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ORIGINAL RESEARCH
Year : 2023 | Volume
: 14
| Issue : 2 | Page : 55-58
Serum and Salivary Vitamin B12 Levels among Iron Deficiency Anemia Patient with Recurrent Aphthous Stomatitis: An Analytical Cross-Sectional Study
Ghaith Majid Al-hamdani1, Layla Sabri Yas2
1 Department of Oral Medicine, College of Dentistry, University of Kufa, Najaf, Iraq
2 Department of Oral and maxillofacial Pathology, College of Dentistry, University of Baghdad, Baghdad, Iraq
Correspondence Address:
Ghaith Majid Al-hamdani
Department of Oral Medicine, College of Dentistry, University of Kufa, Najaf
Iraq
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/denthyp.denthyp_59_23
Introduction: We aimed to evaluate serum and salivary vitamin B12 levels in patients with iron deficiency anemia (IDA) with and without recurring aphthous ulcers (RAS). Methods: Study participants were divided into three groups, clinically healthy control group (n = 25), an IDA patient with RAS (n = 25), and an IDA without RAS (n = 25). The enzyme-linked immunosorbent assay (ELISA) method was used to assess the serum and salivary B-12 levels blindly. Data analyses were performed using one-way ANOVA and the Tukey’s post hoc test using R software. Result: There were statistically significant differences regarding serum and salivary B12 levels among study groups (P<0.001). All pairwise comparisons showed significant differences regarding serum (P<0.001) and salivary (P ≤ 0.002) B12 levels. Conclusion: The Control group (396 ± 18.2 pg/mL) was within the normal serum level of vitamin B12 (200-900 pg/mL), while in IDA with RAS (111 ± 15.4 pg/mL) and IDA without RAS (145 ± 57.2 pg/mL) serum level of vitamin B12 is lower than the normal range and significantly lower than the control group.
Keywords: Aphthae, ELISA assay, iron deficiency anemia, recurrent aphthous stomatitis, salivary biomarkers, Vitamin B12
One of the most prevalent oral mucosal inflammatory ulcerative disorders is recurrent aphthous stomatitis (RAS). Its prevalence is reported to vary from 5% to 60%.[1] The ulcers may be preceded by prodromal regional burning, pain, or discomfort for 24 to 48 hours. Various treatments have been suggested for RAS such as Dexamethasone, Amlexanox, Tricoslan, Levamisole, Benzydamine, Tetracycline,[2] Mastic Orabase,[3] vitamin B12, vitamins B complex, chlorhexidine gluconate,[4] and lavender,[5] etc.
The exact cause of aphthous ulcers is still unknown, even though many factors, including smoking, immunological disorders, stress, hematological abnormalities, hormone imbalances, infections, vitamin deficiencies, and hereditary factors, have been related to the development of RAS.[6],[7] According to certain theories, RAS may be caused by hematinic deficiencies such as ferritin, folate, or vitamin B12. It is unclear how typical hematinic deficiencies, including those in vitamin B12 and ferritin, or their role in preventing and progressing RAS.[8] While some studies have not discovered a link between RAS and a deficiency in iron, folate, or vitamin B12.[8],[9] Other studies have revealed a considerable incidence of hematinic deficiencies in RAS patients.[10],[11]
However, certain groups of RAS patients may experience hematinic (iron, folic acid, or vitamin B12) deficits twice as frequently as healthy control participants. The most often documented pertinent anomaly, occurring in up to 37% of reported patient groups, is reduced iron storage. RAS has been linked to vitamin B12 insufficiency in 1% to 6% of adults or children.[1] Other studies reported anemia and vitamin B12 deficiency in 20.9% and 4.8% of RAS patients.[4] Also, it has been reported that in 13% of RAS patients, a deficiency in multiple areas was found. This included hemoglobin, serum ferritin, hematocrit, RBC folate, and serum B12.[12]
Nevertheless, the current study aimed to evaluate serum and salivary vitamin B12 levels in patients with iron deficiency anemia (IDA) with and without recurring aphthous ulcers.
Material and MethodsThe study protocol was approved by the institutional ethics committee issued by the University of Baghdad, College of Dentistry (Ref. number 750/750722 on December 28, 2022). Sample size was calculated by using G power 3.1.9.7 (http://www.gpower.hhu.de/) with the power of study = 85%, alpha error of probability = 0.05 two-sided, assuming the effect size of F was 0.4, with three groups, the sample size was estimated to be 75 subjects (25 subjects for each group).
The sample collection was conducted for this analytical cross-sectional study at Alsader Medical Center in Najaf City and Alfurat Hospital in Alkufa/Najaf City from February 2022 to the end of August 2022. The laboratory work is done at the International Center for Research and Development (ICRD) in Baghdad, and the operator was blinded to all groups.
A total of 75 subjects signed an informed consent form after receiving a comprehensive explanation of the objectives and scope of the study. Participants are divided into the following three groups:
(1) Control group (n = 25), (2) iron deficiency anemia without aphthous stomatitis (n = 25), and (3) iron deficiency anemia with aphthous stomatitis (n = 25).
Subjects with hemoglobin levels lower than 13.2 g are included in the iron deficiency anemia groups. An expert in oral medicine established the diagnosis through the presence of symmetrical, spherical, yellow-white ulcers, and an erythematous halo ringed by a detached membrane that completely healed without any scarring. Chronic smokers and alcoholics, individuals with a history of autoimmune disease like Behcet’s syndrome, hypertension, cardiac disorders, hepatic, renal, and hematological disorders, as well as those taking medications like methotrexate, non-steroidal anti-inflammatory drugs, sulphonamides, rifampicin, and vancomycin, were excluded from the study.[6]
The subjects in the control group had no history of illness and no RAS lesions at the time of data collection. Five milliter of venous blood was collected in the educational laboratories. Saliva was collected for all participants into plastic containers (CNWTC, Jiangsu, China) between 9 and 11 a.m. The subjects were instructed to avoid any form of oral hygiene 1 hour before saliva sample collection and rinse and wash their mouths with distilled water before collecting saliva. Saliva was collected before or at least 1 hour after a meal.[13] Unstimulated saliva was collected by draining by asking the participant to sit with their head bent with an open mouth. Samples (serum and saliva) were centrifuged (Rotofix 32A, Tuttlingen, Germany) at 3000 rpm for 10 minutes, and the clear supernatants were stored in Eppendorf tubes (CNWTC/OEM, Jiangsu, China) inside a deep freezer at −80 °C (Angelantoni Life Science, Massa Martana PG, Italy) until further analysis.[14]
Human specific enzyme-linked immunosorbent assay (ELISA) kits from (MyBioSource, San Diego, CA, USA) were utilized to determine salivary and serum vitamin B-12 levels per the manufacturer’s instructions. Salivary and serum samples were thawed and labeled before the procedure. Kits’ sensitivity values were 0.469 ng/mL, with a detection range of 0.781 to 50 ng/mL. The optical density was measured at 450 nm. The operator who performed the procedures was blinded to the tested groups.
Data analyses were performed using one-way analysis of variance (ANOVA) and the Tukey’s post hoc using R software (R Foundation for Statistical Computing, Vienna, Austria).
ResultsA total of 25 IDA with RAS patients, 25 IDA without RAS, and 25 healthy controls were included in the final analysis (mean age = 38.3 ± 13.3, range 20-49, females [68%]). The subject flow diagram is available via https://doi.org/10.6084/m9.figshare.22639984.v1.
However, 23 (92%) patients had a minor aphthous ulcer and did not find the herpetic form of an aphthous ulcer. Location was 11 (44%) in the buccal mucosa, 9 (36%) in the lower lip, 3 (12%) in the upper lip, and 2 (8%) in the ventral of the tongue.
There were statistically significant difference regarding serum and salivary B12 levels among study groups (P<0.001). All pairwise comparisons showed significant differences regarding serum (P<0.001) and salivary (P ≤ 0.002) B12 levels [Figure 1].
Figure 1 Box and whisker plot presented deviation in the amount of serum and salivary B12 levels (pg/mL). Discussion In the current study, the serum and saliva levels of vitamin B12 were compared among IDA with RAS, IDA, without RAS patients, and healthy controls.
In this study, the minor aphthous ulcer is the most common (23 of 25 cases), and the lower lip has the greater frequency of recurrent aphthous ulcers. Most studies have extreme variations in results regarding the RAS site. Queiroz et al.,[15] found that most RAS lesions were in the tongue. Chattopadhyay and Chatterjee[16] stated that the lower vestibule is the position most affected by RAS. At the same time, Oyetola et al.,[17] suggested the lower lip as the major position affected by RAS. Therefore, the relationship between RAS and site is mostly variable from one study to another, which could be related to the differences in the sample of the population selected.
In the present study, the control group (396 ± 18.2 pg/mL) was within the normal serum level of vitamin B12 (200-900 pg/mL), while in IDA with RAS (111 ± 15.4 pg/mL) and IDA without RAS (145 ± 57.2 pg/mL) serum level of vitamin B12 was lower than the normal range and significantly lower than the control group.
Olson et al. reported a mean serum level of vitamin B12 among RAS patients of 722 pg/mL which was in the normal range.[18] Also, Piskin et al. showed that the mean serum level of vitamin B12 in the population of RAS patients was 318.81 ± 173.76 pg/mL which was in the normal range, yet significantly lower than the control group.[19] Khan et al. reported significantly lower serum vitamin B12 levels in comparison with the control group among RAS patients.[12] Wray et al. described that 30.4% of RAS patients were deficient in vitamin B12.[20] Ali stated a low serum level of vitamin B12 among 65% of RAS patients (178.58 ± 30.53 pg/mL).[21]
To our knowledge, the level of vitamin B12 in saliva is rarely assessed and published in the literature.
Results regarding serum levels of vitamin B12 among RAS patients were controversial. The heterogeneity may be related to different ages and populations. The conduction of systematic reviews and meta-analysis in this regard is highly recommended.
However, regarding the etiopathogenesis of RAS, there is still no solid conclusion. Within limitations of this cross-sectional study it seems logic to recommend all RAS patients undergo routine hematological screening and tests for serum iron, and vitamin B12 deficiency.
Cross-sectional studies have limitations and cannot establish causality. Our study did not follow up participants over time and only selected a sample from a diverse population.
Financial support and sponsorship
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Conflicts of interest
There are no conflicts of interest.
References
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