The power of this study was fixed at 90% and margin of error being 5%, so around 80 subjects were needed for the study as per statistical sample size.

In order to improve the power of study and to reduce bias 90 samples aged 18-60 years were selected from the outpatient department of Department of Periodontics, P.M. Nadagouda Memorial Dental College, Bagalkot, Karnataka, India. The study was conducted for a period of five months. The protocol for this cross-sectional study was approved by the Institutional Ethical Committee. Prior to enrolment in the study, a written consent was obtained from the candidates who fulfilled the inclusion criteria.

After clinical and radiographic examination, the subjects were divided in three groups. Group I consisted of 30 healthy subjects showing absence of clinical and radiographic manifestations of periodontal disease, at least 20 teeth present. Group II comprised of 30 subjects diagnosed as chronic periodontitis with the presence of bleeding on probing and clinical attachment level of 3 mm or more at more than 30% of all sites in the mouth [20]. Group III consisted of 30 subjects fulfilling the criteria of Group II in addition to being smokeless tobacco users. Various forms of smokeless tobacco such as mishri, gutka, mawa, khaini users were included in the study.

Subjects suffering from systemic conditions (rheumatic fever, heart diseases, hypertension, diabetes, liver and kidney disease), any infection requiring prophylactic antibiotic therapy, pregnant female, lactating women, subjects on hormonal contraceptives or on hormone replacement therapy, on steroids and NSAIDs (for previous three months) or on vitamin supplements, alcoholics and having undergone scaling and root planing in past six months were excluded from the study as they proved to affect the levels of GGT.

After proper grouping of the subjects, a full mouth periodontal examination was performed by a single examiner. The periodontal parameters pocket probing depth; clinical attachment level and gingival index (Loe and Silness 1963) were assessed using a Williams periodontal probe by a single examiner. Plaque index and OHI-S gave information about the amount of debris or calculus present but no information about the amount of inflammation present. Since in this study GGT was hypothesised to increase in inflammatory conditions, gingival index which provides an insight in the amount of inflammation present was assessed.

Using aseptic precautions 2 ml of venous blood was drawn from median cubital vein into a serum separating tube. Serum obtained was immediately centrifuged at 2500 rpm for 15 minutes then transferred to laboratory for analysis of GGT by VITROS 250 fully automated dry chemistry analyzer.

The VITROS GGT slide method is performed using the VITROS GGT slides and the VITROS chemistry products calibrator kit 3 on VITROS 250 /350/950/5, 1 FS and 4600 chemistry systems and the VITROS 5600 integrated system. A drop of the sample was deposited on the slide and was evenly distributed by the spreading layer to the underlying layers. GGT catalyses the transfer of the gamma glutamyl portion of a L-gamma glutamyl-p-nitroanilide to glycylglycine leading to the production of L-gamma glutamyl-glycylglycine (glu-gly-gly) and 3 carboxy-4-nitroaniline. p-nitroaniline at 37°C at 400 nm. The rate of change in reflection density is measured and is used to calculate the enzyme activity of GGT [21].

The data collected was analyzed using computer software, IBM Statistical Package for Social Sciences Version 20.0. Analysis was done using unpaired t-test and Pearson’s correlation coefficient. Data were expressed as mean and standard deviation. A p<0.001 were considered to be statistically significant.

As subjects were recruited in the study, when they came to the college outpatient department for a dental examination, age and sex matching was not possible for the three groups. The mean age of chronic periodontitis subjects is higher than the healthy group [Table/Fig-1].

GGT level was found to be highest in the chronic periodontitis group followed by smokeless tobacco users with chronic periodontitis and then the healthy group [Table/Fig-2].

The mean age of males was higher than the females in the study groups [Table/Fig-1]. Non-significant difference was found in healthy group. The mean gingival index and pocket probing depth was found to be highest in the chronic periodontitis group [Table/Fig-3,4].

These parameters showed a positive correlation with GGT levels in the study groups. Chronic periodontitis group showed a statistically significant difference in the loss of clinical attachment level than the smokeless tobacco group. [Table/Fig-5]. All the three parameters show positive correlation with the GGT levels in the study groups.

Chronic periodontitis is usually seen affecting the higher age group. This fact is very well reflected in the present study [Table/Fig-1]. Neutrophils are at the forefront of diseased periodontium. They face the bacterial challenge by producing proteolytic enzymes and oxygen by inducing oxidative burst. Lipopolysaccharides and DNA from plaque bacteria cause activation of Activator protein-1 (AP-1) and NF-kappa β pathways in gingival fibroblasts via CD14 and Toll like receptor-4 which causes activation of osteoclasts and increases concentration of Matrix-Metallo Proteinase (MMPs) which ultimately causes tissue damage. Recruitment and activation of hyper responsive Polymorpho-Nuclear (PMNs) cells by inflammatory cytokines produced by bacteria hastens the ROS production [22]. Enzymatic antioxidant glutathione plays a major role in maintaining the intracellular redox balance and thus regulating signaling pathways which are affected by OS.

GGT being an intracellular enzyme is a good indicator to assess cellular damage. It reflects the pathological changes occurring in the periodontal tissues [23]. Inflamed gingival tissues undergo various metabolic changes that cause it to be released in high amounts in various body fluids such as blood, saliva, gingival crevicular fluid etc. In the present research chronic periodontitis subjects showed higher levels of GGT as compared to healthy subjects [Table/Fig-2], which is in accordance to a study done by Sreeram M [17]. Other studies showing similar results in saliva and gingival crevicular fluid include those by Todorovic T et al., and Agnihotram G et al., [23,24].

Smokeless tobacco is a known risk factor for periodontitis, affecting the prevalence, extent and severity of periodontal disease. Tobacco in any form is known to cause potential alteration in the systemic as well as oral health [6]. Monocyte and oral keratinocyte production of inflammatory mediators is affected thus eventually leading to development of localized tissue alterations [7]. It’s use also induces dose dependent increase in hepatic mitochondrial and microsomal lipid peroxidation as well as hepatic DNA damage thus producing oxidative tissue damage and apoptosis. This strongly suggests that smokeless tobacco induces an OS [7]. The smokeless tobacco users in the present study also exhibited OS. The results obtained were in accordance to a study done by Alwar V et al., [25]. A study done on experimental rats by Ugbor CI et al., also gave similar results [26].

In this study less clinical periodontal damage was exhibited by smokeless tobacco users with chronic periodontitis which showed relatively lower OS compared to chronic periodontitis indicating the presence of a positive correlation between the amount of destruction and the value of GGT obtained. The vasoconstrictive effects of nicotine interfere with the inflammatory signs and symptoms [4]. The tar products produced due to consumption of tobacco result in black or brown staining on the tooth surface. Smokeless tobacco users being more concerned about the unaesthetic appearance of teeth reported earlier to clinic thereby with less destruction of underlying periodontal tissues. This may be the probable explanation of lower GGT values in the smokeless tobacco subjects compared to the chronic periodontitis subjects.

Currently in the field of OS, biomarkers have been gaining a lot of interest lately. Emphasis is on development of functional biomarkers of OS status, i.e, biomarkers integrating effects of exposure to oxidants along with full range of antioxidant protective mechanisms in vivo [18]. Traditional methods of diagnosis of periodontal disease based on clinical and radiographic parameters are being replaced by newer methodologies wherein the periodontal disease activity can be quantified and differentiation can be done between active and quiescent site. The newer technologies being developed although highly sensitive and specific are invasive and expensive. The development of a simple, less invasive, quicker, easy to interpret, economical and yet confirmatory test is the need of the hour. Radiographs require a longer period to detect measurable changes in bone. The quest for a reliable biochemical test that detects inflammatory changes in a short period is still on. Biochemical investigations are currently preferred due to them being simple, less invasive, less time consuming, comparatively economical and may also be useful for pre and post-treatment evaluation of therapy. Moreover, biochemical alteration reflects tissue changes at cellular level.

According to a study by Lim JS et al., the activity of serum GGT was found to be unchanged in repeated tests during 40 weeks when frozen stored samples were thawed and then frozen again after each testing [18]. The value of their activity can correlate with the amount of damage and can indicate the prognosis of the course of this disease [23].

From the present study, it is evident that by estimation of GGT in circulation of chronic periodontitis patients, the degree of oxidative damage can be assessed. So we speculated that its estimation would have important clinical significance to prevent further periodontal damage and thereby maintain a healthy periodontium in the high risk cases. By correcting the underlying deficiency of antioxidants the treatment plan can be improved. This may be helpful for successful management of periodontitis, thereby arresting it in early stages and avoiding the possible consequences i.e., mobility and thus loss of teeth. The limitations of the present study includes having a smaller sample size. So, further elaborate studies on the estimation of before and after scaling and root planing levels are needed to ascertain the actual role of these parameters in the initiation and promotion of periodontitis.

There is a lack of gold standard methods for measuring OS generated during periodontal tissue destruction. There is a paradigm shift in the techniques and advanced researches for detection of free radicals. Measurement of serum GGT being an easy, reliable and inexpensive test, it can be put to use as a diagnostic chair side test for early detection of OS suggesting high scope for it to be recognized as a biomarker for OS damage. Researchers have confirmed that periodontal diseases are not just confined to the oral cavity but are also known to have an effect on systemic health. Hence, GGT can be a promising option to establish both a good oral and systemic health. Further long term studies are needed to justify its role as a biomarker.