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 Table of Contents  
Year : 2021  |  Volume : 33  |  Issue : 1  |  Page : 66-70

Correlation of Serum Vitamin B12 and Iron as etiologic factors in recurrent aphthous stomatitis: A case-control study

1 Department of Oral Medicine and Radiology, Karpagavinayaga Institute of Dental Sciences, Chengalpet, Tamil Nadu, India
2 Department of Oral Medicine and Radiology, Saveetha Dental College and Hospital, Chennai, Tamil Nadu, India
3 Department of Oral Medicine and Radiology, Rajas Dental College, Tirunelveli, Tamil Nadu, India

Date of Submission23-Jun-2020
Date of Decision26-Nov-2020
Date of Acceptance10-Jan-2021
Date of Web Publication26-Mar-2021

Correspondence Address:
Dr. Subha Manoharan
Professor, Department of Oral Medicine and Radiology, Saveetha Dental College and Hospital, Chennai, Velappanchavadi - 600 077, Chennai, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jiaomr.jiaomr_120_20

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Objective: This study aimed to estimate and correlate the levels of serum vitamin B12, iron, and their association with family history and cigarette smoking in patients with recurrent aphthous stomatitis (RAS). Methods: Sample size was calculated using the earlier prevalence studies with a power of 85% and rounded to 30 considering other confounding factors. Thirty patients with RAS and 30 control subjects were included in this controlled prospective study. A proforma on demographic details and case history was filled followed by serum analysis for Iron and B12. The collected data were analyzed using SPSS 20. Results: Family history is a significant predisposing factor for RAS. Vitamin B12 was found to have a significant positive correlation with RAS. Patients with vitamin B12 deficiency, positive family history, and nonsmoking status have been found to have the highest risk for having RAS. Binary logistic regression analysis revealed a significant relationship between vitamin B12 and RAS (probability P =0.001). Conclusion: Our study had shown a positive correlation of RAS with family history and vitamin B12 deficiency.

Keywords: Aphthous minor, iron, recurrent aphthous stomatitis, vitamin B12

How to cite this article:
Saravanan T, Manoharan S, Bharathi C S, Shakila K R. Correlation of Serum Vitamin B12 and Iron as etiologic factors in recurrent aphthous stomatitis: A case-control study. J Indian Acad Oral Med Radiol 2021;33:66-70

How to cite this URL:
Saravanan T, Manoharan S, Bharathi C S, Shakila K R. Correlation of Serum Vitamin B12 and Iron as etiologic factors in recurrent aphthous stomatitis: A case-control study. J Indian Acad Oral Med Radiol [serial online] 2021 [cited 2021 Jul 23];33:66-70. Available from: https://www.jiaomr.in/text.asp?2021/33/1/66/312190

   Introduction Top

Recurrent aphthous ulcer (RAU) or recurrent aphthous stomatitis (RAS) seems to be as old as humanity itself. The term aphthae was derived from the Greek word aphthi, which means “to set on fire” or “to inflame,” and was thought to have been first used by the philosopher Hippocrates to describe the pain associated with a common disorder of the mouth.[1] It is found in men and women of all ages, races, and geographic regions.[2] Local and systemic conditions, genetic, immunologic, and nutritional factors play a role in the pathogenesis of RAS.[3],[4] Pain, recurrence, self-limitation, and destruction of the epithelium seem to be the ultimate outcomes.[5] Though not life threatening, it diminishes the quality of life.

This study aimed to investigate the possible roles of family history, cigarette smoking, serum vitamin B12 levels, iron levels, and complete blood count in the predisposition of RAS.

   Materials and Methods Top

Patients diagnosed with classical minor RAS and a history of recurrent painful ulcers on nonkeratinized mucosa were included in this study.

Patients without RAS who came to the outpatient department for other dental treatment and patients who were willing to participate in this study were included and written informed consent was obtained. Patients with other ulcerations such as traumatic ulcers, ulcerations in systemic diseases or on any iron and vitamin supplementation, and alcohol were excluded. The sample size was calculated based on the existing literature with a power of 85% and rounded to 30 considering other confounding factors. Ethical committee approval was obtained from the Institutional ethical committee of the institute (Ethical approval no. KIDS/F/2018/6 dated 21/9/2018). Thirty patients with RAS and 30 control subjects were included in this prospective study. A proforma on demographic details and case history was filled followed by the serum analysis for Iron and B12. Patient consent was obtained based on Helsinki Declaration 2013. Venous blood samples were collected in EDTA and unanticoagulated tubes. Serum was separated from unanticoagulated tubes and stored at -20°C. Tests were performed within 10 days of collection. Complete blood count was estimated by flow cytometry. Serum iron was determined by colorimetric method using Ferrimat kit. Serum vitamin B12 was determined by an immunoassay test kit. The collected data were analyzed using SPSS 20. Since the data was non-parametric, demographic details were tested using Ch-square analysis except age which was analyzed using Mann–Whitney U test value. The blood parameters were also analyzed using Mann–Whitney U test value. The correlation between serum vitamin B12 and serum iron with RAS was done using Point Biserial Correlation.

   Results Top

RAS group consisted of (56.67%) 19 men and (43.33%) 11 women and the control group was composed of 15 men and 15 women. Gender distribution was not statistically different (P = 0.29) [Figure 1].
Figure 1: Distribution of study subjects based on Gender

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Age-wise distribution of subjects was not found to be statistically significantly different between the groups, with an average age of distribution of 26.5 years [Figure 2], and a value of P = 0.53.
Figure 2: Distribution of study subjects based on age

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Smoking had a positive correlation with patients with RAS. Patients and controls with history of smoking is expressed in [Figure 3]. Smoking shows positive correlation in the patients with RAS. It proves to be statistically significant with a P value of 0.01. Family history of RAS among the patients and the controls were 56.7% and 5% respectively. On comparing the family history among the RAS and control subjects, it was found to be statistically significant with a value of P = 0.001 [[Table 1]: Comparison of demographic parameters of RAS and Healthy group] [Figure 4].
Figure 3: Distribution of study subjects based on smoking habit

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Figure 4: Distribution of study subjects based on family history

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Table 1: Comparison of Demographic parameters between the groups

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On comparing the other blood parameters lymphocyte levels show marginal significance whereas other blood parameters did not have any significant association [Table 2]. The normal laboratory range of serum vitamin B12 is 200-835 pg/ml. We found 19 of 30 patients to be deficient in vitamin B12, whereas, in the control group, only 8 of 30 patients had a vitamin B12 deficiency. Binary logistic regression analysis revealed a significant relationship between vitamin B12 and RAS probability P = 0.001 [Figure 5] and [Table 3]. However, serum iron levels on Point Biserial Correlation did not have any significant relation with RAS with a P = 0.48 [Table 4] and [Figure 6].
Table 2: Comparison of parameters between healthy and diseased groups

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Figure 5: Scatter plot between serum vitamin B12 and groups

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Table 3: Correlation between serum vitamin B12 and groups

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Table 4: Correlation between serum iron and groups

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Figure 6: Scatter plot between serum iron and groups

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   Discussion Top

RAS is one of the most common oral diseases characterized by multiple recurring ulcers.[2] RAS is caused by a range of etiologies like stress, vitamin B12 deficiency, iron, immunologic abnormalities.[5]

This study highlights the occurrence of RAS in patients with vitamin B 12 deficiency and serum iron deficiency. Patients with vitamin B12 deficiency are prone to RAS when compared to controls. Smoking and family history was also found to influence on RAS.

This study shows male predilection of 63.3% (19 males) as against 36.7% of females (11 females) but the study conducted by Axell T Henricsson,[6] stated the occurrence of RAS in females (57%) and Shang H[7] showed decreased prevalence in males (16.9%), and not in females (23.3%). It may be concluded if an equal number of males and females it would be possible to arrive at the correct gender predilection.

From the study, it was found that about 86.7% of patients with RAS were nonsmokers as against 56.7% in the control subjects with a significant P = 0.010. Hence it is clear that cigarette smoking has a protective effect on RAS. Queiroz SIML[8] suggested that the association of smoking and a decrease in the frequency and severity of RAS lesions in Dorsey's patients lends credence to the concept of protection from trauma by oral hyperkeratosis. Axéll T, Henricsson V[9] found the prevalence of aphthous ulcers in the population as 17.7 with a negative correlation between the prevalence of ulcers and tobacco habit especially pipe smoking and smoking cigarettes without filter. Robertson PB[10] examined the oral mucosa of 1456 professional Baseball players in whom about half of them were smokeless tobacco (ST) users and found that ST use significantly reduced the risk of aphthous ulcers which could be due to increased keratinization of the oral mucosa. Atkin PA[11] compared blood nicotine levels in 84 RAS patients and 81 control subjects and showed that a group of RAS patients is significantly less likely to contain smokers than a matched control population. Pizzorno JE[12] reported that nicotine has been beneficial in RAS and inflammatory bowel disease and its effects may result from influences on nerve function, although these may also exert direct anti-inflammatory effects.

There was about 56.7% of relevant family history in patients of RAS with a significant P value of 0.001. This indicates that family history may have a role to play in the occurrence of RAS.[13] Natah SS[14] stated clear variability in host susceptibility which can be explained by polygenic inheritance, dependent on environmental factors. Ziaudeen S[15] reported in their study the rate of positive family history in patients and control groups as 54.2% and 9% respectively, with statistically significant value.

Among the control subjects 37.5% males and 62.5% females the serum vitamin B12 were below 200 pg/dL, whereas in RAS, 55.5% of males and 44.5% females were having less than 200 pg/dL. The male and female distribution of vitamin B12 was not significant with a P value of 0.926. Ślebioda Z[16] referred to a study in which screening a series of 130 consecutive outpatients with RAS for deficiencies in vitamin B12 found 17.7% of them had deficiencies. The 23 deficient patients with recurrent aphthae were treated with specific replacement therapy and 15 showed complete remission of ulceration and 8 had definite improvement. Aynali G[17] had reported significantly lower levels of vitamin B12 in RAS in 36 patients when compared with 26 healthy controls. They have concluded that vitamin B12 deficiency may be an etiological factor in RAS. Vigarios E[17],[18] stated that there was a dramatic response to vitamin B12 replacement therapy and a higher incidence of RAS in cases that have vitamin B12 deficiency suggest a direct role of this vitamin on the pathogenesis of RAS. The mechanism of role of Vitamin B 12 deficiency in the pathogenesis of RAS remains unclear. A definite correlation of vit B12 levels as a causative factor has been found in this study which could be seen from the studies of the above authors, when they were supplemented with vitamin B12, the remission of lesions could be observed. Considering all these findings, we can clearly state that RAS is a multifactorial disease and we can conclude that positive family history and vitamin B12 deficiency are among the studied possible etiologic factors. The outcome of this study is that it is always essential to do a routine vitamin B12 and serum iron analysis rather than treating the patient with multivitamins.

Limitations and future prospects

The limitation of this study is that the sample size is small and the follow-up analysis of vitamin B12 and serum iron had not been done. The future scope would be that we should routinely do the analysis of patients with RAS and also do an analysis after the supplementation of the vitamin to ensure their relationship and to rule out other possible causes.

   Conclusion Top

As it is one of the common oral diseases in our practice, it needs an insight for proper management. Since the etiology is multifactorial and the disease is self-limiting many times it is overlooked by the patients and the practitioner as well. Stress is one of the factors positively attributed to the disease, we neglect other factors. However, the underlying etiology goes unnoticed and never attended. Vitamin B12 and Iron are few of the among the many factors when observed shows their relation to the disease and the importance to treat it. India, being a country with a significant amount of malnourishment subjects, it is important to look into it and rule it out before treating the patient. Vitamin B12 is also not a single entity; it has numerous functions from iron absorption to myelination of nerves. If this study is performed on large scale the association with iron could also be established as long-term deficiency of Vitamin B12 will affect iron and folic acid levels also. Hence, we conclude that it is essential to rule out Vitamin B12 deficiency in patients with RAS before starting the management. When the deficiency of Vitamin B12 is identified and managed, it would protect the patient from long term consequences/sequelae.

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

Cash JC, Cook M, Miller L. Stomatitis, Minor Recurrent Aphthous Stomatitis, Canadian Guideline. Canadian Family Practice Guidelines 2019. doi: 10.1891/9780826194985.0008f.  Back to cited text no. 1
Hamedi S, Sadeghpour O, Shamsardekani MR, Amin G, Hajighasemali D, Feyzabadi Z. The most common herbs to cure the most common oral disease: Stomatitis recurrent aphthous ulcer (RAU). Iran Red Crescent Med J 2016;18:e21694.  Back to cited text no. 2
Ślebioda Z, Dorocka-Bobkowska B. Systemic and environmental risk factors for recurrent aphthous stomatitis in a Polish cohort of patients. Adv Dermatol Allergol 2019;36:196-201.  Back to cited text no. 3
Manthiram K, Preite S, Dedeoglu F, Demir S, Ozen S, Edwards KM, et al. Common genetic susceptibility loci link PFAPA syndrome, Behçet's disease, and recurrent aphthous stomatitis. Proc Natl Acad Sci U S A 2020;117:14405-11.  Back to cited text no. 4
Gomes CC, Gomez RS, Zina LG, Amaral FR. Recurrent aphthous stomatitis and helicobacter pylori. Med Oral Patol Oral Cir Bucal 2016;21:e187-91.  Back to cited text no. 5
Axéll T, Henricsson V. The occurrence of recurrent aphthous ulcers in an adult Swedish population. Acta Odontol Scand 1985;43:121-5.  Back to cited text no. 6
Shang H, Ye JJ, Ji M, Wang FF, Zhu YY, Qi XM. Anticoagulant and fibrinolytic disorders in patients with Behçet's disease and recurrent aphthous ulcer. Chin J Physiol 2011;54:235-40.  Back to cited text no. 7
Queiroz SIML, Silva MVAD, Medeiros AMC, Oliveira PT, Gurgel BCV, Silveira ÉJDD. Recurrent aphthous ulceration: An epidemiological study of etiological factors, treatment and differential diagnosis. An Bras Dermatol 2018;93:341-6.  Back to cited text no. 8
Bardellini E, Amadori F, Conti G, Majorana A. Oral mucosal lesions in electronic cigarettes consumers versus former smokers. Acta Odontol Scand 2018;76:226-8.  Back to cited text no. 9
Robertson PB, Walsh MM, Greene JC. Oral effects of smokeless tobacco use by professional baseball players. Adv Dent Res 1997;11:307-12.  Back to cited text no. 10
Atkin PA, Xu X, Thornhill MH. Minor recurrent aphthous stomatitis and smoking: An epidemiological study measuring plasma cotinine. Oral Dis 2002;8:173-6.  Back to cited text no. 11
Pizzorno JE, Murray MT, Joiner-Bey H. The Clinician's Handbook of Natural Medicine E-Book. Elsevier Health Sciences; 2016 Jan 5.  Back to cited text no. 12
Okoh ME, Ikechukwu O. Presentation of recurrent aphthous ulcer among patients in a tertiary hospital. African Journal of Oral Health. 2019 Apr 17;8 (2).  Back to cited text no. 13
Natah SS, Häyrinen-Immonen R, Hietanen J, Malmström M, Konttinen YT. Quantitative assessment of mast cells in recurrent aphthous ulcers (RAU). J Oral Pathol Med 2007;27124-9.  Back to cited text no. 14
Ziaudeen S, Ravindran R. Assessment of oxidant-antioxidant status and stress factor in recurrent aphthous stomatitis patients: Case control study. J Clin Diagn Res 2017;11:ZC01-4.  Back to cited text no. 15
Ślebioda Z, Krawiecka E, Szponar E, Dorocka-Bobkowska B. Haematinic deficiencies and patient clinical profiles in Polish patients with recurrent aphthous stomatitis (RAS). J Oral Pathol Med 2018;47:531-7.  Back to cited text no. 16
Aynali G, Ozkan M, Aynali A, Ceyhan B, Armağan H, Yarıktaş M, et al. The evaluation of serum vitamin B12, folic acid and hemoglobin levels in patients with recurrent minor aphthous stomatitis]. Kulak Burun Bogaz Ihtis Derg 2013;23:148-52.  Back to cited text no. 17
Vigarios E, Comont T, Piroth M, Cougoul P, Sibaud V. Severe aphthous stomatitis secondary to vitamin B12 deficiency with isotretinoin therapy. JAAD Case Rep 2019;5:563-5.  Back to cited text no. 18


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1], [Table 2], [Table 3], [Table 4]


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