Vitamin E Supplementation, Superoxide Dismutase Status, and Outcome of Scaling and Root Planing in Patients With Chronic Periodontitis: A Randomized Clinical Trial

Journal of Periodontology; Copyright 2013 DOI: 10.1902/jop.2013.120727

Vitamin E Supplementation, Superoxide Dismutase Status and Outcome of Scaling and Root Planing in Chronic Periodontitis Patients: A Randomized Clinical Trial

Dr Neha Singh, Post Graduate Student*, Dr S C Narula, MDS, Senior Professor*, Dr R K Sharma, MDS, Professor*, Dr Shikha Tewari, MDS, Professor*, Dr P K Sehgal, MD, Professor†

*Department of Periodontology and Implantology, Post Graduate Institute of Dental Sciences, Rohtak, Haryana, India

†Department of Blood & Transfusion Medicine, Pt. B.D. Sharma, University of Health Sciences, Rohtak, Haryana, India

Background: This study was aimed to investigate the levels of superoxide dismutase (SOD) activity in serum and saliva of patients with chronic periodontitis. Also the outcome of scaling and root planning (SRP) with and without vitamin E supplementation in terms of changes in periodontal parameters and SOD activity was evaluated in patients with chronic periodontitis.

Method: Serum and salivary SOD activity in 38 chronic periodontitis patients were assayed and compared to 22 systemically and periodontally healthy subjects (Control Group). Periodontal examination was performed and serum and saliva samples were obtained. Patients with chronic periodontitis were randomly divided into treatment group-1 (TG-1) and treatment group-2 (TG-2). SRP was performed for both TG-1 and TG-2 and TG-2 was supplemented with 200mg (300 IU) of vitamin E every other day. Follow-up for periodontal parameters and SOD activity was done after 3 months. SOD activity was determined using SOD assay kit and ELISA reader at 450 nm.

Results: SOD activity both in serum (P<.05) and saliva (P<.001) was lower in chronic periodontitis patients compared to control group. After 3 months follow-up SOD activity improved in both the treatment groups however, the improvement in TG-2 was high compared to TG-1 along with better improvement in periodontal parameters in TG-2 compared to TG-1. Serum SOD levels in TG-2 raised even above the level of control group.

Conclusion: Systemic and local SOD levels are lowered in chronic periodontitis. Adjunctive vitamin E supplementation improved periodontal healing as well as antioxidant defense.

KEY WORDS Antioxidants, Periodontitis, Reactive Oxygen Species (ROS), Scaling and root planing.

Periodontitis is an inflammatory disorder of the periodontium that affects the supporting tissues of teeth. Primer etiology of the disease is the interaction between the pathogenic microorganism and the host defensive mechanisms. Phagocytosis of invading micro-organisms that involve oxidative and non-oxidative killing of the ingested microorganism is one important mechanism of host defense. Oxidative killing leads to the formation of toxic, highly reactive metabolites such as superoxide anion (O2

-) which are collectively known as reactive oxygen species (ROS).1

Role of ROS in tissue destruction, characteristic of periodontal disease has been widely discussed.2-6 Pathological reactions that excessive ROS can initiate in the tissues include: lysis of the cell membrane, DNA fragmentation (mutagenic response), inactivation of certain proteolytic enzyme inhibitors e.g. tissue inhibitor of matrix metalloproteinase (TIMP) and α-1 antiproteinase, activation of proteolytic enzymes such as collagenase, gelatenase and matrix metalloproteinase (MMP) resulting in collagenolysis and degradation of specific extracellular matrix components such as hyaluronic acid and proteoglycan.7 These events may partly be responsible for the periodontal destruction in periodontitis.3,6

1


Antioxidants, which are present in all the aerobic cells and extracellular fluids, provide protection against ROS.4,8 In healthy state, homeostasis is maintained by the interaction of oxidants and antioxidants. Under pathological conditions, the balance may be tilted towards the oxidative side generating oxidative stress. Superoxide dismutase (SOD) has been known to be one of the most prominent antioxidant enzymes in the body.8,9 SOD concentration has been estimated in relation to periodontitis in various body fluids demonstrating inconclusive results.9-

11 Therefore, there arises a need for careful assessment of SOD levels and its association with periodontitis.

Vitamin-E is a group of about 8 naturally occurring tocopherols. This vitamin is essential in humans for normal reproduction, development of muscles, resistance of RBCs (red blood cells) to hemolysis and a number of other physiological and biochemical functions. It is a potent antioxidant12-14 and has anti-inflammatory properties15-18 as well.

Vitamin E administration has been reported to significantly improve the levels of platelet anti-oxidant enzymes and retard lipid per-oxidation.19-21 Furthermore increase in the activities of antioxidant enzymes eg. SOD has been demonstrated in myocardial infarction patients as well as healthy controls.12 Perusal of the available English literature reveals no study concerning the influence of vitamin E administration during scaling and root planning (SRP) on periodontal healing. Taking this into consideration the present study was designed with an aim to evaluate the effect of vitamin E administration as an adjunct to SRP on the outcome of periodontal therapy as well as on oral and systemic anti-oxidant status in terms of SOD activity. This study was performed in two parts. First part, a cross-sectional study, to investigate the levels of salivary and serum SOD enzyme activity in patients with chronic periodontitis compared to healthy individuals and second part, a prospective randomized parallel clinical trial, to explore the effect of periodontal treatment on local and systemic SOD activity levels of patients with chronic periodontitis. Additionally, an effort is made in present study to explore the effect of adjunctive vitamin E supplementation on the SOD levels and periodontal healing in terms of improvement in clinical parameters.

MATERIAL & METHOD The study was conducted in the Department of Periodontics and Oral Implantology, Post Graduate Institute of Dental Sciences, Rohtak. The study protocol was carried out in accordance with the ethical standards outlined in the 1964 Declaration of Helsinki, as revised in 2008. The protocol was approved by the Institutional Review Board. First part is a cross-sectional study and second part is a prospective randomized parallel clinical trial. Participants for the study were recruited from the patients attending our outpatient department (OPD). Duration of the study was from March 2011 to October 2012. Prior written informed consent was taken from each patient after explaining the procedure along with the risks and benefits in their own language.

Total sixty individuals were recruited in the study. Twenty two periodontally and systemically healthy individuals were recruited in the control group (CG). Out of the 106 subjects assessed for eligibility thirty eight patients with chronic periodontitis were included in the test group depending on the inclusion, exclusion criteria and willingness to participate.

CG participants had no evidence of interproximal attachment loss, no pocket probing depth ≥ 3 mm at any site on any tooth and whole mouth bleeding score (from base of the sulcus) of ≤ 10%.6

2


Inclusion criteria for the test group patients were, presence of at least 20 teeth and at least two or more interproximal sites with attachment loss ≥4 mm, or two or more interproximal sites with probing depths ≥5 mm, not on the same tooth.22

Individuals with any of the following conditions were excluded from the study: Patients who have undergone oral prophylaxis in last 6 months, have taken vitamins, antioxidant supplements, anti-inflammatory or antibiotics in previous 3 months, patient using mouthwashes regularly, pregnant or lactating females, smokers in any form, patients diagnosed with cystic fibrosis, fat mal-absorption and any form of chronic liver disease and patient with other medical condition that could influence the results e.g.: Diabetes, Rheumatoid arthritis and any other chronic inflammatory disease.

Patients in the test group were randomly assigned to one of the two treatment groups (allocation ratio 1:1) by computer generated table. A balanced random permuted block approach (4-unit block size) was used to prepare the randomization tables in order to avoid unequal balance between the two treatments.

Patients in treatment group-1 (TG-1) received scaling and root planing and in treatment group-2 (TG-2) received scaling and root planing along with oral administration of 200mg (300 IU) of vitamin E‡ in the form of capsule on every alternate day along with the meal for three months).

Periodontal Examination Enrollment of the participants was done by one investigator (SN), random allocation sequence was generated by second (RS) and assignment of participants to intervention was done by third investigator (ST). Clinical periodontal examination and scaling and root planing was carried out by one trained examiner (NS) throughout the study to preclude inter-examiner variability whereas vitamin E was supplemented by another investigator to ensure blinding. Periodontal parameters which included plaque index (PI),23 gingival index (GI),24 bleeding on probing (BOP), probing pocket depth (PPD) and clinical attachment level (CAL) were recorded. PI and GI were recorded on four sites while rests were recorded at six sites on each tooth. Determination of the examiner reproducibility was done by carrying out double clinical periodontal data recording on ten patients. Each participant was assessed twice in one appointment, over a one-hour interval and the repeat measurements were carried out masked to the first measurement. Reproducibility of data collection was found to be with > 90% accuracy. All the examinations were performed at baseline for all the participants and at three month recall for subjects belonging to TG-1 and TG-2.

Sampling All the samples, prior to and after periodontal therapy i.e. at baseline and 3 month follow-up, were collected after 48 hrs of the clinical measurements. All participants, after an overnight fast were told to abstain from eating, drinking anything except water and brushing prior to sample collection the next morning. The participants were enquired whether they had followed these instructions before sample collection.

Unstimulated whole saliva samples were used in this study. Patients were instructed to allow saliva to pool in the bottom of the mouth and drain into a collecting tube in a seated position. 2 ml of whole saliva was collected in disposable, sterile and clean tubes and centrifuged

3


immediately to remove cell debris at 1000 g for 10 min at 40C. The supernatant was removed and stored in small aliquots at -80oC until analysis.

Venous blood from anticubital vein was collected in plain vacutainer tubes without additive and was centrifuged at 3500 g for 5 min to separate serum. Serum aliquots were stored at -80oC until analysis.

Treatment Patients in TG-1and TG-2 received non-surgical periodontal therapy, one week after the sample collection, in the form of full mouth SRP using manual instruments§ and an ultrasonic scaler║. The patients were further provided information on the periodontal disease and oral hygiene instructions were given at each appointment. The patients were instructed to use only mechanical methods i.e. toothbrushes and interdental cleaning aids during the study period and mouthwashes and /or antimicrobials were not prescribed. Patients in TG-2 were supplemented with vitamin E (200 mg every other day along with meal for three months) adjunctive to SRP. Vitamin E was started immediately after completion of SRP. At the end of each month the patients were asked to return the remaining tablets. Compliance was estimated by counting the number of tablets remaining.

Analysis of SOD Activity Biochemical estimation of SOD activity was performed using SOD Assay Kit¶ according to manufacturer’s instructions and an ELISA reader at 450 nm. A fixed volume of 20µl was used for SOD estimation at each stage for all the patients. The levels of SOD activity were calculated by the manufacturer’s formula using the values from ELISA. The unit of measurement was percentage (%).

Statistical Analysis Minimum sample size of 15 per group is required for detection of a significant difference in BOP% with 80% power and two-sided 0.05 level of significance.25 Data for all groups was tested for normal distribution by Kolmogorov-Smirnov test and was found to be non-normally distributed. Differences among groups were assessed Mann-Whitney-U analysis. Comparison of periodontal and biochemical parameters between baseline and 3 months were analyzed using Wilcoxon-signed rank test. Partial correlation was applied after adjusting for PI to determine correlation among periodontal parameters and SOD activity. All statistical analyses were 2-tailed with significance level at 0.05 and were calculated using statistical software#.

RESULTS 22 individuals were recruited in the CG. 38 patients with chronic periodontitis were randomly assigned to TG-1 and TG-2 (n=19 in each group). Duration of the study was from March 2011 to October 2012. Trial ended in October 2012 after completion of the follow-up. All the patients reported to 3 month follow-up. No untoward complication or undesirable response was observed in any of the participants with the treatment regime provided.

4


Cross-Sectional Findings Table 1 displays the demographic and clinical findings of CG and disease group at baseline. Biochemical parameters in control and test group at baseline are shown in Table 2. Evaluation of this cross-sectional data revealed that the SOD activity in saliva (P<.05) and serum (P<.001) was markedly lower in disease samples as compared to the periodontally and systemically healthy subjects.

Interventional Study Findings Both the TG-1 and TG-2 demonstrated significant improvement in the periodontal and biochemical parameters after therapy at 3 months follow-up compared to baseline (Table 3). However, improvement in periodontal parameters and SOD activity in serum (P<.001) was significantly high in TG-2 as compared to TG-1(Table 4). Table 4 also demonstrates that intergroup comparison among TG-1 and TG-2 show improvement in all the periodontal parameters after SRP was significantly high in TG-2 as compared to TG-1 (P< 0.05, 0.001, 0.001, 0.05and 0.05 for PI, GI, BOP, PPD and CAL respectively). The level of SOD activity in saliva was raised comparable to the CG while the SOD activity in serum increased to a level above that of the CG (Table 5). Results for partial correlation, after adjusting for plaque as possible confounder, demonstrated serum SOD level to be significantly and inversely correlated with GI (r: -.454, P< 0.05).

DISCUSSION In our body an oxidant-antioxidant balance exists in healthy state. When this balance is disrupted and shifts towards the oxidant side “oxidative stress” results. Oxidative stress has been found to be related with many systemic conditions and diseases such as diabetes,26 rheumatoid arthritis,27 ulcerative colitis,16 oral lichen planus28 etc. Estimation of ROS-related tissue destruction products such as malonaldialdehyde (MDA) {lipid peroxidation product}, total oxidant status or anti-oxidant levels such as SOD, glutathione etc. may act as an indicator for measuring the oxidative stress. Although, intracellular SOD is the most prominent antioxidant in mammalian tissue,29,30 several authors have proven significance of extracellular SOD in plasma and other body fluids.31 SOD activity in periodontal health and disease in serum and saliva has been evaluated with inconclusive results.8,9 Thus, the present study was aimed to evaluate firstly the significance of SOD as antioxidant in serum and saliva by estimating their levels in patients with chronic periodontitis and periodontally healthy individuals and secondly the interventional part of the study deals with the analysis of salivary and serum SOD levels along with the periodontal parameters following SRP. Additionally, this study is the first attempt to evaluate the effect of systemic supplementation of antioxidants in the form of vitamin E as adjunct to SRP on antioxidant status in periodontitis patients.

In the present study, even distribution of age among all the groups along with a narrow range allowed us to minimize the bias in the estimation of SOD as the level of antioxidants in human body is affected by aging.32 Smoking, one of the factors negatively influencing the antioxidant levels,33 was ruled out in the study population. Thus careful selection of study population helped us to rule out the prejudice for systemic SOD level estimation. Sampling, storage of samples and processing was performed according to the recent recommendations. Moreover, use of highly sensitive SOD assay kit enhanced the quality of the study as the estimation of SOD activity was precise. Additionally, randomization of the disease group (patients with chronic periodontitis)

5


and blinding was done for elimination of selection bias that could affect the results. There were no specific limiting condition, other than chronic periodontitis, for inclusion in test group thus the findings of the study are applicable to a large proportion of the population.

Evaluation of the data at baseline revealed that SOD levels in serum (P<0.05) and saliva (P<0.001) were significantly lower in patients suffering from chronic periodontitis as compared to age matched periodontally and systemically healthy subjects (Table 2). It indicates a compromised oxidant-antioxidant balance in chronic periodontitis patients. Our results are in accordance with previous studies who demonstrated a lower antioxidant activity in saliva of chronic periodontitis patients compared to periodontally healthy subjects.6,8,34,35 Depressed levels of serum antioxidant levels were recorded in chronic periodontitis patients in comparison to periodontally healthy subjects by various authors.8,10,11,36 The alteration in serum antioxidant capacity can be explained by the fact that chronic periodontal disease is associated with peripheral neutrophils that are hyper-reactive and responsible for the production of reactive oxygen species in response to Fc gamma-receptor stimulation.10 Moreover, partial correlation indicated that SOD activity in serum is strongly and negatively correlated with gingival inflammation after controlling plaque level as potential confounder.

It is required to be highlighted here that level of salivary SOD (Table 2) was within detectable range even during lowered levels in diseased subjects, pointing towards the efficient diagnostic utility of salivary SOD levels.

Study population in the test group was randomly divided into treatment group 1(TG-1) and treatment group 2 (TG-2). Non-surgical periodontal treatment in the form of SRP in TG-1 resulted in significant improvement in clinical as well as biochemical parameters (Table 3). The improvement in SOD levels can be attributed to reduction in the levels of inflammatory burden as a result of SRP. These results are in accordance with Sang-Chul Kim et al,8 who also observed significant improvement in the SOD levels in saliva. However, in their study the SOD levels at follow-up in test group remained inferior to those of the control group. In our study, the SOD levels in saliva and serum were improved and were statistically comparable to the baseline values of healthy group.

Vitamin E is a naturally occurring antioxidant of significance as literature provides evidence on negative association of vitamin E levels with oxidative stress28 and its efficacy in the treatment of various oxidative stress induced diseases due to its ability to increase antioxidant levels, especially SOD.12,16,17 However its effect in periodontal healing is not explored so far. Therefore vitamin E was assessed as adjunct to non-surgical periodontal therapy in present study.

The recommended dose of vitamin E for an adult is 200-400 IU once daily as routine nutritional supplement. Due to non availability of 150 IU formulation for daily supplementation, patients belonging to TG-2 were supplemented with a moderate dose of 200 mg (300 IU) of vitamin-E on alternate days along with meal for three months of observation period. There are studies with alternate day use of vitamin E without any untoward response.37,38

Intra-group comparison for TG-2 at baseline and at follow-up revealed all the periodontal parameters (P<.001) as well as SOD levels (P<.05 for saliva and P<.001 for serum) to be significantly improved.

Intergroup comparison among TG-1 and TG-2 demonstrated that improvement in all the periodontal parameters after SRP was significantly high in TG-2 as compared to TG-1 (P< 0.05,

6


0.001, 0.001, 0.05and 0.05 for PI, GI, BOP, PPD and CAL respectively). Adjunctive vitamin E administration may have played an additional role in resolution of the periodontal inflammatory burden which manifested in the form of better healing response in TG-2, though the contribution of better plaque control in TG-2 cannot be ignored. Contribution of vitamin E towards inhibition of inflammation may be due to its various properties which include inhibition of biosynthesis of prostaglandins,15,39 suppression of pro-inflammatory cytokine production,17 decreasing C-reactive protein levels17 and prevention of activation of nuclear factor kappa B (NFκB) by free radicals.17,40 It has been reported that vitamin E has inhibitory effect on wound healing in fresh cut wounds in animal studies,18 which may be due to the anti-inflammatory nature of this compound. Since moderate inflammation is mandatory for providing necessary chemical mediators for wound healing in fresh wounds, inhibition of inflammation may lead to poor wound healing. However, chronic periodontitis is a chronic lesion with established inflammation and preponderance of pro-inflammatory destructive mediators, overpowering the abundant anti-inflammatory reparative mediators, which are responsible for the clinical presentation of the disease.

Since resolution of inflammation is one of the important factors influencing the outcome of non-surgical periodontal therapy therefore the anti-inflammatory properties of vitamin E are found to be beneficial in the management of chronic inflammatory diseases.16,17

When improvement in SOD levels was compared among TG-1 and TG-2 only SOD serum demonstrated significant difference. This may be partly due to the resolution of periodontal inflammation and partly because of the additional antioxidant effect of vitamin E. Vitamin E is a major lipophilic antioxidant that is known to be a lysosomal stabilizer.18 It prevents peroxidation of lipids and results in improved stability and integrity of biological membranes.41 Stable biological membranes inhibit release of ROS18,41 thus lowering the oxidant status and also preventing the damage caused by ROS. This includes prevention of DNA degradation and cell lysis,42 inactivation of tissue degradation enzymes and activation of inhibitors of tissue degradation enzymes.43 Results of our study in terms of increase in the levels of serum SOD are in agreement with Dwivedi et al12 as they demonstrated a significant increase in the activities of antioxidant enzymes including SOD in patients of myocardial infarction as well as healthy subjects after vitamin E administration compared to no vitamin E group. The concentration of vitamin E has been reported to be significantly low in saliva compared to serum.44 As vitamin E is fat soluble and thus not secreted in saliva, therefore the additional antioxidant effect of vitamin E could not be appreciated in saliva in contrast to serum. This explains why no significant difference in salivary SOD level change was observed in comparison to SOD serum.

Components of the antioxidant system work in synergy and not isolation,20 assaying other components of lipid peroxidation and antioxidant network would have improved the quality of the study. However, non-inclusion of other antioxidant assays in the methodology does not dilute the quality of findings of effectiveness of adjunctive vitamin E supplementation in periodontal therapy. There are studies that demonstrate a concomitant rise in the levels of glutathione peroxidase (GPx) and catalase (CAT) activity along with SOD activity following vitamin E administration.19,21 Thus, the basis of effectiveness of adjunctive vitamin E supplementation is the total antioxidant system working in harmony rather than SOD component alone. Moreover, literature also provides evidence of reduction in the levels of lipid peroxidation after vitamin E administration19,20 and negative correlation between lipid peroxidation and SOD activity.45

7


Therefore these results must be comprehended as pioneer findings which only opens a gateway for future research involving assays of other markers of lipid peroxidation and antioxidants.

Also, vitamin E levels at baseline and follow-up were not assessed which might have further improved the quality of the study.

CONCLUSION Therefore, in light of the limitations, the following findings may be comprehended. Effect of vitamin E supplementation as adjunct to SRP is demonstrated by significant improvement in clinical parameters and SOD levels compared to SRP alone. Thus it could be concluded that vitamin E may be effective as an adjunct to SRP in the management of periodontitis and in improving the systemic antioxidant status. Possible recommendation of vitamin E supplementation with routine SRP can only be considered after the validation of these preliminary findings by multiple studies with larger sample size.

CONFLICT OF INTEREST: The authors have no conflict of interest and all the facilities were provided by the institution itself.

The authors have no conflict of interest and all the facilities were provided by the institution itself.

REFERENCES 1. Keith L. Kirkwood. Immunity and inflammation: basic concepts. In: Carranza FA, Cochran DL, Kenney EB,

Giannobile WV, Novak MJ. eds. Carranza’s Clinical Periodontology. Saunders: An imprint of Elsevier 11830 Westline industrial drive, St. Louis, Missouri 63146; 2006:209-227.

2. Panjamurthy K, Manoharan S, Ramachandran C.R. Lipid peroxidation and antioxidant status in patients with periodontitis. Cell Mol Biol Lett 2005;10:255-264.

3. Buduneli N, Kardesler l, Isik H, Wilis C.S. Effects of smoking and gingival inflammation on salivary antioxidant capacity. J Clin Periodontol 2006;33 :159-164.

4. Akalin F.A, Baltacioglu E, Alver A, Karabulut A. Lipid peroxidation levels and total oxidant status in serum, saliva and gingival crevicular fluid in patients with chronic periodontitis. J Clin Periodontol 2007;34:558-565.

5. Gustafsson A, Asman B. Increased release of free oxygen radicals from peripheral neutrophils in adult periodontitis after Fca-receptor stimulation. J Clin Periodontol 1996;23:38-44.

6. Brock G.R, Butterworth M.J, Matthews J.B, Chapple I.L.C. Local and systemic total antioxidant capacity in periodontitis and health. J Clin Periodontol 2004;31:515-521.

7. Jacoby BH, Davis L. the electron microscopic immunolocalization of a copper-zinc superoxide dismutase in association with collagen fibres of periodontal soft tissues. J Periodontol 1991;62:413-420.

8. Kim S.C, Kim O.K, Kim O.J, Kim Y.J. Antioxidant profile of whole saliva after scaling and root planing in periodontal disease. J Periodontol Implant Sci 2010;40:164-171.

9. Akalin F.A, Toklu E, Renda N, Analysis of superoxide dismutase activity levels in gingiva and gingival crevicular fluids in patients with chronic periodontitis and periodontally healthy controls. J Clin Periodontol 2005;32:238-243.

10. Canakci V, Yildrim A, Canakci F.C, Eltas A. Total antioxidant capacity and superoxide dismutase activity levels in serum, saliva and gingival crevicular fluid in preeclamptic women with and without periodontal disease. J Periodontal 2007;78:1602-1611.

11. Akalin F.A, Baltacioglu E, Alver A, Karabulut E. Total antioxidant capacity and superoxide dismutase activity levels in serum and gingival crevicular fluid in pregnant women with chronic periodontitis. J Periodontol 2009;80:457-467.

8


12. Dwivedi V.K, Chandra M, Misra P.C, Misra M.K. Effects of vitamin E on platelet enzymatic antioxidant in the patients of myocardial infection. Indian J Clin Biochem 2005;20:21-25.

13. Bhartiya U.S, Yogita R.S, Joseph L.J, Hawaldar R.W. Evaluation of radioprotective effect of turmeric extract and vitamin E in mice exposed to therapeutic dose of radioiodine. Indian J Clin Biochem 2008;23:382-386.

14. Saxena R, Garg P. Vitamin E provides protection against in vitro oxidative stress due to pesticide (chlorphrifos and endosulphan) in goat. Gerf Bull Biol 2010;1:1-6.

15. Likoff R.O, Guptill D.R, Lawrence L.M, Mckay C.C. Vitamin E and aspirin depress prostaglandins in protection of chickens against Escherichia coli infection. Am J Clin Nutr 1981;34:245-251.

16. Tahan G, Aytac E, Aytekin H, et al. Vitamin E has a dual effect of anti-inflammatory and antioxidant activities in acetic acid-induced ulcerative colitis in rats. Can J Surg, 2011;54(5):333-338.

17. Nazrun A.S, Norazlina M, Norliza M, Nirwana S.I. The anti-inflammatory role of vitamin E in prevention of osteoporosis. Advances in Pharmacological Sciences 2012, article ID 142702. doi:10.1155/2012/142702.

18. Ehrlich H.P, Tarver H, Hunt T.K. Inhibitory effects of vitamin E on collagen synthesis and wound repair. Ann Surg 1972;175:235-40.

19. El-Azab SM, Morsy HK, El-Adawy EH, Seleem AK. Does vitamin E influence oxidative stress and renal anaemia in haemodialysed patients? Mansoura J Forensic Med Clin Toxicol 2007; july 15(2):129-146.

20. Seven A, Guzel S, Seymen O et al. Effects of vitamin E supplementation on oxidative stress in streptozotocin induced diabetic rats: investigation of liver and plasma. Yonsei Med J 2004;45(4):703-710.

21. Liu Y, Wang WN, Wang AL, Wang JM, Sun RY. Effects of dietary vitamin E supplementation on antioxidant enzyme activities in Litopenaeus vannamei (Boone, 1931) exposed to acute salinity changes. Aquaculture 2007;265(1-4):351-358.

22. Page RC and Eke PI. Case definitions for use in population based surveillance of periodontitis. J Periodontol 2007:78;1387-1399.

23. Silness J, Loe H. Periodontal disease in pregnancy. II. correlation between oral hygiene and periodontal condition. Odontol Scand 1964; 22:121-135.

24. Loe H, Silness J. Periodontal disease in pregnancy. I. prevalence and severity. Odontol Scand. 1963; 21:533-551.

25. Tamaki N, Tomofuji T, Ekuni D et al. Short term effects of non-surgical periodontal treatment on plasma levels of reactive oxygen metabolites in patients with chronic periodontitis. J Periodontol 2009;80:901-906.

26. SA Moussa. Oxidative stress in diabetes mellitus. Rom J Biophys 2008;18(3):225-236.

27. Compan VN, Madrid EM, Cruz BH, et al. Interaction between oxidative stress and smoking is associated with an increased risk of rheumatoid arthritis: A case control study. Rheumatology (Oxford)2012;52(3):487-493.

28. Rai B., Kharb S, Jain R, Anand S.C. Salivary vitamin E and C in lichen planus. Gomal J Med Sci .2008;6:91-2.

29. Waddington RJ, Moseley R, Embery G. Reactive oxygen species: a potential role in the pathogenesis of periodontal diseases. Oral Dis 2000;6:138-151.

30. Fridovich I. Superoxide anion radical (O2-), superoxide dismutases, and related matters. J Biol Chem 1997;272:18515-18517.

31. Wei D, Zhang X.L, Wang Y.Z, Yang C.X. Lipid peroxidation levels, total oxidant status and superoxide dismutase in serum, saliva and gingival crevicular fluid in chronic periodontitis patients before and after periodontal therapy. Aust Dent J 2010;55:70-78.

32. Culter R.G. Antioxidant and aging. Am J Clin Nutr 1991;53:373-379.

33. Guentsch A, Preshaw PM, Sybille BS, Klinger G, Glockmann E, Sigusch BW. Lipid peroxidation and antioxidant activity in saliva of periodontitis patients: effect of smoking and periodontal treatment. Clin Oral Investig 2008;12:345-352.

9


34. Sculley DV, Simon C. Periodontal disease is associated with lower antioxidant capacity in whole saliva and evidence of increased protein oxidation. Clin Sci 2003;105, 167-172.

35. Ladki RD, Pellat B, Chahine R. Decrease in the total antioxidant activity of saliva in patients with periodontal diseases. Clin Oral Investig 2003;7:103-107.

36. Sobaniec S, Sobaniec W, Sendrowaski K. Sobaniec S. Antioxidant activity of blood serum and saliva in patients with periodontal disease treated due to epilepsy. Adv Med Sci 2007;52:204-206.

37. Sesso HD, Buring JE, Christen WG et al. Vitamins E and C in the prevention of cardiovascular disease in men. J Am Med Assoc.2008;300(18):2123-2133.

38. Lee IM, Cook NR, Gaziano JM et al. Vitamin E in the primary prevention of cardiovascular disease and cancer. J Am Med Assoc.2005;294(1):56-65.

39. Majagi S.I, Bhosle T.N, Patil P.A. Anti-inflammatory and analgesic activity of d-alpha-tocopherol acetate and its interaction with aspirin in wister rats. Int J Drug Dev Res 2011;3:86-93.

40. Maniam S, Mohamed N, Shuid A. N, Soelaiman I. N. Palm tocotrienol exerted better antioxidant activities in bone than α-tocopherol. Basic Clin Pharmacol Toxicol,2008;103(1): 55-60.

41. Mackay D, Miller AL. Nutritional support for wound healing. Altern Med Rev 2003; 8(4):359-377.

42. Elkashef A.M, Wyatt R.J. Tardive dykinesia: possible involvement of free radicals and treatment with vitamin E. Schizophr Bull 1999;25:731-740.

43. Asman B, Wijkander P, Hjerpe A. Reduction of collagen degradation in experimental granulation tissue by vitamin E and selenium. J Clin Periodontol 1994; 21: 45-47.

44. Saral Y, Coskun B K, Ozturk P, Karatas F, Ayar A. Assessment of salivary and serum antioxidant vitamins and lipid peroxidation in patients with recurrent aphthous ulceration. Tohoku. J. Exp. Med., 2005, 206, 305-312.

45. Canakci CF, Cicek Y, Yildirim A, Sezer U, Canakci V. Increased levels of 8-hydroxydeoxyguanosine and malondialdehyde and its relationship with antioxidant enzymes in saliva of periodontitis patients. Eur J Dent 2009;3:100-106.

CORRESPONDING AUTHOR: Dr Neha Singh, Post Graduate Student, Department of Periodontics and Oral Implantology, Post Graduate Institute of Dental Sciences, Rohtak,

Haryana, India, Postal code-124001, E-Mail Address: [email protected] Submitted December 17, 2012; accepted for publication April 20, 2013.

10


Table 1:

Demographic and Clinical parameters for control and test group at baseline.(median with minimum – maximum)

Control group (n=22) Test group (n=38) Parameter Median Minimum - Maximum Median Minimum - Maximum 27.50 37.50 Age (in years) 22 - 50 17 - 58

Male:female 1:3 4:15 .22 1.75 PI 05 - .55 .81 – 2.80 .20 1.80 GI .04 – 1.28 1.16 – 2.93 2.60 65.59 BOP (%) .00 – 5.10 51.46 – 78.86 1.19 3.61 PPD (mm) .17 – 2.18 2.31 – 4.96 .15 3.99 CAL (mm) .04 – 1.44 1.66 – 6.79

Table 2:

Biochemical parameters for control and test group at baseline (Mann-Whitney-U test) Control group (n=22) Test group (n=38) SOD activity (%) Median Minimum - maximum

Median Minimum - maximum

21.6600 10.9500* Saliva 9.14 – 56.04 0.73 – 55.68 69.3550 59.8150† Serum 53.85 – 91.31 10.31 – 85.18

* significantly different between groups(p<0.05)

† significantly different between groups(p<0.001)

Table 3:

Periodontal and Biochemical parameters at baseline and after 3 month follow-up (Wilcoxon signed-rank test) Parameter Treatment group-1 (TG-1) (n=19) Treatment group-2 (TG-2) (n=19) Baseline Follow-up Baseline Follow-up

1.6200 0.6200* 1.8300 0.4000* PI 0.81 – 2.80 .25 – 1.91 1.11 – 2.64 .40 – 2.50 1.7200 1.0400* 1.9400 0.6700* GI 1.21 – 2.35 .24 – 1.40 1.16 – 2.93 .20 – 2.41 64.2800 18.5400* 67.3500 6.1800* BOP (%) 57.14 – 78.36 10.34 – 23.69 51.46 – 76.39 1.28 – 12.69 3.5500 2.0600* 3.7300 1.8500* PPD (mm) 2.31 – 4.33 1.12 – 3.03 2.38 – 4.96 1.14 – 2.98 3.8200 2.8500* 4.1200 2.3400* CAL (mm) 1.91 – 6.79 .57 – 5.59 1.66 – 6.50 .95 – 6.20 10.7100 17.2700† 11.1900 14.3600† Saliva SOD

activity (%) 0.73 – 42.15 6.67 – 55.23 1.75 – 55.68 3.53 – 57.93 59.4100 62.0600† 61.0600 85.9000* Serum SOD

activity (%) 23.43 – 83.65 55.19 – 90.92 10.31 – 85.18 49.06 – 95.63

* significantly different between baseline and follow-up (p<0.05)

11


3

† significantly different between baseline and follow-up (p<0.001)

Table 4:

Improvement in periodontal and biochemical parameters (Mann-Whitney-U test) Parameter TG-1 (n=19) TG-2 (n=19)

1.0200 1.4100* Δ PI .03 – 1.88 -.06 – 2.17 0.6800 1.1700* Δ GI .15 – 1.33 .37 – 2.26 47.8000 60.7600† Δ BOP (%) 38.12 – 58.67 46.30 – 73.01 1.1900 1.6800* Δ PPD (mm) .40 – 2.05 .27 – 3.44 1.1300 1.7600* Δ CAL (mm) .23 – 2.41 .04 – 3.57 6.7800 3.6800 Δ Saliva SOD activity (%) -14.08 – 36.77 -9.76 – 39.98 4.8400 25.1300† Δ Serum SOD activity (%) -11.94 – 16.54 -2.60 – 68.68

* significantly different between treatment groups (p<0.05)

† significantly different between treatment groups (p<0.001)

Table 5:

Comparison of biochemical parameters of TG-1 and TG-2 at follow-up with control group (Mann-Whitney-U test) Δ SOD activity (%) Control group (n=22) TG-1 (n=19) TG-2 (n=19)

21.6600 17.2700 14.3600 Saliva 9.14 – 56.04 6.67 – 55.23 3.53 – 57.93 69.3550 62.0600 85.9000* Serum 53.85 – 91.31 55.19 – 90.92 49.06 – 95.63

* significantly different between control group and treatment group (p<0.05)

‡ : capsule Evion, Merck India Pvt. Ltd., India.

§ : Hu-Freidy scalers and curettes, USA

║ : P5 booster, Suprasson, Satelec

¶ : SOD assay kit, Sigma Aldrich Pvt. Ltd., USA.

# : SPSS, version 17.0 for windows, SPSS, Chicago, IL.

12

Documents

Vitamin E Supplementation, Superoxide Dismutase Status, and Outcome of Scaling and Root Planing in Patients With Chronic Periodontitis: A Randomized Clinical Trial