Biomarker Analysis of Blood Samples

Blood samples were collected into serum- and plasma-separator tubes at the specified time points, centrifuged for 15 min at 4000 rpm and 4 °C, aliquoted into 1 mL microcentrifuge tubes, and stored at −80 °C as previously described [13 (link)]. Serum samples were analyzed for glucose by photometric assay (COBAS Integra 400 Analyzer; Roche Diagnostic Systems, Indianapolis, IN, USA). Insulin was assayed using an electroluminescence immunoassay on a COBAS e411 analyzer (Roche Diagnostic Systems, Indianapolis, IN, USA). Plasma amino acid (AA) concentrations were determined using High-Performance Liquid Chromatography (Agilent 1260 Infinity High-Performance Autosampler, Agilent Technologies, Santa Clara, CA, USA) with the Agilent Amino Acid Protocol [14 ]. Twenty AAs were reported (Ala, Arg, Apn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val).