Macro and trace minerals are functional to periodontal maintenance, and their alteration in biofluids can represent a sign of pathological conditions.34 Our study was the first systematic review to evaluate the salivary ionic profile of subjects with different periodontal statuses. Results for 22 specific elements were sorted from 13 included reports. Consistent outcome measures were found for those elements related to the pathways of immune-modulation and bone tissue homeostasis.

4.1 Outcomes for specific metals

Periodontitis results as the consequence of a chronic immune-inflammatory reaction to the microbial challenge that brings to the destruction of tooth-supporting apparatus and to the discharge of inflammatory markers and breakdown products in gingival crevicular fluid (GCF) and saliva.5, 35 Therefore, it is rationale to hypothesize that perturbations in ionic content of these biofluids would be suggestive of the presence and severity of periodontitis.

Na concentrations were found consistently higher in saliva of patients with chronic or aggressive periodontitis with respect to individuals with periodontal health. Na represents one of the most abundant elements in humans, mainly distributed in blood, bone, and connective tissues where it mainly set the balance of fluids and nutrients throughout the cellular membrane.36 Following the destruction of the alveolar bone tissue, Na can be released into the extracellular compartment and hence into the GCF and saliva.19, 37 Some authors have reported that Na levels tend to increase proportionally according to the severity of the clinical attachment level loss and bleeding,38, 39 while others failed to confirm it.32 An antibacterial function of salivary Na ions has also been recognized, possibly suggesting its elevation in saliva as a mechanism of defense.26

Increased salivary Ca concentrations have been widely related to periodontitis in the present review. Several biological processes may be accounted for this shift. Calcium is an essential mediator for intracellular signaling pathways, which during the development of periodontal inflammation is upregulated to stimulate reactive oxygen species (ROS) production and the expression of cytokine mediators, such as tumor necrosis factor-alpha (TNF-α).40 Therefore, both intracellular calcium increment and hard tissue breakdown could contribute to the elevated concentration of salivary calcium. Four authors showed a strong significant difference in the salivary levels of ions between healthy and periodontal subjects, with a higher concentration in P.21, 27, 32, 33 Increased levels of this metal in saliva influence the mineralization of dental plaque and therefore calculus formation, which is a risk factor for the development of gingivitis that can later evolve into periodontitis.41 The authors also found a significant correlation between Na, Ca, and clinical attachment levels, supporting a possible dose-dependent effect between ion imbalance and the severity of the disease.

Increased Cu levels were also associated with periodontal breakdown. It is known how Cu and Zn functions can be closely related, being key constituents of antioxidant enzymes like Cu–Zn superoxide dismutase (SOD).42 The results from the present review indicate that there is a tendency for an excess of Cu and Zn decrease within the saliva of periodontitis patients. This can be due to the fact that elevated levels of Cu can alter the permeability of the gingival epithelium and impair Zn mucosal absorption.43 Notably, Zn concentration was restored to physiologic levels after successful periodontal treatment.22 Moreover, salivary histatins are relevant antimicrobial enzymes within the oral cavity and they are composed of copper.44 Therefore, increased salivary copper levels might indicate that either SOD and/or histatins are not well functioning. Accordingly, histatin 3 gene was revealed under expressed in gingival smears of P compared to H, possibly indicating a host defense dysregulation.45

Among the controversies, magnesium was the element which was examined in most studies. Three of them reported a decreased Mg concentration in the saliva of P. Notably, a close association between Mg deficiency and macrophage and leukocyte overactivation has been demonstrated across multiple conditions, as Mg can elicit oxidative-inflammatory processes by impairing intracellular Ca.46 Conversely, Mg is abundantly represented in calcified tissues, and when periodontal breakdown occurs, the release of Mg can produce an increase in its salivary concentration, even in presence of Mg deficiency.47 As for other ions, some authors found a direct correlation between salivary Mg levels and severity of periodontal diseases observing an increase in salivary Mg levels in patients with gingivitis. Magnesium can also have a negative influence on intracellular and salivary K concentration. This was suggested by Kaslick et al.48 and Aun32 which found an enhanced salivary K concentration in P associated with magnesium deficiencies, although further studies are required to provide mechanistic explanation.

Notably, the study by Huang et al.31 reported outcomes in marked contrast with the rest of the included works, despite using the same technique (ICP-MS) and comparable case definitions. The authors found a higher level of all analyzed chemical elements in the unstimulated saliva of healthy subjects compared to P. These discrepancies can be attributed to ethnic aspects related to genetic features and dietary habits, being the level of these micronutrients strictly associated with the food frequency questionnaire of the population analyzed.

More than per se variations, it would be relevant to understand whether metal ion concentration could serve for diagnostic purposes. To this regard, only two studies implemented ionomic analysis into discriminating models. Romano et al.22 developed a cluster analysis which was able to correctly separate active periodontitis and periodontally healthy individuals, whereas treated periodontitis individuals were classified as H. Also, Herman et al.29 successfully applied such a statistical approach to distinguish CP from H.

4.2 Methodologic issues

In the last years, ionomics has received an impressive development owing to the implementation of reliable measurement technologies and instruments for large-scale data analysis. Mineral element levels in saliva have been traditionally measured by different methods, such as AAS, ICP-optical emission spectrometry (OES), potentiometry, thermal neutron activation analysis, and gamma ray spectrometry. In particular, the most commonly used in ionomics is ICP-MS, because it allows fast and accurate routine multi-element determination with improved sensitivity for biological samples.49 Considering the high heterogeneity of findings for each metal concentration, the employment of different detection method could be a major determinant.

Also, circadian and seasonal variations are of utmost importance when interpreting the findings from metabolites and ions in saliva, as well as for the effect of tobacco smoking.50 Standardization of sampling procedures, in accordance to dietary restrictions, home oral hygiene restraints, and timing of collection are of primary importance.19

A thorough assessment of periodontal status represents the reference standard for periodontal diagnosis; nonetheless, only few studies accurately reported how periodontal parameters were recorded and the definition of periodontal cases largely varied across the included studies. It has been shown that patients with untreated periodontitis had values above those of H and patients with restored healthy periodontium at the completion of active periodontal therapy, while levels of treated periodontitis patients were not statistically different from those of H.22 Ethnic background and variations in sample sizes can also account for a large part of the variability.51, 52 Finally, most of the included studies did not get high scores in the quality assessment.

4.3 Future research directions

Despite ionomics being rapidly developing in different fields of medicine through the last years, our understanding of the relationship between different ions and the pathogenesis of complex diseases is still limited. This review critically examined the available evidence on ionomics as a new platform to improve early diagnosis, prognosis evaluation, and therapy of periodontal diseases. Meaningful reduction and integration of big data arising from ion-based high-throughput techniques is highly demanded to find the signal among the noise and to successfully translate biochemical signatures into clinically relevant information. Whether significant differences in macro and trace elements between patients will become evident, a microfluidic chair-side test could be developed in both early diagnosis and/or monitoring of the clinical progression/response to the treatment. To this regard, only one study considered the elemental profiles of successfully treated periodontitis patients.

From the present systematic review, ionomics emerges as a promising landscape for biomarkers discovery and implementation for diagnostic purposes applied to periodontology, despite its preliminary results could be hampered by environmental and technical biases in elemental composition and detection. Sodium and potassium were the elements found consistently and significantly higher in saliva of P compared to H. Due to high concerns in developing more accurate, non-invasive, and portable diagnostic tools for individual use and population-based strategies, the results from the present work indicate a great need to identify and quantify macro and trace elements in saliva for periodontitis by providing measures of diagnostic accuracy.