2.4.3. Blood Sample Collection and Analyses
BLOOD, SEPARATION
Following an overnight fast, the participants visited the laboratory at 08:00 and rested for 10 min before the first blood sample collection. Blood samples were collected before, and 0, 1, and 3 h after exercise sessions using serum separation tubes (for serum samples), tubes containing EDTA-2Na, and tubes containing EDTA-2K (for plasma and whole blood samples). Serum and plasma samples were centrifuged (10 min, 3000 rpm, 4 °C) and stored at −80 °C until subsequent analysis. Whole blood samples were stored in the refrigerator and sent to a clinical laboratory (SRL Inc., Tokyo, Japan).Blood glucose and lactate levels were measured immediately after blood collection using a blood glucose analyzer (Freestyle; Nipro Co., Osaka, Japan), and a lactate analyzer (Lactate Pro; Arkray Co., Kyoto, Japan). Serum ferritin, iron, and total ketone body (acetoacetic acid and β-hydroxybutyric acid) were measured in the serum samples, and a complete blood count (white blood cells, red blood cells, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet) from whole blood samples were measured in a clinical laboratory (SRL Inc.). Serum hepcidin and glycerol, as well as plasma IL-6 levels were analyzed using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA; Cayman Chemical, Ann Arbor, MI, USA). The intra-assay coefficients of variation were 3.87%, 4.92%, and 3.56% (hepcidin); 0.88% and 1.19% (glycerol); and 2.00% and 2.88% (IL-6).