Supplementary material Gut Supplemental laboratory methods The samples were sent to Metabolon, Inc. (Durham, NC, USA) on dry ice with the ATBC samples sent prior to that of the PLCO samples. The serum samples were assayed using untargeted ultrahigh performance liquid chromatography-tandem mass spectrometry and/or gas chromatography mass spectrometry. Metabolites were measured using either the Orbitrap Elite or Q-Exactive platforms. Peaks were identified via linkage to Metabolon’s known chemical reference library. Ultrahigh performance liquid chromatography/Mass Spectroscopy (UPLC/MS/MS). The LC/MS portion of the platform was based on a Waters ACQUITY ultra-performance liquid chromatography (UPLC) and a ThermoFisher Scientific Orbitrap Elite or Q-Exactive high resolution/accurate mass spectrometer, which consisted of a heated electrospray ionization (HESI) source and orbitrap mass analyzer operated at 30,000 or 35,000 mass resolution, respectively. The sample extract was dried then reconstituted in acidic or basic LC-compatible solvents, each of which contained 8 or more injection standards at fixed concentrations to ensure injection and chromatographic consistency. Data was acquired over the course of 4 years; over which time Metabolon’s methodology has evolved. For the first ATBC studies, one sample aliquot was acquired using acidic positive ion optimized conditions and another using basic negative ion optimized conditions in two independent injects over separate dedicated columns. Subsequent studies (i.e. PLCO) increased the number of chromatographic columns as methodology advanced. Details in methodology can be found in Evans et al, 2009, Evans et al 2014, and Long et al, 2017 1, 2, 3. Gas chromatography/Mass Spectroscopy (GC/MS). The ATBC samples but not PLCO had GC/MS analysis. The samples were re-dried under vacuum desiccation for a minimum of 18 hours prior to being derivatized under dried nitrogen using bistrimethyl-silyl-triflouroacetamide (BSTFA). The GC column was 5% phenyl and the temperature ramp was from 40° to 300° C in a 16 minute period. Samples were analyzed on a Thermo-Finnigan Trace DSQ fast-scanning single-quadrupole mass spectrometer using electron impact ionization. Data Extraction and Compound Identification: Raw data was extracted, peaks identified and QC processed using Metabolon’s hardware and software. Compounds were identified by comparison to library entries of purified standards or recurrent unknown entities. Metabolon maintains a library based on authenticated standards that contains the retention time/index (RI), mass to charge ratio (m/z), and chromatographic data (including MS/MS spectral data) on all molecules present in the library. Biochemical identifications are based on three criteria: retention index within a narrow RI window of the proposed identification, accurate mass match to the library +/- 0.005 amu, and the MS/MS forward and reverse scores between the experimental data and authentic standards. The MS/MS scores are based on a comparison of the ions present in the experimental spectrum to the ions present in the library spectrum. While there may be similarities between these molecules based on one of these factors, the use of all three data points can be utilized to distinguish and differentiate biochemicals. More than 3300 commercially available purified standard compounds have been acquired and registered into LIMS for distribution to both the LC-MS and GC-MS platforms for determination of their analytical characteristics. Additional mass spectral entries have been created for structurally unnamed biochemicals, which have been identified by virtue of their recurrent nature (both Stolzenberg-Solomon R, et al. Gut 2020;0:1–8. doi: 10.1136/gutjnl-2019-319811 Supplementary material Gut chromatographic and mass spectral). These compounds have the potential to be identified by future acquisition of a matching purified standard or by classical structural analysis. Curation: A variety of curation procedures were carried out to ensure that a high quality data set was made available for statistical analysis and data interpretation. The QC and curation processes were designed to ensure accurate and consistent identification of true chemical entities, and to remove those representing system artifacts, mis-assignments, and background noise. Metabolon data analysts use proprietary visualization and interpretation software to confirm the consistency of peak identification among the various samples. Library matches for each compound were checked for each sample and corrected if necessary. 1 Evans AM, DeHaven CD, Barrett T, et al. Integrated, nontargeted ultrahigh performance liquid chromatography/electrospray ionization tandem mass spectrometry platform for the identification and relative quantification of the small-molecule complement of biological systems. Anal Chem 2009;81:6656-67. 2 Evans AM, Bridgewater BR, Liu Q, et al. High resolution mass spectrometry improves data quantity and quality as compared to unit mass resolution mass spectrometry in high-throughput profiling metabolomics Metabolomics 2014;4:7. 3 Long T, Hicks M, Yu HC, et al. Whole-genome sequencing identifies common-to-rare variants associated with human blood metabolites. Nature genetics 2017;49:568-78. Stolzenberg-Solomon R, et al. Gut 2020;0:1–8. doi: 10.1136/gutjnl-2019-319811 Supplementary material Gut Supplementary methods: pathway groups for pathway analysis 1. Alanine and aspartate metabolism group Alanine and aspartate metabolism Alanine and aspartate metabolism; pyrimidine metabolism, uracil containing 2. Benzoate metabolism Benzoate metabolism; phenylalanine & tyrosine metabolism Benzoate metabolism Benzoate metabolism; bacterial 3. Butanoate metabolism; cysteine, methionine, SAM, taurine metabolism group Butanoate metabolism; cysteine, methionine, SAM, taurine metabolism Cysteine, methionine, SAM, taurine metabolism Cysteine, methionine, SAM, taurine metabolism; endocannabinoid 4. Bile acids group Secondary bile acid metabolism Primary bile acid metabolism 5. Carnitine metabolism group Carnitine metabolism Carnitine metabolism; fatty acid synthesis Acetylated peptides 6. Food component/plant group Chemical; food component/plant Food component/plant Food component/plant; krebs cycle, dicarboxylic acid Glycolysis, gluconeogenesis, pyruvate metabolism; food component/plant Pentose metabolism; food component/plant Sugar, sugar substitute, starch; food component/plant Tryptophan metabolism; food component/plant Valine, leucine and isoleucine metabolism; food component/plant 7. Chemical group Chemical Chemical; food component/plant Chemical; ketone bodies Detoxification metabolism; chemical Valine, leucine and isoleucine metabolism; chemical 8. Dipeptide group/polypeptide Dipeptide Dipeptide derivative Dipeptide derivative; glutathione metabolism Dipeptide; urea cycle; arginine and proline metabolism Polypeptide 9. Drug Drug Stolzenberg-Solomon R, et al. Gut 2020;0:1–8. doi: 10.1136/gutjnl-2019-319811 Supplementary material Gut 10. Polyunstaturated (n3 and n6) Essential fatty acid; polyunsaturated fatty acid (n3 and n6) Long chain fatty acid; polyunsaturated fatty acid (n3 and n6) Polyunsaturated fatty acid (n3 and n6) 11. Fatty acid metabolism Fatty acid, dicarboxylate; lysine metabolism Fatty acid metabolism (also BCAA metabolism) Fatty acid metabolism; carnitine metabolism Fatty acid metabolism; valine, leucine and isoleucine metabolism Fatty acid, amino Fatty acid, branched Fatty acid, methyl ester Fatty acid, monohydroxy Fatty acid metabolism (acyl choline) Fatty acid metabolism (acyl glutamine) Fatty acid, dicarboxylate 12. Fibrinogen cleavage peptide Fibrinogen cleavage peptide 13. Gamma-glutamyl amino acid metabolism group Gamma-glutamyl amino acid 14. Glutamate metabolism Glutamate metabolism 15. Glutathione metabolism Glutathione metabolism Dipeptide derivative; glutathione metabolism 16. Glycerolipid metabolism group Glycerolipid metabolism Glycerolipid metabolism; phospholipid metabolism 17. Glycine, serine and threonine metabolism group Glycine, serine and threonine metabolism 18. Glycolysis, gluconeogenesis, pyruvate metabolism group Glycolysis, gluconeogenesis, pyruvate metabolism Glycolysis, gluconeogenesis, pyruvate metabolism; food component/plant 19. Hemoglobin and porphyrin metabolism Hemoglobin and porphyrin metabolism 20. Histidine metabolism Histidine metabolism 21. Krebs cycle / TCA cycle Krebs cycle / TCA cycle 22. Long chain fatty acid group Long chain fatty acid 23. Lysine metabolism Lysine metabolism Stolzenberg-Solomon R, et al. Gut 2020;0:1–8. doi: 10.1136/gutjnl-2019-319811 Supplementary material Gut 24. Lysolipid group Lysolipid; lysoplasmalogen Lysophospholipid Lysolipid Glycerophosphodiester /lysolipid 25. Medium and short chain fatty acid group Medium chain fatty acid Medium chain fatty acid; fatty acid, amino Short chain fatty acid 26. Mono and diacylglycerol group Monoacylglycerol Diacylglycerol 27. Pentose metabolism group Pentose metabolism Pentose metabolism; food component/plant 28. Phenylalanine & tyrosine metabolism Tyrosine metabolism Phenylalanine & tyrosine metabolism Phenylalanine metabolism Tyrosine metabolism; phenylalanine & tyrosine metabolism Phenylalanine & tyrosine metabolism; acetylated Phenylalanine & tyrosine metabolism; acetylated peptides Phenylalanine metabolism; phenylalanine & tyrosine metabolism 29. Polyamine metabolism group Polyamine metabolism Guanidino and acetamido metabolism Guanidino and acetamido metabolism; polyamine metabolism 30. Purine metabolism