Abstract.BACKGROUND: Salivary markers of oxidative stress and antioxidant status represent promising tool for the research of oral diseases. One of the criteria is the validation of these biomarkers from the perspective of the confounding and modifying factors. AIM: To examine the effect of circadian rhythm, tooth-brushing and ascorbic acid treatment on selected salivary markers of oxidative and carbonyl stress, and antioxidant status. SUBJECTS AND METHODS: Whole unstimulated saliva samples were collected from 19 healthy participants three times during a day, before and after tooth-brushing, and before and after the administration of vitamin C (250 mg). Advanced oxidation protein products (AOPP), thiobarbituric acid reactive substances (TBARS), advanced glycation end products (AGEs), ferric reducing antioxidant power (FRAP) and total antioxidant capacity (TAC) were measured. RESULTS: Salivary AGEs levels varied significantly during the day (p < 0.05) with the highest concentrations in the morning. FRAP levels varied during the day (p < 0.01) with the highest concentrations in the afternoon. Tooth-brushing decreased AGEs (p < 0.05) and TBARS levels (p < 0.01) and increased FRAP levels (p < 0.05). Single intake of vitamin C significantly decreased AGEs (p < 0.001) and increased both FRAP (p < 0.01) and TAC (p < 0.01) concentrations. CONCLUSION: Significant daily variations were observed in salivary AGEs and FRAP levels. Tooth-brushing and treatment with vitamin C decreased carbonyl stress and increased the antioxidant status. These results are important from the perspective of using saliva for the research of oral diseases.

Background: Previous studies have shown that salivary thiobarbituric acid reactive substances are related to the periodontal status in adults. Such an analysis has not been done on children yet. The aim of our study was to analyze salivary markers of oxidative stress in relation to periodontal and dental status in children. Methods: The periodontal and dental status of 82 consecutive pediatric dental patients was assessed. The oral hygiene index (OHI), the papillary bleeding index (PBI) and the caries index (CI) were assessed as clinical parameters. Markers of oxidative stress and antioxidant status were measured in whole saliva samples. Results: Multivariate analysis of covariance showed that the variability of PBI explains 10.9% of the variance of salivary thiobarbituric acid reacting substances (TBARS). Advanced oxidation protein products (AOPP) were related to CI (eta 8.6%). Measures of antioxidant status (total antioxidant capacity and ferric reducing ability of saliva) were partially determined by OHI (13.6% and 7.2%) and PBI (16.9% and 7.9%). Conclusions: Antioxidant status in saliva is related to oral hygiene and periodontal status. Salivary TBARS are a potential sensitive marker of periodontitis in children, similarly to adults, at least on a population level. Salivary AOPP are related to caries. Potential diagnostic value of the analyzed markers should be analyzed in further interventional studies.

Introduction: Salivary TBARS are a potential marker of oxidative stress in the oral cavity. Previous studies have found increased levels of salivary TBARS in various diseases. The aim of this study was to assess the variability of salivary TBARS in both genders. Subjects & Methods: Saliva samples from thirty-eight healthy volunteers (18F & 20M) were collected every day during 30 day period. TBARS levels were measured spectrophotometrically using a high-throughput 96-well plate method. Time series analysis was performed using standard statistical methods. Results: Repeated measures ANOVA showed a significant variation of salivary TBARS within day and subjects (p < 0.001). The dynamics did not differ between genders. Intraindividual variability was very high in both genders with coefficients of variation of more than 60%. Interindividual variability was higher in men than in women (73% vs. 46%; p < 0.01). Discussion: The relatively high intraindividual variability indicates that the use of salivary TBARS will be limited to research on a population level, although some informative value might be gained by repeated samplings. Factors influencing the biological variability of salivary TBARS should be identified in further studies.

e12558 Background: Circulating tumor cells (CTCs) contributing to the metastatic cascade and represent an independent survival predictor in breast cancer (BC) patients. Vitamin D has pleiotropic effects and its low concentrations are associated with infections, cancer, but also other pathologic conditions. The aim of this study was to assess plasma vitamin D level in primary BC patients in relation to CTCs. Methods: This study included 91 non-metastatic breast cancer patients (stage I-III) with plasma samples available in the biobank of the National Cancer Institute in Slovakia. Blood samples for the analyses were drawn at the time of surgery. Peripheral blood mononuclear cells (PBMCs) were depleted of hematopoetic cells using RossetteSepTM negative selection kit. RNA extracted from CD45-depleted PBMCs was analyzed for the expression of transcription factors (TWIST1, SNAIL1, SLUG, ZEB1) that induce epithelial-to-mesenchymal transcription (EMT-TF) and epithelial ( CK19) gene transcripts using qRT-PCR. Patient samples with higher epithelial and/or mesenchymal gene transcripts than those of healthy donors (n = 60) were considered CTCs positive. Total 25-OH vitamin D was measured in plasma using ELISA. Results: CTCs were detected in 30 (33%) patients, 14 (15.4%) patients had detectable CTCs with epithelial characteristics, 13 (14.3%) patients had CTCs with an EMT phenotype, while peripheral blood of 3 (3.3%) patients exhibit both CTCs subtypes. Patients with detectable CTCs in peripheral blood had significantly lower vitamin D concentrations in comparison to patients without detectable CTCs (mean ± SD) 8.50 ± 3.89 µg/L for CTCs positive vs. 9.69 ± 3.49 µg/L for CTCs negative patients respectively, P = 0.03). There was no association between plasma vitamin D and other patient/tumor characteristics. At a median follow-up of 96.5 months (range: 8.4 - 109.4 months), 28 patients (30.8%) had experienced a progression and 24 patients (26.4%) had died. Patients with vitamin D above median had better overall survival (HR = 0.36, 95%CI 0.16 – 0.80, P = 0.017) compared to patients below median vitamin D. Conclusions: According to our knowledge, this is the first report of an association between plasma vitamin D concentrations and CTCs in primary breast cancer patients. We suggest that vitamin D could play a direct role in tumor dissemination and its supplementation might affect metastatic cascade. Alternatively, decreased vitamin D could be a consequence and hence a biomarker of a more invasive disease. Animal experiments and interventional studies are needed to prove the causality of the observed association.