Blood samples were collected from the antecubital vein between 9:30 and 10:30 am. Peripheral blood tests were performed to determine red blood cell (RBC) counts, hemoglobin (Hb) and hematocrit (Hct) levels, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. To quantify the active form of hepcidin, hepcidin-25, serum samples were mixed with synthetic human hepcidin (Peptide Institute, Osaka, Japan) as an internal standard and applied to a reverse-phase PLRP-S column (5 mm, 300 Å, 150 × 3 × 2.1 mm; Varian, Inc, Palo Alto, CA, USA). Levels of hepcidin-25 in the eluate were measured using a 4000 QTRAP liquid chromatography tandem mass spectrometry system (Applied Biosystems, Foster City, CA) (25 (link)). A LABOSPECT 008α automatic colorimetric analyzer (Hitachi High-Technologies Corporation, Schaumburg, IL, USA) was used to measure serum levels of iron. To test levels of iron stored in the body, we measured the concentrations of ferritin in serum using an iatro ferritin kit (LSI Medience Corporation, Tokyo, Japan). The leptin levels were quantified using a human leptin radioimmunoassay kit (EMD Millipore Corporation, Billerica, MA) (26 (link)). The adipokine adiponectin was measured using a human adiponectin latex kit (LSI Medience Corporation) (27 (link)).
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