxide levels with plaque Streptococcus mutans, plaque pH and decayed, missing and filled teeth index of children of different age groups. J Indian Soc Pedod Prev Dent 2016;34:17-22
|How to cite this URL:|
Saini S, Noorani H, Shivaprakash P K. Correlation of plaque nitric oxide levels with plaque Streptococcus mutans, plaque pH and decayed, missing and filled teeth index of children of different age groups. J Indian Soc Pedod Prev Dent [serial online] 2016 [cited 2021 Mar 1];34:17-22. Available from: https://www.jisppd.com/text.asp?2016/34/1/17/175505
| Introduction|| |
Caries is said to be a multifactorial disease with more prevalence in children. Different individuals of the same age, sex, race, and geographic area sustaining on the similar diets under the same living conditions accentuate the complexity of the caries problem. Nitrate is an essential plant nutrient, taken up by plants in large quantities from the soil as their principle nitrogen source.  Nitrate is, therefore, a natural component of all cereals, fruit, and vegetables. Dietary nitrate was described to form the basis for a nonimmune system mediated mechanism of defense against gastrointestinal and oral pathogens in animals and humans. 
Numerous factors appear to be elemental in the salivary defense system. Saliva and its organic and inorganic components have therefore been extensively studied to evolve newer preventive measures. There has been growing interest in the role of nitrate and nitrites level of saliva in protecting against oral and gastrointestinal disease,  which led us to consider the possible relevance of salivary nitric oxide (NO) levels and its role in dental caries.
The acidification of nitrite produces a complex mixture of nitrogen oxides as well as nitrous acid. Nitrous acid is unstable and will spontaneously decompose to produce NO and nitrogen dioxide.  Nitrites acidification occurs in acid environment of teeth tissues.
Acid surrounding is obtained by existing microflora including Lactobacillus, Streptococcus mutans, Actinomyces, microorganisms implied in dental caries,  as well as Staphylococcus aureus, and Staphylococcus epidermidis.  These local sites of extreme pH depression make nitrite conversion to antimicrobial components possible. As the dentition makes transition from primary to permanent, microflora, and dietary patterns changes, three groups were included in the study.
Plaque is considered the most important factor in initiation and progression of carious lesion compared to saliva.  Aim of the study was to investigate relationship between the plaque NO, plaque S. mutans levels, plaque pH, and different level of dental caries.
| Materials and Methods|| |
The study was conducted on the children reporting to the outpatient Department of Pedodontics and Preventive dentistry, from which 75 healthy children between ages ranging from 3 to 14 years were selected for this study.
These children were divided into three groups comprising 25 children in each group according to dentition exhibited.
- Group 1: 25 children with primary dentition only.
- Group 2: 25 children with mixed dentition.
- Group 3: 25 children with permanent dentition.
General inclusion criteria
- Children who had no history of any medical disorder.
- Children who refrained from eating at least 2 h before the plaque sample collection. 
- Children having a minimum decayed, missing and filled teeth/decayed, extracted or filled deciduous teeth (DMFT/deft) of 1 (according to World Health Organization criteria). 
- Children suffering from any medical disorder.
- Children with DMFT/deft-0.
- Subjects who had their meal <2 h before the plaque sample collection. 
In subjects of all the three groups, a thorough medical history and dental examination were done. Healthy children with a minimum of DMFT/deft-1 were selected for the study.
The age, state of dentition, and the level of caries in all individuals were determined and categorized by the same examiner using DMFT/deft index  while seated on the dental chair [Figure 1].
The plaque samples were collected from the maxillary teeth by means of toothpick  by scraping the buccal surface of the maxillary second primary molar (Group 1) and maxillary first permanent molar (Group 2 and Group 3) dipped in the sterilized vial containing 1 mL saline [Figure 2] and [Figure 3].
|Figure 2: Collection of plaque from the buccal surface of maxillary tooth|
Click here to view
|Figure 3: Transfer of plaque sample to 1 mL of normal saline (transport media)|
Click here to view
After the collection of sample, they were divided into 2 vials of 0.5 mL sample each. A volume of 0.5 mL of the sample collected was used for the determination of the NO in Pharmacology laboratory whereas the other 0.5 mL was cultured for the S. mutans count [Figure 4] using Mitis Salivarius Agar Media  (Himedia M259, Lot no Y1100) with potassium tellurite (FD 052) and Bacitracin. The determination of the pH in the Microbiology Laboratory was carried out using digital pH meter (Equitronics, model no - EQ-612). Estimation of the NO concentration was done by the Griess reaction method  which is as follows-mix the following in a spectrophotometer cuvette (1 cm pathlength): 1 mL of Griess Reagent (G4410-10G, Sigma Aldrich), 3 mL of the nitrite-containing sample, and 2.6 mL of deionized water. Incubate the mixture for 30 min at room temperature. Prepare a photometric reference sample by mixing 1 mL of Griess Reagent (above) and 2.9 mL of deionized water. Measure the absorbance of the nitrite-containing sample at 550 nm using spectrophotometer (Shimadzu, UV-1601), relative to the reference sample [Figure 4]. Convert absorbance readings to nitrite concentrations using standard graph and calibrated in micronmeters.
| Results|| |
Totally, 75 healthy children, ages ranging from 3 to 14 years, were selected for the study, which consisted of 45 males and 30 females. Mean age in Group 1 is 4.64 years, Group 2 is 8.72 years, and Group 3 is 13.24 years. Scores of each variable in comparison to groups are presented in [Table 1], [Table 2], [Table 3] and [Table 4], respectively.
|Table 1: Comparison of three Groups (1, 2, and 3) with respect to DMFT scores by one-way ANOVA|
Click here to view
|Table 2: Comparison of three Groups (1, 2, and 3) with respect to NO level estimation (micrometer) scores by one-way ANOVA|
Click here to view
|Table 3: Comparison of three Groups (1, 2, and 3) with respect to log S. mutans in plaque (colony/unit) scores by one-way ANOVA|
Click here to view
|Table 4: Comparison of three Groups (1, 2, and 3) with respect to pH of plaque scores by one-way ANOVA|
Click here to view
DMFT scores in Group 1, 2, and 3 were 3.64, 4.12, and 4.09, respectively, which was nonsignificant. Similarly, Tukeys post-hoc tests were nonsignificant with respect to plaque NO, plaque S. mutans, and plaque pH on comparing different groups. Correlations were estimated with Karl Pearson's correlation coefficient method [Table 5].
|Table 5: Correlations among different variables in total samples of all three groups by Karl Pearson's correlation coeffi cient method|
Click here to view
In Groups 2 and 3, DMFT had very strong positive correlation with NO level estimation and strong positive correlation with S. mutans in plaque (colony/unit), moderate negative correlation with pH of plaque (r = −0.5127). All of these individual variables were statistically significant in relation to each other with P < 0.05 [Table 5].
NO levels with S. mutans in plaque (colony/unit) showed strong positive correlation, which was statistically significant (P < 0.05); when compared to NO level with pH of plaque, no significant weak negative correlation was seen. In Group 2, moderate correlation in Groups 1 and 3 was noted, which was statistically significant [Table 5].
| Discussion|| |
Saliva is often referred to as the "mirror of the body" as it is the indicator of health not just in the oral cavity but also throughout the body.  Saliva consists of therapeutic, hormonal, immunologic, and toxicological molecules, which can provide vital clues to systemic health. 
The elements of salivary defense system, i.e., organic and inorganic compounds, are the significant factors in caries. A large number of salivary substances have direct or indirect role in caries onset. ,
Several inter-related factors such as tooth and saliva (host), microorganisms and cariogenic substrate result in dental caries. These factors interact in a certain period, causing an imbalance in the demineralization and remineralization between tooth surface and the adjacent plaque (biofilm). 
Plaque is considered a more important factor in initiation and progression of carious lesion compared to saliva.  As reported in experimental studies,  NO levels were significantly higher in the dental plaque than in saliva. Thus, dental plaque was evaluated for assessing the effect of NO on the teeth along with its inhibition capacity to eliminate the S. mutans. Similarly, plaque pH has direct effect on demineralization process that is related with the dmft and S. mutans count.
Dietary nitrates (principally derived from green, leafy vegetables) are rapidly absorbed from the gastrointestinal tract and mix with endogenously synthesized nitrate, which mainly comes from the L-arginine-NO pathway.  Most nitrate is ultimately excreted in the urine. Up to 25% of plasma nitrate is actively taken up by the salivary glands and secreted with saliva,  and the resulting salivary nitrate concentrations can be at least 10 times higher than the concentrations.
Three different age groups included in the study showed similar pattern of result although the statistically significant correlation among the variables were high in the Group 2.
In the present study, irrespective of the group considering the NO and DMFT/deft strong significant positive correlation was seen. These results are in accordance with the results by Bayindir et al.,  Mobarak,  and Javandenizad  leading to consideration that NO production might be a host defense mechanism when dental caries increases. Whereas the study by Hegde et al.  and Hegde et al.  achieved strong negative correlation between the salivary NO and DMFT scores concluding that caries incidence might be low in subjects with high NO levels. In them, plaque deposition might have permitted conversion of nitrite at acidity levels below pH-7 and activated inducible NO synthase (iNOS), promoting the conversion of L-arginine to NO and its compounds.  Results in contrast to other studies may be first, due to the intake of nitrate-rich foods causing transient increase in the salivary NO but also of the NO in exhaled air, , which reverses within 4 h. The level can increase some 30-fold following ingestion of food or drink rich in nitrate containing compounds, mostly derived from leafy vegetables.  Large inter individual variations in nitrate and nitrite concentrations similar to the present results have been observed previously.
Second, increase in the bacterial count due to increased DMFT might have resulted in the increased production of the NO by nitrate reductase enzyme synthesized by facultative anaerobic bacteria when exposed to low oxygen tension, , allowing nitrite ion to be metabolized.  NO here is derived from enzymatic pathway. Plaque deposition might have activated the iNOS promoting the conversion of L-arginine into NO and its compounds. 
Results in Group 2 and Group 3 are in strong positive correlation whereas in Group 1, moderate positive correlation was observed between NO and S. mutans that were highly significant. Reason for this could be the change in the dietary patterns as the age increases and the eruption of the permanent teeth results in tight contacts leading to more caries risk.
Results here are in accordance with Hegde et al.  and in contrast to Doel et al.  in vitro studies demonstrated that at acidity levels of below pH 7, low concentrations of nitrite caused effective complete killing of S. mutans.  In other study by Radcliffe et al.,  level of NO more than 20 μM was both bactericidal and antiacidogenic with respect to S. mutans .
Correlating DMFT with the S. mutans and pH provided strongly positive highly significant correlation (P < 0.05) and negative highly significant correlation, respectively in all groups. Results are in accordance with the study conducted by Jafar et al.,  Alaluusua and Renkonen  where the high S. mutans in plaque was seen in caries active children (dmft >3). In contrast, Palmerini et al.  concluded that the presence of wide range of bacteria in oral cavity can produce/synthesize NO even at neutral pH value.
Statistically significant negative correlation was obtained with the NO and plaque pH. Results are in accordance with Smith et al.  and Pannala et al. 
Intraoral production of the NO may depend on many factors, such as nitrate intake, salivary flow rates, specific nitrate reductase activities of the microbial flora, their carriage rate, and spatial distribution in relation to the salivary nitrate flow. 
For future studies, reactive nitrogen species, such as N 2 O 3 formed from NO and NO 2 , have been shown to react with thiols to form S-nitrosothiols,  which could have longer half-lives and greater stability than NO, while exerting similar biological activity. 
No single salivary antibacterial component in vivo is particularly significant in determining caries risk.  More nitrous containing product can be consumed for better protection. Thus, further experimental and clinical studies are needed for progress to assess the potential activity of nitrite against dental caries.
| Conclusion|| |
The major conclusions drawn from our study are:
- Strong positive significant correlation between plaque NO levels (micronmeter) and DMFT/deft were obtained. Increase in the NO levels is directly proportional to the DMFT/deft. As the transition from primary (Group 1) to permanent dentition (Group 2) occurs, there is a rise in the NO levels.
- There is positive strong significant correlation between plaque NO levels (micronmeter), and S. mutans were obtained which signifies the activation of the host defense mechanism for protection.
- Inverse relationship was observed between the plaque NO and plaque pH stating that as the pH drops. the plaque NO level increases. More of the nitrate synthase is released from the bacteria when pH drops. Thus, increasing the NO levels in plaque.
- Positive direct correlation was seen between the DMFT/deft scores and S. mutans count. As the colony forming units of S. mutans increases, the caries incidence also rises.
- NO estimation can be a screening tool to predict level of dental caries.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Duncan C, Li H, Dykhuizen R, Frazer R, Johnston P, MacKnight G, et al.
Protection against oral and gastrointestinal diseases: Importance of dietary nitrate intake, oral nitrate reduction and enterosalivary nitrate circulation. Comp Biochem Physiol A Physiol 1997;118:939-48.
Duncan C, Dougall H, Johnston P, Green S, Brogan R, Leifert C, et al.
Chemical generation of nitric oxide in the mouth from the enterosalivary circulation of dietary nitrate. Nat Med 1995;1:546-51.
Lundberg JO, Weitzberg E, Lundberg JM, Alving K. Intragastric nitric oxide production in humans: Measurements in expelled air. Gut 1994;35:1543-6.
De Soet JJ, Nyvad B, Kilian M. Strain-related acid production by oral streptococci. Caries Res 2000;34:486-90.
Radcliffe C, Vierjoki T, Stahlber T. Effects of nitrite and nitrate on the growth and acidogenicity of Streptococcus mutans
. J Dent 2002;30:325-31.
Carossa S, Pera P, Doglio P, Lombardo S, Colagrande P, Brussino L, et al.
Oral nitric oxide during plaque deposition. Eur J Clin Invest 2001;31:876-9.
Bayindir YZ, Polat MF, Seven N. Nitric oxide concentrations in saliva and dental plaque in relation to caries experience and oral hygiene. Caries Res 2005;39:130-3.
World Health Organization. Oral Health Survey: Basic Methods. 3 rd
ed. Geneva: World Health Organization; 1997.
Wennerholm K, Lindquist B, Emilson CG. The toothpick method in relation to other plaque sampling techniques for evaluating mutans streptococci. Eur J Oral Sci 1995;103: 36-41.
Gold OG, Jordan HV, Van Houte J. A selective medium for Streptococcus mutans
. Arch Oral Biol 1973;18:1357-64.
Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and (15N)nitrate in biological fluids. Anal Biochem 1982;126:131-8.
Ahmadi-Motamayel F, Davoodi P, Dalband M, Hendi SS. Saliva as a mirror of the body health. DJH 2010;1:1-15.
Zhang L, Xiao H, Wong DT. Salivary biomarkers for clinical applications. Mol Diagn Ther 2009;13:245-59.
Slavkin HC. Protecting the mouth against microbial infections. J Am Dent Assoc 1998;129:1025-30.
Surdilovic DS, Stojanovic I, Apostolovic M. Salivary nitric oxide as biomarker of caries risk in children. Acta Stomatol Croat 2009;43:39-44.
Harris R, Nicoll AD, Adair PM, Pine CM. Risk factors for dental caries in young children: A systematic review of the literature. Community Dent Health 2004;21 1 Suppl:71-85.
Leaf CD, Wishnok JS, Tannenbaum SR. L-arginine is a precursor for nitrate biosynthesis in humans. Biochem Biophys Res Commun 1989;163:1032-7.
Spiegelhalder B, Eisenbrand G, Preussmann R. Influence of dietary nitrate on nitrite content of human saliva: Possible relevance to in vivo
formation of N-nitroso compounds. Food Cosmet Toxicol 1976;14:545-8.
Mobarak E, Abdallah D. Saliva nitric oxide levels in relation to caries experience and oral hygiene. J Adv Res 2011;2:357-62.
Javadinejad SH, Talebi M, Aslani G. Nitric oxide concentrations in saliva in relation to caries; experience in 6-12 years old children. J Isfahan Dent Sci 2008;3:71-5.
Hegde AM, Neekhra V, Shetty S. Evaluation of levels of nitric oxide in saliva of children with rampant caries and early childhood caries: A comparative study. J Clin Pediatr Dent 2008;32:283-6.
Hegde M, Kumari M, Hegde N, Shetty S. Evaluation of the status of salivary nitric oxide in patients with dental caries. Nitte Univ J Health Sci 2012;2:6-9.
Olin AC, Aldenbratt A, Ekman A, Ljungkvist G, Jungersten L, Alving K, et al.
Increased nitric oxide in exhaled air after intake of a nitrate-rich meal. Respir Med 201;95:153-8.
Rolla G, Bucca C, Brussino L, Dutto L, Colagrande P, Polizzi S. Pentoxifylline attenuates LPS-induced bronchial hyperresponsiveness but not the increase in exhaled nitric oxide. Clin Exp Allergy 1997;27:96-103.
Tannenbaum SR, Weisman M, Fett D. The effect of nitrate intake on nitrite formation in human saliva. Food Cosmet Toxicol 1976;14:549-52.
Forman D, Al-Dabbagh S, Doll R. Nitrates, nitrites and gastric cancer in Great Britain. Nature 1985;313:620-5.
Zetterquist W, Pedroletti C, Lundberg JO, Alving K. Salivary contribution to exhaled nitric oxide. Eur Respir J 1999;13:327-33.
Klebanoff SJ. Reactive nitrogen intermediates and antimicrobial activity: Role of nitrite. Free Radic Biol Med 1993;14:351-60.
Matejka M, Partyka L, Ulm C, Solar P, Sinzinger H. Nitric oxide synthesis is increased in periodontal disease. J Periodontal Res 1998;33:517-8.
Doel JJ, Hector MP, Amirtham CV, Al-Anzan LA, Benjamin N, Allaker RP. Protective effect of salivary nitrate and microbial nitrate reductase activity against caries. Eur J Oral Sci 2004;112:424-8.
Silva Mendez LS, Allaker RP, Hardie JM, Benjamin N. Antimicrobial effect of acidified nitrite on cariogenic bacteria. Oral Microbiol Immunol 1999;14:391-2.
Jafar ZJ, Al-Bayati YA, Taha GI. Correlation between caries related bacteria in plaque and saliva in different age group children. J Baghdad Coll Dent 2012;24:140-4.
Alaluusua S, Renkonen OV. Streptococcus mutans
establishment and dental caries experience in children from 2 to 4 years old. Scand J Dent Res 1983;91:453-7.
Palmerini CA, Palombari R, Perito S, Arienti G. NO synthesis in human saliva. Free Radic Res 2003;37:29-31.
Smith AJ, Benjamin N, Weetman DA, Mackenzie D, MacFarlane TW. The microbial generation of nitric oxide in the human oral cavity. Microb Ecol Health Dis 1999;11: 23-7.
Pannala AS, Mani AR, Spencer JP, Skinner V, Bruckdorfer KR, Moore KP, et al.
The effect of dietary nitrate on salivary, plasma, and urinary nitrate metabolism in humans. Free Radic Biol Med 2003;34:576-84.
Stamler JS, Simon DI, Osborne JA, Mullins ME, Jaraki O, Michel T, et al.
S-nitrosylation of proteins with nitric oxide: Synthesis and characterization of biologically active compounds. Proc Natl Acad Sci U S A 1992;89:444-8.
Hogg N. The biochemistry and physiology of S-nitrosothiols. Annu Rev Pharmacol Toxicol 2002;42:585-600.
Kirstilä V, Häkkinen P, Jentsch H, Vilja P, Tenovuo J. Longitudinal analysis of the association of human salivary antimicrobial agents with caries increment and cariogenic micro-organisms: A two-year cohort study. J Dent Res 1998;77:73-80.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]