Serum tumor necrosis factor α (TNF-α) levels and other biochemical markers
TNF-α
BLOOD
Blood samples were collected from each participant in the morning after fasting for 12 h overnight. Each blood sample was placed in a serum separator tube and centrifuged at 2,000 rpm for 15 minutes at 4°C. The serum was stored at −80°C until further analysis. Serum TNF-α levels were measured by ELISA using the Human TNF-α Assay kit (#KHC3011, Invitrogen – Thermo Fisher scientific, Tokyo, Japan), according to the manufacturer's protocol. Serum levels creatine kinase (CK), hs-CRP, and creatinine were determined using standard enzymatic techniques. These analyses were outsourced to Tsukuba i-Laboratory LLP (Tsukuba, Japan).