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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 pa¬rameters 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 Trust’s 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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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 anti-inflammatory 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 2°C- 4°C.

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

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: 650nmTemperature: 37°CSensitivity: the lower limit of detection is 0.04mg/dl.

REAGENTS USED:

Phosphate buffer, pH7.58- hydroxyquinoline-5-sulfonic acidArsenazo IIIDetergents

PROCEDURE:

All reagents were brought to room temperature.

Pipette into test tubes blank (µl) standard

(µl) sample (µl)

reagent 1000 1000 1000

sample - - 10

standard - 10 -

water 10 - -

It was then mixed well and incubated for 5 min at 20-25°C/ 37°C.

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. * Diagnostic kit, Dialab Productions.

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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). PD’s 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 1: DEMOGRAPHIC DATA SHOWING STUDY POPULATION

SUBJECTS DIAGNOSISN=60

MALES= 42 FEMALES=18

HEALTHY= 20 GINGIVITIS=20

PERIODONTITIS=20 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

TABLE 2: MEAN AGE OF SUBJECTS

TABLE 3: MEAN PROBING DEPTH AT SAMPLE SITES

GROUPS MEAN AGEHEALTHY 27.15±5.03

GINGIVITIS 29±6.3PERIODONTITIS 33.75±6.27

GROUP MEAN (mm) STANDARD DEVIATION

HEALTHY 1.54 0.08

GINGIVITIS 1.63 0.25

PERIODONTITIS 4.91 1.2

TABLE 4: COMPARISION OF CALCIUM LEVELS IN HEALTHY, GINGIVITIS AND PERIODONTITIS

GROUPS *

HEALTHY GINGIVITIS PERIODO NTITIS

p VALUE

CALCIUM LEVELS (mmol/l)

0.51 ± 0.27 0.97 ± 0.52 1.54 ±

0.84 p=0.00

TABLE 5: INTERGROUP COMPARISION OF SALIVARY CALCIUM LEVELS *

p VALUEHEALTHY VS GINGIVITIS 0.001

HEALTHY VS PERIODONTITIS 0.000

GINGIVITIS VS PERIODONTITIS 0.015

Comparision of Salivary Calcium Levels in Health, Ginvitis and Periodontitis

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with no signs of periodontal disease. The results however were in contrast with the results obtained by Sewon and Makela in 1990 11 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.

REFERENCES:

1. Lavelle Christopher L.B. Applied oral physiology. Second edition. Butterworth and co Ltd;1988:133-134

2. Erdemir E, Erdemir A. The detection of salivary minerals in smokers and non smokers with chronic periodontitis

by inductively coupled plasma atomic emission. J Periodontol 2006;77:990-995.

3. Otman Z, Machtei EE, Ben-Aryeh H, Ardekian L, Peled M, Laufer D. The effect of smoking and periodontal treatment on salivary composition in patients with established Periodontitis. J Periodontol 1999;70:1240-1246.

4. Griffiths GS, Sterne JA, Wilton JM, Eaton KA, Johnson NW. Association between volume and flow rate of gingival crevicular fluid and clinical assessment of gingival inflammation in a population of british male adolescent. J Clin Periodontol 1992;19:464-474.

5. Miller CS, King P Jr, Langub C, Kryscio J, Thomas MV. Salivary biomarkers of existing periodontal disease. J Am Dent Assoc 2006;137:322-329.

6. Sewon LA, Karjalainen SM, Soderling E, Lapinleimu H, Simell O. Associations between salivary calcium and oral health. J Clin Periodontol 1998;25:915-919.

7. Silness J, Loe H. Periodontal disease in Pregnancy. Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 1964;22:112-135.

8. Bauer PJ. Affinity and stoichiometry of calcium binding by arsenazo III. Anal Biochem 1981;110:61-72.

9. Cortelli SC, Feres M, Rodrigues AA, Aquino DR, Shibli JA, Cortelli JR. Detection of Actinobacillus actinomycetemcomitans in unstimulated saliva of patients with chronic periodontitis. J Periodontol 2005;76:204-209.

10. Sewon L, Soderling E, Karjalainen S. Comparative study on mineralization related intra oral parameters in periodontitis affected and perioodontitis free adults. Scand J Dent Res 1990;98:305-312.

11. Sewón L, Mäkelä M. A study of the possible correlation of high salivary calcium levels with periodontal and dental conditions in young adults. Arch Oral Biol 1990;35(Suppl): 211S-212S.

12. Hassan ShA, Al Sandook TA. Al-Rafidain. Salivary calcium concentration in patients with high incidence of calculus formation. Al–Rafidain Dent J 2005;5:88-90.

CORRESPONDENCE: Dr Nupur Sah

A-404 , mehek chs, plot -17a Sec- 12, kharghar,

Navimumbai-410210 E mail: sahnupur02@gmail.com

Nupur Sah et al.