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 Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 27  |  Issue : 3  |  Page : 349-353

Estimation of serum antioxidant enzymes in histopathological grades of oral leukoplakia, oral submucous fibrosis, and oral cancer: A clinicopathologic study


1 Department of Oral Medicine and Radiology, Institute of Dental Studies and Technologies, Modinagar, India
2 Department of Oral Medicine and Radiology, Lala Lajpat Rai Memorial Medical College, Meerut, Uttar Pradesh, India

Date of Submission05-Nov-2014
Date of Acceptance17-Nov-2015
Date of Web Publication25-Nov-2015

Correspondence Address:
Shalu Rai
Department of Oral Medicine and Radiology, Institute of Dental Studies and Technologies, Kadrabad, Modinagar, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-1363.170444

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   Abstract 

Introduction: This study was undertaken to assess the levels of malondialdehyde (MDA), superoxide distmutase (SOD), beta carotene, and vitamin E in oral submucous fibrosis (OSMF), oral leukoplakia (OL), and oral cancer patients. Though several studies have been done on the levels of antioxidants in these disorders, there are very few available reports in patients with various histological staging of these disorders. Materials and Methods: Blood samples were collected from 40 patients who were divided into three groups: Group I as control with 20 normal individuals, group II with histopathologically confirmed different stages of OSMF and OL, and group III with histopathologically confirmed different stages of oral squamous cell carcinoma (OSCC). Standard methods for estimation of serum values were used and statistical comparisons were performed using SPSS software 16.0 version and the level of significance was calculated. Results: The mean levels of vitamin E, beta carotene, MDA, and SOD among Group I subjects were 9.89 ± 0.75, 112.10 ± 11.97, 2.92 ± 0.36, and 189.45 ± 14.17, respectively. In group II patients with OL having mild dysplasia, serum vitamin E, beta carotene, MDA, and SOD levels were found to be 7.89 ± 0.87, 70.75 ± 2.82, 9.89 ± 0.92, and 119.63 ± 24.97, respectively. In moderate dysplasia, these values were 7.16 ± 0.55, 69.80 ± 5.54, 10.56 ± 0.46, and 115.20 ± 23.03, respectively. Serum vitamin E, beta carotene, MDA, and SOD levels were 7.70 ± 0.57, 70.50 ± 3.54, 9.81 ± 0.11, and 115.00 ± 8.49, respectively, in grade I OSMF and 7.32 ± 0.29, 69.67 ± 3.43, 9.76 ± 0.38, and 113.67 ± 8.32, respectively, in grade II OSMF. In group III patients with well-differentiated squamous cell carcinoma (WDSCC), serum vitamin E, beta carotene, MDA, and SOD levels were 7.01 ± 0.36, 64.32 ± 3.42, 12.98 ± 0.67, and 59.22 ± 4.01, respectively, and in patients with moderately differentiated squamous cell carcinoma (MDSCC), the values were 6.54 ± 0.41, 61.87 ± 2.03, 13.34 ± 0.42, and 58.43 ± 2.26, respectively. The P-value was calculated and the results were highly significant. Conclusion: A decrease in the levels of antioxidants like beta carotene, vitamin E, and SOD and an increase in MDA level in these disorders have been previously reported in the literature.

Keywords: Beta carotene, MDA, oral leukoplakia, oral squamous cell carcinoma, oral submucous fibrosis, SOD, vitamin E


How to cite this article:
Rai S, Sharma A, Ranjan V, Misra D, Panjwani S. Estimation of serum antioxidant enzymes in histopathological grades of oral leukoplakia, oral submucous fibrosis, and oral cancer: A clinicopathologic study. J Indian Acad Oral Med Radiol 2015;27:349-53

How to cite this URL:
Rai S, Sharma A, Ranjan V, Misra D, Panjwani S. Estimation of serum antioxidant enzymes in histopathological grades of oral leukoplakia, oral submucous fibrosis, and oral cancer: A clinicopathologic study. J Indian Acad Oral Med Radiol [serial online] 2015 [cited 2022 Jul 1];27:349-53. Available from: https://www.jiaomr.in/text.asp?2015/27/3/349/170444


   Introduction Top


Oral malignancies are debilitating disorders of the oral cavity. Etiopathogenesis of these disorders is multifactorial. The prognosis of these disorders depends upon early diagnosis and management. [1] Diagnosis is made by biopsy of these disorders, which has certain shortcomings in the form of being invasive and a time-consuming technique. Sometimes due to incorrect site selection or insufficient tissue depth, the results of biopsy are misleading. Therefore, continuous research in this issue has raised interests of the scientific community to find an accurate, yet simpler, less invasive, less time consuming, easily interpretable, and economical technique. Biochemical investigations are one such alternative and have the above-mentioned advantages. They may also be used to monitor the response of therapy for these disorders. These investigations reflect tissue changes at a cellular level and thus aid in early diagnosis of these conditions. [2]

Oxidative damage as a result of reactive oxygen species (ROS) is responsible for these disorders. The extent of oxidative damage caused by ROS directly depends on body antioxidant defense mechanism. [3] Literature reports reveal reduction in the levels of nonenzymatic antioxidants like vitamin E and beta carotene and the enzymatic antioxidant superoxide dismutase (SOD), and an increase in antioxidants like malondialdehyde (MDA) in these disorders. [4],[5] To the best of our knowledge, in spite of the high prevalence of these disorders, very few studies have been done to assess the oxidative damage caused by ROS and the role of antioxidants and their relation with histopathologic grading of these disorders. Moreover, no study has been done to assess the levels of MDA, SOD, vitamin E, and beta carotene together in different histopathologic grades of oral leukoplakia (OL), oral submucous fibrosis (OSMF), and oral squamous cell carcinoma (OSCC). There are very few reports in which these parameters have been together analyzed in patients with various histological staging of these disorders. Keeping this in mind, the present study was undertaken to estimate the serum levels of these antioxidants in these disorders and to assess the relation with histopathological grading.


   Materials and Methods Top


Study design

A total of 40 patients that consisted of 20 patients with OSMF and OL (n = 20) and 20 patients with OSCC (n = 20) who reported to the Department of Oral Medicine and Radiology were included in the study. Clinical diagnosis of OL, OSMF, and OSCC was made on the basis of history and characteristic clinical features. Only those patients who were not suffering from any systemic disease and/or had not received any form of treatment for OL, OSMF, and OSCC prior to the study were subjected to biopsy for histopathological examination. The clinico-histopathological grading of OL was in accordance with the modified classification and staging system (OLEP) given by van der Wall (2000). [6] For OSMF, grading was done according to Khanna et al. (1995). [7] For OSCC, grading was done according to Broder's classification. [8] Twenty individuals without any tissue abusive habit and without any clinically obvious oral lesion or systemic disease were selected as controls. The subjects were grouped as:

Group I: Control group, 20 healthy individuals

Group II: 20 patients with OSMF and OL

Group III: 20 patients with OSCC

Method of blood collection

After obtaining permission from the institutional ethical committee, informed consent was obtained from the patients. The blood samples were collected with a tourniquet applied 1½-2 inch above the antecubital fossa. The area was rendered aseptic with 70% alcohol, and using 24 gauge needles, 6 ml of blood was drawn. The collected blood was centrifuged and the plasma separated was analyzed for vitamin E, beta carotene, MDA, and SOD.

Beta carotene was estimated by the method given by Levinson (1969), in which a mixture of serum, water, ethanol, and petroleum ether was centrifuged to obtain an absorbance which was determined at 440 nm and the concentration of beta carotene was calculated by reference to a standard curve. [9] Vitamin E was estimated by the calorimetric method given by Nair and Magar (1955) in which the color density of the obtained solution was read in a Beckman model DU quartz spectrophotometer at 725 mm with corex absorption cells and distilled water as a reference blank. [10] MDA was calculated by the method given by Kei Satoh in 1978 [11] in which trichloroacetic acid was added to the mixture which was centrifuged at 3300 rpm and measured at 530 nm. SOD was estimated by the method given by Marklund and Marklund (1974) in which the content of test tube was mixed and absorbance was measured at 420 nm. [11]

Statistical analysis

The software used for the statistical analysis was Statistical Package for Social Sciences (SPSS) version 16.0 and "IBM USA" Epi-info version 3.0. Chi-square test was used to find the level of significance (P-value), where P < 0.001 was considered to be highly significant.


   Results Top


In group I, the mean vitamin E level was 9.89 ± 0.75, beta carotene level was 112.10 ± 11.97, MDA level was 2.92 ± 0.36, and SOD level was 189.45 ± 14.17. In group II patients with OL having mild dysplasia, serum vitamin E was 7.89 ± 0.87, beta carotene was 70.75 ± 2.82, MDA was 9.89 ± 0.92, and SOD level was found to be 119.63 ± 24.97. In patients with moderate dysplasia, the mean vitamin E level was 7.16 ± 0.55, beta carotene level was 69.80 ± 5.54, MDA level was 10.56 ± 0.46, and SOD level was 115.20 ± 23.03 [Table 1] and Graph 1]. In grade I OSMF, the mean serum vitamin E was 7.70 ± 0.57, beta carotene was 70.50 ± 3.54, MDA level was 9.81 ± 0.11, and SOD level was 115.00 ± 8.49. In grade II OSMF, the mean vitamin E level was 7.32 ± 0.29, beta carotene was 69.67 ± 3.43, MDA level was 9.76 ± 0.38, and SOD was 113.67 ± 8.32 [Table 2] and Graph 2]. In group III patients with well-differentiated squamous cell carcinoma (WDSCC), serum vitamin E was 7.01 ± 0.36, beta carotene was 64.32 ± 3.42, MDA was 12.98 ± 0.67, and SOD level was 59.22 ± 4.01. In group III patients with moderately differentiated squamous cell carcinoma (MDSCC), serum vitamin E level was 6.54 ± 0.41, beta carotene was 61.87 ± 2.03, MDA level was 13.34 ± 0.42, and SOD level was 58.43 ± 2.26 [Table 3] and Graph 3]. The level of significance was determined and the P-value was calculated and was found to be highly significant (P < 0.001) for all the parameters except for beta carotene which was found to be nonsignificant (P < 0.687) in cases of mild and moderate dysplasia and again nonsignificant (P < 0.888) in cases of grades of OSMF. Beta carotene levels were found to be significant (P < 0.021) in cases of different grades of OSCC.
Table 1: Comparison of values in different types of dysplasia (OL) (n = 15) divided into mild and moderate dysplasias and the control group

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Table 2: Comparison of values in different grades of OSMF (n = 5) divided into grade I and grade II and the control group

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Table 3: Comparison of values in different grades of OSCC (n = 20) divided into WDSCC and MDSCC and the control group

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   Discussion Top


This study was carried out to assess the levels of serum beta carotene, vitamin E, SOD, and MDA in patients with histopathologically different grades of OL, OSMF, and OSCC. In our study, the mean vitamin E and beta carotene levels were decreased in group II patients with mild and moderate dysplasia and group III patients with WDSCC and MDSCC when compared to group I patients, and the results were statistically significant. Similar results were obtained by Manoharan et al., [12] Kumar et al., [13] and Lawal et al. [1] in oral cancer patients when compared to healthy patients and the reports suggested that host tumor cells in oral cancer patients seize essential nutrients from the circulation to meet the damage caused by the growing tumor. They also described normal role of vitamin E as a free radical scavenger in maintaining membrane integrity and inhibiting cancer cell growth and differentiation.

A study was conducted in 1997 by Barth et al. to assess different treatment modalities of OL where the authors administered beta carotene and vitamin E to 24 patients with histopathologically different grades of leukoplakia. The outcome of their study showed that 97.5% of patients with different grades of dysplasia responded to their treatment and they concluded that any specific treatment modality is independent of the grades of dysplasia. [14] In our study, beta carotene level was highest in group I and showed a reduction in different grades of dysplasia, OSMF, and OSCC, with the lowest value obtained as the severity of disease increased. However, the results were statistically nonsignificant. Our study was contrary to a similar study conducted by Aggarwal et al. in 2011, in which 45 patients with histopathologically different grades of OSMF were studied and the authors found that reduction in beta carotene levels was maximum in grade III in comparison to grade II and grade I patients and concluded that beta carotene plays an important role in the pathogenesis of OSMF, and their results were statistically significant. [15] This could be attributed to the fact that the sample size was small in our study.

The mean serum SOD levels were highest in group I patients and gradually decreased in groups II and III, and the results were found to be statistically significant (P < 0.001). Similar results were obtained by Gurudath et al. [5] in 2012. They described SOD as a decisive antioxidant enzyme in aerobic cells that is responsible for the elimination of superoxide radicals. Gurudath et al., in their study of oral cancer group, showed a statistically significant (P < 0.001) decrease in the level of SOD when compared to the control group and also found it to be at the lowest level among all groups.

The mean MDA level gradually increased when healthy individuals, potentially malignant and OSCC patients were compared, which was statistically highly significant (P < 0.001). It was highest in group III patients and lowest in group I patients. Hegde et al. [16] compared vitamin C and MDA levels in oral precancerous patients and healthy controls, and found increased MDA levels which was statistically significant (P = 0.05). Khanna et al. [17] found that serum MDA levels were highest in oral cancer patients compared to controls (P < 0.001), whereas the levels of MDA in leukoplakia patients were similar to those of controls (P > 0.05). In a similar study, Chole et al. [4] found that serum MDA levels were increased in OSSC as compared to healthy controls due to increased oxidative stress of the body.

Gupta et al. showed in their study statistically significant increases in plasma MDA in all grades of OSMF (P < 0.001) and decreases in plasma vitamin E level and beta carotene level in various grades of OSMF, whereas the SOD activity did not show any significant change in any stage of disease. But our results showed statistically significant difference between grade I and grade II OSMF, which was more in grade I OSMF and less in grade II OSMF for vitamin E, beta carotene, and SOD activity, whereas MDA level was found to be less in grade I and more in grade II OSMF. [18]

In 2007, Metkari et al. [19] conducted a study to correlate serum MDA, SOD, vitamin A levels in relation to clinical and histopathological grading of OSMF. They found a positive correlation with clinical grades, but no significant correlation with histopathological grading. They also concluded that histopathological examination shows localized tissue changes, whereas OSMF may affect various parts of the oral cavity to a different extent. On the other hand, changes in blood are seen consistently even though they are secondary to the tissue changes taking place anywhere in the body. They further emphasized the need of histopathological grading criteria that include epithelial changes along with connective tissue changes, which would better correlate biochemical parameters to the histopathological grading.


   Conclusion Top


Thus, this study gives the benefits of enzymatic and nonenzymatic antioxidants as valid biomarkers for patients who are at risk of developing cancer. However, a study with larger sample size would yield a better understanding of the role of antioxidants and their action on premalignant and malignant lesions of the oral cavity. Also, it can be concluded that the deranged values of these variables can be used as a predictor of development of OSMF and OL. Furthermore, a diet rich in antioxidants or supplements of antioxidants may play an important role in managing these patients with oxidative stress and preventing the progression of premalignant disorders into malignancy.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3]


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