Multiplex luminometry

All measurements were made in duplicate using commercially available kits and manufacturer's guidelines. Serum interleukin- (IL-) 1β, IL-4, IL-6, IL-8, IL-10, IL-13, IL-17, granulocyte/macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)α were simultaneously measured by multiplex luminometry (Bio-Rad, Hemel Hempstead, UK). Samples we0re analysed using a Bio-Plex Luminex²⁰⁰ platform equipped with a 635 nm red and 532 nm green laser using Bio-Plex Manager™ software. Detection of C-reactive protein (CRP) was by high-sensitivity enzyme-linked immunosorbent assay (ELISA) using a commercial kit (IBL International, Hamburg, Germany). Plasma cortisol and DHEAs were assessed separately by ELISA (IBL International, Hamburg, Germany) and plasma adiponectin and leptin were assessed separately by solid phase sandwich ELISA (R&D Systems, Abingdon, UK).

All measurements were made in duplicate using commercially available kits and manufacturers guidelines. Serum IL-1β, IL-4, IL-6, IL-8, IL-10, IL-13, IL-17, GMC-SF, TNFα and MCP-1 were simultaneously measured by multiplex luminometry (Bio-Rad, Hemel Hempstead, UK). Samples were analysed using a Bio-Plex Luminex²⁰⁰ platform equipped with a 635 nm red and 532 nm green laser using Bio-Plex Manager™ software. Detection of C-reactive protein (CRP) was by high sensitivity ELISA using a commercial kit (IBL International, Hamburg, Germany). Plasma adiponectin and leptin were assessed separately by solid phase sandwich ELISA (R&D Systems, Abingdon, UK). Plasma insulin levels were determined by sandwich ELISA (Life Technologies, Paisley, UK). Plasma glucose, cholesterol and triglycerides (Instrumentation Laboratories, Warrington, UK) were determined photometrically on an automated chemistry analyser (iLab 650, Instrumentation Laboratories, Warrington, UK).