Journal of South Asian Association of Pediatric Dentistry
Volume 6 | Issue 3 | Year 2023

Comparative Assessment of the Association between Salivary Vitamin D Levels and Early Childhood Caries Using Enzyme-linked Immunosorbent Assay

Umme Azher1, Aishwarya Chidambareshwar2, Santhosh T Paul3, Divya Reddy4

1,3,4Department of Pediatric and Preventive Dentistry, Sri Rajiv Gandhi College of Dental Sciences & Hospital, Bengaluru, Karnataka, India

2Department of Pediatric & Preventive Dentistry, Hyderabad, Telangana, India

Corresponding Author: Umme Azher, Department of Dental, Faculty of Paedodontics and Preventive Dentistry, Sri Rajiv Gandhi College of Dental Sciences & Hospital, Bengaluru, Karnataka, India, Phone: +91 9945352984, e-mail: drummeazher@yahoo.com

Received: 19 June 2023; Accepted: 11 July 2023; Published on: 30 December 2023


Aim: Evaluation of interrelation between vitamin D levels in saliva and early childhood caries (ECC) utilizing enzyme-linked immunosorbent assay (ELISA).

Materials and methods: The sample selection in the cross-sectional, case-control study involved—(1) an interview with the parents/caregivers through a questionnaire; (2) a clinical examination of children to ascertain dental decay status; those with severe ECC (S-ECC) were enlisted in group I (n = 15) and children with a decayed, missing, and filled surfaces (dmfs) score of 0 to group II (n = 15). The samples of unstimulated whole saliva (5 mL) were collected, and the salivary vitamin D levels were measured using a human 25-dihydroxy vitamin D3 (25-OHD) ELISA kit.

Results: Subjects in groups I and II showed mean salivary vitamin D levels of 8.8 + 4.92 and 16.8 + 10.8 ng/mL, respectively. The vitamin D levels in saliva exhibited a difference statistically.

Conclusion: Variations in vitamin D scores in the saliva of children with S-ECC and those caries-free demonstrate that vitamin D in saliva can serve as a marker of caries risk in children.

Clinical significance: The simplicity and noninvasiveness of saliva collection, especially in children, confers the advantage of a more acceptable and cost-effective biofluid for caries-risk factors assessment.

How to cite this article: Azher U, Chidambareshwar A, Paul ST, et al. Comparative Assessment of the Association between Salivary Vitamin D Levels and Early Childhood Caries Using Enzyme-linked Immunosorbent Assay. J South Asian Assoc Pediatr Dent 2023;6(3):114–117.

Source of support: Nil

Conflict of interest: None

Keywords: Biofluid, Dental decay, Early childhood caries, Saliva, Vitamin D


Early childhood caries (ECC) is the occurrence of one or more noncavitated or cavitated, missing, or filled tooth surfaces due to decay in any primary tooth in a child <6 years of age,1 and in its severe form, impacts the health and welfare of children.2 ECC being multifactorial in origin, its prevalence has been attributed to socioeconomic status, constricted access to dental services, sugar consumption, oral hygiene maintenance, fluoride intake, and regular dental examination.3-7 The interconnection of severe ECC (S-ECC) with deficiency of essential vitamins and nutrients is plausible.8 Vitamin D has been viewed as an essential part of amelogenesis, dentinogenesis, and osteogenesis as their precursor cells are target cells for 1,25-dihydroxycholecalciferol.9

Vitamin D deficiency during tooth development may lead to developmental imperfections encompassing enamel hypoplasia, a notable risk factor for S-ECC.9 It is thus pivotal in the management of dental decay, presumably by intensifying calcium absorption, which extends serum calcium levels and thus promotes tooth remineralization.10 Although blood serum is the benchmark for the detection of diseases and drugs, saliva presents as a noninvasive biotic substitute. The recognition of the importance of vitamin D in facilitating general and oral health has prompted the exploration of easy, noninvasive procedures to gauge its level in human biotic fluids. The current study, hence, aimed to discover the interrelation between vitamin D levels in saliva and ECC employing enzyme-linked immunosorbent assay (ELISA).


The cross-sectional, case-control research was approved by the Institutional Ethical Committee, and informed consent was obtained from the participants in accordance with the Helsinki Declaration. The study design involved—(1) an interview with the parents/caregivers through a questionnaire and (2) a clinical examination of the subjects to determine the status of the dental caries. A structured questionnaire (in both English and vernacular language) was randomly distributed among the parents. The questionnaire was assessed for adequacy by randomly distributing it to about five parents following prevalidation by specialists in pediatric and preventive dentistry.

The questionnaire comprised six parts—part I: general information about the child; part II: educational level, occupation, and socioeconomic status of the parents/caregivers; part III: natal history; part IV: a medical history of the child; part V: feeding and dietary practices of the child, which included information on the frequency and manner of milk consumption, frequency of sleeping with a nursing bottle and intake of sugary food, milk fortification with vitamin D, lactose intolerance, intake of vitamin D supplements, and duration of exposure to sunlight; part VI: oral hygiene practices, which included information on the age of initiation of tooth brushing, method and frequency of tooth brushing, type of dentifrice (fluoridated/nonfluoridated), intake of fluoride supplements, and if the tooth brushing was done by the child/parent/under parental supervision.

A trained and calibrated examiner performed the clinical examination on children of 3–5 years, and a recording assistant was trained to precisely record the clinical examination details. The decayed, missing, and filled surfaces (dmfs) was determined using a plane mouth mirror and the Community Periodontal Index probe under the operatory light. Children’s dmfs scores of >4 (3 years), >5 (4 years), and >6 (5 years) were assigned to group I (n = 15) and those with dmfs scores of 0 to group II (n = 15).

The collection of unstimulated whole saliva (5 mL) was done in a sterile polypropylene graduated tube by spitting method. The partakers did not eat and drink within 30 minutes and did not brush their teeth within 2 hours prior to sample collection. The subjects were seated comfortably, inclined slightly forward, and advised to rest for 5 minutes and minimize orofacial contractions. The subjects were instructed to expectorate in the graduated tube every 60 seconds. The salivary samples were frozen (−20°C) until use, and then brought to room temperature (1 hour) and centrifuged for 10 minutes at 2500 rpm to isolate debris and squamous cells. The salivary vitamin D levels were measured using a human 25-dihydroxy vitamin D3 (25-OHD) ELISA kit, utilizing an antibody sandwich enzyme-linked immunosorbent assay.


The Chi-squared (χ2) test compared the study parameters, and the student t-test ascertained the existence of a statistical difference between the study and control groups. The receiver operating characteristic (ROC) curve assessed the optimal cutoff of vitamin D levels.

The study sample comprised of participants aged 3–5 years. The mean age in groups I and II was 4.2 + 0.61 and 4.7 + 0.41 years, respectively. Group I (S-ECC) comprised of 46.7% boys and 53.3% girls. Group II (control) comprised of 53.3% boys and 46.7% girls. The educational status of the mothers showed that 66.7% had high school education in both groups I and II, 20% of the mothers in group I and 33.3% in group II had a diploma level of education, and 13.3% of the mothers in group I had university level of education and nil in group II. However, none of them were uneducated. Socioeconomic status, gestational age, birth weight, and medical history showed no significant difference between the two groups. Group I exhibited a higher frequency of sugary snack consumption in comparison to group II. Furthermore, a significant difference was observed between the groups in the use of the tooth-brushing method. In terms of the use of dentifrices and fluoride supplements, both groups used a fluoridated dentifrice and were not in any fluoride supplements. The subjects in both groups brushed only once/day.

It was observed that the majority (66.67%) of the subjects in group I demonstrated vitamin D levels in saliva lower than 12 ng/mL in comparison to 33.33% in group II. About 33.33% of subjects in group I exhibited vitamin D levels between 12.0 and 20.0 ng/mL in comparison to 46.67% in group II. About 20% of the subjects in group II demonstrated salivary vitamin D levels greater than 20 ng/mL as against 0% in group I. A significant difference was observed in the salivary vitamin D levels of both groups statistically (Table 1 and Fig. 1).

Table 1: Salivary vitamin D levels among children of groups I and II
Vitamin D level range Group Total p-value*
Group I Group II
<12.0 ng/mL 10 5 13 0.00967*
66.67% 33.33% 50%
12.0–20.0 ng/mL 5 7 14
33.33% 46.67% 46.7%
>20.0 ng/mL 0 3 3
0% 20.0% 10.0%

*p < 0.05, significance; ng, nanogram; mL, milliliter

Fig. 1: Distribution of the salivary vitamin D levels among children of groups I and II

The subjects in group I showed average salivary vitamin D levels of 8.8 + 4.92 ng/mL in comparison to 16.8 + 10.8 ng/mL in group II. A significant difference was observed in the vitamin D levels of saliva in both groups (Table 2 and Fig. 2).

Table 2: Comparison of the mean salivary vitamin D levels (ng/mL) among children of groups I and II
N Mean SD Median Minimum Maximum p-value*
Group I 15 8.8 4.923 8.5 1.0 17.5 0.015
Group II 15 16.8 10.865 14.25 2.3 45.5

*p < 0.05, significance; ng, nanogram; mL, milliliter; N, number of samples; SD, standard deviation

Fig. 2: Mean salivary vitamin D levels in groups I and II

Receiver Operating Characteristics (ROC) Analysis

According to the analysis, the optimum salivary vitamin D cutoff value was ≤9.5 ng/mL for group I and >9.5 ng/mL for group II (control). Figure 3 depicts the area under the ROC curve [area under the curve (AUC)]. The AUC value of 0.762 indicates an acceptable discriminative ability of salivary vitamin D level between two groups.

Fig. 3: Area under the ROC curve


The prevalence of dental caries as the most persistent malady of childhood warrants the determination of the contributory factors to facilitate its prevention. Research evidence suggests a correlation between ECC and vitamin deficiency, specifically vitamin D.11,13 Saliva has been observed to be a good indicator of plasma levels of various substances such as drugs and hormones.14 Furthermore, as salivary assays are noninvasive, current research is focused on investigating its possible use for disease assessment and identification of disease biomarkers.15

The 25-hydroxycholecalciferol levels are proven to be a reliable index for estimating an individual’s vitamin D score.16 Serum 25-hydroxycholecalciferol, the major circulating figure of vitamin D with a t1/2 of 2–3 weeks, reflects both cutaneous synthesis and absorption from the gut. Thus, the salivary 25-hydroxycholecalciferol levels were measured. The study involved the collection of unstimulated whole saliva, as it represents secretions from major to minor salivary glands.

The spitting method was employed for saliva collection due to its reproducibility and reliability, contrary to suction and swab methods, which are associated with some degree of stimulation and variability.13 Although serum samples are the gold standard, in the present research, unstimulated whole salivary samples were employed due to the noninvasiveness of the collection method. Furthermore, the whole saliva may be used for the diagnosis of systemic diseases because it contains serum constituents. According to Bahramian et al.,17 salivary and serum vitamin D levels exhibited notable association. Fairney et al.18 observed that salivary 25-OHD levels represented 1.2% of the total serum values. In the present research, the serum vitamin D values were thus extrapolated to the salivary vitamin D levels.

According to the Indian Academy of Pediatrics, >20 ng/mL serum vitamin D concentrations are considered sufficient, 12–20 ng/mL are insufficient, and <12 ng/mL are deficient.19,20

About 66.67% of the children with S-ECC had salivary vitamin D levels of <12 ng/mL, depicting deficiency. Mellanby and Pattison,21 observed that the incorporation of vitamin D into the diet in the form of irradiated ergosterol prevented the onset of new carious lesions, retarded the spread of old carious lesions, and limited the infective process. A study by Brown et al.22 elicited that a high percentage of children younger than 5 years with dental caries were deficient in vitamin D. Similarly, children with S-ECC had lower concentrations of 25(OH)D and were twofold susceptible to deficiency.8 Ali et al.23 observed that dental caries and vitamin D levels in the serum were closely related. Furthermore, vitamin D deficiency is a risk factor both for the incidence of dental caries and its severity in children.24 According to Zhan et al.,25 there is an inverse relation between serum vitamin D levels and incidence of tooth loss and caries status.

On the contrary, Dudding et al.26 found no evidence of an inverse causal effect of vitamin D on dental caries but observed an association between low vitamin D levels and early dental caries onset. Herzog et al.27 observed no significant association between vitamin D levels and caries experience. However, the results of the present research displayed an inverse correlation between salivary vitamin D levels and ECC in children. In the current study, the sample population was matched with regards to intake of vitamin D supplements and geographical location.

Temperature, cloud coverage, total hours of sunshine, and humidity remained similar for both groups as the study population was selected from areas near and around the institution where the study was conducted. However, as the duration for which the subjects get exposed to sunlight cannot be controlled, a statistically significant difference was observed between the groups in terms of duration of exposure to sunlight. According to Casey et al.,28 a direct relationship exists between vitamin D obtained from the sun, bone health, and dental caries.

The other factors attributable to the onset and progression of dental caries include frequency of sleeping with a feeding bottle/sippy cup, frequency of consumption of sugary snacks, and method of tooth brushing.


  • Sample size.
  • Study design does not facilitate the determination of the causal effect of the parameter under study.
  • The lack of radiographic examination may be associated with the possibility of undiagnosed caries in group II.


Within limitations, a difference in vitamin D levels in the saliva of caries-free children and those with S-ECC suggests the effectiveness of salivary vitamin D levels as a biomarker of caries risk in children. Nonetheless, the complexity and etiology of dental decay mandate the assessment of other contributing factors in addition to vitamin D status, consequently leading to the use of appropriate preventive strategies.

Clinical Significance

Based on the literature review and the outcome of the present study, vitamin D levels are a valuable indicator of dental caries. Hence, it is imperative to conduct further research and establish an acceptable technique to estimate salivary vitamin D levels, as saliva has the potential to be an admissible and cost-effective method owing to its noninvasiveness.


1. American Academy of Pediatric Dentistry (AAPD). Policy on early childhood caries (ECC): classifications, consequences and preventive strategies. Pediatr Dent 2016;38(6):71–73. PMID: 27931420.

2. Low W, Tan S, Schwartz S. The effect of severe caries on the quality of life in young children. Pediatr Dent 1999;21(6):325–326. PMID: 10509332.

3. Tagliaferro EP, Pereira AC, Meneghim Mde C, et al. Assessment of dental caries predictors in a seven-year longitudinal study. J Public Health Dent 2006;66(3):169–173. DOI: 10.1111/j.1752-7325.2006.tb02575.x

4. Pereira SM, Tagliaferro EP, Ambrosano GM, et al. Dental caries in 12-year-old schoolchildren and its relationship with socioeconomic and behavioural variables. Oral Health Prev Dent 2007;5(4):299–306. PMID: 18173091.

5. Gedicke K. Rickets and dental caries; possibilities of dental caries prevention with vitamin D preparations with reference to social hygiene uses. Offentl Gesundheitsdienst 1959;20(10):419–432. PMID: 13633162.

6. Petersen PE. The World Oral Health Report 2003: continuous improvement of oral health in the 21st century – the approach of the WHO Global Oral Health Programme. Community Dent Oral Epidemiol 2003;31(Suppl 1):3–23. DOI: 10.1046/j..2003.com122.x

7. World Health Organization. Oral Health Surveys – Basic Methods, 4th edition. Geneva: WHO; 1997. pp. 39–44.

8. Schroth RJ, Jeal NS, Kliewer E, et al. The relationship between vitamin D and severe early childhood caries: a pilot study. Int J Vitam Nutr Res 2012;82(1):53–62. DOI: 10.1024/0300-9831/a000094

9. Berdal A, Bailleul-Forestier I, Davideau J. Vitamin D. Elsevier Academic Press; 2005. pp. 599–607.

10. Haussler MR, Whitfield GK, Kaneko I, et al. Molecular mechanisms of vitamin D action. Calcif Tissue Int 2013;92(2):77–98. DOI: 10.1007/s00223-012-9619-0

11. Schroth RJ, Levi JA, Sellers EA, et al. Vitamin D status of children with severe early childhood caries: a case-control study. BMC Pediatr 2013;13(1):174. DOI: 10.1186/1471-2431-13-174

12. Hu XP, Li ZQ, Zhou JY, et al. Analysis of the association between polymorphisms in the vitamin D receptor (VDR) gene and dental caries in a Chinese population. Genet Mol Res 2015;14(3):11631–11638. DOI: 10.4238/2015.September.28.15

13. Sobel AE, Hanok A. Calcification. XVI. Composition of bones and teeth in relation to blood and diet in the cotton rat. J Dent Res 1958;37(4):631–637. DOI: 10.1177/00220345580370040901

14. Ezhil I, Savitha G, Kumar MPS. Saliva as a diagnostic tool: a review. Drug InventToday 2018;10(11):2188–2193. Available at https//:jprsolutions.info.

15. Costantini E, Sinjari B, Piscopo F, et al. Evaluation of salivary cytokines and vitamin D levels in periodontopathic patients. Int J Mol Sci 2020;21(8):2669. DOI: 10.3390/ijms21082669

16. Khadilkar AV. Vitamin D deficiency in Indian adolescents. Indian Pediatr 2010;47(9):755–756. DOI: 10.1007/s13312-010-0110-6

17. Bahramian A, Falsafi P, Abbasi T, et al. Comparing serum and salivary levels of Vitamin D in patients with recurrent aphthous stomatitis and healthy individuals. J Dent (Shiraz) 2018;19(4):295–300. PMID: 30680302.

18. Fairney A, Saphier PW. Studies on the measurement of 25-hydroxy vitamin D in human saliva. Br J Nutr 1987;57(1):13–25. DOI: 10.1079/bjn19870005

19. Munns CF, Shaw N, Kiely M, et al. Global consensus recommendations on prevention and management of nutritional rickets. J Clin Endocrinol Metab 2016;101(2):394–415. DOI: 10.1210/jc.2015-2175

20. Khadilkar A, Khadilkar V, Chinnappa J, et al. Prevention and treatment of vitamin D and calcium deficiency in children and adolescents: Indian Academy of Pediatrics (IAP) Guidelines. Indian Pediatr 2017;54(7):567–573. DOI: 10.1007/s13312-017-1070-x

21. Mellanby M, Pattison CL. The action of vitamin D in preventing the spread and promoting the arrest of caries in children. Br Med J 1928;2(3545):1079–1082. DOI: 10.1136/bmj.2.3545.1079

22. Brown T, Creed S, Alexander S, et al. Vitamin D deficiency in children with dental caries - a prevalence study. Arch Dis Child 2012;97(1):A103. DOI: 10.1136/archdischild-2012-301885.243

23. Ali N, Rahim A, Ali S, et al. Impact of vitamin D on development of early childhood caries. Pak Armed Force Med J 2017;67(3):429–433. PMID: emr-188573.

24. Chhonkar A, Gupta A, Arya V. Comparison of vitamin D level of children with severe early childhood caries and children with no caries. Int J Clin Pediatr Dent 2018;11(3):199–204. DOI: 10.5005/jp-journals-10005-1511

25. Zhan Y, Samietz S, Holtfreter B, et al. Prospective study of serum 25-hydroxy vitamin D and tooth loss. J Dent Res 2014;93(7):639–644. DOI: 10.1177/0022034514534985

26. Dudding T, Thomas SJ, Duncan K, et al. Re-examining the association between Vitamin D and childhood caries. PLoS One 2015;10(12):e0143769. DOI: 10.1371/journal.pone.0143769

27. Herzog K, Scott JM, Hujoel P, et al. Association of vitamin D and dental caries in children: findings from the National Health and Nutrition Examination Survey, 2005-2006. J Am Dent Assoc 2016;147(6):413–420. DOI: 10.1016/j.adaj.2015.12.013

28. Casey CF, Slawson DC, Neal LR. Vitamin D supplementation in infants, children, and adolescents. Am Fam Physician 2010;81(6):745–748. PMID: 20229973.

© The Author(s). 2023 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and non-commercial reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.