The Use of the 25 (OH) D Saliva Test as a Substitute for the 25 (OH) D Serum Test in Healthy People
Phase: NA
Conditions: Nutritional Deficiency
Sponsor: Universitas Sumatera Utara

The Use of the 25 (OH) D Saliva Test as a Substitute for the 25 (OH) D 
Serum Test in Healthy People 
Dina Keumala Sari1*, Liza Meutia Sari2, Lidya Imelda Laksmi3, Farhat4 
1Tropical Medicine Program Study, Faculty of Medicine, Universitas Sumatera 
Utara, Medan, Indonesia, dina@usu.ac.id 
2Oral Medicine Department, Faculty of Dentistry, Universitas Syah Kuala, Banda 
Aceh, Indonesia, lizameutiasari@unsyiah.ac.id 
3Anatomy Pathology Faculty of Medicine, Universitas Sumatera Utara, Medan, 
Indonesia, lidya@usu.ac.id 
4Ear, Nose, Throat, Head and Neck Department, Faculty of Medicine, Universitas 
Sumatera Utara, Medan, Indonesia, farhat@usu.ac.id 
*Correspondence author: dina@usu.ac.id; Phone: +6281397177693 
 
Date: November 18th, 2020 
 

The Use of the 25 (OH) D Saliva Test as a Substitute for the 25 (OH) D 
Serum Test in Healthy People 
 
 
 
ABSTRACT 
Background: Routine examination of vitamin D levels is carried out by checking 
serum 25 (OH)D levels, which indicate circulating vitamin D levels. While serum 
1.25 (OH) D levels are less frequently utilized, they represent the active form of 
vitamin D and could be a substitute for checking vitamin D levels. 
Objective: This study aims to find the correlation between vitamin D levels, namely 
25 (OH) D and 1.25 (OH) D saliva, which correlate with serum 25 (OH) D and 1.25 
(OH) D levels in the examination of salivary vitamin D, and which could be a 
substitute for checking serum vitamin D levels. 
Design: This study is a cross-sectional study involving healthy men and women, 
aged 20–50 years, from Lima Puluh Village, Batubara District, North Sumatra 
Province, Indonesia. The parameters studied were 25 (OH) D and 1.25 (OH) D levels 
of saliva and serum. 
Results: This study involved 56 study subjects, male and female, with a percentage 
of deficiency of 78.6% found by examining 25 (OH) D saliva, and 76.8% found by 
examining 25 (OH) D serum. For the 1.25 (OH) D examination of saliva and serum, 
all were within normal limits. The analysis showed that a moderate correlation was 
obtained for levels of 25 (OH) D saliva with serum 25 (OH) D (p = 0.424), and a weak 
correlation for levels of 1.25 (OH) D saliva with serum 25 (OH) D (p = 0.339). 
Conclusions: Salivary 25 (OH) D assay can be used to replace serum 25 (OH) D 
assay in healthy people, as a non-invasive alternative. 
Keywords: vitamin D; cross-sectional; non-invasive; correlation; deficiency; 
insufficiency 
 
 

Introduction  
Vitamin D deficiency occurs in various parts of the world, including in the tropics, 
and occurs in both healthy and sick people.1, 2 Examination of serum 25 (OH) D 
levels is needed to determine the level of vitamin D in circulation and in the active 
form, namely 1.25 (OH) D serum.3 However, this examination often causes 
discomfort in the patient, so an easier examination is needed during which the 
patient does not feel pain. 
This examination of vitamin D is necessary due to the role of vitamin D, 
which can increase body immunity through its role as an endocrine. 4-7 It also acts as 
an anti-inflammatory and has a regulatory effect on the immune system. The effects 
of vitamin D therapy can be felt in various metabolic diseases and cancer. 7, 8  
 
Examination of vitamin D levels in the form of 25 (OH) D and 1.25 (OH) D in 
saliva has not been used often for diagnostic purposes of vitamin D levels, because 
these levels may not show the actual levels in the body.9 Saliva examination is more 
focused on hormonal, immunological, and infection tests, but it is rarely used.10, 11 In 
addition, saliva examination may reveal many other contamination factors, so saliva 
examination in diagnostic tests is often neglected.11 However, if done according to 
the correct procedure, then the risk of contaminants can be removed. 
Based on the results of the above research, we conducted a study that looks at 
the correlation between levels of 25 (OH) D and 1.25 (OH) D of saliva and serum. 
The goal was to find an alternative for serum testing, which is invasive and causes 

discomfort to the patient. It is hoped that with the findings of this research, saliva 
examination could replace serum testing. 
Methods  
This research was conducted following the ethics committee protocol, and was 
approved by the ethics committee of the Universitas Sumatera Utara (number 63 / 
KEP / USU / 2020). The subjects of this study have their signed informed consent 
prior to being included. During the study, no therapy or intervention was carried 
out and the research subjects were not charged any fees for the laboratory 
examination. 
This research was conducted in Lima Puluh Village, Batubara District, 
Simalungun Regency, North Sumatra, Indonesia. This area is about 153 kilometers 
from the city center of Medan City, near rubber and oil palm plantations, and the 
hope was that the research subjects would be healthier because of high activity 
levels. This study included 56 study subjects: the inclusion criteria were men and 
women aged 18–60 years, not currently experiencing chronic pain, kidney problems, 
liver problems, or other hormone disorders. Exclusion criteria were study subjects 
who consumed vitamin D supplements regularly, were pregnant, or were 
breastfeeding. 
The tests carried out were examination of 25 (OH) D and 1.25 (OH) D levels of 
serum and saliva, examination of demographic data, and other anthropometries. The 
examination was carried out by taking 5 mL of blood and 2 mL of saliva, and then 

checking the serum and saliva levels of 25 (OH) D and 1.25 (OH) D. Before the 
examination, the research subjects were asked not to consume food or drink for at 
least 90 minutes before being examined. Centrifugation was carried out (2500 g, 10 
minutes) and the product immediately stored at –20 oC. In the serum examination, 
after the blood was drawn, centrifugation was carried out and the product stored at 
–20 oC for further determination of the levels of 25 (OH) D and 1.25 (OH) saliva. 
 
The serum and salivary 25 (OH) D level is defined as a deficiency if below 10 
ng/mL, insufficiency if between 11–20 ng/mL, and optimal if 20 ng/mL. The 1.25 
(OH) D serum and saliva are deemed deficient if 48 pmol/L and normal if >48 
pmol/L.12, 13 Examination of 25 (OH) D and 1.25 (OH) D serum and saliva was carried 
out using the Bio-Rad ELISA technology tool, California, United States of America, 
using the Enzyme-Linked Immunosorbent Assay (ELISA) kit, Brand Bioassay, 
Bioassay Technology Laboratory, Shanghai, China. 
Statistical analysis was performed by presenting the data in the form of 
standard deviations if the data was normally distributed, but if the data was not 
normally distributed, then it was presented in the form of a minimum, maximum, 
and median. For correlation analysis with a normal distribution, the Pearson 
correlation test was used, whereas if the data was not normally distributed, the 
Spearman correlation test was used. A weak correlation is 0.2 to <0.4, a moderate 
correlation is 0.4 to <0.6, and a strong correlation is 0.6 to <0.8. 
Results 

Based on the demographic data, it can be seen that in the research location there are 
more people in their mid-30s, and women are more likely to work as housewives, 
while men are most likely to be self-employed (Table 1). Based on the 
anthropometric examination, most of the study subjects were categorized as obese, 
however, based on the criteria for abdominal circumference, a greater percentage of 
women experienced central obesity (Table 2). 
Table 3 shows that the percentage of deficiency was 78.6% when testing with 
25 (OH) D saliva and 76.8% when testing with serum 25 (OH) D. As for the 1.25 
(OH) D examination of saliva and serum, it was shown that 100% of the study 
subjects were within normal limits. 
The results of the study shown in Table 4 suggest that there is a moderate 
correlation for levels of 25 (OH) D saliva with 25 (OH) D serum (p = 0.424), and a 
weak correlation for levels of 1.25 (OH) D saliva with 25 (OH) D serum (p = 0.339) 
using the Spearman test. 
Discussion  
This study shows that vitamin D deficiencies occur in groups of healthy research 
subjects, although they do not tend to be accompanied by diseases caused by 
vitamin D deficiency. 1, 14 Examination of vitamin D has become routine, and most 
often uses serum. 3, 15, 16 This examination is often uncomfortable and invasive. 
Various studies have also been conducted to compare this examination with 
examination of other body fluids. 17, 18 

 
In addition to serum examinations, some studies have discussed the diagnosis 
of vitamin D status using a questionnaire. 19 This suggests that an invasive 
examination is not required to establish vitamin status, but the study focused only 
on the elderly. 19 Other studies have shown that serum levels give more precise 
results compared to other body fluids. 20-22 
 
The saliva examination in this study showed a higher level than the serum 
level, which was probably due to the different sensitivities in detecting 25 (OH) D in 
serum and saliva. Saliva examination is also considered to be heavily influenced by 
contaminants, so these results cannot be adjusted to the levels in serum. However, 
this study showed that there was a moderate correlation between saliva and serum 
levels, especially in 25 (OH) D levels. 
The examination of 1.25 (OH) D serum showed a higher limit compared to 
serum, so with a cutoff point of 48 pmol/L, it showed that no study subject had a 
vitamin D deficiency. All study subjects belonged to the normal group. This result is 
certainly different from other studies, which showed a deficiency both through 
serum and saliva. 2, 5, 17, 22-24 
This study showed a moderate strength of correlation between saliva and 
serum for 25 (OH) D levels, and this moderate correlation shows that saliva 
assessment can be used as the same test as that for serum. Examination of 1.25 (OH) 
D showed a weak correlation; this needs further analysis, and indicates that salivary 
examination cannot reveal the correlation between saliva and serum. 

This study also has limitations, particularly around the abnormal data for 
vitamin D levels that were very high and very low. This study also does not assess 
the levels of calcium and parathyroid hormone, which would have allowed 
discussion of the relationship between the three nutrients, and this study did not 
compare vitamin D levels in people experiencing disease, which would have shown 
a more pronounced difference. 
Conclusions  
Salivary 25 (OH) D assay can be used to replace serum 25 (OH) D assay in healthy 
people as a non-invasive alternative. Examination using saliva as a substitute for 
serum testing is expected to facilitate the examination of 25 (OH) D. 
Acknowledgements 
The authors would like to thank all research subjects in Lima Puluh Village, 
Batubara District, Simalungun Regency, North Sumatra, Indonesia. 
Funding 
Funded by the Department for Strengthening Research and Development, Ministry 
of Research and Technology/National Research and Innovation Agency for the 2020 
Fiscal Year in accordance with the Research Contract Amendment Number: 11 / 
AMD / E1 / KP.PTNBH / 2020, dated 11 May 2020. 
Availability of data and materials 
All data generated or analyzed during this study are included in this article. 
Ethics approval and consent to participate 
All participants knowledgeably consented to participate in this study. This research 
was conducted following the ethics committee protocol, and was approved by the 
ethics committee of the Universitas Sumatera Utara (number 63 / KEP / USU / 2020). 
Competing interests 
The authors declare that they have no competing interests. 
References  

1. Sari DK, Mega JY and Harahap J. Nutrition Status Related to Clinical 
Improvement in AFB-Positive Pulmonary Tuberculosis Patients in Primary 
Health Centres in Medan, Indonesia. Open Access Maced J Med Sci 2019; 7: 1621-
1627. Researc article. DOI: 10.3889/oamjms.2019.338. 
2. Chaiprasongsuk A, Janjetovic Z, Kim TK, et al. Protective effects of novel 
derivatives of vitamin D3 and lumisterol against UVB-induced damage in 
human keratinocytes involve activation of Nrf2 and p53 defense mechanisms. 
Redox Biol 2019; 24: 101206. 2019/05/01. DOI: 10.1016/j.redox.2019.101206. 
3. Alshahrani FM, Almalki MH, Aljohani N, et al. Vitamin D: Light side and best 
time of sunshine in Riyadh, Saudi Arabia. Dermatoendocrinol 2013; 5: 177-180. 
2014/02/05. DOI: 10.4161/derm.23351. 
4. Ganmaa D, Munkhzul B, Fawzi W, et al. High-Dose Vitamin D3 during 
Tuberculosis Treatment in Mongolia. A Randomized Controlled Trial. Am J 
Respir Crit Care Med 2017; 196: 628-637. 2017/07/12. DOI: 10.1164/rccm.201705-
0936OC. 
5. McCullough PJ and Lehrer DS. Vitamin D, cod liver oil, sunshine, and 
phototherapy: Safe, effective and forgotten tools for treating and curing 
tuberculosis infections - A comprehensive review. J Steroid Biochem Mol Biol 2018; 
177: 21-29. 2017/08/02. DOI: 10.1016/j.jsbmb.2017.07.027. 
6. Wahyunitisari MR, Mertaniasih NM, Amin M, et al. Vitamin D, cell death 
pathways, and tuberculosis. Int J Mycobacteriol 2017; 6: 349-355. 2017/11/25. DOI: 
10.4103/ijmy.ijmy_120_17. 
7. Bochen F, Balensiefer B, Korner S, et al. Vitamin D deficiency in head and neck 
cancer patients - prevalence, prognostic value and impact on immune function. 
Oncoimmunology 2018; 7: e1476817. 2018/09/20. DOI: 
10.1080/2162402X.2018.1476817. 
8. Fanidi A, Muller DC, Midttun O, et al. Circulating vitamin D in relation to 
cancer incidence and survival of the head and neck and oesophagus in the EPIC 
cohort. Sci Rep 2016; 6: 36017. 2016/11/05. DOI: 10.1038/srep36017. 
9. Fairney A and Saphier PW. Studies on the measurement of 25-hydroxy vitamin 
D in human saliva. Br J Nutr 1987; 57: 13-25. 1987/01/01. DOI: 
10.1079/bjn19870005. 
10. Gottlieb CW, Retief FP and Herbert V. Blockade of vitamin B12-binding sites in 
gastric juice, serum and saliva by analogues and derivatives of vitamin B12 and 
by antibody to intrinsic factor. Biochim Biophys Acta 1967; 141: 560-572. 
1967/08/29. DOI: 10.1016/0304-4165(67)90185-7. 
11. Flusser J, Chvojkova V and Kozicka V. [Relationship between saliva and vitamin 
B12. (Contribution to the problem of the intrinsic factor)]. Cas Lek Cesk 1961; 100: 
199-205. 1961/02/17. 
12. Heaney RP and Holick MF. Why the IOM recommendations for vitamin D are 
deficient. J Bone Miner Res 2011; 26: 455-457. 2011/02/22. DOI: 10.1002/jbmr.328. 
13. Grant WB and Holick MF. Benefits and requirements of vitamin D for optimal 
health: a review. Altern Med Rev 2005; 10: 94-111. 2005/07/02. 

14. Baggerly CA, Cuomo RE, French CB, et al. Sunlight and Vitamin D: Necessary 
for Public Health. J Am Coll Nutr 2015; 34: 359-365. 2015/06/23. DOI: 
10.1080/07315724.2015.1039866. 
15. Cannell JJ, Grant WB and Holick MF. Vitamin D and inflammation. 
Dermatoendocrinol 2014; 6: e983401. 2014/01/01. DOI: 
10.4161/19381980.2014.983401. 
16. Cannell JJ, Vieth R, Umhau JC, et al. Epidemic influenza and vitamin D. 
Epidemiol Infect 2006; 134: 1129-1140. 2006/09/09. DOI: 
10.1017/S0950268806007175. 
17. Evans LW and Omaye ST. Use of Saliva Biomarkers to Monitor Efficacy of 
Vitamin C in Exercise-Induced Oxidative Stress. Antioxidants (Basel) 2017; 6 
2017/01/14. DOI: 10.3390/antiox6010005. 
18. Rakshasbhuvankar AA, Clarke MW, Simmer K, et al. Saliva for Assessing 
Vitamin A Status in Extremely Preterm Infants: A Diagnostic Study. Neonatology 
2020: 1-4. 2020/03/03. DOI: 10.1159/000506132. 
19. Annweiler C, Kabeshova A, Callens A, et al. Self-administered Vitamin D Status 
Predictor: Older adults are able to use a self-questionnaire for evaluating their 
vitamin D status. PLoS One 2017; 12: e0186578. 2017/11/02. DOI: 
10.1371/journal.pone.0186578. 
20. Biancuzzo RM, Clarke N, Reitz RE, et al. Serum concentrations of 1,25-
dihydroxyvitamin D2 and 1,25-dihydroxyvitamin D3 in response to vitamin D2 
and vitamin D3 supplementation. J Clin Endocrinol Metab 2013; 98: 973-979. 
2013/02/07. DOI: 10.1210/jc.2012-2114. 
21. Cocate PG, Kac G, Heitmann BL, et al. Calcium and vitamin D supplementation 
and/or periodontal therapy in the treatment of periodontitis among Brazilian 
pregnant women: protocol of a feasibility randomised controlled trial (the 
IMPROVE trial). Pilot Feasibility Stud 2019; 5: 38. 2019/03/16. DOI: 10.1186/s40814-
019-0417-6. 
22. Cunha Figueiredo AC, Trujillo J, Freitas-Vilela AA, et al. Association between 
plasma concentrations of vitamin D metabolites and depressive symptoms 
throughout pregnancy in a prospective cohort of Brazilian women. J Psychiatr 
Res 2017; 95: 1-8. 2017/07/30. DOI: 10.1016/j.jpsychires.2017.07.009. 
23. Isola G, Polizzi A, Muraglie S, et al. Assessment of Vitamin C and Antioxidant 
Profiles in Saliva and Serum in Patients with Periodontitis and Ischemic Heart 
Disease. Nutrients 2019; 11 2019/12/11. DOI: 10.3390/nu11122956. 
24. Balcells ME, Garcia P, Tiznado C, et al. Association of vitamin D deficiency, 
season of the year, and latent tuberculosis infection among household contacts. 
PLoS One 2017; 12: e0175400. 2017/04/14. DOI: 10.1371/journal.pone.0175400. 
 
 

Table 1. Characteristics of the subjects. 
 
 
Mean 
n (%) 
Age (years) 
41.32 ± 10.68 
Minimum: 18 
Maximum: 58 
Median: 42 
 
 
Age classification: 
     18–25 years 
     26–35 years 
     36–45 years 
     46–60 years 
 
 
7 (12.5) 
6 (10.7) 
19 (33.9) 
24 (42.9) 
 
Genders: 
Male 
Female 
 
 
 
 
23 (41.1)  
33 (58.9) 
Ethnic:  
       Batak  
       Melayu 
 
 
 
26 (46.4) 
30 (53.6) 
 
Occupation: 
       
       Housewife 
       Entrepreneur 
       State civil apparatus 
Farmer 
       Student 
 
 
 
 
 
21 (37.5) 
14 (25) 
9 (16) 
8 (14.3) 
4 (7.2) 
 
Education 
Strata 1 
Diploma 
Senior High School 
Junior High School 
Primary School 
 
 
9 (16.1) 
2 (3.6) 
33 (58.9) 
8 (14.3) 
4 (7.1) 
 
Vitamin D supplementation 
No 
Yes 
 
 
 
56 (100) 
0 (0) 
Continuous variable: mean ± SD; categorical variable: n (%); SD= standard deviation 
 
 

 
Table 2. Anthropometry parameters of the subjects. 
Variable 
Mean 
n (%) 
Body Mass Index (BMI) (kg/m2) 
26.71±11.76 
Minimum: 16.69 
Maximum: 88.95 
Median: 24.62 
 
BMI classification: 
     <18 kg/m2 
     18–22.9 kg/m2 
     23–24.9 kg/m2 
     >25 kg/m2 
 
 
3 (5.4) 
17 (30.4) 
11 (19.6) 
25 (44.6) 
 
Waist circumference measurement 
and  classification: 
 
Men (cm): 
     <90 cm 
     >90 cm 
 
Women (cm): 
     <80 cm 
     >80 cm 
 
 
 
 
 
83.5711.07 
 
 
 
82.3612.14 
 
 
 
 
 
16 (69.6) 
7 (30.4) 
 
 
13 (39.4) 
20 (60.6) 
Continuous variable: mean ± SD; categorical variable: n (%); SD= standard deviation 
 
 
 
 
 

Table 3. Vitamin D saliva and serum level. 
Variable 
Saliva 
Serum 
25 (OH) D level (ng/mL) 
 
16.545.01 
Minimum: 2.05 
Maximum: 25.1 
Median: 17.45 
15.0715.34 
Minimum: 2.32 
Maximum: 80.1 
Median: 8.7 
 
25 (OH) D categorized n(%): 
10 ng/mL (Deficiency) 
11–20 ng/mL (Insuficiency) 
20 ng/mL (Optimal) 
 
6 (10.7) 
38 (67.9) 
12 (21.4) 
 
30 (53.6) 
13 (23.2) 
13 (23.2) 
1.25(OH)D level (pmol/L) 
 
201.1550.58 
Minimum: 52.7 
Maximum: 285 
Median: 221.5 
268.31219.26 
Minimum: 51.7 
Maximum: 884.2 
Median: 182 
 
1.25(OH)D categorized n(%): 
48 pmol/L (Deficiency) 
>48 pmol/L (Normal) 
 
 
0 (0) 
56 (100) 
 
0 (0) 
56 (100) 
Continuous variable: mean ± SD; categorical variable: n (%); SD=standard deviation 
 
 
 

 
Table 4. Correlation between vitamin D saliva and serum level. 
Variable 
25 (OH) D level (ng/mL) in 
serum 
1.25 (OH) D level 
(pmol/L) in serum 
25 (OH) D level (ng/mL) in 
saliva 
 
r = 0.424 (positive-moderate) 
p = 0.001 (significant) 
n = 56 
 
 
1.25 (OH) D (pmol/L) in 
saliva 
 
 
r=0.339(positive-
weak) 
p = 0.01 (significant) 
n = 56 
Analysis: Spearman test 
 
 
 
 

Approval Sheet After Explanation (Informed Consent) 
 
My Informed Consent Sheet signed below,  
Name :  
Age :  
Gender :  
 
 Has been clearly explained by the researcher about the study “The Use of the 25 (OH) D 
Saliva Test as a Substite for the 25 (OH) D Serum Test in Healthy People”, so I hereby 
voluntarily and without coercion stated : 
 
Willing to be included in the study.  
 
Such is this statement to be used as necessary. 
 
Sincerely, 
 
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