2.3.4. Serum Biomarkers

Fasted blood samples were collected from the antecubital vein at baseline and at 4 weeks. Serum samples were collected using silicone-coated tubes and stored at −80 ◦C until subsequent analysis of the biomarkers. Glucose (EIAGLUC; Invitrogen, Carlsbad, CA, USA) was measured using the glucose oxidase method. Insulin was measured using an ELISA kit (80-INSHUE01.1, ALPCO Diagnostics, Salem, NH, USA). The homeostatic model assessment for insulin resistance was calculated as [fasting glucose (mg/dL) × fasting insulin (μIU/mL)]/405. Glutathione peroxidase (ab102530; Cambridge, UK), malondialdehyde (Abcam 233571; Cambridge, UK), and superoxide dismutase (ab119520; Cambridge, UK) were analyzed using the colorimetric method. Arginase-1 (BMS2216; Invitrogen, Carlsbad, CA, USA) and oxidized low-density lipoprotein (CSB-E07931h; Cusabio Biotech, Houston, TX, USA) were measured using ELISA kits. Serum ARG, CIT, ORN, and ADMA were analyzed using an AbsoluteIDQ p400 HR kit (Biocrates, Innsbruck, Austria), which is a combined flow injection and high-performance liquid chromatography–tandem mass spectrometry assay. The tandem mass spectrometry platform consisted of a Thermo Scientific Vanquish HPLC coupled to the Thermo Scientific Q-Exactive HF Quadrupole-Obitrap mass spectrometer.
