Salivary biomarkers of inflammation and oxidative stress in healthy adults
Introduction
There have been numerous efforts to utilize saliva for diagnostic purposes. The advantages of saliva sampling, such as non-invasiveness, cost-effectiveness, safety, and the ease of repetitive collection have facilitated its use as a diagnostic modality (Yoshizawa et al., 2013). Saliva-based diagnostics are advantageous for the elderly, physically handicapped individuals, and non-cooperative subjects such as children. Saliva contains components directly produced by the salivary glands and those transported from blood via passive diffusion, ultrafiltration, and active transport (Lee & Wong, 2009; Pfaffe, Cooper-White, Beyerlein, Kostner, & Punyadeera, 2011). These components can be used as biomarkers to diagnose oral and systemic diseases and for health surveillance (Prior & Cao, 1999; Sezer, Çiçek, & Çanakçi, 2012; Trivedi, Lal, Mahdi, Singh, & Pandey, 2015).
However, the use of salivary components as diagnostic biomarkers has many limitations. First, scientific consensus is lacking regarding the standardization of collection, storage, and analysis procedures for saliva samples (Palmieri & Sblendorio, 2007; Wang, Schipper, Velly, Mohit, & Gornitsky, 2015). Second, because the levels of salivary biomarkers are highly variable in patients and controls, it is difficult to determine the normal or pathologic range for each salivary biomarker (Kamodyová, Červenka, & Celec, 2015), e.g., according to previous studies, the normal level of salivary IL-6 ranges widely from 0.89 ± 1.72 (Riis et al., 2014) to 47.46 ± 18.74 pg/mL (Rhodus et al., 2005). Therefore, standardization of collection, storage, and analysis procedures for saliva samples and establishment of the concentrations of major salivary components with a diagnostic value in healthy individuals are crucial for the utilization of saliva as a reliable diagnostic medium.
The clinical value of salivary biomarkers highly depends on the reproducibility of results and predictable correlations between the same biomarkers in the blood and saliva, e.g., since the salivary levels of various stress and gonadal hormones are relatively good reflections of their blood concentrations, salivary analyses for these hormones have been widely employed for research purposes (Lee, Kim, Chang, & Kho, 2015). To extend the applicability of salivary diagnostics, “reproducibility” and “correlation” should be tested for each biomarker in different sexes and age groups, in healthy populations, and in various disease conditions. To this end, studies on the salivary biomarkers of healthy adults to rule out the influence of pathology-related factors and establish a reference are essential.
The development of methods to determine the blood contamination level in saliva is also necessary. Since the concentrations of some analytes in saliva are much lower than those in blood, leakage of blood into saliva due to gingival inflammation and deterioration of oral mucosal integrity may increase the level of salivary analytes (Behr et al., 2017; Kivlighan et al., 2004; Schwartz & Granger, 2004). Therefore, knowing whether changes in the concentrations of salivary biomarkers are due to the pathogenesis of targeted diseases or the effects of blood contamination is essential for accurate salivary diagnostics.
To investigate the reliability, blood reflection, and blood contamination issues in salivary diagnostics, we focused on inflammatory and oxidative stress biomarkers in saliva in healthy young adult males. Increased inflammation and the resultant increase in reactive oxygen species can induce or worsen the symptoms of many chronic diseases and accelerate the aging process. Therefore, numerous studies on diagnostics and health surveillance using saliva have been conducted to investigate inflammatory and oxidative stress biomarkers (Cullen, Thomas, Webb, & Hughes, 2015; Heitzer, Schlinzig, Krohn, Meinertz, & Münzel, 2001; Tulunoglu, Demirtas, & Tulunoglu, 2006). Only healthy young adult males were recruited as subjects in our study to limit the uncontrollable variables such as the menstrual cycle and chronic diseases.
The purposes of this study were to investigate the reliability of saliva samples in repeated collections, the blood reflectance of saliva, and the effects of blood contamination on salivary biomarkers. Our study can provide a basic but essential reference for monitoring the health status of patients with chronic diseases and elderly individuals experiencing difficulties with repetitive blood sampling.
Section snippets
Participants
Thirty-seven healthy young men (26.7 ± 2.2 years) participated in this study; all of them were nonsmokers, had no specific oral diseases, and had no history of serious illness. To control for the influence of the menstrual cycle, female participants were excluded. In addition, participants with the following conditions were excluded: those taking medications that can affect salivary secretion in the past three months (e.g., psychiatric and neurological medications, antihistamines,
Results
Salivary flow rate, mean concentrations, and the distributions of all examined biomarkers in the saliva and blood samples are shown in Table 1 and Fig. 1. The secretion rates and the ratios of biomarkers out of the total protein in saliva are shown in Supplemental Tables 1, respectively. There were no significant differences (P = 0.110 - 0.891) in the concentrations of all examined biomarkers between the saliva samples collected on two different visits. When comparing the concentrations in
Discussion
In this study, we evaluated the reliability of saliva samples, correlations between the biomarkers in saliva and blood, and the influence of blood contamination on the saliva/blood ratios of biomarkers.
The ICC coefficients of every biomarker except TNF-α in the saliva samples ranged from “fair” to “excellent” (Yen & Lo, 2002). Inflammatory biomarkers showed higher ICC coefficients in saliva samples than oxidative stress biomarkers. Our findings were consistent with previous studies. Although
Conclusions
The analyses of salivary levels of inflammatory and oxidative stress as well as blood contamination biomarkers were reproducible. However, the investigated salivary inflammatory and oxidative stress biomarkers, with the exception of IL-6, did not reflect the biomarkers present in blood. The level of IL-6 showed a significant, but relatively low, correlation between saliva and blood. The saliva/blood ratios of the biomarkers more concentrated in blood than saliva tended to increase as the
Author contributions
Yoon Nam designed the study, collected the samples, analyzed the data, and drafted the manuscript. Yoon-Young Kim and Ji-Youn Chang performed the experiments on saliva and blood samples. Hong-Seop Kho designed the study, supervised all procedures, and wrote the manuscript. All authors revised the manuscript and approved the final version.
Funding
This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2016R1A2B4007286).
Conflict of interest statement
The authors have no conflicts of interest to report.
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