European Journal of Cardiovascular Medicine

Oral Submucous Fibrosis, (OSMF) with a definite etiological link to use of arecanut in betel quid or smokeless tobacco preparations, has a high malignant transformation rate of 7.6%. mediated by various factors including Reactive Oxygen Species (ROS) such as superoxide anion, hydroxyl radical and hydrogen peroxide generated from the constituents of betel quid or smokeless tobacco preparations.1 Generally, these ROS are countered by the natural antioxidants including non- enzymatic antioxidants such as uric acid, glutathione,vitaminE (α–tocopherol), vitamin A (retinol), vitamin C (ascorbic acid) carotenoids, thioredoxin, lipoic acid, and ubiquinone and the enzymatic antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT).2 Uric acid is a predominant antioxidant that removes various ROS for example perooxynitrite, hydroxyl radical, singlet oxygen and lipid peroxides. It reduces the potential for peroxynitrite‑induced oxidative damage by promoting vasodilatation through interaction with peroxynitrite to form a stable nitric oxide donor, thus protecting against oxidative stresses. It also acts as a substrate for glutathione transferase (GST) in the detoxification of peroxides and other ROS.3 increased production of ROS and/or reduced elimination of natural antioxidants can create an imbalance leading to oxidative stress (OS). Assessment of antioxidant levels in saliva can be used to establish the extent of cellular injury, the burden of oxidative stress and in the case of oral potentially malignant disorders like OSMF; it can additionally serve to recognize the risk of malignant transformation. Although antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione transferase (GSH) have been studied at length in OSMF in tissue, serum and saliva, there is hardly any literature available regarding levels of the antioxidants, uric acid and catalase in saliva of OSMF subjects. Therefore, in view of this background, the present study was planned to evaluate the levels of antioxidants catalase and uric acid in saliva of patients with OSMF.

AIMS & OBJECTIVE

To evaluate and compare the levels of catalase and uric acid in saliva of patients with Pre-malignant condition (Oral sub mucous fibrosis)

This comparative case control study was conducted in the Department of Dentistry associated with the department of pathology, Autonomous State Medical College, Kushinagar, Uttar Pradesh, on 60 subjects between the age groups of 20-50 years. The present study comprised of 60 subjects (Group 1 (Study group) 30 OSMF cases and Group 2 consist 30 controls) who were selected on the basis of set inclusion such as age group considered 20 to 50 years diagnosed with OSMF followed by Pindborg’s classification of OSMF 1989 and those subjects excluded who’s suffering from systematic diseases, taken treatment for osmf and pregnant women. Group 1, further subdivided into three groups as follows: Group1A: 10 Stage I OSMF study subjects (5 males and 5 females). Group1B: 10 Stage II OSMF study subjects (5males and 5 females).  Group1C:10 Stage III OSMF study subjects (5 males and 5 females). Following this subjects were advised to undergo oral prophylaxis for the improvement of periodontal status. After 1 week, subjects were evaluated for the periodontal conditions which were evidenced by decrease in bleeding on probing and probing depth before the collection of the saliva.

METHOD OF COLLECTION OF SALIVARY SAMPLE:

Whole unstimulated saliva was collected using Navazesh spitting method. Saliva sampling was carried early morning to avoid diurnal variation. The subjects were asked not to have any oral stimulus and requested not to eat, drink, or perform oral hygiene methods or chew or smoke 60 min before and during the entire procedure. Subjects were then be made to sit comfortably in dental chair and explained regarding the details of procedure and asked to swallow all the saliva in the mouth prior to the test, and not to swallow anymore during the test. Saliva was allowed to pool in the bottom of the mouth and 5 ml of unstimulated saliva was collected in sterile Falcon tubes.4

METHOD FOR SALIVA PREPARATION (CENTRIFUGATION AND STORAGE)- Collected saliva samples was appropriately labeled, kept in a transport (ice box) and immediately transported to laboratory where it was centrifuged (1000 g, 10 min) at 4°C to remove squamous cells and cellular debris. The resulting supernatant was isolated and immediately frozen at -80°C until the sample collection period was completed. The salivary samples were then brought to room temperature before the analysis of catalase and uric acid.

STATISTICAL ANALYSIS

Data was entered in Microsoft excel and analysed using SPSS (Statistical Package for Social Science, Ver.10.0.5) package. 

The results were averaged (mean + standard deviation) for continuous data and number and percentage for dichotomous data are presented in Tables and Figures. Age-wise distribution of subjects in study group (OSMF) group and control group (Table 1) - All the 30 OSF subjects and 30 controls were in the age range of 21 to 50 years with mean age of 38.90±9.56 years with minimum and maximum age of 21 years and 50 years respectively. The age groups were stratified in both the study and control groups as 21-30 years, 31-40 years and 41-50 years. While 8 subjects (26.7%) were in age group of 21-30 years and 9 subjects (30%) were in the age range of 31-40 years, the highest number of subjects were in theage group of 41-50 years (n=13, 43.3%) for both study group and control group (n=13, 43.3%). No statistical significance was noted between the mean ages of study group and control group (p value > 0.05)

Table1-Showing age-wise (years) distribution of subjects in study group (OSF) group and control group

p >0.05; Not significant

Age wise and gender wise distribution of subjects in study group (Table 2): However, among the 30 subjects in study group-out of 8 subjects: 3 were females and 5 were males in the age group 21-30 years, out of 9 subjects: 4 were females and 5 were males in the age group 31-40 years and out of 13 subjects: 8 were females and 5 were males in the age group 41-50 years.

 Table 2-Showing age-wise (years) and gender-wise distribution of subjects in study group (OSMF)

Comparison of mean salivary CAT and mean salivary UA in study group (OSF) and control groups (Table 3)

The mean catalase values in study and control groups were 1.42±2.42 and 1.48±1.92 mg/dl respectively. The minimum and maximum values were -0.3ng/dl and 8.3 ng/dlin study group and -0.1mg/dl and 8.8mg/dl in control group. The mean uric acid values in study and control groups were 2.85±2.31 and 4.18±2.19 mg/dl respectively. The minimum and maximum values were 0.01 mg/dl and 8.36 mg/dl in study group and 0.02 mg/dl and 8.48 mg/dl in control group. No statistically significant difference ((p value- >0.05) was obtained regarding the comparison of catalase between study and control group although the mean catalase levels in the study group was lower than the control group. However, there was a statistically significant difference (p value- 0.025*) for uric acid noted between the study and control group with uric acid being lower in study group than control group.

Table 3 - Showing comparison of mean salivary CAT (mg/dl) and mean salivary UA (mg/dl) in study group (OSF) and control group -

    p >0.05; Not significant

    p<0.05; Significan

Comparison of mean salivary catalase, mean salivary uric acid, mean frequency and mean duration between males and females in study group (OSF) and control group (Table 4) Among study(OSF)group-The mean catalase levels in female sand males were 1.75±2.58 ng/dl and 1.09±2.29 ng/dl respectively, with no statistical significant difference between the gender (p-0.465). The minimum and maximum catalase values were -0.3 ng/dl and 8.5 ng/dl for females and -0.3 ng/dl and 7.4 ng/dl for males. In females and males, the mean uric acid levels were 2.06±1.40 mg/dl and 2.64±1.88 mg/dl respectively and showed no statistical significant difference between the gender (p- 0.347). The minimum and maximum uric acid values were 0.01 mg/dl and 8.36 ng/dl for females and 1.85 mg/dl and 8.15 mg/dl for males. Among control group- The mean catalase levels in females and males were 1.65±1.91ng/dl and 1.3±2.25 ng/dl respectively, with a statistical significant difference between the gender (p-0.001*). The minimum and maximum catalase values were -0.1 ng/dl and 8.8 ng/dl for females and 0 ng/dl and 5.7 ng/dl for males. In females and males, the mean uric acid levels were 3.35±2.30 mg/dl and 2.35±1.94 mg/dl respectively and showed statistical significant difference between the gender (p- 0.001*). The minimum and maximum uric acid values were 0.02 mg/dl and 8.49 ng/dl for females and 0.02 mg/dl and 7.34 mg/dl for males.

Table 4: Showing comparison of mean salivary catalase (mg/dl) and mean salivary uric acid (mg/dl) between males and females in study group (OSF) and control group -

p >0.05; Not significant

p<0.05; Significant

Employing saliva for such an assessment is highly advantageous in that saliva is an excellent reflector of physiological or diseased state of human body, is rich in antioxidants and provides for economical and non-invasive assessment of the same. Moreover, there is a dearth of literature regarding salivary levels of cyto-protective enzyme catalase and non-enzymatic antioxidant, uric acid in OSMF subjects. In the present study, the overall mean age of both study and control subjects were 38.90±9.56  years ranging from 18 to 50 years, with maximum number of subjects between 41-50 years and least number of subjects between 21-30 years. Though, this finding was similar to Aliet al (2013) who reported a mean age of 38.8 years with age ranging from 22 to 61 years in OSF subjects and to Nigam et al (2014) who found majority of OSF cases between 36-40 years  

Saliva constitutes the first line of defense responsible for protecting the oral cavity from oxidative stress by external agents including the ingredients of betel quid and tobacco by way of its antioxidant activity contributed by uric acid, albumin, as part ate, thioredoxin, lipoic acid, ubiquinone, vitamin E, vitamin C, vitamin A, carotenoids and enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx).

Catalase acts as a secondary free radical scavenger by eliminating H2O2 formed as a by-product of dismutation reaction of SOD in removing O2− radicals. H2O2 is a potent reactive radical, also generated in considerable amounts within the cell. With the increasing levels of H2O2, the CAT levels get depleted. Oxidative stress can be imposed on cells due to increase in oxidant generation, a decrease in antioxidant protection, and failure in the repair of oxidative damage.5

In the present study, the mean salivary catalase levels assessed in the study group (1.42±2.42 ng/dl) were lower than in controls (1.48±1.92 ng/dl), although not statistically significant (p = 0.925). This finding demonstrates the depletion of catalase and a possible presence of oxidative stress in OSMF. Tarboush et al 2018 demonstrated a statistically significant reduction in salivary catalase levels in Khat (an evergreen shrub found in southern Arabia and eastern Africa practised as a chewing habit) chewers and suggested that impaired CAT levels can lead to build-up of H2O2 paving the way for pathological conditions such as cancer.6 Begum et al 2018 showed decreased levels of salivary catalase and other antioxidants in smokeless tobacco users compared to controls attributing it to increased production of reactive oxygen species (ROS).7

Statistically non significant higher catalase levels were noted in females in this study. This is in agreement with the studies by Ide et al 2002 and Tsuber et al 2014 who showednodifferenceincatalaseactivitylevelsbetweenmalesandfemales suggesting that catalase activity and thus hydrogen peroxide degradation may not be affected by gender.8,9 Also the studies by Gomez-Perez et al 2011 and Barp et al 2002 conducted were in agreement with the present study.10

Male OSF subjects showed higher mean salivary uricacid levels compared to females in the current study. Nosratzehi et al 2017 and Almadori et al 2007 found significantly higher salivary uric acid in males as it is influenced by serum levels and hyperuricemia is strongly associated with male sex.11,12 WG Barr 1990 reported that many additional factors, including exercise, diet, drugs, and state of hydration, may result in transient fluctuations of uric acid levels.13Among controls a statistically significant increase in the mean salivary catalase anduric acid levels in females were noted than males. Kander et al 2016 suggested an apparent association between gender and oxidative stress. Higher catalase levels in females could beat tribute to the fact that women seem to be less susceptible to oxidative stress.14 Various literatures have suggested a linear relationship between serum and salivary uric acid. Okada et al 1973 obtained a higher level of serum uric acid in females than males which is in accordance with our study. This might be due to the fact that the uric acid levels are influenced by drugs, serum creatinine levels, obesity and menopause. Also the diet with high purine content will cause variations in the uric acid levels.

The mean salivary uric acid levels were higher in the age group between 41-50 years in both OSF subjects and controls. De Oliveira and Burini 2012 reviewed on causes and consequences of high plasma uric acid levels and stated that uric acid levels physiologically increase with the age.15

The mean salivary catalase levels were higher in the age group between 21-30 years and 31-40 years among OSMF subjects and control group respectively. Mean salivary catalase levels in the study (OSMF) and control subjects had statistically insignificant varied distribution in different age groups with least levels in younger age groups (21- 30 years and 31-40 years among OSMF subjects and control group respectively). Although many studies have depicted an age- wise decrease in mean serum catalase levels, Goth et al 1989 showed low catalase activity with aging process.16

Gideon Bahar et al 2006 reported a decreased concentration of salivary uric acid levels in premalignancy and OSCC.17Joanna et al 2011 reported that the concentration of salivary uric acid significantly decreases in oral squamous cell carcinoma. Thus, previous literatures have established a decrease in catalase and uric acid levels in serum and saliva of subjects with oral squamous cell carcinoma. Early detection of these antioxidants and its depletion in the oral potentially malignant condition like OSMF would be helpful to know the extent of tissue injury and the ability for development of oral carcinogenesis.12

The strengths of the current study were that besides evaluating the salivary levels of catalase and uric acid in OSMF subjects, comparison of their levels among different stages of OSMF was done, there by quantitatively determining their level which can help us to recognize the extent of oxidative stress and tissue injury in each stage of OSMF as the disease advances. Although other salivary antioxidants in OSMF such as SOD, ascorbate, GPx and so on, have been studied extensively, a glimpse into the role of antioxidants, catalase and uric acid can provide further inputs to the impact of oxidative stress in OSMF. 

After completion of the study we were concluded, the results of our study suggest that a decrease in mean salivary CAT activity and UA levels in OSMF could be related to increased production of reactive oxygen species by the habit of chewing areca nut and tobacco preparations leading to oxidative stress depleting more CAT and UA. Elevated consumption of these antioxidants in advanced stages of OSMF reveals the higher free radical damage associated with progress in the severity of the disease and a possible indication of the extent of cell injury with a potential for malignant transformation.

However, studies with larger sample size by recruiting a greater number of OSMF subjects in each stage is required to document other clinical findings present in different stages of OSMF and to conclusively prove the role of salivary CAT and UA as predictors of malignant transformation in OSMF.

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