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 Table of Contents  
Year : 2021  |  Volume : 33  |  Issue : 4  |  Page : 442-446

Comparative estimation of serum levels of vitamin A, vitamin B12, vitamin D and vitamin E in patients with recurrent aphthous stomatitis and normal individuals – A case-control study

1 Department of Health and Family Welfare, Government of West Bengal, Kolkata, West Bengal, India
2 Department of Oral Medicine and Radiology, Haldia Institute of Dental Science and Research, Haldia, West Bengal, India
3 Department of Oral Medicine and Radiology, Dr. R. Ahmed Dental College and Hospital, Kolkata, West Bengal, India
4 Department of Periodontics, Dr. R. Ahmed Dental College and Hospital, Kolkata, West Bengal, India
5 Department of Community Dentistry, Dr. R. Ahmed Dental College and Hospital, Kolkata, West Bengal, India

Date of Submission21-Feb-2021
Date of Decision28-Sep-2021
Date of Acceptance21-Oct-2021
Date of Web Publication27-Dec-2021

Correspondence Address:
Dr. Anindya P Saha
RA- 323, Saltlake City, Sector 4, Kolkata, West Bengal - 700 105
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jiaomr.jiaomr_50_21

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Background: Recurrent aphthous stomatitis (RAS) is a recurrent acute ulcerative lesion of oral mucosa. Till now, the exact cause and mechanism of this condition isn't very much clear; it is however believed to be multifactorial. Various studies indicate that genetically mediated disturbances of the innate and acquired immunity play an important role in the disease development. Vitamins and micro-elements deficiencies are one of the principal factors that modify the immunologic response in RAS. Aim: To probe for correlation between serum levels of Vitamin A, B12, D3, E, and RAS. Methods and Materials: The cross-sectional clinical study was conducted on persons seeking dental service at some private dental college in West Bengal, India between January 2017 and June 2018. Forty patients suffering from RAS were compared to forty normal individuals. Applying ELISA method, serum concentration of vitamins A, vitamin B12, vitamin D3 and vitamin E were measured in two groups. Statistical Analysis: The means between two groups were compared by Student's unpaired 't' test. A value of P < 0.05 was considered as statistically significant. Results: RAS patients had a significantly lower serum level of vitamin A, B12, D3 and E than normal individuals; and there were a positive relation between the serum level of vitamin B12 and vitamin E in RAS patients. Conclusion: Vitamin A, B12, D3 and E deficiency have a significant bearing on RAS; and serum level of vitamin B12 and E of RAS patients have a significant interrelationship.

Keywords: Recurrent Aphthous Stomatitis, ulcer, vitamin

How to cite this article:
Mustafi S, Sinha R, Sarkar S, Giri D, Saha AP, Yadav P. Comparative estimation of serum levels of vitamin A, vitamin B12, vitamin D and vitamin E in patients with recurrent aphthous stomatitis and normal individuals – A case-control study. J Indian Acad Oral Med Radiol 2021;33:442-6

How to cite this URL:
Mustafi S, Sinha R, Sarkar S, Giri D, Saha AP, Yadav P. Comparative estimation of serum levels of vitamin A, vitamin B12, vitamin D and vitamin E in patients with recurrent aphthous stomatitis and normal individuals – A case-control study. J Indian Acad Oral Med Radiol [serial online] 2021 [cited 2022 Dec 6];33:442-6. Available from: http://www.jiaomr.in/text.asp?2021/33/4/442/333877

   Introduction Top

The Father of Medicine, Hippocrates (460 to 370 BC) first coined the term “aphthai” in relation to focal acute inflammatory condition of the oral mucosa; although a detailed clinical description of 'Recurrent Aphthous Stomatitis' (RAS) only appeared in 1898 after a paper had been published by Mikulicz and Kümmel. RAS represents one of the most challenging, yet common, lesions of oral mucosa for the clinicians, with 1.5% point prevalence in India, as reported by Mirowski (2020), entailing great discomfort for patients interfering with their regular activities like eating, drinking, swallowing, and speaking.[1]

RAS is defined as an acute, non-traumatic, inflammatory condition of idiopathic origin, characterized by painful, recurrent ulcerative lesion without being preceded by vesiculation. RAS presents as well-demarcated, round or oval, shallow/moderately deep ulcers of varying size, appearing as a single lesion or multiple lesions in a cluster, involving the movable oral mucosa, with the surrounding regions being clinically unaffected. The lesions consist of a central area of necrosis, covered by a yellow-greyish pseudo-membrane, and a raised halo of erythema at periphery. This is classified as major aphthous, minor aphthous and herpetiform aphthous, according to clinical characteristics.[2],[3]

Till now, the exact pathosis of RAS remains dubious; studies suggest its multi-factorial in origin. Recent researches indicate that disturbances in the regulation of immune-inflammatory response of oral mucosa play a pivotal role.[4],[5]

Again, vitamins play an important role in immunological events, as they act as catalyst for certain biochemical reactions. Vitamin A maintains integrity of oral epithelium (that serves as primary barrier against external assaults), and also maintains function of polymorph, macrophages and NK cells (those represent the immune-competent cells involved in innate/non-specific immunity). Besides, Vitamin A up-regulates CD4 + cells and B-cell functions, and thus promotes antibody-mediated adaptive immunity.[6] Vitamin B12 acts as immune-modulator by maintaining CD4/CD8 ratio and NK cells in cellular immunity.[7] Vitamin D3 controls CD8 + cells proliferation and T-cell mediated cytotoxicity.[8] Vitamin E decreases the discharge of reactive oxygen species by macrophages and the expression of CD11b cells and very late antigen 4 (VLA4), preventing its adhesion to the endothelium. Vitamin E also reduces the production of pro-inflammatory cytokines, including IL-1, IL-6, IL-8TNF by macrophages.[9] Since vitamins are among the major factors that influence the immunological reactions, vitamin deficiency can possibly trigger uncoordinated immune response resulting RAS.

The current clinical study was designed to probe the inter-relationship among different Vitamins and RAS.

   Aims and Objectives Top

The aim of the study was to find the correlation, if any, between vitamin A, B12, D3, E and RAS.

Hence, the study was carried out with the objective to estimate the serum levels of Vitamin A, B12, D3 & E among RAS patients and normal individuals; compare the same between the two groups, and probe for the relationship of vitamin levels and RAS.

   Materials and Methods Top

The clinical study was conducted on persons seeking dental service at private dental college in West Bengal, India between January 2017 and June 2018; and were citizens of India, residents of Bengal, living in and around the town, speaking Bengali as mother language; from wide socio-economical level; belonging to age group 5-75 years and both the sex. The subjects were randomly selected from the outpatients reporting to the Department of Oral Medicine, qualifying the inclusion and exclusion criteria.

The subjects were enrolled in the study after signing written informed consent. This study received necessary approval from the institutional ethics committee (dated February 10, 2017). All the procedures were in accordance with 'Helsinki declaration' maintaining the standard ethical principles.

The samples were divided into two groups, as 'Case' and 'Control'. Case was defined as patients clinically diagnosed of having Recurrent Aphthous Stomatitis (RAS) and with minimum 3 episodes of ulceration within last 3 months, while patients not suffering from RAS were control.

Sample size estimation

Taking 95% confidence level, and 5% margin of error, and 1.5% point prevalence of the disease, and adjusting for 30% non-response rate, the minimum quantifiable sample size for each group was estimated as 31. Hence, total sample size was taken 80, including 40 cases and 40 control (1:1 matched ratio), as estimated by www.surveysystem.com/sscalc.com.

Aphthous ulcers were clinically diagnosed by painful, recurrent, ulcer involving mobile oral mucosa, not preceded by vesiculation, appearing as single lesion or clusters of multiple lesions, round or oval, shallow or moderately deep, measuring within 1 cm in diameter, with a central area of necrosis, and a halo of erythema at periphery, and covered by a yellow-grayish pseudo-membrane. The lesions were furthermore categorized into minor aphthous, major aphthous and herpetiform variety.

Inclusion criteria

  • Voluntary enrollment of the subjects.
  • Age between 5 & 75 years.
  • Patients with aphthous ulcer (diagnosed by clinical history and findings)
  • At least 3 episodes of ulceration within 3 months.

Exclusion criteria

  • Patients suffering from other ulcerative oral lesion except RAS.
  • Patients with auto-immune diseases.
  • Patients with systemic illness, hormonal imbalances.
  • Patient with known history of recent viral infection.
  • Patients with allergic reactions.
  • Patients already under treatment for RAS.

The data, thus obtained, were entered into Microsoft Excel spreadsheet. Means of two groups were compared by Student's unpaired 't test for samples with normal distribution, while Non-parametric Mann–Whitney U t-test was used for comparison of the samples with no normal distribution. Pearson's product–moment correlation coefficient or Spearman nonparametric correlation coefficient was used for determining the correlation between two variables. A value of P < 0.05 was considered as statistically significant. The direct or inverse correlation was indicated by a positive or negative 'r' value respectively. The numerical value of 'r' indicated the degree of correlation: weak (0.1 to 0.3), moderate (0.3 to 0.5) and strong (0.5 to 1.0). The statistical analysis was performed by Graph Pad prism software (version 5, 2007, California, USA).

After detailed clinical examination and establishment of diagnosis of recurrent aphthous, 4 ml of venous blood was aspirated by vein-puncture using 23 G needle and 5 ml syringe from ante-cubital fossa of RAS patients and collected in a clot vial and fluoride vial, labelled and stored in an ice box containing dry ice and finally sent to the Department of Biochemistry within an hour. The same procedure was also repeated for the normal individuals. After coagulating and separating the clot of blood from the samples, separating serum from the clot using centrifuge machine at a speed of 3000 rpm for period of 5 minutes. Serum samples of control and RAS patients were transferred to aliquots vials to store at - 20°C for the assay of 25-hydroxyvitamin D, vitamin B12, vitamin E and vitamin A.

For estimation of vitamin A, vitamin B12, vitamin D3 and vitamin E at first, all the reagents were brought to room temperature for at least 30 min before use. For dilution of the serum and plasma samples, 200 μl of biotinantibody was added, incubated for 1 hour at 37°C. After that 90 μl of Tetramethylbenzidine (TMB) substrate was added and incubated for 30 minutes. Finally, the optical density was determined within 30 minutes using a micro plate reader set at 450 nm. Serum vitamin A was assayed by solid-phase sandwich ELISA technique by kit following the manufacturer's protocol. The methods of estimation of vitamin B12, vitamin D3, and vitamin E were nearly similar as the aforementioned method.

Here following kits are used.

  • Serum vitamin A ELISA Kit (Cusabio, USA)
  • Serum vitamin B12 ELISA Kit (Accubind, USA)
  • Serum 1, 25-hydroxy vitamin D3 ELISA Kit (Calbiotech, USA)
  • Serum Vitamin E ELISA Kit (Cusabio, USA).

   Results Top

80 Subjects were registered for the study, consisting of 40 RAS patients (case) and 40 normal individual (control) with 1:1 matched ratio. The case group had the mean age of 34.32 ± 16.56 years with range 5-72 years and median age of 32 years; while the control group had the mean age 33.43 ± 15.12 years with age range 6-74 years and the median age of 33.50 years [Figure 1]. In both of the groups, there were 22 male and 18 females. Among 40 case population, 7 (17.5%) had the aphthous ulcers on buccal mucosa, 10 (25%) on tongue, 2 (5%) on gingiva and 21 (52.5%) on labial mucosa [Figure 4]. Again among the RAS patients 34 (85%) presented with minor RAS type, 5 (12.5%) major RAS type and only 1 (2.5%) herpetiform variety [Figure 3]. [Figure 1] and [Figure 2] represent the distribution of different types of RAS in different age groups and sex respectively.
Figure 1: Distribution of sex among the RAS subjects

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Figure 2: Distribution of ages among RAS subjects

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Figure 3: Distribution of types of lesion

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Figure 4: Distribution of sites of development of the lesions

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Our study found, RAS patients had a significantly lower serum level of vitamin A, B12, D3 and E than normal individuals as shown in [Table 1]. The results indicated that there was a correlation between those vitamins and the disease.
Table 1: Comparison of the serum level of vitamin A, B12, D3, E between RAS patients and control

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No significant correlation was found among the serum level of each individual vitamin except between vitamin B12 and vitamin E in RAS patients as shown in [Table 2]. The results reported that there was an inter-relationship between Vitamin B12 and Vitamin E in the pathosis of RAS (p-value = 0.045), as showed in [Figure 5].
Table 2: Correlation between Vitamin B12 and Vitamin A/Vitamin D3/Vitamin E IN RAS & normal subjects

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Figure 5: Relation between Vitamin E and Vitamin B12 in RAS subjects

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   Discussions Top

Crispian Scully and Stephen Porter (1989)[10] opined that minor RAS, the most common variety, usually has onset after puberty (usually in the second decade of life) and gradually it becomes chronic, with a predilection for men; while a herpetiform lesion, the least common variety, possess a predisposition for women and a delayed age of onset than other types of aphthous ulcerations. All the three types of ulcers were included in our study among total 40 RAS cases (male = 22, female = 18). 18 females RAS patients were enrolled for the study, among which only 1 (5.55%) was having herpetiform RAS. The minor variety showed the maximum prevalence, with 81.81% occurrence for men and 88.88% occurrence for women. 4 males were suffering from major variety, while only1 female was having major ulcer. Hence results from our study resonated with Crispian Scully and Stephen Porter.[10]

Saral et al. (2005)[11] reported that serum level of vitamin A was significantly lower in aphthous cases than healthy individuals. However, Khademi et al. (2012)[12] did not agree with this. The results of our study is similar with Saral et al. (2005)[11],[13] and Azizi et al. (2012),[14] proclaiming that vitamin A deficiency has a bearing on RAS.

Our study showed that RAS patients had significantly lower serum level of vitamin B12, which was in accordance with studies conducted by Piskin et al. (2002),[13] Sari et al. (2016),[15] Burgan et al. (2006),[16] Khan et al. (2008),[17] Ghafoor and Khan (2012),[18] Lopez-Jornet et al. (2012),[19] Sun et al. (2014),[20] Tidgundi et al. (2017)[21]; indicating that vitamin B12 deficiency has a role in RAS pathogenesis. This went against the studies conducted by Porter and Scully (1988),[22] Barnadas et al. (1997)[23] and Kalati et al. (2013).[24]

Results from our study supports Khabbazi et al. (2014),[25] who reported that the 25-hydroxy vitamin D level in the minor RAS group was lower than the normal individuals, suggesting a significant correlation.

This study came in agreement with studies by conducted Azizi et al. (2011)[26] and Li et al. (2016),[27] who showed that vitamin E level was decreased in RAS patients, pointing a significant correlation.

This case-control study further suggested a relation between the serum level of vitamin B12 and vitamin E in RAS patients. It reported, the serum level of vitamin B12 is directly proportional to serum level of vitamin E. Such interrelationship was postulated by Hoe and Hardin (1952), and established by Pappu et al. (1978).[28]


Stress and anxiety also have bearing on aphthous ulcer. Through activating ANS, CNS and HPA (Hypothalamic-Pituitary-Adrenal axis), stress triggers a cascade of events, that influence immune-inflammatory response. Again, emotional stress is thought to affect daily diet, often leading to vitamin deficiency and aphthous ulcer. Hence, stress and anxiety play an obvious role in the causation of aphthous, which was not addressed, assessed and matched here among the samples. In addition, the small sample size also represented the limitation of a conclusive study.

Future prospect

This clinical study enlightened the role of serum level of vitamins in pathology of aphthous, and provides a thought for future research work (such as intervention study with administration of vitamin) to confirm the causal relationship, and for development of a therapeutic regimen for the RAS.

   Conclusion Top

Recurrent aphthous ulcer is a recurrent, painful, acute, non-traumatic, inflammatory, ulcerative oral lesion, which interferes with regular activities, like food and beverage intake and speech, causing discomfort to patient, presenting serious clinical concern. Many researchers through various studies have suggested that vitamin A, B12, D3 and E deficiency have a definite role in the aetio-pathogenesis, but the exact mechanism has not been yet clear.

This clinical case control study tried to search for a correlation between vitamin deficiency and recurrent aphthous ulcer. From this study, it can be concluded that vitamin A, B12, D3 and E deficiency have a significant bearing on RAS; and serum levels of vitamin B12 and E of RAS patients have a significant proportional interrelationship; though prospective, multi-centric, extensive controlled trials on a diverse study population are required to establish a causal relationship.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient (s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

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Slebioda Z, Szponar E, Kowalska A. Etiopathogenesis of recurrent aphthous stomatitis and the role of immunologic aspects: Literature review. Arch Immunol Ther Exp 2014;62:205-15.  Back to cited text no. 2
Maqbool MA, Aslam M, Akbar W, Iqbal Z. Biological importance of vitamins for human health: A review. J Agric Basic Sci 2018;2:50-8.  Back to cited text no. 3
Stephensen CB. Vitamin A infection and Immune function. Annu Rev Nutr 2001;21:167-92.  Back to cited text no. 4
Tamura J, Kubota K, Murakami H, Swamura M, Matsushima T, Tamura T, et al. Immunomodulation by vitamin B12: Augmentation of CD8+ T lymphocytes and natural killer (NK) cell activity in vitamin B12-deficient patients by methyl-B12 treatment. Clin Exp Immunol 1999;116:28-32.  Back to cited text no. 5
Alroy I, Towers TL, Freedman LP. Transcriptional repression of the interleukin-2 gene by vitamin D3: Direct inhibition of NFATp/AP-1 complex formation by a nuclear hormone receptor. Mol Cell Biol 1995;15:5789-99.  Back to cited text no. 6
Cippitelli M, Santoni A. Vitamin D3: A transcriptional modulator of the interferon-γ gene. Eur J Immunol 1998;28:3017-30.  Back to cited text no. 7
Meehan MA, Kerman RH, Lemire JM. 1,25-dihydroxyvitamin D3 enhances the generation of Non-specific suppressor cells while inhibiting the induction of cytotoxic cells in a human MLR. Cell Immunol 1992;140:400-9.  Back to cited text no. 8
Jialal I, Devaraj S, Kaul N. The effect of α-tocopherol on monocyte proatherogenic activity. J Nutr 2001;131:389S-94S.  Back to cited text no. 9
Scully C, Porter S. Recurrent aphthous stomatitis: Current concepts of etiology, pathogenesis and management. J Oral Pathol Med 1989;18:21-7.  Back to cited text no. 10
Saral Y, Coskun BK, Ozturk P, Karatas F, Ayar A. Assessment of salivary and serum Antioxidant vitamins and lipid peroxidation in patient with recurrent Aphthous ulceration. Tohoku J Exp Med 2005;206:305-12.  Back to cited text no. 11
Khademi H, Khozeimeh F, Tavangar A, Amini S, Ghalayani P. The Serum and salivary level of malondialdehyde, vitamins A, E, and C in patient with recurrent aphthous stomatitis. Adv Biomed Res 2014;3:246.  Back to cited text no. 12
[PUBMED]  [Full text]  
Piskin S, Sayan C, Durukan N, Senol M. Serum iron, ferritin, folic acid, and vitamin B12 levels in recurrent aphthous stomatitis. J Eur Acad Dermatol Venereol 2002;16:66-7.  Back to cited text no. 13
Azizi A, Shahsiah S, Ahmadi M. Serum vitamin A level in patients with recurrent aphthous stomatitis compared with healthy individuals. J Islamic Dent Assoc Iran 2012;24:177-80.  Back to cited text no. 14
Sari K, Yildirim T, Sari N. The level of Vitamin B12 and hemoglobin in patients with recurrent aphthous stomatitis. Med J DY Patil Univ 2016;9:605-8.  Back to cited text no. 15
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Burgan SZ, Sawair FA, Amarin ZO. Hematologic status in patients with recurrent aphthous stomatitis in Jordan. Saudi Med J 2006;27:381-4.  Back to cited text no. 16
Khan NF, Saeed M, Chaudhary S. Haematological parameters and recurrent aphthous stomatitis. J Coll Physicians Surg Pak 2013;23:124-7.  Back to cited text no. 17
Ghafoor F, Khan AA. Association of vitamin B12, serum ferritin and folate levels with recurrent oral ulceration. Pak J Med Res 2012;51:132-5.  Back to cited text no. 18
Lopez-Jornet P, Camacho-Alonso F, Martos N. Hematological study of patients with aphthous stomatitis. Int J Dermatol 2014;53:159-63.  Back to cited text no. 19
Sun A, Chen HM, Cheng SJ, Wang YP, Chang JYF, Wu YC, et al. Significant association of deficiencies of hemoglobin, iron, vitamin B12, and folic acid and high homocysteine level with recurrent aphthous stomatitis. J Oral Pathol Med 2014;1-5.  Back to cited text no. 20
Tidgundi MS, Moinuddin K, Baig MSA. Ferritin and vitamin B12 levels in patients with recurrent aphthous ulcers. Int J Clin Biochem Res 2017;4:136-9.  Back to cited text no. 21
Porter SR, Scully C, Flint S. Hematologic status in recurrent aphthous stomatitis compared with other oral disease. Oral Surg Oral Med Oral Pathol 1988;66:41-4.  Back to cited text no. 22
Barnadas MA, Remacha A, Condomines J, de Moragas JM. Hematologic deficiencies in patients with recurrent oral aphthai. Med Clin (Barc) 1997;109:85-7.  Back to cited text no. 23
Kalati FA, Nosratzehi T, Sarabadani J, Niazi A, Elham Y. Evaluation of hematologic status in patients with recurrent aphthous stomatitis in an Iranian population. Zahedan J Res Med Sci 2014;16:21-5.  Back to cited text no. 24
Khabbazi A, Ghorbanihaghjo A, Fanood F, Kolahi S, Hajialiloo M, Rashtchizadeh N. A comparative study of vitamin D serum levels in patients with recurrent apthous stomatitis. Egypt Rheumatol 2015;37:133-7.  Back to cited text no. 25
Azizi A, Shahsiah S, Katanbaf N. Assessment of serum vitamin E levels in patients with recurrent apthous stomatitis. J Res Dent Sci 2011;7:51-5.  Back to cited text no. 26
Li X-Y, Zhang Z-C. Assessment of serum malondialdehyde, uric acid, and vitamins C and E levels in patients with recurrent aphthous stomatitis. J Den Sci 2016;11:401-4.  Back to cited text no. 27
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

  [Table 1], [Table 2]


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