#METABOLOMICS WORKBENCH mcglinchey_aidan_20230217_054653 DATATRACK_ID:3748 STUDY_ID:ST002480 ANALYSIS_ID:AN004050 PROJECT_ID:PR001602 VERSION 1 CREATED_ON February 17, 2023, 7:26 am #PROJECT PR:PROJECT_TITLE Impact of in-utero exposures to per- and polyfluoroalkyl substances on the human PR:PROJECT_TITLE fetal liver metabolome PR:PROJECT_SUMMARY Background Per- and polyfluoroalkyl substances (PFAS) are classed as Endocrine PR:PROJECT_SUMMARY Disrupting Compounds (EDCs) but continue to be used in many products. This PR:PROJECT_SUMMARY includes firefighting foams, flame retardants, utensil coatings and PR:PROJECT_SUMMARY waterproofing of food packaging. PFAS exposure aberrantly modulates lipid, PR:PROJECT_SUMMARY metabolite and bile acid (BA) levels, increasing susceptibility to onset and PR:PROJECT_SUMMARY severity of metabolic diseases, such as diabetes and non-alcoholic fatty liver PR:PROJECT_SUMMARY disease (NAFLD). To date, most studies in humans have focused on PFAS-exposure PR:PROJECT_SUMMARY effects in adults. In this study we now demonstrate that PFAS are present in the PR:PROJECT_SUMMARY human fetal liver and that they have metabolic consequences for the human fetus. PR:PROJECT_SUMMARY Methods Human fetal livers from elective termination of pregnancies between PR:PROJECT_SUMMARY 11-19 weeks of gestation (n = 78) were analysed by both targeted and untargeted PR:PROJECT_SUMMARY metabolomic analyses of lipids, polar metabolites, BAs and PFAS. Stringent PR:PROJECT_SUMMARY bioinformatic and statistical methods were applied to this data to generate a PR:PROJECT_SUMMARY network of interacting molecules. Findings Metabolites associated with PFAS were PR:PROJECT_SUMMARY identified in the fetal liver and these varied with gestational age . Conjugated PR:PROJECT_SUMMARY BAs were markedly positively associated with fetal age. Several amino acids, PR:PROJECT_SUMMARY fatty acids and sugar derivatives in fetal livers were inversely associated with PR:PROJECT_SUMMARY PFAS exposure, while the BA glycolithocholic acid (GLCA) was markedly positively PR:PROJECT_SUMMARY associated with all quantified PFAS. Furthermore, 7α-hydroxy-4-cholesten-3-one PR:PROJECT_SUMMARY (C4), a marker of BA synthesis rate, was strongly positively associated with PR:PROJECT_SUMMARY PFAS levels and was detectable as early as gestational week 12. Interpretation PR:PROJECT_SUMMARY The data show direct evidence for in-utero effects of PFAS exposure on specific PR:PROJECT_SUMMARY key hepatic products. Our results provide evidence that PFAS exposure, with PR:PROJECT_SUMMARY potential future consequences, manifests in the human fetus as early as the PR:PROJECT_SUMMARY first trimester of gestation. Furthermore, the profiles of metabolic changes PR:PROJECT_SUMMARY resemble those observed in perinatal PFAS exposures. Such exposures are already PR:PROJECT_SUMMARY linked with susceptibility, initiation, progression and/or exacerbation of a PR:PROJECT_SUMMARY wide range of metabolic diseases. PR:INSTITUTE Örebro University PR:LAST_NAME McGlinchey PR:FIRST_NAME Aidan PR:ADDRESS School of Medical Sciences, Örebro, Örebro, 70281, Sweden PR:EMAIL aidan.mcglinchey@oru.se PR:PHONE +46736485638 #STUDY ST:STUDY_TITLE Impact of in-utero exposures to per- and polyfluoroalkyl substances on the human ST:STUDY_TITLE fetal liver metabolome ST:STUDY_SUMMARY Background Per- and polyfluoroalkyl substances (PFAS) are classed as Endocrine ST:STUDY_SUMMARY Disrupting Compounds (EDCs) but continue to be used in many products. This ST:STUDY_SUMMARY includes firefighting foams, flame retardants, utensil coatings and ST:STUDY_SUMMARY waterproofing of food packaging. PFAS exposure aberrantly modulates lipid, ST:STUDY_SUMMARY metabolite and bile acid (BA) levels, increasing susceptibility to onset and ST:STUDY_SUMMARY severity of metabolic diseases, such as diabetes and non-alcoholic fatty liver ST:STUDY_SUMMARY disease (NAFLD). To date, most studies in humans have focused on PFAS-exposure ST:STUDY_SUMMARY effects in adults. In this study we now demonstrate that PFAS are present in the ST:STUDY_SUMMARY human fetal liver and that they have metabolic consequences for the human fetus. ST:STUDY_SUMMARY Methods Human fetal livers from elective termination of pregnancies between ST:STUDY_SUMMARY 11-19 weeks of gestation (n = 78) were analysed by both targeted and untargeted ST:STUDY_SUMMARY metabolomic analyses of lipids, polar metabolites, BAs and PFAS. Stringent ST:STUDY_SUMMARY bioinformatic and statistical methods were applied to this data to generate a ST:STUDY_SUMMARY network of interacting molecules. Findings Metabolites associated with PFAS were ST:STUDY_SUMMARY identified in the fetal liver and these varied with gestational age . Conjugated ST:STUDY_SUMMARY BAs were markedly positively associated with fetal age. Several amino acids, ST:STUDY_SUMMARY fatty acids and sugar derivatives in fetal livers were inversely associated with ST:STUDY_SUMMARY PFAS exposure, while the BA glycolithocholic acid (GLCA) was markedly positively ST:STUDY_SUMMARY associated with all quantified PFAS. Furthermore, 7α-hydroxy-4-cholesten-3-one ST:STUDY_SUMMARY (C4), a marker of BA synthesis rate, was strongly positively associated with ST:STUDY_SUMMARY PFAS levels and was detectable as early as gestational week 12. Interpretation ST:STUDY_SUMMARY The data show direct evidence for in-utero effects of PFAS exposure on specific ST:STUDY_SUMMARY key hepatic products. Our results provide evidence that PFAS exposure, with ST:STUDY_SUMMARY potential future consequences, manifests in the human fetus as early as the ST:STUDY_SUMMARY first trimester of gestation. Furthermore, the profiles of metabolic changes ST:STUDY_SUMMARY resemble those observed in perinatal PFAS exposures. Such exposures are already ST:STUDY_SUMMARY linked with susceptibility, initiation, progression and/or exacerbation of a ST:STUDY_SUMMARY wide range of metabolic diseases. ST:INSTITUTE Örebro University ST:LAST_NAME McGlinchey ST:FIRST_NAME Aidan ST:ADDRESS School of Medical Sciences, Örebro, Örebro, 70281, Sweden ST:EMAIL aidan.mcglinchey@oru.se ST:PHONE +46736485638 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male and female #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - Sample.1 AGE:19 | SEX:F BMI=30.078125; RAW_FILE_NAME=0059_LC_20190913_Sample_0001.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.2 AGE:12 | SEX:F BMI=21.63114885; RAW_FILE_NAME=0059_LC_20190913_Sample_0002.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.3 AGE:14 | SEX:F BMI=24.69040546; RAW_FILE_NAME=0059_LC_20190913_Sample_0003.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.4 AGE:19 | SEX:M BMI=17.78970694; RAW_FILE_NAME=0059_LC_20190913_Sample_0004.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.5 AGE:18 | SEX:F BMI=22.40878677; RAW_FILE_NAME=0059_LC_20190913_Sample_0005.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.6 AGE:14 | SEX:F BMI=20.90288866; RAW_FILE_NAME=0059_LC_20190913_Sample_0006.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.7 AGE:16 | SEX:F BMI=25.92866535; RAW_FILE_NAME=0059_LC_20190913_Sample_0007.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.8 AGE:19 | SEX:F BMI=21.09619051; RAW_FILE_NAME=0059_LC_20190913_Sample_0008.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.9 AGE:12 | SEX:F BMI=26.87958738; RAW_FILE_NAME=0059_LC_20190913_Sample_0009.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.10 AGE:17 | SEX:M BMI=20.32443826; RAW_FILE_NAME=0059_LC_20190913_Sample_0010.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.11 AGE:15 | SEX:F BMI=28.76397107; RAW_FILE_NAME=0059_LC_20190913_Sample_0011.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.12 AGE:17 | SEX:F BMI=22.30814991; RAW_FILE_NAME=0059_LC_20190913_Sample_0012.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.13 AGE:12 | SEX:M BMI=24.31412397; RAW_FILE_NAME=0059_LC_20190913_Sample_0013.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.14 AGE:14 | SEX:M BMI=21.56552602; RAW_FILE_NAME=0059_LC_20190913_Sample_0014.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.15 AGE:13 | SEX:F BMI=20.31221304; RAW_FILE_NAME=0059_LC_20190913_Sample_0015.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.16 AGE:15 | SEX:F BMI=25.31545429; RAW_FILE_NAME=0059_LC_20190913_Sample_0016.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.17 AGE:19 | SEX:M BMI=25.59833507; RAW_FILE_NAME=0059_LC_20190913_Sample_0017.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.18 AGE:13 | SEX:M BMI=21.01186848; RAW_FILE_NAME=0059_LC_20190913_Sample_0018.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.19 AGE:19 | SEX:F BMI=24.76756592; RAW_FILE_NAME=0059_LC_20190913_Sample_0019.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.20 AGE:17 | SEX:F BMI=26.7802994; RAW_FILE_NAME=0059_LC_20190913_Sample_0020.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.21 AGE:14 | SEX:F BMI=24.9107674; RAW_FILE_NAME=0059_LC_20190913_Sample_0021.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.22 AGE:17 | SEX:M BMI=24.38237298; RAW_FILE_NAME=0059_LC_20190913_Sample_0022.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.23 AGE:14 | SEX:M BMI=24.7955; RAW_FILE_NAME=0059_LC_20190913_Sample_0023.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.24 AGE:18 | SEX:M BMI=20.68747281; RAW_FILE_NAME=0059_LC_20190913_Sample_0024.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.25 AGE:14 | SEX:M BMI=23.87511478; RAW_FILE_NAME=0059_LC_20190913_Sample_0025.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.26 AGE:13 | SEX:F BMI=30.77409913; RAW_FILE_NAME=0059_LC_20190913_Sample_0026.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.27 AGE:17 | SEX:M BMI=28.28125; RAW_FILE_NAME=0059_LC_20190913_Sample_0027.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.28 AGE:15 | SEX:F BMI=23.14049587; RAW_FILE_NAME=0059_LC_20190913_Sample_0028.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.29 AGE:13 | SEX:F BMI=21.04402142; RAW_FILE_NAME=0059_LC_20190913_Sample_0029.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.30 AGE:17 | SEX:F BMI=18.90203813; RAW_FILE_NAME=0059_LC_20190913_Sample_0030.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.31 AGE:14 | SEX:F BMI=21.07719523; RAW_FILE_NAME=0059_LC_20190913_Sample_0031.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.32 AGE:14 | SEX:F BMI=31.24876389; RAW_FILE_NAME=0059_LC_20190913_Sample_0032.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.33 AGE:14 | SEX:M BMI=21.1552942; RAW_FILE_NAME=0059_LC_20190913_Sample_0033.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.34 AGE:13 | SEX:F BMI=23.69708214; RAW_FILE_NAME=0059_LC_20190913_Sample_0034.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.35 AGE:14 | SEX:M BMI=25.76298058; RAW_FILE_NAME=0059_LC_20190913_Sample_0035.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.36 AGE:13 | SEX:F BMI=29.0687733; RAW_FILE_NAME=0059_LC_20190913_Sample_0036.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.37 AGE:12 | SEX:M BMI=21.77843524; RAW_FILE_NAME=0059_LC_20190913_Sample_0037.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.38 AGE:13 | SEX:M BMI=31.20917405; RAW_FILE_NAME=0059_LC_20190913_Sample_0038.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.39 AGE:14 | SEX:F BMI=23.03004535; RAW_FILE_NAME=0059_LC_20190913_Sample_0039.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.40 AGE:15 | SEX:M BMI=20.86111966; RAW_FILE_NAME=0059_LC_20190913_Sample_0040.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.41 AGE:16 | SEX:M BMI=28.66889244; RAW_FILE_NAME=0059_LC_20190913_Sample_0041.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.42 AGE:13 | SEX:F BMI=27.74498229; RAW_FILE_NAME=0059_LC_20190913_Sample_0042.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.43 AGE:17 | SEX:M BMI=31.99217133; RAW_FILE_NAME=0059_LC_20190913_Sample_0043.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.44 AGE:14 | SEX:F BMI=20.28479857; RAW_FILE_NAME=0059_LC_20190913_Sample_0044.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.45 AGE:12 | SEX:F BMI=19.83516296; RAW_FILE_NAME=0059_LC_20190913_Sample_0045.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.46 AGE:14 | SEX:M BMI=24.3902439; RAW_FILE_NAME=0059_LC_20190913_Sample_0046.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.47 AGE:17 | SEX:M BMI=22.60026298; RAW_FILE_NAME=0059_LC_20190913_Sample_0047.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.48 AGE:15 | SEX:M BMI=40.97529312; RAW_FILE_NAME=0059_LC_20190913_Sample_0048.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.49 AGE:16 | SEX:F BMI=22.85289116; RAW_FILE_NAME=0059_LC_20190913_Sample_0049.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.50 AGE:13 | SEX:F BMI=23.81496723; RAW_FILE_NAME=0059_LC_20190913_Sample_0050.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.51 AGE:13 | SEX:M BMI=25.3142397; RAW_FILE_NAME=0059_LC_20190913_Sample_0051.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.52 AGE:12 | SEX:M BMI=26.484375; RAW_FILE_NAME=0059_LC_20190913_Sample_0052.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.53 AGE:17 | SEX:F BMI=22.03856749; RAW_FILE_NAME=0059_LC_20190913_Sample_0053.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.54 AGE:12 | SEX:M BMI=20.76124567; RAW_FILE_NAME=0059_LC_20190913_Sample_0054.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.55 AGE:14 | SEX:M BMI=29.43213296; RAW_FILE_NAME=0059_LC_20190913_Sample_0055.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.56 AGE:12 | SEX:F BMI=28.53541543; RAW_FILE_NAME=0059_LC_20190913_Sample_0056.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.57 AGE:17 | SEX:M BMI=25.10239133; RAW_FILE_NAME=0059_LC_20190913_Sample_0057.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.58 AGE:14 | SEX:M BMI=30.69387755; RAW_FILE_NAME=0059_LC_20190913_Sample_0058.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.59 AGE:14 | SEX:M BMI=26.1748959; RAW_FILE_NAME=0059_LC_20190913_Sample_0059.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.60 AGE:12 | SEX:F BMI=18.81892431; RAW_FILE_NAME=0059_LC_20190913_Sample_0060.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.61 AGE:19 | SEX:M BMI=24.34175829; RAW_FILE_NAME=0059_LC_20190913_Sample_0061.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.62 AGE:13 | SEX:M BMI=20.78826393; RAW_FILE_NAME=0059_LC_20190913_Sample_0062.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.63 AGE:16 | SEX:M BMI=25; RAW_FILE_NAME=0059_LC_20190913_Sample_0063.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.64 AGE:14 | SEX:F BMI=26.00151172; RAW_FILE_NAME=0059_LC_20190913_Sample_0064.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.65 AGE:13 | SEX:F BMI=20.28650791; RAW_FILE_NAME=0059_LC_20190913_Sample_0065.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.66 AGE:19 | SEX:M BMI=18.93877551; RAW_FILE_NAME=0059_LC_20190913_Sample_0066.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.67 AGE:13 | SEX:M BMI=23.05175491; RAW_FILE_NAME=0059_LC_20190913_Sample_0067.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.68 AGE:14 | SEX:M BMI=23.828125; RAW_FILE_NAME=0059_LC_20190913_Sample_0068.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.69 AGE:14 | SEX:F BMI=26.85440557; RAW_FILE_NAME=0059_LC_20190913_Sample_0069.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.70 AGE:16 | SEX:F BMI=29.02069603; RAW_FILE_NAME=0059_LC_20190913_Sample_0070.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.71 AGE:13 | SEX:M BMI=27.13500257; RAW_FILE_NAME=0059_LC_20190913_Sample_0071.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.72 AGE:14 | SEX:M BMI=26.59320478; RAW_FILE_NAME=0059_LC_20190913_Sample_0072.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.73 AGE:18 | SEX:M BMI=22.67995241; RAW_FILE_NAME=0059_LC_20190913_Sample_0073.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.74 AGE:19 | SEX:F BMI=42.36028537; RAW_FILE_NAME=0059_LC_20190913_Sample_0074.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.75 AGE:13 | SEX:M BMI=20.78826393; RAW_FILE_NAME=0059_LC_20190913_Sample_0075.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.76 AGE:11 | SEX:F BMI=24.6366782; RAW_FILE_NAME=0059_LC_20190913_Sample_0076.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.77 AGE:12 | SEX:F BMI=20.3125; RAW_FILE_NAME=0059_LC_20190913_Sample_0077.mzdata.xml SUBJECT_SAMPLE_FACTORS - Sample.78 AGE:15 | SEX:M BMI=32.7727778; RAW_FILE_NAME=0059_LC_20190913_Sample_0078.mzdata.xml #COLLECTION CO:COLLECTION_SUMMARY The collection of fetal material involved was approved by the National Health CO:COLLECTION_SUMMARY Service (NHS) Grampian Research Ethics Committees (REC 04/S0802/21) and the CO:COLLECTION_SUMMARY study was conducted according to the guidelines laid down in the Declaration of CO:COLLECTION_SUMMARY Helsinki. In all cases, women seeking elective terminations of pregnancy were CO:COLLECTION_SUMMARY recruited with full written, informed consent by nurses working independently of CO:COLLECTION_SUMMARY the study at Aberdeen Pregnancy Counselling Service. Maternal data, medications CO:COLLECTION_SUMMARY used and self-reported number of cigarettes smoked per day were recorded. Only CO:COLLECTION_SUMMARY fetuses from normally-progressing pregnancies (determined at ultrasound scan CO:COLLECTION_SUMMARY prior to termination) from women over 16 years of age, and between 11-21 weeks CO:COLLECTION_SUMMARY of gestation, were collected following termination by RU-486 (Mifepristone) CO:COLLECTION_SUMMARY treatment (200mg) and prostaglandin induced delivery, as detailed previously CO:COLLECTION_SUMMARY [REF-https://doi.org/10.1210/jc.2007-1690]. Fetuses were transported to the CO:COLLECTION_SUMMARY laboratory within 30 minutes of delivery, weighed, sexed and the crown-rump CO:COLLECTION_SUMMARY length recorded. Fetal tissues were snap-frozen in liquid-nitrogen, and stored CO:COLLECTION_SUMMARY at -80°C, or fixed in 10% neutral buffered formalin. Maternal smoking status CO:COLLECTION_SUMMARY was confirmed by measurement of fetal plasma cotinine using a commercially CO:COLLECTION_SUMMARY available kit (Cozart Plc, Abingdon, Kent, UK). CO:SAMPLE_TYPE Blood (serum) CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY No treatment was applied. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Sample preparation and mass spectrometric analyses All solvents were HPLC grade SP:SAMPLEPREP_SUMMARY or LC-MS grade, from Honeywell (Morris Plains, NJ, USA), Fisher Scientific SP:SAMPLEPREP_SUMMARY (Waltham, MA, USA) or Sigma-Aldrich (St. Louis, MO, USA). Mass spectrometry SP:SAMPLEPREP_SUMMARY grade ammonium acetate and reagent grade formic acid were also from SP:SAMPLEPREP_SUMMARY Sigma-Aldrich (St. Louis, MO, USA). The lipid standards were from Avanti Polar SP:SAMPLEPREP_SUMMARY Lipids Inc. (Alabaster, AL, USA). 13C-labeled PFAS internal standards (IS), SP:SAMPLEPREP_SUMMARY 13C-labeled performance standards, and native calibration standards SP:SAMPLEPREP_SUMMARY (perfluorocarboxylic acids (PFCA) and perflurosulfonic acids (PFSA)) were SP:SAMPLEPREP_SUMMARY purchased from Wellington Laboratories (Guelph, Ontario, Canada). One native SP:SAMPLEPREP_SUMMARY performance standard, 7H-dodecafluoroheptanoic acid, was purchased from ABCR SP:SAMPLEPREP_SUMMARY (Karlsruhe, Germany). CA, CDCA, DCA, DHCA, GCA, GCDCA, LCA, TCA, TCDCA, TDCA, SP:SAMPLEPREP_SUMMARY TDHCA, THCA, THDCA, TLCA, and TUDCA were obtained from Sigma-Aldrich (St. Luis, SP:SAMPLEPREP_SUMMARY MO, USA), HDCA, HCA, αMCA, βMCA, ωMCA, 7-oxo-HDCA, 7-oxo-DCA, 12-oxo-LCA, SP:SAMPLEPREP_SUMMARY TαMCA, TβMCA, TωMCA, GDHCA, GHCA, and GHDCA from Steraloids (Newport, RI, SP:SAMPLEPREP_SUMMARY U.S.A), GLCA and GUDCA from Calbiochem (Gibbstown, NJ, U.S.A), and GDCA and UDGA SP:SAMPLEPREP_SUMMARY from Fluka (Buchs, Switzerland). Internal standards CA-d4, LCA-d4, UDCA-d4, SP:SAMPLEPREP_SUMMARY CDCA-d4, DCA-d4, GCA-d4, GLCA-d4, GUDCA-d4 and GCDCA-d4 were obtained from Qmx SP:SAMPLEPREP_SUMMARY laboratories Ltd. (Essex, UK). For quality assurance (QA), standard reference SP:SAMPLEPREP_SUMMARY material serum SRM 1950 (for lipidomics and metabolomics) and 1957 (for PFAS and SP:SAMPLEPREP_SUMMARY BAs) was purchased from the National Institute of Standards and Technology SP:SAMPLEPREP_SUMMARY (NIST) at the US Department of Commerce (Washington, DC, USA). All samples were SP:SAMPLEPREP_SUMMARY randomized before preparation and again before the analysis. The liver samples SP:SAMPLEPREP_SUMMARY were weighted and phosphate-buffered saline (PBS) was added so that the ratio of SP:SAMPLEPREP_SUMMARY tissue to buffer was 1 mg tissue to 10 mL buffer and the samples were SP:SAMPLEPREP_SUMMARY homogenized manually. Two extraction methods described below were applied, the SP:SAMPLEPREP_SUMMARY first one for extraction of BAs, PFAS and other semipolar compounds and the SP:SAMPLEPREP_SUMMARY second one for extraction of lipids. For analysis of BAs, PFAS and polar SP:SAMPLEPREP_SUMMARY metabolites, three methods were used, applying targeted method for PFAS and BAs SP:SAMPLEPREP_SUMMARY as well as for 7α-hydroxy-4-cholesten-3-one (C4), using method described SP:SAMPLEPREP_SUMMARY previously1 and a combined target-non-target method for the analysis of SP:SAMPLEPREP_SUMMARY semipolar metabolites and pollutants. 40 µl of liver homogenate was extracted SP:SAMPLEPREP_SUMMARY with 400µL of cold MeOH/H2O containing the internal standard mixture SP:SAMPLEPREP_SUMMARY (Valine-d8, Glutamic acid-d5, Succinic acid-d4, Heptadecanoic acid, Lactic SP:SAMPLEPREP_SUMMARY acid-d3, Citric acid-d4. 3-Hydroxybutyric acid-d4, Arginine-d7, Tryptophan-d5, SP:SAMPLEPREP_SUMMARY Glutamine-d5, SP:SAMPLEPREP_SUMMARY 1-D4-CA,1-D4-CDCA,1-D4-CDCA,1-D4-GCA,1-D4-GCDCA,1-D4-GLCA,1-D4-GUDCA,1-D4-LCA,1-D4-TCA, SP:SAMPLEPREP_SUMMARY 1-D4-UDCA). The tube was vortexed and ultrasonicated for 3 min, followed by SP:SAMPLEPREP_SUMMARY centrifugation (10000 rpm, 5 min). After centrifuging, 350 µl of the upper SP:SAMPLEPREP_SUMMARY layer of the solution was transferred to the LC vial and evaporated under the SP:SAMPLEPREP_SUMMARY nitrogen gas to the dryness. After drying, the sample was reconstituted into 60 SP:SAMPLEPREP_SUMMARY µl of MeOH: H2O (70:30). The targeted analysis of BA and PFAS was performed SP:SAMPLEPREP_SUMMARY using automated column-switching ultra-performance liquid chromatography-tandem SP:SAMPLEPREP_SUMMARY mass spectrometry (UPLC-MS/MS) (Waters, Milford, USA) using an ACQUITY C18 BEH SP:SAMPLEPREP_SUMMARY 2.1×100mm×1.7µm column and a gradient with 30% methanol in 2mM NH4Ac water SP:SAMPLEPREP_SUMMARY and 2mM NH4Ac in methanol with a flow rate of 0.3 mL/min. Quantitative analysis SP:SAMPLEPREP_SUMMARY of the selected analytes was performed using the isotope dilution method; all SP:SAMPLEPREP_SUMMARY standards (i.e., internal standards, recovery standards, and native calibration SP:SAMPLEPREP_SUMMARY standards) were purchased from Wellington Laboratories (Guelph, Ontario, SP:SAMPLEPREP_SUMMARY Canada). The method’s detection limits ranged between 0.02-0.19 ng/mL, SP:SAMPLEPREP_SUMMARY depending on the analyte. NIST SRM 1957 reference serum as well as in-house SP:SAMPLEPREP_SUMMARY pooled plasma samples were used in quality control. The analysis of C4 was done SP:SAMPLEPREP_SUMMARY using the same method but using a positive ion mode in MS. The analysis of SP:SAMPLEPREP_SUMMARY semipolar metabolites was performed from the same extract that was used for SP:SAMPLEPREP_SUMMARY target analyses using an ultra-high-performance liquid chromatography quadrupole SP:SAMPLEPREP_SUMMARY time-of-flight mass spectrometry (UHPLC-QTOFMS). Briefly, the UHPLC system used SP:SAMPLEPREP_SUMMARY in this work was a 1290 Infinity II system from Agilent Technologies (Santa SP:SAMPLEPREP_SUMMARY Clara, CA, USA). The system was equipped with a multi sampler (maintained at 10 SP:SAMPLEPREP_SUMMARY °C), a quaternary solvent manager and a column thermostat (maintained at 50 SP:SAMPLEPREP_SUMMARY °C). Injection volume was 1 µL and the separations were performed on an SP:SAMPLEPREP_SUMMARY ACQUITY UPLC® BEH C18 column (2.1 mm × 100 mm, particle size 1.7 µm) by SP:SAMPLEPREP_SUMMARY Waters (Milford, MA, USA). The mass spectrometer coupled to the UHPLC was a 6545 SP:SAMPLEPREP_SUMMARY QTOF from Agilent Technologies interfaced with a dual jet stream electrospray SP:SAMPLEPREP_SUMMARY (Dual ESI) ion source. Chromatographic separation was carried out using an SP:SAMPLEPREP_SUMMARY Acquity UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm particle size), SP:SAMPLEPREP_SUMMARY fitted with a C18 precolumn (Waters Corporation, Wexford, Ireland). Mobile phase SP:SAMPLEPREP_SUMMARY A consisted of H2O:MeOH (v/v 70:30) and mobile phase B of MeOH with both phases SP:SAMPLEPREP_SUMMARY containing 2mM ammonium acetate as an ionization agent. The flow rate was set at SP:SAMPLEPREP_SUMMARY 0.4 mLmin-1 with the elution gradient as follows: 0-1.5 min, mobile phase B was SP:SAMPLEPREP_SUMMARY increased from 5% to 30%; 1.5-4.5 min, mobile phase B increased to 70%; 4.5-7.5 SP:SAMPLEPREP_SUMMARY min, mobile phase B increased to 100% and held for 5.5 min. A post-time of 5 min SP:SAMPLEPREP_SUMMARY was used to regain the initial conditions for the next analysis. The total run SP:SAMPLEPREP_SUMMARY time per sample was 18 min. The dual ESI ionization source was settings were as SP:SAMPLEPREP_SUMMARY follows: capillary voltage was 4.5 kV, nozzle voltage 1500 V, N2 pressure in the SP:SAMPLEPREP_SUMMARY nebulised was 21 psi and the N2 flow rate and temperature as sheath gas was 11 SP:SAMPLEPREP_SUMMARY Lmin-1 and 379 °C, respectively. In order to obtain accurate mass spectra in MS SP:SAMPLEPREP_SUMMARY scan, the m/z range was set to 100-1700 in negative ion mode. MassHunter B.06.01 SP:SAMPLEPREP_SUMMARY software (Agilent Technologies, Santa Clara, CA, USA) was used for all data SP:SAMPLEPREP_SUMMARY acquisition. For lipidomics, the samples were extracted using a modified version SP:SAMPLEPREP_SUMMARY of the previously Folch procedure.2 In short, 10 µL of 0.9% NaCl and, 120 µL SP:SAMPLEPREP_SUMMARY of CHCl3: MeOH (2:1, v/v) containing the internal standards (c = 2.5 µg/mL) was SP:SAMPLEPREP_SUMMARY added to 10 µL of sample homogenate. The standard solution contained the SP:SAMPLEPREP_SUMMARY following compounds: 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine SP:SAMPLEPREP_SUMMARY (PE(17:0/17:0)), N-heptadecanoyl-D-erythro-sphingosylphosphorylcholine SP:SAMPLEPREP_SUMMARY (SM(d18:1/17:0)), N-heptadecanoyl-D-erythro-sphingosine (Cer(d18:1/17:0)), SP:SAMPLEPREP_SUMMARY 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (PC(17:0/17:0)), SP:SAMPLEPREP_SUMMARY 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(17:0)) and SP:SAMPLEPREP_SUMMARY 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine (PC(16:0/d31/18:1)), were SP:SAMPLEPREP_SUMMARY purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), and, SP:SAMPLEPREP_SUMMARY triheptadecanoylglycerol (TG(17:0/17:0/17:0)) was purchased from Larodan AB SP:SAMPLEPREP_SUMMARY (Solna, Sweden). The samples were vortex mixed and incubated on ice for 30 min SP:SAMPLEPREP_SUMMARY after which they were centrifuged (9400 × g, 3 min). 60 µL from the lower SP:SAMPLEPREP_SUMMARY layer of each sample was then transferred to a glass vial with an insert and 60 SP:SAMPLEPREP_SUMMARY µL of CHCl3: MeOH (2:1, v/v) was added to each sample. The samples were stored SP:SAMPLEPREP_SUMMARY at -80 °C until analysis. Calibration curves using SP:SAMPLEPREP_SUMMARY 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine SP:SAMPLEPREP_SUMMARY (PC(16:0e/18:1(9Z))), SP:SAMPLEPREP_SUMMARY 1-(1Z-octadecenyl)-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine SP:SAMPLEPREP_SUMMARY (PC(18:0p/18:1(9Z))), 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine SP:SAMPLEPREP_SUMMARY (LPC(18:0)), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(18:1)), SP:SAMPLEPREP_SUMMARY 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (PE(16:0/18:1)), SP:SAMPLEPREP_SUMMARY 1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphocholine SP:SAMPLEPREP_SUMMARY (PC(18:0p/22:6)) and 1-stearoyl-2-linoleoyl-sn-glycerol. (DG(18:0/18:2)), SP:SAMPLEPREP_SUMMARY 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (LPE(18:1)), SP:SAMPLEPREP_SUMMARY N-(9Z-octadecenoyl)-sphinganine (Cer(d18:0/18:1(9Z))), SP:SAMPLEPREP_SUMMARY 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(16:0/18:1)) SP:SAMPLEPREP_SUMMARY from Avanti Polar Lipids, 1-Palmitoyl-2-Hydroxy-sn-Glycero-3-Phosphatidylcholine SP:SAMPLEPREP_SUMMARY (LPC(16:0)), 1,2,3 trihexadecanoalglycerol (TG(16:0/16:0/16:0)), SP:SAMPLEPREP_SUMMARY 1,2,3-trioctadecanoylglycerol (TG(18:0/18:0/18:)) and SP:SAMPLEPREP_SUMMARY 3β-hydroxy-5-cholestene-3-stearate (ChoE(18:0)), SP:SAMPLEPREP_SUMMARY 3β-Hydroxy-5-cholestene-3-linoleate (ChoE(18:2)) from Larodan, were prepared to SP:SAMPLEPREP_SUMMARY the following concentration levels: 100, 500, 1000, 1500, 2000 and 2500 ng/mL SP:SAMPLEPREP_SUMMARY (in CHCl3:MeOH, 2:1, v/v) including 1250 ng/mL of each internal standard. The SP:SAMPLEPREP_SUMMARY samples were analyzed by ultra-high-performance liquid chromatography quadrupole SP:SAMPLEPREP_SUMMARY time-of-flight mass spectrometry (UHPLC-QTOFMS). Briefly, the UHPLC system used SP:SAMPLEPREP_SUMMARY in this work was a 1290 Infinity II system from Agilent Technologies (Santa SP:SAMPLEPREP_SUMMARY Clara, CA, USA). The system was equipped with a multi sampler (maintained at 10 SP:SAMPLEPREP_SUMMARY °C), a quaternary solvent manager and a column thermostat (maintained at 50 SP:SAMPLEPREP_SUMMARY °C). Injection volume was 1 µL and the separations were performed on an SP:SAMPLEPREP_SUMMARY ACQUITY UPLC® BEH C18 column (2.1 mm × 100 mm, particle size 1.7 µm) by SP:SAMPLEPREP_SUMMARY Waters (Milford, MA, USA). The mass spectrometer coupled to the UHPLC was a 6545 SP:SAMPLEPREP_SUMMARY QTOF from Agilent Technologies interfaced with a dual jet stream electrospray SP:SAMPLEPREP_SUMMARY (Dual ESI) ion source. All analyses were performed in positive ion mode and SP:SAMPLEPREP_SUMMARY MassHunter B.06.01 (Agilent Technologies) was used for all data acquisition. SP:SAMPLEPREP_SUMMARY Quality control was performed throughout the dataset by including blanks, pure SP:SAMPLEPREP_SUMMARY standard samples, extracted standard samples and control serum samples. Mass SP:SAMPLEPREP_SUMMARY spectrometry data processing was performed using the open-source software SP:SAMPLEPREP_SUMMARY package MZmine 2.18.3 The following steps were applied in this processing: (i) SP:SAMPLEPREP_SUMMARY Crop filtering with a m/z range of 350 – 1200 m/z and an RT range of 2.0 to 12 SP:SAMPLEPREP_SUMMARY minutes, (ii) Mass detection with a noise level of 750, (iii) Chromatogram SP:SAMPLEPREP_SUMMARY builder with a minimum time span of 0.08 min, minimum height of 1000 and a m/z SP:SAMPLEPREP_SUMMARY tolerance of 0.006 m/z or 10.0 ppm, (iv) Chromatogram deconvolution using the SP:SAMPLEPREP_SUMMARY local minimum search algorithm with a 70% chromatographic threshold, 0.05 min SP:SAMPLEPREP_SUMMARY minimum RT range, 5% minimum relative height, 1200 minimum absolute height, a SP:SAMPLEPREP_SUMMARY minimum ration of peak top/edge of 1.2 and a peak duration range of 0.08 - 5.0, SP:SAMPLEPREP_SUMMARY (v), Isotopic peak grouper with a m/z tolerance of 5.0 ppm, RT tolerance of 0.05 SP:SAMPLEPREP_SUMMARY min, maximum charge of 2 and with the most intense isotope set as the SP:SAMPLEPREP_SUMMARY representative isotope, (vi) Peak filter with minimum 12 data points, a FWHM SP:SAMPLEPREP_SUMMARY between 0.0 and 0.2, tailing factor between 0.45 and 2.22 and asymmetry factor SP:SAMPLEPREP_SUMMARY between 0.40 and 2.50, (vii) Join aligner with a m/z tolerance of 0.009 or 10.0 SP:SAMPLEPREP_SUMMARY ppm and a weight for of 2, a RT tolerance of 0.1 min and a weight of 1 and with SP:SAMPLEPREP_SUMMARY no requirement of charge state or ID and no comparison of isotope pattern, SP:SAMPLEPREP_SUMMARY (viii) Peak list row filter with a minimum of 10% of the samples (ix) Gap SP:SAMPLEPREP_SUMMARY filling using the same RT and m/z range gap filler algorithm with an m/z SP:SAMPLEPREP_SUMMARY tolerance of 0.009 m/z or 11.0 ppm, (x) Identification of lipids using a custom SP:SAMPLEPREP_SUMMARY database search with an m/z tolerance of 0.009 m/z or 10.0 ppm and a RT SP:SAMPLEPREP_SUMMARY tolerance of 0.1 min, and (xi) Normalization using internal standards SP:SAMPLEPREP_SUMMARY PE(17:0/17:0), SM(d18:1/17:0), Cer(d18:1/17:0), LPC(17:0), TG(17:0/17:0/17:0) SP:SAMPLEPREP_SUMMARY and PC(16:0/d30/18:1)) for identified lipids and closest ISTD for the unknown SP:SAMPLEPREP_SUMMARY lipids followed by calculation of the concentrations based on lipid-class SP:SAMPLEPREP_SUMMARY concentration curves. An aliquot of each sample was collected and pooled and SP:SAMPLEPREP_SUMMARY used as quality control sample, together with NIST SRM1950 reference plasma SP:SAMPLEPREP_SUMMARY sample, an in-house pooled serum sample. Relative standard deviations (% RSDs) SP:SAMPLEPREP_SUMMARY for peak areas for pooled samples were on average 12.1% for lipidomics and 12-0 SP:SAMPLEPREP_SUMMARY % for PFAS and BAs SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Sample preparation and mass spectrometric analyses All solvents were HPLC grade CH:CHROMATOGRAPHY_SUMMARY or LC-MS grade, from Honeywell (Morris Plains, NJ, USA), Fisher Scientific CH:CHROMATOGRAPHY_SUMMARY (Waltham, MA, USA) or Sigma-Aldrich (St. Louis, MO, USA). Mass spectrometry CH:CHROMATOGRAPHY_SUMMARY grade ammonium acetate and reagent grade formic acid were also from CH:CHROMATOGRAPHY_SUMMARY Sigma-Aldrich (St. Louis, MO, USA). The lipid standards were from Avanti Polar CH:CHROMATOGRAPHY_SUMMARY Lipids Inc. (Alabaster, AL, USA). 13C-labeled PFAS internal standards (IS), CH:CHROMATOGRAPHY_SUMMARY 13C-labeled performance standards, and native calibration standards CH:CHROMATOGRAPHY_SUMMARY (perfluorocarboxylic acids (PFCA) and perflurosulfonic acids (PFSA)) were CH:CHROMATOGRAPHY_SUMMARY purchased from Wellington Laboratories (Guelph, Ontario, Canada). One native CH:CHROMATOGRAPHY_SUMMARY performance standard, 7H-dodecafluoroheptanoic acid, was purchased from ABCR CH:CHROMATOGRAPHY_SUMMARY (Karlsruhe, Germany). CA, CDCA, DCA, DHCA, GCA, GCDCA, LCA, TCA, TCDCA, TDCA, CH:CHROMATOGRAPHY_SUMMARY TDHCA, THCA, THDCA, TLCA, and TUDCA were obtained from Sigma-Aldrich (St. Luis, CH:CHROMATOGRAPHY_SUMMARY MO, USA), HDCA, HCA, αMCA, βMCA, ωMCA, 7-oxo-HDCA, 7-oxo-DCA, 12-oxo-LCA, CH:CHROMATOGRAPHY_SUMMARY TαMCA, TβMCA, TωMCA, GDHCA, GHCA, and GHDCA from Steraloids (Newport, RI, CH:CHROMATOGRAPHY_SUMMARY U.S.A), GLCA and GUDCA from Calbiochem (Gibbstown, NJ, U.S.A), and GDCA and UDGA CH:CHROMATOGRAPHY_SUMMARY from Fluka (Buchs, Switzerland). Internal standards CA-d4, LCA-d4, UDCA-d4, CH:CHROMATOGRAPHY_SUMMARY CDCA-d4, DCA-d4, GCA-d4, GLCA-d4, GUDCA-d4 and GCDCA-d4 were obtained from Qmx CH:CHROMATOGRAPHY_SUMMARY laboratories Ltd. (Essex, UK). For quality assurance (QA), standard reference CH:CHROMATOGRAPHY_SUMMARY material serum SRM 1950 (for lipidomics and metabolomics) and 1957 (for PFAS and CH:CHROMATOGRAPHY_SUMMARY BAs) was purchased from the National Institute of Standards and Technology CH:CHROMATOGRAPHY_SUMMARY (NIST) at the US Department of Commerce (Washington, DC, USA). All samples were CH:CHROMATOGRAPHY_SUMMARY randomized before preparation and again before the analysis. The liver samples CH:CHROMATOGRAPHY_SUMMARY were weighted and phosphate-buffered saline (PBS) was added so that the ratio of CH:CHROMATOGRAPHY_SUMMARY tissue to buffer was 1 mg tissue to 10 mL buffer and the samples were CH:CHROMATOGRAPHY_SUMMARY homogenized manually. Two extraction methods described below were applied, the CH:CHROMATOGRAPHY_SUMMARY first one for extraction of BAs, PFAS and other semipolar compounds and the CH:CHROMATOGRAPHY_SUMMARY second one for extraction of lipids. For analysis of BAs, PFAS and polar CH:CHROMATOGRAPHY_SUMMARY metabolites, three methods were used, applying targeted method for PFAS and BAs CH:CHROMATOGRAPHY_SUMMARY as well as for 7α-hydroxy-4-cholesten-3-one (C4), using method described CH:CHROMATOGRAPHY_SUMMARY previously1 and a combined target-non-target method for the analysis of CH:CHROMATOGRAPHY_SUMMARY semipolar metabolites and pollutants. 40 µl of liver homogenate was extracted CH:CHROMATOGRAPHY_SUMMARY with 400µL of cold MeOH/H2O containing the internal standard mixture CH:CHROMATOGRAPHY_SUMMARY (Valine-d8, Glutamic acid-d5, Succinic acid-d4, Heptadecanoic acid, Lactic CH:CHROMATOGRAPHY_SUMMARY acid-d3, Citric acid-d4. 3-Hydroxybutyric acid-d4, Arginine-d7, Tryptophan-d5, CH:CHROMATOGRAPHY_SUMMARY Glutamine-d5, CH:CHROMATOGRAPHY_SUMMARY 1-D4-CA,1-D4-CDCA,1-D4-CDCA,1-D4-GCA,1-D4-GCDCA,1-D4-GLCA,1-D4-GUDCA,1-D4-LCA,1-D4-TCA, CH:CHROMATOGRAPHY_SUMMARY 1-D4-UDCA). The tube was vortexed and ultrasonicated for 3 min, followed by CH:CHROMATOGRAPHY_SUMMARY centrifugation (10000 rpm, 5 min). After centrifuging, 350 µl of the upper CH:CHROMATOGRAPHY_SUMMARY layer of the solution was transferred to the LC vial and evaporated under the CH:CHROMATOGRAPHY_SUMMARY nitrogen gas to the dryness. After drying, the sample was reconstituted into 60 CH:CHROMATOGRAPHY_SUMMARY µl of MeOH: H2O (70:30). The targeted analysis of BA and PFAS was performed CH:CHROMATOGRAPHY_SUMMARY using automated column-switching ultra-performance liquid chromatography-tandem CH:CHROMATOGRAPHY_SUMMARY mass spectrometry (UPLC-MS/MS) (Waters, Milford, USA) using an ACQUITY C18 BEH CH:CHROMATOGRAPHY_SUMMARY 2.1×100mm×1.7µm column and a gradient with 30% methanol in 2mM NH4Ac water CH:CHROMATOGRAPHY_SUMMARY and 2mM NH4Ac in methanol with a flow rate of 0.3 mL/min. Quantitative analysis CH:CHROMATOGRAPHY_SUMMARY of the selected analytes was performed using the isotope dilution method; all CH:CHROMATOGRAPHY_SUMMARY standards (i.e., internal standards, recovery standards, and native calibration CH:CHROMATOGRAPHY_SUMMARY standards) were purchased from Wellington Laboratories (Guelph, Ontario, CH:CHROMATOGRAPHY_SUMMARY Canada). The method’s detection limits ranged between 0.02-0.19 ng/mL, CH:CHROMATOGRAPHY_SUMMARY depending on the analyte. NIST SRM 1957 reference serum as well as in-house CH:CHROMATOGRAPHY_SUMMARY pooled plasma samples were used in quality control. The analysis of C4 was done CH:CHROMATOGRAPHY_SUMMARY using the same method but using a positive ion mode in MS. The analysis of CH:CHROMATOGRAPHY_SUMMARY semipolar metabolites was performed from the same extract that was used for CH:CHROMATOGRAPHY_SUMMARY target analyses using an ultra-high-performance liquid chromatography quadrupole CH:CHROMATOGRAPHY_SUMMARY time-of-flight mass spectrometry (UHPLC-QTOFMS). Briefly, the UHPLC system used CH:CHROMATOGRAPHY_SUMMARY in this work was a 1290 Infinity II system from Agilent Technologies (Santa CH:CHROMATOGRAPHY_SUMMARY Clara, CA, USA). The system was equipped with a multi sampler (maintained at 10 CH:CHROMATOGRAPHY_SUMMARY °C), a quaternary solvent manager and a column thermostat (maintained at 50 CH:CHROMATOGRAPHY_SUMMARY °C). Injection volume was 1 µL and the separations were performed on an CH:CHROMATOGRAPHY_SUMMARY ACQUITY UPLC® BEH C18 column (2.1 mm × 100 mm, particle size 1.7 µm) by CH:CHROMATOGRAPHY_SUMMARY Waters (Milford, MA, USA). The mass spectrometer coupled to the UHPLC was a 6545 CH:CHROMATOGRAPHY_SUMMARY QTOF from Agilent Technologies interfaced with a dual jet stream electrospray CH:CHROMATOGRAPHY_SUMMARY (Dual ESI) ion source. Chromatographic separation was carried out using an CH:CHROMATOGRAPHY_SUMMARY Acquity UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm particle size), CH:CHROMATOGRAPHY_SUMMARY fitted with a C18 precolumn (Waters Corporation, Wexford, Ireland). Mobile phase CH:CHROMATOGRAPHY_SUMMARY A consisted of H2O:MeOH (v/v 70:30) and mobile phase B of MeOH with both phases CH:CHROMATOGRAPHY_SUMMARY containing 2mM ammonium acetate as an ionization agent. The flow rate was set at CH:CHROMATOGRAPHY_SUMMARY 0.4 mLmin-1 with the elution gradient as follows: 0-1.5 min, mobile phase B was CH:CHROMATOGRAPHY_SUMMARY increased from 5% to 30%; 1.5-4.5 min, mobile phase B increased to 70%; 4.5-7.5 CH:CHROMATOGRAPHY_SUMMARY min, mobile phase B increased to 100% and held for 5.5 min. A post-time of 5 min CH:CHROMATOGRAPHY_SUMMARY was used to regain the initial conditions for the next analysis. The total run CH:CHROMATOGRAPHY_SUMMARY time per sample was 18 min. The dual ESI ionization source was settings were as CH:CHROMATOGRAPHY_SUMMARY follows: capillary voltage was 4.5 kV, nozzle voltage 1500 V, N2 pressure in the CH:CHROMATOGRAPHY_SUMMARY nebulised was 21 psi and the N2 flow rate and temperature as sheath gas was 11 CH:CHROMATOGRAPHY_SUMMARY Lmin-1 and 379 °C, respectively. In order to obtain accurate mass spectra in MS CH:CHROMATOGRAPHY_SUMMARY scan, the m/z range was set to 100-1700 in negative ion mode. MassHunter B.06.01 CH:CHROMATOGRAPHY_SUMMARY software (Agilent Technologies, Santa Clara, CA, USA) was used for all data CH:CHROMATOGRAPHY_SUMMARY acquisition. For lipidomics, the samples were extracted using a modified version CH:CHROMATOGRAPHY_SUMMARY of the previously Folch procedure.2 In short, 10 µL of 0.9% NaCl and, 120 µL CH:CHROMATOGRAPHY_SUMMARY of CHCl3: MeOH (2:1, v/v) containing the internal standards (c = 2.5 µg/mL) was CH:CHROMATOGRAPHY_SUMMARY added to 10 µL of sample homogenate. The standard solution contained the CH:CHROMATOGRAPHY_SUMMARY following compounds: 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine CH:CHROMATOGRAPHY_SUMMARY (PE(17:0/17:0)), N-heptadecanoyl-D-erythro-sphingosylphosphorylcholine CH:CHROMATOGRAPHY_SUMMARY (SM(d18:1/17:0)), N-heptadecanoyl-D-erythro-sphingosine (Cer(d18:1/17:0)), CH:CHROMATOGRAPHY_SUMMARY 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (PC(17:0/17:0)), CH:CHROMATOGRAPHY_SUMMARY 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(17:0)) and CH:CHROMATOGRAPHY_SUMMARY 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine (PC(16:0/d31/18:1)), were CH:CHROMATOGRAPHY_SUMMARY purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA), and, CH:CHROMATOGRAPHY_SUMMARY triheptadecanoylglycerol (TG(17:0/17:0/17:0)) was purchased from Larodan AB CH:CHROMATOGRAPHY_SUMMARY (Solna, Sweden). The samples were vortex mixed and incubated on ice for 30 min CH:CHROMATOGRAPHY_SUMMARY after which they were centrifuged (9400 × g, 3 min). 60 µL from the lower CH:CHROMATOGRAPHY_SUMMARY layer of each sample was then transferred to a glass vial with an insert and 60 CH:CHROMATOGRAPHY_SUMMARY µL of CHCl3: MeOH (2:1, v/v) was added to each sample. The samples were stored CH:CHROMATOGRAPHY_SUMMARY at -80 °C until analysis. Calibration curves using CH:CHROMATOGRAPHY_SUMMARY 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine CH:CHROMATOGRAPHY_SUMMARY (PC(16:0e/18:1(9Z))), CH:CHROMATOGRAPHY_SUMMARY 1-(1Z-octadecenyl)-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine CH:CHROMATOGRAPHY_SUMMARY (PC(18:0p/18:1(9Z))), 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine CH:CHROMATOGRAPHY_SUMMARY (LPC(18:0)), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC(18:1)), CH:CHROMATOGRAPHY_SUMMARY 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (PE(16:0/18:1)), CH:CHROMATOGRAPHY_SUMMARY 1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphocholine CH:CHROMATOGRAPHY_SUMMARY (PC(18:0p/22:6)) and 1-stearoyl-2-linoleoyl-sn-glycerol. (DG(18:0/18:2)), CH:CHROMATOGRAPHY_SUMMARY 1-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (LPE(18:1)), CH:CHROMATOGRAPHY_SUMMARY N-(9Z-octadecenoyl)-sphinganine (Cer(d18:0/18:1(9Z))), CH:CHROMATOGRAPHY_SUMMARY 1-hexadecyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (PE(16:0/18:1)) CH:CHROMATOGRAPHY_SUMMARY from Avanti Polar Lipids, 1-Palmitoyl-2-Hydroxy-sn-Glycero-3-Phosphatidylcholine CH:CHROMATOGRAPHY_SUMMARY (LPC(16:0)), 1,2,3 trihexadecanoalglycerol (TG(16:0/16:0/16:0)), CH:CHROMATOGRAPHY_SUMMARY 1,2,3-trioctadecanoylglycerol (TG(18:0/18:0/18:)) and CH:CHROMATOGRAPHY_SUMMARY 3β-hydroxy-5-cholestene-3-stearate (ChoE(18:0)), CH:CHROMATOGRAPHY_SUMMARY 3β-Hydroxy-5-cholestene-3-linoleate (ChoE(18:2)) from Larodan, were prepared to CH:CHROMATOGRAPHY_SUMMARY the following concentration levels: 100, 500, 1000, 1500, 2000 and 2500 ng/mL CH:CHROMATOGRAPHY_SUMMARY (in CHCl3:MeOH, 2:1, v/v) including 1250 ng/mL of each internal standard. The CH:CHROMATOGRAPHY_SUMMARY samples were analyzed by ultra-high-performance liquid chromatography quadrupole CH:CHROMATOGRAPHY_SUMMARY time-of-flight mass spectrometry (UHPLC-QTOFMS). Briefly, the UHPLC system used CH:CHROMATOGRAPHY_SUMMARY in this work was a 1290 Infinity II system from Agilent Technologies (Santa CH:CHROMATOGRAPHY_SUMMARY Clara, CA, USA). The system was equipped with a multi sampler (maintained at 10 CH:CHROMATOGRAPHY_SUMMARY °C), a quaternary solvent manager and a column thermostat (maintained at 50 CH:CHROMATOGRAPHY_SUMMARY °C). Injection volume was 1 µL and the separations were performed on an CH:CHROMATOGRAPHY_SUMMARY ACQUITY UPLC® BEH C18 column (2.1 mm × 100 mm, particle size 1.7 µm) by CH:CHROMATOGRAPHY_SUMMARY Waters (Milford, MA, USA). The mass spectrometer coupled to the UHPLC was a 6545 CH:CHROMATOGRAPHY_SUMMARY QTOF from Agilent Technologies interfaced with a dual jet stream electrospray CH:CHROMATOGRAPHY_SUMMARY (Dual ESI) ion source. All analyses were performed in positive ion mode and CH:CHROMATOGRAPHY_SUMMARY MassHunter B.06.01 (Agilent Technologies) was used for all data acquisition. CH:CHROMATOGRAPHY_SUMMARY Quality control was performed throughout the dataset by including blanks, pure CH:CHROMATOGRAPHY_SUMMARY standard samples, extracted standard samples and control serum samples. Mass CH:CHROMATOGRAPHY_SUMMARY spectrometry data processing was performed using the open-source software CH:CHROMATOGRAPHY_SUMMARY package MZmine 2.18.3 The following steps were applied in this processing: (i) CH:CHROMATOGRAPHY_SUMMARY Crop filtering with a m/z range of 350 – 1200 m/z and an RT range of 2.0 to 12 CH:CHROMATOGRAPHY_SUMMARY minutes, (ii) Mass detection with a noise level of 750, (iii) Chromatogram CH:CHROMATOGRAPHY_SUMMARY builder with a minimum time span of 0.08 min, minimum height of 1000 and a m/z CH:CHROMATOGRAPHY_SUMMARY tolerance of 0.006 m/z or 10.0 ppm, (iv) Chromatogram deconvolution using the CH:CHROMATOGRAPHY_SUMMARY local minimum search algorithm with a 70% chromatographic threshold, 0.05 min CH:CHROMATOGRAPHY_SUMMARY minimum RT range, 5% minimum relative height, 1200 minimum absolute height, a CH:CHROMATOGRAPHY_SUMMARY minimum ration of peak top/edge of 1.2 and a peak duration range of 0.08 - 5.0, CH:CHROMATOGRAPHY_SUMMARY (v), Isotopic peak grouper with a m/z tolerance of 5.0 ppm, RT tolerance of 0.05 CH:CHROMATOGRAPHY_SUMMARY min, maximum charge of 2 and with the most intense isotope set as the CH:CHROMATOGRAPHY_SUMMARY representative isotope, (vi) Peak filter with minimum 12 data points, a FWHM CH:CHROMATOGRAPHY_SUMMARY between 0.0 and 0.2, tailing factor between 0.45 and 2.22 and asymmetry factor CH:CHROMATOGRAPHY_SUMMARY between 0.40 and 2.50, (vii) Join aligner with a m/z tolerance of 0.009 or 10.0 CH:CHROMATOGRAPHY_SUMMARY ppm and a weight for of 2, a RT tolerance of 0.1 min and a weight of 1 and with CH:CHROMATOGRAPHY_SUMMARY no requirement of charge state or ID and no comparison of isotope pattern, CH:CHROMATOGRAPHY_SUMMARY (viii) Peak list row filter with a minimum of 10% of the samples (ix) Gap CH:CHROMATOGRAPHY_SUMMARY filling using the same RT and m/z range gap filler algorithm with an m/z CH:CHROMATOGRAPHY_SUMMARY tolerance of 0.009 m/z or 11.0 ppm, (x) Identification of lipids using a custom CH:CHROMATOGRAPHY_SUMMARY database search with an m/z tolerance of 0.009 m/z or 10.0 ppm and a RT CH:CHROMATOGRAPHY_SUMMARY tolerance of 0.1 min, and (xi) Normalization using internal standards CH:CHROMATOGRAPHY_SUMMARY PE(17:0/17:0), SM(d18:1/17:0), Cer(d18:1/17:0), LPC(17:0), TG(17:0/17:0/17:0) CH:CHROMATOGRAPHY_SUMMARY and PC(16:0/d30/18:1)) for identified lipids and closest ISTD for the unknown CH:CHROMATOGRAPHY_SUMMARY lipids followed by calculation of the concentrations based on lipid-class CH:CHROMATOGRAPHY_SUMMARY concentration curves. An aliquot of each sample was collected and pooled and CH:CHROMATOGRAPHY_SUMMARY used as quality control sample, together with NIST SRM1950 reference plasma CH:CHROMATOGRAPHY_SUMMARY sample, an in-house pooled serum sample. Relative standard deviations (% RSDs) CH:CHROMATOGRAPHY_SUMMARY for peak areas for pooled samples were on average 12.1% for lipidomics and 12-0 CH:CHROMATOGRAPHY_SUMMARY % for PFAS and BAs. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Waters ACQUITY UPLC BEH C18 (100 x 2.1mm,1.7um) CH:SOLVENT_A 30% water/70% methanol CH:SOLVENT_B 100% methanol CH:FLOW_GRADIENT 0-1.5 min, mobile phase B was increased from 5% to 30%; 1.5-4.5 min, mobile CH:FLOW_GRADIENT phase B increased to 70%; 4.5-7.5 min, mobile phase B increased to 100% and held CH:FLOW_GRADIENT for 5.5 min CH:FLOW_RATE 0.4 mLmin-1 CH:COLUMN_TEMPERATURE 50°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6545 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS Mass spectrometry data processing was performed using the open-source software MS:MS_COMMENTS package MZmine 2.18. The following steps were applied in this processing: (i) MS:MS_COMMENTS Crop filtering with a m/z range of 350 – 1200 m/z and an RT range of 2.0 to 12 MS:MS_COMMENTS minutes, (ii) Mass detection with a noise level of 750, (iii) Chromatogram MS:MS_COMMENTS builder with a minimum time span of 0.08 min, minimum height of 1000 and a m/z MS:MS_COMMENTS tolerance of 0.006 m/z or 10.0 ppm, (iv) Chromatogram deconvolution using the MS:MS_COMMENTS local minimum search algorithm with a 70% chromatographic threshold, 0.05 min MS:MS_COMMENTS minimum RT range, 5% minimum relative height, 1200 minimum absolute height, a MS:MS_COMMENTS minimum ration of peak top/edge of 1.2 and a peak duration range of 0.08 - 5.0, MS:MS_COMMENTS (v), Isotopic peak grouper with a m/z tolerance of 5.0 ppm, RT tolerance of 0.05 MS:MS_COMMENTS min, maximum charge of 2 and with the most intense isotope set as the MS:MS_COMMENTS representative isotope, (vi) Peak filter with minimum 12 data points, a FWHM MS:MS_COMMENTS between 0.0 and 0.2, tailing factor between 0.45 and 2.22 and asymmetry factor MS:MS_COMMENTS between 0.40 and 2.50, (vii) Join aligner with a m/z tolerance of 0.009 or 10.0 MS:MS_COMMENTS ppm and a weight for of 2, a RT tolerance of 0.1 min and a weight of 1 and with MS:MS_COMMENTS no requirement of charge state or ID and no comparison of isotope pattern, MS:MS_COMMENTS (viii) Peak list row filter with a minimum of 10% of the samples (ix) Gap MS:MS_COMMENTS filling using the same RT and m/z range gap filler algorithm with an m/z MS:MS_COMMENTS tolerance of 0.009 m/z or 11.0 ppm, (x) Identification of lipids using a custom MS:MS_COMMENTS database search with an m/z tolerance of 0.009 m/z or 10.0 ppm and a RT MS:MS_COMMENTS tolerance of 0.1 min, and (xi) Normalization using internal standards MS:MS_COMMENTS PE(17:0/17:0), SM(d18:1/17:0), Cer(d18:1/17:0), LPC(17:0), TG(17:0/17:0/17:0) MS:MS_COMMENTS and PC(16:0/d30/18:1)) for identified lipids and closest ISTD for the unknown MS:MS_COMMENTS lipids followed by calculation of the concentrations based on lipid-class MS:MS_COMMENTS concentration curves. MS:MS_RESULTS_FILE ST002480_AN004050_Results.txt UNITS:Raw quantification Has m/z:Yes Has RT:Yes RT units:Minutes #END