Elsevier

Archives of Oral Biology

Volume 97, January 2019, Pages 215-222
Archives of Oral Biology

Salivary biomarkers of inflammation and oxidative stress in healthy adults

https://doi.org/10.1016/j.archoralbio.2018.10.026Get rights and content

Highlights

  • Diagnostic value of saliva depends on reproducibility of analyzed results.

  • The presence of blood in saliva samples affects the analyzed results of saliva.

  • Analyses of inflammatory and oxidative stress biomarkers in saliva are reproducible.

  • The level of IL-6 in saliva reflects that of blood.

  • Transferrin is a reliable biomarker of blood contamination in saliva samples.

Abstract

Objectives

Diagnostic value of saliva depends on the reproducibility of data in repeatedly collected samples and predictable correlations between saliva and blood. We aimed to investigate the reliability, blood reflectance, and influence of blood contamination in the analysis of inflammatory and oxidative stress biomarkers in saliva samples.

Design

In total, 37 healthy young male participants (26.7 ± 2.2 years) were included. Unstimulated whole saliva and blood samples were collected on the first visit, and saliva samples were collected again after 2–3 days. The concentrations of total protein and inflammatory [C-reactive protein (CRP), IL-1β, IL-6, and TNF-α] and oxidative stress [8-hydroxy-2’-deoxyguanosine (8-OHdG), malondialdehyde (MDA), and total antioxidant capacity (TAC)] biomarkers in saliva and blood, and as well as blood contamination biomarkers (transferrin and hemoglobin) in saliva were analyzed.

Results

The intra-class correlations of all examined biomarkers except TNF-α were fair to excellent. Significant positive correlations between CRP and IL-6 and between total protein and TAC were stable in the saliva samples collected on different days. Notably, IL-6 was the only biomarker that showed a significant correlation between saliva and blood. As the concentration of salivary transferrin increased, the saliva/blood ratios of total protein and TAC also increased. The concentration of salivary hemoglobin did not affect the saliva/blood ratios of biomarkers.

Conclusions

The findings of this study are limited to healthy young males. For clinical applications, studies on salivary diagnostics should be performed for individual disease and health conditions, demographic characteristics, and biomarkers.

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.

References (44)

  • N.L. Rhodus et al.

    A comparison of the pro-inflammatory, NF-κB-dependent cytokines: TNF-alpha, IL-1-alpha, IL-6, and IL-8 in different oral fluids from oral lichen planus patients

    Clinical Immunology

    (2005)
  • E. Sjögren et al.

    Interleukin-6 levels in relation to psychosocial factors: Studies on serum, saliva, and in vitro production by blood mononuclear cells

    Brain, Behavior, and Immunity

    (2006)
  • K.I. Suh et al.

    Salivary levels of IL-1β, IL-6, IL-8, and TNF-α in patients with burning mouth syndrome

    Archives of Oral Biology

    (2009)
  • J. Wang et al.

    Salivary biomarkers of oxidative stress: A critical review

    Free Radical Biology & Medicine

    (2015)
  • Y. Zhang et al.

    NF-κB-dependent cytokines in saliva and serum from patients with oral lichen planus: a study in an ethnic Chinese population

    Cytokine

    (2008)
  • P. Zukowski et al.

    Sources of free radicals and oxidative stress in the oral cavity

    Archives of Oral Biology

    (2018)
  • A. Aleksandra Nielsen et al.

    Saliva interleukin-6 in patients with inflammatory bowel disease

    Scandinavian Journal of Gastroenterology

    (2005)
  • M. Behuliak et al.

    Variability of thiobarbituric acid reacting substances in saliva

    Disease Markers

    (2009)
  • J.M. Bland et al.

    Statistical methods for assessing agreement between two methods of clinical measurement

    Lancet

    (1986)
  • R.W. Browne et al.

    Performance of multiplex cytokine assays in serum and saliva among community-dwelling postmenopausal women

    PLoS One

    (2013)
  • J.S. Choi et al.

    Relationship between salivary haemoglobin and number of remaining teeth in older Koreans

    Gerodontology

    (2018)
  • P. Gümüş et al.

    Saliva and serum levels of B‐cell activating factors and tumor necrosis factor‐α in patients with periodontitis

    Journal of Periodontology

    (2014)
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