|Year : 2016 | Volume
| Issue : 2 | Page : 119-123
Analysis of iron levels in the mucosal tissue and serum of oral submucous fibrosis patients
Basavaraj N Kallalli1, Pavan K Gujjar2, Kamala Rawson3, Sudhir Bhalerao4, Treville Pereira4, Jyoti Zingade5
1 Department of Oral Medicine and Radiology, Hazaribag College of Dental Sciences, Hazaribag, Jharkhand, India
2 Department of Oral Pathology and Microbiology, Narsinhbhai Patel Dental College and Hospital, Visnagar, Gujarat, India
3 Department of Oral Medicine and Radiology, Rama Dental College, Kanpur, Uttar Pradesh, India
4 Department of Oral and Maxillofacial Pathology, D.Y. Patil School of Dentistry, Navi Mumbai, Maharashtra, India
5 Department of Oral Medicine and Radiology, Narsinhbhai Patel Dental College and Hospital, Visnagar, Gujarat, India
|Date of Submission||21-Oct-2015|
|Date of Acceptance||21-Nov-2016|
|Date of Web Publication||02-Dec-2016|
Department of Oral Medicine and Radiology, Rama Dental College, Lakhanpur, Kalyanpur, Kanpur - 208 024, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Oral submucous fibrosis (OSMF), being a chronic insidious oral mucosal condition, affects most parts of the oral cavity and has a high malignant transformation rate, which is triggered by areca nut chewing, nutritional deficiencies, immunologic processes, and genetic predisposition. Aims and Objectives: The objective of the present study was to evaluate the levels of iron, as a trace element of patients with OSMF, and its association with areca nut and betel quid chewing. Materials and Methods: The study sample comprised 30 patients who were clinically diagnosed with OSMF and 10 healthy controls. OSMF patients were categorized by clinical staging. Tissue and serum estimation of iron levels was done using Vitros 250 photometry. Results: The levels of tissue and serum iron was significantly decreased in OSMF patients when compared to controls, which was statistically significant. Conclusion: Most of the patients with OSMF chewed gutkha, as compared to the transitional form of pan masala. The betel quid and areca nut chewing habits are associated with the disease state, which may play a role in altering the tissue and serum iron levels.
Keywords: Oral cancer, oral disease, OSMF, serum iron, tissue iron
|How to cite this article:|
Kallalli BN, Gujjar PK, Rawson K, Bhalerao S, Pereira T, Zingade J. Analysis of iron levels in the mucosal tissue and serum of oral submucous fibrosis patients. J Indian Acad Oral Med Radiol 2016;28:119-23
|How to cite this URL:|
Kallalli BN, Gujjar PK, Rawson K, Bhalerao S, Pereira T, Zingade J. Analysis of iron levels in the mucosal tissue and serum of oral submucous fibrosis patients. J Indian Acad Oral Med Radiol [serial online] 2016 [cited 2021 Apr 13];28:119-23. Available from: https://www.jiaomr.in/text.asp?2016/28/2/119/195084
| Introduction|| |
Oral cancer is one of the 10 most common forms of cancer in developed countries and is ranked as the sixth most common cancer around the world. , It accounts for approximately 4% of all cancers and 2% of all cancer deaths worldwide. The World Health Organization (WHO) reported that oral squamous cell carcinoma (OSCC) has one of the highest mortality ratios among all malignancies.  In India it accounts for 50-70% of all cancers.  Approximately 90-95% of oral cancers occur predominantly in tobacco and alcohol users, between the 6 th and 7 th decades of life.  In spite of the large amount of research data in cellular and molecular biology as well as the advances in oncology and surgery, the mortality and morbidity rates in OSCC patients remain unchanged. 
Oral submucous fibrosis (OSMF) is classified under potentially malignant disorders. This entity is associated with chewing areca nut. In India, there has been a consistent rise in the number of patients with OSMF. Many attempts have been made to understand the processes involved, and as concurrent knowledge of molecular technology increased, newer and advanced modes of cytological, histological, immunohistochemical and functional analysis were applied. 
Trace elements are required in small concentrations as essential components of biological enzyme systems or structural portions of biologically active constituents. Many metabolic disorders, potentially malignant disorders and oral cancer are accompanied by alterations in the concentration of one or more trace elements such as copper, iron, zinc and magnesium in body fluids, especially blood serum or plasma.  Trace element levels also give an idea about necessary elements to be given as supplements to counteract the oxidative stress induced by free radicals, which can cause serious damage to cells in oral potentially malignant disorders and cancer. 
Among many trace elements, iron has been recognized as an important element for maturation of epithelium and it is well documented that iron deficiency is associated with epithelial abnormalities in addition to malignancies such as post-cricoid carcinoma and tumors of the pharynx and mouth. Thus, biochemical alterations of iron concentrations in pateint's with potentially malignant disorders can help not only in early diagnosis but also as an indicator for prognosis. 
The populations of the Mumbai and Navi Mumbai exhibit predominant use of areca nut due to their cultural and social backgrounds. There is need to study whether areca nut causes local alteration in serum and tissue iron levels, as well as its effect in different age and gender. Thus, the aim of the present study is to evaluate the alteration of serum and tissue iron levels in OSMF and its comparison with healthy individuals. There is a need to study whether the areca nut habit could cause local alteration in the tissue and serum iron and are acted upon by factors such as age, gender and the type of habit practiced. Thus, the need to understand the etiopathogenesis and progression of these conditions of whether the tissue and serum iron levels in OSMF are altered from those in the normal subject will be investigated in this study.
| Materials and Methods|| |
This prospective case control study was designed and carried out in the Department of Oral and Maxillofacial Pathology. The study consisted of 40 participants, 31 males and 9 females, with the age range of 20-60 years; the participants were divided into two groups. Group I consisted of 30 clinically and histopathologically diagnosed OSMF participants and group II 10 healthy participants.
A through history and clinical examination of all the participants were carried out. Patients with the habit of tobacco chewing, smoking, arecanut chewing and alcohol consumption were included in the study group. All the healthy participants without any habits were included in the control group. Participants who had taken antibiotics in the course of the previous month for any tooth infection, those on anticoagulant therapy, chemotherapy, radiation therapy, and with malignant lesions were excluded from the study. Written informed consent and institutional ethical committee clearance was obtained for the study.
The staging of the OSMF was done according to the criteria of clinical staging by Haider et al. Clinical staging was done depending on the topography and the extent of distribution of bands. Stage 1 participants had faucial bands, whereas those in stage 2 presented faucial and buccal bands, and among individuals in stage 3, faucial, buccal, and labial bands were involved.  Ten millilitres of blood was drawn from the median cubital vein under aseptic precautions, from which subsequently serum was obtained. An incisional biopsy was performed under local anesthesia from the affected site in the study group and from the retromolar area of the controls, who were undergoing pericoronal flap removal. The tissue and serum obtained were preserved in a frozen state until analysis. Frozen specimens of OSMF and controls were measured, weighed in a microbalance, dried in a desiccator overnight and reweighed to estimate the net dry weight; thereafter the specimens were immersed in a solution containing 10 volume of nitric acid. After 72 h in the solution, the tissues were completely dissolved; however, fats present on the surface of the aqueous layer were removed. The blood was centrifuged at 1000-1500 RPM for 15 min. Subsequently, 5 ml of serum and tissue was collected, which were analyzed for iron with VITROS 250 PHOTOMETRY CLINICAL DIAGNOSTICS (by Johnson and Johnson). The data obtained from the procedures were tabulated and analyzed using statistical methods (descriptive analysis and paired t-test).
| Results|| |
There were 30 participants in study group and 10 participants in the control group. The mean tissue iron level in the study group was calculated to be 73.79 ± 7.50 μg/100 mg, whereas in the control group was 119.67 ± 8.38 μg/100 mg, and the mean serum iron level in the study group was calculated to be 69.26 ± 5.28 μg/100 ml, whereas in the control group was 110.77 ± 7.26 μg/100 ml [Table 1], Graph 1. The mean tissue and serum levels of iron were noted to be different among the patients with three clinical stages [Table 2] and [Table 3], Graphs 2 and 3. This shows a high negative relationship between the serum iron level and clinical staging. It implies that, as the clinical staging increases, the tissue and serum iron level decrease. The statistical correlation of tissue and serum iron in the study groups were significantly different with a P value of 0.0001 [Table 4].
|Table 1: Mean serum and tissue levels of iron in both the study and control groups |
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|Table 2: Mean tissue levels of iron were noted to be different among patients with three clinical stages |
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|Table 3: Mean serum levels of iron were noted to be different among patients with three clinical stages |
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|Table 4: The statistical correlation of tissue and serum iron in study groups |
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| Discussion|| |
OSMF is a chronic, insidious oral mucosal condition that occurs predominantly among Indians and occasionally in Asians. In the Indian continent alone, the statistics for OSMF is approximately 5 million affected people, i.e., 0.5% of the population.  The reasons for the rapid increase of the disease are reported to be an upsurge in the popularity of commercially prepared areca nut preparations (pan masala) in India as well as an increased uptake of this habit by young people due to easy access, effective price changes and marketing strategies.  The hallmark of the disease is submucosal fibrosis that affects the oral cavity and progressively involves the pharynx and the upper esophagus. This leads to a burning sensation in the oral cavity, blanching, and stiffening of oral mucosa and oropharynx, resulting in restricted mouth opening, which in turn causes limited food consumption, difficulty in maintaining oral health, and impairs the ability to speak.  The rate at which oral precancerous and cancerous lesions are spreading like an epidemic is alarming. The prevalence of oral potentially malignant disorders is significantly higher than that of oral cancer, and these disorders provide useful clinical markers for oral cancer. 
Over the years, awareness that trace elements play a very important role, either beneficial or harmful, in human health has increased. Any constituent whose concentration is equal to or less than 0.01% (100 parts per million) of the total matrix has been defined as a "trace constituent." In many studies, it was found that three-dimensional active conformations of many proteins such as thymidylate synthetase, dihydrofolate reductase, p53, p16, and K-ras, are maintained by trace elements. Many metabolic disorders in humans are accompanied by alterations in the concentration of one or more trace elements in some body fluids, especially blood serum or plasma.  The mean age of the OSMF group in a study conducted by Karthik H et al.  was 26.85 years, 29.04 years by Katharia et al.  and 30 years by Maher et al.,  which is consistent with the findings in our study with the mean age of 31.5 years.
A male predominance was seen in the OSMF group with 51 males and 9 females by Karthik H et al.  The findings of a male predominance in the present study are consistent with that conducted by Ranganathan et al.,  where 25 males and 5 females were found to be dominating because they were using gutkha and other related products more because of easy availability in all the places. Females being more conscious about their health and esthetic value probably feel uncomfortable to ask the vendors in getting the gutkha products. This may be one of the reasons, which may be responsible for a higher male-to-female ratio.
Stevens et al.  , (1988-1994), reported cancer incidence in the First National Health and Nutrition Examination Survey of 41276 men and women aged between 20 and 74 years and its follow-up study. They found a positive association of cancer risk with lowered serum iron levels. Serum iron levels are considered to be biochemical indicators for nutritional assessment. Utilization of iron in collagen synthesis (Huang et al.)  by the hydroxylation of proline and lysine leads to decreased serum iron levels in OSMF patients. Occurrence of iron deficiency is known to be present in oral cancer, which may be due to malnutrition caused by the tumour burden. In the present study, we considered 30 participants in the study group and 10 participants in the control group. We found mean tissue and serum iron levels (P = 0.000), which was far less than 0.05 and 0.025. This indicates that there is a significant difference between the mean tissue and serum iron in the study group. Khanna et al.  conducted a study in which they considered 30 study test participants and 30 control participants (P = 2.35e-90), which was found to be statistically significant with a mean SI in the study group being 101.13 μg and in control group being 138.10 μg per 100 ml, which is far more significant than the present study. Tadakamadla et al.  evaluated serum copper and serum iron levels in OSMF patients, in which mean iron level differed significantly between the patients and controls. The patients exhibited lower iron (114.78 ± 23.47) than control group (138.86 ± 31.01), which is far more than the present study. Furthermore, lack of iron in the tissues results in decreased vascularity, which facilitates percolation of arecoline. In vitro studies on human fibroblasts observed that arecoline causes increased fibroblastic proliferation and collagen formation which is a hallmark of OSMF.  Similar to the present study, the mean tissue iron level in the study group was calculated to be 73.79 ± 7.50 μg/100 mg, whereas the control group was 119.67 ± 8.38 μg/100 mg; hence, we can state that there is a significant decrease in tissue iron levels in OSMF patients.
| Conclusion|| |
In the present study, we undertook the analysis of tissue and serum concentration of iron in clinically diagnosed cases of OSMF. After statistically analyzing the data, the following inferences could be drawn. OSMF manifests more commonly in young age group of 26-35 years, with a definite male predilection. Majority of the patient's with OSMF chewed gutkha compared to the transitional forms of pan chewing. There is a decrease in tissue and serum iron levels in OSMF patient, and hence there is significant alteration from the normal ranges. Thus, the present study forms a nidus for further research to be conducted to affirm the absolute utility of serum iron estimation in OSMF. Above all, studies including the follow-up of patients are necessary to understand the true value of tissue and serum iron as a prognostic parameter in OSMF.
We would like to thank STATSMASTER TEAM (www.statsmaster.tk; [email protected]) for their valuable contribution for statistical analysis for this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]