In this hospital based cross-sectional comparative study the samples were collected from the Department of Periodontology, SDM College of Dental Sciences and Hospital, Dharwad, and study was conducted in Department of Biochemistry, S.D.M. College of Medical sciences and Hospital, Dharwad over a period of one year.

The study included a total of 100 subjects who were categorized as follows:

Based on our pilot study, a sample of 25 subjects for each group was estimated to achieve a 95% power at α error of 5% in each of the groups at a gross precision rate of 20%.

Group 1 – Age and sex matched controls were taken in group 1.

Group 2 – Clinically, gingivitis can be diagnosed using certain subjective (colour and presence of edema and ulcerations) and objective (presence of bleeding on probing) criteria. Besides having the advantages of being an objective criterion, bleeding on probing is considered to be one of the first signs of gingivitis [10]. Sulcus Bleeding Index (SBI) score of ≥2 was used to diagnose gingivitis [11]. The reason for using the Sulcus Bleeding Index (SBI) was that it had a greater scoring range (0-5), and could be considered more sensitive. This index assesses the presence or absence of bleeding from the gingival tissues along with changes in the colour and consistency of the gingival tissues.

Gingival bleeding could be a result of haematological disorders, use of anticoagulant medications, local trauma and hormonal changes. Thus, the subjects included in Group 2 were systemically healthy individuals, non pregnant/lactating women, with no current use of any medications. They were diagnosed with plaque induced gingivitis.

Group 3- Chronic periodontitis was diagnosed as the presence of probing pocket depth (PPD) of ≥5mm along with the presence of attachment loss of ≥2mm with at least 3 teeth assessed at four sites per tooth, using a UNC-15 periodontal probe [12].

Group 4- Freshly diagnosed type 2 diabetes mellitus subjects who had periodontitis were selected in group 4. Uncontrolled diabetes mellitus is considered to be a risk factor for periodontal diseases [13] and at the same time, subjects with well-controlled diabetes have reported to show no significant increase in the risk for periodontitis [14]. Hence, freshly diagnosed diabetics were included who were not on any antidiabetic treatment. The diagnosis of type 2 diabetes mellitus was done based on fasting blood sugar more than 126mg/dl and postprandial blood sugar of more than 200mg/dl for three consecutive times. They had not started taking any antidiabetic medication at the time of sample collection for our study.

Absence of any other chronic systemic illness, any other source of infection, and intake of any medication affecting immune/inflammatory reactions or any vitamin supplementations. A detailed patient history was taken to rule out any diagnosed medical condition. Smokers were also excluded from the study. Only never smokers were included in the study and the smoking status was ascertained by detailed history.

All the patients signed for informed consent prior to the study. Approval for the study was taken from the institutional ethical committee.

Sample collection and processing: A 5 ml of fasting venous blood was collected aseptically in EDTA bulb. The sample was centrifuged at 3000 rpm for 15 min. Plasma and Red blood corpuscles (RBCs) were separated and the samples were analysed for all the parameters immediately. RBCs were washed in 0.9% NaCl for three times. For MDA analysis, 1:1 RBC haemolysate was used and for determination of superoxide dismutase (SOD) and catalase activity, the haemolysate was further diluted 1:50 with 0.9% normal saline. Total antioxidant capacity and Vitamin C were analysed in plasma. Analysis was performed using UV Visible spectrophotometer (Shimadzu).

Free radical level was analysed as MDA (Malondialdehyde) or TBARS (Thiobarbituric acid reacting substances) in RBC haemolysate.

Antioxidant status was analysed by

MDA levels in erythrocytes were measured by the method of Ohkawa et al., [15]. MDA is estimated as thiobarbituric acid reacting substances (TBARS). 0.75ml of 1:1 RBC haemolysate was added to 3ml of MDA reagent (75mg Thiobarbituric acid and 15g Trichloroacetic acid in 2.08ml of 0.2N HCl made upto 100ml with distilled water) and kept in boiling water bath for 20 min, cooled and centrifuged at 3000 rpm for 10 min. Optical density (O.D) of supernatant was read against reagent blank. MDA reacts with thiobarbituric acid at 100^oC in acidic medium to form a pink coloured complex which was measured at 535 nm. MDA concentration was calculated using the molar extinction coefficient of MDA-TBA complex (1.5×10⁵L/mol/cm). The result is expressed as nmol/g haemoglobin.

Total Antioxidant Capacity (TAC) was measured by method of Koracevic et al., [16]. The assay measured the capacity of biological fluids to inhibit the production of TBARS from sodium benzoate under the influence of free oxygen radicals derived from Fenton’s reaction. A solution of 1 mmol/L uric acid was used as standard.

Vitamin C was measured by Dinitro phenyl hydrazine (DNPH) method [17]. Ascorbic acid is initially oxidized by cupric ions to dehydroascorbate. This reacts with 2,4-Dintrophenyl hydrazine to form a red bishydrazone, and the intensity of colour developed is measured at 520 nm.

SOD was measured in RBC haemolysate using the method of Marklund and Marklund [18]. A 50μl of 1:100 haemolysate was added to 2.85ml of 0.05M Tris buffer (pH 8.5) and 0.1ml of 20mM pyrogallol. The principle involves the inhibition of autoxidation of pyrogallol by SOD which was measured as increase in absorbance at 420 nm for 3 min. One unit of SOD is defined as the amount of enzyme required to cause 50% inhibition of pyrogallol autoxidation and results were expressed as U/g haemoglobin.

Catalase activity was determined by the method of Aebi [19]. A 10μl of 1:100 RBC haemolysate was added to 3ml of 0.05M phosphate buffer and 1ml of 30mM H₂O₂. The decomposition of H₂O₂ by catalase was measured at 240 nm as decrease in absorbance for 3 min. One unit of catalase activity is expressed as mM of H₂O₂ decomposition/mg haemoglobin/min. The molar extinction coefficient of H₂O₂ is 71 L/mol/cm.

All the findings are expressed as Mean ± Standard deviation. The p-value of <0.05 was considered statistically significant, p<0.01 as highly significant and p<0.001 as very highly significant. Analysis was performed by various statistical tests like ANOVA, Tukey’s post-hoc tests, Pearson’s correlation using IBM^®-SPSS^®-statistics-version 20.0.

[Table/Fig-1] shows, the results of one-way ANOVA for comparison of means of biomarkers between overall groups under study. The mean values of free radical marker MDA was highly significant between all the groups (p<0.01). The difference in the mean values of total antioxidant capacity and the catalase were statistically significant between all groups (p<0.05). Plasma antioxidant vitamin C and erythrocyte SOD levels showed highly significant difference between all the study groups (p<0.01).

[Table/Fig-2] shows, the Tukey’s Post-hoc analysis results for comparison of means of biomarkers between individual groups. The MDA levels were significantly increased among patients with gingivitis (p=0.04), chronic periodontitis (p=0.003) and periodontitis with type 2 diabetes (p=0.022) compared to the healthy control group. TAC was significantly decreased among periodontitis with type 2 diabetes in comparison with healthy controls (p=0.012). Decreased plasma vitamin C levels and erythrocyte SOD levels found in periodontitis with type 2 diabetes as compared to controls were highly significant (p=0.004 and 0.003 respectively). Catalase levels showed significantly low levels among periodontitis patients with type 2 diabetes in comparison with gingivitis and chronic periodontitis patients without diabetes (p=0.034 and 0.029 respectively).

We performed Pearson’s correlation between all the analysed parameters with the probing pocket depth (PPD) which indicated the severity of the disease [Table/Fig-3]. Overall MDA values among both periodontitis group with and without type 2 diabetes together (n=50) were positively correlated with PPD (r=0.275, p=0.05). Whereas, there was a very significant positive correlation between the MDA values and PPD among periodontitis patients with diabetes mellitus (r=0.566, p=0.003).

Lipid peroxidation causes profound alteration in the structural integrity and function of cell membranes. The extent of tissue damage could be assessed by measuring the concentrations of lipid peroxidation products and antioxidants in erythrocytes and plasma as the lipid peroxidation products leak from the site of inflammation into the plasma [20]. In our study, erythrocyte MDA levels were significantly increased in all the patient groups when compared to healthy controls as observed in previous studies [21,22]. ROS causes periodontal tissue destruction either by degrading the ground substance or by release of collagenases or by release of various inflammatory mediators [23].

The occurrence of periodontitis in Type 2 diabetes commonly suggests that both the conditions have common elements of pathogenesis as both have inflammation in common. This is supported by the fact that Type2 diabetes is a significant risk factor for the development of periodontal disease and aggravates the severity of periodontal infections [24]. Our results show excessive free radical activity (MDA) among diabetics as observed in previous study [25] and decrease in antioxidant capacity (TAC, Vitamin C, SOD) which is highly significant among diabetic periodontitis compared to healthy controls. MDA levels correlated positively with probing pocket depth in all periodontitis patients. But there was a strong positive correlation between excessive ROS generation and the periodontal pocket depth in diabetic periodontitis subjects indicating that there is more tissue destruction in diabetics with excessive generation of ROS. Periodontitis is a low grade inflammatory condition associated with systemic inflammation supported by the association of C reactive protein levels. Studies have shown that the C reactive protein levels mediate the association between probing pocket depths and oxidative stress [26]. ROS compromises the body’s ability to fight infection and repair the tissue in periodontal disease. Hence, worst periodontal health status is associated with greater oxidative injury [27]. Since the final result of lipid peroxidation, MDA is considered a biomarker of oxidative stress, the hypothesis of oxidative stress implication in pathogenesis of periodontal disease in diabetic patients can be supported by elevated MDA levels [28].

Increased destruction of periodontal tissue among those with diabetes may be due to altered host susceptibility to periodontal pathogens mediated by accumulation of advanced glycation end products (AGEs). The inflammatory cells such as monocytes and macrophages have receptors for AGEs. Hence, accumulation of AGEs in diabetic gingiva increases the intensity of the immune-inflammatory response to periodontal pathogens. Interactions between AGEs and their receptors on inflammatory cells result in increased production of proinflammatory cytokines leading to enhanced oxidant stress and accelerated tissue damage [29]. The periodontal tissues are highly sensitive to the products derived from oxidative damage supported by the presence of a high RAGE (receptor for AGEs) expression in periodontal tissues [30]. The interaction between AGE formation, increased ROS by both diacylglycerol and protein kinase C pathway and activation of polyol pathway provides insight to exaggerated periodontal destruction among diabetics.

Exaggeration in innate immune response; impaired polymorphonuclear leukocyte function facilitate bacterial persistence in tissues; impaired bone healing due to accumulation of AGEs; reduction in collagen synthesis and increase in collagenase activity may collectively lead to excessive periodontal tissue breakdown in diabetes [31].

Inadequate total antioxidant defense enhances tissue injury due to excessive free radical production in individuals with periodontal disease [32]. Significantly decreased Total antioxidant capacity and SOD activity was observed in periodontitis patients with diabetes; although lower mean levels were observed in patients with gingivitis and periodontitis as compared to healthy controls. Both cross-sectional and longitudinal experimental studies have also shown a reduction in TAC and SOD both within the local periodontal tissues and in plasma compared to controls [9,33,34]. Reduced plasma total antioxidant defense could result from low grade inflammation induced by the host response to periodontal bacteria [34]. The disturbed antioxidant status may be explained on the basis of increased ROS production by phagocytic cells in periodontitis [35, 36].

The mean vitamin C levels in our study decreased in a graded manner from group1 to group 4 study subjects but significant decrease was noted in diabetic periodontitis group as compared with healthy controls. Low serum vitamin C levels were reported in periodontitis patients with diabetes by Thomas B et al., [37]. Patients with periodontitis tend to have a low level of plasma ascorbate due to gingival bleeding [38]. A 30-40% reduction in plasma vitamin C levels among periodontitis patients were reported by Panjamurthy et al., [21]. Vitamin C deficiency increases susceptibility to infection, impairs the function of neutrophils and macrophages, and also depletes its role as a radical scavenger. Vitamin C is also known to reduce the CRP mediated inflammatory/ immune responses and hence decrease likelihood of tissue damage [9].

SOD and catalase respectively protect the cell against ROS by scavenging superoxide radical and H₂O₂, which cause damage to the structure and function of membrane. The increased mean catalase levels in the gingivitis and periodontitis groups may be explained on the basis that increased free radical production may enhance the antioxidant defense system which counterbalances the pro-oxidant environment [21, 25, 39]. Significantly decreased catalase levels among periodontitis patients with type 2 diabetes in comparison with other patient groups may be indicative of a stage of excessive free radical generation and consumption of antioxidant species which could be mediated by severe hyperglycemia and systemic inflammation [40]. The results of similar studies published in literature in the recent past have been summarized in [Table/Fig-4].

The present study had a few limitations like the group of only Type 2 Diabetic individuals without periodontal disease for comparison was not included.

Strict control of blood sugar levels may decrease the severity of periodontitis in uncontrolled diabetics since tissue destruction is more with excessive oxidative stress in these patients. This can be a starting point for further research on the efficiency of different antioxidant agents for prevention and treatment of periodontal disease in diabetic patient.

Excessive ROS associated with decreased antioxidant defense mechanisms results in tissue destruction in periodontal diseases. The severity of tissue destruction due to excessive ROS is more when periodontal disease is associated with type 2 DM, indicating that oxidative stress is a common factor involved in tissue destruction. Several more controlled interventional studies are warranted to understand the possible role of antioxidant therapy in either preventing the disease or in controlling the progression of disease.