Introduction

Periodontal diseases are pathogen-induced inflammatory conditions of multifactorial nature. They are characterized by constituting the most widespread form of bone pathology in humans, destroying the tooth-supporting structures and a modifying factor of the systemic health of individuals [1, 2]. Periodontitis is categorized as mild to severe form depending on the rate of progression, clinical signs, symptoms of inflammation, and extent of periodontal breakdown [3]. Tobacco smoking plays a significant etiological role in the development of periodontal disease. The periodontal breakdown is more pronounced among current smokers than former smokers and gingival bleeding has been constantly reported to occur less in smokers due to nicotine-induced vasoconstriction [4]. Also, greater pocket depth measurements are found in smokers due to increased alveolar bone loss [5].

During periodontitis, cigarette smoking may differentially influence neutrophil function, generally preventing the abolition of periodontal pathogens but in heavy smokers. Also, it stimulates the release of oxidative stress-mediated tissue damage reactive and oxygen species [6, 7]. Tobacco smoke contains a rich source of oxidants. It has been a dilemma that, the increased production of reactive oxygen species associated with smoking may beat the capacity of the oxidant defense system, which leads to oxidative damage [8]. The potential damage that the free radicals can cause is normally minimized by combining biological antioxidant systems including non-enzymatic and enzymatic reactions [9-12].

Many reasearch correlate periodontal diseases with an imbalance between antioxidants and oxidants, thus suggesting that this imbalance can be because of an a defect in antioxidant activity and/or increase in free radical production [13, 14]. Glutathione reductase (GR) is one of the antioxidants in reductive processes that is essential for the regulation of enzymes, synthesis and degradation of proteins, and free radicals produced even in normal metabolism and protection of the cells against ROS [15, 16].

As far as we know, among various antioxidant enzymes, salivary GR is an important, but least studied enzyme. So far, there is little scientific documentation about fluctuations in smokers and GR activity in normal individuals. Furthermore, none of the previous studies have identified if the salivary levels of GR are linked with the degree of periodontal breakdown in terms of extent/severity, or neither their association with different periodontal conditions. Very few reports are available regarding the range of activity for glutathione reductase. Hence, the present study was carried out to evaluate & compare the salivary GR levels following NSPT in smokers and non-smokers with and without periodontitis.

Materials and Methods

Study design, study population, and inclusion/exclusion criteria

This cross-sectional study was carried out in the Department of Periodontology, School of Dental Sciences, Krishna Institute of Medical Sciences, Karad. Ethical committee approval (0328/2018-2019) from Krishna Institute of Medical Sciences Deemed University was obtained. The purpose and nature of the study were explained to the subjects and written consent was obtained before commencing the study. A total of 76 patients reporting to the Department of Periodontology, SDS, KIMSDU, and Karad were included in the study. According to the power analysis, the minimum number of study subjects required in each group was 19. This minimum number is determined on basis of 95% confidence and 80% power. Based on the status of smoking and periodontal parameters patients were categorized into 4 groups. 1] Group I: smokers with periodontitis, 2] Group II: smokers without periodontitis, 3] Group III: non-smokers’ with periodontitis. 4] Group IV: non-smokers without periodontitis. Subjects were considered non-smokers if they had never smoked, or had stopped smoking for more than one year before the date of examination. Chronic generalized gingivitis subjects are considered a non-periodontitis group. All clinical periodontal parameters were based on data derived from the full-mouth examination and performed by a single trained and calibrated observer. Clinical periodontal status was determined by measuring both the probing depth (PD) and clinical attachment level (CAL) on six sites of teeth (midbuccally, midlingually, and proximally both buccally and lingually) using a calibrated periodontal probe (HU-FRIEDY UNC 15). Subsequently, these measurements were used to calculate the extent and severity according to the new classification of periodontal diseases which is given by the American Academy of Periodontology and European Federation of Periodontology 2017. All the clinical parameters including the Plaque index (PI) and Gingival index (GI) were recorded at the baseline, 14th day, and after 1 month.

Patients with other systemic diseases or conditions (e.g. uncontrolled Diabetes Mellitus) that are known as risk factors for periodontitis and also patients with connective tissue diseases (such as systemic lupus erythematosus, Sjögren's syndrome) were excluded from the study. Subjects with a history of periodontal therapy within the past 6 months, pregnant and lactating women were also excluded from study groups.

Saliva collection method

The whole saliva was collected using the Spitting method by Navazesh et al. (1993) [17]. At Baseline all samples were collected within 48 hours of clinical measurement in the morning following an overnight fast. Before the non-surgical periodontal treatment, 1ml of unstimulated whole saliva was collected in a centrifuge tube before breakfast intake and any dental hygiene procedure. After collection samples were centrifuged for 10 min at 2500-3000 rpm to remove cell debris, and supernatants were collected in the Eppendorf tubes and stored at -20ºC until processed. On the 14th day and at the 1st month, the same procedure of saliva sample collection was carried out for evaluation of salivary glutathione reductase level measurement and clinical periodontal parameters.

Non-surgical periodontal treatment

At the baseline, after the collection of unstimulated whole saliva, patients were treated with complete full-mouth ultrasonic scaling and root planning. After 14th day and 1 month, only saliva collection and periodontal parameter evaluation were repeated.

Glutathione reductase analysis

The GR level was estimated in saliva by using a glutathione reductase assay commercial kit (abcam, ab83461) according to the manufacturer’s protocol and was analyzed by spectrophotometry at the baseline, at 14th day and 1 month in the Department of Biochemistry, KIMSDU, Karad.

Statistical methods and data management

All the collected data were statistically analyzed using SPSS software version 20. Results were reported as mean ± standard deviation, or number (percentage). The continuous variables between the inter groups and between subgroups were compared using the ordinary one-way ANOVA test and repeated measures ANOVA respectively. The P value <0.05 was considered statistically significant. The correlation between periodontal parameters and GR disease activity was analyzed by Pearson Correlation Coefficient. The correlation coefficient (r) was also said to be significant if p was <0.05 in addition to this it was categorized as ‘Poor’ (r-<0.3), ‘Small’ (r=0.3-<0.5), ‘Moderate’ (r=0.5-<0.7) and ‘High’ (r=≥0.7).

Results and Discussion

Demographic, clinical, and biochemical characteristics of the study population

Seventy-six patients were included in the present study. The mean and standard deviation of the age range of smokers with periodontitis, smokers without periodontitis, non-smokers with periodontitis, and non-smokers without periodontitis were 40.2±8.1, 31.7±8.1, 42.5±7.5 and 25.1±6.5 respectively. Out of 76 patients, 55 were male patients and 21 were female patients. A total of 19 current smokers, 19 former smokers, and 38 never-smokers were included in this study.

Comparison of clinical parameters pre and post-NSPT at baseline, on the 14th day, and at 1 month among study groups

In non-smokers without periodontitis, the mean and standard deviation of PI and GI were 0.89±0.30 and 1.22±0.32 respectively at baseline. Their values were significantly less than other groups (Table 1). On the 14th day post-NSPT and 1 month follow up PI ( p‹0.001) was significantly less in non-smokers without periodontitis as compared to other groups, but GI values were not significantly different among all the study groups (p‹0.065) (Table 1).

Table 1. Comparison of Mean PI and GI score pre and post-NSPT at Baseline, 14^th Day, and at 1 Month

Among all the study groups non smokers without periodontitis group had the least mean PPD values while smokers with periodontitis had the highest mean PPD values. Intragroup revealed that there was a significant reduction in PPD on 14th day and at 1 month as compared to baseline in each study group (Table 2).

Intergroup comparison demonstrated that after the 14th day and 1-month post NSPT significant PPD reduction was observed in smokers with periodontitis group as compared to other groups ( p‹0.001) (Table 2).

Table 2. Inter and Intra Group Comparison of PPD at Baseline, on 14th Day, and at 1 Month

Among all the study groups non-smokers without periodontitis, had the least mean CAL values while smokers with periodontitis had the highest mean CAL values. Intra group revealed that there was a significant gain in CAL on the 14th day and at 1 month as compared to baseline in each study group (Table 3).

Intergroup comparison demonstrated that after the 14th day and 1-month post NSPT significant CAL gain was observed in smokers with periodontitis group as compared to other groups ( p‹0.001) (Table 3).

There was no significant correlation of Glutathione reductase (GR) activity with PI and GI score at baseline, on the 14th day, and at 1 month (Table 4).

Table 3. Inter and Intra Group Comparison of CAL at Baseline, at 14th Day and 1 Month

Table 4. Correlation between GI, PI, and GR activities at Baseline, 14th Day, and 1 Month

Comparison of glutathione reductase (GR) activity and mean GR pre and post-NSPT among study group subjects.

The regression model was developed to determine the concentration of GR on basis of OD value. It was determined by applying the cubic regression approach as follows.

Using this equation the GR (concentration) activities for four study groups at baseline, at 14 days, and at 1 month was computed.

These GR activities were calculated and are depicted in Table 5. However, on the intragroup comparison, there was a significant increase in GR activities from baseline to 1 month in each study group after non-surgical periodontal treatment (NSPT) (p<0.001). On intergroup comparison, there was no significant difference in GR activities among the study groups. Maximum improvement in GR concentration was found in non-smokers without periodontitis group followed by smoking without periodontitis group and non-smokers with periodontitis group at 14thday and 1 month follow up post-NSPT. Among the entire study group, the least improvement in GR activity was observed in smokers with the periodontitis group (Table 5). The standard concentration of GR and its respective OD is graphically presented in Figure 1.

Table 5. Comparison of Mean and Standard Deviation of GR activities at Baseline, on 14th Day, and at 1 Month pre and post NSPT