ABSTRACT
Introduction:
In recent decades, biomarkers have been used to predict the progression of chronic periodontitis. One of these biomarkers is alkaline phosphatase (ALP). Due to limitations of the performed studies, this study was performed to determine the amount of salivary ALP and gingival crevicular fluid in patients with chronic periodontitis and healthy individuals.
Materials and Methods:
Twenty-three patients with severe chronic periodontitis and 23 healthy individuals referred to the Periodontology Department of Ahvaz Jundishapur School of Dentistry were evaluated in this analytical epidemiological study. Salivary ALP and gingival crevicular fluid (GCF) were measured using ALP measuring kit and Hitachi device.
Results:
Mean (standard deviation) of ALP enzyme was 19.43 (12.5) in GCF of patients with chronic periodontitis and 12 (1.48) in the healthy group, and it was 80.17 (23.9) in the saliva of patients with periodontitis and 24.78 (4.37) units per litre in the healthy group. There was a significant difference in the mean of this enzyme in GCF and saliva of patients with chronic periodontitis and healthy individuals (P < 0.001).
Conclusion:
The results showed that mean of ALP enzyme is significantly higher in GCV and saliva of patients with chronic periodontitis than in healthy individuals. Therefore, it seems that this parameter can be used as a useful biochemical parameter for the diagnosis of periodontal disease.
Keywords: Alkaline phosphatase, chronic periodontitis, gingival crevicular fluid, saliva
Introduction
Periodontal disease is an inflammatory disease of the supporting tissues around the tooth. The most common form of this disease is chronic periodontitis, which is considered an infectious disease leading to inflammation in the supporting tissue of the tooth, progressive attachment loss, and bone loss.[1] Although subgingival plaque biofilm is the main cause of the disease, the interference between microbial plaque and the response of host tissues affected by environmental factors such as age, systemic conditions, and genetics, plays an important role in pathogenesis.[2] Periodontal disease is diagnosed based on clinical parameters such as bleeding on probing, clinical attachment loss, probe depth, and bone loss seen on radiography.[1] Another advanced diagnostic method for periodontal disease is the evaluation of the host response, which is performed by studying specific or nonspecific mediators by biochemical or immunological methods. Recently, saliva has been considered an important biological substance in new diagnostic tests.[3] Gingival crevicular fluid (GCF) is also considered the most important source for analysis of inflammatory and destructive mediators of periodontal disease. Alkaline phosphatase (ALP) is one of the enzymes in GCF and saliva that can be tested for periodontitis.[4] ALP is an intracellular enzyme produced by many cells such as polymorphonuclear leukocytes (PMNs), macrophages, fibroblasts, osteoblasts, and white blood cells in the periodontium and gingival sulcus. When periodontal tissue is destroyed, secretion of this enzyme actively increases into GCF and saliva.[2] As ALP in saliva has been determined in limited studies and there is no available study that simultaneously evaluated this enzyme in GCF and saliva, the objective of this study is to determine ALP level in GCF and saliva of patients with periodontitis and healthy individuals.
Materials and Methods
Population
The population included 23 patients with chronic periodontitis who referred to the Periodontology Department of Ahvaz Jundishapur School of Dentistry, and 23 healthy individuals from other departments of the school from July to September 2018.
Procedure
In this analytical epidemiological study, first, a written consent form was obtained from each patient. Initial clinical examination, including bleeding on probing (BOP), probing pocket depth (PPD), plaque index (PI), and clinical attachment loss, was performed for each patient. All measurements were performed by Williams probe at six dental surfaces (midbuccal, midlingual, buccal, lingual, mesial, and distal) and recorded in the patient file. All samples were examined and approved by a periodontist. To collect saliva, patients were asked to avoid eating and drinking for 2 h before collecting the sample. Before collecting saliva, people rinsed their mouths with water for 1 min; after 15 min, they swallowed all their saliva and immediately emptied their nonstimulating saliva into sterile containers for 2 min. For this purpose, 3 mL of saliva was collected from each person.[5]
GCF of the patient group was collected from areas with deep pockets (5–7 mm) and from gingival crevices of healthy individuals by paper point. To do this, the collection area was isolated by a cotton roll and dried under gentle air pressure. Paper point No. 30 was then inserted into the gingival crevice to feel resistance. The paper point remained in the crevice for 30 s and was then transferred to a microtube containing 500 mL of phosphate-buffered saline (PBS).[6] In the laboratory, salivary ALP and GCF were measured using Pars Azmoun ALP measurement kit (made in Iran) and Hitachi autoanalyzer (made in Japan).[4] The microtubes containing the sample were placed in a centrifuge for 3–5 min at a speed of 4000–4500 rpm.[7] Analysis was performed based on the reaction between ALP with P-Nitrophenyl phosphate (colorless) and the formation of P-Nitrophenol (yellow).[8] Paranitrophenyl phosphate was used as a substrate.
Sample size
The sample size was calculated by the Cochran formula as follows:
(Z1-α2 + Z1-β)2 (S12 + Sc2) (1.96+1.28)2 (7.62 + 4.32)
N= =23
Z1-α = % (X̄l - X̄c)2 621.96
Z1-β= 1.28
S1 = 7.6(4)
S2 = 4.3
X̄1 - X̄2 = 6
where Z1−α = 1.96 for 95% confidence; Z1−β for 90% power; S1 = 7.6; S2 = 4.3; X̄1 − X̄2 = 6. In this empirical study, 23 patients with severe chronic periodontitis and 23 healthy individuals were studied. Samples were selected from individuals referring to the Periodontology Department of Ahvaz Jundishapur School of Dentistry.
Inclusion criteria included:
Absence of any systemic disease
No medication in the past 6 months
No history of smoking and alcohol use
No periodontal treatment and scaling in the past 6 months.
Exclusion criteria included:
Pregnancy.
Previous history of smoking and alcohol use
Aphthous ulcer.
Systemic diseases, particularly those that affect periodontal conditions, such as diabetes and AIDS, patients who need antibiotic therapy (such as people with artificial heart valves).
Data analysis
Using descriptive statistics such as mean and standard deviation to compare the mean of quantitative variables in the two groups, an independent t-test was run, all of which were performed with Statistical Package for Social Sciences (SPSS) software version 22.
Ethical considerations
At the beginning of the work, informed consent was obtained from the participants and objectives of the project were explained and it was emphasized that participation was voluntary and free of charge and information were confidential and all ethical principles were adhered.
Results
Measurement and comparison of ALP enzyme in GCF of patients with chronic periodontitis and healthy individuals
Mean and standard deviation of ALP enzyme were 19.43 (12.5) units per litre in GCF of patients with chronic periodontitis and 12 (1.48) units per litre in healthy individuals [Figure 1]. To compare two groups in terms of ALP enzyme level in GCF, t-test for independent groups with moderating degree of freedom was used [Table 1] because the distribution of variables was normal based on Kolmogorov–Smirnov test in the patient group (P = 0.661) and the healthy group (P = 0.498), as shown in Table 2, and variances in the ALP enzyme level in GCF of both groups were not equal based on Levene test (P < 0.01), as shown in Table 3.
Figure 1.
Distribution of means of alkaline phosphatase enzyme in GCF of the studied groups
Table 1.
Independent t-test for comparing the studied groups in pretest of dependent variables
| Dependent variable | Patient | Healthy | Independent t-test | ||||
|---|---|---|---|---|---|---|---|
|
|
|
|
|||||
| n | M±SD | n | M±SD | t | Df | P | |
| ALP enzyme in GCF | 23 | 19.43±5.12 | 23 | 1.48±12 | −6.69** | 25.64 | 0.001 |
**P<0.001. ALP, alkaline phosphatase; GCF, gingival crevicular fluid.
Table 2.
Normal distribution of ALP enzyme in GCF
| Variable | Patient K-S |
Healthy K-S |
||
|---|---|---|---|---|
|
|
|
|||
| Z | P | Z | P | |
| ALP enzyme in GCF | 0.565 | 907 | 0.730 | 0.661 |
ALP, alkaline phosphatase; GCF, gingival crevicular fluid.
Table 3.
Homogeneity of variances of ALP in GCF of people with chronic periodontitis and normal individuals
| Variable | Levene test | Df1 | Df2 | P |
|---|---|---|---|---|
| ALP enzyme in GCF | 20.1 | 1 | 44 | 0.001 |
ALP, alkaline phosphatase; GCF, gingival crevicular fluid.
Measurement and comparison of ALP enzyme in saliva of patients with chronic periodontitis and healthy individuals
Mean (standard deviation) of ALP enzyme was 80.17 (23.9) units per litre in saliva of patients with chronic periodontitis and 24.78 (4.37) units per litre in healthy individuals [Figure 2]. To compare two groups in terms of ALP enzyme level in saliva, t-test for independent groups with moderating degree of freedom was used [Table 4] because the distribution of variables was normal based on Kolmogorov–Smirnov test in the patient group (P = 0.613) and the healthy group (P = 0.907), as shown in Table 5, and variances in ALP enzyme level in the saliva of both groups were not equal based on Levene test (P < 0.01), as shown in Table 6.
Figure 2.
Distribution of means of alkaline phosphatase enzyme in saliva of the studied groups
Table 4.
Independent t-test for comparing the studied groups in pretest of dependent variables
| Dependent variable | Patient | Healthy | Independent t-test | ||||
|---|---|---|---|---|---|---|---|
|
|
|
|
|||||
| n | M±SD | n | M±SD | t | Df | P | |
| ALP enzyme in saliva | 23 | 80.17±23.9 | 23 | 24.78±4.37 | −10.93** | 23.47 | 0.001 |
**P<0.001. ALP, alkaline phosphatase.
Table 5.
Normal distribution of ALP enzyme in saliva
| Variable | Patient K-S |
Healthy K-S |
||
|---|---|---|---|---|
|
|
|
|||
| Z | P | Z | P | |
| ALP enzyme in saliva | 0.759 | 0.613 | 0.565 | 0.907 |
ALP, alkaline phosphatase.
Table 6.
Homogeneity of variances of ALP in saliva of people with chronic periodontitis and normal individuals
| Variable | Levene test | Df1 | Df2 | P |
|---|---|---|---|---|
| ALP enzyme in saliva | 28.8 | 1 | 44 | 0.001 |
ALP, alkaline phosphatase.
As shown in Table 6, the mean level of ALP enzyme was significantly higher in the saliva of patients with chronic periodontitis than in healthy individuals (P < 0.001).
Discussion
Periodontal disease is an inflammatory disease of the supporting tissues of the tooth caused by specific microorganisms that leads to progressive destruction of the periodontal ligament, alveolar bone, with the pocket formation or gingival loss, or both.[1,9] Diagnosis is usually based on clinical parameters such as bleeding on probing, clinical attachment loss and probing depth, and bone loss seen on radiography.[3,10] Due to limitations of diagnostic methods and the nature of sedation and activity of periodontitis, identification of inflammatory and destructive markers of gingival crevicular fluid and saliva has been considered in recent years and various studies have used these biomarkers to predict the progression of chronic periodontitis.[10] One of these biomarkers is ALP. Findings of this study showed that the mean (standard deviation) of ALP enzyme was 19.43 (12.5) units per litre in GCF of patients with chronic periodontitis and 12 (1.48) units per litre in healthy individuals. These results show that the mean amount of ALP enzyme is significantly higher in GCF of people with chronic periodontitis than healthy people (P < 0.001). Measurement and comparison of ALP enzyme in saliva of patients with chronic periodontitis and healthy individuals in this study showed that the mean (standard deviation) of ALP was 80.17 (23.9) units per litre in saliva of patients with chronic periodontitis and 24.78 (4.37) units per litre in healthy individuals and the mean amount of ALP enzyme was significantly higher in the saliva of patients with chronic periodontitis than in healthy individuals (P < 0.001). Overall, these findings indicate a higher level of ALP in saliva and GCF of people with chronic periodontitis, which could be evidence that the activity of this enzyme can be used as an indicator in the early diagnosis of periodontitis.
In this regard, various studies have been conducted in Iran and abroad. For example, Sanikop et al.[11] (2012), in a study to evaluate the level of ALP activity in GCF of three groups of samples including healthy individuals, patients with gingivitis and chronic periodontitis, concluded that there is a significant relationship between ALP levels and periodontal disease and the ALP level in GCF can be used as a biochemical marker to determine the progression of periodontal diseases. Mohammad and Aziz[12] (2018), in a study that investigated the effect of scaling and root planning treatment on the level of alkaline and acid phosphatase activity in patients with chronic periodontitis, reported that alkaline and acid phosphatase enzymes can be used as a marker in the monitoring of periodontal disease, its recovery status, and follow-up. In addition, Dabra and Singh[5] (2012) examined the levels of ALP and salivary acid phosphatase in three groups, namely, periodontitis, gingivitis, and healthy groups and the results showed a significant increase in these two enzymes in patients with periodontitis compared with the control group. There was also a significant decrease in salivary levels of these two enzymes after routine periodontal treatments. The researchers concluded that salivary ALP and ACP levels could be used to assess periodontal tissue damage. Nomura et al.[10] (2012) used biomarkers to predict the progression of periodontitis; the results showed that the salivary level of ALP and P. gingivalis could be used to increase the accuracy of periodontitis prediction along with other biochemical markers. Azizi et al.[3] (2011) measured and compared the level of nonstimulatory salivary ALP in the group with periodontitis and healthy group; they concluded that the level of salivary ALP was significantly higher in people with periodontitis compared with healthy people and that this enzyme could be a good marker for determining the extent of periodontal tissue damage. Ranjan et al.[13] (2010) also concluded in their study that ALP levels increase in periodontitis. Jaiswal et al.[14] (2011) also reported that ALP is an important indicator for assessing the status of periodontitis in patients with liver cirrhosis.
In general, the results of previous studies indicate an increase in ALP in periodontal disease. The similarity of the current results with previous studies is that the rate of release of enzymes from tissue cells and their entry into saliva increases in periodontal disease with spread of tissue destruction and increased inflammatory process.[11,15,16] Differences in the results of these studies with the present study can be attributed to cases such as characteristics of the samples in terms of disease status and their different demographic characteristics, as well as different methods of measurement and clinical calculation of ALP levels in different studies.
Conclusion
In general, the findings of this study showed that the mean amount of ALP enzyme in GCF and saliva of people with chronic periodontitis was significantly higher than in healthy people (P < 0.001). Therefore, it seems that this parameter can be used as a useful biochemical marker for diagnosis of periodontal disease and monitoring of the response to treatment in patients, particularly in the early stages of the disease, which are less detectable by clinical parameters.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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