1. INTRODUCTION:

Periodontal disease is an immune-inflammatory lesion of the oral cavity^1,2. Although traditionally regarded as a disease confined to the oral cavity, inflammatory molecules associated with periodontitis is now suggested to affect systemic health. Periodontitis is the sixth most prevalent complication of diabetes mellitus (DM), with a two-way relationship between the two chronic diseases³which induce the production of various inflammatory mediators. Current evidence suggests that the prevalence, extent, and severity of periodontal disease are increased in DM type II patients².

Reactive oxygen species (ROS) is a term commonly used to describe chemically reactive molecules containing oxygen, which includes free radicals as well as other reactive species that can cause formation of free radicals. ROS production can cause extensive tissue damage and cell death^4,5. Antioxidants (AO) are substances that occur at low concentrations compared to those of oxidizable substrates but help to markedly delay or inhibit substrate oxidation⁶. Under normal physiological conditions, there is a balance between ROS activity and defense capacity of the AO system. An imbalance in favor of ROS results in oxidative stress. Hyperglycemia has been identified as a factor responsible for activating oxidative stress^7,8.However, the role of periodontitis in increasing oxidative stress is not fully understood.

Human body maintains a complex system of AOs, including glutathione (GSH) and enzymatic AOs, such as superoxide dismutase (SOD) and catalase (CAT), to protect against the actions of ROS^9,10. There is a need to understand the role of AOs in the development of periodontal disease and to determine whether they can serve as possible markers of periodontal disease. This study evaluated AO (GSH, SOD, CAT, and TAOC) levels in chronic periodontitis patients with or without DM type II.

2. MATERIALS AND METHODS:

2.1. Subjects:

As per the prepared case history format, case history was recorded for all subjects who were part of the study.

This was a randomized clinical trial involving 300 subjects, 30–60 years of age. The participants were selected from the outpatient dental department of AB Shetty Dental College during the period from February 2019 to October 2019. The power of the study was calculated as 80% and the error was set at 5%.

Subjects who were overweight and obese, with a BMI >25, were not included in the study¹¹.Of the 300 eligible subjects, 100 with chronic periodontitis who did not have any systemic diseases were included in group I, 100 with chronic periodontitis and DM type II who were not suffering from any other systemic diseases were included in group II, and 100 systemically healthy individuals without any periodontal disease were included in group III. The diabetic patients in group II were on oral medication (metformin derivatives) and appropriate diet control measures for a period of at least 1 year but not for more than 5 years. Group I and II patients had chronic generalized severe periodontitis, as assessed by a. single trained investigator.

Subjects were excluded if they had taken antibiotics and vitamins or mineral supplements any time in the past 4 weeks or had undergone periodontal treatment procedures during the last 6 months. Tobacco users, alcoholics, and pregnant or lactating women were also excluded from the study. Only subjects who had an HbA1c score of less than 7 were accepted in the study.

Patients were referred to a physician to determine their systemic condition, and later, they were allotted to the three study groups by the single trained investigator as per the inclusion criteria. Patients not fulfilling the various criteria were excluded from the study. Peripheral blood (5ml) was collected from the antecubital vein from each study subject and then analyzed for various antioxidants, as described below.

2.2. Biochemical protocols:

Approximately 5ml of venous blood collected from each study subject, 2.5ml in EDTA-coated vacutainer tubes, and 2.5ml in uncoated tubes. Blood in the EDTA-coated tubes were used to assess GSH and SOD. Blood in the uncoated tubes were used to assess CAT and total antioxidant levels. The tubes were centrifuged, and the sera were stored at −20°C until further analysis. SOD, GSH, and CAT were assessed with a UV double beam spectrophotometer, and total antioxidant levels were assessed with a double UV beam spectrophotometer. The TAOC of serum was determined via the phosphomolybdenum assay. The detailed protocols are explained in subsequent sections.

2.3. Total antioxidant assay:

The first step was the standardization of vitamin C, followed by the estimation of TAOC in the samples. Exactly 100µl of the sample (serum from non-coated tubes) was pipetted into a clean test tube, and 100µl of 5% TCA was added to precipitate the proteins. The mixture was then allowed to stand for approximately 5 min and centrifuged. Then, 100µl of the clear supernatant was transferred to a clean test tube, 1ml of TAC reagent was added, and the mixture was then incubated in a water bath at 90°C for 90 min. A blank or control tube contained 100µl of water instead of sample in the reaction mixture. Following incubation, the reaction mixture was cooled (development of greenish-blue color was observed in the experimental tubes), and the optical density was read at 695nm, relative to the blank reading. The concentration of the total antioxidants in the serum was calculated by plotting the absorbance of the test against the standard graph and expressed as µg/ml.

2.4. Superoxide dismutase assay:

SOD enzyme activity was determined via the Beauchamp and Fridovich assay. For sample preparation, 500µl of EDTA-treated blood was centrifuged at 344g for 10 min, and the upper plasma layer was separated; approximately 500µl of normal saline was added to the erythrocyte layer, mixed well, and centrifuged, again discarding the upper layer and adding fresh normal saline to the erythrocytes. This step was repeated two more times to wash the erythrocytes. Finally, 200µl of the washed erythrocytes was taken and lysed with 600µl cold distilled water. The prepared erythrocytes (1:4 dilution) were stored at 0–4°C until analysis. Then, 100µl RBC lysate was further diluted by adding 400µl 0.05 M phosphate buffer to obtain a final erythrocyte dilution of 1:20. The hemoglobin level in the 1:20 diluted sample was determined using the cyanmethemoglobin method as previously described¹². Units of enzyme present in the sample were expressed as U/mg Hb (whole blood).

2.5. Catalase assay:

CAT activity was assessed using the hydrogen peroxide reduction assay. The UV light absorption of hydrogen peroxide solution can be easily measured at 240nm. CAT present in the RBC lysate decomposes hydrogen peroxide, thereby decreasing the absorption over time. The activity of CAT is expressed as the rate constant for the exponential decay of H₂O₂ calculated by linear regression analysis relative to the activity of hemoglobin. For this procedure, 3ml distilled water was used as the blank, and 3ml 0.3% hydrogen peroxide was the reagent blank. For the test, 10μl 1:10 diluted RBC lysate was added to 3ml 0.3% hydrogen peroxide, and 10μl 1:10 diluted RBC lysate was added to 3ml distilled water as the positive and negative controls, respectively. Eight readings were taken at 240nm at a time interval of 15 s, immediately after the addition of RBC lysate to the test/control. The decrease in the OD was calculated, and the enzyme activity was expressed as U/mg hemoglobin.

2.6. Glutathione estimation assay:

GSH was estimated using the reagent DTNB [sulfhydryl reagent 5,5ʹ-dithio-bis (2-nitrobenzoic acid)]. Exactly 500µl of whole blood, anticoagulated with EDTA and fluoride, was centrifuged, and 100µl of the erythrocytes was removed and diluted to 1ml with distilled water (dilution 1:10). The diluted samples were treated with 1.5ml of precipitating solution¹² and let sit at room temperature for 10 min to complete the precipitation. The solutions were then filtered through Whatman Grade 1 filter paper. Next, 500µl of the filtrate was mixed with 2ml of phosphate solution and 250µl of DTNB solution. Simultaneously, a blank solution was prepared containing 200µl of distilled water, 300µl of precipitating solution, 2 ml of phosphate solution, and 250µl of DTNB. The intensity of the yellow color formed was spectrophotometrically read immediately (within 10 min) at 412nm against the blank. The optical densities obtained were then plotted against the standard graph. The concentration of GSH was calculated graphically and multiplied by the respective dilution factors. GSH concentration in the samples were expressed as µg/ml.

2.7. Statistical analysis:

Statistical analysis of the data was performed using SPSS version 17 software (IBM Software). Comparisons of the means of two groups were performed using the Student’s t test. A two-tailed analysis of variance (ANOVA) was used to compare the means of more than two groups. An HSD Tukey test with ANOVA was used to find means that were significantly different from one another. A p value ≤ 0.05 was considered statistically significant.

3. RESULTS:

3.1. Reduced level of various antioxidants in the sera of periodontitis patients, with or without DM, compared to the levels in healthy subjects.

Sera from patients with periodontitis, with or without DM, were analyzed for GSH levels and compared with that in healthy subjects. We observed that the mean serum GSH levels, expressed in µg/ml, were the highest in group III (healthy control, 86.58±12.29) followed by group I (periodontitis, 64.41±13.43) and group II (periodontitis with diabetes, 57.72±9.02) (Table 1 and Figure 1). Mean serum GSH levels were significantly lower (p < 0.001) in groups I and II compared to the level in group III.

Table 1. Comparison of antioxidant levels among the three groups using one-way ANOVA^*

		N	Mean	Standard Deviation	Minimum	Maximum	F	p
SOD^†	Group 1	100	26.2977	6.71905	17.08	39.35
	Group 2	100	34.3364	7.14435	14.14	49.64	97.883	< 0.001
	Group 3	100	39.9557	6.94548	25.68	56.45
TAOC^‡	Group 1	100	0.5865	0.17011	0.35	1.44
	Group 2	100	0.6207	0.11240	0.42	0.88	229.068	< 0.001
	Group 3	100	1.1028	0.26000	0.22	1.56
GSH^§	Group 1	100	64.4014	13.43737	25.50	93.40
	Group 2	100	57.7208	9.02135	42.20	82.10	165.693	< 0.001
	Group 3	100	86.5800	12.29954	37.00	115.80
CAT^‖	Group 1	100	2239.03	581.723	1139	3472
	Group 2	100	2049.86	566.563	1014	3349	112.033	< 0.001
	Group 3	100	3282.28	721.231	1870	4881

^*Analysis of variance;^†superoxide dismutase; ^‡total antioxidants; ^§glutathione; ^‖catalase

Table 2. Post hoc tests (multiple comparison of the antioxidant status between groups)

Tukey HSD
Dependent Variable	(I) group	(J) group	Mean Difference (I-J)	p	95% Confidence interval
Dependent Variable	(I) group	(J) group	Mean Difference (I-J)	p	Lower bound	Upper Bound
SOD	Group 1	Group 2	−8.03870^*	0	−10.3501	−5.7273
	Group 1	Group 3	−13.65800^*	0	−15.9694	−11.3466
	Group 2	Group 3	−5.61930^*	0	−7.9307	−3.3079
TAOC	Group 1	Group 2	−0.03419	0.415	−0.0977	0.0294
	Group 1	Group 3	−0.51630^*	0	−0.5798	−0.4528
	Group 2	Group 3	−0.48211^*	0	−0.5457	−0.4186
GSH	Group 1	Group 2	6.68065^*	0	2.771	10.5903
	Group 1	Group 3	−22.17855^*	0	−26.0882	−18.2689
	Group 2	Group 3	−28.85920^*	0	−32.7688	−24.9496
CAT	Group 1	Group 2	189.17	0.085	−19.71	398.05
	Group 1	Group 3	−1043.250^*	0	−1252.13	−834.37
	Group 2	Group 3	−1232.420^*	0	−1441.30	−1023.54

*Mean difference is significant at 0.05 level. ^†superoxide dismutase; ^‡total antioxidants; ^§glutathione; ^‖catalase

Figure 2. Catalase (CAT) levels in the sera of periodontitis patients with or without DM.

The sera derived from subjects in the three study groups were analyzed for CAT levels as described in the Methods section. Group I, periodontitis patients; group II, diabetic patients with periodontitis; group III, controls; Results are presented as the mean ± SD.

Figure 3. Superoxide levels in the sera of periodontitis patients with or without DM.

The sera derived from subjects in the three study groups were analyzed for superoxide levels as described in the Methods section. Group I, periodontitis patients; group II, diabetic patients with periodontitis; group III, controls; Results are presented as the mean (units/ml) ± SD.

Figure 4. Total antioxidant levels in the sera of periodontitis patients with or without DM.

The sera derived from subjects in the three study groups were analyzed for total antioxidant levels as described in the Methods section. Group I, periodontitis patients; group II, diabetic patients with periodontitis; group III, controls; Results are presented as the mean ± SD.

4. DISCUSSION:

The inflammatory process in periodontal disease is initiated by bacterial infection and exacerbated by the infiltration of bacterial toxins and enzymes through the junctional epithelium lining. When the host defense mechanism is unable to subdue the disease process, pathogenic microflora continue to multiply, leading to periodontal tissue destruction^13,14. This local disruption in homeostasis can eventually extend beyond the confines of the periodontium. Evidence suggests that the immune system can possibly transmit inflammatory responses to distant tissues and organs of the body, when stimulated by the local periodontal environment¹⁵. This is postulated to occur in two possible ways, either through migration of the pathogenic microflora and the colonization of distant sites and organs, causing a local inflammatory reaction in the new site, or via a systemic inflammatory response caused by a systemic oxidative stress resulting from the periodontal assault¹⁵. Therefore, chronic periodontitis patients with DM type II were considered a separate group, in this study, to assess the combined effect of the two diseases on the systemic antioxidant status.

Studies on the role of ROS in tissue destruction are mainly indirect and circumstantial. However, it is accepted that chronic inflammatory conditions can lead to enhanced oxidative stress with neutrophils being implicated in the pathogenesis of disease. This is possibly caused by an oxidative burst during the process of phagocytosis^15,16. Neutrophils generate ROS in the presence of a minimum oxygen tension of approximately 1% and a pH of 7.0–7.5. This is similar to that found in periodontal pockets, thus indicating that ROS production is possible in these sites. Studies have also revealed that peripheral neutrophils in patients with chronic periodontitis show increased ROS production¹⁶.

Most studies to date on the serum concentrations of total AOs have reported that total AO capacity is inversely associated with periodontitis and is directly correlated with disease severity^{16, 17}. However, in the present study, total AO levels were only mildly compromised in chronic periodontitis patients as compared to those in the healthy control group. Enzymes that neutralize ROS include glutathione peroxidase and CAT. Low levels of oxidative stress are addressed by the GSH redox cycle, whereas CAT is thought to be effective against severe oxidative stress^9,10. Hence serum levels of GSH and CAT are expected to be inversely related to periodontal disease activity. Our results on serum GSH and CAT levels are consistent with recent reports showing that their low serum levels are associated with periodontitis^9,10.

Free radicals play an important role in the pathogenesis of DM, and there is a relationship between oxidative stress and secondary complications of diabetes^17-19. Due to the increased production of free radicals and the decreased capacity of the antioxidant systems in such individuals, it was stated that diabetic individuals might require more antioxidants than healthy individuals^19,20.Advanced glycation end products are highly prevalent with DM type II. This can cause an increased adhesion of neutrophils, enhanced chemotaxis, and priming of hyperactive neutrophils, which can lead to an increase in ROS and oxidative stress mediated by periodontal pathogens. This can explain the enhanced risk of periodontitis with DM type II^19-21.

The results of this study are consistent with the outcomes of other investigations that reported low levels of GSH and CAT in DM patients with periodontitis^10,12,22. However, in our study, although serum CAT levels in periodontitis patients were lower than those in healthy controls, this was not statistically significant. SOD levels in this study are in line with those of other studies showing that the SOD enzyme level can adapt to conditions of chronic oxidative damage^22,23. A previous study on diabetic rats showed that SOD activity decreases in the beginning of hyperglycemia, and then increases over time to counter the effects of oxidative stress¹⁹.

Hyperglycemia induces overproduction of O₂⁻, which is central to the major molecular mechanisms that lead to glucose-mediated damage to the vascular system^24,25. The SOD enzyme is an important antioxidant, which catalyzes dismutation of the O₂⁻ radical in all oxygen-consuming cells. Overexpression of SOD can compensate for various diabetic complications in target cells, which are caused by hyperglycemia-induced phenotypes^23,25,26.

In this study, serum SOD levels in DM type II patients were found to be increased, when compared to those in chronic periodontitis cases. This can be explained as a protective and adaptive mechanism developing in the tissues and can indicate increased ROS generation in DM patients. Within periodontal tissues, SOD is localized predominantly in the periodontal ligament in association with collagen fibrils and fibroblasts²⁷. SOD levels in the periodontitis group of this study were significantly lower than those in the non-periodontitis control group. Decreased SOD activity could lead to increased ROS levels, eventually causing tissue breakdown, as seen in chronic periodontitis^28,29. Petelin et al, in a study on beagle dogs, showed that the local delivery of liposome-encapsulated SOD can suppress inflammation of the periodontium and can also lead to maximum reductions in probing depth and gains in clinical attachment levels^22,29. However, it is difficult to extrapolate these results to human studies because the SOD enzyme has a slow rate of activity compared to that of the radical scavenging species of the extracellular environment^29–31. Further, CAT was found to provide no additional benefit. Misaki et al, in an interventional study performed on rats, concluded that SOD had a beneficial effect on inflammation induced by Porphyromonas gingivalis and on periodontal wound healing²⁹. In contrast to the findings of our study, a study by Akalin et al suggested that SOD activity in chronic periodontitis patients was increased in the gingiva. The authors suggested that this could be due to a higher need for SOD activity and protection in the gingiva in chronic periodontitis cases²³.

Our results with total AO levels confirmed the study performed by Chapple et al, which showed a reduction in systemic and local antioxidant defense during periodontitis¹². In our study, total AO levels in diabetic patients with periodontitis were elevated significantly when compared to those in the periodontitis group. Evidence available suggests that hyperglycemia can result in an increased generation of ROS. The stress-sensitive intracellular signaling pathways can be activated due to this, which can cause the expression of gene products that lead to cellular damage and the late complications of diabetes.

The diabetic patients included in this study were all on oral hypoglycemic drugs, specifically, a metformin derivative. Metformin is used to normalize glucose concentrations in type II diabetes. Studies have reported added antioxidant benefits in diabetic patients treated with metformin^24,32.

However, this study was performed without any case control assessment. Therefore, it is possible that several confounding factors could have affected the outcome. Another limitation of this study is that only serum was analyzed. Therefore, a comparative study could be carried out, wherein the AO levels in serum, saliva, gingival crevicular fluid, and gingival tissue samples are compared to better understand both the local and systemic effects of chronic periodontitis.

Memisogullari et al²⁴, in a study assessing the effect of metformin on lipid peroxidation and antioxidant levels in patients with DM type II, reported that the regular use of metformin can cause a decrease in oxidative stress. Pavlović et al reported that metformin leads to a decrease in lipid peroxidation and an increase in AO enzyme activities²⁶.This might explain the higher TAOCs observed in DM type II patients when compared to those in periodontitis patients^30–32.

5. CONCLUSION:

Serum levels of the antioxidant enzymes, SOD, GSH, CAT, and TAOC, were found to be significantly reduced in chronic periodontitis patients with or without DM type II, compared to those in healthy controls. An assessment of serum AO profiles showed that they have the potential to be used as a biomarker for chronic periodontitis and that periodontitis might not be a localized phenomenon, but rather can possibly have systemic effects.

6. LIST OF ABBREVIATIONS:

If abbreviations are used in the text either they should be defined in the text where first used, or a list of abbreviations can be provided.

7. ETHICS APPROVAL AND CONSENT TO PARTICIPATE:

The research protocol was approved by the ethical committee NU/CEC/Ph. D04/2010 and registered in ClinicalTrials.gov ID: NCT04180332. The research manuscript follows consolidated standards of reporting trials (CONSORT) guidelines, as well as the Helsinki declaration for human research as revised in 2013. A consent form was given to all subjects, which was explained to them, and written consent was obtained before enrolling them.

8. CONFLICT OF INTEREST:

The authors report no conflicts of interest related to this study.

9. ACKNOWLEDGEMENTS:

The authors would like to extend their gratitude to all the technical staff of the Central Research Laboratory, K S Hegde Medical Academy, Nitte (deemed to be university), Mangalore, who have helped in conducting the tests. No external funding was obtained for this study.

10. AUTHOR CONTRIBUTION:

All authors have made substantial contributions to conception and design of the study. BT, SV and RP have been involved in design, literature search, clinical study, data analysis, interpretation and manuscript preparation. MS, and MK have been involved in literature search, data analysis and interpretation. AE and EE has been responsible for drafting the manuscript, editing, revising, and has given final approval of the version to be published.

11. SUPPORTIVE/SUPPLEMENTARY MATERIAL:

Supportive/Supplementary material intended for publication must be numbered and referred to in the manuscript but should not be a part of the submitted paper. In-text citations as well as a section with the heading "Supportive/Supplementary Material" before the "References" section should be provided. All Supportive/Supplementary Material must be listed and a brief caption line for each file describing its contents should be included.

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Received on 09.04.2020 Modified on 18.05.2020

Research J. Pharm. and Tech. 2021; 14(2):1025-1032.

DOI: 10.5958/0974-360X.2021.00183.9