Influence of Salivary PH and Urea Level on Calculus Formation – A Clinico – Biochemical Study

Introduction

Dental plaque and calculus are considered to be primary etiological factors for periodontal disease. Calculus does not contribute directly to gingival inflammation,but it provides a fixed surface conducive for the continued accumulation of plaque and its retention in close proximity to the gingiva.Plaque commence gingival inflammation, which progress to pocket formation and periodontal destruction.The saliva is a complex fluid that includes a number of mucosal host defense factors from the various salivary glands. The salivary ph has a wide range of 5-8. Studies have documented the importance of alkaline pH for calcium phosphate deposition, thus facilitating the mineralization of plaques. For maintaining oral homeostasis, bicarbonate along with phosphate and other compounds andenzymes acts as buffers which includes urea, salivary amylases and fluorides. The significance of salivary urea was accepted early in dentalliterature. Stefan first gave an account of the pH-raising effects of intraoral urea application.¹ He concluded that both in vivo and in vitro urea could raise plaque pH up to pH 9 and that the addition of 40–50% urea to carbohydrates to a great extent overcame the pH-lowering effect for up to 24 h. The normal levels of urea in unstimulated and stimulated whole saliva are 3.3 and 2.2 mmol/l, respectively. Urea has a dual effect: it impedes the metabolism and growth of bacteria in the saliva and it also participatesin maintaining the salivary acidobasic balance by neutralizing the acids in the oral environment which it actually owes to its buffer capacity. Some oral microbes hydrolyze salivary and dietary urea via the enzyme urease to produce ammonia and carbon dioxide, which results in an increase in plaque pH.^2,3 The ureolytic pH response (an increase in plaque pH by the formation of ammonia from urea) promotes calculus formation by increasing the saturation degree of calcium phosphate in plaque fluid.⁴ It has been concluded previously that saliva is the source of mineralization for supragingival calculus. Therefore, it is logical to evaluate the salivary components in heavy, moderate and light calculus formers as a method to recognize factors responsible for individual susceptibility. This study was carried out to evaluate the effect of salivary pH and urea level in calculus formation.

MATERIALS AND METHODS:

The subjects were included from among the patients coming to the out-patient unit of Department of Periodontics, Sri Rajiv Gandhi College of Dental Sciences and Hospital.The inclusion criteria for selecting patients were:Age group 18-55 years,nohistory of systemic disease, not undergoing any drug therapy,not had any periodontal therapy in the past 3 months.

Based on the Calculus Index (part of OHI-S index), subjects were split into three groups:

Group A- Mild calculus formers (score of 0.0 to 0.6)

Group B- Moderate calculus formers (score of 0.7 to 1.8)

Group C- Heavy calculus formers (score of 1.9 to 3.0)

10 subjects were included in each group. Informed consent for participation was taken from each subject. The subjects were advised to refrain from any oral hygiene procedures, eating, drinking, or smoking for minimum 2 hours before clinical parameters were recorded.

Saliva Sampling:

The subject was asked to sit in a relaxed position, with head tilted down, with minimal oral musculature activity for 5 minutes and was asked to expectorate the pooled unstimulated whole saliva in a sterile container as passively as possible. Salivary pH was recorded by using pH indicator strips [Dental Saliva pH indicator strips pH 6.5 - 9.0; gradation 0.5; color coded] as soon as saliva was collected in a sterile container. Semiautoanalyser was used for the biochemical estimation of salivary urea. The calculus scores were determined according to Calculus index (part of Oral Hygiene index- Simplified). Plaque was disclosed (using Alphaplac®DPIIndia) and plaque scores recorded based on Plaque index by Silness & Loe.

STATISTICAL ANALYSIS

Statistical Analysis:

SPSS for Windows, Version 22.0 was used to perform statistical analyses.

Descriptive Statistics:

Descriptive analysis includes expression of categorical data in terms of number & percentage, whereas for continuous data, it will be expressed in frequency, mean & standard deviation (SD).

Inferential Statistics:

One-way ANOVA Test followed by Tukey's Post hoc Analysis was performed to compare the mean values of different parameters between 03 study groups.

The level of significance [P-Value] was set at P<0.05.

RESULTS:

There was no significant relation between salivary pH value and calculus formation in the present study. Salivary urea values were significantly higher in Group C, suggesting that salivary urea has an effect on calculus formation. Groups A and B reported no statistically significant difference between pH levels (p=0.97) but there was statistically significant difference between Group A and Group C (p= 0.01). TABLE 1 There was a highly significant difference in the urea levels between Group A and Group C (p<0.001).

DISCUSSION:

This study was conducted to evaluate the influence of salivary pH and urea level in calculus formation. There was statistically significant difference in the salivary urea concentrations of the groups. There was a notable correlation between urea concentration in saliva and corresponding pH values in heavy calculus formers, which reflected in substantial amount of calculus formation in these subjects. The noteworthy role of alkaline pH for the deposition of calcium phosphate salts and thereby enhancing plaque mineralization was brought forward by Wong L et al.⁵ The salivary pH in chronic generalized periodontitis was found to be high compared to healthy gingiva.⁶ There was a positive and significant association between urea levels and calculus formation in both the groups in this study which is in concord with the study carried out by Gupta et al, Dipika K et al and ArpitaR^7,8,9. Thenitrogenous substances in saliva is broken down to smaller units, such as, urea & amino acids is transformed to ammonia.⁵ The ph may be elevated by the formation of ammonia by the dental plaque bacteria.Oral ureolytic activity varies quantitatively between individuals' plaque. This dissimilarity is probably related to variations in the flora, since common plaque microflora differ to a great extent in their capacity to degrade urea.¹⁰ An increase in the pH of saliva lowers the precipitation constant causing precipitation of calcium phosphate salts.¹¹ Application of urea solutions to plaque in situ causes a rapid rise in supragingival plaque pH. This rise has been imputed to the generation of ammonia by the ureolytic activity of the plaque bacteria. Both the levels of urea in saliva and of ammonia in plaque accounts for the high pH of fasting plaque.¹² In contradiction to our study, Fure and Lingstorm reported that three months’ frequent use of sugar free chewing gum-with or without ureaneither promotes nor inhibits calculus formation. However, the variation in calculus formation seen in different individuals may also be related to variation in salivary flow rate in different parts of the oral cavity.¹³

A study done by Charles M. Belting to evaluate in Vitro Effect of Urea on Artificial Calculus Formation the study was done on preformed artificialcalculus on the glass slides, it wasfound that increased concentrations of urea resulted in a progressive removal of calciumdeposits up to a maximum of 62 percent removal at 30 percent concentrationof urea. Above 30 percent concentration adecreased removal of calcium deposits wasnoted. It is believed that urea interferes withartificial calculus formation, and also reducespreformed artificial calculus, by dissolving the mucoproteinaceous material within which the calcium salts are deposited,and/or by increasing the solubilityof calcium salts in the saliva. It was found that increased concentrationsof urea resulted in a progressive inhibitionof calcium deposition up to a maximum.¹⁴

Another study by Belting and Gordon concluded that a significant degree of calculus formation can be inhibited in the human mouth by the use, twice daily, of a dentifrice containing 30 percent urea. Among the experimental group who used the dentifrice containing 30 percent urea, a 36 percent reduction in calcium accumulation was found which was statistically significant at the 0.005 level of probability. In the control group who used the placebo dentifrice during both test periods, an inherent variability in calcium accumulation of ± 5 percent was found.¹⁵

CONCLUSION:

These results suggest that the salivary urea has a major role in calculus formation, and even though evidence shows that pH has a role in calculus formation, there was no significant relation between pH and calculus formation was observed in this study, which can be due to the small sample size. Therefore, further studies should be done with a larger sample size and taking into consideration the other factors which effect salivary pH (e.g., dietary habits, oral microbiota, oral hygiene measures, smoking) is required to establish the role of salivary pH in calculus formation.