*4.2. Laboratory Data*

The blood samples were collected from all the study participants, after signing the informed consent, who fasted 12 h, using a holder-vacutainer system and ×3000 g, for ten minutes, after one hour of keeping at room temperature. The sera were separated and frozen at −80 degrees before analyzing.

4-HNE and MDA were assessed by competitive ELISA method (semi-automatic Tecan analyzer). The wells were pre-coated with substrate and the final product colorimetric evaluation was made at 450 nm. The results were expressed as microgram/mL serum for 4-HNE and as nanogram/mL serum for MDA. MDA forms a complex with thiobarbituric acid reactive substances (TBARS) that is measured using the spectrophotometric method (BS-3000M Semi-Automatic Chemistry Analyzer) and read at a wave-length of 532 nm. The results were expressed as µmol/L serum. 8-Isoprostan was determined by competitive ELISA method (semi-automatic Tecan analyzer). The wells were pre-coated with substrate to acetylcholinesterase and the final product colorimetric evaluation was made at 412 nm. Results were expressed in pg/mL. ImAnOx was assessed by spectrophotometry (semiautomatic Tecan analyzer). by the reaction of antioxidants in the sample with a defined amount of exogenously provided hydrogen peroxide. The antioxidants in the sample

eliminate a certain amount of the provided hydrogen peroxide. The residual H2 O2 is determined photometrically at 450 nm. Results were expressed in µmol/L.

8-OHdG was determined by competitive ELISA method (semi-automatic Tecan analyzer). The wells were pre-coated with a target specific capture antibody and the final product colorimetric evaluation was made at 450 nm. Results were expressed in ng/mL. OGG1 was determined by ELISA method, quantitative sandwich (semi-automatic Tecan analyzer). The wells were pre-coated with a target specific capture antibody and the final product colorimetric evaluation was made at 450 nm. Results were expressed in pg/mL.

Pentosidine and AGEs were assessed by ELISA method (semi-automatic Tecan analyzer). The wells were pre-coated with a target specific capture antibody and the final product colorimetric evaluation was made at 450 nm. Results were expressed in ng/mL. sRAGE was determined using the sandwich ELISA method (immunoenzymatic kits-R&D SYSTEMS (DR600 and SRG00 kits), USA), the results were read at 450 nm, using a TECAN analyzer (Tecan, Switzerland). The sensitivity of the method was 16.14 pg/mL and assay range was between 78 and 5000 pg/mL.

Circulant 3-NT was determined by ELISA method using the R&D systems reactive (MAB3248 kit) and a TECAN analyzer (Tecan, Switzerland).

Carbonyl groups were assessed by spectrophotometric methods, in reaction with 2,4-dinythrophenylhydrasine that generated hydrazone, employing the HumanStar300 analyzer (HUMAN Gesellschaft für Biochemica und Diagnostica mbH, Weisbaden, Germany) and Merck reactives (MAK094 kit).

Thiol disulphide homeostasis parameters (TDHPs) were determined using a spectrophotometric method The dynamic and reducible disulfide bonds were transformed into free functional thiol groups by using sodium borohydride (NaBH4, 10 mM) as follows:

R-S2-R '+ NaBH4 → 2 R-SH + BH3 + Na.

Subsequently, the amount of NaBH4, which have not participated in the reaction, was removed with formaldehyde (10 mM, pH 8.2). The levels of native thiol (TN) and total thiol (TT) were assessed using 5,5′ -dithiobis-2-nitrobenzoic acid (DTNB, 10 mM) as follows:

R-SH + DTNB → R-TNB + TNB.

The final product, 2-nitro-5-thiobenzoate (TNB), ionized at alkaline pH and turned yellow. An automatic biochemistry analyzer (HumaSTAR 300, (HUMAN Gesellschaft für Biochemica und Diagnostica mbH, Weisbaden, Germany) and Merck reactives were used. This technique allows for the assessment of functional disulfide bonds in the sample. Disulfide (DS) level was calculated as half of the difference between TT and NT. The levels of TT, NT and DS were expressed as µmol/L serum. The disulfide/native thiol ratio (DS/NT), disulfide/total thiol ratio (DS/TT), and native thiol/total thiol ratio (NT/TT) were calculated and expressed as %. TDHPs were represented by:


#### *4.3. Statistical Analysis*

We used mean and standard deviation for data presentation. We compared the data using either the analysis of variance (ANOVA) with Tukey post hoc test or Kruskal–Wallis test with Dunn's post hoc test for normally and non-normally distributed data. The relation between the studied parameters was assessed by Pearson's correlation coefficient, but before the assessment, data normality by the Kolmogorov–Smirnov test was evaluated. The level of significance (p) chosen was 0.05 (5%) and the confidence interval was 95% for hypothesis testing and the corresponding ethical approval code.
