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232]

Original Article

Estimation and comparison of salivary calcium levels in

healthy controls and patients with generalized gingivitis and
chronic periodontitis
Madhura Vijay Rane, Girish Suragimath1, Siddhartha Varma1, Sameer Anil Zope1, S. R. Ashwinirani2
Intern, 1Departments of Periodontology and 2Oral Medicine and Radiology, School of Dental Sciences, KIMSDU, Karad, Maharashtra, India

Abstract Aim: To evaluate salivary calcium levels in healthy subjects and patients with chronic gingivitis and Chronic
Periodontitis.
Materials and Methods: One fifty subjects in the age range between 20 – 45 years were randomly selected
and subjected to periodontal examination using gingival index, plaque index, oral Hygiene Index and
clinical attachment loss. Following periodontal examination, subjects were divided in three groups of 50
patients each: Group A: healthy subjects, Group B: gingivitis patients, Group C: periodontitis patients. Saliva
samples from the study subjects was collected and subjected to estimation of salivary calcium levels. The
obtained results were subjected to statistical analysis. The significance of difference in means was tested
by ANOVA test.
Results: The levels of salivary calcium increased as the disease progressed from healthy to gingivitis and
periodontitis. There was statistically significant difference observed between healthy to gingivitis group
and gingivitis to periodontitis group.
Conclusion: Salivary calcium levels can be used as a biomarker to assess the periodontal disease progression.
Early diagnosis of periodontal disease by estimation of calcium levels in saliva can help in prevention of
gingivitis or periodontitis by various therapeutic measures.

Keywords: Gingivitis, periodontitis, saliva, salivary calcium

Address for correspondence:

Dr. Girish Suragimath, Department of Periodontology, School of Dental Sciences, KIMSDU, Karad ‑ 415 110, Maharashtra, India.
E‑mail: drgirishsuragimath@gmail.com

INTRODUCTION Periodontal disease results from a complex interplay

between subgingival biofilm and the host immune
Periodontitis is defined as “an inflammatory disease of inflammatory events that develop in the gingival and
the supporting tissues of the teeth caused by specific periodontal tissues in response to the challenge presented
microorganisms, or group of specific microorganisms, by the bacteria. Plaque consists of different proteins
resulting in progressive destruction of the periodontal derived from saliva and gingival crevicular fluid  (GCF),
ligament and alveolar bone with increased probing depth, in which a number of microorganisms are embedded to
recession or both.”
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How to cite this article: Rane MV, Suragimath G, Varma S, Zope SA,
DOI: Ashwinirani SR. Estimation and comparison of salivary calcium levels
10.4103/jorr.jorr_36_16 in healthy controls and patients with generalized gingivitis and chronic
periodontitis. J Oral Res Rev 2017;9:12-5.

12 © 2017 Journal of Oral Research and Review | Published by Wolters Kluwer - Medknow
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Rane, et al.: Salivary calcium levels in health and disease
form a highly organized matrix. Plaque accumulation is the
first step toward initiation of periodontal problems.[1] The
primary cause of gingival inflammation is bacterial‑therapy,
self‑inflicted injuries, use of tobacco and others. Calculus
is one of the major predisposing factors. It consists of
mineralized bacterial plaque that forms on the surface
of natural teeth and dental prosthesis. The soft plaque is
hardened by the precipitation of mineral salts present in
the saliva.
Saliva is one of the most complex, versatile, and important
body fluids supplying large range of physiological needs.
Saliva is an exocrine secretion consisting of approximately
99% water and organic and inorganic molecules. It
contains a variety of electrolytes such as calcium,
magnesium, potassium, sodium, chloride, bicarbonate,
and phosphate.[2] Among these factors, some of them can
be used as biomarkers to assess the periodontal status.
Biomarkers are measurable and quantifiable biological
parameters that have an important impact on clinical
situations.[3] Some studies have assessed the relationship
existing between periodontal disease and these ions.
Among these components, elevated levels of calcium are
one of the factors seen to be associated with periodontal
diseases.
Salivary calcium plays a major role in the formation of supra
and subgingival calculus in the presence of unmineralized
dental plaque. The dental calculus formed act as niches
for further plaque accumulation which are the causative
agents of periodontal diseases. As the mineral content
increases in saliva, the plaque mass becomes calcified to
form calculus.[4] This is difficult to remove through routine
oral hygiene measures. It is hypothesized that high salivary
calcium content in saliva would result in a more rapid rate
of plaque mineralization leading to periodontal diseases.
There is a need to estimate the salivary calcium levels and
compare them between the healthy controls and patients
with generalized gingivitis and chronic periodontitis to
determine whether calcium levels in saliva can be used
as an early diagnostic marker and help in the prevention
of periodontal diseases. The objective of the study is to
estimate, compare, and correlate salivary calcium levels
in healthy controls, generalized gingivitis, and chronic
periodontitis patients.
MATERIALS AND METHODS
A total of 150 patients reporting to the Department of
Periodontology were enrolled for the study after due
approval of the ethical committee.
Journal of Oral Research and Review | Volume 9 | Issue 1 | January-June 2017 13
The subjects in the study were explained about the
objectives of the study, and informed consent was obtained
before commencing the study.
The study subjects were divided equally into three groups
according to the following parameters:
• Group  A  (50): Healthy controls with absence of
gingival inflammation and bleeding on probing[3]
• Group  B  (50): Generalized gingivitis patients with
gingival inflammation and bleeding on probing without
clinical attachment loss (CAL)[5]
• Group C (50): Chronic periodontitis patients who had
bleeding on probing with CAL.
Exclusion criteria
• Subjects who had received any periodontal treatment
during the past 3 months
• Subjects who had taken any antibiotic therapy during
the past 3 months
• Subjects who were suffering from any known systemic
diseases or conditions
• Tobacco users in smoked and smokeless form
• Pregnant and lactating women
• Subjects with less than 20 teeth.
Clinical examination
All the subjects were interviewed, and their general
information, medical history, and periodontal parameters
were recorded in a specially designed proforma. The
enrolled subjects were examined by a single calibrated
examiner under the supervision of a senior Periodontist and
categorized as a healthy, generalized gingivitis or chronic
periodontitis according to consensus of World Workshop
for the classification of periodontal diseases  (1999).[6]
Clinical parameters assessed:
• Gingival index (Loe and Sillness 1963)
• Plaque index  (Turesk y–Gilmore–Glickman
modification of the Quigley‑Hein Plaque Index,
1970)
• Oral Hygiene Index‑Simplified (John C.Greene, 1960)
• CAL.
After a detailed dental examination, the subjects were
rescheduled for the collection of saliva samples.
Saliva collection
Saliva samples were collected from 10:00 am, 2 h after the
last meal to standardize the collections according to the
circadian rhythm. Two milliliter unstimulated whole saliva
samples were collected following a brief rinsing of the
mouth with distilled water. The subjects were refrained
from talking and asked to drop down the head. They were
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Rane, et al.: Salivary calcium levels in health and disease

then asked to let the saliva pool in their floor of the mouth no statistical difference observed between Group B and
to their maximum extent and then expectorate into the Group C [Figure 1].
collecting vessel till the desired quantity was collected. The
samples were then transferred into sterile eppendorf tube, DISCUSSION
and 2 drops of 1% sodium azide was added which served
Saliva and GCF not only play a decisive role in preventing
as an antibacterial agent. The saliva samples were then
periodontal disease but also ironically in the induction of
transported to the laboratory immediately using standard
periodontal pathology.[7,8] Salivary calcium has more affinity
gel coolant packs to maintain the temperature between
toward plaque. Therefore, it is an important factor not only with
2°C and 4°C.
regard to periodontitis but also with regard to dental health.[9]
Saliva analysis
Calcifying plaque increases plaque retention limiting oral
The biochemist who analyzed the salivary samples was
hygiene and hence causing gingivitis. The continuous,
unaware of the periodontal status of the patients. The
apically growing, calcifying plaque may be sufficient
estimation of inorganic salivary calcium was carried out
enough to cause periodontitis, despite further efforts to
using Arsenazo reagent (Accucare Calcium Arsenazo III
improve oral hygiene. The mineralized plaque acts as niche
Lab Care Diagnostics, India Pvt. Ltd.). Biochemical assay
where further plaque accumulation takes place, and due to
of saliva samples was carried out to quantify the salivary
constant irritation caused by the increasing bacterial load,
calcium using a calcium test kit O‑Cresolphthalein complex
the healthy gingiva starts to show signs of inflammation
one) and end point assay (Span Cogent Diagnostic Ltd.
further leading to periodontitis.
India Erba EM 360 fully automated auto analyzer (Erba
Diagnostic, Mannheim, Germany). In most of the previous studies stimulated saliva was used
for analysis.[10] but in the present study, we used unstimulated
Statistical analysis
All the data collected was statistically analyzed using
Table 1: Demographic distribution
Statistical Package for the Social Sciences software
Healthy gingiva Gingivitis Periodontitis Total
version 19 (Armonk, New York: IBM. Corporation, USA). Group A Group B Group C
Study 50 50 50 150
The results were expressed in means and percentages; sample (n)
P ≤ 0.05 was considered statistically significant. Male (%)
Female (%)
19 (8)
31 (62)
28 (56)
22 (44)
35 (70)
15 (30)
82 (54.67)
68 (45.33)

The significance of difference in means was tested by

ANOVA test. Table 2: Salivary calcium levels
Healthy gingiva Gingivitis Periodontitis
RESULTS Group A (mg/dl) Group B (mg/dl) Group C (mg/dl)
Mean±SD 4.84±1.07 5.43±0.90 5.91±1.46
The 150 enrolled subjects were equally divided into three SD: Standard deviation

groups of fifty patients. In Group A, 19 patients were male

and 31 were female, in Group B, 28 were male and 22 were 6
female whereas in Group C, 35 were male and 15 were
female patients. Therefore, out of 150 patients 82 (54.67%) 5

patients were males and 68 (45.33%) patients were females.

The above data suggest that more number of males are 4

affected by periodontitis than the females [Table 1].

3

The mean salivary calcium in group A was found to be

4.84  ±  1.07  mg/dl, in Group  B is 5.43  ±  0.90  mg/dl 2

and in Group C is 5.91 ± 1.46, which showed that as the

periodontal disease progressed there was an increase in 1

the calcium levels in saliva [Table 2]. The comparison of

salivary calcium in Group A, B, and C revealed that there 0
HEALTHY GINGIVA GINGIVITIS PERIODONTITIS
was a statistically significant difference observed between
Group A and Group B: group A and Group C. There was Figure 1: Salivary calcium levels

14 Journal of Oral Research and Review | Volume 9 | Issue 1 | January-June 2017

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Rane, et al.: Salivary calcium levels in health and disease
whole saliva for analysis as it predominantly bathes the oral
cavity most of the time, and is a representative of pooled
subgingival plaque samples.[11]
The results of the present study revealed that the subjects
in the periodontitis group had significantly higher levels of
salivary calcium than gingivitis and healthy group. There
was a linear increase in the salivary calcium levels as the
disease progressed from healthy gingiva to periodontitis.
Similar results were reported by several authors (Sewon and
Karjalaine et al. and Fiyaz et al.,[9,12] Rajesh et al.[13]).
Sewon and Karjalaine et al. suggested that higher calcium
concentration of plaque is associated with low caries
incidence. The periodontitis‑affected group seemed to
have more intact teeth than the controls. It supports the
view that periodontitis‑affected subjects may have a higher
remineralization potential than individuals with no signs
of periodontitis. This is partly due to the excess of salivary
calcium.[9] However, in contrast, the results obtained by
Sewon and Makela, found that higher calcium level was
related to good dental health, but there was no relation to
periodontal bone destruction.[10]
In the current study population, the prevalence of gingivitis
and periodontitis was more in male subjects than female
subjects. These results were similar to study conducted by
Shaju et al. who reported more prevalence of periodontal
disease in male subjects than female subjects in the rural
population.[14]
Limitation
The calcium level of saliva may reflect fluctuations in
both dietary calcium and general calcium turnover but
the mechanisms are far from clear. In the present study,
the effect of diet and age wise calcium changes was not
considered. Larger sample size could be considered to
substantiate the results.
Future perspective
Further studies with larger sample size can be undertaken
to prove that salivary calcium levels can be used as a risk
biomarker for periodontal disease.
CONCLUSION
It can be concluded that the levels of salivary calcium
increased as the disease progressed from healthy to
Journal of Oral Research and Review | Volume 9 | Issue 1 | January-June 2017 15
gingivitis and periodontitis. Salivary calcium levels can
be used as a biomarker to assess the periodontal disease
progression. Early diagnosis of periodontal disease by
estimation of calcium levels in saliva can help in the
prevention of gingivitis or periodontitis by various
therapeutic measures.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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