4.3. Saliva Collection

The study material was whole saliva, both non-stimulated (NWS) and stimulated (SWS), collected from patients via the spitting method. The subjects had not consumed food or beverages other than pure water, and had refrained from oral hygiene activities for at least 2 h prior to saliva collection. Moreover, the patients in the study/control group had not taken any medicines for at least 8 h before saliva collection.

Saliva was always collected in the same circumstances: between 8 a.m. and 10 a.m. (to minimize the effect of daily rhythm on salivation) in the same, separate and quiet room so that the patients did not feel uncomfortable or upset. After at least 5 min of adaptation to the room environment and upon rinsing the mouth with distilled water at room temperature three times, the patients had their saliva collected in a seated position, with the head slightly inclined downwards, trying to refrain from facial and lip movements. The saliva gathered at the bottom of the oral cavity was spat into a sterile centrifuge tube placed in a container with ice. Saliva collected in the first minute was discarded. The NWS collection time was 10 min [15]. After a 5 min break, the collection of SWS begun. Salivation was stimulated by sprinkling the tongue every 30 s with 10 μL of 2% citric acid. SWS was collected for 5 min to a maximum volume of 5 mL, in the same manner as the NWS [15]. The volume of each saliva sample was measured with a pipette calibrated to 100 μL. The minute flow of NWS and SWS was calculated by dividing the saliva volume by the time necessary for its secretion. Upon collection, saliva was immediately centrifuged (20 min, 3000× g, +4 °C; MPW 351, MPW Med. Instruments, Warsaw, Poland). In order to protect the obtained supernatants against oxidation processes, butylated hydroxytoluene (BHT, Sigma-Aldrich, Sigma-Aldrich, Saint Louis, MO, USA; 5 μL 0.5 M BHT in acetonitriles per 0.5 mL saline fluid) was added and the supernatants were frozen at −80 °C. The samples were stored at that temperature until the performance of an assay [14]. Saliva samples for pH determination were analysed immediately after collection with the SevenMulti Mettler Toledo pH meter (Mettler-Toledo, Columbus, OH, USA).