2.4. Laboratory Analyses
Gas chromatography (GC) was used for the analysis of fatty acids. Technical details included a gas chromatograph mass spectrometer (GCMS-QP2010Plus) and auto injector and autosampler (all from Shimadzu, Tokyo, Japan); a high polarity capillary column (internal diameter 15 m × 0.10 mm, film thickness 0.10 µm) (Suprawax-280, Teknokroma Analítica, S.A., Barcelona, Spain); and GCMS solution software for data acquisition. In order to optimize the whole spectrum of fatty acid analysis, functioning conditions of MS operating parameters were optimized (10,000 amu/s for scan rate, 40–400 Total antioxidant capacity (TAC) expressed as µM copper-reducing equivalents (Cat. No. STA-360) and lipid peroxidation (thiobarbituric acid reactive substances (TBARS)) assessed as malondialdehyde (MDA) levels (Cat. No. STA-330) were measured in plasma samples using the OxiSelect™ assay kit (Cell Biolabs, San Diego, CA, USA) following the manufacturer’s instructions.The OxiSelect™ assay (Cell Biolabs, San Diego, CA, USA) was used to measure total antioxidant capacity (TAC) and malondialdehyde (MDA) levels in plasma samples. MDA levels were indicative of lipid peroxidation (thiobarbituric acid reactive substances (TBARS)). The manufacturer’s instructions were followed. TAC levels were expressed as µM copper-reducing equivalents and MDA as µM.A fluorescently labeled microsphere-based multiplex immunoassay was used for simultaneous analysis of IL-1β, IL-4, IL-6, IL-10, IL-18, TNF-α, and VEGF-A in plasma samples. Fluorescence was read on the Luminex-100 ISv2 system (Cat. No. HCYTOMAG-60K-05, Milliplex Map Human Cytokine/Chemokine; Linco Research/Millipore, Saint-Charles, MO, USA). The intra- and inter-assay coefficient of variation for each cytokine was: IL-1β: 7 and 12%; IL-4: 3 and 11%; IL-6: 2 and 10%; IL-10: 2 and 11%; IL-18: 2 and 11%; TNF-α: 3 and 19%; and VEGF-A: 3 and 15%, respectively.To determine glutathione levels, the DetectX