Salivary Vitamin D and Glutathione Peroxidase as Biomarkers for Caries in Children

Abstract

Context:

Saliva is rich in antioxidants, a natural defense system. Vitamin D, a steroid hormone and an antioxidant, is required in optimal quantities to maintain normal mineralization of teeth. The enzyme, glutathione peroxidase (GPx) is an antioxidant having an essential role in neutralizing free radicals. An imbalance in such salivary antioxidants causes an increase in reactive oxygen species, leading to oxidative stress that affects intracellular signals and promotes the growth of cariogenic bacteria.

Aim:

The aim of this study was to evaluate and compare the levels of salivary Vitamin 25(OH)D and GPx in children with and without caries.

Settings and Design:

A cross-sectional study was conducted with 120 children, between 3 and 10 years of age.

Subjects and Methods:

Salivary samples of children with and without caries were collected to assess the levels of Vitamin D and GPx through commercially available enzyme-linked immunosorbent assay kits.

Statistical Analysis Used:

Descriptive mean, along with Mann–Whitney test.

Results:

Salivary Vitamin 25(OH) D levels were significantly higher in caries-free children, showing a strong inverse correlation with caries risk. This suggests a protective role of Vitamin D. Although the mean GPx levels were higher in children with caries, the difference was not statistically significant, indicating a possible oxidative stress response but limited diagnostic value. Overall, Vitamin D showed a more consistent association with caries status than GPx.

Conclusions:

This study focused on individual antioxidant biomarkers, thus allowing applications for tailor made preventive strategies for early intervention of caries progression in pediatric population.

Introduction

Saliva influences various physiological and biochemical processes and serves as the first line of defense against dental caries.[1,2] Thus, the ability of saliva to maintain an appropriate balance in the ecosystem of the oral cavity is of high importance.[3] Recently, saliva has emerged as a promising alternative for clinical diagnostics.[4]

Antioxidants in saliva help counteract oxidative stress caused by reactive oxygen species (ROS), in response to an infection, inflammation, or disease.[4] The recent advancements in the field of microbiological aspects of dental caries, argues that oxidative stress in saliva plays an important role in onset and progression of the lesion.[5] Oxidative stress affects microbial virulence, host immune response, and enamel integrity. Cariogenic bacteria’s ability to tolerate oxidative environments may also contribute to disease progression.[6]

Among salivary antioxidants, Vitamin D and glutathione peroxidase (GPx) have gained attention for their roles in maintaining redox balance. Vitamin 25(OH)D/D3 not only regulates calcium metabolism but also modulates oxidative stress by enhancing antioxidant defenses.[7] Worldwide, Vitamin D deficiency is common in children and is linked to increased caries risk.[8,9] GPx, an enzyme responsible for reducing hydrogen peroxide and lipid peroxides, plays a crucial role in protecting oral tissues from oxidative damage.[10] In microbial communities, glutathione also contributes to resistance against acid and osmotic stress.[11]

Despite evidence of their individual roles, the combined evaluation of salivary Vitamin D and GPx in children with and without dental caries remains underexplored.

Thus, this study aims to compare salivary levels of Vitamin D and GPx in pediatric population with differing caries status to assess their potential as biomarkers for caries risk.

Subjects and Methods

This research study was conducted on children visiting the outpatient department of Pediatric and Preventive Dentistry of our institute in collaboration with the Department of Biochemistry for analysis of salivary samples.

The study was approved by the Institutional Review Board (IRB No: 2023/PG/PEDO/47). The parents of the children who were willing to participate in the study were provided with a patient information sheet and were asked to provide written consent and assent.

Study type

The study employed a cross-sectional design with simple randomization. A computerized randomization was used where random segregation of children in each group was done.

Distribution of the samples

• Group 1: Children in the age group of 3–6 years without caries

• Group 2: Children in the age group of 3–6 years with caries

• Group 3: Children in the age group of 7–10 years without caries

• Group 4: Children in the age group of 7–10 years with caries.

As the effect of Vitamin D and GPx were to be assessed with caries in both primary and mixed dentition periods, including a wide range of age group, that is, 3–10 years children were taken.

Inclusion criteria

(1) Children between the age group of 3 and 10 years without caries, (2) Children between the age group of 3 and 10 years with moderate and high caries risk category based on American Academy of Pediatric Dentistry (AAPD) 2022 caries risk assessment tool for 0–5 years and >6 years of age.[12]

Exclusion criteria

(1) Patients who do not consent to participate in the study, (2) Children with current or past history of medical illnesses/nutritional deficiencies (assessed through medical history or diagnostic reports) (3) Children on any long-term medications such as antifungal agents, corticosteroids, antihistamines, (4) Children with any salivary gland diseases or disorders, (5) Children with congenital malformations of the teeth.

The inclusion criteria included children with moderate to high-risk caries according to the AAPD caries risk assessment tool (AAPD CAT) (2022).[12] AAPD CAT is a highly sensitive tool for assessing caries risk category in children. The AAPD CAT gives a classification of caries risk as low, moderate, and high based on the children’s age, physiological factors, preventive factors, environmental factors, and clinical parameters.[13] Children with 5–8 decayed, missing, filled teeth in primary/permanent dentition (dmft/DMFT) were categorized as moderate caries risk and children with >8 dmft/DMFT were considered being at high-risk category and were included in the study. Children with 0 dmft/DMFT scores were considered to be in the caries free group.

Blinding: The inter- and intraexaminer reliability were assessed between the examiner and the supervisor for examination of caries using caries risk assessment tool by AAPD guidelines. The lab analyst was also blinded to the groups while testing the salivary samples, to reduce the bias.

Saliva collection and processing method

Two to five milliliters of whole unstimulated saliva were collected in a sterile disposable test tube [Figure 1] container using spit method. The samples were stored in an air tight container and refrigerated at 4°C. The collected saliva samples were transferred within 1–4 h to a laboratory and were centrifuged at 2000 rotations per minute for 5 min at 4° to collect the supernatant saliva and then were stored in −20°C refrigeration until salivary sample analysis using commercially available enzyme-linked immunosorbent assay (ELISA) KITS [Figures 2 and 3]. The microtiter plate was read with an ELISA reader at 450 nm absorbance within 15 min of color change to obtain the results [Figure 4].

Figure 1.

Collection of 2 ml of saliva samples in sterile 15 ml test tubes

Figure 2.

Ten micro-litres of biotinylated antibodies (Vitamin D antibody and glutathione antibody) and streptavidin horse raddish peroxidase added to all the wells

Figure 3.

The wells turning yellow after adding stop solution

Figure 4.

The results being analysed by EPOCH enzyme-linked immunosorbent assay microplate reader at 450 nm

Results

The present study included a total of 120 participants. In the 3–6-year-old age group, 24 children had caries and 34 were caries-free. In the 7–10-year-old age group, 36 children had caries and 26 were caries-free. The gender distribution in the carious group was 31 males and 29 females, and in the noncarious group, it was 26 males and 34 females. Before examination of the children, inter and intra-examiner reliability were assessed between the examiner and the supervisor for examination of caries using caries risk assessment tool by AAPD guidelines.

The intergroup mean comparison of Vitamin D and glutathione peroxidase levels in carious and noncarious groups

Table 1, Graphs 1 and 2: The intergroup mean comparison of Vitamin D and GPx levels in carious and noncarious groups were as follows:

Table 1.

The Intergroup mean comparison of Vitamin D and glutathione peroxidase level in carious and noncarious groups

	Groups	Minimum concentration	Maximum concentration	Mean value	Standard deviation
Carious	Vitamin D	12.10 nmol/L	296.92 nmol/l	91.1914 nmol/L	66.41
	Glutathione	2.85 ng/ml	38.58 ng/ml	12.6043 ng/ml	8.69
Non-carious	Vitamin D	37.66 nmol/L	359.58 nmol/L	171.2710 nmol/L	58.68
	Glutathione	0.68 ng/ml	16.78 ng/ml	9.9381 ng/ml	3.59

P<0.05 is statistically significant

Graph 1.

Graphical representation of inter-group mean comparison between caries and noncaries groups for Vitamin D levels. Note: The mean values are represented in nmol/L for Vitamin D

Graph 2.

Graphical representation of inter-group mean comparison between caries and noncaries groups for glutathione peroxidase levels. Note: The mean values are represented in ng/ml for glutathione peroxidase

In carious group: the Vitamin D mean level was 91.19 nmol/L and GPx mean levels were found to be 12.60 ng/ml.

In noncarious group: the Vitamin D mean level was 171.27 nmol/L and Glutathione mean levels were found to be 9.93 ng/ml.

Inference: the Vitamin D levels showed a higher mean value in noncarious group and GPx showed a higher mean value in carious group.

Statistical analysis for inter-group comparison

Table 2: The Mann–Whitney U-test gave the statistical significance of Vitamin D with and without caries and GPx with and without caries. The P < 0.05 for Vitamin D levels in carious and noncarious groups was statistically significant. However, the P = 0.6 for GPx levels indicated no statistical significance.

Table 2.

Statistical analysis with significance test

	Vitamin D	Glutathione
Mann–Whitney U	616.000	1717.500
P	0.000	0.665

P<0.05 is statistically significant

Confidence intervals for groups were set at 95%:

• 95% Confidence Interval for mean of Vitamin D with caries:

  • Lower Bound: 74.0337

  • Upper Bound: 108.3491.

• 95% Confidence Interval for mean of Vitamin D without caries:

  • Lower Bound: 156.1111

  • Upper Bound: 186.4309.

• 95% Confidence Interval for mean of GPx with caries:

  • Lower Bound: 10.3594

  • Upper Bound: 14.8493.

• 95% Confidence Interval for mean of GPx without caries:

  • Lower Bound: 9.0092

  • Upper Bound: 10.8670.

Correlation between Vitamin D and caries risk category

Table 3: The spearman’s correlation was used to assess the correlation between Vitamin D and caries risk category. The correlation coefficient of 0.840 depicted a very strong negative correlation between both these parameters. The P < 0.05 which indicates a highly significant correlation.

Table 3.

Correlation between Vitamin D levels and caries risk category

Salivary parameter	Spearman’s correlation	Caries risk category	P
Vitamin D	Correlation coefficient	−0.840	<0.05

P<0.05 is statistically significant

Discussion

Traditional diagnostic methods often detect carious lesions at advanced stages, making treatment more complex. This highlights the need for research on salivary biomarkers as a noninvasive, efficient tool for early caries detection and prevention. Saliva contains enzymes that can signal demineralization before visible cavities form. ELISA, quantify such specific biomarkers, hence is a far more sensitive testing. This study utilized commercially available ELISA kits from KRISHGEN BIOSYSTEMS Pvt Ltd for Vitamin D and GPx biomarkers to be tested.

Oxidative stress is defined as an “imbalance between the body’s free radicals and antioxidants.”[14] Oxidative stress may cause physiological imbalances, leading to cellular and DNA damage.[14,15]

Modulation of oxidative stress pathways are shown to be done by Vitamin D which enhances the expression of genes involved in antioxidant defense, thereby indirectly reducing oxidative stress caused by ROS[16,17] and by GPx which are direct scavengers of ROS.[18] Thus, this study aimed to assess these two parameters and their effect on caries.

The Vitamin D levels in this study showed the mean value of 91.19 nmol/L in caries group and 171.27 nmol/L in the noncarious group indicating a wide range. The result showcases that caries free children had the higher mean concentration of Vitamin D levels in saliva compared to caries group.

The explanation for Vitamin D levels to be lower in carious group in our study could be because electrolytic secretions from various salivary glands have a direct dependence on Vitamin D levels. Studies done by Peterfy et al., Moneem and Mohammed reported that low levels of Vitamin D are said to reduce the salivary flow parameters and alter its composition by decreasing the amount of calcium ions.[19,20] This fact is agreed upon by other studies by Stumpf and He et al. where a significant positive correlation between serum Vitamin D concentration and salivary flow rate was assessed.[21,22]

Catalase, superoxide dismutase, and GPx are key salivary antioxidants that protect against ROS, which serve as signaling molecules for bacterial destruction by phagocytic cells.[23]

The GPx mean levels between the groups were – 12.60 ng/ml for caries group and 9.93 ng/ml for noncarious group showing some amount of variation. The result although suggests that the carious children had a higher mean concentration of GPx levels in saliva compared to the caries-free group, it showed no statistical significance (P = 0.6).

Hendi et al., compared caries-active and caries-free individuals, found that the caries-active group exhibited higher levels of peroxidase, uric acid, catalase, and GPx, whereas superoxide dismutase was lower.[24] The elevated GPx levels in caries-active subjects supports our observation of a trend toward higher levels.

Another cross-sectional study among 90 children aged 7–12, researchers measured salivary GPx (along with Catalase and Super-oxide Dismutase). Although GPx activity was higher in caries-active children, no significant correlation was found between GPx and the DMFT/dmft index.[25] This is in direct accordance with our study where a higher mean concentration in the caries group was found, but lacking statistical significance.

A comprehensive review and meta-analysis assessed multiple salivary oxidative stress markers in children with carious lesions versus caries-free controls. Although there were observable differences in biomarkers, methodological variability across studies meant that the evidence lacked consistency and statistical robustness. Specifically, GPx was noted in some studies to be elevated in caries-active subjects, but without uniform statistical significance.[26]

The plausible reasons for an increase trend in GPx levels in carious children could be as follows: The salivary antioxidant system encompasses the salivary peroxidase system as one of the pivotal entities. The bacteria in the oral cavity are controlled by salivary peroxidase by affecting the dental plaque associated bacteria, to altering the eco-system, and thus leading to caries. Salivary peroxidase acts as a catalyst in peroxidation of the thiocyanate ion (SCN-) to generate a more stable oxidation result. This inhibits microbial metabolism and growth, thereby halting caries progression.[27]

To assess the relationship between salivary glutathione levels, cariogenic bacteria, and dental caries, Han et al., in 6–14 years children saw an increased salivary glutathione levels with elevated lactobacilli levels and higher DMFT scores.[28] Thus, an increase in the GPx may suggest that the oxidant system may be produced in excess to counteract the carious microorganisms.

Another significant finding in the present study is attributed to the correlation between Vitamin D and caries risk category. A strong negative correlation (r = −0.840) between salivary Vitamin D levels and caries risk was seen, indicating that children with higher caries risk had significantly lower Vitamin D levels. This inverse relationship may be explained by Vitamin D’s role as an immune modulator. Through its receptor, Vitamin D stimulates the production of antimicrobial peptides such as cathelicidins and defensins, which protect against oral pathogens by disrupting bacterial biofilms and modulating the host immune response. Hung et al. also found a strong correlation between low Vitamin D levels and higher caries risk, highlighting Vitamin D’s preventive role in dental health.[29]

Strengths and limitations

This study presents a promising, noninvasive approach for early detection of dental caries by analyzing salivary biomarkers–Vitamin D and GPx–using sensitive ELISA kits. A key merit is the identification of a strong negative correlation between Vitamin D levels and caries risk, where supporting its role as a preventive factor. The dual focus on immune modulation and oxidative stress adds depth to understanding caries pathogenesis. However, the study’s cross-sectional design and the lack of statistical significance in GPx findings limit its ability to establish causality. In addition, potential confounding factors such as diet and sunlight exposure, were not accounted for, which may influence biomarker levels.

Conclusions

This study acts as a framework for further longer longitudinal studies and can integrate the role of a wider range of salivary biomarkers with traditional clinical indices. Salivary Vitamin D levels could be used as a biomarker for caries risk. The salivary GPx and caries although showed the carious group with a higher mean value, a weak correlation was found with the caries risk category. Glutathione may indicate an adaptive response to oxidative stress which is induced by the caries-causing bacteria and this relationship can be solidified using further studies. Hence, caries activity can also be assessed by these salivary factors therefore underscoring the importance of lifestyle/nutritional factors to prevent the occurrence of caries in children.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.