Human insulin specific

Two samples (12 ml each one) of peripheral venous blood were obtained using a vacutainer system. To the first baseline sample, the participants reported to the laboratory at 8:00 h following an overnight fast and 48-h abstention from vigorous physical activity. The second sample was obtained 30 min after finishing the exercise session. Blood samples were treated to measure plasma irisin by ELISA with the Irisin kit (Cat.# EK-067-29 (Phoenix Pharmaceuticals, Burlingame, CA, USA), with a coefficient of variations (CVs) for intra assay: 5–7%; CVs for inter-assay: 12–15%; detection limit range: 0.1–1000 ng/ml. The assays used to detection of irisin were previously validated.
Blood samples were also treated to measure secondary outcomes such as glucose, insulin, and lipid profile. Glucose was determined using the enzymatic colorimetric method: glucose oxidase/peroxidase (BioSytems, USA). Insulin levels were determined by radioimmunoassay (Human Insulin Specific, MILLIPORE. Darmstadt, Germany). Lipid profile was determined using the enzymatic colorimetric methods CHOD-POD and GPO-POD (SPINREACT, Spain). Index of insulin resistance (HOMA-IR) was calculated according to Matthews et al. [22 (link)].

Plasma glucose concentrations were determined by the glucose oxidase test (Glucose assay; Randox Laboratories Ltd), using semi-automatic glucose equipment (Randox Laboratories Ltd). Plasma fructosamine concentrations were determined by the kinetic method of fixed time (Frutosamina test; Labtest Diagnóstica S.A.) in the Multiuser Laboratory of the College of Veterinary Clinics of Food Engineering and Animal Science at the University of São Paulo. Insulin analysis was performed using human insulin containing ¹²⁵I as a tracer hormone and 100 % of specificity for canine insulin (Human Insulin Specific; Millipore Corporation) at Genese laboratory (São Paulo, Brazil).