A total of 18 saliva samples were used in this study. For the SDS-PAGE analysis, saliva samples from eight donors (4 females and 4 males) aged 20-25 years were used. Whole saliva samples were collected by passive drooling method following previously reported methodologies3. Briefly, participants were instructed not to eat for at least one hour prior and to rinse their mouths thoroughly 5 min prior to saliva sample collection to avoid possible contamination of the sample. Saliva samples obtained (about 5 ml) were lightly vortexed and centrifuged (3000×g for 10 min at 4°C) and the supernatant was aliquoted. Samples for proteomics were stored at -30°C until analysis (no longer than two months).
For biochemical analysis, saliva samples were collected from ten subjects (5 females and 5 males) aged 25-35 years, as described above, and processed and analyzed fresh.
In the present study, saliva samples from subjects who tested positive for SARS-CoV-2 were not included, as our aim was to assess the specific effect of thermal or chemical methods in the analysis of salivary molecules and this effect should not be affected by the presence or absence of virus.
All procedures were in accordance with the Declaration of Helsinki given by the World Medical Association, guaranteeing the non-existence of an invasive character, and included in a project approved by the Ethics Committee of the University of Evora (GD /6543/2021). Informed consent was obtained from all participants, after receiving information about the purpose of the study and the type of data to be obtained from the collected saliva samples.
Six aliquots of each sample were prepared and simultaneously subjected to the following treatment protocols: TT1 – control (no inactivation treatment); TT2—heated at 65°C for 30 min; TT3—heated at 92°C for 15 min; TT4 – mixed with SDS (10% w/v) to a final concentration of 0.5% SDS followed by immediate incubation at room temperature for 30 min; TT5 – treated with NP-40 (10% v/v) to a final concentration of 0.5% NP-40 followed by immediate incubation for 30 min; TT6 – treated with Triton X-100 (10% v/v) to a final concentration of 0.5% Triton X-100 followed by immediate incubation at room temperature for 30 min. All of these inactivation procedures were performed in duplicate.
Due to the different possible effects, of each inactivation protocol, in the results obtained from the total protein assays17, the total protein concentration of the controls (TT1) was used for the calculation of the volume to be used in the SDS-PAGE analyses. Each saliva sample was analyzed in duplicate and, for each, a volume corresponding to 7.5 µg of total protein was mixed with sample buffer and analyzed on each row of 14% polyacrylamide mini-gels (Protean xi, Bio-Rad, CA, USA) using a Laemmli buffer system, as described elsewhere2. In each gel, one of the lanes was used for the molecular mass standard (Bio-Rad Precision Plus Protein Dual Color 161-0394). The electrophoretic cycle was performed at a constant voltage of 140 V until the forward dye reached the end of the gel. Gels were fixed for 1 h in 40% methanol/10% acetic acid, followed by staining for 1 h with Coomassie Brilliant Blue (CBB) R-250 and destained in several acetic acid washes at 10%. Gel images were acquired using a Molecular Dynamics scanning densitometer with internal calibration and LabScan software (GE Healthcare), and images were analyzed using GelAnalyzer software (GelAnalyzer 2010a by Istvan Lazar , https://www.gelanalyzer.com) for the volume percentage of each protein band.
Biochemical Analytes and Quantification of Antioxidants
Total saliva protein, alpha-amylase (sAA), cholinesterase (ChE), total esterase activity (TEA), creatinine kinase (CK), aspartase transaminase (AST), lactate dehydrogenase (LDH), lactate, iron reducing capacity of Saliva (FRAS), Trolox Equivalent Antioxidant Capacity (TEAC), Copper Reducing Antioxidant Capacity (CUPRAC), Uric Acid, Catalase (CAT), Advanced Oxidation Protein Products (AOPP), Hydrogen Peroxide (H2O2), glucose, triglycerides, creatinine, calcium (Ca), total cholesterol, phosphorus (P), alanine aminotransferase (ALT) and urea were determined using previously validated commercial kits or homemade tests for use in saliva samples5.16. An automated biochemistry analyzer was used for all measurements (Olympus AU600, Olympus Diagnostica GmbH, Freiburg, Germany).
Total IgA was assessed with a commercial ELISA kit (Bethyl, Montgomery, TX, USA) previously validated for use with human saliva samples18. Cortisol was analyzed with an immunoassay system (Immulite, Siemens Healthcare Diagnostics. Deerfield, IL)19.
All data were tested for normal distribution using Shapiro-Wilk. Since most of the parameters were not normally distributed and due to the small number of samples (N=8, for SDS-PAGE analysis and N=10 for biochemical parameters), non-parametric tests were used. Friedman’s test was used to compare each salivary parameter among the six protocols used, followed by Dunn’s post hoc pairwise tests with Bonferroni’s correction for multiple tests. Due to the independence of the inactivation treatments from each other, in cases where Friedman caused nonsignificant variations between protocols and therefore Dunn’s pairwise post-hoc tests were not performed, the d inactivation were compared to the control using the Wilcoxon test. Statistical analysis was performed using SPSS v. 24, with a significance level set at 5%.