Nefa assay

Blood samples collected into vacutainers containing either sodium fluoride, EDTA or silica were centrifuged at 1000g and 4°C for 10 minutes. Aliquots of plasma (and serum) were frozen and stored at -80°C until analysis. An ILab-600 semi-automatic spectrophotometric analyser was used to determine plasma glucose during the OGTT and fasting serum non-esterified fatty acid (NEFA), triglyceride (TG), total cholesterol (TC), LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C) concentrations, in combination with the appropriate assay kit (all obtained from Instrumentation Laboratory Ltd UK, Warrington, UK, except for the NEFA assay, which was obtained from Randox, London, UK). Plasma insulin concentrations were determined using a commercially available direct insulin ELISA kit (Invitrogen, UK). Area under the curve (in response to the OGTT) for glucose, insulin and NEFA was calculated using the conventional trapezoid rule. Insulin sensitivity was assessed using the homeostatic model assessment (HOMA) and Matsuda [30 (link)] insulin sensitivity index.

Blood samples were taken three times from the antecubital vein into the vacutainer tubes with a silica clot activator before, 15 min, and one hour after the tests. Additionally, in the WAnT group, the blood samples were taken 3 min after the first and second bout for lactate measurement. The samples were centrifuged at 2000g for 10 min at 4 °C. The separated serum and plasma samples were frozen and kept at − 80 °C until later analysis. The tubes containing the samples were number-coded to blind the laboratory personnel regarding the treatment group and the sequence of sample collection.
Serum 25(OH)D₃ concentration was determined by enzyme immunoassay method using 25-OH Vitamin D total ELISA kit (DE1971, Demeditec Diagnostics, Germany), according to the manufacturer’s instructions.
Plasma interleukin 6 (IL-6) concentration was determined by ELISA using a commercial kit (HS600, R&D Systems, USA).
Plasma parathyroid hormone (PTH) was determined using PTH intact ELISA kit (DE 3645, Demeditec Diagnostics, Germany).
Plasma non-esterified fatty acids and glycerol concentrations were determined by direct colorimetric methods using NEFA assay (FA115, Randox, United Kingdom) and Glycerol assay (GY105, Randox, United Kingdom).