Quantikine human total adiponectin acrp30 immunoassay

Whole-blood Se and plasma glutathione peroxidase 3 (GPx3) activity at 12 and 35 weeks were determined by inductively coupled plasma MS and a spectrophotometric assay, respectively, as previously described (17, 28) . The CV for the whole-blood Se assay was 0•25 % at 1•4 mmol/l and 0•17 % at 3•0 mmol/l (28) . For GPx3, the inter-assay variation was 0•43 % and the intra-assay variation was 1•25 % (29) .
Toenail Se at 16 weeks was measured using instrumental neutron activation analysis at the Interfaculty Reactor Institute in Delft, as previously described (17, 30) . The laboratory has an embedded quality-control system for quality assurance and management, which complies with the requirements of the International Standard ISO/IEC 17025:2005 and has been accredited by the Dutch Council for Accreditation since 1993.
Plasma SEPP1 concentration at 35 weeks was measured by ELISA in the laboratory of Raymond Burk, as previously described (17, 30) . For quality control, each ELISA was run with two standards: purified SEPP1 and a standard plasma sample (31) .
Total plasma adiponectin concentration at 12 and 35 weeks was measured using a commercial ELISA kit (Quantikine ® , Human Total Adiponectin/Acrp30 Immunoassay; R&D Systems Europe Ltd) according to the manufacturer's instructions (http://www.rndsystems.com/pdf/drp300.pdf). The intra-and inter-assay CV were 2•5 and 6•8 %, respectively.

Body weight and height were measured using a Wedderburn Chair Scale and a Holtain Stadiometer, respectively. Body mass index (BMI) was calculated as weight (kg)/height 2 (m). Waist circumference was measured at the umbilicus level with a tape measure (to the nearest 0.1 cm). Information on smoking was obtained from questionnaires at 17-and 20-years. Resting systolic and diastolic BP were measured after 5 mins supine rest using an oscillometric sphygmomanometer (DINAMAP 8100, DINAMAP XL or DINAMAP ProCare 100 vital signs monitors; GE Healthcare, USA).
Six readings were recorded, each two minutes apart, with the last five readings averaged.
Fasting bloods were analysed for serum insulin, glucose, triglycerides, total cholesterol, HDL-cholesterol, and high-sensitivity C-reactive protein (hsCRP) (PathWest Laboratory, Royal Perth Hospital). LDL-cholesterol was calculated using the Friedewald equation. Insulin resistance was estimated using HOMA-IR calculated as fasting insulin [µU/ml] x fasting glucose [mmol/L]/22.5). Serum leptin was measured by the ACTIVE® Human Leptin ELISA kit (DSL-10-23100, Diagnostic Systems Laboratories, Webster, TX, USA) and adiponectin by the Quantikine® Human Total Adiponectin/Acrp30 Immunoassay (R&D Systems, Minneapolis, MN, USA).