Determination of Cytokines
10 ml of peripheral blood were centrifuged at 2500 rpm for 10 min at 4 ° C. Then, 500 µl aliquots of serum were prepared and stored at -75 ° C until cytokine determination. Subsequently, endothelin 1 (ET-1), IL-1ß, IL-1, IL-6, OPN, OPG, RANK, RANK-L, and TGF-α were determined by an enzyme-linked immunosorbent assay (ELISA) in DuoSet sandwich (R&D Systems, Minneapolis, MN), according to the instructions provided by the manufacturer18.