Blood Sampling and Metabolic Analysis Protocol

Blood samples (10 mL) were collected in EDTA-containing tubes and centrifuged at 1000× g and 4 °C for 10 min. Aliquots of plasma were frozen in liquid nitrogen and stored at −80 °C until analysis. Plasma glucose and lactate were analyzed with a COBAS FARA semiautomatic analyzer (Roche). Plasma insulin concentrations were analyzed using commercially available kits (Elecsys Insulin assay, Roche, Ref: 12017547122; Mannheim, Germany). Plasma I-FABP levels were measured using an in-house developed enzyme-linked immunosorbent assay. The detection window of the I-FABP assay is 12.5–800 pg·mL⁻¹, with an intra-assay and inter-assay coefficient of variation of 4.1% and 6.2%, respectively [23 (link),29 (link)]. Breath samples were analyzed for ¹³C/¹²C ratio by gas chromatography continuous flow isotope ratio mass spectrometry (GC/C/IRMS; Finnigan, Bremen, Germany). From indirect calorimetry (VO₂ and VCO₂) and stable isotope measurements (breath ¹³CO₂/¹²CO₂ ratio), oxidation rates of total fat, total carbohydrate and exogenous carbohydrate were calculated.