Assessment of Alkaline Phosphatase , Salivary Flow Rate and Salivary Potential of Hydrogen in Relation to Severity of Chronic Periodontitis

Background: The cells of periodontium contain many intracellular enzymes like (alkaline phosphatase ALP) that are released outside into the saliva and gingival crevicular fluid (GCF) after destruction of periodontal tissue. The aim of study was to determine the activity of this enzyme in saliva and its relation to the salivary flow rate, PH and clinical periodontal parameters in patients with chronic periodontitis. Subject, Materials and methods: Sample population consist of 75 individuals ;divided into four groups , the first group (15):control subject, the second group (20):mild chronic periodontitis, the third group(20) moderate chronic periodontitis and the fourth group (20) sever chronic periodontitis, Measurements of plaque index (PLI), gingival index (GI), bleeding on probing (BOP), probing pocket depth (PPD) and clinical attachment level (CAL), only male were included and saliva was collected from them and subjected to biochemical analysis of the alkaline phosphatase enzyme (ALP), and also measurement of salivary flow rate(FR) and PH. Results: Statistical analysis of the results revealed the presence of a highly significant difference in the enzymatic activity between healthy and chronic periodontitis subjects (mild, moderate, severe) with positive correlation between the activity of this enzyme and the clinical periodontal parameters, and negative correlation between this enzyme and Salivary flow rate and pH. Conclusion: From this study it can be concluded that a number of markers show promise as sensitive measures of disease and the effectiveness of therapy. At this time enzymes such as alkaline phosphatase ALP, is good biochemical markers of screening chronic periodontitis. Also ALP can be used as a monitor for healthy individuals and patients with different periodontal diseases. Furthermore, analysis of saliva may offer a cost effective approach to assessment in controlling progression of chronic periodontitis in large populations. Keyword: Chronic periodontitis, saliva, flow rate, alkaline phosphatase. (J Bagh Coll Dentistry 2016; 28(3):125-131).


INTRODUCTION
Periodontal disease (PD) is one of the common inflammatory diseases within complex etiology and multifactorial in origin.Diagnosis of periodontal disease depended on clinical and radiographic measures of periodontal tissue destruction.These parameters provide measures of past destruction and are of limited use in early diagnosis (1) .Genetics and molecular biology in advances stage lead to a better knowledge of the pathways and mechanisms through which microorganism maintain the host immune/inflammatory response (2) .
Chronic periodontitis (CP) is very common disease (3) and it is a slowly progressing form of PD, but may have periods of rapid progression (4) .Saliva is an important biological material that got in new diagnostic tests which may contribute in the diagnosis and explaining the pathogenesis of some diseases (5) .
Saliva plays an important role in discovery of periodontal disease because it is easily collected and allows analysis of several biological markers such as proteins, enzymes, host cells, hormones, and bacterial products; therefore, no specific laboratory devices are necessary and this approach may be suitable for public health use (6) .The amount of saliva naturally produced by the salivary glands is called salivary flow rate.Production of saliva is increased by the presence of food or irritating substances, such as vomit, in the oral cavity (7) .
Mean daily saliva production for healthy person ranges from 1 to 1.5L (8) .The Salivary Flow Index is a parameter allowing stimulated and unstimulated saliva flow to be classified as normal, low, or very low (hyposalivation) (9) .Normal total stimulated Salivary flow rate (FR) ranges from up to 3 ml/ min.
A measure of the acidity or alkalinity of a solution, is defined salivary PH, the normal PH range for saliva is considered to be 5.6 to 7.9, depend on international journal of drug testing's above 7 scale usually indicate alkalinity and when below 7 scale indicate Acidity (10) .
Alkaline phosphatase (ALP) is hydrolase enzyme responsible for removing phosphate groups from many of types of molecules, including nucleotides, proteins, and alkaloids.The process of removing phosphate group is called dephosphorlation, ALP is effective in an alkaline environment (11) .This enzyme is an important indicator of bone formation and considered as marker for osteoblast cells.ALP was detected inthe parotid, submandibular and minor salivary glands (12) .Assessment of Alkaline Oral and Maxillofacial Surgery and Periodontics 126

MATERIALS AND METHODS
The sample consisted of 75 patients, subjects' collection from the department of Periodontics, at teaching hospital, College of Dentistry, University of Baghdad as well as from blood bank.all subject enrolled voluntarily in the study after a well explanation about the aim and purposes of the study and gave informed consent to participate in the study.
The subjects were divided into four groups according to American Academy of Periodontology 1999 (13) .

1-Control group I (healthy periodontium):
Consisted of fifteen (15) apparently systemically healthy without history of any systemic disease and with healthy periodontium, this was defined by GI scores <0.5 (14) .andwithout periodontal pockets or clinical attachment loss.

Exclusion criteria:
1-Patients who have undergone periodontal treatment in the 3 month period prior to the study.

Clinical periodontal parameter examination: 1-Assessment of Plaque Index System (PLI):
This was done by using plaque index system which was introduced by Silness (15) .

2-Assessment of Gingival Inflammation by the Gingival Index System (GI):
This was assessed using the criteria of the gingival index system that modified by Löe (14) .

3-Assessment of Gingival Bleeding on Probing (BOP):
If bleeding occurs within 30 seconds the site was given a positive score (1) and a negative score (0) for the non -bleeding site (16) .

4-Assessment of Probing Pocket Depth (PPD):
This is defined as the distance from gingival margin to the most apical penetration of the periodontal probe inserted into the gingival crevice or pocket without force or pressure

5-Assessment of Clinical Attachment Level (CAL):
It is defined as the distance from the cementoenamel junction (CEJ) to the location of the inserted probe tip (bottom of gingival crevice or pocket).

Measurement of salivary flow rate and pH:
The volume of unstimulated saliva that collected firstly from each subject at 5 min was recorded; through using graduated syringes, the saliva collected firstly was aspirated from the collection receptacle with a disposable 5 mL sterile syringe avoiding contact with the epithelium.The amount of saliva in mL, divided by the time of duration of the collection was recorded as the mean salivary flow rate.Only the liquid component of the saliva, not the foam, was measured.Where estimation of flow rate (ml/min) was made according to this equation: Flow rate (FR): Volume (ml) / Time (min)﴿ .
Samples containing blood were discarded.The samples were put in a small cooling box after collection to stop bacterial growth.The tube was labeled with the number of the subject corresponding to that written previously on the case sheet.The salivary pH was measured by using the (DP universal test paper) by immersing the strip into the saliva for about 2 seconds, then waited for color changes for 15 seconds and compared to the color chart present on the plastic case of the product.

Biochemical Analysis: Alkaline Phosphatase (ALP) Enzyme:
For ALP enzyme analysis we used kit manufactured by (BIOMEREIUX ®Sa) which is one of the French leaders of reagents (R) for medical biochemistry, All the kits used for enzymes analysis subjected to modification by a specialist (biochemist) in the laboratories of the poisons center to measure the activity of these enzymes in saliva.Principle: according to Biomérieux Kit for Alkaline phosphatase enzyme.
Colorimetric determination of ALP activity according to the following reaction:

Phenyl phosphate ALP phenol + phosphate
The liberated phenol is measured in the presence of 4-aminoantipyrine and potassium ferricyanide.The presence of sodium arsenate in the reagent stops the enzymatic reaction.

RESULTS
The current results revealed that mean values of (PLI, GI, BOP, PPD and CAL)were higher in severe group than other groups, while the mean values of the (FR, pH)were higher in control than other groups and the mean value of (ALP) was higher in sever group than other groups, these results show in table1and 2. Inter-groups comparisons by Least significant difference (LSD) test for testing equality of variances illustrated in the table (3, 4), The statistical analysis using the LSDtest to compare mean values of (PI, GI, BOP, PPD, CAL),(ALP, FR, PH) between study groups, revealed highly significant differences at p<0.01 .Regarding the correlation coefficient between level of ALP enzyme and PLI, GI Parameters for control group strong highly-significant positive correlation was found, while with Salivary FR, it showed a strong highly -significant negative correlation, these results were demonstrated in table (5).

Table 5: Pearson's Correlation Coefficient (r) between ALP and PL, GI, FR and PH of the control group
Variables PI GI FR PH ALP r 0.903 0.930 -0.970 p-value 0.000 0.000 0.000 - The results of correlation coefficient between PLI, GI, BOP, PPD, CAL Parameters and level of the ALP enzyme for mild group, revealed a strong highly-significant positive correlation.The results between FR, PH Parameters and ALP, revealed a strong highly-significant negative correlation.These results were demonstrated in table (6).Regarding the correlation coefficient between PLI, GI, BOP, PPD, CAL Parameters and level of the ALP for moderate group, the results revealed a strong highly-significant positive correlation.The results of correlation coefficient between FR, PH Parameter and ALP, revealed a strong highlysignificant negative correlation, these results were demonstrated in table (7).The results of correlation coefficient between PLI, GI, BOP, PPD, CAL Parameters and level of the ALP for severe group, revealed a strong highly-significant positive correlation.The result between FR, PH Parameters and ALP, showed a strong highly-significant negative correlation, these results were demonstrated in table (8).

DISCUSSION
The findings of the study that a highly significant difference in PLI, GI,BOP, PPD, CAL between the study and control groups, these findings were due to increase in the bacterial invasion and the amount of plaque that caused destruction of the sulcular and junctional epithelium and surrounding alveolar bone.
This findings revealed that the level of Alkaline Phosphatase ALP in the study group higher than control groups these results were in agreement with Nakamura (17) .Also accordance with many studies (18,19) .
The explanation for this difference in the enzyme activity between the two groups may be due to the fact that ALP is present at or near the cell membrane of alveolar bone osteoblasts and fibroblasts of the periodontal ligaments (20- 22) .During the active stages of periodontitis, there will be destruction of alveolar bone osteoblasts and fibroblasts and their cell membrane will be ruptured releasing their intracellular contents outside.So ALP will be released into saliva and GCF and the level of ALP will increase in saliva (6,23,24)   .
In the present study there was statistically high-significant differences in unstimulated salivary flow rate among either groups (25) indicate that Periodontitis induces an increase in the output of proteins, thereby enhancing the protective potential of saliva, but this is accompanied by a decrease in flow rate.
But disagree with Fiyaz (26) who found that individuals who have increased salivary inorganic calcium, phosphate, pH, FR and maintain poor oral hygiene could be at ahigher risk for developing periodontitis and may have less dental caries and more number of intact teeth.
The findings of the study showed that there was a highly-significant difference in term of salivary PH, between study and control groups, Baliga (27) observed that there is a correlation between pH of saliva and periodontal diseases when compared with healthy groups.Salivary pH in patients with chronic generalized periodontitis is more acidic than the control group, increase acidity (pH below 7) as the increasing severity of periodontal condition.
This results revealed that a strong highlysignificant positive correlation between ALP and PL, GI index in control group, also between ALP

( 1 )
Master Student.Department of Periodontics, College of Dentistry, University of Baghdad (2) Assistant Professor, Department of Periodontics, College of Dentistry, University of Baghdad.
Analyses a) One-way ANOVA test: to compare the measured variables among the groups.b) LSD test: to test any statistically significant difference between each two groups.c) Pearson's correlation coefficient test (r): to test the relation between ALP and other Oral and Maxillofacial Surgery and Periodontics 127 clinical periodontal parameter and the measured variables in each group.