ABSTRACT:Aim: with present history of smoking. The subjects

 ABSTRACT:Aim: To estimate and correlate level of ALP in smokers and non-smokers with chronic periodontitis. Materials and Methods: Study population included 30 patients in the age group of 18-60 years suffering from moderate generalized chronic periodontitis with present history of smoking. The subjects were randomly divided into three groups: (1).Group I: Healthy patients. (2).Group II: Non-smokers with Chronic Periodontitis.  (3).Group III: Smokers with Chronic Periodontitis. Following parameters were evaluated were probing pocket depth (PD), plaque index, bleeding index. A higher level of ALP in saliva samples of smokers with periodontitis was observed.Key words:Alkaline phosphatase, chronic periodontitis, saliva, smoking. INTRODUCTIONPeriodontitis is a chronic inflammatory disease of periodontium, which destroys the connective tissue and bone that supports teeth.1 Periodontal disease  generally occurs due to an imbalance between pathogenic microbes and the local and systemic host responses. However, chronic periodontitis is known to vary by race, gender, and socioeconomic status, suggesting that factors related to the social environment may also have major role in terms of disease development.2Biomarkers can be a useful tool in predicting ongoing or future disease activity. They may also possess the ability to determine the current activity status of historically diseased sites.The possible biomarkers are:      1.Bacteria and their products,      2.Inflammatory and immune products,      3.Enzymes released from host cells.      4.Connective tissue degradation products,      5.Products of bone resorptionWhole saliva, gingival crevicular fluid, plaque and serum can be used as source of specimen for these markers. The enzymes released from host cells can be easily obtained within the oral cavity either from gingival crevicular fluid or from whole saliva. As the whole saliva contains secretions from gingival crevicular fluid, it contains enzymes released by host cells in periodontal pocket during periodontal infections. Alkaline phosphatase is an important indicator of bone formation and is a phenotypic marker for osteoblast cells. The first one identified host enzymes was ALP. ALP is detected in the parotid, submandibular, and minor salivary glands, as well as in desquamated epithelial cells, leukocytes, and bacteria from dental plaque. 3 It is released by many cells polymorphonuclear neutrophils during inflammation, osteoblasts during bone formation and periodontal ligament fibroblasts during periodontal regeneration. It is most effective in an alkaline environment. The optimal pH for the ctivity of ALP is 8.0-8.5 depending on the source.4 ALP is stored in specific granules as well as secreted by vesicles of neutrophils and is mainly released during their migration to the site of infection.5 Cigarette smoking is a risk factor for many diseases, and mounting evidence suggests that smoking adversely influences periodontal health. Many authors supported the role of smoking as a risk factor for periodontitis and stated that the potential risk reduces with cessation of smoking.6 The response of an organism to the periodontal infection includes production of several enzymes and inflammation markers which can be analyzed both in serum as well as saliva.According to Centers for Disease Control (CDC) and Prevention, the smokers are classified as:i. Current smokers: Those that had smoked ? 100 cigarettes over their lifetime and smoked at the time of interview.ii. Former smokers: Those that had smoked ? 100 cigarettes over their lifetime but were not currently smoking.iii. Non-smokers: Those that had not smoked ? 100 cigarettes in their lifetime.Gao et al (1999) found that ALP activity was highest in osteoblasts, moderate in periodontal ligament, fibroblasts, and lowest in gingival fibroblasts. No activity was detected in cementoblasts .7Aim of this study is to estimate and correlate level of ALP in smokers and non-smokers with chronic periodontitis. Material & Methodology:Sample size : 10 Patients were selected per group:The subjects were randomly divided into three groups:(1).Group I: Healthy patients(2).Group II: Non-smokers with Chronic Periodontitis (3).Group III: Smokers with Chronic Periodontitis Inclusion Criteria:1.    Patients with moderate generalized chronic periodontitis.2.    According CDC classification of smokers, patient must be a current smoker.3.    Subject age between 18 to 60 years.4.    Subjects must have 20 teeth.5.    Systemic healthy individual.6.    No antibiotic consumption in last 6 months.7.    Patients who have not undergone any periodontal treatment in last 6 months.  Exclusion Criteria:1.    Patients with regular medications for any systemic diseases that might alter ALP concentration.2.    Patients with mental or physical disability.3.    Patients on antibiotics or anti-inflammatory drugs in last 3 months.4.    Betel nut users. Materials& Methods:The study was conducted in the Department of Periodontology & Implantology of among the patients visiting the daily OPD of the same college. A total of 30 subjects aged between 30 and 60 years were included in this study. Of these subjects 10 were healthy (Group I) and 10 were chronic periodontitis devoid of smoking (Group II). For comparison 10 healthy individuals were smokers with chronic periodontitis (Group III). The following standardized materials and equipment used for the purpose of study:1. AutoZyme Alkaline Phosphatase Kinetic2. Semi autoanalyzer3. Remi Centrifugal machine4. Eppendorff tube5. Ice bag6. UNC 15 probe7. Mouth mirror8. Tweezer9. Cotton          Centrifugal macchine       AutoZyme ALP kinetic       Eppendroff tube                                                                                                                                                            Semi autoanalyzerSaliva Collection:Participants were instructed to refrain from eating, drinking, and practicing oral hygiene procedures 2 hours before saliva collection. Whole unstimulated saliva was collected from all patients using expectoration into eppendroff tube. Collected samples were immediately placed on ice and transported to the laboratory, where they were centrifuged at 5,000 rpm for 10 minutes and the clear supernatants were procured.Enzyme assay:For the estimation of ALP, a diagnostic kit was used. The kit consisted of two reagents:Reagent 11. p-Nitrophenyl phosphate2. Magnesium ion (Mg2+)s3. Tris/carbonate buffer (pH 10.2±0.2 at 25ºC)Reagent 2 (Aqua-4)1. Distilled water Measurement of enzyme level was performed by a enzyme activity measurement kit. The measurement of ALP enzyme level in saliva was done with dilution of the mentioned liquid. The samples (5ml) collected from saliva were diluted in 250 ?l of 20 mMMgC12, 200 mM Tris (pH 9.8± 0.1) and 1 mg/ml of p-nitro phenol phosphate buffer and were incubated at 37 ºC for 3 hours. The interaction was then terminated by addition of 5 ?l of NaOH and the amount of absorption was determined by spectrophotometry and recorded in the form of enzyme activity unit.ALP level in saliva was measured with the automatic biochemistry analyzer (Furuno, Japan).Discussion:Periodontitis is a chronic destructive category of periodontal disease that leads to the alveolar bone resorption, which leads to bone and tooth loss. As a result of resorption, breakdown of products are released into periodontal tissues, migrating toward the gingival sulcus, and finally reaches saliva and then they are collected there. 8, 9The response of an organism to the periodontal infection includes production of several enzymes and inflammation markers which can be analyzed in saliva.10 In peridontitis, one of the mechanism of collagen loss is fibroblast phagocytize collagen fibers which contributes to the total ALP level.11 ALP is found primarily in the liver (isoenzyme ALP 1), in the bone (isoenzyme ALP 2) and small amount produced by cell lining the instestine (isoenzyme ALP 3), the placenta and the kidney. 12-14 Cigarette smoking is associated with periodontal disease, is a major risk factor that lead to increased severity of periodontal disease, rates of disease progression .15Gingival redness was lower in smokers as compared to non- smokers due to suppression of the normal inflammatory response to plaque and calculus. The reduced gingival bleeding was due to the vasoconstrictive effects of nicotine may lead to misdiagnosed of periodontitis.16,17 The vasoconstriction of peripheral blood vessels which occurs due to smoking can also affect the periodontal tissue and can lead to less overt signs of gingivitis such as redness, bleeding and exudation.17 Smoking creates an environment that favors colonization of pathogens in shallow sites and could help to explain the initiation of disease at new sites and the development of periodontitis in young smokers. Higher levels of ALP in saliva of smokers with periodontitis could explain the higher rate of alveolar bone destruction in smokers. The mean ALP levels of group 1 (healthy) were found to be lower than other two groups (with & without chronic periodontitis).Among the three groups, the mean values of ALP levels were highest in group 3. ALP showed a significant rise in both diabetic and non-diabetic patients with periodontitis as compared to control.18 Most of the studies are related to levels of ALP in GCF of patients with periodontal disease. Very few studies have evaluated saliva ALP levels in patients with chronic periodontitis. The results of the present study indicate the positive correlation between the periodontitis patients having habit of smoking and salivary ALP activities. The tendency of linear increase in the level of ALP activity in saliva reflects the advancing periodontal tissue injury and damage.Conclusion:In view of the above findings of the present study, we cannot confirm a clear cause- effect relationship between ALP and smokers /non-smokers with chronic periodontitis at this state. In order to explore the actual relationship, further prospective studies and clinical trails with larger sample size would be necessary. Higher levels of ALP in saliva samples of smokers with periodontitis could explain the higher rate of alveolar bone destruction in smokers.