*2.3. Saliva Collection*

The research material was non-stimulated (NWS) and stimulated (SWS) whole saliva collected by the spitting method. In order to eliminate the influence of physical exercise and daily rhythm on saliva secretion, samples were taken from subjects who were not physically active for the last 24 h, after an all-night rest, always between 7 a.m. and 9 a.m. Subjects did not consume any meals or drinks (other than water), and refrained from performing any oral hygiene procedures at least 2 h before

saliva collection. Additionally, children did not take any medications for at least 8 h prior to saliva collection [16,28].

The subjects were instructed to rinse their mouth two times with distilled water and to spit saliva into a sterile Falcon tube placed in an ice container. Saliva collection was done by the patient when sitting with the head down (with minimized facial and lip movements), after at least a 5-min adaptation, always in the same child-friendly room. The time of NWS collection was 15 min. Then, saliva was stimulated by dropping 10 μl of citric acid (2%, w/v) solution on the tip of the tongue every 30 s [16,17,26,28]. The time of SWS collection was 5 min [16,28].

Immediately after collection, the volume of saliva was measured with a pipette set to 100 μL. The salivary flow rate was calculated by dividing the volume of saliva by the time necessary for its secretion (mL min<sup>−</sup>1). The pH of saliva was also analyzed using Seven Multi pH meter (Mettler Toledo, Greifensee, Switzerland).

After measuring the salivary pH, the samples were immediately centrifuged (3000× *g*, 4 ◦C, 20 min) and the supernatant was preserved for further analysis [32]. To protect against sample oxidation, butylated hydroxytoluene (BHT, Sigma-Aldrich, Nümbrecht, Germany) was added (10 μL 0.5 M BHT in acetonitrile (ACN)/1 mL NWS/SWS) [32]. The samples were portioned into 200 μL aliquots and frozen at −80 ◦C. Frozen samples were stored for no more than six months.

In order to identify samples contaminated with blood, the concentration of transferrin in saliva was assessed (Human Transferrin ELISA Kit; Abcam; Cambridge, UK). However, no blood contamination was confirmed in any of the samples.

The activity of salivary amylase (EC 3.2.1.1) was assessed for the evaluation of salivary gland function [33,34]. A spectrophotometric method with 3,5-dinitrosalicylic acid (DNS) was used and absorbance was measured at 540 nm.
