*2.3. Saliva Collection*

The assessment of the antioxidant barrier of saliva is complete when it includes analysis of both stimulated and unstimulated saliva. It has been shown that antioxidants produced by the parotid gland are aimed at combating deleterious foreign ROS that may penetrate oral cavity during eating, and antioxidants present in NWS for the rest of the time [22]. The studied material was non-stimulated (NWS) and stimulated (SWS) whole saliva collected via the spitting method. Participants were advised to refrain from consuming meals and drinks other than clean water, performing oral hygiene procedures for 2 h and taking any medications for 8 h before saliva collection. Saliva was collected between 8 a.m. and 10 a.m. to minimize the effect of daily changes on its secretion. The material was taken in a separate room so that patients did not feel uncomfortable or nervous. Participants had their saliva

collected in a sitting position, with the head slightly inclined downwards, with minimized face and lip movements, upon a 5-min adaptation period. After that time, every patient rinsed their mouth three times with water at room temperature. The saliva collected during the first minute was discarded. Subsequent batches of saliva (the patient actively spat out the saliva accumulated in the bottom of the oral cavity) were collected into plastic centrifuge tubes placed in ice containers. The time of NWS collection was 15 min [15,16,22]. SWS was collected after a 5-min break, for 5 min. Its stimulation was triggered by dripping 100 μL 2% citric acid under the tongue every 20 s [23]. To avoid sample oxidation, 0.5 M BHT was added to the saliva (Sigma-Aldrich, Saint Louis, MO, USA; 10 μL/mL saliva) [24]. The volume of each sample was measured with a pipette calibrated to 0.1 mL. Saliva secretion was calculated by dividing the volume of the obtained saliva by the number of minutes of its collection. Then saliva was centrifuged (20 min, 4 ◦C, 10,000× *g*). Further tests were performed using the preserved supernatant fluid, which was frozen at −80 ◦C until assayed. Frozen samples were stored for no longer than 6 months.

**Table 1.** Clinical characteristics of the patients and control group.


Abbreviations: HT—Hashimoto thyroiditis, C—control, BMI—body mass index, TSH—thyrotropic hormone, anty TPO—thyroid peroxidase antibody, anty TG—thyroid peroxidase antibody, PTH—parathyroid hormone.

## *2.4. Dental and Periodontal Examination*

Dental examination was performed on the day of and immediately after saliva collection using a mirror, an explorer and a periodontal probe, in artificial light, by one calibrated dentist (K.M.). The study included dental evaluation, caries severity index (DMFT) as well as approximal plaque index (API), periodontal probing depth (PPD) and gingival index (GI). DMFT is an index that evaluates the condition of teeth, which consists in counting teeth with caries, removed due to caries or filled because of caries. GI is the assessment of the gingiva for possible inflammation. API is an index used to assess plaque located in interdental spaces. Finally, PPD is an index of the depth of probed gingival pockets. In 20 participants, the study was conducted by another experienced dentist (A. Z.) and the results were compared with those obtained by the head doctor (K. M.). The interrater reliability for DMFT was *r* = 0.92, for GI *r* = 0.94, for API *r* = 0.98 and for PPD *r* = 100.
