The Role and Impact of Salivary Zn Levels on Dental Caries

Introduction Minimal attention has been given to the role of salivary microelements, the importance they have in reducing the intensity of caries, and the effect of caries prophylaxes. Aim This research aimed to determine the concentration and quantity of Zn and its impact on the prevention and the reduction of the intensity of caries in schoolchildren aged 12-13 years with permanent dentition. Methods For this research, we analyzed the stimulated and nonstimulated full saliva of 106 schoolchildren divided into three groups by mean decayed, missing, and filled teeth (DMFT) index. The control group consisted of 25 caries-free children, the second group had 47 children with mean DMFT index of 1 to 6, and the third group had 34 children with DMFT index of ≥ 6. Complete saliva was collected from all children in a sterile test tube. Results The concentration of Zn in saliva before stimulation in caries-free children has variations of the order of 0.001+ to 0.01 mmol/l. The maximum concentration after stimulation is 6.72 mmol/l, while the maximum value is 64.38 mmol/l. Conclusion The Zn concentration in the stimulated saliva showed a significant increase in the group of caries-free children and could be described as a positive value for the reduction of caries.


Introduction
A number of theories attempt to explain the mechanism of initiation of the appearance of caries. Recent research shows that caries have multicausal aetiology, mostly under the in uence of general factors but especially under the in uence of local factors.
Dental caries is perhaps the most ubiquitous disease that has a icted mankind. While it is not normally a fatal condition, it can cause a great deal of pain and distress, and the loss of teeth has profound consequences in terms of eating, speaking, and social behavior in general [1]. In recent years, particular importance in the appearance of caries has been devoted to saliva because of the impact of its chemical components and immunology [2,3].
Although it is known that the basic prerequisites for the appearance of cavities are de ned by the presence of microorganisms, the substrate, and the tooth itself for a certain period of time, all of this occurs under the in uence of the liquid media of the mouth saliva. In their researches, Gamershtein and Maksimovski [4] and Tvinnereim et al. [5] showed that the appearance of caries is directly a ected by the presence of salivary components, speci cally the amount of microelements that are present. Saliva, with the presence and composition of its immunochemistry, enables a large number of functions within oral homeostasis such as maintenance of oral cavity humidity and its self-cleaning ability, bu er of oral media, stabilization and preservation of bacterial ora, maintenance and preservation of the surrounding tooth minerals, digestive activity, control of pH, and many other functions. According to Dawes [6], Mason and Chisholm [7], and Schmidt [8], during high salivary ow, osmolarity can reach plasma osmolarity, while the ow of nonstimulated saliva can be so low that it can reach 1/20 of the plasma osmolarity. Research regarding salivary electrolytes shows that the saliva is saturated with some of the ions. Between the components of salivary electrolytes and those deposited in the enamel of the tooth, it has been found that there are certain equilibrium and controlled report [6][7][8].
e most important microelements that are present include calcium, sodium, magnesium, zinc, and uoride; these are of great importance for the mineralization and maturation of hard tooth tissue [9]. e relationship that trace elements in saliva might have with dental caries activity has interested scientists for many years [10].
Qualitative and quantitative analysis (EDS X-rays) shows evidence that the lowest content of the macroelements Ca, P, C, and O and the microelements Al, Cl, In, Mg, Si, Na, S, and W was found in carious enamel layers compared with normal enamel layers [11].
ere is an evident di erence in salivary electrolyte concentrations from di erent sources of saliva. Parotid saliva contains fewer Zn electrolytes of Zn and is the opposite of the concentration of Zn 2+ in the mixed saliva; the concentration of Zn 2+ varies signi cantly. e research regarding mineral components in saliva is scarce and has contradictory results with respect to their role in the process of demineralization, remineralization, and dental maturity [12]. In saliva, Zn plays multiple roles and a ects many metabolic processes. Its role in the metabolism of protein is so important that it is compared with essential amino acids. It is found in the composition of many enzymes where their activation depends on the presence of Zn. e impact and the amount of Zn in the tooth enamel are more in the outer layer (200-900 ppm) compared to the inner layer (up to 200 ppm) [13][14][15]. Curzon has noted that zinc and calcium showed promise as antiplaque agents, whereas Sr and Zn may enhance remineralization in enamel [16].
Research has shown that Zn is easily incorporated as a substitute for Ca ++ ions. Its incorporation in the enamel helps decrease its solubility. In many publications, the role of Zn in dental plaque and oral tissue has been described as an important factor in reducing the ability of bacteria, especially anaerobic bacteria [2,9].
Zinc salts have antibacterial actions due to their ability to inhibit bacterial adhesion, metabolic activity, and growth [17].
Relatively large amounts of zinc are incorporated into enamel prior to eruption, but after eruption, zinc concentration at the surface of the teeth apparently increases further, suggesting that some incorporation does occur during posteruptive exposure to the oral uids [18].
Zinc competes with calcium for bacterial-binding sites in model bio lms, and it has been proposed that half of the bound zinc would be released under cariogenic conditions through, for example, protonation of carboxylate and phosphate groups in bacterial lipoteichoic acid [19]. e aim of this research was to nd the Zn values in a group of caries-free children and two other groups with vulnerability to caries to determine the concentration and volume of Zn in the full stimulated and nonstimulated saliva through chemical and immunochemical analysis.
Also, this research aimed to de ne the in uence of this microelement in preventing or reducing the rate of the incidence of caries in schoolchildren aged 12-13 years with permanent dentition.

Materials and Methods
is research was conducted on 106 schoolchildren aged 12 to 13 years with permanent dentition. is was a crosssectional study where all children were divided into three groups (control group and two groups with vulnerability). e control group was composed of children with all cariesfree teeth (DMFT index � 0), healthy oral tissues, and good oral hygiene (25 children). e second group were children with a mean DMFT index of 1 to 6 (47 children), and the third group were children with a mean DMFT index of > 6 (34 children). Nonstimulated saliva was taken from all of the children in the morning because of the circadian rhythm for ve-minute duration. For obtaining the stimulated saliva, a clean para n wax bone was used for chewing for the duration of ve minutes. All samples were taken in sterile test tubes that were graded; until the analysis, the samples were stored in chambers at a temperature of −20°C. Chemical and immunochemical tests were conducted at the Faculty of Science, Ss. Cyril and Methodius University in Skopje. Analyses were performed by ame atomic absorption spectrometer model Solar S4 from ermo Elemental (UK), at a wavelength of 213.9 nm, spectral slit of 0.5 nm, and lamp current of 10 mA, representing a method with relatively high sensitivity. For the determination of zinc, 1 ml of saliva was diluted with redistilled water in a 10 ml volumetric ask. Statistical analyses were processed with Statistics for Windows/Release 7.0, at the Institute of Statistics of the Faculty of Medicine in Skopje. We obtained permission for this research from the corresponding institutions of our country.

Results and Discussion
Publications describing the mineral composition of native nonstimulated saliva are few; in the research that has been published, the results are often contradictory. Fluctuations of the volumetric physiological sphere of studied electrolytes are caused by the speed of saliva ow and the composition changes of the various secretions of salivary glands. Table 1 shows the concentration values of the examined samples and the amount of Zn in saliva before and after stimulation with para n. e rst group (control) had a concentration of Zn in stimulated saliva that varied in the interval of 0.01 ± 0.01 mmol/l, with a con dence interval of −0.01 + 0.01, a minimum value of 0.0002, and a maximum value of 0.03 mmol/l. e Zn concentration in the stimulated saliva showed variations in the interval of 0.28 ± 1.35, with a con dence interval of −0.27 + 0.83 mmol/l. e minimum value was 0.002, while the maximum value was 6.78 mmol/l. e Zn amount before stimulation was 0.02 ± 0.02 µmol/l. e minimum value is 0.0001 µmol/l, while the maximum value is 0.07 µmol/l. e amount of Zn after stimulation showed a more pronounced change with values of 2.65 µmol/l, with a standard deviation of ± 12.86, a con dence interval of −2.66 + 7.96, and a maximum value of 64.38 µmol/l. Table 2 shows the di erences in the concentration and quantity of Zn before and after stimulation. e concentration of Zn after stimulation was Z � 0.55 and showed no increase compared with that prior to stimulation (p < 0.05), while the amount of Zn was Z � 3.56 and showed a signicant increase after salivary stimulation (p < 0.001).
A reported decrease in the molarities of Ca and Zn that are found in caries can be very important in acknowledging the risk of caries, namely, to have an important role in the demineralization process of tooth decay. A relative surplus of Zn over Ca can be more closely associated with the occurrence of caries because it is validated with statistical tests. Table 3 shows the values of Zn concentration and the quantity before and after stimulation in children with DMFT index of 1-6. e Zn concentration varies before stimulation at intervals of 0.01 ± 0.01 mmol/l, with a con dence interval of −0009 + 0.01, a minimum value of 0.0002, and a maximum value of 0.08 mmol/l. e Zn concentration after stimulation has variations with intervals of 0.01 ± 0.01 mmol/l, with a con dence interval of −0005 + 0.01, a minimum value of 0.0005, and a maximum value of 0.07 mmol/l. e Zn amount before the stimulation varied at intervals of 0.02 ± 0.03 µmol/l, with a con dence interval of −0.01 + 0.03, a minimum value of 0.0005, and a maximum value of 0.16 µmol/l. e amount of Zn after stimulation varies, ranging between 0.07 ± 0.12 µmol/l, with a con dence interval of −0.03 + 0.11, a minimum value of 0.002, and a maximum value of 0.71 µmol/l. e highest incidence of enamel lesions was observed in the mandibular molars in rats fed with Zn-de cit diets compared with mice doubly fed with supplementary Zn diets. Furthermore, the e ects of Zn de ciencies in caries in young mice were observed in a greater mass in the smooth surface of molar zinc. Dietary Zn may be a trace mineral that is important during the posteruptive process of enamel mineralization; it could reduce a tooth's sensitivity to caries. Table 4 shows the di erences in Zn concentration and quantity before and after saliva stimulation. For Z � 1.84, no signi cant changes were observed in the Zn concentration after stimulation (p < 0.05). e amount of Zn for Z � 3.36 is signi cantly higher after stimulation with high valuation (p < 0.001). Table 5 shows the values of Zn concentration and quantity before and after stimulation of the third group of researched children with a mean DMFT index of > 6. e Zn concentration before and after stimulation in the saliva did not show signi cant di erences; the amount of Zn before and after stimulation showed signi cance in the saliva after stimulation for the interval of 0.04 ± 12.08 µmol/l, with a con dence interval of −0.01 + 0.07, a minimum value of 0.0006, and a maximum value of 12.48 µmol/l.
Di erences between Zn concentration and quantity before and after stimulation are presented in Table 6. e concentration of Zn after stimulation for Z � 0.84 did not show important di erences (p < 0.05); the amount of Zn for Z � 3.65 signi cantly increased, showing statistical signicance (p < 0.001).
Di erences were observed among the three groups in this research (Table 7) for H � 2.54 (p < 0.05); for H � 3.56, no signi cant di erence was found in the Zn concentration before and after saliva stimulation nor in the nonstimulated saliva (for H � 5.66 and p < 0.05). A di erence was observed among the three groups in the Zn amount after stimulation for H � 7.99 (p < 0.05). e amount of Zn for U � 568.00 (Table 8) among the groups before stimulation is obviously higher in the second   group compared to the third group (p < 0.05), while for U � 240, the amount of Zn after stimulation has had an ascendance of great importance in the rst group compared to the third group (p < 0.01). Although we expected that there will be di erences in the three groups for H � 5.66, we did not nd a statistical signi cance during the comparison. Di erences for U � 568 and U � 240 in the three groups showed a statistical signi cance at p < 0.01 and p < 0.05, respectively.
During the research, we saw that Zn was easily incorporated in the hydroxyapatite, exchanging with the Ca +2 ions. With the incorporation of Zn, the dissolution of enamel decreases, but this does not a ect the appearance of caries [20]. Insu cient data to describe the role of Zn in the process of caries have been presented in the literature. Quantitative analysis of ions released into solution following the demineralization of samples con rmed that Zn reduces the rate of demineralization as a function of concentration. To in uence enamel demineralization under cariogenic conditions, Zn must be available in the plaque uid at a concentration su cient to reduce or inhibit tooth mineral loss [21]. Contrary to the research done by Mohammed et al. [21], a study by Duggal et al. found that the concentration of Zn had no relationship with dental caries [22]. e results of Bales and Freeland-Graves [23], Fang et al. [14], and Gregory et al. [24] showed that, in the mice fed with a diet de cient in Zn, the incidence of caries has been high in the molars compared to the mice fed with normal food. is shows a close connection of Zn with proteins. Although there is little research on the role of Zn in the process of decay, in our research, the concentration of Zn is signicantly higher in the saliva of children with caries where      International Journal of Dentistry quantitative growth of Zn is evident in the two groups of children; this could, together with the "empty spaces," have the potential to show the demineralization role of saliva. It is assumed that Zn is easily released from the crystalline structure by leaving "empty space." Our results are similar to those of Tvinnerein et al. [25]. Regarding the clinical e ects of zinc on de-and remineralization, it seems unlikely that potentially bene cial e ects, such as reductions in solubility and enhanced/prolonged lesion porosity to mineral ingress, will counter any possible negative e ects [26]. In light of the current ndings, it would appear that there is scope for exploring and optimizing the therapeutic potential of zinc, not only as an antibacterial agent but also as a possible preventive treatment for caries [21]. Distinguished from the results of other authors [27,28], our data do not match since they have worked in selective saliva (from the saliva of the selected gland, with incomplete saliva) and because of the indirect in uence of saliva on dental plaque. Di erences were also prescribed to children's age and the presence of mixed dentition. According to Hussein et al., salivary Cu and Zn levels were signi cantly higher in children with dental caries compared to those who were caries-free [29]. Also, it is found that the use of toothpaste containing nanocrystals of carbonate hydroxyapatite replaced with Zn can produce mimicking e ect of morphology, structure, and composition of biological hydroxyapatite of enamel [30].

Conclusion
Based on the results obtained with the chemical and immunochemical analysis of whole saliva, we can obtain the following conclusions: (i) After the stimulation, we found that the Zn concentration in the rst group was higher. (ii) e quantity of Zn before and after the stimulation in the second and third groups with caries showed statistically signi cant di erences. (iii) e quantity of Zn after the stimulation showed signi cant di erences among the three groups. ese di erences are higher in the rst group in comparison to the second and third groups. (iv) e increase in Zn concentration and quantity in the rst group (caries-free) in comparison with the second and third groups indicates the positive e ect on reducing caries.
From these ndings, we can conclude that Zn has an impact in reducing the appearance of caries that is proved with the Zn quantity di erences found in the three groups that were investigated.
Ethical Approval e protocols and human data that the authors used in this study were approved by the Ethical Board of the Faculty of Chemistry at the University of "St. Cyrus and Methodius" in Skopje, which gave permission for this research.