Apelin 12

Blood samples were collected into EDTA and serum tubes and were centrifuged for 15 min at 1000g at 4°C and 21°C, respectively. Aliquots were immediately frozen in liquid nitrogen and subsequently stored at −80°C until analysis. Plasma glucose, FFA, TAG and total cholesterol were analyzed with standard enzymatic methods (ABX Pentra 400 autoanalyzer, Horiba ABX, Montpellier, France). Plasma glycerol was analyzed with an enzymatic assay (Enzytec Glycerol, Roche Biopharm, Basel, Switzerland) automated on a Cobas Fara spectrophotometric autoanalyzer. Plasma insulin, leptin, and adiponectin concentrations were analyzed with commercially available radioimmunoassay (RIA) kits (Human insulin specific RIA, human leptin RIA, human adiponectin RIA, Millipore Corporation, Billerica, MA). Plasma concentrations of IL‐6 (Meso Scale Discovery, Gaithersburg, MD), apelin‐12 (Phoenix pharmaceuticals Inc., Burlingame, CA), RBP4 (R&D systems, Minneapolis, MN) and Vaspin (Adipogen, San Diego, IL) were determined by ELISA. The apelin‐12 ELISA has 100% cross‐reactivity with human apelin‐12, apelin‐13, and apelin‐36. Serum ACE activity was measured with a standard enzymatic assay (Bühlmann, Basel, Switzerland) while serum nesfatin‐1 (Phoenix pharmaceuticals Inc., Burlingame, CA) concentrations were measured by ELISA.

The peripheral blood samples were collected from all participants after 12-h overnight fast. The serum Apelin concentration was measured by an ELISA kit (Apelin-12, Phoenix pharmaceuticals, Belmont, USA) according to manufacturer’ protocol. The sensitivity was 0.05 ng/mL, and intra- and inter-assay variations were <5 and <10 %, respectively.