Type of the Paper (Article Journal of Baghdad College of Dentistry, Vol. 34, No. 3 (2022), ISSN (P): 1817-1869, ISSN (E): 2311-5270 1 Research Article Serum ferritin level and B12 in a sample of Iraqi re- current aphthous stomatitis patients Noor S. Mohammed Ali 1* 1 Assistant Lecturer, Department of oral diagnosis, College of Dentistry, University of Baghdad. * Correspondence: noorsaad2011@codental.uobaghdad.edu.iq Abstract: Background: With a frequency of 50–66%, recurrent aphthous stomatitis (RAS) is one of the most prevalent conditions affecting the oral mucosa. It is unknown how common hematinic deficiencies, such as those in vitamin B12 and ferritin, affect the pre- vention and progression of RAS. Numerous investigations have shown that individuals with RAS have a significant frequency of hematinic deficits. This research compared pa- tients with recurrent aphthous ulcers and healthy controls' serum levels of ferritin and vitamin B12.Subjects, Materials and Methods: Patients who need blood testing to rule out anemia between November 2020 and May 2021 provided the data. The institutional ethics committee gave its approval to the project. 5ml of blood was taken from patients and con- trols in educational labs after they had provided their demographic information (age, gen- der, occupation, and residance). The serum was then centrifuged at 3000 rpm for 10 minutes before being stored at -20°C until serum ferritin and vitamin B12 levels were de- termined. The information was presented as Mean ± SD. when comparing biochemical parameters between patients and controls using Students unpaired t-test.A p-value of< 0.05 was deemed statistically significant, while a p-value of< 0.001 was deemed highly sta- tistically significant, Results: A total of 30 RAS patients and 30 healthy control with age and gender matches were included. 40% of the patients had low serum ferritin levels and 56.6% of the patients had low serum vitamin B12 levels, according to statistical analysis of the current study. Significant differences were also seen between the two groups' serum levels of ferritin and vitamin B12.Conclusion: Patients with recurrent aphthous stomatitis require serum ferritin and vitamin B12 measurements. In order to stop the recurrence of aphthous ulceration, it's crucial for people with recurrent aphthous ulcers to consume a balanced di- et rich in iron and vitamin B12. Keywords: ferritin, Serum B12, recurrent aphthous ulceration. Introduction One of the most prevalent conditions affecting the oral mucosa, recurrent aphthous stomatitis (RAS) is defined as the occurrence of reoccurring ulcerations exclusive to the oral mucosa. It affects 20% of the general population and up to 60% of some areas of populations (1). According to its clinical characteristics, RAS is typically divided into three clinical forms: minor, major, and herpetiform ulcers. More than 80% of patients with the minor form of RAS are vulnerable to recurrences, with estimates for three-month re- currence rates as high as 50%(2). they typically appear as little round or oval ulcers with erythromatous "halos" and a yellow-grey tint. They typically heal without leaving any permanent scars (3). Despite numerous circumstances, the precise cause of aphthous ulcers is still unknown . Although numerous factors, including smoking, immunological issues, stress, hematological problems, hormone imbalances, infections, vitamin deficiencies, and hereditary factors, have been linked to the pathogenesis of RAS, the actual cause of aphthous ulcers is still unknown (3,4). It has been suggested that hematinic de- ficiencies, such as a deficiency in ferritin, folate, or vitamin B12, may be the cause of RAS. It is unknown how common hematinic deficiencies, such as those in ferritin and vitamin B12, are or what role they play in the prevention and progression of RAS(5). While some research have found no connection between RAS and a deficit in iron, folate, or vitamin B12 (5, 6), other investigations have shown a significant incidence of hematinic deficits in RAS patients (7-9). Received date: 13-12-2021 Accepted date: 14-1-2022 Published date: 15-9-2022 Copyright: © 2022 by the authors. The article is published under the terms and conditions of the Crea- tive Commons Attribution (CC BY) license. (https://creativecommons.org/licens es/by/4.0/). https://doi.org/10.26477/jbcd .v34i3.3211 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://doi.org/10.26477/jbcd.v34i3.3211 https://doi.org/10.26477/jbcd.v34i3.3211 J. Bagh. Coll. Dent. Vol. 34, No. 3 2022 Ali 2 A globular intracellular protein called ferritin accumulates iron and releases it gradually over time. Although ferritin is mostly present in the cytosol of most tissues, it is also released in minute amounts into the serum, where it serves as an iron carrier. Serum ferritin is utilized as a diagnostic test for iron-deficiency anemia because plasma ferritin is also an indirect indicator of the total amount of iron stored in the body (10). Since the discovery and identification of vitamin B12 more than 60 years ago and the realization of its essential role in the serious condition known as pernicious anemia, much has been learned about B12 deficiency. Vitamin B12, also known as cobalamin, is one of the eight B vitamins (11). RAS's pathogenesis is un- clear, hence diagnosis is solely reliant on history and clinical criteria since there are no available labora- tory tests to back up the finding (12). Because there have been few studies on the association between RAS and serum ferritin and vitamin B12 levels, the current study seeks to assess ferritin and vitamin B12 levels in patients with recurrent aphthous ulcers and healthy controls. Subjects , Materials and Methods The data was collected from patients who needed blood analysis to exclude anemia from November 2020 till May 2021. The study was approved by the institutional ethics committee. An oral medicine spe- cialist established the diagnosis based on the existence of round, symmetrical, yellow-white ulcers with a diameter of less than 1 cm and also an erythematous halo surrounded by a detachable membrane that healed completely without leaving any scars. There were exclusion criteria including chronic smokers and alcoholism, patients with a history of medical diseases such as Behcet's disease, hypertension, cardiac diseases, hepatic, renal, hematological disorders, Crohn's disease and ulcerative colitis, subjects on med- ications such as cytotoxic agents such as methotrexate, non-steroidal anti-inflammatory drugs, sul- phonamides, rifampicin and vancomycin (3). The subjects in the control group without a history of illness and without any RAS lesions at the time of data collection; these were collected from previously documented data. demographic information on patients, including their residence, gender, age, and occupation, From patients and controls, 5ml of ve- nous blood was collected in the educational laboratories, Serum was centrifuged at 3000 rpm for 10 minutes, and it was kept at -20°C until the evaluation. Serum ferritin levels were estimated using ELISA kits from Biocheck USA and vitamin B12 was estimated using IBL kits from the USA. SPSS software, version18.0 (Chicago, Illinois, USA) was used for the statistical analysis. The data was presented as Mean± SD. when comparing biochemical parameters between patients and controls using the unpaired t-test, A P-value of < 0.05 was regarded as statistically significant while a P-value of <0.001 was consid- ered a highly statistically significant. Results A total of 30 RAS patients and 30 healthy control were included in the final analysis. Table 1 displays the demographic information of the subjects. Table 1: Demographic information of the RAS patients and controls. RAS Patients Healthy control Number of subjects 30 30 Age (mean± SD) 31.86 ± 5.88 32.4 ± 5.96 Male : Female 14 : 16 13 : 17 J. Bagh. Coll. Dent. Vol. 34, No. 3 2022 Ali 3 Low ferritin levels were seen in 12 RAS patients and 5 controls (p>0.05), While the mean estimates of normal levels of serum ferritin between patients and control were highly significant difference p<0.001. 17 RAS patients and 6 controls had serum vitamin B12 levels that were ≤ 220pg/ml. (p<0.05), It was demonstrated that there was a highly significant difference in the mean estimations of serum B12 and ferritin between RAS patients and healthy controls (p<0.001) as shown in table 2. Table2: A comparison of serum ferritin levels and vitamin B12 levels in RAS patients and controls. Variable RAS Patients Healthy control Number Mean ± SD Number Mean ± SD Serum ferritin ( ng/ml) Low 12 9.86 ± 4.34 5 8.98 ± 0.93 Normal 18 59.44 ± 26.63 25 91.8 ± 34.16** Total 30 39.62 ± 32.14 30 77.99 ± 44.17** Serum B12 ( pg/ml) Low 17 178.58 ± 30.53 6 208.66 ± 11.57* Normal 13 396.15 ± 154.31 24 691.5 ± 138.7** Total 30 272.86 ± 149.6 30 580.33 ± 230.7** Cut-off values for low levels B 12 ≤ 220 pg/ml Ferritin Male ≤ 10ng/ml Female ≤ 20 ng/ml *=p<0.05 **=p<0.001 Discussion In the current study, the serum level of ferritin and vitamin B12 were compared between RAS pa- tients and healthy controls.. The demographic characteristics of the patients and controls showed no sta- tistically significant difference. The most common RAS patients affected were females and between 32- 41 years of age. The age and gender incidence are similar to other studies. The fact that women have a significant propensity to become anemic may help to explain the in- creased incidence of RAS in females as indicated by previous research (13). Several investigations have shown no link between RAS and iron, folate, or vitamin B12 deficiency (5, 6). While other research has J. Bagh. Coll. Dent. Vol. 34, No. 3 2022 Ali 4 shown that RAS patients have a significant frequency of hematinic deficits (7-9) . In the present research, statistically significant variations in ferritin and vitamin B12 serum levels were found between the two groups. 40% of the patients in the current research showed low serum ferritin levels., other studies have reported similar but with varying percentages of serum ferritin levels. Some have reported 60% of pa- tients having low serum ferritin levels, while others have reported as low as 20% (3, 14, 15, 16) . Serum B12 is required for DNA synthesis, and its insufficiency causes megaloblastic anemia, par- ticularly in impoverished nations. Vitamin B12 deficiency inhibits cell-mediated immunity and causes abnormalities in the tongue epithelium and buccal mucosa (3, 17). Serum vitamin B12 levels were low in 56.6% of the patients in this investigation. Other studies have also reported similar findings but with varying percentages of vitamin B12 deficiency (15, 18, 19, 20) . Vitamin B12 insufficiency interferes with the metabolism of folate, which can cause folate deficiency (21, 22). Therefore, in addition to Vitamin B12 measurement, serum folate level should also be measured in cases of RAS.This study also tried to classify the patients and controls into subjects with normal values or low values by specified cut-off values of kits used. Many studies had used their cutoff values in accord- ance with the local laboratory outcomes. However, most studies only relied on the proportions having low or high positions in the patients and control as whole groups (23, 24). 70% of patients with recurrent aphthous ulcers improved with hematinic replacement therapy(25). Future research should ideally have a large sample size and measure the levels of serum folate to investigate the association between RAS and serum folate. Conclusion In the current study, low serum ferritin levels were found in 40% of patients, while low serum vitamin B12 levels were observed in 56.6% of patients.. Serum ferritin and vitamin B12 levels must be measured in individuals with recurring aphthous stomatitis. Recurrent aphthous ulcer sufferers must also follow a healthy diet rich in iron and vitamin B12 to avoid aphthous ulcer recurrence. Conflict of interest: None. References 1. Ujević A, Lugović-Mihić L, Situm M, Ljubesić L, Mihić J, Troskot N. Aphthous ulcers as a multifactorial problem. Acta Clin Cro- at. 2013;52:213-21. 2. Tarakji B, Gazal G, Al-Maweri SA, Azzeghaiby SN, Alaizari N. Guideline for the diagnosis and treatment of recurrent aphthous stomatitis for dental practitioners. J Int Oral Health. 2015;7:74-80. 3. Moin Sabeer Tidgundi , Khaja Moinuddin, , Mirza Sharif Ahmed Baig. Ferritin and vitamin b12 levels in patients with recurrent aphthous ulcers. International Journal of Clinical Biochemistry and Research, April-June 2017;4(2):136-139. 4. Fischman SL. Oral ulcerations. Semin Dermatol 1994;13(2):74-7. 5. Koybasi S, Parlak AH, Serin E, et al. Recurrent aphthous stomatitis: Investigation of possible etiologic factors. Am J Otolaryngol 2006;27(4):229-32. 6. Carrozzo M, Bone MC, Gandolfo S. Recurrent aphthous stomatitis: current etiopathogenetic and therapeutic concepts. Minerva Stomatol 1995;44(10):467-75. 7. Challacombe SJ, Scully C, Keevil B et al. Serum ferritin in recurrent oral ulceration. J Oral Pathol. 1983;12(4):290-9. J. Bagh. Coll. Dent. Vol. 34, No. 3 2022 Ali 5 8. Porter SR, Scully C, Flint SR, et al. Haematological status in recurrent aphthous stomatitis compared with other oral disease. Oral Surg Oral Med Oral Pathol 1988;66(1):41-4. 9. Field EA, Rotter E, Speechley JA, Tyldesley WR. Clinical and haematological assessment of children with recurrent aphthous ulceration. Br Dent J 1987;163(11):19-22. 10. Wang W, Knovich MA, Coffman LG, Torti FM, Torti SV ."Serum ferritin: Past, present and future" Biochimica et Biophysica Ac- ta (BBA) 2010 Aug;1800 (8): 760–9. 11. Green, R. & Miller, J. W. in Handbook of Vitamins 5th edn (eds Zempleni, J. et al.) 447–489 (Taylor & Francis, 2014). A compre- hensive review of B12 biochemistry, nutrition and metabolism. 12. Natah SS, Konttinen YT, Enattah NS, Ashammakhi N, Sharkey KA, Hayrinen-Immonen R. Recurrent aphthous ulcers today: a review of the growing knowledge. Int J Oral Maxillofac Implants 2004;33(3):221-34. 13. Sumathi K, Shanthi B, Subha Palaneeswari M, Manjula Devi A.J. Significance of Ferritin in Recurrent Oral Ulceration. J Clin D i- agn Res 2014;8(3):14-15. 14. Nabiha Farasat Khan, Mohammad Saeed, Saima Chaudhary, et al. Haematological Parameters and Recurrent Aphthous Stoma- titis. Journal of the college of Physicians and Surgeons Pakistan 2013;23(2):124-7. 15. Farkhanda Ghafoor , Ayyaz A Kha. Association of Vitamin B12, Serum Ferritin and Folate Levels with Recurrent Oral Ulcera- tion. Pak J Med Res 2012; 51 (4):132-135. 16. Rogers RS, Hutton KP. Screening for haematinic deficiencies in patients with recurrent aphthous stomatitis. Aust I Derm 1986;27(3):98-103. 17. Volkov I, Press Y, Rudoy I. Vitamin B12 could be a “Master Key” in the regulation of multiple pathological processes. J Nippon Med Sch 2006;73(2):65-9. 18. Thongprasom K, Youngnak P, Aneksuk V. Hematologic abnormalities in recurrent oral ulceration. South Asian J Trop Med Public Health 2002; 33: 872-7. 19. Wary D, Ferguson MM, Hutcheon WA, Dagg JH. Nutritional deficiencies in recurrent aphthae. J Oral Pathol 1978;7:418-23. 20. Olson JA, Feinberg I, Silverman S Jr, Abrams D, Greenspan JS. Oral Surg Oral Med Oral Pathol 1982; 54: 571-20. 21. Nolan A, Lamey PJ, Milligan KA, Forsyth A. Recurrent aphthous ulceration and food sensitivity. J Oral Pathol Med 1991;20(10):473-5. 22. Weusten Bl, Van De Wiel A. Aphthous ulcers and vit B12 deficiency. Neth J Med 1998;53:172-5. 23. Burgan SZ, Sawair FA, Amarin ZO. Hematologic status in patients with recurrent aphthous stomatitis in Jordan. Saudi Med J. 2006; 27: 381-4. 24. 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. J. Bagh. Coll. Dent. Vol. 34, No. 3 2022 Ali 6 25. Volkov IIIA, Rudoy I, Freud T, Sardal G, Naimer S, Peleg R, et al. Effectiveness of vitamin B12 in treating recurrent aphthou s stamatitis: A randomized, double blind placebo controlled trial. J Am Board Fam Med 2009; 22: 9-1. في عينة من المرضى العراقيين المصابين بالتهاب الفم القالعي المتكرر 12ين في الدم و فيتامين ب مستوى الفيريت نور سعد محمد علي : المستخلص ٪. إن انتشار نقص الدم بما في ذلك 66-50( هو أحد أكثر اضطرابات الغشاء المخاطي للفم شيوعًا بمعدل انتشار يقارب RASالخلفية :التهاب الفم القالعي المتكرر ) . كان RASغير معروف جيدًا. أظهرت العديد من الدراسات انتشاًرا كبيًرا لنقص الدم في مرضى RASودورها في الوقاية وتطوير B12نقص الفيريتين وفيتامين في المرضى الذين يعانون من القرحة القالعية المتكررة والضوابط الصحية. 12الدراسة هو مقارنة مستويات مصل الفيريتين وفيتامين ب الهدف من الموافقة على . تمت 2021إلى مايو 2020االشخاص والمواد والطريقة: تم جمع البيانات من المرضى الذين يحتاجون إلى تحليل الدم الستبعاد فقر الدم من نوفمبر للمرضى السكانية التركيبة تسجيل بعد المؤسسية. األخالقيات لجنة قبل من والعنوان (الدراسة والمهنة والجنس سحب )العمر تم المرضى 5، من الدم من مل درجة مئوية لتقييم مصل الفيريتين وفيتامين 20-عند تم تخزين المصل ثم دقائق ، 10دورة في الدقيقة لمدة 3000والضوابط في المعامل التعليمية التي تم طردها عند مات البيوكيميائية بين المرضى وعناصر التحكم ، واعتبرت و. يستخدم اختبار الطالب غير المقيدين لمقارنة المعل Mean ± SD. تم التعبير عن البيانات على أنها 12ب ذات داللة إحصائية عالية. P <0,001بينما اعتبرت قيمة ذات داللة إحصائية P <0,05قيمة من الضوابط الصحية العمرية والجنس ، في الدراسة الحالية لوحظت فروق ذات داللة إحصائية في 30و RASمريًضا من 30لنتائج: تم تضمين ما مجموعه ا ٪ من المرضى لديهم مستويات 56,6مستويات الفيريتين و ٪ من المرضى لديهم مصل منخفض 40بين المجموعتين ، B12مستويات المصل من الفيريتين وفيتامين في الدم. 12منخفضة من فيتامين ب في الدم ضروري لمرضى التهاب الفم القالعي المتكرر. من المهم أيًضا لمرضى القرحة القالعية المتكررة اتباع 12الخالصة: قياس مستويات الفيريتين وفيتامين ب لمنع تكرار التقرح القالعي. 12د وفيتامين ب نظام غذائي يحتوي على الحدي