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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 9  |  Issue : 2  |  Page : 190-195

Evaluation of salivary nitric oxide levels in caries-free children and children with early childhood caries: An in vivo study


1 Department of Pediatric Dentistry, KLE VK Institute of Dental Sciences, Belagavi, Karnataka, India
2 Department of Biochemistry, JN Medical College, KLE University, Belagavi, Karnataka, India
3 Department of Pediatric Dentistry, MM University, Mullana, Haryana, India

Date of Web Publication29-Sep-2016

Correspondence Address:
Harsha Gope Assudani
House No. 30, Sector 7, Panchkula - 134 109, Haryana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2349-5006.191270

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  Abstract 

Background and Objectives: Early childhood caries (ECC) is one of the most prevalent diseases of childhood. Pediatric dentists must make conscious efforts to prevent this condition for optimal oral health. Normal salivary function is considered critical for the maintenance of a healthy oral cavity. Saliva provides an easily available, noninvasive medium for the diagnosis of wide range of diseases and clinical conditions. The objective of the present study was to estimate and compare salivary nitric oxide (NO) levels in caries-free children and children with ECC.
Methodology: The children were divided into two groups. Group I comprised thirty caries-free children and Group II comprised thirty children with ECC. Saliva was collected by suction method. Griess reaction was used to estimate the NO levels. Unpaired t-test was used for comparing and evaluating the NO levels in both the groups.
Results: Mean salivary Nitric Oxide level is significantly higher in caries free children as compared to that of children with early childhood caries (ECC).
Interpretation and Conclusion: The present study clearly indicates a significant increase in salivary NO levels in caries-free children as compared to children with caries. This may be attributed to the antimicrobial action of NO.

Keywords: Early childhood caries, nitric oxide, saliva


How to cite this article:
Assudani HG, Hugar SM, Patil A, Sogi SH, Dhariwal NS, Thakkar P. Evaluation of salivary nitric oxide levels in caries-free children and children with early childhood caries: An in vivo study. Indian J Health Sci Biomed Res 2016;9:190-5

How to cite this URL:
Assudani HG, Hugar SM, Patil A, Sogi SH, Dhariwal NS, Thakkar P. Evaluation of salivary nitric oxide levels in caries-free children and children with early childhood caries: An in vivo study. Indian J Health Sci Biomed Res [serial online] 2016 [cited 2022 May 17];9:190-5. Available from: https://www.ijournalhs.org/text.asp?2016/9/2/190/191270


  Introduction Top


Dental caries is a multifactorial local disease that involves destruction of the hard tissues of the teeth by metabolites produced by oral microorganisms. It is a communicable and infectious disease. The uniqueness of dental caries makes it a fascinating study from a scientific standpoint. The disease of early childhood caries (ECC) is the presence of 1 or more decayed (noncavitated or cavitated lesions), missing (due to caries), or filled tooth surfaces in any primary tooth in a child 71 months of age or younger. The main cariogenic microorganisms responsible for ECC are Streptococcus mutans and Streptococcus sobrinus. [1]

Recently, it has been claimed that oxidative stress plays an important role in the onset and development of dental caries. Nitric oxide (NO) is a labile and highly reactive gas, and it has been reported that NO plays an important role in immune response, neurotransmission, and vasodilation in a variety of biological tissues. In humans, ingested nitrate is concentrated in the salivary glands up to ten times of that found in the plasma. [2],[3] The use of saliva for diagnosis of oral diseases has been a subject of considerable research activity. The markers include enzymes and immunoglobulins (proteins of host defense), epithelial keratins (phenotypic markers), host cells, hormones such as cortisol, bacteria and bacterial products, volatile compounds, ions, and free radicles such as peroxide and NO. [4],[5] Saliva could be considered as constituent which forms a first line of defense against free radical-mediated oxidative stress. This is the why antioxidant capacity of saliva has led to increasing interest and thus the need for this study. [6]


  Methodology Top


An in vivo study was designed to evaluate and compare the salivary NO levels in caries-free children and children with ECC. The required sample for the study was obtained from the Outpatient Department of Pedodontics and Preventive Dentistry, KLE Vishwanath Katti Institute of Dental Sciences, Belagavi. Ethical clearance for the study was obtained from the Ethical Committee of the college.

Source of data

The study was conducted in the Department of Pedodontics and Preventive Dentistry KLE VK Institute of Dental Sciences, Belagavi, with the assistance from the Department of Biochemistry, JN Medical College, KLE University, Belagavi.

Selection of subjects

Inclusion criteria

  • Caries-free children constituted the control group
  • Children with ECC constituted the experimental group
  • Children free from systemic or local diseases which affect salivary secretions
  • Children residing in Belagavi district and consuming water from the same municipal source.
Exclusion criteria

  • Children with special health-care needs
  • Children who were on medications (4 weeks before study)
  • Children unwilling to participate in the study
  • Children whose parents were not willing to give informed consent.
Methods

Procedure used in the study

  • The study was carried out on sixty subjects who fulfill the inclusion criteria of the study. All potential participants were explained the need and design of the study. The informed consent was obtained from the parent/guardian was take. Assent was obtained from all the children participating in the study. Data were collected from the subjects who were fulfilling all the criteria defined in the study.


Selection of case and recording of case history

  • A case history was taken for the present study so as to have a systematic and methodological recording of all the observations and information. The relevant data comprising details of the chief complaint, preliminary history, brushing habits, etc., were recorded. After recording of preliminary information, clinical examination was carried out on a dental chair.


Study groups

The selected sixty samples were divided into two groups.

  • Group I (control group): Thirty Caries-free children
  • Group II (experimental group): Thirty Children with ECC.
Procedure for saliva collection

  • Children in both the groups were asked not to have anything to eat or drink 2 h before collection of saliva. Furthermore, they should not have used any mouthwash in that duration. The saliva collection was done during morning hours between 9 am to 10 am. Two milliliters of unstimulated whole saliva was collected for the study from both the groups. Saliva collection was done by suction method. Patient was made to sit, head slightly down, and asked not to swallow during the period of collection. The saliva was allowed to accumulate in the patients mouth for 2 min and was aspirated directly from the floor of the mouth using a disposable syringe (needle removed). The saliva was then transferred to Department of Biochemistry for analysis of salivary NO levels.


Biochemical analysis



NO levels were estimated using classical Griess reaction in the Department of Biochemistry, KLE Jawaharlal Nehru Medical College, Belagavi. The Griess reagent was prepared using 1% sulfanilamide, 1% naphthyl ethylenediamine dihydrochloride, and 2.5% phosphoric acid. The samples of the sixty subjects were added to Griess reagent, transferred to spectrophotometer and their optical densities were recorded. The optical densities were then correlated in the standard curve [Figure 1], and the corresponding concentrations of nitrite were observed. NO estimation was done which is the sum of NO 2 and NO 3 as mentioned by Miranda et al., with some modification. [7]
Figure 1: Calculation of total NO in salivary sample using standard value as reference


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Preparation of calibration curve

The concentration of NaNO 2 solution is plot on X-axis, and the absorbance value is plot on Y-axis and the calibration curve is prepared.

Calibration curve





Where, As T is absorbance of test sample

As S is absorbance of standard sample [Figure 2].
Figure 2: Standardization of reagents


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Statistical analysis

  • All the data were entered on Microsoft Excel sheet and subjected to statistical analysis using statistical package for social sciences version 18. Unpaired t-test was used to compare the NO levels between the two groups.

  Result Top




All the data was entered on Microsoft Excel sheet and subjected to statistical analysis using statistical package for social sciences (SPSS) version 18. Unpaired 't' test was used to compare the Nitric oxide (NO) levels between the two groups. ANOVA - analysis of variance was used to compare the difference in the Nitric oxide levels in different groups, gender as well as age.

The mean age of participants in Group I was found to be 3.3 +/- 0.80 while that in group II was found to be 3.2 +/- 0.69. [Table 1] shows the mean optical density of saliva samples of participants in Group I and Group II. Mean salivary nitric oxide levels of participants in Group I and Group II has been depicted in [Table 2].
Table 1: Mean optical density of saliva samples of participants in Group I and Group II


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Table 2: Mean salivary nitric oxide levels of participants in Group I and Group II


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  Discussion Top


ECC is an alarming problem as the disease is widespread and if untreated; it can lead to serious disability. ECC results in pain, impairment of function, deleterious influence on the child's growth rate, body weight, and ability to thrive, hence reducing quality of life. [8] Hence, the caretakers as well as the pediatric dentists must aim at prevention of this debilitating disease. Since NO has been known to have antibacterial properties in the oral cavity, the aim of this study was to evaluate the salivary NO levels in children with and without ECC.

The prevalence of ECC in preschool children in Bangalore city was 27.5%. ECC increases significantly with age. Children whose mothers had no schooling and those who belonged to low socioeconomic strata showed a higher prevalence of caries. A significant increase in caries prevalence was found in children who were accustomed to the practice of on-demand breastfeeding and bottle feeding at night. [9]

For caries prevention or reversal, it is necessary to effectively increase the effect of one or more protective factors or to decrease the effect of one or more pathological factors. Thus, caries can be controlled with reduction in acid-producing bacteria such as S. mutans and increasing the protective factors. [10] Therefore, researchers are inclined toward finding protective factors that prevent dental caries.

NO, a short living product of nitrogen metabolism, is produced by many cells in the organism. It is an endogenous mediator of various physiological processes that range from neurotransmission to cardiovascular function. Furthermore, it has antipathogenic and tumoricidal responses. [7] Since NO has been known to have antibacterial properties in the oral cavity, the aim of our study was to evaluate the salivary NO levels in children with and without ECC.

Saliva is a multiconstituent oral fluid regulates and maintains the integrity of the oral mucosa and therefore plays a very important role in maintaining the oral health. Saliva being noninvasive and easily obtainable is gaining attention by various researchers as a diagnostic tool. Most compounds found in the blood are also present in saliva; therefore, saliva is functionally equivalent to serum in reflecting the physiological state of the body including hormonal, emotional, nutritional, and metabolic variations. Salivary diagnosis is anticipated to be particularly useful in cases where repeated samples of body fluids are required but drawing blood is impractical unethical or both. [11] Hence, the NO level of saliva was tested in our study.

Salivary flow rate shows a circadian rhythm. Hence, the time of day for saliva collection must be standardized. It is best to collect saliva while the subject is sitting upright with the head slightly tilted forward and the eyes open. [12]

In this study, the suction method was used for the collection of unstimulated whole saliva. Salivary samples were taken in the morning between 9 am and 10 am in to minimize the effect of circadian rhythm on the samples.

Hegde et al. had concluded that NO (NO 2 + NO 3 ) is a potential biomarker of caries risk in adults which has antimicrobial activity. [13] In another study, it was concluded that salivary nitrite levels in caries-free children were higher as compared to the children with caries. [14] These results were similar to the present study [Table 2]. Our study showed a negative correlation of NO levels and ECC. The range of salivary NO level in this study was 38.02 μM (decayed, missing, and filled teeth [dmft] index score 5) to as high as 752.11 μM (dmft index score zero).

Salivary nitrate is derived from both, the dietary as well as metabolic sources. Dietary nitrate is present in large quantities in green leafy vegetables. Many children in the industrialized countries are indulged in eating fast food that lacks green vegetables. One way of reducing caries in these children may be by increasing nitrate intake so that nitrate reducing bacteria are established in the oral cavity. These bacteria (Veillonella dispar and Veillonella atypica) may suppress the growth of acid-forming bacteria and thus protect teeth against caries. In addition, probiotic therapy at an early age that encourages colonization of oral cavity with these microorganisms may be a potential therapy against dental caries. [15]

Silva Mendez et al. have stated that nitrite acidified artificially with hydrochloric acid had a dramatic effect on the growth and survival of exposed bacteria. A low nitrite concentration of 0.02 mM (the normal fasting salivary level) was inhibitory when compared with the negative controls. This indicates that some amount of inhibition of acid production by acidified nitrite may occur naturally, and hence NO as a supplement in mouth rinse may merely suffice. [16]

Not only bacteria but also yeast such as Candida albicans is sensitive to acidified nitrite. The incidence of oral thrush is increased by antibiotic treatment, and it has been suggested that this may be due to diminution of salivary nitrite concentration. Moreover, nitrite-containing toothpaste delays tooth decay, whereas medications that inhibit salivary nitrite secretion accelerate tooth decay. [17]

Various methods to assess nitric oxide levels in saliva

Saliva NO test Strips, ELISA, calorimetric NO assay kit. However, Griess reaction is used in the present study since it is a cost-effective and sensitive method of detecting salivary NO levels. The conventional Griess reaction had limitations regarding both inability to detect nitrate as well as sensitivity.

Miranda et al., in 2001, proposed a modification of Griess reaction wherein simultaneous evaluation of nitrate and nitrite could be done. This assay allows large batch analysis of minimal samples and does not require specialized equipment. A similar method for NO detection was used in our study. [7]

There has been a growing interest in the role of nitrates and nitrites in protecting humans from oral and gastrointestinal disease. It has been indicated that acidified nitrite inhibits acid production. Nitrite in concentrations of 20 and 200 mM is highly bactericidal for S. mutans and inhibits acid production by strains of Actinomyces naeslundii, Lactobacilli casei, and S. mutans.[13]

Human odontoblasts show a marked immunoreactivity for 3-nitrotyrosine (a biomarker for NO-derived peroxynitrite) in the inflamed pulp which suggests that these cells release NO upon NOS2 activation. The NOS2 gene is upregulated in the inflamed pulp of carious teeth compared to that of healthy ones. NO production by odontoblasts might be an important defense mechanism against dentin-invading oral microorganisms because it inhibits S. mutans growth. In addition, NO may exert antiviral actions on particular viruses (such as herpes simplex virus-1, hepatitis virus type 3, and Coxsackie group B virus). [18]

In a study done by Reher et al., it was reported that NO levels were elevated in patients with chronic generalized periodontitis (GCP). The severity was related to salivary nitrite concentration which indicated that NO may serve as an important biological marker for detection and monitoring GCP. [19]

It has also been noted that concentrations of NO lower than 1 ppm have been shown to inhibit the growth of several fungi as well as bacteria such as C. albicans and Escherichia coli. This raises the possibility of the fact that the anaerobic flora in the posterior dorsum of tongue and also within the depths of the periodontal pocket generates enough NO to inhibit colonization by other microbial species of this niche. Intraoral generation of NO may also influence the inflammatory response (vasodilation) in the oral cavity particularly in the gingival margins in gingivitis as well as periodontitis. [14] The stimulated release of endogenous NO may also be involved in the control of the dental pulp vascular tone. [20]

NO pathway plays a potential role in the regulation of pulpal blood circulation. Nitric-oxide-dependent basal vasodilator tone exists in the dental pulp. Dental pulp vasculature appears to be highly responsive to exogenous NO. The stimulated release of endogenous NO may also be involved in the control of the dental pulp vascular tone. [20]

It has been postulated that pharmacological inhibition of NO or its actions may be therapeutically valuable in the disease management. Levels of NO may also provide clues about severity and the state of underlying disease process. It could serve as an inflammatory biomarker that might enable clinicians to direct environmentally based prevention and treatment programs. Although the role of NO in systemic diseases must further be evaluated. [21]

Salivary diagnostics would enable the clinicians to monitor diseases frequently and more easily. This would also have an impact on the future medical research and therapy. The effect of nitrite containing toothpaste should be studied in more detail. Furthermore, efforts of incorporating NO in a mouth rinse agent can be made after further research on a larger sample size. Moreover, the diet modifications must be made by the parents and caretakers to incorporate more amount of green leafy vegetables.

The limitations of this study are a small sample size. Furthermore, only the children of Belagavi district were included in the study. Hence, a larger area should be covered to gain more evidence regarding the possible antimicrobial action of NO in the oral cavity.


  Conclusion Top


With a high prevalence of early childhood caries (ECC), all attempts should be made by the Pediatric dentists to prevent this condition. We conclude from our study that: the levels of salivary Nitric Oxide (NO) decrease with the increase in caries. As the dmft score increases, the salivary nitric oxide (NO) level decreases. This may be attributed to the antimicrobial action of Nitric Oxide (NO).

In future, further studies should be carried in order to test the nitric oxide (NO) levels in certain systemic conditions so that the relationship can be correlated. Also, the effect of nitric oxide on caries must be tested by dietary modification and by using mouth rinse as well as toothpastes containing nitrates.

Acknowledgments

Dr. Gope Assudani, Dr. Neeraj Gupta, Mr. Mallapur, Dr. Pratibha Kukreja, Dr. Divyata Kohli.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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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.  Back to cited text no. 13
    
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Radcliffe CE, Akram NC, Hurrell F, Drucker DB. Effects of nitrite and nitrate on the growth and acidogenicity of Streptococcus mutans. J Dent 2002;30:325-31.  Back to cited text no. 14
    
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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.  Back to cited text no. 15
    
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Silva Mendez LS, Allaker RP, Hardie JM, Benjamin N. Antimicrobial effect of acidified nitrite on cariogenic bacteria. Oral Microbiol Immunol 1999;14:391-2.  Back to cited text no. 16
    
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]


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