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 Table of Contents  
Year : 2022  |  Volume : 34  |  Issue : 3  |  Page : 268-271

Liver-Expressed antimicrobial peptide as an early predictor of dysplasia in oral submucous fibrosis: An experimental trial

1 Department of Oral Medicine and Radiology, Sri Rajiv Gandhi College of Dental sciences and Hospital, Cholanagar, Bangalore, Karnataka, India
2 Department of Oral Medicine and Radiology Jawaharlal Nehru Institute of Medical Sciences Dental College, Imphal Manipur, India

Date of Submission27-May-2022
Date of Decision03-Sep-2022
Date of Acceptance04-Sep-2022
Date of Web Publication26-Sep-2022

Correspondence Address:
Bhavana T Veerabasavaiah
Senior Lecturer, Department of Oral Medicine and Radiology, Sri Rajiv Gandhi College of Dental Sciences and Hospital, Cholanagar, Bangalore - 560 032, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jiaomr.jiaomr_161_22

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Background: Liver-expressed antimicrobial peptide 1(LEAP1) plays a major role in dysplasia. Oral potentially malignant disorders (OPMDs) are on the rise, and the rate of malignant transformation is alarming. Iron (Fe) deficiency in anemia of chronic disease may be because of the presence of oral submucous fibrosis (OSMF). The metabolism of Fe is altered in dysplasia, leading to dysregulation of Fe homeostasis. Aim and Objective: This study aimed to estimate the levels of serum LEAP1 in subjects having OSMF and OSMF with dysplasia and to establish the role of LEAP1 in dysplasia associated with OSMF and in iron homeostasis. Materials and Methods: This study was registered with the CTRI [Clinical Trial Registry of India (REF/2019/06/026566)] as a clinical trial. Twenty participants were selected. Ten participants with OSMF and 10 with OSMF along with dysplastic changes were chosen and categorized as group I and group II. Serum LEAP was estimated in the 20 subjects with clinically diagnosed OSMF. Baseline hematologic investigations like complete blood count (CBC), peripheral smear, and LEAP1 were done. Statistics: Statistical analysis was done using the Statistical Package for the Social Sciences (SPSS) software version 11.5 (IBM, New York, USA). To assess the correlation between serum LEAP1 and OSMF, the one-way analysis of variance (ANOVA) test was used. To obtain the interconnection between serum LEAP1 and dysplasia, independent t-test was used. By calculating the effect size, clinical significance was established. Results: Serum LEAP1 levels in group I (OSMF with dysplasia) showed a remarkable increase in the value in comparison with group II (OSMF without dysplasia). The correlation between the values of serum LEAP1 and dysplasia was significant with P < 0.001. Clinical Significance: Alterations in the iron metabolism are observed in dysplasia; hence, LEAP1 can be a novel marker in the early detection of cancer and can lead to effective treatment and increased survival rate in oral cancer. Conclusion: This research explores new avenues by linking LEAP1 levels to the presence of dysplasia. We can conclude that improvement in the body's iron stores leads to a decrease in serum LEAP1. Therefore, to assess iron storage in OSMF serum, LEAP1 can be used as a novel diagnostic marker.

Keywords: Anemia of chronic disease, dysplasia, liver-expressed antimicrobial peptide 1, oral submucous fibrosis

How to cite this article:
Nagaraj T, Veerabasavaiah BT, Santosh H N. Liver-Expressed antimicrobial peptide as an early predictor of dysplasia in oral submucous fibrosis: An experimental trial. J Indian Acad Oral Med Radiol 2022;34:268-71

How to cite this URL:
Nagaraj T, Veerabasavaiah BT, Santosh H N. Liver-Expressed antimicrobial peptide as an early predictor of dysplasia in oral submucous fibrosis: An experimental trial. J Indian Acad Oral Med Radiol [serial online] 2022 [cited 2022 Dec 1];34:268-71. Available from: http://www.jiaomr.in/text.asp?2022/34/3/268/356953

   Introduction Top

Oral submucous fibrosis (OSMF) occurs mainly because of elevated collagen turnover rate, and iron is utilized more for hydroxylation of proline, and lysine in OSMF. This will increase the total ironbinding capacity (TIBC) and decrease serum iron, hemoglobin (Hb), and serum ferritin. Iron-deficiency anemia is synonymic with OSMF.[1],[2] In chronic kidney disease (CKD), anemia of inflammation, and iron refractory iron deficiency anemia, serum liver-expressed antimicrobial peptide 1 (LEAP1) levels are increased. Hepatitis C infection, iron loading anemia, and hereditary hemochromatosis are characterized by LEAP1 deficiency.[1],[3] Defects in iron (Fe) metabolism can be attributed to the inadequate Fe in OSMF; therefore, levels of LEAP1 may indicate the Fe metabolism. During iron deficiency, LEAP1 hepatocytes release LEAP1 as an iron-regulatory protein. The enzyme ferric reductase helps in the reduction of the dietary ferric ion to its ferrous form. With the help of a divalent metal transporter 1, it is transported across the cell membrane. The internalization of Ferro protein (iron transporter protein) is caused by LEAP1.[1] With this, transport of iron from enterocytes to the plasma is blocked. Therefore, chronic inflammatory conditions like OSMF and regulation of iron recycling are noted with elevated LEAP activator of transcription (STAT) pathway, and Janus kinase (JAK) signal transducer regulates the release of LEAP1. In chronic inflammation, the JAK-STAT3 pathway is upregulated by interleukin 6, which leads to an increase in LEAP1 production. In neoplastic disease, the hepcidin axis pertinence is multifaceted. Overexpressed LEAP1 is associated with many types of cancer and is also accountable for anemia in neoplastic disease. Inflammatory cytokines, especially IL-6, cause elevation in LEAP1 and anemia in cancer.[4] After iron deficiency, anemia of chronic disease (ACD) is the second most frequent form of anemia. ACD is known as the presence of anemia in inflammatory conditions/neoplasia/chronic infections, which may occur in spite of sufficient iron and vitamins but which is not due to marrow deficiencies.[5] ACD is commonly associated with acute and chronic infections, CKD, and autoimmune disorders. Chronic inflammation contributes about 25%–30% of ACD. Thus, the condition is known as “anemia of inflammation”.[5] ACD is characterized by reduced reticulocyte (RBC) counts, decreased or within the normal range of TIBC, reduced level transferrin saturation, and reduction of serum iron concentration.[5] In ACD, iron get accumulate in reticuloendothelial macrophages even though there is decreased circulating iron levels. For Hb synthesis, a reduced circulating iron is present despite a normal – high level of circulating iron in ACD. For the survival and growth of certain pathogens, iron is required. Therefore, as a result of sequestration of iron, the invading pathogens are destroyed. In functional iron deficiency present in ACD, LEAP1 plays an important role which is indicated in the current clinical studies.[4],[5] Thus the abstraction of ACD in OSMF and dysplasia can be made stronger by establishing a link between OSMF and LEAP1 levels. This study aimed to estimate the levels of serum LEAP1 in subjects having OSMF without dysplasia and OSMF with dysplasia and establish the vital role of LEAP1 in dysplasia associated with OSMF and in iron homeostasis.

   Materials and Methods Top

The institutional ethical clearance number (No. SRGCDS/2019/631) dated 22/10/2019 was obtained from the Institutional Ethical Committee of Sri Rajiv Gandhi College of Dental Sciences and Hospital, Bangalore, and also the study was registered with the CTRI [Clinical Trial Registry of India (REF/2019/06/026566)]as a clinical trial. Twenty clinically established OSMF subjects were included in the study. Informed consent as per Helsinki declaration in appropriate format was obtained from the participants selected for the study. Using G power software (Version 2.1.3) (Heinrich-Heine Universität Düsseldorf, Germany) sample size was estimated as 10 in each group; a total of 20 was the sample size with α error taken into consideration, and the confidence interval (CI) of 95%. Participants with OSMF within the age of 18–50 years and subjects without pre-existing CKD, cardiovascular ailment, arthritis, or other chronic inflammatory conditions were included in the study. Patients who underwent treatment for OSMF, patient who underwent chemotherapy, radiotherapy, recent blood transfusion, and iron therapy were excluded. This study was conducted for a period of years in Sri Rajiv Gandhi College of dental sciences and Hospital, Bangalore. Clinical criteria of OSMF were based on the presence of a burning sensation in the mouth, history of chewing areca nut, blanching of the oral mucosa, restricted mouth opening, fibrous bands, and impaired tongue movements. Appropriate biopsy sites with fibrosis/erythema were chosen. The specimen was sent for histopathological examination, and routine hematoxylin and eosin (H and E) staining was done. The OSMF cases were clinically and histopathologically confirmed. Depending on the presence of dysplasia, participants were divided into groups: group I (10 subjects of OSMF without dysplasia) and group II (10 subjects of OSMF with dysplasia). Evaluation of hematocrit and serum LEAP1 was done at baseline. Serum LEAP1 was estimated using an enzyme-linked immunosorbent assay (ELISA).

Statistical analysis

Using the Statistical Package for the Social Sciences (SPSS) software version 11.5 (IBM, New York, USA), statistical analysis was performed. To assess the correlation between serum LEAP1 and OSMF, one-way analysis of variance (ANOVA) was used. To obtain the interconnection between serum LEAP1 and dysplasia, independent t-test was used. By calculating the effect size, clinical significance was established. The study was statistically significant with P < 0.0001 at a 95% confidence interval (CI).

   Results Top

After randomization, 20 subjects participated in this study. There were two groups that were divided into group I (case) and group II (control), with 10 subjects in each group. Serum LEAP1 levels showed remarkable increase in group I (OSMF with dysplasia). The study was statistically significant with a P value of <0.0001. Unpaired t-test and Pearson correlation coefficient (r = 0.8945) were done to find the correlation between dysplasia and serum LEAP1 values. Mean variables were calculated and increased value were obtained in group I compared to group II [Table 1].
Table 1: The calculation of clinical significance in the study

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P < 0.0001 [Table 1] represents the clinical significance calculation in the study with reliable change index statistics. The calculation for the effect size of LEAP1 and dysplasia was done. This indicates a significant increase of serum LEAP1 in OSMF (68.52 ng/ml) subjects compare to OSMF without dysplasia subjects (28.2 ng/ml). The Pearson correlation coefficient value (r) = 0.8945 [Table 1] signifies a strong correlation between serum LEAP1 values in the case and control groups.

   Discussion Top

In ancient medicine (600 B.C), Shushrutha described OSMF as “Vidari” (mouth and throat diseases). OSMF in India was first described by Joshi.[6] In Indians of Africa, Schwartz[6] termed OSMF as “atrophia idiopathic tropica mucosae oris.” Pindborg and Sirsat defined OSMF as a chronic, insidious disease that affects any part of the oral cavity and sometimes the pharynx. Although occasionally preceded by or associated with the formation of the vesicle, it is always associated with juxta-epithelial inflammatory reaction followed by fibroelastic changes of the lamina propria with epithelial atrophy that leads to stiffness of mucosa and causing trismus and inability to eat.[7] With a prevalence rate of 0.20%, OSMF has now been considered the brand in the Indian subcontinent. Different part of india contributes 5% and Kerala being the highest with the rate of 0.4% due to its captivating low prices and the unlimited use. OSMF has a multifactorial etiopathogenesis.[8],[9]

The relation between iron and dysplasia in OSMF has been debated tremendously. Alteration in trace elements level is characteristically seen in dysplasia. Therefore, the utilization of LEAP1 as a diagnostic marker in detecting a few aspects of cancer and precancer. Since few studies are done worldwide to know the importance of LEAP1 in OSMF, knowing the importance of iron in the etiology of OSMF is required.[4],[5],[6],[7] Areca nut is mainly composed of arecoline and guvacoline, cholinergic muscarinic alkaloids, with diverse effects of para sympathomimetic changes.[10] In the pathogenesis of OSMF, arecoline plays an important role in the excessive abnormal production of collagen. The stabilization of the collagen fibers resists degradation by collagenase with the help of flavonoids (catechin and tannins).[11],[12] Decrease in hemoglobin has been associated with the preferred usage of iron in the hydroxylation of proline to hydroxyproline and reduced nutritional support.[4]

However, there is a temporal relation between iron metabolism and stages of OSMF in this study. In the early stages of OSMF, epithelial atrophy is a characteristic feature. Iron-dependent enzymes such as cytochrome oxidase mediate the epithelium maturation. In iron deficiency anemia, cytochrome oxidase levels are deficient. Excessive fibrosis in OSMF will lead to increased consumption of iron for the hydroxylation of lysine and proline. Consequently, there is a reduction in the serum iron level.[4],[11] Crosslinking of the collagen fibers aids in the progression of OSMF to its higher grade. The level of lysyl oxidase contributes to cross-linking of collagen fibers. The progression of the OSMF is indicated by the up-regulation of lysyl oxidase.[4] Hence, it can be concluded that the levels of serum iron deplete with the progression of OSMF from grade I to grade II, marked by dysplastic changes and fibrosis. The progress of OSMF to cancer can be linked to the body's iron levels. Hence, the results in this study obtained are statistically significant. The alteration in the level of serum LEAP1 with the improvement of hemoglobin can be an indicator of the anemic status being improved. Evaluation of serum LEAP1 gives perspicacity into the physiology of iron absorption.[11]

In our study, a majority of the subjects affected with OSMF were males. This data agrees with studies done by Karthik et al.[13] The mean serum LEAP1 levels are higher in the group having OSMF with dysplasia. This proves our hypothesis that LEAP1 plays a role in carcinogenesis. However, the role of LEAP1 in carcinogenesis has been proven in other cancers. This implies that LEAP1 can be used as a biomarker in identifying OSMF having an increased risk for dysplastic changes. The same can be translated to other OPMD like leukoplakia also. There is a strong correlation between LEAP1 values in OSMF with dysplasia and OSMF without dysplasia. This implies a concomitant rise in LEAP1 with the presence of dysplasia, thus further establishing the role of LEAP1 in carcinogenesis. However, more research must be done to prove this at a molecular level. The concept of iron as a promutagen is primitive. Iron, due to its ability as a strong reducing agent, can catalyze carcinogenesis. The Iron levels are regulated by the master regulator- LEAP1. Thus, evaluation of LEAP1 holds the key to predicting the burden of cancer.


This study was performed on a small-sized sample and for the purpose of diagnosis. The stringent methodology and rigorous randomization process is lacking in aspect of OSMF management. Iron and its relation to carcinogenesis have to be evaluated further for a reduction in malignant transformation of OSMF in the most prevalent population.

Future prospects

OSMF is a potentially malignant disorder that has a high malignant transformation and morbidity risk with no permanent treatment. Further studies are required in the field of early diagnosis, management, and patient education for cessation of the habit and prevention of malignant potential of OSMF.

   Conclusion Top

Serum LEAP 1 levels has a vital role in assessing the dysplastic changes of OSMF along with ACD thus laying the foundation stone for future research in the field of iron and carcinogenesis. There is a temporal relation between LEAP1 and dysplasia. With this study, it can be concluded that LEAP1 is vital in carcinogenesis associated with OSMF. It is not untrue to say LEAP1 is the new biomarker in oral cancer. However, further studies must be done with a larger sample size and at the molecular level.

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

Rajiv Gandhi University of Health Sciences, Karnataka, sponsored this study in 2019. The grants were sanctioned vide order no. Adv.Res/BR-19/2019-20, dated 11/04/2019.

Conflicts of interest

There are no conflicts of interest.

   References Top

Nagaraj T, Santosh HN, Tagore S, Sasidharan A. Hepcidin as a marker of iron stores in oral submucous fibrosis. J Med Radiol Pathol Surg 2016;2:23-6.  Back to cited text no. 1
Ganz T. Hepcidin and its role in regulating systemic iron metabolism. Hematol Am Soc Hematol Educ Program 2006;29-35. doi: 10.1182/asheducation-2006.1.29.  Back to cited text no. 2
Ganapathy KS, Gurudath S, Balikai B, Ballal S, Sujatha D. Role of iron deficiency in oral submucous fibrosis: An initiating or accelerating factor. J Indian Acad Oral Med Radiol 2011;23:25-8.  Back to cited text no. 3
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Kali A, Charles MV, Seetharam RS. Hepcidin - A novel biomarker with changing trends. Pharmacogn Rev 2015;9:35-40.  Back to cited text no. 4
Weiss G. Pathogenesis and treatment of anemia of chronic disease. Blood Rev 2002;16:87-96.  Back to cited text no. 5
Joshi SG. Submucous fibrosis of the palate and pillars. Indian J Otolaryngyol 1953;4:1-4.  Back to cited text no. 6
Pindborg JJ, Mehta FS, Gupta PC, Daftary DK. Prevalence of oral submucous fibrosis among 50,915 Indian villagers. Br J Cancer 1968;22:646-54.  Back to cited text no. 7
Khanna SS, Karjodkar FR. Circulating immune complexes and trace elements (Copper, Iron and Selenium) as markers in oral precancer and cancer: A randomised, controlled clinical trial. Head Face Med 2006;2:33. doi: 10.1186/1746-160X-2-33.  Back to cited text no. 8
Santosh HN, Nagaraj T, Sasidharan A. Anemia of chronic disease: A comprehensive review. J Med Radiol Pathol Surg 2015;1:13-6.  Back to cited text no. 9
Papanikolaou G, Tzilianos M, Christakis JI, Bogdanos D, Tsimirika K, MacFarlane J, et al. Hepcidin in iron overload disorders. Blood 2005;105:4103-5.  Back to cited text no. 10
Trivedy C, Warnakulasuriya KA, Hazarey VK, Tavassoli M, Sommer P, Johnson NW. The upregulation of lysyl oxidase in oral submucous fibrosis and squamous cell carcinoma. J Oral Pathol Med 1999;28:246-51.  Back to cited text no. 11
Deepalakshmi R, Sakarde SK, Sur J, Singh AP, Jain S, Mujoo S. Altered taste perception in oral submucous fibrosis: A research. J Indian Acad Oral Med Radiol 2012;24:288-91.  Back to cited text no. 12
Karthik H, Nair P, Gharote HP, Agarwal K, Ramamurthy Bhat G, Kalyanpur Rajaram D. Role of hemoglobin and serum iron in oral submucous fibrosis: A clinical study. Sci World J 2012;2012:254013. doi: 10.1100/2012/254013.  Back to cited text no. 13


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