A total number of 50 male patients were selected from the out–patients reporting to Department of Periodontics, Yenepoya Dental College & Hospital Mangalore, who gave consent to participate in the study. Sample size was determined based on the pilot study and estimated to be 50 with 90% power and 95% confidence interval. Subjects who fulfilled selection criteria for the study were equally divided into two groups; Group I smokers with chronic generalized periodontitis (25) and Group II non smoker/ non-tobacco users with chronic generalized periodontitis (25). Ethical clearance was duly obtained from the institutional ethics committee.

Patient population: 50 males

All subjects followed a regular oral hygiene regimen, brushing the tooth once daily in the morning with tooth brush and tooth paste using horizontal/ scrub technique of brushing. None of the subjects reported using any form of interdental aids/ mouth washes.

Armamentarium used includes mouth mask, disposable latex gloves, mouth mirror, tweezers, No.5 explorer (Shepard’s hook), CPITN-probe, kidney tray, cotton rolls, 2ml disposable plastic vials for transfer of specimen to the laboratory, saliva measuring unit, digital stopwatch, salivary calcium estimation kit (Bhat Bio-Tech India (Pvt) Ltd.), sterilized test-tubes, spectrophotometric unit.

The clinical parameters assessed included Calculus component of Oral Hygiene Index Simplified (OHI-S) and Community Periodontal Index (CPI) [6,7].

Subsequently a 2 ml of unstimulated whole saliva was collected; during 8 am-12 pm; at least 30 min after the last meal and also restraining from smoking for the past one hour. Initially the subjects were asked to swallow the residual saliva and allow the saliva to be accumulated in the floor of the mouth in the next two minutes. The sample collected in a vial was subjected to biochemical analysis by spectrometric method for the estimation of salivary calcium which was carried out in the next 20 min.

The same operator recorded all the clinical data which was entered in Microsoft excel sheet. The data in this study was analyzed by using unpaired t-test and Mann-Whitney test by SPSS 17. A p-value of <0.05 was considered to be statistically significant.

The examinations on the 50 male subjects revealed that the mean calcium content of the saliva was significantly higher (p< 0.0005) for the Group I (2.2700) than for the Group II (1.7260) as demonstrated in [Table/Fig-1]. [Table/Fig-2] highlights the elevated extent of calculus in Group I (p<0.0005). On comparing the CPI scores and the loss of attachment, a statistically significant relationship were found between Group I and Group II [Table/Fig-3,4].

Although salivary calcium is increased in periodontitis [8], its significantly higher rates seen in smokers compared to non-smoker, highlights the role of smoking in the progression of periodontitis; as it seems that salivary calcium, due to its affinity for being readily taken up by plaque, is an important factor with regard to calculus formation and also the level of attachment loss as revealed by the present study.

Cigarette tobacco varies in its nicotine content, but common blends contain 15-25 mg per cigarette. Cotinine, an alkaloid, is one of the metabolite of nicotine. Robson et al., stated that collection of unstimulated saliva produced highest salivary nicotine concentration, whereas stimulated technique results in the highest salivary nicotine concentration [9]. Because of the ease of collection and storage, effect of calcium on stimulated saliva of smokers over non-smokers has well been documented. Far less attention has been paid with respect to unstimulated saliva, hence this study was carried out to quantify the calcium profile in whole saliva, from smokers and non-smokers with chronic generalized periodontitis; followed by comparison of the salivary calcium and the periodontal status between both the groups.

Analysis of saliva may offer a cost-effective approach to assess periodontal disease in large populations (Kaufman and Lamster,) and salivary calcium may be important with regard to both dental and gingival health by way of effect on mineralization of plaque (White) [10].

Plaque with high calcium content rapidly hardens and is therefore an indirect cause of poor oral hygiene. Calcium concentration of plaque significantly increases due to smoking. It has been shown previously that smokers have difficulty in performing efficient tooth brushing. Bergström et al., observed supragingival calculus was strongly associated with smoking [11]. A dose-dependent relation exists between the influences of smoking on the amount of supragingival calculus. Supra-and sub gingival calculus is known to be specifically favourable for microbial growth and retention [12].

Sevón states, subgingivally retained, rapidly mineralizing plaque may be a significant reason for periodontitis susceptibility [13]. Hence, it appears that one of the main oral side effects of smoking is rapid mineralization of plaque and associated disease progression when compared to non-smokers [12].

Salivary calcium, due to its affinity to be readily taken up by plaque, is a principle factor not only with regard to the initiation of periodontitis but also significantly with regard to dental health [14]. The calcium levels of saliva may also reflect the fluctuations in dietary calcium and general calcium turnover. Thus monitoring for change in salivary calcium might be a useful tool to establish favourable response to periodontal therapy (Zuabi et al.,) [15]. The assay of salivary calcium level could perhaps be also used in future risk assessment of periodontitis (Sewon et al.,) [16]. Assessment of salivary calcium from both Group I and Group II revealed increased salivary calcium in both the groups compared to the health. Although calcium levels were significantly higher (p<0.0005) in Group I compared to Group II. This difference was statistically significant, which suggests an existence of an altered calcium metabolism and absorption among the periodontitis subjects and is seen pronounced among the smokers. This is in accordance with the findings of Sevón et al., and Macgregor et al., though the assessment was done using stimulated saliva [5,17]. A positive correlation was also exhibited by female smoker patients with periodontitis and higher salivary calcium (Endre Kiss), which may also be an influence of the hormonal shifts exhibited by the females [12]. Since the subject population was of middle aged females (30-62 years), decline in bone mass as a consequence of osteoporosis is accelerated with the onset of menopause and hormonal variations in these age group individuals. Salivary sample collected is of stimulated in origin. Hence the saliva obtained depends on which gland is been stimulated. Calcium concentration is about 45% higher in the submandibular glands than in the parotid glands. A study by Mandel et al., has shown high concentration of plaque and submandibular saliva has been found in heavy calculus formers than in light calculus formers, signifying the need for unstimulated salivary sample [18]. Whereas Zuabi et al., stated decreased calcium concentration of non-stimulated saliva of tobacco smokers. According to Sewón diminished skeletal bone density, a known adverse effect of smoking, may reflect elevated levels of salivary calcium highlighting the existence of disturbances in calcium metabolism and absorption [12,15,16].

In health, secretion of saliva occurs in two stages-first, secretion occurs into the glandular acini which is approximately similar to extra cellular fluid (ECF) and then this primary secretion flows into the acinar duct where reconditioning occurs, some substances are actively reabsorbed while some are actively secreted but during maximal salivation, there is not much time for the reconditioning process to occur and hence, the total concentration of solute in saliva reduces [19]. Smoking in general increases the activity of salivary glands and a level of tolerance develops to the salivatory effects of smokers resulting in habitual smokers to salivate minimal compared to novice smokers [20]. Hence elevated salivary calcium may also be linked to decreased salivary flow among the smokers and may also predict calcium dissolution from teeth matrix. Bone resorption is also directly regulated locally by ionized calcium generated as a result of osteoclastic resorption [21].

On comparing the calculus scores (Simplified Oral Hygiene Index), the mean value for the Group I was 2.472 and for Group II, it was 1.501 which is statistically significant (p<0.0005); emphasizing that plaque sample with increased calcium content mineralizes rapidly. The community periodontal index score and loss of attachment were statistically significant among both Group I and Group II, in which smokers showed increased periodontal pockets and these results are in accordance with the study by Baharin B, et al. It was found that smokers demonstrated higher proportion of sites probing ≥ 5mm and more bone loss in all regions. In a longitudinal study, Machtei et al., states that smokers had three times more attachment loss and two times more alveolar bone loss than non smokers [22,23].

Within the limitations, the findings of the study support the view that increased salivary calcium reflecting altered calcium turnover, fluctuations in dietary calcium, enhanced mineral dissolving action of osteoclast induced bone resorption among the smokers could act as a risk factor for the development and progression of periodontal diseases, possibly by raising the mineralization potential of dental plaque and hampering the normal structure and function of alveolar bone. The clinical significance of these findings needs, however, to be determined in further, large scale controlled studies.

Cigarette smoking is a major risk factor for periodontitis. It is often associated with high susceptibility, comparatively early onset disease, and more severe and wide spread periodontal destruction and treatment failure. Elevated salivary calcium in smokers, emphasizes the role of smoking in the progression of periodontitis; as it seems that salivary calcium plays a significant role with regard to calculus formation and also the level of attachment loss revealed by our present study. Whilst the limitations of this study include severity of periodontitis was not standardized and serum calcium level was not assessed.

Hence, the usage of salivary biomarkers appears promising for future application to diagnose periodontal diseases and to prognosticate periodontal treatment outcomes. The present study clearly demonstrates that high salivary calcium concentration seems to be a characteristic feature of periodontitis, which is worsened by the synergistic action of smoking, revealing the significance of habit cessation. This highlights the supportive role of dentist in an attempt to improve the oral and general health status of an individual.