2.4. Glutathione Assay

The measurements of the oxidized (GSSG) and the reduced glutathione (GSH) were performed in the heart homogenates with Ellman’s reagent [26]. Hearts were removed from Langendorff apparatus, washed with cold (+4 °C) 0.9% KCl solution, weighed and homogenized in the isolation buffer based on 0.1 M potassium phosphate buffer (KPE) with the addition of 5 mM EDTA, pH = 7.5, 0.1% Triton X-100 and 0.6% sulfosalicylic acid. The ratio of isolation buffer and tissue was 1:9. The homogenized tissue was centrifuged (Allegra X-22R, Beckman Coulter, Brea, California, USA) at +4 °C, 8000× g for 10 min. After this stage, the precipitate was leached into clean microtubes. 1 mL of precipitate for GSH measurement was immediately frozen (−20 °C). For GSSG measurement, 1 mL of precipitate was mixed with 30 μL of 97% 2-vinylpyridine in KPE solution (1:10), stirred, and 60 μL of 98% triethanolamine in KPE solution (1:6) was added after an hour, stirred and frozen. The measurements were performed using a BiosanHiPo MPP-96 microplate reader (Lithuania). 60 μL of 500 units glutathione reductase in KPE solution (1:150), 60 μL of 0.8 mmol/L cofactor β-NADPH reduced tetrasodium salt and 60 μL of 1.68 mmol/L dithiobisnitrobenzoic acid were added to initiate the reaction. Optical density was measured immediately and for 2 min each 30 s at 405 nm. The concentrations of GSH and GSSG were calculated according to the linear regression equation obtained from the calibration curve of the standard GSH and GSSG solutions.