Keywords
atomic absorption spectrophotometry - calcium - dental health survey - edentulous
- mouth - saliva
Introduction
Saliva and its components play a significant role in oral defense and ecological balance,
and in maintaining oral and dental health.[1]
[2]
[3]
[4] Saliva has a complex and unique composition of water, proteins, glycoproteins, and
ions responsible for its various properties.[1]
[2] Recently, saliva has become an efficient diagnostic tool for scientists in oral
and systemic health and diseases.[2]
[3]
[5] The idea of using saliva for diagnostic purposes was first suggested in a late twentieth
century.[2]
[6] This method is noninvasive, simple, safer, and painless, and does not require a
skilled workforce.[2]
[3]
[7] The main electrolytes in the whole saliva include sodium, potassium, calcium, magnesium,
chloride, bicarbonate, phosphate, thiocyanate, and fluoride. Of salivary electrolytes,
calcium, phosphate, bicarbonate, and fluoride are highly crucial for oral and dental
health.[8] Saliva serves as an irrigating solution for teeth and oral mucosa. It also has the
buffering capacity and serves as a reservoir of calcium and phosphate ions, which
are required for remineralization of initial enamel lesions.[9]
Calcium is an inorganic component of the saliva, and quantitatively, it is considered
as the main mineral component of the human skeletal system.[6] Calcium plays a significant role in the physiological processes of the human body.
Calcium is present in different forms in oral fluid. A large portion of salivary calcium
ions is bonded to inorganic components such as orthophosphate or carbonate or macromolecules
such as proteins. Almost half the calcium present in the saliva is in ionized form.
Most studies have shown that the concentration of calcium present in dental plaque
is significantly higher than that in the saliva.[10] The concentration of calcium ion in the saliva is much lower than its plasma concentration.[2]
[3]
[7]
Hormonal factors can affect saliva composition in the long term. For instance, the
salivary concentration of calcium ions is higher in the mid-menstrual period compared
to premenstrual and postmenstrual phases.[11] Estrogen plays an important role in calcium metabolism in menopausal women by inhibiting
bone decalcification.[5] Saliva also protects the tooth structure by providing calcium and phosphate ions.
According to the chemical law of mass action, these ions affect driving forces responsible
for deposition or dissolution of calcium hydroxyapatite, which is the primary mineral
component of dental hard tissue.[12]
[13]
Change in the level of calcium present in the fluid surrounding enamel crystals, which
are mainly composed of hydroxyapatite, affects the dissolution of minerals by changing
the degree of saturation of hydroxyapatite. It appears that the salivary calcium level
affects the calcium concentration of dental plaque by the diffusion phenomenon. These
changes can consequently impact on the activity of calcium in demineralization and
remineralization processes that occur in tooth structure.[10]
In general, low concentration of calcium translates to low thermodynamic driving force
for deposition of hydroxyapatite at normal oral pH. Driving force for dissolution
of hydroxyapatite is higher at pH values lower than the critical pH.[12] According to Dawes, calcium is the only salivary electrolyte at a constant level.
In contrast to other salivary electrolytes, the concentration of calcium is not affected
by changes in saliva stimulation.[14] Dawes presumed that salivary calcium is remaining stable through interaction by
teeth surface which undergoes dissolution and deposition to reach to an equilibrium.[10]
This study aimed to compare unstimulated salivary calcium level of dentulous and edentulous
patients to find out whether the salivary calcium level remains constant in edentulous
patients or not.
Materials and Methods
The protocol of this case-control study was approved in the Ethics Committee of Guilan
University, School of Dentistry (IR.GUMS.REC.1394.463).
Sample size was calculated to be 32 patients in each group for the comparison of salivary
calcium level considering 95% confidence interval (CI) and 80% study power with expected
clinical difference equally for 4 teeth in dichotomous variables (high and low calcium
level).[11] Thirty-two dentulous and 32 edentulous patients, matched in terms of age and sex,
were selected using convenience sampling and included in the study. Patients were
selected among those presenting to the Dental Clinic of Guilan University, School
of Dentistry. Patients with osteoporosis, hyperparathyroidism, Paget's disease, renal
disorders, and systemic conditions affecting calcium level were not included in the
study. Participants were divided into two groups of dentulous and edentulous, and
their demographic information and salivary calcium level were assessed. Informed consent
was obtained from all individual participants included in the study.
Patients were briefed about the study and written informed consent was obtained from
them. Furthermore, participants were provided with a brochure containing information
about the study.
Saliva Collection
Saliva was collected in the morning between 8:30 a.m. and 12:30 p.m. Patients were
requested to have breakfast and not to eat or drink anything after that until saliva
collection. Before saliva collection, patients were asked to rinse their mouth with
deionized water and remove their removable partial or complete denture (if any). Saliva
collection tubes and funnels were acid-washed to increase accuracy. Unstimulated saliva
was collected in tubes using the spitting method. Salivary flow rate was also measured
in gram per minute. The saliva samples were transferred to a laboratory in a cold
box.[7]
Measurement of Salivary Calcium Level
After transfer to a laboratory, the samples were frozen at –20°C. Before experiment,
the samples were removed from the freezer and thawed to 25°C. They were then centrifuged
at 2,000 g for 10 min. The samples were transferred to BT3500 autoanalyzer (Biotecnica
Instruments, Rome, Italy) for measurement of salivary calcium level, which was determined
by colorimetry (atomic absorption spectrometry). In this method, salivary calcium
causes a color change when exposed to methylene blue. The degree of color change is
proportionate to the concentration of calcium ions. To prevent interference by magnesium
ions, 8-hydroxyquinoline is added.
Statistical Analysis
The data were analyzed using SPSS version 21 (Chicago, Illinois). The Shapiro–Wilk
test was used to assess the distribution of unstimulated salivary calcium level in
terms of normality in the two groups, which showed that it did not have a normal distribution
(p < 0.0001). Nonparametric Mann–Whitney U-test was applied to compare the salivary
level of calcium between the two groups of dentulous and edentulous patients. Spearman's
rho was used to measure the strength of association between salivary calcium and flow
rate. Multivariate analysis was exerted for the effect of study group (dentulous/edentulous)
on unstimulated salivary calcium.
Results
This study was conducted on 72 patients in two groups of 36. [Table 1] shows the comparison of unstimulated salivary calcium level, salivary flow rate,
and number of teeth in the two groups of dentulous and edentulous patients.
Table 1
Comparison of calcium level, salivary flow rate, and number of teeth in the two groups
of dentulous and edentulous patients
|
Mean ± SD
|
p
|
|
Dentulous
|
Edentulous
|
|
Abbreviation: SD, standard deviation.
|
|
Calcium concentration
|
0.61 ± 0.39
|
0.80 ± 0.6
|
0.213
|
|
Salivary flow rate
|
0.33 ± 0.20
|
0.39 ± 0.25
|
0.241
|
|
Number of teeth
|
18.9 ± 7.5
|
0±0
|
0.000
|
Both groups included 31 males and 5 females. The mean age (± standard deviation) was
56.36 ± 10.58 years in dentulous and 56.69 ± 10.99 years in edentulous patients.
The results showed that the two groups were not significantly different in terms of
unstimulated salivary concentration of calcium (p > 0.05). No significant difference was noted in unstimulated salivary calcium level
of males (0.725 ± 0.525 mmol/L) and females (0.5625 ± 0.145 mmol/L) either (p > 0.05). The mean (± standard deviation) number of teeth in dentulous patients was
18.9 ± 7.5 teeth (range: 3–30).
Salivary concentration of calcium was not significantly different in edentulous and
dentulous patients (p > 0.05). However, in the 50- to 60-year-old group, the mean unstimulated salivary
level of calcium was significantly different between edentulous and dentulous patients
such that edentulous patients had higher level of unstimulated salivary calcium (p = 0.04; [Table 2]).
Table 2
Comparison of unstimulated salivary calcium level (mmol/L) in dentulous and edentulous
patients based on the age groups
|
Age group (years)
|
Mean ± SD
|
Total
|
p*
|
p**
|
|
Dentulous
|
Edentulous
|
|
Abbreviation: SD, standard deviation.
*Mann–Whitney.
**Kruskal–Wallis.
|
|
< 50
|
0.68 ± 0.34
|
0.68 ± 0.25
|
0.68 ± 0.29
|
0.999
|
0.301
|
|
50–60
|
0.63 ± 0.46
|
1.01 ± 0.745
|
0.82 ± 0.6
|
0.04
|
|
> 60
|
0.54 ± 0.36
|
0.69 ± 0.59
|
0.61 ± 0.5
|
0.836
|
According to the Spearman's rho, the results showed that in dentulous patients, unstimulated
salivary calcium concentration had an inverse correlation with unstimulated salivary
flow rate (r = –0.370, p = 0.027) such that by an increase in salivary flow rate, salivary level of calcium
decreased. As seen in [Fig. 1], by 1-unit increase in salivary flow rate of dentulous patients, their salivary
level of calcium decreased by 3.85 units. The regression formula for this inverse
correlation was as follows:
Fig. 1 The scatter plot of the correlation of salivary flow rate with unstimulated salivary
calcium level in dentulous patients.
Furthermore, the R
2 coefficient in [Fig. 1] shows that 24.3% of changes in unstimulated salivary calcium level in dentulous
patients depend on their salivary flow rate; however, no such significant correlations
were noted in edentulous patients ([Fig. 2]). There was no correlation between salivary calcium and the number of teeth.
Fig. 2 The scatter plot of the correlation of salivary flow rate with unstimulated salivary
calcium level in edentulous patients.
Multivariate analysis of the effect of study group (dentulous/edentulous) on unstimulated
salivary calcium level after controlling for the effects of age, sex, and salivary
flow rate by multivariate linear regression model with backward stepwise technique
and possibility of inclusion and exclusion of variable from the model yielded 0.05
and 0.1 values, indicating that study group (p = 0.05) and salivary flow rate (p = 0.038) were among the predictors of unstimulated salivary calcium level, such that
edentulous patients had averagely higher unstimulated salivary calcium level compared
to dentulous patients (0.9 ± 0.47). Furthermore, by 1-unit increase in salivary flow
rate, after controlling for the effects of age, gender, and study group, unstimulated
salivary calcium level decreased by 2.2 units.
The final regression formula for the correlation of unstimulated salivary calcium
level and study variables was found to be as follows:
Multivariate logistic regressions verify the findings. The odds of salivary calcium
level up to mean in edentate rising (odds ratio [OR]: 2.6, 95% CI: 8.16–8.4) versus
dentate patients (p = 0.1). Salivary flow rate had inverse correlation with salivary calcium level (OR:
0.048, 95% CI: 0.002–1.28) (p = 0.07; [Table 3]).
Table 3
Regression coefficients of the predictors of unstimulated salivary calcium level according
to the multiple linear regression model with backward stepwise method
|
Model
|
Unstandardized coefficients
|
Significant
|
95% CI for B
|
|
B
|
SE
|
Lower bound
|
Upper bound
|
|
Dependent variables are salivary calcium level.
Abbreviations: CI: confidence interval; SE: standard error.
|
|
Final model
|
|
|
|
|
|
|
Constant
|
2.282
|
0.787
|
0.005
|
0.712
|
3.851
|
|
Edentulous versus dentulous
|
0.900
|
0.468
|
0.058
|
–0.033
|
1.833
|
|
Salivary flow rate (g/min)
|
–2.215
|
1.045
|
0.038
|
–4.300
|
–0.131
|
Discussion
This study aimed to compare unstimulated salivary calcium level in dentulous and edentulous
patients. The main finding of this study was the absence of a significant difference
in this respect between the two groups.
A previous study showed no significant association between the salivary calcium level
and salivary flow rate.[14] However, calcium ions are in constant reaction with the tooth structure. Thus, in
lower salivary flow rate and lower than critical pH, calcium concentration may be
different based on the ionization coefficient since salivary calcium constantly reacts
with hydroxyapatite in tooth structure.[13] Another finding of the current study was that the salivary level of this ion was
almost similar in dentulous and edentulous patients. It should be noted that due to
the absence of teeth in edentulous patients, enamel pellicle and dental plaque do
not exist.[15] A reduction in salivary calcium level was noted in dentulous patients with higher
salivary flow rate, which also suggests that the level of calcium ion remains constant.
In other words, in higher salivary flow rates, the concentration of calcium decreases
in order to maintain a constant level in the saliva.[13] It should be noted that the increase we are talking about in this study is an increase
in unstimulated salivary flow rate. Stimulated saliva is more serous and contains
higher levels of proline-rich proteins, statherin, and some other compounds that prevent
deposition of calcium on teeth. Different results may be obtained if stimulated saliva
is evaluated, which needs further assessment in future studies. In the other hand,
point-of-care technology is a potential for biomarker identification through salivary
diagnostic toolboxes.[16]
[17]
[18] This method brings accuracy, repeatability, and reproducibility. This method will
be suggested to detecting biomarkers as salivary calcium in further studies.[18]
The absence of any difference in unstimulated salivary calcium concentration of dentulous
and edentulous patients suggests many other theories raising the possibility of involvement
of many other factors, which may play a role in maintaining this balance. For instance,
bicarbonate, phosphate, and urea may have a more significant role in this respect.
Enamel pellicle inhibits the uptake of hydrogen ions and prevents the release of calcium
and phosphate ions. Saliva is supersaturated with calcium ions compared to hydroxyapatite.
In the absence of pellicle, hydroxyapatite crystals tend to expand over the surface
(calculus formation).[19]
[20] However, it should be noted that some studies have shown a correlation between low
concentrations of calcium and phosphate and occurrence of caries,[9]
[21] while some others found no such association.[22] Some authors believe that the role of calcium and phosphate concentrations in this
respect is more important than that of pH. Studies have indicated that patients with
low concentrations of calcium and phosphate have a critical pH of 6.5, while patients
with higher levels of these minerals have a critical pH of 5.5.[12] Maier et al evaluated the excretion of ionized calcium in pilocarpine-stimulated
saliva. The concentration of ionized calcium significantly increased in higher salivary
flow rates, but the total calcium concentration had no association with salivary flow
rate.[19] Salivary calcium level of menopausal women with xerostomia can be higher than that
of women without xerostomia.[5]
[6]
[10]
[20]
[23]
Lack of a significant difference in unstimulated salivary calcium level of edentulous
and dentulous patients and no association between salivary calcium level and number
of teeth in our study question the common belief and the existing literature, stating
that individuals with higher salivary calcium level have higher number of sound teeth
than those with lower salivary calcium level.[17] Moreover, the current study found no significant difference in salivary calcium
concentration of males and females or different age groups, which was in contrast
to the findings of previous studies.[9]
[11]
[24]
[25] Rabiei et al suggested the use of increase in salivary calcium concentration higher
than the cutoff point of 6.1 mg/dL for efficient osteoporosis screening of menopausal
women. They showed that each 1-unit increase in salivary calcium concentration increased
the risk of osteoporosis by 1.6 folds.[7]
With regard to the critical pH for enamel dissolution in the oral environment, the
reaction between salivary calcium and calcium in tooth structure is a balanced forward–backward
reaction, which constantly changes. When saliva is supersaturated with calcium, its
deposition rate exceeds its dissolution rate. When saliva is not saturated with calcium,
calcium tends to release from the tooth structure into the saliva and dissolution
of minerals occurs at a faster rate. These reactions continue until the saliva is
saturated with calcium. At this point, the forward reaction equals the backward reaction.
However, on the other hand, the salivary calcium concentration remains constant.
Conclusion
The results of this study showed that irrespective of the presence/absence of teeth,
salivary calcium level remains constant. It appears that only salivary flow rate can
change the salivary concentration of calcium and presence/absence of teeth plays no
role in this respect.
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