Cobas mira analyser

Blood was collected into BD Vacutainer Plus plasma tubes (catalogue number 367960, BD Biosciences) by cardiac puncture and centrifuged at 1,300g for 10 min at 4 °C, to collect the plasma. The plasma cytokine levels were measured using their respective ELISA kits (MEM-004A, Multi-Analyte ELISArray kit, Qiagen). The total cholesterol, HDL, LDL and TGs levels in the plasma were measured using Roche Diagnostics COBAS MIRA analyser using the manufacturer's kits.

Serum insulin was measured by an AutoDelfia fluoroimmunometric assay (PerkinElmer, Waltham, MA, USA). Fasting plasma glucose was analysed using the HemoCue Glucose System (HemoCue, Ängelholm, Sweden). Serum total cholesterol, HDL-cholesterol and triacylglycerol concentrations were measured first on a Cobas Mira analyser (Hoffman-La Roche, Basel, Switzerland) and LDL-cholesterol concentrations were calculated using the Friedewald formula. Since January 2006, total cholesterol, LDL-cholesterol, HDL-cholesterol and triacylglycerol concentrations have been measured using an enzymatic method (Konelab 60i analyser; Thermo Electron Oy, Vantaa, Finland).

Serum total cholesterol and triacylglycerols were determined enzymatically on a Cobas MIRA analyser (Roche Diagnostics, Basel, Switzerland) with reagents from Trace Scientific (Melbourne, Australia). High density lipoprotein-cholesterol (HDL-C) was determined on a heparin-manganese supernatant [9 (link)]. The intra-assay coefficients of variation were 2.2% at 4.2 mM and 1.4% at 10.5 mM for total cholesterol, 1.6% at 4.0 mM and 2.5% at 1.2 mM for triacylglycerols, and 1.9% at 1.1 mM for HDL-C. In each case all samples were measured in a single assay. Serum levels of a range of fatty acids were analysed by gas chromatography as previously described [10 (link)]. Briefly, serum (200 μL) was extracted with 2 ml chloroform/methanol (2:1, v/v). Fatty acid methyl esters were prepared by treatment of total lipid extracts with 4% H₂SO₄ in methanol at 90°C for 20 min and analysed by gas liquid chromatography using a Hewlett-Packard model 5980A gas chromatograph (Hewlett Packard, Rockville, MD). The samples were resolved on a BPX70 column (25 cm × 0.32 mm, 0.25 μm film thickness; SGE, Ringwood, Victoria, Australia) with a temperature program increasing from 150°C to 210°C at 4°C/min and using N₂ as the carrier gas at a split ratio of 30:1. Peaks were identified by comparison with a known standard mixture.