Glucose oxidase kit

Blood samples were centrifuged and aliquots of plasma and serum were stored at -80 O C until analysis. Plasma glucose (glucose oxidase kit; Instrumentation Laboratory, Monza, Italy, Interassay CV: 4.9%, Intra assay CV: 2.3%) and plasma lactate (Lactate kit, Randox, County Antrim, UK, Inter CV: 4.5%, Intra CV: 2.7%) concentrations were analysed by spectrophotometry (iLab 300 plus, iLab, UK). Serum insulin was analysed using a chemoilumino-metric immunoassay (ADIVA Centaur, Bayer diagnostics, Berkshire, UK, Inter CV: 3.2-4.6%, Intra CV: 2.6-5.9%). Serum FFA concentration was analysed by an acyl-CoA synthetase and oxidase assay (NEFA-HR2, Wako Chemicals GmbH, Germany, Inter assay CV: 1.5%). Isotope ratio mass spectrometry (IRMS; AP2003, GVI Instruments Ltd, Manchester, UK) were used to determine the 13 CO2: 12 CO2 in expired air as described previously (36) . The 13 C: 12 C in plasma glucose was determined using liquid chromatography linked-isotope ratio mass spectrometry (LC-IRMS), as previously described (35) . Briefly, plasma samples were spiked with an internal standard (L-fucose, Sigma Aldrich, Poole, UK) and prepared by ultrafiltration (30000 MWCO, Amicon Ultra 4, Millipore, Watford, UK) for LC-IRMS analysis of 13 C-glucose enrichment (d 13 C-glucose).