Macro and trace elements signature of periodontitis in saliva: A systematic review with quality assessment of ionomics studies

Abstract Objectives The present systematic review examined the available evidence on distinctive salivary ion profile in periodontitis compared to periodontal health and provided a qualitative assessment of the literature. Background Macro and trace elements are essential for cellular physiology, and their changes in biological fluids can be revelatory of an underlying pathological status. Methods Data from relevant studies identified from PubMed, Embase, and Scopus databases were retrieved to answer the following PECO question: “In systemically healthy individuals, are there any differences in any salivary macro or trace element concentration between periodontally healthy subjects (H) and patients with periodontitis (P)?” Quality of included studies was rated using a modified version of the QUADOMICS tool. A consistency analysis was performed to identify significantly discriminant chemical elements. Results After the screening of 873 titles, 13 studies were included reporting data on 22 different elements. Among them, levels of sodium and potassium were consistently and significantly higher in P compared to H. Conflicting results were found for all the other elements, despite concentration of calcium, copper, and manganese mostly increased in saliva of P. Levels of magnesium were found higher in P than in H in 2 studies but lower in 3. Zinc resulted significantly increased in saliva from H compared to P individuals in 2 studies, but one study reported opposite results. Four studies were considered as high quality, while reporting of operative protocols and statistical analysis was a major limitation for the others. Due to high methodologic heterogeneity, meta‐analysis was not performed. Conclusions Levels of macro or trace elements were differentially identified in saliva across diverse periodontal conditions, having a major potential for investigation of oral homeostasis and for high‐resolution periodontal diagnosis. Products of inflammatory physiologic cellular impairment, such as sodium and potassium, were the most consistently associated with periodontitis (PROSPERO CRD42021235744).


| INTRODUC TI ON
Periodontitis is one of the most prevalent chronic non-communicable diseases worldwide, 1 affecting up to 20% of the people over 40 years of age in its severe form. 2 When periodontitis occurs, the tissues that surround and support the teeth undergo an irreversible destruction that may progress over time, eventually leading to tooth migration or tooth loss with consequent disability in terms of altered masticatory function and aesthetics, and impairment of general health. 3,4 Early detection of periodontitis would be the key to preventing a more severe illness for the patient and to reducing the economic burden for the society. Indeed, treatment of periodontitis at its initial stages is easy and successful, whereas at more advanced stages, it requires surgical procedures performed by trained specialists to arrest disease progression. Therefore, non-invasive accurate diagnostic methods are sought to discriminate periodontitis from healthy subjects in a rapid way which may be accessible to large proportions of the population. 5 Salivary diagnostics is evolving as a highly potential field for early diagnosis and monitoring of oral and systemic diseases. 6,7 Saliva can be obtained by inexpensive sampling methods that cause minimal patient discomfort and contains a vast variety of biomolecules, such as DNA, mRNA, microRNA, protein, metabolites, microbiota, and ions. 8 Thus, it represents an ideal fluid for the discovery and validation of biomarkers. 9 Although many clinical applications of salivary diagnostics are available to date, the lack of definite biomarkers for periodontal disease and portable, inexpensive, user-friendly diagnostic platforms still prevent salivary diagnostics from being fully clinically translated.
Ionomics is the study of the ionome, defined as "the mineral nutrient and trace element composition of an organism, representing the inorganic component of cellular and organismal systems." 10 Some macro elements are essential for the human life, and they are needed in more amount because they control important biological processes, such as hormone synthesis, cellular signaling, and enzymatic catalysis (such as magnesium, calcium, potassium, and sodium). Some other elements (such as zinc, iron, selenium, and iodine) are required in very small amounts and, therefore, are known as trace elements. 11 In the last years, ionomics has been increasingly applied to the investigation of human physiologic and pathological conditions. 12,13 Imbalance in essential elements has been consistently associated with human diseases, such as diabetes mellitus, neurodegenerative diseases, cancer, and periodontitis. 14 Recently, research interest has been directed toward the analysis of the salivary mineral profile across different periodontal conditions considering that macro and trace mineral deficiency or excess could be related to both inflammation and oxidative damage, and they may be associated with periodontal tissue breakdown. Since no systematic reviews have been conducted to date, the present study aimed at summarizing the available evidence regarding the feasibility of salivary ionomic analysis for the diagnosis of periodontitis. Moreover, a methodologic quality assessment of the included studies was provided in order to evaluate risk of bias and favor a standardization of future research endeavors.

| MATERIAL S AND ME THODS
This research has been conducted according to the Cochrane Handbook and reported according to the PRISMA statement. 15,16 The protocol was registered on PROSPERO (CRD42021235744).

| Focused questions
This systematic review was designed to answer the following focused PECO question: In systemically healthy individuals, are there any differences in any salivary macro or trace element concentration between periodontally healthy subjects and patients with periodontitis? (O) Type of outcome measures. Difference in salivary macro or trace element levels between P patients and G or H controls.

| Types of studies
Original studies in humans with observational design (cross-sectional, case-control, and cohort) reporting data on salivary chemical elements. Both prospective and retrospective studies were included.

| Control and target condition
The target conditions were chronic (CP) and aggressive (AgP) forms of P, regardless of severity and extent, according to the classification of Armitage 17 or P of any stage and grade according to the current classification. 18 As controls, G and H conditions were considered. The diagnosis was considered both at patient and site level. Only studies comparing P patients versus H or G subjects were selected.

| Exclusion criteria
Studies investigating salivary ionic profiling in specific medical conditions (i.e., diabetes), as well as studies evaluating elemental changes after periodontal treatment, were excluded. In vitro studies, animal studies, editorials, clinical case reports, and literature reviews were not included, as well as articles not written in English.

| Search methods for the identification of studies
Reviewers were calibrated for study screening, data extraction, and risk of bias assessment against another experienced reviewer.
Literature searches were performed on three databases (Medline via PubMed, Embase, and Scopus) in duplicate by 2 reviewers. Studies were collected between January 1, 2010, and May, 31 2021. In addition, the references of all included studies and relevant reviews were manually cross-checked to ensure complete data collection. The detailed search strategy is reported in Appendix S1.

| Study selection
Titles and abstracts were screened independently by two reviewers (G.I. and M.C.), and full text of potentially eligible studies was obtained for independent assessment by the same reviewers. Any disagreement was solved by discussion until consensus was reached.
Where resolution was not possible, a third reviewer was consulted (G.B.). The reasons for exclusion of studies after full-text analysis were recorded. The inter-reviewer reliability (percentage of agreement and kappa correlation coefficient) of the screening method was calculated.

| Data extraction and management
Data were extracted by two authors independently using specially designed templates (G.I. and M.C.). In case of a prospective clinical study, only data recorded before treatment were collected.
Similarly, in case of a study comparing periodontal status in patients with and without specific medical conditions, only data of systemically healthy individuals were extracted. Disagreement was resolved through discussion with a third reviewer (G.B.).

| Risk of bias in the included studies and quality assessment
The quality assessment of the included studies was carried out independently and in duplicate by two reviewers (G.I. and M.C.) using a modified version of the NIH Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies and the QUADOMICS tool, which has been developed for omics research. 9,19 This tool includes 13 questions specifically addressed to evaluate the research question, study population, exposure, outcomes, and statistics in omics studies. Every item was given either 1 or 0 point, and, consequently, studies were rated based on their total score, where 0 < 3 was considered very low, 4 < 6 as low, 7 < 11 as moderate, and ≥11 as high.

| Strategy for data synthesis
Element concentration was originally reported as mean ± standard deviation (SD), median (interquartile range, IQR), or median (minmax). Effort was made to convert the level of each chemical element to mean ± SD using the same unit of measure to allow comparisons.
Where data were reported as median (IQR) or median (min-max), the correspondent mean and SD were calculated according to the method described by Hozo et al. 20 Due to very high heterogeneity in methodologies and outcomes among the included studies, metaanalyses of data were attempted but did not deserve publication.
However, a consistency analysis was presented focusing on chemical elements that were significantly discriminant between P and H groups.

| Quality assessment
Appendix S4 reports the results of the methodological quality assessment using the QUADOMICS tool. While the almost totality of the studies adopted well-defined clinical criteria for P, G, and H subject selection (Items 2 to 7), operative protocols and reporting were less rigorous for saliva sampling methods and statistical analysis. Four studies were considered as high quality, 5 studies as moderate quality, while the others were rated as low or very low quality.   The high heterogeneity in the outcome measures for each chemical element did not render meta-analyses reliable, and therefore, they were not presented.

| DISCUSS ION
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.

| Outcomes for specific metals
Periodontitis results as the consequence of a chronic immuneinflammatory 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 (TNFα). 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 break- 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.

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no competing interests.