ORIGINAL
RESEARCH

 

ABSTRACT:

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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, smokers.

 

INTRODUCTION

Periodontitis 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.2

Biomarkers 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 resorption

Whole 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 alkaline phosphatase. Alkaline
phosphatase is detected in the parotid, submandibular, and minor salivary
glands, as well as in desquamated epithelial cells, leukocytes, and bacteria
from dental plaque. 3

 

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.4 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 .5

Aim of this
study is to estimate and correlate level of alkaline phosphatse 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 30 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 alkaline
phosphatase 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 Kinetic
2. Semi autoanalyzer
3. Remi Centrifugal machine
4. Eppendorff tube
5. Ice bag
6. UNC 15 probe
7. Mouth mirror
8. Tweezer
9. Cotton

 

      

    

Centrifugal macchine           Eppendroff tube            AutoZyme ALP kinetic      

                                                                  

 

                          

         

                                                 
Semi autoanalyzer

Saliva
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 alkaline phosphatase, a diagnostic kit was used. The kit consisted
of two reagents:

Reagent 1

1. p-Nitrophenyl
phosphate

2. Magnesium ion
(Mg2+)s

3.
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 alkaline phosphatase 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).

 

Statistial Analysis:

Data were summarised as Mean ± SD
(standard deviation). Data were analyzed with ANOVA and Tukey’s test. Groups
were compared by one way analysis of variance (ANOVA) and the significance of
mean difference between the groups was done by Tukey’s HSD (honestly
significant difference) post hoc test. A two-tailed (?=2) P Group II > Group
I). Comparing the mean alkaline phosphatase of the groups, ANOVA showed
significantly different ALP among the groups (F=45.40, P0.05) between Group II and Group III though it was 7.1% higher in Group
III as compared to Group II.

 

Group

Mean ±
SD (n=10)

F value

P value

Group I

68.20 ±
42.08

45.40

0.05

-62.42 to 30.02

Table 2: Comparison of
mean alkaline phosphatase between the groups by Tukey test

***P Group II > Group I). Comparing the mean PD of the groups,
ANOVA showed significantly different PD among the groups (F=13.48, P0.05) between Group II and Group III though it was 4.7% higher
in Group III as compared to Group II.

Group

Mean ±
SD (n=10)

F value

P value

Group I

2.50 ±
0.53

13.48

0.05

-1.14 to 0.74

Table 4: Comparison of
mean probing depth between the groups by Tukey test

***P