Plasma metabolome analysis
COLD
Before and after completion of cold exposure blood was sampled into serum gel tubes. After 30 min samples were centrifuged at 4 °C. Serum was separated and frozen at −80 °C until analysis.Metabolite extraction was performed on the Hamilton STAR M liquid handling robot with the following protocol. In brief, 10 μl of the sample was aliquoted into 1.2 ml 96-deep well plates. Afterward, 300 ul of cold extraction solvent Methanol: H2O, (4:1 v:v) was added. After brief vortexing on a plate shaker, the samples were kept at −20 °C for 2 h. The samples were then centrifuged at 4000 g for 20 min. After centrifugation, the supernatant was transferred to another deep-well plate, and dried under NFlow injection mass spectrometry was performed as described previously.Raw mass spectrometric data was converted to an open source format (mz5) and then processed using an in-house data processing pipeline (FiaMiner) in Matlab. The m/z axis for the whole dataset was recalibrated using expected masses. Afterwards, annotation was performed based on m/z, with mass accuracy of 0.001 Da matching to HMDB. Differential analysis statistics were performed in Matlab for pairwise comparisons followed by correction of multiple comparisons. Pathways enrichment analysis was performed in Matlab using the HMDB pathway definition v3.