2.8.2. Non-Enzymatic Antioxidants

The non-enzymatic antioxidant barrier was assessed by measuring GSH and UA concentrations in NWS, SWS and plasma.

GSH concentration was determined colorimetrically based on the reaction with DTNB. Absorbance of the samples was measured at 412 nm wavelength [31].

UA concentration was determined spectrophotometrically at 490 nm using the ability of 2,4,6-Tris(2-pyridyl)-s-triazine to form a blue complex with iron ions in the presence of UA. We used a commercial set of reagents (QuantiChromTM Uric Acid Assay Kit DIUA-250; BioAssay Systems, Hayward, CA, USA).

Total antioxidant capacity (TAC) level in plasma was determined spectrophotometrically at 660 nm wavelength using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ATBS). The intensity of the color resulting from the reaction of ABTS radical cation was proportional to the content of antioxidants in the tested samples [32].

2.8.3. Total Oxidant Status (TOS) and Oxidative Stress Index (OSI)

TOS and OSI levels were determined in NWS, SWS and plasma.

TOS level was assayed bichromatically (560/800 nm) based on the oxidation of Fe2<sup>+</sup> to Fe3<sup>+</sup> in the sample. TOS level was calculated from the standard curve for H2O2 [33].

OSI was calculated based on the formula OSI = (TOS/TAC)/100 [34].

2.8.4. Oxidative Damage to Proteins and Lipids

Oxidative damage to proteins and lipids was assessed by measuring the concentration of advanced glycation end products (AGE) of proteins, advanced oxidation protein products (AOPP), lipid hydroperoxides (LOOH) and malondialdehyde (MDA) in saliva and plasma samples.

AGE content was assessed by measuring AGE-specific fluorescence at 350/440 nm wavelength, as described by Kalousová et al. [35].

The colorimetric measurement of AOPP content was determined fluorimetrically as described by Kalousová et al. [35] based on the oxidative capacity of iodine ions at 340 nm wavelength.

To measure AGE and AOPP concentrations, NWS, SWS and plasma samples were diluted with phosphate-buffered saline (PBS, pH 7.2) at a ratio of 1:5 (*v*/*v*).

The concentration of LOOH was evaluated spectrophotometrically from the reaction of iron ions (3+) with xylenol orange (XO). The absorbance of Fe-XO complex was measured at 560 nm wavelength [36].

MDA concentration was assessed colorimetrically based on the reaction with thiobarbituric acid (TBA) at 535 nm wavelength. 1,1,3,3-Tetraethoxypropane was used as a standard [24].

## *2.9. Statistical Analysis*

Statistical analysis was performed using GraphPad Prism 8.3.0 for MacOS (GraphPad Software, Inc. La Jolla, CA, USA). The distribution of the obtained results was assessed using the Shapiro–Wilk test. Due to the lack of normal distribution, the Mann–Whitney U test was used for quantitative comparisons. Chi-square test with Yates's modification was used to analyze the differences in the prevalence of qualitative variables. Correlations of the results were assessed using the Spearman rank correlation coefficient. The statistical significance level was set at *p* < 0.05.

The number of subjects was determined based on our previous experiment, assuming that the power of the test would be equal to 0.9 (ClinCalc sample size calculator).
