Cobas e11

Glucose (C311 autoanalyzer, Roche, Mannheim, Germany) and insulin (cobas e11, Roche, Mannheim, Germany) were measured in plasma and the homeostatic model assessment (HOMA) was used to estimate insulin resistance (HOMA2-IR2; The Oxford Centre for Diabetes, Endocrinology and Metabolism, UK) [48 (link)]. As described previously [14 (link)], MOTS-c was extracted from plasma in acetonitrile-hydrochloric acid and measured using an in-house sandwich ELISA developed at the University of Southern California by the laboratory that first described MOTS-c in human and murine plasma. This ELISA has been extensively validated, and peptide blocking experiments have been undertaken to show specificity of the antibody [14 (link)].

At each visit a blood sample was obtained after an overnight fast. We measured parathyroid hormone (PTH) (Immunolite 2000, Siemens), follicle stimulation hormone (FSH), luteinizing hormone (LH) (both AutoDelfia, Perkin Elmer) and 25-hydroxy vitamin D (Cobas e11, Roche Diagnostics), and the coefficient of variation (CV) for these analyses ranged from 4 to 6% while measurements of procollagen type I amino-terminal propeptide (P1NP) and C-terminal telopeptide of type 1 collagen (CTX) (iSYS, Immunodiagnostic Systems, Tyne and Wear, UK) had CV’s ranging from 8 to 10%. CTX and P1NP from all visits were analysed in one batch.