|Year : 2016 | Volume
| Issue : 3 | Page : 254-259
Correlation of alkaline phosphatase activity to clinical parameters of inflammation in smokers suffering from chronic periodontitis
Vishakha Grover1, Ranjan Malhotra1, Anoop Kapoor2, Rupika Bither3, Sonia Sachdeva4
1 Department of Periodontology and Oral Implantology, National Dental College and Hospital, Dera Bassi, Punjab, India
2 Department of Periodontology and Oral Implantology, M N DAV Dental College, Solan, Himachal Pradesh, India
3 Department of Periodontology and Oral Implantology, Laxmi Bai Dental College, Patiala, Punjab, India
4 Department of Periodontology and Oral Implantology, Himachal Dental College, Sundernagar, Himachal Pradesh, India
|Date of Submission||27-Nov-2014|
|Date of Acceptance||30-Mar-2016|
|Date of Web Publication||4-Jul-2016|
3192, Ground Floor, Sector 37-D, Chandigarh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: Current clinical periodontal diagnostic techniques emphasize the assessment of clinical and radiographic signs of periodontal diseases which can provide a measure of history of disease. Hence, new methodologies for early identification and determination of periodontal disease activity need to be explored which will eventually result in expedited treatment. Aim: To evaluate the correlation of alkaline phosphatase (ALP) activity in gingival crevicular fluid (GCF) to clinical parameters of periodontal inflammation in smokers with chronic periodontitis. Materials and Methods: Study population included 15 smoker male patients in the age group of 35–55 years suffering from moderate generalized chronic periodontitis with history of smoking present. Following parameters were evaluated at baseline, 1 month and 3 months after scaling and root planing: plaque index, bleeding index, probing pocket depth (PD), relative attachment level (RAL), and GCF ALP activity. Statistical Analysis Used: Independent variables for measurements over time were analyzed by using Wilcoxon signed rank test. Results: A statistically significant reduction in all the clinical parameters and GCF ALP activity was observed from baseline to 1 month and 3 months. A correlation was observed between change in GCF ALP activity and PD reduction as well as gain in RAL at 3 months. Conclusion: The present study emphasizes that total ALP activity could be used as a marker for periodontal disease activity in smokers. Estimation of changes in the levels of this enzyme has a potential to aid in the detection of progression of periodontal disease and monitoring the response to periodontal therapy.
Keywords: Alkaline phosphatase, chronic periodontitis, gingival crevicular fluid, smoking
|How to cite this article:|
Grover V, Malhotra R, Kapoor A, Bither R, Sachdeva S. Correlation of alkaline phosphatase activity to clinical parameters of inflammation in smokers suffering from chronic periodontitis. J Indian Soc Periodontol 2016;20:254-9
|How to cite this URL:|
Grover V, Malhotra R, Kapoor A, Bither R, Sachdeva S. Correlation of alkaline phosphatase activity to clinical parameters of inflammation in smokers suffering from chronic periodontitis. J Indian Soc Periodontol [serial online] 2016 [cited 2019 Nov 19];20:254-9. Available from: http://www.jisponline.com/text.asp?2016/20/3/254/183099
| Introduction|| |
The diagnosis and need for the treatment of periodontal diseases have been determined traditionally on the basis of clinical evaluation comprising the presence of microbial deposits, extent of inflammation, probing pocket depth (PD), attachment loss, and radiographic assessment of alveolar bone loss. However, the conventional methods for periodontal disease diagnosis have a few limitations associated in terms of accuracy, the ability to predict ongoing or future disease activity, and ability to reflect even marked histological changes., An era of “enlightened diagnosis” has thus arrived that has spawned many new methodologies for early identification of periodontal disease which will eventually result in expedited treatment. These include genetic susceptibility analysis, microbiologic analysis, and biochemical analysis.
Biochemical methods in periodontal diagnosis have centered primarily on gingival crevicular fluid (GCF) and its constituents. The collection of GCF is a minimally invasive procedure and the analysis of specific constituents in the GCF provides a quantitative biochemical indicator for the evaluation of the local cellular metabolism that reflects the periodontal status.
Among the host enzymes in GCF, alkaline phosphatase (ALP) (orthophosphoric-monoester phosphohydrolase) was one of the first to be identified. It is a membrane-bound glycoprotein produced by many cells within the periodontal environment. It is released from polymorphonuclear neutrophils during inflammation, osteoblasts during bone formation, and periodontal ligament fibroblasts during periodontal regeneration. Since increase in ALP in serum has been associated with bone disease, local elevations in GCF could also reflect local soft and hard tissue alterations in active periodontal disease. The potential value of ALP as a marker of periodontal disease activity has been identified by many researchers, markedly by Ishikawa and Cimasoni, Binder et al., Chapple et al.,,, Nakashima et al., and many others.
Smoking as a significant environmental risk factor has a negative effect on the periodontal treatment approaches, ranging from mechanical debridement, local and systemic antimicrobial therapy, surgery, regenerative procedures, and implants.,, It has been reported that smoking affects the levels of some well-known markers of gingival health., Limited number of studies have evaluated the effect of smoking on the levels of ALP in GCF, which is a potentially powerful marker of periodontal disease activity.
In light of the above facts, the present study had been designed to evaluate the correlation of ALP activity in GCF to clinical parameters of periodontal inflammation in smokers with chronic periodontitis.
| Materials and Methods|| |
Fifteen male patients in the age group of 35–55 years suffering from moderate generalized chronic periodontitis with history of smoking present were selected among the patients visiting the Department of Periodontology and Oral Implantology, National Dental College and Hospital, Dera Bassi (Punjab). Study method was approved by the Institutional Ethical Committee. The subject selection was done on the basis of following inclusion and exclusion criteria.
- Patients suffering from generalized chronic periodontitis (minimum four sites of probing PD of 5 to 7 mm)
- Smokers who smoked 15–20 cigarettes/beedi per day
- Systemically healthy individuals
- Patients should not have undergone any periodontal treatment within 6 months prior to this study
- No antibiotic use in the previous 3 months and no history of chronic antibiotic use
- Cooperative patients showing acceptable oral hygiene.
- Patients having any history of chronic systemic disease
- Presence of removable prosthesis
- Furcation sites
- Patients on any antimicrobial drug in the previous 3 months
- Patients on nonsteroidal anti-inflammatory drugs in the past 1 month
- Patients on regular medication for any systemic disease that might alter the ALP concentration
- Patients with physical or mental disability.
The parameters selected for the study were clinical parameters including plaque index (PI), bleeding index (BI), probing PD and relative attachment level (RAL), and biochemical parameters including GCF ALP activity. These were recorded at the selected experimental sites.
For each patient, screening visit was scheduled 1 week before the baseline visit.
At the time of screening visit, the patients were enrolled in the study if they met the enrollment criteria. Qualifying patients received verbal information about the study and were asked about their interest to participation. Informed consent for participating in the study was obtained from each patient. Procedures undertaken at the screening visit were as follows:
- Medical and dental history
- Extraoral and intraoral hard and soft tissue examination
- Full mouth supragingival scaling
- Alginate impression
- Fabrication of acrylic stent
- Oral hygiene instructions
- Patient recalled after 1 week for the baseline visit.
Baseline visit (on 7th day after screening visit)
Based on the periodontal status, four individual tooth sites with the required probing depths were selected for sampling GCF. Before starting any treatment,
- PI was recorded on the selected teeth
- The sites to be sampled were then isolated with cotton rolls, and supragingival plaque was carefully removed from each tooth with cotton pellets
- The individual tooth site was gently air dried with the three-way air syringe for 5 s before the collection of GCF
- GCF was collected before probing the site by placing color-coded 1–5 µl calibrated volumetric microcapillary pipettes for 30 s at the gingival margin of selected individual site (mesial/distal midpoint), and a pooled sample was obtained from four selected sites. Samples of GCF contaminated by blood or saliva were discarded. The GCF in the microcapillary pipettes was forced out with a jet of air from the three-way air syringe into plastic vial containing 250 µl of normal saline. The plastic vial containing the sample was stored at −20°C, till the time of assay. It was then transported to the in-house laboratory under controlled temperature conditions maintained with polar pack and was centrifuged at 5000 rpm for 5 min before the assay for the total ALP activity.
The ALP enzyme assay was carried out at temperature 37°C by using semiautomated analyzer (RMS Daksh, Model no. CCA068I06, CSIR, Chandigarh, India) at a wavelength of 405 nm in kinetic mode. This semiautomated analyzer works on the principle of absorbance transmittance photometry. It is a high-performance, microcontroller-based, photometric biochemical analyzer used to measure various blood biochemical parameters and to determine both enzyme activity and substrate concentration in biological fluids at different temperatures (25°C, 30°C, and 37°C) by initial rates using fixed time, end point, and kinetic methods.
Principle of the assay
ALP cleaves p-nitrophenyl phosphate (p-NPP) into p-nitrophenol and phosphate.
p-NPP + H2Op-nitrophenol + phosphate
Under alkaline conditions, a colorless p-nitrophenol is converted to 4-nitrophenoxide which develops a very intense yellow color and absorbs light at 405 nm. The rate of increase in absorbance at 405 nm is proportional to the activity of ALP in the sample.
Working solution composition
- Diethanolamine buffer pH 9.8-1 mol/L
- p-NPP - 10 mMol/L
- Magnesium chloride - 0.5 mMol/L
- The factor for the assay was 2720. Exactly 20 µl of diluted sample was added to 1 ml of reagent, and average change in absorbance per minute (ΔAbs/min) was recorded. The total ALP activity in GCF was calculated by using the formula:
- ALP concentration = ΔAbs/min × factor × dilution factor (250/vol. of GCF)
- Total ALP activity = ALP concentration × vol. of GCF
- After the collection of GCF, recording of the clinical parameters, viz., BI, probing PD, and RAL was done.
Full mouth scaling and root planing (SRP) was performed for the selected subjects. Patients were instructed for meticulous oral hygiene maintenance and were advised not to use any chemotherapeutic mouthwash during the study period. Patients were recalled for follow-up at 1 month and 3 months from the baseline visit for reevaluation of GCF total ALP activity and the clinical parameters.
The data so obtained were subjected to statistical analysis using the statistical software, namely Statistical Package for Social Sciences program version 15.0 (SPSS Inc., Chicago, IL, USA). Mean and standard deviation for all parameters were calculated. The statistical significance of differences in independent variables for the intragroup measurements over time was analyzed by using Wilcoxon signed rank test. Spearman rank correlation coefficient was used to examine the relationship between total ALP activity and clinical parameters.
A P < 0.05 was considered statistically significant.
| Results|| |
The age for study population ranged from 35 to 55 years. The mean age for study population was 40.53 ± 5.62 years. The mean and standard deviation for all study parameters at the baseline and follow-up visits have been summarized in [Table 1]. From baseline to 1 month and 3 months, mean reduction observed in ALP activity was 327.80 ± 147.38 IU and 331.23 ± 182.82 IU, respectively. A mean reduction of 0.71 ± 0.61 mm and 0.98 ± 0.62 mm in PD and a mean gain of 0.16 ± 0.41 mm and 0.51 ± 0.65 mm in RAL were observed from baseline to 1 month and 3 months, respectively. Mean reduction of 0.50 ± 0.65 and 0.48 ± 0.65 was found in PI, and 0.46 ± 0.65 and 0.68 ± 0.85 in BI from baseline to 1 month and 3 months, respectively. The mean change in study parameters was statistically significant at each reevaluation period [Table 2].
|Table 1: Mean values of the study parameters at different periods of observation|
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|Table 2: Mean change in the study parameters at different periods of observation|
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Correlations were also observed between mean change in clinical parameters and mean reduction in GCF ALP activity over a period of 1 month and 3 months [Table 3], [Figure 1] and [Figure 2].
|Table 3: Correlation of changes in clinical parameters with changes in gingival crevicular fluid total alkaline phosphatase activity|
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|Figure 1: Scattered diagram showing correlation of change in relative attachment level and change in total alkaline phosphatase activity at 3 months|
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|Figure 2: Scattered diagram showing correlation of change in pocket depth and change in total alkaline phosphatase activity at 3 months|
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| Discussion|| |
Smoking has been implicated as a strong risk factor for periodontitis which contributes to the etiology of the most severe cases of periodontal disease. It has been found to affect its prevalence, progression, and periodontal treatment outcome, which might be related to the adverse impact of smoking on microbial and host factors., 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. There are also reports of higher proportions and/or the prevalence of exogenous or commensal flora in moderate to deep probing depths in smokers that point toward an adverse effect of smoking on the host response. The preponderance of evidence suggests diminished gingival bleeding and changes in the proportion of small to large blood vessels in the gingiva of smokers. Smoking impairs various aspects of the innate and adaptive host responses including alterations in neutrophil function, antibody production, fibroblast activities, vascular factors, and inflammatory mediator production. Smokers exhibit elevated total white blood cell and granulocyte counts in their systemic circulation; however, the influence of cigarette smoking on polymorphonuclear leukocyte cell numbers in the gingival crevice is not clear. Polymorphonuclear leukocyte viability and functions such as phagocytosis, superoxide and hydrogen peroxide generation, integrin expression, and protease inhibitor production can be altered by cigarette smoking or various tobacco components. Neutrophils play a key role in both host protection and tissue destruction. Smoking appears to elicit the more destructive activities of polymorphonuclear leukocytes. Smoking may affect GCF by the release of proteases like collagenase and elastase or the activity of protease inhibitors such as alpha-1-antitrypsin and alpha-2-macroglobulin from neutrophils. Tumor necrosis factor-α and interleukin (IL)-8 levels are reported to be depressed; however, IL-1 receptor antagonist and IL-1β levels are not altered in smokers. Many investigations have been carried out to explore the potential of immune-inflammatory mediators to aid in periodontal disease diagnosis., The potential value of ALP as a marker of periodontal disease activity has been identified by many researchers.,,,,,
The present study was designed to evaluate the correlation of clinical parameters such as PI, BI, probing PD, and RAL, and total ALP activity in GCF of smokers suffering from chronic periodontitis. Previous studies have shown positive correlations of ALP levels in GCF harvested from periodontitis patients with PD, attachment loss, and percentage of bone loss.,
GCF ALP levels are presented in this study as total ALP activity/four sites, rather than as final concentrations, to avoid the small errors in volume determination that can lead to large errors in estimates of final concentrations when the total volumes collected are small. There are studies ,, suggesting that total enzyme activities would be more useful than enzyme concentrations in studying the relationship of GCF enzymes to periodontal conditions. Binder et al. observed in their study that total ALP did not differ significantly from sample to sample. Chapple et al. stated that total GCF ALP levels might serve as a predictor of future or current disease activity.
Due to practical difficulty encountered during collection of GCF from healthy subjects, a range for normal GCF ALP activity as reported in previous studies was kept as a reference range. According to the studies by several investigators, mean total ALP activity in the GCF from healthy sites was found to be 65 ± 76 µIU/site, 10 µIU/sample, and 46 ± 26 µIU/sample. As in the present investigation, total mean ALP activity at baseline was 1339.10 ± 375.10 IU, which is considerably lesser than the value reported in nonsmokers suffering from chronic periodontitis (unpublished data). This might be attributed to the effect of smoking on the functions and products of neutrophils, which are the main sources of ALP. Mean values for probing PD and RAL were found to be higher in smokers, which might be due to the effects of smoking on periodontium that contributes to the severity of periodontal disease. Minimal PI scores as observed in this study could be related to meticulous tooth brushing by smokers in an attempt to get rid of halitosis or staining of teeth. BI scores were also minimal which might be due the fact that gingival inflammatory response is dampened because of profound influence of smoking on vascular dynamics and cellular metabolism. The observations of this study are in accordance with the previous studies., Feldman et al. also demonstrated that smokers had similar or lesser gingival inflammation, greater attachment loss and deeper pockets associated with comparable plaque levels as compared to nonsmokers.
A significant reduction in total ALP activity in GCF was observed at 1 month and 3 months after phase I therapy. However, the reduction in total ALP activity from 1 to 3 month period was nonsignificant. The plausible explanation for this could be the resolution of inflammation which leads to a resultant decrease in ALP from neutrophils. The reduction in total ALP activity at all reevaluation periods was higher in nonsmokers suffering from chronic periodontitis as compared to smokers suffering from chronic periodontitis (unpublished data). Erdemir et al. also observed that in both smokers and nonsmokers, total ALP activity in GCF decreased at 3 months period after phase I therapy, difference between the two groups being statistically nonsignificant. In contrast, Perinetti et al. reported that GCF ALP activity significantly decreased at 15 days after SRP and returned to around the initial levels at day 60. Bergström and Boström stated that smoking-associated inadequate inflammatory response might be accompanied by an inadequate regenerative capacity of the periodontium, retarded healing following therapeutic intervention, and accelerated progression rate in smokers. Mouzakiti et al. reported increased tissue inhibitor of matrix metalloprotease (TIMP)-1 expression and decreased the ratios of matrix metalloproteases (MMPs)/TIMP-1 after periodontal treatment with smokers showing significantly higher TIMP-1 expression and lower MMP-8/ TIMP-1 ratio than nonsmokers.
A significant reduction in PD and gain in RAL were observed at all reevaluation periods after phase I therapy compared to the baseline levels. Altered host response in smokers might be implicated as a factor in the less favorable clinical response to the treatment. Previous studies have shown similar results regarding PD reduction in smokers after initial periodontal treatment.,,
For PI scores, significant reduction was observed at 3 months as compared to baseline and similar findings have been reported by Pucher et al., Boström et al., and Erdemir et al. (2004, 2006)., There was a minimal reduction in BI levels at 3 month period as compared to baseline scores and similar findings have been reported by Ah et al., Kaldahl et al., Kinane and Radvar, Machtei et al., and Darby et al., who also reported a poorer response to SRP in smokers. Bergström and Boström observed significantly lower hemorrhagic responsiveness in smokers and suggested vasoconstrictive action of nicotine and its influence on vascular dynamics and cellular metabolism as the possible mechanism.
At the end of study period, a positive correlation was observed between change in RAL and change in total GCF ALP activity. Rest of the parameters, viz., PI, BI, and PD had a nonsignificant correlation with change in total GCF ALP activity. This finding should be interpreted in light of the fact that the gingival inflammatory response is dampened in smokers., The present study emphasizes the significance of ALP activity in diagnosis, progression of periodontal disease, and monitoring the response to periodontal therapy in smokers but as it was an initial study, sample size and time period were limited. Hence, further studies with larger sample size and extended time period are advocated.
| Conclusion|| |
The present study emphasizes that total ALP activity could be used as a fairly accurate risk marker of disease activity since a correlation had been observed between change in total ALP activity and PD reduction as well as gain in RAL at 3 months. Estimation of changes in the levels of this enzyme has a potential to aid in detecting progression of periodontal disease and monitoring the response to periodontal therapy. However, to further validate the association between ALP levels in GCF and disease activity, there is need for further longitudinal studies with larger sample size for extended time period.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]