Periodontitis is an oral multifactorial disorder leading to progressive destruction of the periodontal attachment apparatus. Interventional and cohort studies indicate that periodontitis may be an independent risk factor for diabetes, atherosclerotic cardiovascular diseases (ACVDs) and low birth weight in infants; whereas cross-sectional and case-control studies show periodontitis as a possible risk factor for metabolic syndrome, chronic renal failure, rheumatoid arthritis and neurodegenerative diseases [1
]. A new classification of periodontal diseases, developed in 2017 during the World Workshop on the Classification of Periodontal and Peri-implant Diseases and Conditions, underlines the association between periodontal diseases and systemic diseases affecting the host immune response. This classification aims to identify individual patients in a targeted manner and to indicate those who require greater efforts to control periodontal disease. However, it hinders the extrapolation of previous research results to the periodontal diagnoses described nowadays [2
Oxidative stress plays a vital role in the etiopathogenesis of all systemic diseases. Periodontitis, like any chronic inflammatory disease, is also inextricably linked with oxidative-reductive imbalance [4
]. Indeed, stimulation of neutrophils by periopathogens leads to a “respiratory burst” resulting in increased formation of reactive oxygen (ROS) and nitrogen (RNS) species mainly hypochlorous acid, superoxide anion, and hydrogen peroxide. Many studies have shown that oxidative stress is directly responsible for the degradation of extracellular matrix components of periodontal tissue, including collagen, elastin, proteoglycans, and glycosaminoglycans (e.g., hyaluronic acid) [4
]. This leads to the destruction of the periodontal attachment apparatus [5
]. Nevertheless, oxidative stress also initiates/promotes the inflammatory response in periodontitis. Under the influence of ROS, an increase in cytokine production and growth factors (e.g., interleukin-6 (IL-6) and -8 (IL-8), tumor necrosis factor α (TNF-α), and nuclear factor κB (NFκB)) has been shown [7
]. Indeed, in patients with periodontal disease, the activity of NADPH oxidase (NOX) increases, which not only enhances the production of free radicals but is also an important source of pro-inflammatory cytokines [8
]. The NADPH oxidase NOX2 plays a role in periodontal pathologies. Oxidative stress also leads to the release of lysosomal enzymes responsible for local tissue destruction. Interestingly, the results of recent studies indicate that in periodontitis patients with comorbidities, salivary redox disturbances are exacerbated. In cases with periodontitis and coronary heart disease (CKD) it has been demonstrated enhanced levels of salivary and serum malondialdehyde (lipid peroxidation marker) as well as asymmetric dimethylarginine (ADMA, an endogenous inhibitor of nitric oxide) compared to healthy subjects and CHD cases. This was accompanied by a decrease in vitamin C, one of the most important oral antioxidants, whereas C-reactive protein (CRP) has been found to be a significant predictor of enhanced malondialdehyde and ADMA levels [9
It is believed that the disruption of the antioxidant barrier is directly responsible for oxidative/nitrosative modifications of cell components [12
]. Indeed, when the antioxidant reserves are exhausted, there is no scavenging of oxygen free radicals nor their neutralization. However, in the course of periodontal disease, both an increase/decrease in activity and concentrations of several free radical scavengers were observed [4
]. The multitude of enzymatic and non-enzymatic antioxidants as well as their synergistic effects cause an interest in the evaluation of the total oxidative and antioxidative capacity. Indeed, sometimes much more information is provided by the total free radical scavenging capacity than the evaluation of a concentration for each antioxidant separately. Although this is some simplification, it allows a less complicated comparison of the intensity level of redox imbalance. Spectrophotometric, electrochemical, and chromatographic methods are used for this purpose [13
]. Although these methods have a similar measuring principle, the contribution of individual antioxidants to the total antioxidant potential varies. It is therefore necessary to evaluate several parameters characterizing the total antioxidant potential of the body [16
]. The recent research results indicate that the antioxidant/oxidant capacity of saliva is used in the diagnosis of such systemic diseases as chronic renal disease, hypertension, chronic heart disease, or psoriasis [17
]. Nevertheless, reports on the use of these biomarkers in the diagnosis of periodontopathy are incomplete and contradictory. There is also no research comparing (non-)stimulated saliva and gingival crevicular fluid (GCF).
Therefore, our research aimed to evaluate the total oxidative and antioxidative activity of gingival crevicular fluid and saliva in periodontitis, as well as to relate these biomarkers to clinical markers of periodontopathy (according to the most recent classification of periodontal diseases). Additionally, by calculating the oxidative stress index (OSI), the intensity level of redox imbalance was evaluated.
Despite the increasing knowledge of the etiopathogenesis of inflammatory periodontal diseases, the diagnostics and classification of these diseases are almost exclusively based on traditional clinical evaluation. Since periodontal diagnosis is largely subjective and retrospective, it is not surprising that biochemical biomarkers of periodontopathy are constantly being sought. The indicators evaluated in GCF/saliva could be useful in objectivizing the diagnosis and determining the severity of the disease, as well as in evaluating treatment results.
We have shown a decrease in the total antioxidant potential (↓TAC, ↓FRAP) and an increase in total oxidant activity (↑TOS, ↑OSI). This study is the first one where TOS/TAC were compared to GCF and both stimulated and non-stimulated saliva in patients with periodontitis. We have not found any correlations between the biomarkers measured in NWS, SWS, and GCF. Although the examined parameters correlated with the periodontium’s clinical condition, TAC, TOS, OSI, and FRAP did not differentiate individual stages of periodontitis.
TOS expresses the total amount of oxidants in a test sample. In our study, we found significantly higher concentrations of TOS in GCF and both types of saliva in the periodontitis when compared to the clinically healthy periodontium. In stimulated saliva, mean TOS values were the highest for the other two fluids tested, as well as the highest TOS values in the most advanced stage of the disease. This fact is not surprising since the parotid gland secreting stimulated saliva is the most important source of free radicals among all salivary glands [31
]. These observations are consistent with the findings of Wei et al. [33
] and Baltacioğlu et al. [34
]. In the former research, patients with CP (chronic periodontitis) showed significantly higher mean TOS values in order of CGF, blood serum, and non-stimulated saliva when compared to the control group without any pathological changes in the periodontium. In the latter one, the higher levels of TOS were also significantly higher in the order of serum and non-stimulated saliva of patients with periodontitis (significantly higher as regards aggressive periodontitis than chronic one) when compared to the control group comprising people with clinically healthy periodontium. Zhang et al. [35
] did not find any significant difference in salivary TOS concentration between patients with periodontitis and the control group; however, the high percentage of active nicotinists in both groups could significantly affect the result of this observation. All these studies confirm significantly higher ROS concentrations in GCF, saliva, and blood serum during periodontitis once again. Our patients with periodontitis showed a positively significant correlation between TOS in non-stimulated saliva and the number of teeth only (an understandable covariation of the number of “giving” sites of hydrogen peroxide and its concentration) and, in stage IV, between TOS activity in GCF and the number of regions with at least 3-mm clinical loss of attachment on interproximal surfaces. This is in some opposition to the findings of Baltacioğlu et al. [34
], who showed strong correlations between salivary/serum TOS levels and clinical parameters of periodontal condition (PI, GI, PD, and CAL), as well as to the findings of Wei et al. [33
], who found weaker but also significant correlations between TOS in GCF/saliva/serum and the same clinical markers of periodontopathy. Additionally, no link between salivary TOS and the amount of periopathogens in saliva (P. gingivalis
, T. denticola
, T. forsythia
, A. actinomycemecomitans
, and F. nucleatum
) has been found [35
]. The analysis of own research results does not confirm the suggestion of other authors [33
] that the TOS levels in non-stimulated saliva would be a good marker, stratifying the severity of periodontitis.
compares the most significant studies on the evaluation of the total antioxidant capacity of GCF, saliva, and serum (plasma) in the periodontitis [34
In our research, we found a significant reduction in the exclusively endogenous TAC in GCF of periodontal pockets when compared to gingival fissures, and the extent of this reduction did not coincide with the division into stages and grades of periodontitis. The point of reference in the literature is the study by Becerika et al. [52
], who, using the same methodology of TAC evaluation, also found its reduction, but it did not reach the level of statistical significance. This was probably due to the significantly smaller group investigated in Turkish observation when compared to our own (20 vs. 60 subjects). When comparing the TAC evaluation, identically methodological studies should be compared to each other, as each method prefers other non-enzymatic antioxidants in the overall capacity assessment. In the TEAC method by Erel, the activity of ROS scavengers—in the form of proteins containing thiol groups at the expense of uric acid—is measured to a greater extent [56
]. Similarly, the TAC activity spectrum (in GCF, 75% of it depends on thiol proteins) means the enhanced chemiluminescence (ECL) method used by Chapple et al. [37
]; they also found a significant reduction in the TAC levels in GCF from periodontal pockets when compared to the control group. On the other hand, in our study, after applying the FRAP method, there was no significant difference in the antioxidant capacity of GCF between the study and control groups. This method is only slightly sensitive to the activity of thiol proteins and prefers uric acid as the mainly assessed antioxidant [56
]; therefore, in such a compartment, its concentration is not the most important element in ROS inactivation. Becerik et al. [52
] stated, however, that a significant decrease in the antioxidant capacity (FRAP) of gingival crevicular fluid in patients with periodontitis as well as quite incomprehensible inverse covariations with the clinical parameters of periodontal condition —in particular with the pocket depth and the clinical attachment level. These differences probably resulted from the different methodologies of determining the final FRAP concentration (in our study, they were referred to total protein in GCF, whereas in the Turkish study the GCF volume was determined using Periotron 8000). That is why all these above-mentioned studies indicate a local significant reduction in the antioxidant capacity in the periodontitis. The positively significant correlation between the TAC in gingival crevicular fluid and the number of periodontal pockets above 5 mm, demonstrated throughout our study group, proves either a negative effect of this inhibition on the clinical condition of the periodontium or vice versa. The clarification of the nature of this dysfunction is provided by interventional studies, in which the impact of standard non-surgical periodontal treatment on TAC in GCF of periodontal pockets was evaluated. The first study of this type was conducted by Chapple et al. [41
] and showed that such treatment significantly improved TAC in gingival crevicular fluid, measured with ECL, to the control group level. In another two studies, Turkish authors [57
] proved that such treatment significantly improved TAC in the gingival crevicular fluid by means of the TEAC method by Erel. This discovery concerned deep periodontal pockets, and that effect lasted for at least 6 weeks. At the same time, it was indicated that certain general conditions, such as smoking or familial Mediterranean fever (FMF), made such an improvement impossible. In other words, both TAC evaluation methods proved that suppression of the antioxidant activity in pocket compartments results from the inflammatory and immunological process, rather than predisposes to it. In our study, by means of a comprehensive evaluation of the pro- and antioxidant effect, determination of the oxidative stress index has become possible. This indicator is one of the direct parameters of prevalence and measurement of oxidative stress intensity in many systemic diseases [59
]. Due to the fact that TOS was significantly increased and TAC in compartment fluid of the periodontal pockets was lowered when compared to GCF of periodontally healthy subjects, the local OSI value in periodontitis was significantly higher (approx. 4 times).
The total antioxidant capacity of the salivary compartment is significantly different from that of the gingival crevicular fluid. Firstly, the impact of exogenous, non-enzymatic antioxidants contained in food is significant and difficult to control. Secondly, salivary glands, especially during stimulated salivary secretion (in our study: a significantly higher TAC when compared to GCF and non-stimulated saliva), may be a source of many non-enzymatic antioxidants, including, in particular, uric acid, ascorbate, and drugs with such properties, e.g., allopurinol. It was found that both in saliva and urine, uric acid (70%) and ascorbate have the highest percentage share in TAC [60
]. In clinical-control studies, it was found that the consistency between salivary TAC and the concentration of uric acid was 75% [60
]. In the study carried out by Zhang et al. [35
], a multifactorial analysis showed that out of 9 variables only the diagnosis of periodontitis was significantly related to salivary TAC (regardless of age, sex, smoking or semi-quantitative occurrence of 5 periopathogens in saliva). In the vast majority of studies [34
], as well as in our study, the salivary TAC profile in patients with periodontitis was significantly reduced in ABTS oxidation method. Only one study [47
] did not prove the statistical significance of this difference, although the mean difference between the control and study groups was large (0.59 vs. 0.4 μM). Meta-analysis by Chen et al. [61
], involving 4 studies described in Table 2
] and 3 others [62
] as well as referring to 556 subjects, showed a significant reduction in the salivary TAC for periodontitis in relation to the control group (p
= 0.003, inhomogeneity index: 98.3%). In our study of periodontitis, the total antioxidant capacity of saliva (mainly non-stimulated one) was significantly reduced, as also described earlier by Dhotre et al. [62
] and Baňasova et al. [50
], but only in women. The significant effect of periodontitis on the antioxidant capacity of saliva in the FRAP method was not proved in two determinations [38
]. Such discrepancy may result from gender. The authors of this method, Benzie and Chung [15
], have already described the significantly higher antioxidant capacity of plasma in women, which they explained with higher concentrations of uric acid (UA). In the context of testing the antioxidant capacity in periodontal patients, it seems that a better way to obtain saliva is non-stimulated saliva—less exposed to the fluctuations of its various components during stimulation of large salivary glands, as several authors have pointed out [38
]. After using other methods to evaluate the total antioxidant capacity of saliva in periodontopathies, the results significantly differed from those presented above. After using the ECL method, no effect of periodontal diagnosis on TAC in stimulated and non-stimulated saliva was observed [40
]. After using the TRAP method in chronic and aggressive periodontitis, the significantly higher antioxidant capacity of non-stimulated saliva was described in relation to periodontally healthy control groups, suitable for these diagnoses [51
]. This result explains the sensitivity of this method to the concentration of antioxidants—proteins and polyphenols were responsible for 57%, whereas ascorbate, UA, and tyrosine were responsible for 37% in plasma [65
], which can be with high probability extrapolated to saliva. If analytical methods sensitive to the detection of uric acid are used, a significant reduction in the total antioxidant capacity is observed—mainly in the non-stimulated saliva of patients with periodontitis. It is probably a transfer of this antioxidant deficit from the pockets themselves, together with the combination of several confounding factors (e.g., exogenous supply of polyphenols), weakening this effect. In our patients, such a dependence was confirmed in stimulated saliva with plaque index. A significant increase in OSI in periodontitis was also observed in our study and the salivary compartment as well (the highest mean value was found in stimulated saliva, in the most advanced stage of the disease), which confirms earlier observations [34
]. Thus, quite intense oxidative stress associated with periodontitis is transferred from the pockets to the saliva, including the weakening of non-stimulated saliva. This oxidative stress is primarily caused by a significant increase in the total oxidant status (a very high positive correlation in the whole study group between OSI and TOS in non-stimulated saliva, with no OSI-TAC relationship and no positive OSI-teeth number relationship, i.e., the number of pockets emitting ROS).
It is suggested that shifting the salivary/GCF redox balance in favor of the oxidative reactions (↓TAC, ↓FRAP, ↑TOS, ↑OSI) predisposes to oxidative damage to proteins, lipids, and DNA in the periodontal tissue. It was shown that cell oxidation products stimulate the synthesis of arachidonic acid derivatives (especially prostaglandin (Pg)E2), pro-inflammatory cytokines (e.g., IL-1, IL-6) as well as cell adhesion molecules (e.g., intercellular adhesion molecule 1, ICAM-1). Macrophages and fibroblasts accumulate in such altered periodontium. These cells are also the source of numerous cytokines, metalloproteinases (MMPs), and proteolytic enzymes, which leads to progressive destruction of the periodontal attachment apparatus [4
The studies of the total antioxidant capacity of plasma or serum in patients with periodontitis are a complement of great importance to these determinations carried out at the site of the disease (tissue, gingival crevicular fluid) and saliva. This is because they indicate the potential for the interaction of periodontal oxidative stress with the systemic inflammatory process and its endpoints. In this case, as in saliva, the tests sensitive to the detection of antioxidant properties of uric acid and ascorbates would be the most effective. The use of oxidation methods (by Miller, Erel) or ABTS cationic radical reduction methods quite clearly demonstrated a significant decrease in TAC in peripheral blood, in the periodontitis [34
]. Only two observations have not found any effect of periodontal diagnosis on the change of TAC in blood serum or plasma [52
]. Out of 9 studies of TAC in plasma, serum, and blood, a meta-analysis by Liu et al. [66
], involving 248 subjects in the study group and 238 in the control group, proved a significant decrease in the total antioxidant capacity in periodontitis (p
= 0.000, inhomogeneity index: 95.5%). This result should be approached with caution since 5 studies have been incorrectly included due to different methodologies of the TAC test and the occurrence of exclusionary general conditions. Despite a significant decrease in the total antioxidant activity in peripheral blood of patients with periodontitis, it does not seem possible that the magnitude of the related oxidative stress might have systemic implications. Alas, we did not assess in our study the total antioxidant capacity of plasma/blood serum in patients with periodontitis, which makes it impossible to conclude central redox homeostasis, thus it constitutes a limitation of the study.
Initially, the diagnosis of periodontal diseases was based exclusively on the examination of GCF taken with the use of filters from periodontal pockets. This technique, however, is very time-consuming as well as technically demanding, and the filters are easily contaminated with blood or bacterial plaque. The ease and noninvasiveness of the collection make saliva a biological fluid that can be applied in the diagnosis of oral diseases. Nonetheless, as our studies have shown, none of the evaluated biomarkers in saliva/GCF differentiates the stages of periodontitis. TAC, TOS, and OSI also correlate weakly with clinical periodontal markers. Although salivary antioxidant/oxidant status is used in the diagnosis of many systemic diseases (chronic renal disease, hypertension, psoriasis), it should be remembered that changes in salivary redox homeostasis may result from changes at the systemic level [9
]. Indeed, salivary redox biomarkers faithfully reflect their plasma/blood serum content as well as they correlate with classical disease progression indicators (e.g., creatinine in chronic renal disease or diastolic pressure in hypertension). Low diagnostic usefulness of redox biomarkers in periodontal diseases may result from the fact that the oral cavity is constantly exposed to many environmental factors, such as air pollution, tobacco smoke, food, and microorganisms [68
]. Dental materials and dental procedures performed within the oral cavity are also of great importance. They may destabilize local redox homeostasis, making saliva/GCF useless in the diagnosis of oral diseases. Furthermore, there is a lack of reference values for the redox salivary/GCF biomarkers assessed, which makes it difficult to compare the results obtained in different centers.