2.3. Biochemical Procedures

All samples were then simultaneously tested. The following parameters were assessed: Total antioxidant capacity (TAC), catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), reduced glutathione (GSH), total oxidant status (TOS), 3-nitrotyrosine (3-NT), advanced glycoxidation end products (AGEs), advanced oxidation protein products (AOPP), N-formylkynurenine (NFKN), kynurenine (KN), tryptophan (TRY), and β-amyloid.

All reagents for the biochemical assays were obtained from Sigma-Aldrich (Saint Louis, MO, USA). The list of reagent catalog numbers is listed in the Supplementary Materials. The fluorescence/absorbance was measured using an Infinite M200 PRO Multimode Microplate Reader, Tecan (Tecan Group Ltd., Männedorf, Switzerland). All determinations were standardized to 1 mg of total protein.

Catalase (CAT) activity was assessed spectrophotometrically by measuring the decomposition rate of hydrogen peroxide. The absorbance was measured at 240 nm [31]. It was assumed that 1 unit of CAT decomposes 1 mmol hydrogen peroxide per 1 minute.

Superoxide dismutase-1 (SOD) activity was assayed spectrophotometrically by measuring the inhibition of adrenaline oxidation to adrenochrome at 480 nm [32]. A quantity of 1 unit of SOD activity was defined as a quantity of enzyme necessary to inhibit the oxidation of adrenaline by 50%.

Glutathione peroxidase (GPx) activity was assessed spectrophotometrically in the presence of NADPH and glutathione reductase, based on the reduction of organic peroxides. The absorbance was estimated at 340 nm [33]. A quantity of 1 unit of GPx activity was defined as catalyzing the oxidation of 1 μmol of NADPH per 1 minute.

Reduced glutathione (GSH) concentration was assessed spectrophotometrically based on the reaction with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB). The absorbance of the resulting complex was estimated at 412 nm [34]. The standard curve for GSH (0–50 umol/L) was used.

Total antioxidant capacity (TAC) levels were estimated spectrophotometrically based on the reaction with 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid radical cation (ABTS*+) [35]. Absorbance changes were measured at 660 nm. TAC levels were calculated with the use of the calibration curve for 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox; 0–3 mmol/L).

The content of advanced oxidation protein product (AOPP) was estimated spectrophotometrically at 340 nm with the use of the iodine ion by measuring its oxidative capacity. For AOPP assessment, urine samples were diluted 1:50 (v/v) in phosphate-buffered saline, pH 7.2 [36]. The concentration of AOPP was calculated from the standard curve for chloramine T (0–100 umol/L).

The presence of advanced glycation end-products (AGE) was assessed fluorometrically by measuring AGE-specific fluorescence at 350 nm/440 nm. For AGE assessment, urine samples were diluted 1:50 (v/v) in phosphate-buffered saline, pH 7.2 [36].

For KN, NFKN, TRY, and 3-NT determinations, urine samples were diluted (1:10, v/v) in 0.1 M H₂SO₄. Fluorescence was analyzed at 365/480, 325/434, 95/340, and 330/415 nm, respectively, [37], and all results were normalized to a fluorescence of 0.1 mg/mL quinine sulfate (in 0.1 M H₂SO₄) [38].

The level of TOS was determined colorimetrically based on the oxidation of Fe²⁺ to Fe³⁺ ions in the presence of the oxidants in the sample [35]. The absorbance was measured bichromatically at 560/800 nm. The TOS level was calculated from the standard curve for H₂O₂ (0–200 umol/L).

The content of total protein in the samples was estimated by the bicinchoninic acid (BCA) method [39] using the commercial kit-Thermo Scientific Pierce BCA Protein Assay (Rockford, IL, USA), in accordance with the manufacturer’s instructions.

The formation of beta-amyloid was evaluated by adding 10 uL of Thioflavin T to 90 uL of the urine sample. Thioflavin T fluorescence was measured at 385/485 nm [40].