|Year : 2012 | Volume
| Issue : 3 | Page : 212-217
Evaluation of non-microbial salivary caries activity parameters and salivary biochemical indicators in predicting dental caries
A Kaur, KS Kwatra, P Kamboj
Paediatric and Preventive Dentistry, B.R.S. Dental College and Hospital, Sultan Pur, Panchkula, Haryana, India
|Date of Web Publication||21-Dec-2012|
B.R.S. Dental College and Hospital, Sultan Pur, Panchkula, Haryana
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: The aim of the present study was the evaluation of non-microbial salivary caries activity parameters and salivary biochemical indicators in predicting dental caries. Materials and Methods: The present study was carried out on 60 children, aged 4-6 years, selected from the schools of Panchkula district, Haryana, on the basis of their caries status. Level of hydration, flow rate, pH, buffering capacity, relative viscosity, calcium, phosphorus and alkaline phosphatase levels in caries-free and caries-active children were evaluated. Results: Results showed that 90% of subjects in the caries-free group and 30% of subjects in the caries-active group had normal level of hydration value of less than 60 s and the difference was found to be statistically very highly significant. Normal flow rate of stimulated saliva was found in 90% of the subjects in caries-free group and 33.3% subjects in the caries active group and difference was found to be statistically very highly significant. Adequate salivary pH was found in 100% subjects in caries-free group and 30% in caries-active group and the difference was statistically very highly significant. Conclusion: To conclude, within limitations of this study, it became clear that normal level of hydration and higher values for flow rate, pH, buffering capacity of saliva lead to good oral health and a reduced caries occurrence. Increased salivary viscosity plays a role in increasing caries incidence. Salivary biochemical indicators like calcium, phosphorus and alkaline phosphatase also play their respective role in determining caries susceptibility of an individual. These salivary parameters can be used as diagnostic tool for caries risk assessment.
Keywords: Alkaline phosphatase, buffering capacity, calcium, caries-active, caries-free, flow rate, level of hydration, pH, phosphorus, relative viscosity
|How to cite this article:|
Kaur A, Kwatra K S, Kamboj P. Evaluation of non-microbial salivary caries activity parameters and salivary biochemical indicators in predicting dental caries. J Indian Soc Pedod Prev Dent 2012;30:212-7
|How to cite this URL:|
Kaur A, Kwatra K S, Kamboj P. Evaluation of non-microbial salivary caries activity parameters and salivary biochemical indicators in predicting dental caries. J Indian Soc Pedod Prev Dent [serial online] 2012 [cited 2019 Sep 19];30:212-7. Available from: http://www.jisppd.com/text.asp?2012/30/3/212/105013
| Introduction|| |
Saliva is a complex biological fluid containing several compounds which collaborate to prevent dental caries by mechanical washing, antimicrobial function, remineralization and regulating oral pH by its buffering capacity.  Saliva not only physically removes dietary substrates and acids produced by plaque from the mouth, but also has an important role in buffering the pH of saliva and plaque. Fast-flowing saliva is alkaline reaching pH values of 7.5-8.0 and is vitally important in raising the pH of dental plaque previously lowered by exposure to sugar and carbohydrates.  Maintenance of physiologic hydrogen ion concentration at the mucosal epithelial cell surface and the tooth surface is an important function of salivary buffers. In saliva, the most important salivary buffer is the bicarbonate-carbonic acid system. 
Saliva consists of various organic and inorganic compounds like bicarbonate, calcium, phosphate ions etc., to maintain its saturation. The consequence of this saturation establishes thermodynamic driving force, which is favorable for remineralization and unfavorable for demineralization. Low salivary buffering capacity, low calcium and phosphate levels show a pronounced link to increased caries. Maintenance of the equilibrium between demineralization and remineralization depends on the ionic concentration of calcium and phosphate in saliva, which in turn is influenced by alkaline phosphatase levels. On the surface of tooth, a high concentration of calcium and phosphate causes post-eruptive maturation of enamel, increases surface hardness and resistance to demineralization. Variations in alkaline phosphatase levels cause changes in phosphate levels, which lead to initiation and progression of dental caries. Alkaline phosphatase is a non-specific enzyme that reacts between pH 9 and 10 and is associated with the calcification process. 
Salivary flow will have a caries-preventive effect by influencing the clearance rate of the substrate: The higher the flow rate, the faster is the clearance rate (Miura et al., 1991). 
Inverse relation exists between salivary viscosity, hydration level and salivary flow rate. Saliva that is more viscid is less effective in clearing the mouth. The process of dental caries can be minimized if high caries risk factors as well as individuals at risk are identified at the earliest, in order to implement the preventive and interceptive procedures. Keeping this in mind, the present study was undertaken to evaluate the level of hydration, flow rate, pH, buffering capacity, relative viscosity, calcium, phosphorus and alkaline phosphatase levels in caries-free and caries-active children.
| Materials and Methods|| |
The present study was carried out on 60 children, aged 4-6 years, selected from the schools of Panchkula district, Haryana, on the basis of their caries status. Written consent to examine the schoolchildren was obtained from the District Education Officer of Panchkula. The final selection of schools was done after obtaining permission from the respective school Principals, who in turn took the consent from the parents of children. The study was approved by ethic committee.
The study was carried out on a total of 60 healthy children, aged 4-6 years, selected from different schools, on the basis of their dental caries status and divided into two groups, which are as follows:
The children were excluded from the study if they had any special healthcare needs, suffering from any systemic or metabolic diseases, on any medication for last two weeks and undergoing orthodontic treatment.
A self-prepared proforma was filled by the examiner, which consisted of six parts; first part consisted of personal history, second part consisted of medical history, which included any history of hospitalization, history of intake of any medication and any systemic diseases, third part consisted of caries status of child (according to WHO criteria, 1997),  fourth part consisted of diet history with special emphasis on in-between meals and snacking, fifth part comprised of oral hygiene procedures practiced by the child and sixth part consisted of the values of various salivary parameters including level of hydration, pH, relative viscosity, buffering capacity, flow rate, level of calcium, phosphorus and alkaline phosphatase.
Recording procedure for different salivary parameters
Selected children were brought to Department of Paediatric and Preventive Dentistry at B.R.S. Dental College and Hospital for thorough intraoral examination and estimation of various salivary parameters. Saliva sample was collected by making the child sit on the dental chair, two hours after intake of any food or drink, since any food ingestion is likely to alter the physiochemical properties of saliva.
Testing of resting saliva
Visual inspection of the level of hydration was assessed by averting the lower lip, and gently blotting the labial mucosa with a small piece of gauze and observing the mucosa under good light. Droplets of saliva formed at the orifices of the minor glands. The time taken for visible production of saliva if greater than 60 s indicated low resting flow whereas flow was considered to be normal when time taken was less than 60 s. pH measurement of resting/unstimulated saliva was assessed using the GC Saliva Check Kit (commercial name). Highly acidic resting saliva was indicated by red section (pH 5.0-5.8), moderately acidic saliva by the yellow section (pH 6.0-6.6), healthy saliva by the green section (pH 6.8-7.8).
Biochemical analysis of unstimulated saliva
To carry out the Biochemical Assay, the saliva was prepared by centrifugation for 15 minutes and the supernatant was used for biochemical estimation of levels of calcium, phosphorus and alkaline phosphatase. Calcium levels were measured by Orthocresolphtalein Complexone (OCPC) method. OCPC reacts with calcium at pH 10.0 to form a purple colored complex. This method did not require pre-treatment of the saliva sample. Phosphorus levels were measured by Ammonium Molybdate method, which works on the principal that ammonium molybdate in the presence of H 2 SO 4 reacts with phosphorus to form an unreduced complex of phosphomolybdate. The amount of complex formed is proportional to the concentration of phosphorus present in the sample. Measurement of alkaline phosphatase levels was done using the p-Npp method with stabilized substrate. Alkaline phosphatase hydrolyses paranitrophenol phosphate into paranitrophenol and phosphate in the presence of magnesium ions. The rate of increase in absorbance of the reaction mixture at 405 nm due to liberation of paranitrophenol is proportional to the alkaline phosphatase activity.
Testing of stimulated saliva
The measurement of the salivary flow rate and buffering capacity was done using the GC Saliva Check Kit.
Quantity of saliva at 5 min indicated:
Very low salivary flow rate <3.5 mL
Low salivary flow rate between 3.5 and 5.0 mL
Normal salivary flow rate >5.0 mL
Buffering capacity of stimulated saliva was considered to be very low if the value of test was 0-5, low for 6-9 and normal/high for a value of 10-12. Measurement of Salivary Viscosity  was done using the Ostwald viscometer. Recordings of all the variables included in the study were subjected to statistical analysis. In case of level of hydration, pH, buffering capacity and flow rate, data was subjected to Chi-Square statistical analysis and in case of relative viscosity, calcium, phosphorus and alkaline phosphatase, t-test was applied. P value of ≤0.05 is considered statistically significant.
| Results|| |
Results showed that 90% (i.e., 27) of subjects in the caries-free group and 30% (i.e., 9) of subjects in the caries-active group had normal level of hydration value of less than < 60 s and the difference was found to be statistically very highly significant (P < 0.0001). Normal flow rate of stimulated saliva (5 ml/5 min) was found in 90% of the subjects in caries-free group and 33.3% subjects in the caries-active group and difference was found to be statistically very highly significant (P < 0.0001). Adequate salivary pH was found in 100% subjects in caries-free group and 30% in caries-active group and the difference was statistically very highly significant (P < 0.0001).
| Discussion|| |
Etiology and pathogenesis of dental caries is multifactorial. With the identification of the role played by various factors in initiation and progression of caries in primary dentition, it will be possible to prevent and reduce the risk of caries in permanent dentition. In the present study, an attempt was made to compare salivary caries activity parameters i.e., salivary flow rate, pH, viscosity, buffering capacity and levels of hydration and biochemical indicators of dental caries i.e., salivary calcium, phosphorus and alkaline phosphatase in caries-free and caries-active children. The time of collection of saliva was taken between 10 and 11.30 a.m. to prevent circadian variation. The participants fasted for at least one and two hours before saliva collection to avoid influence of immediate food consumption and food contamination.
In the resting saliva, the test for levels of hydration were significantly (P < 0.0001) higher in caries-free group (90%) as compared to the caries-active group (30%). Therefore, normal level of hydration imparts a strong protective effect against dental caries. Lower salivary flow rate found in caries-active group might be associated with a number of predisposing factors such as lack of raw material (water), lack of stimulus to salivary gland or could be a problem with salivary gland itself. Oral cavity is quite frequently exposed to components whose pH differs from normal pH of saliva (6.5-7.5) and these components may cause damage to teeth or mucosal surface.  Studies have shown that patient with low or no caries activity had a resting salivary pH of around 7.0. Those with extreme caries activity had a resting pH below critical pH 5.5. In the present study, percentage of children having healthy saliva (pH≥6.8) in CFG was 100% as compared to 30% in CAG. Therefore, low salivary pH is directly related to caries activity in children with high caries activity.
In stimulated saliva, measurement of salivary flow rate is used as a screening method in a population to identify people with low salivary flow, which is often but not always related to caries susceptibility and activity.  In general, higher the flow rate, faster the clearance and higher the buffer capacity, lesser are the microbial attacks.  In the present study, 90% children in caries-free group had a stimulated salivary flow rate greater than 1 ml/min as compared to 33.3% in caries-active group. Normal salivary flow rate (hydration status and stimulated salivary flow rate) imparts a strong protective effect against dental caries. The patient with stimulated salivary flow rate less than 1.0 ml/min are considered to be at risk to develop dental caries, whereas a stimulated salivary flow rate greater than 1.0 ml/min is considered to be normal. Individuals with reduced salivary flow should be advised to maintain acceptable patterns of oral hygiene and to reduce the frequency of sugar ingestion. 
Buffering agents in saliva try to bring the pH back to the normal range as fast as possible when the oral cavity is exposed to food items whose pH differs from normal pH (6.5-7.5) of saliva. In resting saliva, the major buffering agent is inorganic phosphate and in stimulated saliva it is carbonic acid/bicarbonate system. At very low pH (4-4.5) salivary proteins also display some buffering action. 
In present study, 76.6% children in the caries-free group and only 13.33% subjects in the caries-active group had normal/high buffering capacity (buffering capacity score 10-12), the difference being statistically very highly significant (P < 0.0001) [Table 1]. Therefore, higher the buffering capacity of saliva, lower is the caries activity. Therefore, buffering capacity of saliva can be used as a diagnostic tool for caries risk assessment.
|Table 1: Buffering capacity of stimulated saliva of caries‑free and caries‑active group|
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The lubricating action of saliva is important for oral health. It facilitates the movements of tongue and lips during swallowing and eating and is important for clearly articulated speech (Waterman et al., 1988). The efficacy of saliva as a lubricant depends on its viscosity and how it changes with shear rate.  In present study, relative viscosity of saliva in children belonging to the caries-active group was significantly (P < 0.0001) greater than relative viscosity of saliva in children belonging to the caries-free group [Table 2]. Greater the viscosity of saliva, lesser will be its cleansing action leading to a higher caries rate.
|Table 2: Mean relative viscosity of stimulated saliva of caries‑free and caries‑active group|
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Alkaline phosphatase (ALP, ALKP) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate is called dephosphorylation.  Levels of the enzyme are significantly higher in children and pregnant women. Also, elevated ALP indicates that there could be active bone formation occurring as ALP is a byproduct of osteoblast activity. Lowered levels of ALP are less common than elevated levels.  Abnormally low levels of alkaline phosphatase is a genetic condition called hypophosphatasia, which results in bone deformities. Patients with elevated serum alkaline phosphatase have normal salivary alkaline phosphatase. This effect is attributed to the presence in high concentrations of inorganic phosphate in salivary secretions. , Variations in alkaline phosphatase levels causes changes in phosphate levels, which lead to initiation and progression of caries. 
In present study, it was found that levels of alkaline phosphatase were significantly (P < 0.0001) greater in caries-free group than in caries-active group [Table 3]. The mean relative values for caries-free and caries-active group were calculated to be 13.52 ± 0.98 IU/L and 12.02 ± 0.98 IU/L, respectively. Study done by Shahrabi M et al. also reported the higher level for alkaline phosphatase in the caries-free group (10.04 ± 3.7) than for carious group (7.24 ± 5.2) but the difference was statistically non-significant. (P > 0.05). 
|Table 3: Mean alkaline phosphatase concentration (Iu/L) in unstimulated saliva of caries‑free and caries‑active group|
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One of the factors important for prevention of dental caries includes remineralization of incipient carious lesions. It requires calcium, phosphate and fluoride all of which are present in saliva.  The concentrations of calcium and phosphate in saliva have significant influence on the protective mechanisms of dental hard tissues within the oral cavity.  Saliva contains calcium and phosphate in concentrations rendering it supersaturated with respect to all calcium phosphate salts. Calcium is a bivalent ion excreted together with salivary proteins into the lumen of the acinus. Therefore, the concentration of calcium in the saliva is dependent on the salivary flow rate in the same way as with proteins. The ionized calcium fraction is about 50%, and it is strongly dependent on the salivary pH.
In the present study, the mean calcium concentration in caries-active children (4.96 ± 0.97 mg/dl) was found to be more compared to caries-free children (4.45 ± 1.06 mg/dl) but difference was not statistically significant (P > 0.05) [Table 4]. The mean phosphate concentration was found to be significantly (P < 0.0001) higher in caries-active children (7.09 ± 1.47 mg/dl) as compared to caries-free children (4.99 ± 1.73 mg/dl) [Table 5]. Therefore, there is no correlation between salivary calcium and phosphate levels and dental caries. Other factors like concentration of fluoride, bicarbonate, pH, buffering capacity of saliva have a cumulative effect on caries incidence of individual.
|Table 4: Mean calcium concentration (mg/Dl) in unstimulated saliva of caries‑free and caries‑active group|
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|Table 5: Mean phosphorus concentration (mg/Dl) in unstimulated saliva of caries‑free and caries‑active group|
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The dental profession relies on mechanical plaque control as the most dependable way of achieving oral health benefits for all dental patients including periodontal patients. In the present study, statistically significant (P < 0.0001) difference was found between the children who brushed their teeth more than once per day in caries-free group i.e., 25 (83.3%) and in the caries-active group i.e., 8 (26.6%) [Table 6].
|Table 6: Frequency of tooth brushing of caries‑free and caries‑active group|
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Frequent consumption of sugars is associated with the prevalence of dental caries. Food eaten at meals produces less caries than the same eaten between the meals.
Grand total time of exposure to the acid = Total exposure × 20 minutes.
In our study, no significant (P > 0.05) difference was found between the number of children having more than three sugar exposures in caries-active group (60%) and in caries-free group (50%) [Table 7].
|Table 7: Number of sugar exposures/day of caries‑free and caries‑active group|
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Fluoride's ability to cause remineralization is believed to be more significant than its inhibition of demineralization. The surface and subsurface of the enamel absorb and hold minerals and fluoride, which are present in plaque fluid and enhance the regrowth of partially dissolved crystals. The regrowth by fluoride incorporation chemically forms new crystals which are larger and more acid resistant and contain a higher concentration of fluoride. Also fluoride inhibits growth of Mutans streptococci, thus enhancing its anticariogenic effect. In the present study, no significant (P > 0.05) difference was found regarding brushing with fluoridated toothpaste in caries-free group i.e., 73.3% and in caries-active group i.e., 66.6% [Table 8].
|Table 8: Frequency of use of fluoridated and non fluoridated toothpaste in caries‑free and caries‑active group|
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Therefore, level of hydration, flow rate, pH, buffering capacity, viscosity, levels of calcium, phosphorus and alkaline phosphatase of saliva play an important role in caries risk assessment. As dental caries is a multifactorial disease, other factors like microflora should also be taken into consideration while assessing caries risk.
To conclude, within limitations of this study, it became clear that normal level of hydration and higher values for flow rate, pH, buffering capacity of saliva lead to good oral health and a reduced caries occurrence. Increased salivary viscosity plays a role in increasing caries incidence. Salivary biochemical indicators like calcium, phosphorus and alkaline phosphatase also play their respective role in determining caries susceptibility of an individual. These salivary parameters can be used as diagnostic tool for caries risk assessment.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]
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