An experimental study was planned on a population of smokers with or without periodontitis. A total of 165 patients, 86 males and 79 females (matched), within age range of 30 to 50 years, with Indian nationality and belonging of high, middle and low socioeconomic status were selected from the Outpatient Department of Periodontics and Implantology, Manubhai Patel Dental College and Hospital, Vadodara, Gujarat, India, after fulfilling the inclusion andexclusion criteria. The study protocol was approved by Institutional Research and Ethical Committee (IEC), Bhavnagar University, India. Informed consent was provided by all the participants. Sample size of 165 patients was chosen, to estimate Standard Deviation (SD) of CRP level by 0.05 with 0.01 absolute precision at 1% risk. A pilot study was conducted and based on the finding of the pilot study, statistical software NMASTER2 was used for sample size determination. Patients who underwent periodontal treatment, who were prescribed drugs such as antibiotics, non steroidal anti inflammatory or immune-modulators in last six months, lactating females, obese patients, one having recent history of trauma and on nicotine replacement therapy were excluded. Patients with systemic diseases or infection were also excluded from the study. Based on smoking and periodontal status, the patients were divided into four groups.

Group 1 (45 participants)- non-smokers with chronic periodontitis. The non-smoker participants consisted of patients who never smoked. Patients with moderate generalized chronic periodontitis (i.e., 3-4 mm attachment loss, ≥ 30% sites involved of the mouth) were recruited for the study [8].

Group 2 (45 participants)- current smokers without chronic periodontitis. Patients who have smoked ≥ 5 cigarettes per day for five years or more were considered as current smokers.

Group 3 (45 participants)- current smokers with chronic periodontitis.

Group 4 (30 participants)- former smokers without chronic periodontitis. Patients who quit smoking for ≥ 6 months were considered as former smokers.

Clinical parameters including Plaque Index (PI) [9] (on selected index teeth using mouth mirror and explorer), Bleeding on Probing (BOP) [10] and Clinical Attachment Level (CAL) [11,12] were evaluated.

History of smoking was taken including the number of years of smoking, number of cigarettes per day, type of cigarette and type of smoking habit.

To assess CRP, 5 ml blood was drawn by venipuncture from the median cubital vein which lies within the cubital fossa anterior to the elbow using needle of 23 guage in plain bulb. The blood was subsequently centrifuged for 15 minutes and serum obtained was used for the estimation of CRP. A 5 μl of serum sample was mixed with the working reagent (1 ml of latex reagent and 9 ml of diluent) and the sample was run on a Mindray Semiautomatic Biochemical Analyser. Quantitative measurement of serum CRP is done by turbidimetric test known as turnilatex [13].

Data analysis was done by Post-hoc test and ANOVA.

Mean values, SD and p-value for metabolic parameter of CRP are presented in [Table/Fig-1] and results for clinical parameters CAL, BOP and PI are displayed in [Table/Fig-2]. Multiple intergroup comparisons with p<0.05 using post-hoc test (Tukey HSD) depending on CRP levels showed a statistical significant difference between Group 3 and Group 4 [Table/Fig-3]. An association between cigarette and bidi smoking to CRP level was evaluated using Chi-square test. The test results indicated a non significant relationship (p= 0.367). Corelation of CRP with CAL, BOP and PI evinced CRP having significant corelation with BOP, unlike with PI and CAL [Table/Fig-4]. There was a significant difference observed between mean CRP values of each group (p=0.36). [Table/Fig-5] shows group wise age and gender distribution of 165 participants with mean being 36.80±8.4 years.

Pathologic changes occurring in tissues of peridontium- gingiva, periodontal ligament, cementum and alveolar bone is known as periodontal disease. The branch of “Periodontal Medicine” was introduced to show the relationship between oral infection and systemic disease. APP levels elevates (positive APR) or lessens (negative APR) by minimum 25% in inflammatory reactions, with CRP displaying the quantification of the acute phase response. [14]. The purpose of these responses is to restore homeostasis and to remove the cause of its disturbance [15]. Being the most sensitive APP, CRP describes systemic inflammation by series of cytokines like IL-6 [16]. Old age, smoking, chronic bacterial infections, and chronic bronchial inflammation are the established risk factors for “high-normal” values of CRP [17].

Rise in APR have been found in localized infections due to systemic inflammation [18]. When bacteria, bacterial products and cytokines enter blood circulation, increase in value of CRP, IL-6 and neutrophils in patients with periodontal disease have been recorded [19]. Due to elevation in polymorphonuclear neutrophils released from bone marrow, smokers have higher levels of white blood cells [20]. CRP gene expression is induced during lung inflammation due to the stimulation of bone marrow cells and release of IL-β and IL-6 [21]. A research has shown that, following nine days of abstinence, when levels of inflammatory markers are measured in blood of intermittent smokers 24 hours post two cigarettes, TNF-α, IL-10 and IL-1β were same but IL-8 was elevated after three hours [22].

In the present study, subjects were divided into four groups, and the levels of serum CRP were evaluated and compared following periodontal examination. Analysis of the results showed that highest level of CRP was found in smokers with periodontitis followed by non-smoker periodontitis patients and smoker non periodontitis patients. Former smokers had minimum CRP compared to the other groups. Former smokers who had quit smoking for the longer duration had CRP levels much lower than the ones who had quit recently. Nevertheless in Group 3, it was difficult to substantiate whether the change in CRP is due to smoking or periodontitis. The many fold increase in its proportion cannot be precisely predicted and none of the studies had been able to explain the same.

Difference between Group 1 and Group 2 was not statistically significant evincing the analogous contribution put by smoking and periodontal infection on inflammatory load by elevation of CRP. This finding is strengthened by Vigushin DM et al., depicting oral disease as a risk factor for cardiovascular disease by channel of systemic inflammatory reaction and thus putting “apparently healthy” patients to cardiovascular risk [23].

Additionally, CRP levels were also found to increase with increase in frequency, number of cigarettes smoked per day and duration of time of smoking. Yet, type of smoking did not have much of out-turn. No statistical difference was found in the levels of CRP between bidi and cigarette smokers. This was supported by the Das I. in his study, which did not show any disparity in nicotine concentration of filtered and unfiltered cigarettes (p>0.05) [24]. None of the patient had the habit of reverse smoking. Similar results were found in one of the earlier studies. Male and female smokers had higher CRP compared with non-smokers (median values of 1.0 mg/l and 11.2 mg/ l for male non-smokers and smokers, respectively, and for females 2.0 mg/ l and 11.6 mg/ l, respectively) [25]. Furthermore, in our study CRP had a significant correlation with BOP, establishing the fact that active disease multiplies the inflammatory burden compared to inactive state. Besides this, elevation in CRP was noticed with the increase in extent and depth of periodontal pocket.

‘Speedwell’ survey showed that those who had stopped smoking within a year, CRP levels were 2.10 mg/l, but eventually reduced to 1.34 mg/l 15-9 years and 1.36 mg/l > 10 years post quitting, though this CRP measure was still more than never smokers [26]. Present study showed indistinguishable results with the Speedwell study, in terms of inverse inter-relationship between CRP levels and the stretch of time left for the smoking.

A positive relation between age, severity of smoking, periodontitis and pocket depth on rise of CRP was recently documented. However, BOP had negative effect on the same [27]. Yet another research proved CRP, IL-1β, IL-6, IL-8, Tumour Necrosis Factor-α (TNF-α), and homocysteine (HCY) in the serum of a Korean population were synergistically associated with periodontitis-MetS (metabolic syndrome) coexistence [28].

When measured by high sensitivity –CRP, level < 1.0 mg/l denotes low risk, 1.0 to 3.0 mg/l intermediate risk and 3.0 mg/l high risk. [29]. In current study, CRP levels above 3 mg/l was demonstrated in current smokers and participants with periodontitis, putting patients in the high risk category. Former smokers allied in class of moderate risk. In spite of that, one should always remember that CRP secreted in the body is affected by lifestyle and genetic coding of the person and it also differs between individuals [30]. Findings of our research affirm the result of the studies done by D’ Aiuto F et al., and Azizi A et al., [16,27].

Timely changes in the levels of CRP cannot be understood due to absence of follow up. Longitudinal studies needs to be done to know the phased alterations in CRP. Further, there was sample size disparity in Group 4 due to only sparse number of people quitting the habit, thus, making it difficult to reach the large sample size.

Periodontitis alone and in combination with smoking increases the systemic inflammatory burden and associated cardiovascular risk of patients. This fact should be made aware to the general population, general dentist, physicians and cardiovascular specialist to enhance early screening and multidisciplinary treatment.