Blood sampling and measurement of serum adipokine and myokine levels

Patient medical history and clinical characteristics were collected from medical records. Fasting blood was analyzed to determine glucose (GLU), glycosylated hemoglobin (HbA1c), and immunoreactive insulin (IRI) levels. We evaluated the endogenous effect of insulin resistance on vascular function. Insulin resistance was assessed using the homeostasis model assessment of insulin resistance (HOMA-IR). HOMA-IR was calculated as follows: HOMA-IR = (IRI × fasting plasma GLU) / 405. Additionally, we measured the plasma levels of myokines, adiponectin, leptin, and irisin. Blood samples were stored at − 80 °C, and both myokine and adipokine levels were measured according to the manufacturer’s instructions. Serum myostatin and irisin levels as myokines were measured using the GDF-8/Myostatin Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA) and human EIA Kit (Phoenix Pharmaceuticals Inc., Burlingame, CA, USA). Serum adiponectin and leptin levels as adipokines were measured using the human Quantikine ELISA Kit (R&D Systems, respectively. Minneapolis, MN, USA). The intra- and inter-assay coefficients of variation were 2.5–4.7% and 5.8–6.9% for adiponectin, 3.0–3.3% and 3.5–5.4% for leptin, 1.8–5.4% and 3.6–6.0% for myostatin, and < 10% and < 15% for irisin, respectively.