|Year : 2023 | Volume
| Issue : 4 | Page : 377-383
Correlation between dental caries and salivary pH, mineral content, and salivary immunoglobulin levels in adult population of northern suburban region of Kolkata, India
Sagnik Bhattacharya, Paromita Mazumdar, Sayantan Mukherjee
Department of Conservative Dentistry and Endodontics, Guru Nanak Institute of Dental Sciences and Research, Kolkata, West Bengal, India
|Date of Submission||07-Feb-2023|
|Date of Decision||25-Jun-2023|
|Date of Acceptance||26-Jun-2023|
|Date of Web Publication||31-Aug-2023|
Dr. Sagnik Bhattacharya
Department of Conservative Dentistry and Endodontics, Guru Nanak Institute of Dental Sciences and Research, 18C/2 Kabi Sukanta Road, Kolkata 700075, West Bengal
Source of Support: None, Conflict of Interest: None
Aim: The aim of this study was to evaluate the prevalence of dental caries in the adult population residing in West Bengal and measure the salivary pH, calcium, phosphorus, immunoglobulin A (IgA), and immunoglobulin A (IgG); and to determine a correlation between the caries experience and the variables measured. Materials and Methods: This was a correlative analytical study with a cross-sectional approach where a total of 100 patients who fit the inclusion criteria were included in the study. First, the number of caries teeth of each patient was recorded using the International Caries Detection and Assessment System II (ICDAS II) scoring system, and subsequently unstimulated saliva was collected. The saliva was measured for pH using a pH meter, calcium using the o-Cresolpthalein Complexone method, phosphorus using Molybdate UV method, and IgA and IgG using the immunoglobulin estimation kit. Chi-square test of proportions was employed to test the categorical variables. Parametric tests were carried out for inferential statistics. Pearson’s correlation test was used to correlate the number of carious teeth with that of the salivary pH, calcium levels, phosphorus levels, salivary IgA levels, and salivary IgG levels. Results: There was a strong negative correlation between the calcium levels and the number of carious teeth present, which was highly statistically significant. There was a very strong negative correlation between the salivary pH, phosphorus levels, salivary IgA levels, and the number of carious teeth, which was highly statistically significant. There was a moderately negative correlation between the IgG levels and the number of carious teeth present which was highly statistically significant. Conclusion: There is an inverse relation between the prevalence of dental caries in the adult population of the Northern Suburban area of Kolkata and salivary pH, calcium, phosphorus, IgA, and IgG.
Keywords: Calcium, dental caries, immunoglobulin A, immunoglobulin G, pH, phosphorus
|How to cite this article:|
Bhattacharya S, Mazumdar P, Mukherjee S. Correlation between dental caries and salivary pH, mineral content, and salivary immunoglobulin levels in adult population of northern suburban region of Kolkata, India. J Int Oral Health 2023;15:377-83
|How to cite this URL:|
Bhattacharya S, Mazumdar P, Mukherjee S. Correlation between dental caries and salivary pH, mineral content, and salivary immunoglobulin levels in adult population of northern suburban region of Kolkata, India. J Int Oral Health [serial online] 2023 [cited 2023 Sep 25];15:377-83. Available from: https://www.jioh.org/text.asp?2023/15/4/377/384662
| Introduction|| |
Dental caries is a chronic biofilm-mediated disease of the oral cavity that results from the growth of cariogenic microorganisms due to the presence of fermentable carbohydrates on the tooth surface. It leads to the depletion of mineral content and deterioration of the organic constituents, resulting in the formation of cavitated lesions.
Saliva plays a crucial role in oral health, aiding chewing, swallowing, speaking, removing food debris, sugars, and eliminating microorganisms. In addition, it has a buffering effect that neutralizes acids in the mouth.
Saliva possesses an array of quantifiable attributes that may serve as biomarkers for dental caries and have the capacity to predict, diagnose, and regulate dental caries while also evaluating the effectiveness of therapeutic treatments.
Salivary electrolytes such as fluoride, calcium, phosphate, and bicarbonate are important for protecting teeth from dental caries. The impact of calcium and phosphate in saliva on ion activity, specifically the salivary pH level, is a crucial factor. The ion activity product for hydroxyapatite, a parameter that reflects pH, calcium, and phosphate ion concentrations in saliva, is significant because it regulates the saturation level in relation to tooth mineral dissolution.,
Dental caries is significantly influenced by calcium and phosphorus present in saliva, as shown by most studies. Bevinagidad et al. and Rajesh et al. discovered an inverse relationship between dental caries and salivary calcium and phosphorus levels. Leander-Lumikari et al. found no correlation and Gandhy and Damle found a direct correlation.
More than 85% of immunoglobulins found in saliva are of the immunoglobulin A (IgA) subclass, with smaller amounts of IgG and IgM subclasses also present. These immunoglobulins prevent bacterial growth by preventing microbial flora from adhering to tooth surfaces, obstructing bacterial metabolism, and neutralizing bacterial toxins and enzymes.
There is conflicting literature regarding the anti-carious effect of immunoglobulins. Although some studies report an inverse relation,,, others show a direct relationship., The majority of research has been conducted on children. To date, only one study has been performed in West Bengal by Pal et al. who aimed to investigate the correlation between salivary IgA and its impact on the caries status of children.
Therefore, the research question is: Is there a correlation between the occurrence of dental caries among the adult population of Kolkata and the quantity of salivary calcium, phosphate, IgA, IgG, and pH?
The purpose of this study was to assess the occurrence of dental caries among the adult demographic residing in Kolkata and measure the salivary pH, calcium, phosphorus, IgA, and IgG and determine a correlation between the caries experience and the variables measured.
| Materials and Methods|| |
This was a correlative analytical study with a cross-sectional approach, which was carried out in the Department of Conservative Dentistry and Endodontics from February 2021 to August 2022. The institutional ethics committee gave its approval for the study.
Sample size calculation
Assuming P < 0.05 to be significant and considering the effect to be two-sided, the value of Zα = 1.96; assuming the power of the study to be 90% we get Z1-β = 1.28; and considering an effect size of 1.25 to be statistically significant we get n = 2(Zα + Z1-β)2 x SD2/d2, that is, n = 90. Hence, a minimum of 90 samples are required. The final sample size was 100.
- Patients residing in Panihati Municipality between the ages 18 and 60 years of either gender.
- Patients free from systemic and local diseases.
- Patients who have not taken any nonsteroidal anti-inflammatory or antibiotics in the last 14 days.
- Patients with systemic and local diseases that affect salivary.
- Patients undergoing radiotherapy.
- Patients with a history of medications that can alter salivary secretions.
- Patient with habit history for example, tobacco chewing, smoking, and alcoholism.
A consent form was signed by patients who fulfilled the inclusion criteria and agreed to take part in the study.
Recording caries experience
First, the number of carious lesions in each patient was recorded. The teeth were dried using a three-way syringe for 5 s. When multiple carious lesions were present on the occlusal surface, the lesion with the most severity was classified and recorded using the International Caries Detection and Assessment System II (ICDAS II) scoring system.
Collection of unstimulated saliva
The specimens of unstimulated whole saliva were obtained two hours following oral or visual contact with food items. Saliva samples were obtained in sterile containers during the hours of 10 am to 1 pm to mitigate any potential influence from circadian rhythm. The patients were directed to perform a mouth rinse using a plain glass of water in order to eliminate any loosely attached debris from their teeth. The patients were directed to sit in an upright position on the dental chair and tilt their heads slightly for a duration of three minutes, in order to facilitate the accumulation of saliva on the floor of their oral cavity. Patients were subsequently instructed to expel their saliva into a sterile container that featured milliliter markings. A volume of 5 mL of unstimulated saliva was obtained.
Measurement of pH
With the help of a single-electrode digital pH meter, the pH of saliva was determined. At first, the pH meter was calibrated using a buffer solution of acidic (4), neutral, (7) and basic (10). After this, the pH meter was placed in the saliva sample and the reading was noted. Between each reading, the electrode was rinsed with distilled water and pat dried with tissue paper [Figure 1].
Subsequently, the saliva samples were subjected to centrifugation at a rate of 4000 revolutions per minute for a duration of 10 min. The resultant supernatant was then subjected to analysis for the presence of calcium, phosphorus, immunoglobulin A, and immunoglobulin G [Figure 2].
Measurement of calcium levels in saliva
Calcium levels in saliva were measured using the OCPC (o-Cresolpthalein Complexone) method using the Calcium Kit by Coral Clinical Systems, Panjim, Goa, India. The kit consists of three reagents – Buffer Reagent (L1), Color Reagent (L2), and Calcium Standard (S). Three disposable, clean, and dry microcentrifuge tubes were selected and marked as blank (B), standard (S), and test (T).
A volume of 0.5 mL of buffer reagent (L1), 0.5 mL of color reagent (L2), and 0.02 mL of distilled water were added to the microcentrifuge tube designated as blank (B). In the microcentrifuge tube marked as standard(S), 0.5 mL of buffer reagent (L1), 0.5 mL of color reagent (L2), and 0.02 mL of calcium standard were added. A volume of 0.5 mL of buffer reagent (L1), 0.5 mL of color reagent (L2), and 0.02 mL of the saliva sample were added to the microcentrifuge tube labeled as a test (T). The contents were mixed thoroughly. Subsequently, the three microcentrifuge tubes were subjected to incubation at room temperature (25ºC) for a duration of 5 min. A spectrophotometer was used to measure the optical density of the standard (S) and test (T) against the blank (B) at a wavelength of 570 nm. The formula that was used to measure salivary calcium levels: calcium in mg/dL = (absorption of test/absorption of standard) × 10.
Measurement of phosphorus levels in saliva
The quantity of phosphorus was measured using the phosphorus kit by Coral Clinical Systems. The kit consists of three reagents: acidic reagent (L1), molybdate reagent (L2), and phosphorus standard (S). The contents of bottle L2 (molybdate reagent) were poured into bottle L1 (acidic reagent) to create the working reagent. Three disposable, clean, and dry microcentrifuge tubes were selected and marked as blank (B), standard (S), and test (T).
A volume of 1.0 mL of working reagent and 0.01 mL of distilled water were introduced into microcentrifuge tubes that were appropriately labeled as blank (B). In microcentrifuge tubes labeled standard (S), 1.0 mL of working reagent and 0.01 mL of phosphorus standard were added. Working reagent (1.0 mL) and test solution (0.01 mL) were introduced into microcentrifuge tubes designated as “test” (T). Thorough mixing of the reagents was performed. The absorbance of the standard and the test was measured and recorded at 340 nm relative to the blank. The formula that was used to measure salivary phosphorus levels: phosphorus in mg/dL = (absorption of test/absorption of standard) × 5.
Measurement of levels of IgA and IgG
The total amount of IgA and IgG was measured immunoturbidimetrically. The IgA and IgG were estimated using an immunoglobulin estimation kit (Quantia IgA and Quantia IgG, Coral Clinical Systems). The kit contains reagent 1 (R1)––activation buffer and reagent 2 (R2)––antihuman IgA antibody. A volume of 950 µL of R1 was introduced into the microcentrifuge tube, followed by the addition of 7 µL of the sample. The mixture was subjected to spectrophotometric analysis (Shimadzu UV-1800) [Figure 3] to obtain the absorbance reading at a wavelength of 340 nm, denoted as A1. Subsequently, a volume of 50 µL of R2 was introduced into the microcentrifuge tube and the spectrophotometer was used to measure the absorbance reading A2 at a wavelength of 340 nm after a duration of one minute. The values were substituted using the following formula: A2 – A1 = ΔA, where ΔA is the quantity of IgA in mg/dL present. The same process was repeated to measure the level of IgG in the samples using the Quantia IgG Kit.
|Figure 3: Spectrophotometer to measure salivary IgA, IgG, calcium, and phosphorus|
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The data that were obtained were arranged into a spreadsheet using Microsoft Excel 2019. The statistical analysis was conducted using IBM SPSS Statistics for Windows, version 26.0 (IBM Corporation, Armonk, New York).
The data were normally distributed as shown by the Shapiro–Wilk test and an examination of the histograms, normal Q–Q plots, and box plots. Descriptive statistics were used to report (i) categorical variables in terms of frequencies and percentages and (ii) quantitative variables in terms of mean and standard deviation.
The categorical variables were subjected to a chi-square test of proportions. Parametric tests were carried out for inferential statistics. Pearson’s partial correlation test was used to correlate the number of carious teeth with that of the salivary pH, calcium levels, phosphorus levels, salivary IgA levels, and salivary IgG levels while controlling for age to omit the effect of the confounding variable. A value P ≤ 0.05 was considered as the level of significance.
| Results|| |
The average number of carious teeth per patient was 7.32 ± 3.05 with mean salivary pH 6.92 ± 0.23, calcium 8.37 ± 1.36 mg/dL, phosphorus 13.16 ± 3.28 mg/dL, salivary IgA 17.61 ± 2.9 mg/dL, and salivary IgG 2.21 ± 0.68 mg/dL [Table 1]. After adjusting for age, a strongly negative relationship was observed between the quantity of carious teeth and salivary pH (r = –0.645), calcium levels (r = –0.575), phosphorus levels (r = –0.575), salivary IgA(r = –0.79), respectively, and a moderately negative correlation with salivary IgG (r = –0.386). All the correlations were highly statistically significant (P < 0.001) [Figure 4].
|Figure 4: Scatter plots showing a relationship between the number of carious teeth present and the outcome variables whilecontrolling for age. Partial correlation between number of carious teeth present and (i) Salivary pH, (ii) Calcium levels, (iii) phosphorus levels, (iv) salivary IgA levels, and (iv) salivary IgG levels. r = partial correlation (controlling for age variable). **Highly statistically significant (P < 0.001)|
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| Discussion|| |
Salivary pH, calcium, and phosphorus
The pH level of saliva has a significant impact on oral health as it plays a role in preventing tooth decay. The pH range of saliva typically falls within the range of 6.2–7.6, with a mean value of 6.7. Saliva plays a crucial role in regulating pH levels via two mechanisms. Initially, the flow of saliva serves to eliminate carbohydrates and acids generated by bacterial activity. Furthermore, the salivary buffering capacity serves to counteract the acidic effects of ingested food, drinks, and microbial metabolic processes.
Muchandi et al. performed a study to establish a correlation between the salivary pH levels of children with severe early childhood caries and those without caries. The results indicated that the group without caries had a higher pH level (7.46 ± 0.37) compared with the group with early childhood caries (6.42 ± 0.34). Yamunadevi et al. in their study found that the average salivary pH was 6.9 ± 1.1 among individuals without caries, 6.9 ± 1.2 among those with a decayed missing filled teeth (DMFT) score of less than 5, and slightly lower pH (6.7 ± 1.4) among those with a DMFT score of 5 or higher. Similarly, Prabhakar et al. found that children with dental caries had salivary pH levels between 6.20 and 7.90, which was higher than the threshold pH of 5.5. According to the study’s findings, the measured levels were inadequate to cause demineralization of the tooth’s inorganic substance.
In this study, the mean salivary pH was 6.92. A strong negative correlation (P < 0.001) was observed between the salivary pH and the quantity of carious teeth, indicating that as the number of carious teeth increased, the salivary pH decreased.
Calcium and phosphate ions prevent caries development by maintaining saliva mineral saturation, ensuring equilibrium between enamel dissolution and remineralization. The UV Molybdate method was used to determine the level of phosphorus in saliva. This method entails the reaction of phosphorus and ammonium molybdate to produce a blue phosphomolybdate complex that absorbs ultraviolet light at 340 nm. The quantity of phosphorus present is directly proportional to the potency of the formed complex. Similarly, the O-Cresolpthalein Complexone method (OCPC) was used to measure calcium. The process involves the interaction between calcium ions and the OCPC complex, resulting in the formation of a violet hue. The relationship between the measured calcium quantity and the intensity of the color is directly proportional, as determined by spectrophotometric analysis.
Bevinagidad et al. showed an inverse correlation between dental caries and salivary levels of calcium and phosphorus. Similarly Rajesh et al. showed that individuals with reduced levels of inorganic calcium and phosphate in their saliva were more susceptible to the development of dental caries. But Lenander-Lumikari et al. in their study concluded that no relation exists between the number of carious teeth and salivary calcium and phosphorus levels. Gandhy and Damle showed that there is a greater concentration of phosphorus in the saliva of children who exhibits rampant caries.
In this study, the mean calcium and phosphorus concentration in saliva was 8.37 mg/dLand 13.16 mg/dL, respectively. There was a strong negative correlation between the calcium levels (mean = 8.37 ± 1.36) and the number of carious teeth and a very strong negative correlation between the phosphorus levels (mean = 13.16 ± 3.28) and the number of carious teeth (mean = 7.32 ± 3.05) which was statistically significant.
Salivary immunoglobulin levels
Dental caries is an infectious disease that is capable of being controlled by the host’s immune system, so immunoglobulins play a role in its management. Immunoturbidimetry was used to quantify the immunoglobulin levels in this study. This method entails combining antigens with IgA and IgG antibodies present in the salivary samples, which results in the formation of insoluble complexes that cause a change in optical density, which is then measured with a spectrophotometer. The reading is then compared with a calibrator of known IgA and IgG concentration.
Angarita-Díaz et al. showed that caries-free children had a greater concentration of IgA than their caries-affected counterparts, although this difference was not statistically significant. In a study conducted by Razi et al., a comparison was made between the levels of IgA and IgG in children who were caries-free and those who were caries-active. The results showed that the mean concentration levels of salivary IgA were significantly higher in children who were free from dental caries. The IgG levels were comparatively elevated in children who were free of caries, although the observed difference did not attain statistical significance. Similarly, Mousavizadeh et al. conducted a study to assess the correlation between dental caries and salivary IgA and IgG in the unstimulated saliva of adult patients. They showed that salivary IgA increases with a decrease in caries activity but salivary IgG did not show any correlation with dental caries.
In contrast, Vitorino et al. showed a positive correlation between elevated salivary IgA levels and individuals showing a high DMFT index. Similarly, Thaweboon et al. found that children with rampant caries exhibited elevated levels of salivary IgA. Haeri-Araghi found a positive correlation between salivary IgA levels and DMFT scores in adult patients.
In this study, the IgA and IgG levels were evaluated in adults between the ages of 18 and 60 years. The mean IgA was 17.61 ± 2.90 mg/dL and the mean IgG was 2.21 ± 0.68 mg/dL. There was a very strong negative correlation between the salivary IgA levels and the number of carious teeth which was highly statistically significant, whereas there was a moderately negative correlation between the salivary IgG levels and the number of carious teeth, which was highly statistically significant.
| Conclusion|| |
There is a negative correlation between the prevalence of dental caries in the adult population of Northern Suburban Kolkata and salivary pH, calcium, phosphorus, IgA, and IgG. This study also gives the caries prevalence data of Kolkata which can be used to address the local needs of the population. The data collection was limited to a single center in Kolkata. In order to extrapolate the results to the state of West Bengal multicentric study, a larger population size is required.
Commercially available tools for salivary risk assessment of caries – the salivary biochemical indicators calcium phosphorus IgA and IgG have shown an inverse relation to dental caries prevalence so similar chairside test kits can be developed to assess the caries risk.
We would like to express our gratitude toward all the patients who took part in this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
All authors had equal contribution in study design, data collection, data annotation and manuscript preparation and final editing.
Ethical policy and Institutional Review board statement
The study received ethical approval (GNIDSR/IEC/20-23/22) from the institutional ethical committee on January 21, 2021. All the procedures have been performed as per the ethical guidelines laid down by the Declaration of Helsinki (1964) and its later amendments.
Patient declaration of consent
Informed consent was obtained for participation in the study and publication of the data for research and educational purposes.
Data availability statement
Data is available on request from corresponding author.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]