Global metabolomics and lipidomics analysis

Serum samples were analyzed at Metabolon Inc. (Morrisville, NC, USA). Global metabolomics was performed as previously described^23–26. Briefly, samples were extracted by agitation with methanol. Extracts were then split to enable analysis by four different methods. All methods used a Waters Acquity ultra-performance liquid chromatography (UPLC) system coupled to a Thermo Scientific Q-Exactive high resolution accurate-mass spectrometer. Raw data extraction, peak identification, and quality control processing were carried out using the Metabolon proprietary software. Metabolite identification was done through comparison with a library of chromatographic and MS data from authenticated standards. Metabolite abundances were determined by their area under the curve (AUC). Lipidomics was done as previously described²⁷. Briefly, lipids were extracted from serum samples using a butanol:methanol (BUME) mixture (3:1) followed by two-phase extraction into heptane:ethyl acetate (3:1) using 1% acetic acid as buffer. Reconstituted extracts were infused into a SelexION equipped Sciex 5500 QTRAP. The scan was performed in multiple reaction monitoring (MRM) mode. Individual lipid species were quantified by referencing to known concentration of internal standard. Lipid class concentrations were calculated from the sum of all molecular species within a class, and fatty acid compositions were determined by calculating the proportion of each class comprised by individual fatty acids. Further experimental details and the analysis process are described in Supplementary Methods.