Oxidative stress is defined as an imbalance between excessively reactive oxygen species (ROS) and antioxidant mechanisms (enzymatic and non-enzymatic). ROS, such as the superoxide radical, hydrogen peroxide and hydroxyl radical, are products of cellular metabolism (1). This imbalance may be a result of infection, inflammation or disease (2). Several studies indicate that the pathogenesis of various systemic inflammatory diseases, including oral ones, can be associated with oxidative stress. Many of these include periodontitis and mucosal lesions, but also dental caries (3-6). Salivary antioxidant system is one of the key salivary defence mechanisms against pathogens and a protective factor for oral mucosa (4). There are multiple different antioxidant compounds in human saliva. The salivary antioxidant system is made of enzymes (for example: peroxidase, super oxide dismutase, glutathione peroxidase, catalase), as well as small molecules such as uric acid or ascorbic acid (7, 8). According to Battino el al., these elements have additive effects rather than act alone. For this reason, measuring individual antioxidants separately can be confusing and lead to misinterpretation. Studies should focus on the whole antioxidant status, which can be expressed, for example, as total antioxidant capacity (TAC) or ferric reducing antioxidant power (FRAP) (9-11).

The aim of the study was to investigate the relationship between oral health (hygiene level, gingival and dental health), age and gender and antioxidant capacity para-meters (TAC, FRAP) in children and adolescents with permanent dentition.

The study conformed to the STROBE guidelines (STrengthening the Reporting of OBservational studies in Epidemiology).

The study was conducted in 87 patients aged 11-16 years with permanent dentition. The participants were recruited from the Department of Paediatric Dentistry, Medical University of Warsaw. The research was conducted in full accordance with the World Medical Association Declaration of Helsinki. The Bioethical Commission of Warsaw Medical University (No. KB/194/2015) approved the study and informed written consent was obtained from all parents. The inclusion criteria were as follows: no systemic diseases, no chronic pharmacotherapy, no developmental defects of tooth hard tissues, no previous orthodontic treatment. Patients who did not meet the above mentioned criteria were excluded from the study.

Clinical examinations were conducted in a dental office by one examiner using standardised WHO (World Health Organization) criteria. Caries index, expressed as mean DMFT/DMFS and white spot lesion (WSL) score, was evaluated (12). Oral hygiene level was assessed using ODI-S (the Debris Index), part of Oral Hygiene Index by Greene and Vermillion (13) and Approximal Plaque Index (API%) index by Lange et al. (14). Gingival health was assessed based on the Gingival Index (GI) by SILNESS and Löe (15), on four surfaces of permanent teeth 16, 12, 24, 36, 32, 44.

Saliva samples were collected from all participants in the morning (8.00-10.00 am). The participants were fasted. Salivette collection tube (Sarstedt, Nümbrecht, Germany) was used. The swab was placed in the mouth and allowed to absorb resting saliva for 5 min. Immediately after collection, the tube was stored at +4°C and centrifuged within 2 hours. The centrifugation to recover saliva from the swab was performed at 1000 x g at +4°C for 15 min. Unstimulated salivary flow rate was calculated by dividing the patient’s salivary volume by collection time. The saliva samples were frozen and stored at -80°C until the analysis.

TAC was assessed using a modified method developed by Erel (10). The assay was based on the principle of reduction of 2,2’-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid) cation radical (ABTS •+) (whose solution is blue-green) to ABTS (colorless solution) by antioxidants present in the sample. The reaction changed the colour of the reaction mixture. The green-blue colour disappeared. The reaction was measured spectrophotometrically at 660 nm. The change in absorbance was linked with the level of antioxidants in the sample. Calibration of the results was performed using Trolox. The results of TAC were expressed in mmol Trolox Equiv./L.

FRAP was determined using a method developed by Benzie and Strain (11). The method is based on the ability of antioxidants to reduce the complex of ferric tripyridyltriazine (Fe3+-TPTZ) to Fe2+-TPTZ at low pH (3.6). As a result of reaction, the yellow colour of Fe3+-TPTZ changed during the reduction to Fe2+-TPTZ, developing an intense blue colour with a maximum absorbance at 593 nm. The reaction was monitored spectrophotometrically. The change in absorbance was directly proportional to the level of antioxidants with a reduction potential in the sample. FRAP value of a sample was determined using calibration curve method. Calibration was performed using aqueous solutions of known Fe2+ (iron (II) sulphate heptahydrate (FeSO₄ x 7H₂O)) concentration. The results are expressed in mmol/L.

The analyses were performed using Statistica 12.0 software and R package. For quantitative variables, descriptive statistics characterising their variability were calculated. For binomial variables, the proportions (%) with division into groups were calculated. Comparison of quantitative variables between two groups was performed using the Mann-Whitney U test and the chi-square test, while the comparison of three or more groups was performed using the ANOVA test. In the case of categorical variables (including binomial variables), chi-square test was used to compare the groups. Spearman’s rank correlation coefficient was calculated. In all analyses, the statistical significance level was set at 0.05. Missing data were excluded from the analysis.

A total of 87 patients, including 46 (52.9%) boys and 41 (47.1%) girls, were examined. The mean age ± SD was 13 ± 2 years. Caries (DMFT > 0) was present in 81 patients (93.1%), active caries (DT > 0) in 47 patients (54.3%), and white spot lesions (WSL > 0) in 41 patients (47.1%). The results for the whole group and those depending on sex are presented in table 1.

The mean ODI-S index value was 1.01 ± 0.56 overall (1.06 ± 0.49 and 0.95 ± 0.63 respectively in boys and girls). The mean API% index was 67.5 ± 29.1 overall (72.4 ± 26.8 and 61.9±30.9 respectively in boys and girls). Gingivitis (GI > 0) was present in 60 patients (69%). The characteristics of oral hygiene and gingival state is presented in table 2. Unstimulated salivary flow rate ranged from 0.4 to 2.1 mL/5 minutes (mean 1.31 ± 0.45 mL/5 min). It was considered low in 44 (50.6%), average in 34 (39.1%), and high ? 9 (10.3%) patients.

The mean TAC values were 0.478 ± 0.257 in mmol Trolox Equiv./L (0.490 ± 0.261 mmol Trolox Equiv./L for boys and 0.465 ± 0.250 mmol Trolox Equiv./L for girls), and the mean FRAP values were 0.465 ± 0.171 mmol/L (0.465 ± 0.182 mmol/L for boys and 0.465 ± 0.159 mmol/L for girls). The intersex differences were not statistically significant. The results showed that the antioxidant capacity parameters were lower in patients presenting with caries, active caries, white spot lesions, bad oral hygiene and gingivitis, but the differences were not statistically significant (tab. 3). Oxidative stress parameters were significantly higher in low unstimulated salivary flow (for TAC p = 0.032; for FRAP p = 0.005). Spearman’s rank correlation analysis revealed no relationship between TAC or FRAP values and sex (p = 0.592; p = 0.873, respectively). There was a positive correlation between TAC or FRAP and age (r = 0.253, p = 0.018 for TAC and r = 0.204, p = 0.05 for FRAP).

The salivary antioxidant system is a part of salivary defence against pathogens. It also constitutes a protective factor for oral mucosa. Salivary antioxidants are the first defence mechanism against oxidative stress caused by free radicals (16, 17). Several research described that the initiation of many oral diseases may be connected with decreased antioxidant capacity of saliva and, consequently, oxidative stress (4, 5, 18). According to Krawczyk et al. (19), the presence of cariogenic bacteria forces an increased response of leukocytes (neutrophils and monocytes) which generate ROSs in a respiratory burst process, producing a bactericidal effect. This may be the possible cause of TAC/FRAP decrease in the saliva of patients with dental caries. Increased levels of salivary ROS intensify the oxidative stress as a result of reduction of the total antioxidant capacity of saliva (19). The results of the present study were in accordance with some previous reports. Reduced levels of TAC in patients with high caries experience were reported by Krawczyk et al. (19) and Rahmani et al. (16). However, their results were statistically significant, as opposed to the present study. On the other hand, many studies show that the total antioxidant capacity of saliva may be also increased in dental caries (4, 20-22). Ahmadi et al. (4) showed that the level of TAC was significantly higher in the caries active group than in caries free subjects. According to Silva et al. (21), oxidative damage was significantly lower in S-ECC (Severe-Early Childhood Caries) children than in caries-free children, while salivary total antioxidant capacity was significantly higher in the S-ECC group. The explanation for this can be that TAC level in individuals with high caries intensity is a form of compensation against oxidative stress. In the present study, we also investigated the gingival condition and oral hygiene, and calculated unstimulated salivary flow rate. These parameters were rarely assessed in previous research concerning the relationship between antioxidant capacity and caries prevalence (19, 23), and taking the unstimulated salivary flow rate into consideration is novel. In the present study, we failed to confirm significant relationships between oral hygiene and TAC/FRAP levels, which corresponded with the results presented by Krawczyk et al. (23). However, they performed another investigation in 2014 (19), which revealed a significant negative correlation between OHI-S and TAC (r = -0.4368, p = 0.000001). The significant relationship between TAC/FRAP and unstimulated salivary flow rate is an original finding and has not been reported before. It may be caused by greater dilution of ROS in a higher salivary volume, which may reduce TAC/FRAP levels, which regulate ROS concentration.

Compared with the previous studies, the obtained results indicate that the relationship between dental caries, gingiva and oral hygiene and salivary antioxidant systems is not fully explained. The differences may result from different laboratory methods used for TAC/FRAP evaluation, or differences in patients’ age and, consequently, dentition type (24). In the present study, we observed that the total antioxidant capacity of saliva increases with age, which corresponds with the results obtained earlier by Hegde et al. (2). Ahmadi et al. (4) suggested a relationship with sex (lower TAC values in females), however, neither we nor other researchers confirmed this finding (16).

Some authors suggest that salivary oxidative stress parameters may potentially be used for screening and monitoring oral diseases, including dental caries (3, 21). However, the authors of the present study remain sceptical as the results of the studies are ambiguous. Therefore, further investigations should be conducted, with a participation of larger patient groups of different age, dentition and caries intensity or gingival health.

Practitioners should bear in mind that particular oral conditions (caries, gingivitis, poor oral hygiene) can modify salivary antioxidant capacity parameters as a result of oxidative stress. There is a correlation between salivary antioxidant capacity parameters and patients’ age, but there is no such correlation with patients’ gender.

We wish to acknowledge Joanna Giebułtowicz, MPharm, PhD (Department of Bioanalysis and Drugs Analysis, Faculty of Pharmacy, Medical University of Warsaw) for her essential help and assistance during the research.