Table of Contents    
ORIGINAL ARTICLE
Year : 2021  |  Volume : 12  |  Issue : 1  |  Page : 113-116  

Correlations of oral bacterial urea catabolism with caries experience in normal-weight children and underweight 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 Decision28-Jul-2020
Date of Acceptance10-Aug-2020
Date of Web Publication27-Jan-2021

Correspondence Address:
Aleena Babu
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_91_20

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   Abstract 


Background: Alkali generation by oral bacteria plays a key role in plaque pH homeostasis and may be inhibitory to the initiation and progression of dental caries. A substantial body of evidence is beginning to accumulate, which indicates that the modulation of the Alkali generating potential of dental biofilms may be a promising strategy for caries control. Aim: The aim of the study was to determine the capacity of oral samples to produce ammonia from urea and its relation to caries experience in normal-weight children and underweight children. Materials and Methods: Urease activity was measured in the saliva and plaque of 25 caries-active (CA) individuals who have normal weight and 25 CA individuals who are underweight. Urease activity was obtained from the ammonia produced by the incubation of plaque and salivary samples in urea. Independent sample t-test and Pearson's correlation were used to compare the differences and to correlate the urease levels and caries experience between groups. Results: The results showed that the mean urease levels in saliva were higher in All are positive value-1.2192±1.012 as compared to that of underweight children-0.78-0.676. The mean urease levels in plaque were lower in normal-weight children-0.1120-0.12206 as compared to that of underweight children-0.4824-0.5215. Conclusion: In underweight children, the caries activity was higher along with increased urease activity in saliva and lower urease activity in plaque, whereas in normal-weight children, the caries activity was lower with increased urease activity in saliva and lower urease activity in plaque.

Keywords: Alkali, ammonia, urease


How to cite this article:
Kumar V, Babu A, Bhat K H, Ashrit P, Nanda A, Shakir M K. Correlations of oral bacterial urea catabolism with caries experience in normal-weight children and underweight children. J Nat Sc Biol Med 2021;12:113-6

How to cite this URL:
Kumar V, Babu A, Bhat K H, Ashrit P, Nanda A, Shakir M K. Correlations of oral bacterial urea catabolism with caries experience in normal-weight children and underweight children. J Nat Sc Biol Med [serial online] 2021 [cited 2021 Jun 16];12:113-6. Available from: http://www.jnsbm.org/text.asp?2021/12/1/113/307864




   Introduction Top


The focus on an oral environment with a low pH has been dominant when teaching or researching in dental caries. The acidification of oral biofilm permits the appearance of a microflora that generates acid and is capable to resist this condition, lowering the pH to a level that can lead to tooth demineralization.[1]

The emphasis of dental research has shifted from the causes of dental diseases to how dental diseases affect general health. Current research in dental medicine trends toward identifying the link between oral health and various systemic diseases. Despite the change in emphasis, the growing evidence on the probable effects of untreated dental caries on growth and health has been ignored.[2] A new approach in caries research is focused on the fact that ammonia production from the metabolism of urea by urease enzymes of oral bacteria moderates biofilm pH and homeostasis and may inhibit dental caries. The hydrolysis of urea by bacterial urease enzymes generates ammonia and CO2, and it is considered a major pathway for alkali production in the oral cavity.[3]

Dental caries and weight disorders are two diseases which share several predisposing factors such as diet, lifestyle, genetics, socioeconomic status, and other environmental factors. In order to study the association between weight disorders and dental caries, a lot of research has been conducted, but the association is still controversially discussed in different studies.[4]

The aim of this study was to determine the capacity of oral samples to produce ammonia from urea and its relation to caries experience in normal-weight children and underweight children. The hypothesis is that an association may exist between dental caries and urease activity levels in saliva and/or plaque.


   Materials and Methods Top


The ethical clearance for this cross-sectional study was obtained from the institutional ethical committee (Ethical certificate Number: NDC/IEC/2019/213), and a written consent was taken from the institutional head of the orphanage named Bala Mandir, Raichur, Karnataka, from where the samples were collected.

Fifty caries-active (CA) children in the age group of 6–12 years from an orphanage were selected for the study. Weight and height of the children were recorded, and their body mass index (BMI) was calculated according to the following formula: weight (kg)/height2 (m2). Children with a BMI <18.5 kg/m2 were categorized as underweight and children with a BMI within a range of 18.5–24.9 kg/m2 were categorized as normal weight.[5] Individuals with at least one carious tooth (Decayed Missing Filled Teeth (DMFT/dmft) score >1) and who had normal weight and underweight according to the BMI rating scale were included in the study.

Individuals with low salivary flow (<0.5 ml/gland in 5 min), those with periodontal and systemic diseases, who had used antibiotics or chlorhexidine in the previous month, and who had received dental bleaching or fluoride-releasing restorations in the previous month were excluded from the study. The study participants were equally divided into the following two groups:

  • Group 1 had 25 CA individuals with normal weight
  • Group 2 had 25 CA individuals with underweight.


Procedures

The individuals were refrained from oral hygiene procedures and had fasted overnight before sample collection. A volume of 2 ml of unstimulated saliva was collected by asking the patients to expectorate into a sterile plastic Eppendorf tube. After saliva collection, supragingival plaque was collected from all smooth dental surfaces of incisors and molar regions using sterile Gracey curettes and transferred to the sterile plastic Eppendorf tube.

Samples of both saliva and plaque were stored in Eppendorf tubes until biochemical analysis, which was done at the Central Research Laboratory.

Biochemical measurement of urease

The enzyme activity is determined by measuring the amount of ammonia formed colorimetrically.

Principle

The urease enzyme splits urea-liberating ammonia and carbon dioxide.

Preparation of standard graph

A pure ammonium sulfate solution (20 mg/100 ml) was prepared, its different aliquots were taken, and the volume was made up to 3 mL with distilled water. To this, 1 mL of Nessler's reagent was added. After mixing, the color intensity was measured at 500 nm. A standard graph was plotted with a concentration of standard (μg ml) in the X-axis and optical density (at 500 nm) in the Y-axis.

Enzyme assay

One milliliter of 3% urea solution buffered with 1 mL of 0.2 M phosphate buffer (pH 7) was pipetted out. A volume of 0.5 mL of saliva samples and plaque samples was added in different test tubes and was incubated at 55°C for 15 min. At the end of the incubation time, the tubes were quickly placed in ice. One milliliter of 0.66 N H2SO4 was added to stop the reaction, and 1 mL of 1 M sodium tungstate solution was added to precipitate the protein. They were centrifuged (15 min) to remove the precipitate, and a supernatant was obtained. A volume of 2 mL of the supernatant was pipetted out, to which 250 μL of Nessler's reagent was added. Then, aliquots of the supernatant were assayed for NH3, and the enzyme activity was calculated using a spectrophotometer at 500 nm and plotted in the standard graph.[6]

Descriptive statistics such as mean and standard deviation were calculated. Inferential statistics such as independent sample t-test were used to compare the difference of urease levels between groups. Pearson's correlation was used to correlate the urease levels and caries experience using SPSS (Statistical Package for the Social Sciences) software version 20 (IBM SPASS statistics [IBM corp., Armonk, New York, U.S.A released 2011]).


   Results Top


[Table 1] shows that the mean urease levels in saliva were higher in Positive value-1.2192-1.012 as compared to underweight children-0.78-0.676, with a mean difference of −0.438. The mean urease levels in plaque were lower in normal-weight children-0.1120-0.12206 as compared to underweight children-0.4824-0.5215, with a mean difference of 0.370. 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 and plaque between the groups.
Table 1: Comparison of mean distribution of urease activity in saliva (s) and plaque (p) between the groups using independent sample t-test

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[Table 2] shows the correlation between the number of carious teeth and urease levels in saliva, plaque, and BMI. There was a very weak, negative correlation between the number of carious teeth and urease levels in saliva (r = −0.069) and was not statistically significant (P = 0.63). Pearson's correlation showed a very weak positive correlation between the number of carious teeth and urease levels in plaque (r = 0.146) and was not statistically significant (P = 0.31). There was a very weak, negative correlation between the number of carious teeth and BMI (r = −0.076) and was not statistically significant.
Table 2: Pearson's correlation between the number of carious teeth and urease levels in saliva, plaque, and body mass index

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   Discussion Top


In spite of the development of preventive dentistry in recent years, dental caries remains the most prevalent oral disease, generating an epidemiological problem in all countries. As a result, dental research is constantly exploring new strategies in the prevention and treatment of this disease.[1]

Bacterial urease activity in dental plaque and in saliva generates alkali, which can increase the plaque pH and can protect noncariogenic oral bacteria against acidification; consequently, it has been hypothesized that oral urease activity may be a caries-inhibiting factor, and that loss of this activity may lead to caries development.[7] Margolis et al.[8] have shown that a more alkaline resting plaque exists in caries-resistant individuals compared to caries-susceptible individuals.

One of the strengths of alkali production as a strategy for control of caries is that it attacks the problem in two very important ways. First, it directly increases the pH of dental plaque, which tips the balance in favor of remineralization and away from demineralization. Second, alkali generation favors the persistence of health-associated bacteria while discouraging the outgrowth of those cariogenic bacteria that depend on low pH to gain an ecological advantage, for example, Streptococcus mutans.[9]

Reyes et al.[1] in their study found that caries-free individuals had a high ammonia generation activity by urease system. Gordan et al.[10] showed that a positive relationship exists between high levels of alkali production and caries resistance as significantly higher levels of urease and activities of daily living were seen in individuals experiencing no caries activity, compared with individuals who had active caries.

Measuring urease activity in saliva and plaque using this colorimetric method, which has been used in the present study, is much faster and cost-effective compared to the microbiological tests. In the present study, we have followed the principle that urease enzyme splits urea-liberating ammonia and carbon dioxide. The urease enzyme activity was determined by measuring the amount of ammonia formed colorimetrically, as mentioned in biochemistry laboratory protocols.[10]

In the present study, the CA children with more number of carious teeth showed a decrease in the urease level saliva. This was in accordance with Nascimento et al.,[11] who observed an inverse relationship between salivary urease activity and caries levels. The present study also showed that, in CA children with more number of carious teeth, there was an increase in plaque urease levels. This was in accordance with Frostel,[12] who observed higher plaque urease activity and increased caries levels.

The relationship between dental caries and BMI has been studied by numerous authors, with often controversial and limited results. Empirical findings suggest the existence of such a relationship, but very few studies to date have confirmed and characterized this relationship. Our study shows that children with lower BMI have more caries. This was in accordance with Chibelean M et al.,[13] who observed a higher caries incidence in underweight children.

In the present study, it was found that urease activity in saliva samples of underweight children was lower than that of children with normal weight. As there is a decrease in urease activity, there may be a further reduction in ammonia production. This may be the attributed reason for the increased caries rate in underweight children. However, in plaque samples, underweight children had higher urease levels contrary to the results of saliva. This might be due to the changes in the composition and microbial environment of plaque and saliva.

The results of this study should be considered in light of several limitations, including the small sample size and a few procedural errors.


   Conclusion Top


The current results support that alkali generation is related with caries activity. The amount of urease generated from the test substrates by salivary samples is generally higher than that produced by plaque samples. Ammonia generation from urea may have a great impact in the oral environment; they could be used as a risk predictor of decay. Weight disorders could be considered a risk factor for caries, and calculation of BMI should be included in the examination of pediatric patients. Further studies have to be carried out with larger sample size to rule out the alkali-generating potential of plaque.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
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. 1
    
2.
Sheiham A. Dental caries affects body weight, growth and quality of life in pre-school children. Br Dent J 2006;201:625-6.  Back to cited text no. 2
    
3.
Shu M, Morou-Bermudez E, Suárez-Pérez E, Rivera-Miranda C, Browngardt CM, Chen YY, et al. The relationship between dental caries status and dental plaque urease activity. Oral Microbiol Immunol 2007;22:61-6.  Back to cited text no. 3
    
4.
Chen D, Zhi Q, Zhou Y, Tao Y, Wu L, Lin H. Association between dental caries and BMI in children: A systematic review and meta-analysis. Caries Res 2018;52:230-45.  Back to cited text no. 4
    
5.
Alswat K, Mohamed WS, Wahab MA, Aboelil AA. The association between body mass index and dental caries: Cross-sectional study. J Clin Med Res 2016;8:147-52.  Back to cited text no. 5
    
6.
Assay of Urease Activity; Biochemistry Laboratory Protocols Enzymology Protocols. Available from: http://www.BiochemDen.com.  Back to cited text no. 6
    
7.
Morou-Bermudez E, Elias-Boneta A, Billings RJ, Burne RA, Garcia-Rivas V. Urease activity as a risk factor for caries development in children during a three-year study period: A survival analysis. Approach. Arch Oral Biol 2011;56:1560-8.  Back to cited text no. 7
    
8.
Margolis HC, Duckworth JH, Moreno EC. Composition of pooled resting plaque fluid from caries-free and caries-susceptible individuals. J Dent Res 1988;67:1468-75.  Back to cited text no. 8
    
9.
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. 9
    
10.
Gordan VV, Garvan CV, Ottenga ME, Schulte R, Harris P, 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
    
11.
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. 11
    
12.
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. 12
    
13.
Chibelean M, Pacurar M, Eşian D, Bud E, Anamaria B, Cristina M. Correlation between BMI, dental caries and salivary buffer capacity in a sample of children from Mures county, Romania. Europ Sci J 2015;11:1857-7881.  Back to cited text no. 13
    



 
 
    Tables

  [Table 1], [Table 2]



 

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