Table of Contents    
ORIGINAL ARTICLE
Year : 2021  |  Volume : 12  |  Issue : 1  |  Page : 109-112  

Urease activity in saliva and plaque as endogenous protection against dental caries in institutionalized blind children


1 Department of Pedodontics and Preventive Dentistry, Navodaya Dental College and Hospital, Raichur, Karnataka, India
2 Department of Biochemistry, Navodaya Medical College and Hospital, Raichur, Karnataka, India

Date of Submission30-Apr-2020
Date of Decision16-Jun-2020
Date of Acceptance06-Jul-2020
Date of Web Publication27-Jan-2021

Correspondence Address:
Anisha Nanda
Department of Pedodontics and Preventive Dentistry, Navodaya Dental College and Hospital, Raichur - 584 103, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jnsbm.JNSBM_90_20

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   Abstract 


Background: Urease activity has been proposed to have a significant effect on dental caries, showing reduced caries activity with increased urease levels. Thus, the aim of the study was to evaluate the ureolytic activities of saliva and plaque in caries-active and caries-free institutionalized blind children. Materials and Methods: Fifty-two institutionalized blind children were divided into two equal groups using decayed, missing, and filled Teeth index as Group A – caries free and Group B – caries active. Urease activity was measured by collecting saliva and plaque samples from them. Children were refrained from any oral hygiene procedures during the 12 h preceding the sample collection. Urease enzyme activity was obtained by carrying out biochemical procedures. Mann–Whitney test was used to determine the statistical significance of the data. Results: In saliva, caries-free group had higher mean urease levels of 1.82 ± 0.497 as compared to caries-active group which had mean urease levels of 0.445 ± 0.304. In plaque, caries-active group had lower mean urease levels of 0.877 ± 0.942 as compared to caries-free group which had mean urease levels of 1.570 ± 0.918. The specific urease activity in saliva and plaque was significantly higher in individuals with low caries rates. Conclusion: Caries-free children had a higher ammonia generation activity by urease for both saliva and plaque samples than low caries-active children. High levels of alkali production in the oral environment were related to caries-free children.

Keywords: Alkali, ammonia, caries, urease


How to cite this article:
Kumar V, Nanda A, Bhat K H, Ashrit P, Babu A, Shakir M K. Urease activity in saliva and plaque as endogenous protection against dental caries in institutionalized blind children. J Nat Sc Biol Med 2021;12:109-12

How to cite this URL:
Kumar V, Nanda A, Bhat K H, Ashrit P, Babu A, Shakir M K. Urease activity in saliva and plaque as endogenous protection against dental caries in institutionalized blind children. J Nat Sc Biol Med [serial online] 2021 [cited 2021 Jun 16];12:109-12. Available from: http://www.jnsbm.org/text.asp?2021/12/1/109/307863




   Introduction Top


For years, caries disease has been studied for its acidogenic nature, and only in the last decades, it has been proposed the increase in alkali production as a strategy to reach oral pH homeostasis and prevent dental caries.[1] A new approach in caries research is focused on the fact that alkali generation from salivary substrates, such as urea, plays an important role in biofilm pH, in inhibiting dental caries. The production of alkali in mouth is associated with the hydrolysis of urea by urease enzyme.[2] The increased levels of ammonia from urease activity can produce a significant increase in neutralizing pH of the oral environment.[1] These are the reasons that determined the hypothesis that the activity of urease is associated with a decrease in caries activity. In recent years, alkali production in biofilm and saliva has been studied, showing a positive relation between alkali generation, expressed as high levels of urease activity, and caries resistance in caries-free patients compared to caries-active patients.[3]

Maintenance of oral health is challenging in children with special needs. Visual impairment is an important disability affecting a substantial proportion of people globally. It is estimated that over 1–4 million children worldwide are living with visual impairment.[4] Lack of hand–eye coordination, inadequate parental supervision, and lack of input from peers may have a negative impact on oral health.[5]

The aim of this cross-sectional and randomized study was to assess urease activity in saliva and plaque as endogenous protection against dental caries in institutionalized blind children. We hypothesized that children with higher levels of urease enzyme activity would present lower Decayed, Missing, and Filled Teeth/decayed, missing, and filled teeth (DMFT/dmft) index.


   Materials and Methods Top


A cross-sectional study was designed consisting of 52 blind children aged between 6 and 12 years from Shri Manik Prabhu Academy for blind, Raichur, Karnataka, India. The study protocol was approved by the institutional ethics committee (Ethical certificate No. NDC/IEC/2019/212). The study included children who were blind since birth, acquired, or partially blind. The study excluded children with low salivary flow (<0.5 ml per gland in 5 min), who had used antibiotics or chlorhexidine in the previous month, with systemic diseases that precluded dental treatment, and who had received dental bleaching treatment in the previous month or with periodontal disease.[3] A sample size of 52 children (26 in each group) would yield 80% power to detect significant differences, with an effect size of 0.8 and the significance level at 0.05. A power analysis was established by G*power, version 3.0.1 (Franz Faul universitat, Kiel, Germany).

Children were equally divided into two groups based on the dmft score.

  • Group 1: 26 caries-free blind children (baseline DMFT/dmft score - 0)
  • Group 2: 26 caries-active blind children (DMFT/dmft score >1).


Procedures

Children were instructed not to eat for 12 h before the sample collection, abstaining from any type of oral hygiene practices. 2 ml of unstimulated saliva was collected by expectoration into the sterile Eppendorf tubes. Supragingival plaque was collected from all available surfaces using Gracey curettes and was transferred to separate sterile Eppendorf tubes.[3] All saliva and plaque samples were collected between 7:00 and 8:00 a.m. and were transferred to the Central Research Laboratory of our Institution immediately.

Biochemical measurement of urease

Plotting of standard graph

A pure ammonium sulfate solution (20 mg/100 ml) was prepared, and different aliquots of it were taken, and the volume was made up to 3 ml with distilled water. To this, 1 ml of Nessler's reagent was added, and the color intensity was measured at 500 nm. A standard graph was plotted with a concentration of standard (micro g/ml) on the x-axis and optical density (at 500 nm) on the y-axis.[6]

Enzyme assay

Since there were no direct methods for estimation of urease enzyme activity, a biochemistry protocol was followed. The rationale behind the use of enzyme assay is that the urease enzyme splits urea liberating NH3 and CO2. This reaction is stoichiometric; the enzyme activity is easily determined by measuring the amount of ammonia formed and this can be observed colorimetrically.

To estimate the urease activity, 1 ml of 3% urea solution was pipetted out to all test tubes with 1 ml of 0.2 m phosphate buffer of pH 7.2. To this, 0.5 ml of saliva and plaque samples was added separately to different test tubes and mixed well and was incubated at 55°C for 15 min. After cooling with ice, 1 ml 2/3N of H2SO4 solution was added to the sample tube to stop urease enzyme activity, and 1 ml sodium tungstate was added to perfect H2SO4 work. The test tubes were then centrifuged for 15 min. Two milliliters of the obtained supernatant solution was pipetted out to a new test tube, to which 250 μl of Nessler's reagent was added, and aliquots of supernatant were assayed for NH3. The color change of the solution was observed. The solution uptake was then measured using UV-vis spectrophotometry at λ = 500 nm, and the obtained values were plotted against the graph to obtain the urease enzyme activity.[6],[7]

SPSS version 20 was the statistical software used for the analysis of the data, while Microsoft word and Excel were used to generate graphs, tables, etc SPSS(statistical Package for Social Sciences) version 20. (IBM SPASS statistics [IBM corp. released 2011. Descriptive statistics were carried out. Inferential statistics such as Mann–Whitney test was used for comparison between the groups; Spearman's correlation was used to correlate dmft and urease.

Independent sample t-test was also applied to check the statistical difference between the groups.


   Results Top


From a total of 52 blind children, 33 (63.5%) were male and 19 (36.5%) were female. [Table 1] shows the mean urease activity in saliva between caries-free and caries-active group. Caries-free group had higher mean urease levels of 1.82 ± 0.497 as compared to caries-active group which had mean urease levels of 0.445 ± 0.304 in saliva. Independent sample t-test was applied to check the statistical difference between the groups. t-Test showed a significant statistical difference of urease levels in saliva between the groups (P = 0.00) with a mean difference of −1.37.
Table 1: Comparison of mean distribution of urease activity in saliva between the groups using independent sample t-test

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[Table 2] shows the mean urease activity in plaque between the groups. Caries-active group had lower mean urease levels of 0.877 ± 0.942 as compared to caries-free group which had mean urease levels of 1.570 ± 0.918 in plaque. Independent sample t-test was applied to check the statistical difference between the groups. t-Test showed no significant statistical difference of urease levels in plaque between the groups (P = 0.01) with a mean difference of −0.69.
Table 2: Comparison of mean distribution of urease activity in plaque between the groups using independent sample t-test

Click here to view


The results indicate that the urease activity in saliva and plaque was significantly higher for caries-free children.


   Discussion Top


A caries lesion occurs when acids produced by the bacterial glycolysis of dietary carbohydrates cause tooth demineralization. Current evidence suggests that alkali production from the metabolism of salivary substrates, such as urea, inhibits tooth demineralization by neutralizing glycolytic acids while positively affecting the ecology of oral biofilms.[8],[9] The urea is a nitrogenous substrate that can produce alkali rapidly enough to buffer salivary acids and thereby contribute to a pH rise. The increased levels of ammonia from urease activity can produce a significant increase in neutralizing pH of the oral environment. The main outcome variables of the study were urease activity in saliva and plaque, which was estimated using a spectrophotometric method. This study analyzed the activity of urease enzyme as an ammonia producing source and assessed their association with the level of dental caries lesions in 5–12-year-old institutionalized blind children. The observations of the present study showed that a greater urease activity was found in children with no caries experience, which was in accordance with Nascimento et al.[2] and Gordan et al.[10] In the present study, significantly lower levels of urease activity were observed in the dental plaque of caries-active individuals compared to the caries-free children. Frostel, in 1960,[11] also observed higher urease levels in the plaque of caries-free children compared to a caries-active group but found this difference to be not statistically significant. Recently, it was found that urease activity in the dental plaque of caries-free children was about three-fold higher than in caries-active children, in a study by Shu.[8] Similarly, lower levels of urease activity were observed in saliva of caries-active individuals compared to caries-free children. This observation was contradictory with previous reports which showed higher urease levels in saliva.[11],[12] A study by Morou-Bermudez et al. concluded that there is a positive association between saliva urease activity and dental caries. Salivary urease levels were significantly lower in children who had consumed sugar-containing foods before sample collection compared to children who had not eaten anything since the night before.[9]

The present study is based on the biochemical reaction that the hydrolysis of urea by the enzyme urease produces ammonia and carbon dioxide. This is considered the major pathway for alkali production in the oral cavity, which in the oral environment creates a protective atmosphere that inhibits cariogenic microbiota. The natural endogenous alkalinogenic potential of the oral environment is associated with caries-free children, and they have a high ammonia generation activity by urease. Similarly, in our study, caries-free children showed more ammonia generation than in caries-active children, resulting in higher urease activity for both saliva and plaque samples. Correlation between urease levels in saliva and plaque in both caries-active and caries-free group showed that higher urease activity was found in saliva. This finding is similar to what was found by Sissons and Cutress.[13]

Finally, the most important evidence of this study is the suggestion that the natural endogenous alkalinogenic potential of the oral environment is associated with caries-free children. High levels of alkali activity in saliva could be related to neutralizing acids and possibly stabilizing the oral microbiota, favoring the conditions of maintaining oral health and the possibility to be used as a strategy against dental caries.[14] Despite this evidence, further studies are necessary to determine the best levels of urease that can prevent caries lesions.

The maintenance of oral hygiene among visually impaired children may be quite challenging, and there is an utmost need for oral health supervision to control plaque.[15] The recent World Health Organization estimate of the blind population is 285 million worldwide, and an estimated 19 million of these are children.[16] Recent studies have shown that the prevalence of caries in visually impaired children may be as high as 40%–81.9%.[17],[18] To the best of our knowledge, no previous study has been conducted among visually impaired evaluating the ureolytic activity of saliva and plaque. The limitations of our study are sample size, and minor procedural errors would have occurred during the biochemical procedures.

The clinical implication of the study is that the alkalinogenic potential of dental biofilm may be used as a strategy for caries control. Simple, chair-side tests for measuring urease activity in saliva can be easily developed, and they could be used as an indicator to assess caries risk and also prevention for dental caries. Furthermore, the levels of urease activity in saliva and plaque can be used for caries risk assessment.


   Conclusion Top


Caries-free children had a higher ammonia generation activity by urease for both saliva and plaque samples than caries-active alkalogenic. According to the results, the presented hypothesis is accepted: an inverse association may exist between urease activity levels in plaque and/or in saliva and dental caries.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Moncada G, Maureira J, Neira M, Reyes E, Oliveira Junior OB, Faleiros S, et al. Salivary urease and ADS enzymatic activity as endogenous protection against dental caries in children. J Clin Pediatr Dent 2015;39:358-63.  Back to cited text no. 1
    
2.
Nascimento MM, Gordan VV, Garvan CW, Browngardt CM, Burne RA. Correlations of oral bacterial arginine and urea catabolism with caries experience. Oral Microbiol Immunol 2009;24:89-95.  Back to cited text no. 2
    
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Reyes E, Martin J, Moncada G, Neira M, Palma P, Gordan V, et al. Caries-free subjects have high levels of urease and arginine deiminase activity. J Appl Oral Sci 2014;22:235-40.  Back to cited text no. 3
    
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Al Sadhan SA, Al Jobair AM, Bafaqeeh M, Abusharifia H, Alag M. Dental and medical health status and oral health knowledge among visually impaired and sighted female schoolchildren in Riyadh: A comparative study. BMC Oral Health 2017;17:154.  Back to cited text no. 4
    
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Biochemistry Laboratory Protocols. Enzymology Protocols. Assay of Urease activity. Available from: http://www.BiochemDen.com. [Last accessed on 2019 Oct 05].  Back to cited text no. 6
    
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Zusfahair DR. Determination of Urease Biochemical Properties of Asparagus Bean (Vigna unguiculata ssp sesquipedalis L.). IOP Conf Series Materials Sci Eng 2018;349:12073.  Back to cited text no. 7
    
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Shu M, Morou-Bermudez E, Suárez-Pérez E, Rivera-Miranda C, Browngardt CM, Chen YY, et al. Protection against dental caries in children. The relationship between dental caries status and dental plaque urease activity. Oral Microbiol Immunol 2007;22:61-6.  Back to cited text no. 8
    
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Morou-Bermudez E, Elías-Boneta A, Billings RJ, Burne RA, Garcia-Rivas V, Brignoni Nazario V, et al. Urease activity in plaque and saliva of children during a 3 year study and its relationship with other caries risk factors. Arch Oral Biol 2011;56:1282-9.  Back to cited text no. 9
    
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Gordan VV, Garvan CV, Ottenga ME, Schulte R, Harris, McEdward D, et al. Could alkali production be considered an approach for caries control? Caries Res 2010;44:547-54.  Back to cited text no. 10
    
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Frostel G. Studies on the ammonia production and the ureolytic activity of dental plaque material. Acta Odontol Scand 1960;18:29-65.  Back to cited text no. 11
    
12.
Ballantyne RM, Rae JJ, Lawford FH. Ammonia production and urease activity in saliva. J Dent Res 1952;31:281-3.  Back to cited text no. 12
    
13.
Sissons CH, Cutress TW. pH changes during simultaneous metabolism of urea and carbohydrate by human salivary bacteria in vitro. Arch Oral Biol 1988;33:579-87.  Back to cited text no. 13
    
14.
Liu YL, Nascimento M, Burne RA. Progress toward understanding the contribution of alkali generation in dental biofilms to inhibition of dental caries. Int J Oral Sci 2012;4:135-40.  Back to cited text no. 14
    
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Sanjay V, Shetty SM, Shetty RG, Managoli NA, Gugawad SC, Hitesh D. Dental health status among sensory impaired and blind institutionalized children aged 6 to 20 years. J Int Oral Health 2014;6:55-8.  Back to cited text no. 15
    
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Bimstein E, Jerrell RG, Weaver JP, Dailey L. Oral characteristics of children with visual or auditory impairments. Pediatr Dent 2014;36:336-41.  Back to cited text no. 16
    
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Shetty V, Hegde AM, Bhandary S, Rai K. Oral health status of the visually impaired children – A South Indian study. J Clin Pediatr Dent 2010;34:213-6.  Back to cited text no. 17
    
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Desai M, Messer LB, Calache H. A study of the dental treatment needs of children with disabilities in Melbourne, Australia. Aust Dent J 2001;46:41-50.  Back to cited text no. 18
    



 
 
    Tables

  [Table 1], [Table 2]



 

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