High sensitivity il 6

Blood samples collected in EDTA tubes (Fig.1) were centrifuged at 600 g for 10 min, the plasma was removed, and placed into −80°C until assay day. On assay day, high sensitivity TNF-α (R&D System Minneapolis, MN), high sensitivity IL-6 (R&D System Minneapolis), high sensitivity IL-8 (R&D System Minneapolis), IL-1ra (R&D System Minneapolis), MCP-1 (R&D System Minneapolis) ELISA assay kits were used to measure cytokine content, according to the manufacturer's instructions. In addition, colorimetric assays detecting free fatty acids (ZenBio Research Triangle Park, NC), and triglycerides (BioAssay Systems Hayward, CA) were also perfomed.

Blood samples were collected from the basilic vein after overnight fasting (between 8 a.m. and 9 a.m.) and centrifuged at 4000 rpm and 4°C. The serum was separated from the sample and stored at -70°C. High-sensitivity C-reactive protein (hsCRP) and myoglobin levels were determined using the immunoenzymatic method with commercially available kits (DRG International Inc., Springfield Township, NJ, USA; test sensitivity: 0.1 mg/L and 5 ng/mL, respectively), as were the levels of high-sensitivity IL-6 (R&D Systems Inc., Minneapolis, MN, USA; test sensitivity: 0.039 pg/mL), irisin (Aviscera Bioscience Inc., Santa Clara, CA, USA; test sensitivity: 100 pg/mL), osteoprotegerin (OPG) and high-sensitivity soluble receptor activator of NF-κB ligand (sRANKL) (Biomedica GmbH & Co KG, Wien, Austria; test sensitivity: 0.07 pmol/L and 0.01 pmol/L, respectively), myostatin (Immundiagostic AG, Bensheim, Germany; test sensitivity: 0.37 ng/mL), and 25(OH)D (Demeditec Diagnostic GmbH, Kiel, Germany; test sensitivity: 1.9 ng/mL).