LC-MS analysis from whole blood and urine
Sample analysis was carried out by MS-Omics (Vedbæk, Denmark).Blood sample extraction was carried out by combining 100 µl whole blood with 900 µl ice-cold extraction solvent (Methanol/Acetonitrile/MQW – 10:6:4 fortified with stable isotope internal standards (Supplementary Data Urine sample extraction was carried out by adding 50 μl of urine into a SpinX filter and mixing it with 50 μL eluent B (10 mM ammonium acetate in 90 % acetonitrile). The filters were centrifugated at 3000 × Samples were handled during the whole extraction procedure at 4 °C, and sample analysis was started immediately after sample preparation without any intermediate storage. To ensure high-quality sample preparation, a quality control sample (QC sample) was prepared by pooling small equal aliquots from each sample for all approaches, to create a representative average of the entire set. This sample was treated and analyzed at regular intervals throughout the sequence.Polar metabolite profiling of whole blood and urine was performed using a Thermo Scientific Vanquish LC coupled to Thermo Q Exactive HF MS. An electrospray ionization interface was used as ionization source. Analysis was performed in negative and positive ionization mode. The UPLC was performed as described by Hsiao et al. Semi-polar metabolite analysis of whole blood was carried out using a Thermo Scientific Vanquish LC coupled to Orbitrap Exploris 240 MS, Thermo Fisher Scientific. An electrospray ionization interface was used as ionization source. Analysis was performed in positive and negative ionization mode under polarity switching. The UPLC was performed using a the protocol described by Doneanu et al. For urine samples only polar extraction was performed. For blood samples the applied extraction method is indicated for each reported metabolite in Supplementary Data Identification of compounds for both methods was performed at four levels; Level 1: identification by retention times (compared against in-house authentic standards), accurate mass (with an accepted deviation of 3 ppm), and MS/MS spectra; Level 2a: identification by retention times (compared against in-house authentic standards), accurate mass (with an accepted deviation of 3 ppm). Level 2b: identification by accurate mass (with an accepted deviation of 3 ppm), and MS/MS spectra, Level 3: identification by accurate mass alone (with an accepted deviation of 3 ppm). All standard metabolites used here and the annotation level of the reported metabolites are indicated in Supplementary Data 