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Archives of Oral Sciences & Research

ESTIMATION AND COMPARISION OF SALIVARY CALCIUM


LEVELS IN HEALTHY SUBJECTS AND PATIENTS WITH
GINGIVITIS AND PERIODONTITIS: A CROSS-SECTIONAL
BIOCHEMICAL STUDY
Nupur Sah*, Shobha Pravin More, Hemant Bhutani
ABSTRACT
Objective: Saliva contains a variety of host defense factors. It influences calculus formation and periodontal disease.
This study was conducted to estimate the role of salivary factors such as calcium of periodontal disease. Salivary calcium,
due to its affinity to be readily taken up by plaque, is an important factor not only with regard to the onset of periodontitis but
also significantly with regard to dental health.
Material and Methods: In this study we have examined the levels of calcium in saliva from patients with gingivitis,
periodontitis and in saliva from healthy patients. Periodontal disease was determined based on clinical parameters gingival
index (GI), bleeding on probing (BOP), probing depth (PD).
Results: Results obtained showed a statistically significant increase in the levels of calcium in periodontitis patients in
comparison to healthy people and gingivitis patients.
Conclusion: Based on these results, it can be assumed that there exists a clear and a significant association between high
salivary calcium levels and periodontal diseased states.
AOSR 2012;2(1):13-16.
Key Words: Saliva chemistry, Calcium analysis, Periodontitis, Metabolism, Oral health, Alveolar bone loss/ metabolism.
* Department of Periodontology Yerala Medical Trusts Dental College and Hospital, Navimumbai, Maharashtra, India.
Department of Periodontology, Sinhgad Dental College and Hospital, Pune, Maharashtra, India.
Department of Periodontology and Implantology, Dr. D.y. Patil dental college and hospital, Navimumbai,
Maharashtra, India.

INTRODUCTION:
Saliva is a complex fluid containing a variety of mucosal
host defense factors from the different salivary glands and
the crevicular fluid.1 Compelling reasons exist to use saliva
as a diagnostic fluid. It meets the demands for inexpensive,
noninvasive and easy-to-use diagnostic methods.2 As a
clinical tool, saliva has many advantages over serum,
including ease of collection, storing and shipping, and it can
be obtained at low cost in sufficient quantities for analysis.3
For patients, the noninvasive collection techniques
dramatically reduce anxiety and discomfort and simplify
procurement of repeated samples for monitoring over time.
Saliva also is easier to handle for diagnostic procedures
because it does not clot, thus lessening the manipulations
required.4 Saliva exerts a major influence on plaque
initiation, maturation, and metabolism. Salivary flow and

composition influences calculus formation, periodontal


disease. The inorganic components of plaque are calcium,
phosphorous and other minerals. As the mineral content
increases, the plaque mass become calcified to form
calculus.5 Salivary calcium, due to its affinity to be readily
taken up by plaque, is an important factor not only with
regard to the onset of periodontitis but also significantly
with regard to dental health.6 It is one of the most intensely
studied potential markers for periodontal disease in saliva.
The purpose of this study was to estimate the salivary
concentration of calcium, in the periodontal disease states.
MATERIAL AND METHODS:
The study group comprised of individuals reporting to the
Department of Periodontics and Implantology. The study
included sixty subjects both males and females in the age
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Nupur Sah et al.

group of 20 45 years. The study included those subjects


who had 20 or more intact teeth. They were divided into
three groups comprising of twenty patients each.
Group I: Comprised of healthy patients who had sulcus
depth 3 mm and absence of gingival inflammation and
bleeding on probing.
Group II: Comprised of gingivitis patients with gingival
inflammation and bleeding on probing but sulcus depth
3mm.
Group III: Comprised of periodontitis patients who had
periodontal pockets 4mm as well as clinical attachment
loss and bleeding on probing.
The selection of patients was done on the same day before
the collection of sample. Informed consent was taken from
all the subjects included in the study.
All subjects with a history of a systemic disorder,
history of medication (especially antibiotics and /or antiinflammatory drugs during the past six months) and with
history of periodontal treatment during the past six months
were excluded from the study. Smokers, pregnant women
and patients undergoing orthodontic treatment were also
excluded. At the initial examination, each subject completed
a detailed medical questionnaire and received a complete
periodontal examination, which included: gingival index
(GI), (Loe and Silness 1963),7 bleeding on probing (BOP),
probing depth (PD).
Unstimulated whole saliva samples were collected
following a brief rinsing of the mouth with water. The
saliva samples were collected from the lower vestibular
sulcus by a plastic syringe.
2 ml saliva sample was collected. The sample was then
transferred into sterile eppendorf tube and 2 drops of 1%
sodium azide was added which served as an antibacterial
agent. The saliva samples were then frozen and transported
to the laboratory within 24 hours using standard gel coolant
packs in order to maintain the temperature between 2C4C.
ESTIMATION OF CALCIUM:
Biochemical assays of saliva samples were carried out to
quantify the calcium levels*. The kit consisted of arsenazo
III reagent and calcium standard .
The method was adapted for saliva. The test was based on
the following principles:8 Arsenazo III is chemically stable
and has a very high affinity for calcium in a neutral pH range.
In this assay system, Arsenazo III forms a blue Arsenazo
*

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Diagnostic kit, Dialab Productions.

III calcium complex with an absorbance maximum at


650nm. The concentration of calcium is proportional to
the absorbance of the blue coloured Arsenazo III calcium
complex.
TEST PARAMETERS:
Wavelength: 650nm
Temperature: 37C
Sensitivity: the lower limit of detection is 0.04mg/dl.
REAGENTS USED:
Phosphate buffer, pH7.5
8- hydroxyquinoline-5-sulfonic acid
Arsenazo III
Detergents
PROCEDURE:
All reagents were brought to room temperature.
Pipette into
test tubes

blank (l)

standard
(l)

sample (l)

reagent

1000

1000

1000

sample

10

standard

10

water

10

It was then mixed well and incubated for 5 min at 20-25C/


37C.
The absorbance of standard and sample was then measured
against reagent blank.
The color was stable for one hour.
CALCULATION:
Calcium (mg/dl) = Absorbance of sample X conc. of

standard (mg/dl)
Absorbance of standard
Conversion:
Mg/dl x 0.2495 = mmol/l.
Descriptive data was presented as mean and standard
deviation. T test was used for comparison between means
of two groups. ANOVA test was used to determine the
significance of each parameter under study. For all tests
a p value of less than 0.05 was considered statistically
significant.

Comparision of Salivary Calcium Levels in Health, Ginvitis and Periodontitis

RESULTS:
The mean age range in the healthy group was found to
be 27.15 years with a standard deviation of 5.039. In the
gingivitis group it was found to be 27 years with a standard
deviation of 27 years with a standard deviation of 6.35 while
in the periodontitis group it was 33.75 6.35 years (Table 1
and 2). PDs were assessed on four sites per tooth (mesial,
distal, buccal and lingual) in mm in all the three groups. It
was found that mean probing depth in healthy group was
1.54 mm with a standard deviation of 0.08, in the gingivitis
group it was 1.63 mm with a standard deviation of 0.25,
while in periodontitis group it was 4.91 with a standard
deviation of 1.2 (Table 1 and 3).
When the salivary calcium levels were assessed in all the
groups it was found to be 0.51mmol/l in the healthy group
with a standard deviation of 0.27. In the gingivitis group
the mean calcium level was 0.97mmol/l with a standard
deviation of 0.52 while in the periodontitis group it was the
highest with a mean value of 1.54mmol/l with a standard
deviation of 0.84 (Table 4) (Graph I). This was statistically
significant (p = 0.00). There was a significant increase in
the calcium levels when they were compared between the
periodontitis and the gingivitis group. (p=0.015); healthy
and gingivitis group as well as healthy and periodontitis
group (Table 5).

TABLE 4: COMPARISION OF CALCIUM LEVELS


IN HEALTHY, GINGIVITIS AND PERIODONTITIS
GROUPS *

CALCIUM
LEVELS
(mmol/l)

HEALTHY

GINGIVITIS

PERIODO
NTITIS

p
VALUE

0.51
0.27

0.97 0.52

1.54
0.84

p=0.00

TABLE 5: INTERGROUP COMPARISION OF


SALIVARY CALCIUM LEVELS *
p VALUE
HEALTHY VS
GINGIVITIS
HEALTHY VS
PERIODONTITIS
GINGIVITIS VS
PERIODONTITIS

0.001
0.000
0.015

TABLE 1: DEMOGRAPHIC DATA SHOWING STUDY


POPULATION
SUBJECTS

DIAGNOSIS

N=60
MALES= 42
FEMALES=18

HEALTHY= 20
GINGIVITIS=20
PERIODONTITIS=20

TABLE 2: MEAN AGE OF SUBJECTS


GROUPS

MEAN AGE

HEALTHY

27.155.03

GINGIVITIS

296.3

PERIODONTITIS

33.756.27

TABLE 3: MEAN PROBING DEPTH AT SAMPLE


SITES
GROUP

MEAN (mm)

STANDARD
DEVIATION

HEALTHY

1.54

0.08

GINGIVITIS

1.63

0.25

PERIODONTITIS

4.91

1.2

GRAPH 1: MEAN CALCIUM LEVELS AMONG


GROUPS
DISCUSSION:
The study was undertaken to examine the relationship
of salivary calcium, with regard to periodontal disease.
Unstimulated whole saliva was collected from the lower
lingual sulcus. It has been suggested that in advanced
periodontitis, unstimulated saliva is representative of
pooled subgingival plaque samples.9 A positive correlation
between the level of calcium and the severity of disease
was observed. The results showed that the subjects in
the periodontitis group had the highest levels of salivary
calcium which was statistically significant. There was also
a significant difference in the calcium levels between the
gingivitis and the healthy group with the higher levels being
in the gingivitis group. These results are consistent with the
findings of Sewon and Karjalainen10 who found that there
was a higher calcium concentration in the saliva in the
periodontitis subjects as compared to the periodontitis free
subjects. They opined that periodontitis affected subjects
had a higher re mineralization potential than individuals
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Nupur Sah et al.

with no signs of periodontal disease. The results however


were in contrast with the results obtained by Sewon and
Makela in 199011 who found that higher calcium level was
related to good dental health but there was no relation to
periodontal bone destruction.
The calcium levels of saliva may also reflect the fluctuations
in dietary calcium and general calcium turnover. In our
present study we could not demonstrate the effect of
diet. It seems that salivary calcium, due to its affinity for
being readily taken up by plaque, is an important factor,
with regard to onset of periodontitis.6 In the present study,
age wise calcium changes have not been determined. The
subjects were in the age range of 20-45 years. Salivary
calcium concentration is said to be significantly high
in younger individuals in period of development and
maturation of skeleton and teeth and it tends to decline with
advancing age nearing osteoporosis.12 All subjects with
a history of any known systemic disorder were excluded
from the study design. In this study only one variable is
analysed, further study can be conducted to analyse the
other variables like phosphate (phosphorous) which plays
a major role in plaque mineralization.
CONCLUSION:
Within their limits the study showed a clear and a significant
association between high salivary calcium levels and
periodontal diseased states. It is therefore, suggested that
monitoring for change in salivary composition might be a
useful tool to establish periodontal health status.
ACKNOWLEDGEMENTS: I would like to thank Dr.
Kishore Bhat , Professor and Head of the Department
of Microbiology , Maratha Mandal Dental College and
Hospital, Belgaum, Karnataka, for his invaluable help and
guidance for the completion of the study.
The study was not funded by any agency or institution and
the authors have no commercial relationships.
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4.



Griffiths GS, Sterne JA, Wilton JM, Eaton KA, Johnson


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Cortelli SC, Feres M, Rodrigues AA, Aquino DR,


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10. Sewon L, Soderling E, Karjalainen S. Comparative


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12. Hassan ShA, Al Sandook TA. Al-Rafidain. Salivary
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CORRESPONDENCE:
Dr Nupur Sah
A-404 , mehek chs, plot -17a
Sec- 12, kharghar,
Navimumbai-410210
E mail: sahnupur02@gmail.com
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