Ransel test kit

In the whole blood samples, glutathione peroxidase (GSH-Px) was determined with a commercial kit (Ransel test kit, Randox Laboratories Ltd., Crumlin, UK) adapted from the method of Paglia and Valentine [35 (link)]. For the determination of hemoglobin (Hb), a hematological analyzer (Advia 120, Siemens Healthcare Diagnostics SL, Barcelona, Spain) was required. The assessment of the overall metabolic profile in the plasma samples was performed using an automatic chemistry analyzer (Olympus AU600, Olympus Diagnostica Europe GmbH, Ennis, Ireland), and commercial Beckman kits (Beckman Coulter Inc., Fullerton, CA, USA) were used for the assays. Glucose was measured using the hexokinase G-6-PDH method; total triglycerides (TGs) were hydrolyzed using a combination of microbial lipases to produce glycerol and fatty acids; urea was measured using the enzymatic adapted Talke and Schubert [36 (link)] method; total proteins were measured using the adapted Weichselbaum [37 (link)] method; and cholesterol was determined using the cholesterol dehydrogenase method. TAC (antioxidant capacity) and TOS (oxidative status) were analyzed in the plasma samples using the methods described by Erel et al. [38 (link),39 (link)]. T₄ total hormone was determined with the immunoassay of chemiluminescence (Immulite 1000 Immunoassay System, Siemens Medical Solutions Diagnostics, Deerfield, IL, USA).

Routine laboratory parameters were measured from fasting blood samples by standardized methods for CRP, blood glucose, creatinine, fibrinogen and lipids at enrolment. For additional measurements, samples were aliquotted and stored at −80°C immediately after blood draw. In EDTA plasma, we measured Copeptin (functional assay sensitivity <1 pmoL/L), CT proendothelin-1 (functional assay sensitivity 19 pmoL/L), MR-proADM (functional assay sensitivity 0.25 nmoL/L), MR-proANP (functional assay sensitivity <10 pmoL/L) (Kryptor Immunoassays, B.R.A.H.M.S, GmbH, Germany), myeloperoxidase (intra–/inter-assay coefficient of variation 6.2/8.6) (CardioMPO kit, Prognostix, USA), and serum Nt-proBNP (intra–/inter-assay coefficient of variation 2.6/1.5) (Elecsys proBNP II Roche Diagnostics, Germany) biomarkers using commercially available assays. Sensitive TnI ultra was measured using Dimension RxL TnI (Siemens Healthcare Diagnostics, Germany) with a detection limit of 6 pg/mL and an assay range of 0–50.000 pg/mL. Glutathione-peroxidase-1 activity was determined in washed red cells obtained from whole blood anticoagulated with EDTA. Glutathione-peroxidase-1 was measured as previously described using the Ransel test kit (Randox, UK). [17] (link).

The evaluation of antioxidant capacity and antioxidant enzymes in plasma were performed following the methods described in [31 (link)] Glutathione peroxidase (GPx) assay was performed based on the method described by Paglia and Valentine [32 (link)] with a commercially available kit (Ransel test kit, Randox Laboratories Ltd., Crumlin, County Antrim, UK). Catalase activity was determined according to Johansson and Borg [33 (link)]. Ferric reducing antioxidant power (FRAP) assay was performed according to Benzie and Strain [34 (link)], and paraoxonase 1 (PON1) was measured following the method described by Tvarijonaviciute et al. [35 (link)].