Blood and urine analyses
BLOOD
Plasma concentrations medium chain fatty acids, lactate, and beta-hydroxybutyrate, whole blood acetoacetate pyruvate, and urinary beta-hydroxybutyrate were determined by gas chromatography/mass spectrometryWhole blood (2 mls) was collected into Lithium Heparin tubes. The remaining whole blood was centrifuged at 2500 g for 5 min and supernatant stored at −80 °C prior to UHPLC-MS metabolomics analysis. Blood samples (1.5 mls, in EDTA tubes) and urine samples (in universal tubes) were centrifuged at 2500 g for 5 min and the protein-free supernatant removed and stored at −80 °C, before analysis for plasma medium chain fatty acids, pyruvate, lactate and beta-hydroxybutyrate, and urinary beta-hydroxybutyrate, respectively, by gas chromatography/mass spectrometryFor acetoacetate, which is not stable in plasma, 1.5 mls of whole blood was collected at the recruiting site and added to a pre-weighed plastic tube containing 5 ml of 0.77 mol/l perchloric acid previously cooled to 0 °C. After mixing, the tubes were re-weighed, centrifuged at 2500 g for 5 min and the protein-free supernatant was removed and stored. The dilution of the blood was determined by the weight changes measured. Samples were stored at -80 °C until analysis. 100ul supernatant plus 20ul internal standard mix (100uM 