Oxidative Stress Markers in Adipose Tissue

A piece of frozen eWAT was homogenized in a RIPA lysis buffer (pH 7.5; Cell Signaling Technology®, Beverly, MA) containing protease and phosphatase inhibitor cocktails (Roche®, Mannheim, Germany). Total protein levels were determined by the Bradford assay [21 (link)]. The eWAT catalase activity was measured according to Xu and colleagues [22 (link)], and enzyme activity was expressed in μmol·min·mL⁻¹ per eWAT protein (mg·mL⁻¹) [21 (link)]. Total superoxide dismutase (SOD) activity was assessed with a commercial colorimetric kit (#19160, Sigma®, Seelze, Germany) following the manufacturer's instructions. Lipid peroxidation in eWAT was determined by measuring the thiobarbituric acid reactive substances (TBARS), as a marker of oxidative stress, mainly malondialdehyde (MDA). The quantification was performed according to Ohkawa et al. [23 (link)] with modifications, as previously described [24 (link)]. Data were normalized per total protein concentration, measured by Bradford [21 (link)] and expressed as nM·mg protein⁻¹.
Hydrogen peroxide (H₂O₂) was measured by fluorescence using the Amplex® UltraRed hydrogen peroxide (10-acetyl-3,7-dihydroxiphenoxazine) assay (Invitrogen®, Paisley, United Kingdom) as described [24 (link)].