Plasma Biomarkers of Inflammation and Metabolism

As previously reported^{44 ,92 (link)}, venous blood samples were drawn into 9 ml heparin coated plasma tubes after 12 h overnight fasting and were centrifuged for 10 min at 2000 × g within 1 h from blood collection. Plasma samples were then transferred into 0.5 ml polypropylene microtubes and stored in a freezer at − 80 °C. Circulating levels of high-sensitivity C-Reactive Protein (CRP) in mg/dL were assayed using a human CRP Quantikine enzyme-linked immunosorbent assay (ELISA) kit (R & D Systems, Minneapolis, USA). Six individuals had a CRP value > 10 mg/ml indicative of acute infection and were, therefore, excluded from the statistical analyses testing for moderating effects of inflammation. Leptin concentrations in pg/ml were determined with the DRP300 Quantikine ELISA kit (R & D Systems) and adiponectin in ng/ml with the human total adiponectin/Acrp30 Quantitkine ELISA kit (R & D Systems). Leptin/adiponectin ratios for each participant were calculated. Interleukin IL-8 levels in pg/mL were determined using a high sensitivity CXCL8/ INTERLEUKIN-8 Quantikine ELISA kit (R & D Systems). Determination of interleukin-1β, interleukin-6 and Tumor Necrosis Factor α (TNFα) levels were trialled with high-sensitivity Quantikine ELISA kits but did not result in reliable measurements consistently above the level of detection for each assay.