#METABOLOMICS WORKBENCH mcglinchey_aidan_20210218_011249 DATATRACK_ID:2495 STUDY_ID:ST001964 ANALYSIS_ID:AN003202 PROJECT_ID:000000 VERSION 1 CREATED_ON November 4, 2021, 9:33 am #PROJECT PR:PROJECT_TITLE Quantitative genome-scale analysis of human liver reveals dysregulation of PR:PROJECT_TITLE glycosphingolipid pathways in progressive nonalcoholic fatty liver disease PR:PROJECT_SUMMARY Nonalcoholic fatty liver disease (NAFLD) is a well-defined chronic liver PR:PROJECT_SUMMARY diseases closely related with metabolic disorders. The prevalence of NAFLD is PR:PROJECT_SUMMARY rapidly increasing worldwide, while the pathology and the underlying mechanisms PR:PROJECT_SUMMARY driving NAFLD are not fully understood. In NAFLD, a series of metabolic changes PR:PROJECT_SUMMARY takes place in the liver. However, the alteration of the metabolic pathways in PR:PROJECT_SUMMARY the human liver along the progression of NAFLD, i.e., the transition from PR:PROJECT_SUMMARY nonalcoholic steatosis (NAFL) to steatohepatitis (NASH) through cirrhosis PR:PROJECT_SUMMARY remains to be discovered. Here, we sought to examine the metabolic pathways of PR:PROJECT_SUMMARY the human liver across the full histological spectrum of NAFLD. We analyzed the PR:PROJECT_SUMMARY whole liver tissue transcriptomic (RNA-Seq) and serum metabolomics data obtained PR:PROJECT_SUMMARY from a large, prospectively enrolled cohort of histologically characterized PR:PROJECT_SUMMARY patients derived from the European NAFLD Registry (n=206), and developed PR:PROJECT_SUMMARY genome-scale metabolic models (GEMs) of human hepatocytes at different stages of PR:PROJECT_SUMMARY NAFLD. The integrative approach employed in this study has enabled us to PR:PROJECT_SUMMARY understand the regulation of the metabolic pathways of human liver in NAFL, and PR:PROJECT_SUMMARY with progressive NASH-associated fibrosis (F0–F4). Our study identified PR:PROJECT_SUMMARY several metabolic signatures in the liver and blood of these patients, PR:PROJECT_SUMMARY specifically highlighting the alteration of vitamins (A, E) and PR:PROJECT_SUMMARY glycosphingolipids (GSLs), and their link with complex glycosaminoglycans (GAGs) PR:PROJECT_SUMMARY in advanced fibrosis. The study provides insights into the underlying pathways PR:PROJECT_SUMMARY of the progressive fibrosing steatohepatitis. Furthermore, by applying PR:PROJECT_SUMMARY genome-scale metabolic modeling (GSMM), we were able to identify the metabolic PR:PROJECT_SUMMARY differences among carriers of widely validated genetic variants associated with PR:PROJECT_SUMMARY NAFLD / NASH disease severity in three genes (PNPLA3, TM6SF2 and HSD17B13). PR:INSTITUTE University of Turku PR:LAST_NAME Sen PR:FIRST_NAME Partho PR:ADDRESS Systems Medicine group, Turku Bioscience, University of Turku (UTU), PR:ADDRESS Tykistökatu 6B, P.O. Box 123 FIN-20521 Turku, Finland PR:EMAIL partho.sen@utu.fi PR:PHONE Phone: +358 469608145 #STUDY ST:STUDY_TITLE Quantitative genome-scale analysis of human liver reveals dysregulation of ST:STUDY_TITLE glycosphingolipid pathways in progressive nonalcoholic fatty liver disease ST:STUDY_SUMMARY Nonalcoholic fatty liver disease (NAFLD) is a well-defined chronic liver ST:STUDY_SUMMARY diseases closely related with metabolic disorders. The prevalence of NAFLD is ST:STUDY_SUMMARY rapidly increasing worldwide, while the pathology and the underlying mechanisms ST:STUDY_SUMMARY driving NAFLD are not fully understood. In NAFLD, a series of metabolic changes ST:STUDY_SUMMARY takes place in the liver. However, the alteration of the metabolic pathways in ST:STUDY_SUMMARY the human liver along the progression of NAFLD, i.e., the transition from ST:STUDY_SUMMARY nonalcoholic steatosis (NAFL) to steatohepatitis (NASH) through cirrhosis ST:STUDY_SUMMARY remains to be discovered. Here, we sought to examine the metabolic pathways of ST:STUDY_SUMMARY the human liver across the full histological spectrum of NAFLD. We analyzed the ST:STUDY_SUMMARY whole liver tissue transcriptomic (RNA-Seq) and serum metabolomics data obtained ST:STUDY_SUMMARY from a large, prospectively enrolled cohort of histologically characterized ST:STUDY_SUMMARY patients derived from the European NAFLD Registry (n=206), and developed ST:STUDY_SUMMARY genome-scale metabolic models (GEMs) of human hepatocytes at different stages of ST:STUDY_SUMMARY NAFLD. The integrative approach employed in this study has enabled us to ST:STUDY_SUMMARY understand the regulation of the metabolic pathways of human liver in NAFL, and ST:STUDY_SUMMARY with progressive NASH-associated fibrosis (F0–F4). Our study identified ST:STUDY_SUMMARY several metabolic signatures in the liver and blood of these patients, ST:STUDY_SUMMARY specifically highlighting the alteration of vitamins (A, E) and ST:STUDY_SUMMARY glycosphingolipids (GSLs), and their link with complex glycosaminoglycans (GAGs) ST:STUDY_SUMMARY in advanced fibrosis. The study provides insights into the underlying pathways ST:STUDY_SUMMARY of the progressive fibrosing steatohepatitis. Furthermore, by applying ST:STUDY_SUMMARY genome-scale metabolic modeling (GSMM), we were able to identify the metabolic ST:STUDY_SUMMARY differences among carriers of widely validated genetic variants associated with ST:STUDY_SUMMARY NAFLD / NASH disease severity in three genes (PNPLA3, TM6SF2 and HSD17B13). ST:INSTITUTE University of Turku ST:LAST_NAME Sen ST:FIRST_NAME Partho ST:ADDRESS Systems Medicine group, Turku Bioscience, University of Turku (UTU), ST:ADDRESS Tykistökatu 6B, P.O. Box 123 FIN-20521 Turku, Finland ST:EMAIL partho.sen@utu.fi ST:PHONE Phone: +358 469608145 #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 - 1022384032 NAFLD.Status:NASH_F2 RAW_FILE_NAME=0001_GC_20180723_0321.mzML SUBJECT_SAMPLE_FACTORS - 1022385756 NAFLD.Status:NASH_F3 RAW_FILE_NAME=0001_GC_20180730_0423.mzML SUBJECT_SAMPLE_FACTORS - 1022385774 NAFLD.Status:NASH_F3 RAW_FILE_NAME=0001_GC_20180703_0083.mzML SUBJECT_SAMPLE_FACTORS - 1022385788 NAFLD.Status:NAFL RAW_FILE_NAME=0001_GC_20180720_0300.mzML SUBJECT_SAMPLE_FACTORS - 1022385839 NAFLD.Status:NAFL RAW_FILE_NAME=0001_GC_20180719_0289.mzML SUBJECT_SAMPLE_FACTORS - 1022385844 NAFLD.Status:NASH_F2 RAW_FILE_NAME=0001_GC_20180801_0474.mzML SUBJECT_SAMPLE_FACTORS - 1022385855 NAFLD.Status:NASH_F4 / cirrhosis RAW_FILE_NAME=0001_GC_20180719_0290.mzML SUBJECT_SAMPLE_FACTORS - 1022385860 NAFLD.Status:NAFL RAW_FILE_NAME=0001_GC_20180720_0319.mzML SUBJECT_SAMPLE_FACTORS - 1022385877 NAFLD.Status:NAFL RAW_FILE_NAME=0030_GC_20190205_0053.mzML SUBJECT_SAMPLE_FACTORS - 1022385893 NAFLD.Status:NASH_F2 RAW_FILE_NAME=0001_GC_20180726_0393.mzML SUBJECT_SAMPLE_FACTORS - 1022385911 NAFLD.Status:NASH_F3 RAW_FILE_NAME=0001_GC_20180711_0192.mzML SUBJECT_SAMPLE_FACTORS - 1022385916 NAFLD.Status:NASH_F3 RAW_FILE_NAME=0001_GC_20180803_0515.mzML SUBJECT_SAMPLE_FACTORS - 1022385926 NAFLD.Status:NASH_F0/1 RAW_FILE_NAME=0001_GC_20180711_0195.mzML SUBJECT_SAMPLE_FACTORS - 1022385928 NAFLD.Status:NASH_F3 RAW_FILE_NAME=0001_GC_20180814_0641.mzML SUBJECT_SAMPLE_FACTORS - 1022385934 NAFLD.Status:NASH_F2 RAW_FILE_NAME=0001_GC_20180731_0443.mzML SUBJECT_SAMPLE_FACTORS - 1022386136 NAFLD.Status:NASH_F2 RAW_FILE_NAME=0001_GC_20180720_0305.mzML SUBJECT_SAMPLE_FACTORS - 1022386159 NAFLD.Status:NASH_F3 RAW_FILE_NAME=0001_GC_20180621_0051.mzML SUBJECT_SAMPLE_FACTORS - 1022386190 NAFLD.Status:NAFL RAW_FILE_NAME=0001_GC_20180813_0638.mzML SUBJECT_SAMPLE_FACTORS - 1022386192 NAFLD.Status:NASH_F2 RAW_FILE_NAME=0001_GC_20180815_0675.mzML SUBJECT_SAMPLE_FACTORS - 1022386202 NAFLD.Status:NAFL RAW_FILE_NAME=0001_GC_20180816_0694.mzML SUBJECT_SAMPLE_FACTORS - 1022386214 NAFLD.Status:NASH_F2 RAW_FILE_NAME=0001_GC_20180712_0216.mzML SUBJECT_SAMPLE_FACTORS - 1022386364 NAFLD.Status:NASH_F3 RAW_FILE_NAME=0001_GC_20180730_0421.mzML SUBJECT_SAMPLE_FACTORS - 1022386427 NAFLD.Status:NAFL RAW_FILE_NAME=0001_GC_20180718_0261.mzML SUBJECT_SAMPLE_FACTORS - 1022386429 NAFLD.Status:NASH_F0/1 RAW_FILE_NAME=0001_GC_20180814_0645.mzML SUBJECT_SAMPLE_FACTORS - 1022386446 NAFLD.Status:NASH_F2 RAW_FILE_NAME=0001_GC_20180806_0525.mzML SUBJECT_SAMPLE_FACTORS - 1022386453 NAFLD.Status:NASH_F2 RAW_FILE_NAME=0001_GC_20180816_0683.mzML SUBJECT_SAMPLE_FACTORS - 1022386463 NAFLD.Status:NAFL RAW_FILE_NAME=0001_GC_20180705_0120.mzML SUBJECT_SAMPLE_FACTORS - 1022386485 NAFLD.Status:NASH_F2 RAW_FILE_NAME=0001_GC_20180816_0684.mzML SUBJECT_SAMPLE_FACTORS - 1022386508 NAFLD.Status:NASH_F3 RAW_FILE_NAME=0001_GC_20180727_0417.mzML SUBJECT_SAMPLE_FACTORS - 1022386899 NAFLD.Status:NASH_F3 RAW_FILE_NAME=0001_GC_20180712_0210.mzML SUBJECT_SAMPLE_FACTORS - 1022386907 NAFLD.Status:NASH_F2 RAW_FILE_NAME=0001_GC_20180706_0124.mzML SUBJECT_SAMPLE_FACTORS - 1022386912 NAFLD.Status:NASH_F2 RAW_FILE_NAME=0001_GC_20180807_0546.mzML SUBJECT_SAMPLE_FACTORS - 1022386914 NAFLD.Status:NASH_F0/1 RAW_FILE_NAME=0001_GC_20180712_0201.mzML SUBJECT_SAMPLE_FACTORS - 1022386933 NAFLD.Status:NASH_F2 RAW_FILE_NAME=0001_GC_20180716_0230.mzML SUBJECT_SAMPLE_FACTORS - 1022386957 NAFLD.Status:NASH_F4 / cirrhosis RAW_FILE_NAME=0001_GC_20180711_0188.mzML SUBJECT_SAMPLE_FACTORS - 1022386958 NAFLD.Status:NAFL RAW_FILE_NAME=0001_GC_20180730_0428.mzML SUBJECT_SAMPLE_FACTORS - 1022386960 NAFLD.Status:NASH_F3 RAW_FILE_NAME=0001_GC_20180801_0467.mzML SUBJECT_SAMPLE_FACTORS - 1022386978 NAFLD.Status:NASH_F2 RAW_FILE_NAME=0001_GC_20180727_0409.mzML SUBJECT_SAMPLE_FACTORS - 1022386981 NAFLD.Status:NASH_F3 RAW_FILE_NAME=0001_GC_20180703_0084.mzML SUBJECT_SAMPLE_FACTORS - 1022387147 NAFLD.Status:NASH_F4 / cirrhosis RAW_FILE_NAME=0001_GC_20180815_0671.mzML SUBJECT_SAMPLE_FACTORS - 1022387276 NAFLD.Status:NASH_F4 / cirrhosis RAW_FILE_NAME=0001_GC_20180731_0446.mzML #COLLECTION CO:COLLECTION_SUMMARY Serum samples were randomized and sample preparation was carried out as CO:COLLECTION_SUMMARY described previously (Castilloet al. 2011). In summary, 400 µL of MeOH CO:COLLECTION_SUMMARY containing ISTDs (heptadecanoic acid, deuterium-labeled DL-valine, CO:COLLECTION_SUMMARY deuterium-labeled succinic acid, and deuterium-labeled glutamic acid, c = 1 CO:COLLECTION_SUMMARY µg/mL) was added to 30 µl of the serum samples which were vortex mixed and CO:COLLECTION_SUMMARY incubated on ice for 30 min after which they were centrifuged (9400 × g, 3 min) CO:COLLECTION_SUMMARY and 350 µL of the supernatant was collected after centrifugation. The solvent CO:COLLECTION_SUMMARY was evaporated to dryness and 25 µL of MOX reagent was added and the sample was CO:COLLECTION_SUMMARY incubated for 60 min at 45 °C. 25 µL of MSTFA was added and after 60 min CO:COLLECTION_SUMMARY incubation at 45 °C 25 µL of the retention index standard mixture (n-alkanes, CO:COLLECTION_SUMMARY c=10 µg/mL) was added. The analyses were carried out on an Agilent 7890B GC CO:COLLECTION_SUMMARY coupled to 7200 QTOF MS. Injection volume was 1 µL with 100:1 cold solvent CO:COLLECTION_SUMMARY split on PTV at 70 °C, heating to 300 °C at 120 °C/minute. Column: Zebron CO:COLLECTION_SUMMARY ZB-SemiVolatiles. Length: 20m, I.D. 0.18mm, film thickness: 0.18 µm. With CO:COLLECTION_SUMMARY initial Helium flow 1.2 mL/min, increasing to 2.4 mL/min after 16 mins. Oven CO:COLLECTION_SUMMARY temperature program: 50 °C (5 min), then to 270°C at 20 °C/min and then to CO:COLLECTION_SUMMARY 300 °C at 40 °C/min (5 min). EI source: 250 °C, 70 eV electron energy, 35µA CO:COLLECTION_SUMMARY emission, solvent delay 3 min. Mass range 55 to 650 amu, acquisition rate 5 CO:COLLECTION_SUMMARY spectra/s, acquisition time 200 ms/spectrum. Quad at 150 °C, 1.5 mL/min N2 CO:COLLECTION_SUMMARY collision flow, aux-2 temperature: 280 °C. Calibration curves were constructed CO:COLLECTION_SUMMARY using alanine, citric acid, fumaric acid, glutamic acid, glycine, lactic acid, CO:COLLECTION_SUMMARY malic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, linoleic acid, oleic CO:COLLECTION_SUMMARY acid, palmitic acid, stearic acid, cholesterol, fructose, glutamine, CO:COLLECTION_SUMMARY indole-3-propionic acid, isoleucine, leucine, proline, succinic acid, valine, CO:COLLECTION_SUMMARY asparagine, aspartic acid, arachidonic acid, glycerol-3-phosphate, lysine, CO:COLLECTION_SUMMARY methionine, ornithine, phenylalanine, serine and threonine purchased from CO:COLLECTION_SUMMARY Sigma-Aldrich (St. Louis, MO, USA) at concentration range of 0.1 to 80 µg/mL. CO:COLLECTION_SUMMARY An aliquot of each sample was collected and pooled and used as quality control CO:COLLECTION_SUMMARY samples, together with a NIST SRM 1950 serum sample and an in-house pooled serum CO:COLLECTION_SUMMARY sample. Relative standard deviations (% RSDs) of the metabolite concentrations CO:COLLECTION_SUMMARY in control serum samples showed % RSDs within accepted analytical limits at CO:COLLECTION_SUMMARY averages of 27.2% and 29.2% for in-house QC abd pooled QC samples. CO:SAMPLE_TYPE Blood (serum) #TREATMENT TR:TREATMENT_SUMMARY No treatment applied. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Serum samples were randomized and sample preparation was carried out as SP:SAMPLEPREP_SUMMARY described previously (Castilloet al. 2011). In summary, 400 µL of MeOH SP:SAMPLEPREP_SUMMARY containing ISTDs (heptadecanoic acid, deuterium-labeled DL-valine, SP:SAMPLEPREP_SUMMARY deuterium-labeled succinic acid, and deuterium-labeled glutamic acid, c = 1 SP:SAMPLEPREP_SUMMARY µg/mL) was added to 30 µl of the serum samples which were vortex mixed and SP:SAMPLEPREP_SUMMARY incubated on ice for 30 min after which they were centrifuged (9400 × g, 3 min) SP:SAMPLEPREP_SUMMARY and 350 µL of the supernatant was collected after centrifugation. The solvent SP:SAMPLEPREP_SUMMARY was evaporated to dryness and 25 µL of MOX reagent was added and the sample was SP:SAMPLEPREP_SUMMARY incubated for 60 min at 45 °C. 25 µL of MSTFA was added and after 60 min SP:SAMPLEPREP_SUMMARY incubation at 45 °C 25 µL of the retention index standard mixture (n-alkanes, SP:SAMPLEPREP_SUMMARY c=10 µg/mL) was added. The analyses were carried out on an Agilent 7890B GC SP:SAMPLEPREP_SUMMARY coupled to 7200 QTOF MS. Injection volume was 1 µL with 100:1 cold solvent SP:SAMPLEPREP_SUMMARY split on PTV at 70 °C, heating to 300 °C at 120 °C/minute. Column: Zebron SP:SAMPLEPREP_SUMMARY ZB-SemiVolatiles. Length: 20m, I.D. 0.18mm, film thickness: 0.18 µm. With SP:SAMPLEPREP_SUMMARY initial Helium flow 1.2 mL/min, increasing to 2.4 mL/min after 16 mins. Oven SP:SAMPLEPREP_SUMMARY temperature program: 50 °C (5 min), then to 270°C at 20 °C/min and then to SP:SAMPLEPREP_SUMMARY 300 °C at 40 °C/min (5 min). EI source: 250 °C, 70 eV electron energy, 35µA SP:SAMPLEPREP_SUMMARY emission, solvent delay 3 min. Mass range 55 to 650 amu, acquisition rate 5 SP:SAMPLEPREP_SUMMARY spectra/s, acquisition time 200 ms/spectrum. Quad at 150 °C, 1.5 mL/min N2 SP:SAMPLEPREP_SUMMARY collision flow, aux-2 temperature: 280 °C. Calibration curves were constructed SP:SAMPLEPREP_SUMMARY using alanine, citric acid, fumaric acid, glutamic acid, glycine, lactic acid, SP:SAMPLEPREP_SUMMARY malic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, linoleic acid, oleic SP:SAMPLEPREP_SUMMARY acid, palmitic acid, stearic acid, cholesterol, fructose, glutamine, SP:SAMPLEPREP_SUMMARY indole-3-propionic acid, isoleucine, leucine, proline, succinic acid, valine, SP:SAMPLEPREP_SUMMARY asparagine, aspartic acid, arachidonic acid, glycerol-3-phosphate, lysine, SP:SAMPLEPREP_SUMMARY methionine, ornithine, phenylalanine, serine and threonine purchased from SP:SAMPLEPREP_SUMMARY Sigma-Aldrich (St. Louis, MO, USA) at concentration range of 0.1 to 80 µg/mL. SP:SAMPLEPREP_SUMMARY An aliquot of each sample was collected and pooled and used as quality control SP:SAMPLEPREP_SUMMARY samples, together with a NIST SRM 1950 serum sample and an in-house pooled serum SP:SAMPLEPREP_SUMMARY sample. Relative standard deviations (% RSDs) of the metabolite concentrations SP:SAMPLEPREP_SUMMARY in control serum samples showed % RSDs within accepted analytical limits at SP:SAMPLEPREP_SUMMARY averages of 27.2% and 29.2% for in-house QC abd pooled QC samples. SP:PROCESSING_STORAGE_CONDITIONS -20? SP:EXTRACT_STORAGE -80? #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890B CH:COLUMN_NAME Zebron ZB-SemiVolatiles (20m x 0.18mm, 0.18µm) CH:CHROMATOGRAPHY_COMMENTS Zebron ZB-SemiVolatiles (20m, I.D. 0.18mm, film thickness 0.18µm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 7200 QTOF MS:INSTRUMENT_TYPE GC x GC-TOF MS:MS_TYPE EI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS In summary, 400 µL of MeOH containing ISTDs (heptadecanoic acid, MS:MS_COMMENTS deuterium-labeled DL-valine, deuterium-labeled succinic acid, and MS:MS_COMMENTS deuterium-labeled glutamic acid, c = 1 µg/mL) was added to 30 µl of the serum MS:MS_COMMENTS samples which were vortex mixed and incubated on ice for 30 min after which they MS:MS_COMMENTS were centrifuged (9400 × g, 3 min) and 350 µL of the supernatant was collected MS:MS_COMMENTS after centrifugation. The solvent was evaporated to dryness and 25 µL of MOX MS:MS_COMMENTS reagent was added and the sample was incubated for 60 min at 45 °C. 25 µL of MS:MS_COMMENTS MSTFA was added and after 60 min incubation at 45 °C 25 µL of the retention MS:MS_COMMENTS index standard mixture (n-alkanes, c=10 µg/mL) was added. The analyses were MS:MS_COMMENTS carried out on an Agilent 7890B GC coupled to 7200 QTOF MS. Injection volume was MS:MS_COMMENTS 1 µL with 100:1 cold solvent split on PTV at 70 °C, heating to 300 °C at 120 MS:MS_COMMENTS °C/minute. Column: Zebron ZB-SemiVolatiles. Length: 20m, I.D. 0.18mm, film MS:MS_COMMENTS thickness: 0.18 µm. With initial Helium flow 1.2 mL/min, increasing to 2.4 MS:MS_COMMENTS mL/min after 16 mins. Oven temperature program: 50 °C (5 min), then to 270°C MS:MS_COMMENTS at 20 °C/min and then to 300 °C at 40 °C/min (5 min). EI source: 250 °C, 70 MS:MS_COMMENTS eV electron energy, 35µA emission, solvent delay 3 min. Mass range 55 to 650 MS:MS_COMMENTS amu, acquisition rate 5 spectra/s, acquisition time 200 ms/spectrum. Quad at 150 MS:MS_COMMENTS °C, 1.5 mL/min N2 collision flow, aux-2 temperature: 280 °C. Calibration MS:MS_COMMENTS curves were constructed using alanine, citric acid, fumaric acid, glutamic acid, MS:MS_COMMENTS glycine, lactic acid, malic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, MS:MS_COMMENTS linoleic acid, oleic acid, palmitic acid, stearic acid, cholesterol, fructose, MS:MS_COMMENTS glutamine, indole-3-propionic acid, isoleucine, leucine, proline, succinic acid, MS:MS_COMMENTS valine, asparagine, aspartic acid, arachidonic acid, glycerol-3-phosphate, MS:MS_COMMENTS lysine, methionine, ornithine, phenylalanine, serine and threonine purchased MS:MS_COMMENTS from Sigma-Aldrich (St. Louis, MO, USA) at concentration range of 0.1 to 80 MS:MS_COMMENTS µg/mL. An aliquot of each sample was collected and pooled and used as quality MS:MS_COMMENTS control samples, together with a NIST SRM 1950 serum sample and an in-house MS:MS_COMMENTS pooled serum sample. Relative standard deviations (% RSDs) of the metabolite MS:MS_COMMENTS concentrations in control serum samples showed % RSDs within accepted analytical MS:MS_COMMENTS limits at averages of 27.2% and 29.2% for in-house QC abd pooled QC samples. MS:MS_COMMENTS Data values for final analysis were log2-transformed and scaled to zero mean and MS:MS_COMMENTS unit variance. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS log2 autoscaled abundance MS_METABOLITE_DATA_START Samples 1022384032 1022385756 1022385774 1022385788 1022385839 1022385844 1022385855 1022385860 1022385877 1022385893 1022385911 1022385916 1022385926 1022385928 1022385934 1022386136 1022386159 1022386190 1022386192 1022386202 1022386214 1022386364 1022386427 1022386429 1022386446 1022386453 1022386463 1022386485 1022386508 1022386899 1022386907 1022386912 1022386914 1022386933 1022386957 1022386958 1022386960 1022386978 1022386981 1022387147 1022387276 Factors NAFLD.Status:NASH_F2 NAFLD.Status:NASH_F3 NAFLD.Status:NASH_F3 NAFLD.Status:NAFL NAFLD.Status:NAFL NAFLD.Status:NASH_F2 NAFLD.Status:NASH_F4 / cirrhosis NAFLD.Status:NAFL NAFLD.Status:NAFL NAFLD.Status:NASH_F2 NAFLD.Status:NASH_F3 NAFLD.Status:NASH_F3 NAFLD.Status:NASH_F0/1 NAFLD.Status:NASH_F3 NAFLD.Status:NASH_F2 NAFLD.Status:NASH_F2 NAFLD.Status:NASH_F3 NAFLD.Status:NAFL NAFLD.Status:NASH_F2 NAFLD.Status:NAFL NAFLD.Status:NASH_F2 NAFLD.Status:NASH_F3 NAFLD.Status:NAFL NAFLD.Status:NASH_F0/1 NAFLD.Status:NASH_F2 NAFLD.Status:NASH_F2 NAFLD.Status:NAFL NAFLD.Status:NASH_F2 NAFLD.Status:NASH_F3 NAFLD.Status:NASH_F3 NAFLD.Status:NASH_F2 NAFLD.Status:NASH_F2 NAFLD.Status:NASH_F0/1 NAFLD.Status:NASH_F2 NAFLD.Status:NASH_F4 / cirrhosis NAFLD.Status:NAFL NAFLD.Status:NASH_F3 NAFLD.Status:NASH_F2 NAFLD.Status:NASH_F3 NAFLD.Status:NASH_F4 / cirrhosis NAFLD.Status:NASH_F4 / cirrhosis Catacerebroside A 5.952098457 5.335054789 5.804564475 4.607239754 5.675413151 6.396935407 7.007978095 6.141826764 5.600843006 5.95695852 2.934156774 6.213908696 5.808312451 6.795125342 5.024062539 0.479119819 6.055657271 4.561981036 3.711503717 5.619333772 5.553102417 6.233611049 4.571566389 6.825760192 4.892944681 6.449267028 5.267816295 5.361366931 5.458304565 5.856083101 5.804880382 5.68402369 5.113246669 6.216332437 4.247125456 6.669368401 5.158153608 5.838653065 5.960843433 4.861218449 4.682605319 Catacerebroside B 2.895302621 -0.943416472 1.992768431 2.097610797 3.852997588 2.192194165 4.244125943 2.825785627 2.837943242 3.080657663 1.807354922 1.944858446 2.61117238 3.588564737 1.722466024 2.427606173 1.948600847 2.438292852 1.827819025 2.198494154 3.883620816 2.912649865 1.773996325 2.572889668 1.327687364 2.204766751 2.260025656 1.169925001 3.033863452 2.63691458 1.782408565 1.682573297 2.336283388 3.288358562 0.773996325 2.060047384 2.192194165 2.307428525 1.298658316 1.5360529 1.895302621 Cer(d40:1) 5.211012193 4.96208625 5.171527106 4.382667253 5.452529545 3.97819563 4.83541884 4.599912842 3.465974465 5.978882028 4.062639828 4.055716264 4.276496666 4.487357715 3.735522177 4.945794957 3.47118746 2.90303827 2.35614381 2.737686761 4.529196268 5.57198012 3.448900951 4.48864354 3.109360559 3.559491813 3.807354922 3.64385619 5 4.910252963 4.947666157 3.650764559 3.657640005 5.308521062 2.513490746 3.691534165 3.803227036 5.136683578 4.14974712 3.220329955 3.503348735 Cer(d18:1/16:0) 2.794935663 2.485426827 1.678071905 2.063502942 2.304511042 1.580145484 2.087462841 2.538538164 1.910732662 2.709290636 1.731183242 2.3950628 2.451540833 2.50589093 1.731183242 2.408711861 1.432959407 1.691534165 1.948600847 1.565597176 2.565597176 2.815575429 2.053111336 2.207892852 1.464668267 1.15704371 2.618238656 1.748461233 2.769771739 2.974529312 2.897240426 1.974529312 2.301587647 2.021479727 1.22650853 1.786596362 2.14404637 2.533563348 1.438292852 1.891419187 1.974529312 Cer(d18:1/22:0) 6.364747506 5.886306263 6.225930411 5.516015147 6.386293635 4.970853654 5.883376437 5.646450706 4.550900665 6.927303898 5.197314999 5.037382222 5.621758857 5.545350645 4.817111573 5.848497755 4.580748492 3.929790998 3.211012193 3.641546029 5.583759754 6.498570013 4.57167681 5.607034346 4.262282806 4.566206293 4.974529312 4.645009884 6.054197294 5.991408467 6.017253839 4.578938713 4.820178962 6.374517881 3.624100895 4.9202933 4.815575429 6.221490455 5.458447627 4.409390936 4.483493351 Cer(d18:1/23:0) 6.088311236 5.809928866 5.80451827 5.097189387 6.367021533 4.567423758 6.106850796 5.334854269 4.231893162 6.675957033 5.401903472 5.28132733 5.486392594 5.34020618 4.940636607 5.903520374 4.463360886 4.21490191 3.23572706 3.821710215 5.402926781 6.446917865 4.166715445 5.571373436 4.456806149 4.646162657 5.112283109 4.72410452 6.018812186 5.945560886 5.821710215 4.530445355 4.661635602 6.252097703 3.447579197 4.828326926 4.782932705 6.057883449 5.076815597 4.363171077 4.170726276 Cer(m42:0) 3.820689561 3.791814071 2.813524689 2.23878686 2.042644337 2.82984956 3.221877081 3.667892125 2.480265122 4.776103988 3.023255352 2.773996325 3.237257771 3.608809243 2.244887059 3.852997588 2.166715445 1.176322773 1.15704371 1.201633861 2.508428653 3.767654798 3.252476214 2.897240426 0.963474124 2.432959407 1.555816155 1.855989697 3.340562269 3.595742339 3.187451054 2.427606173 2.063502942 3.745237332 1.298658316 2.518535139 2.073820233 3.634593268 4.202417722 2.718087584 2.14404637 Cer(d18:1/24:0) 8.111448698 7.368506462 7.196725061 6.954777925 7.822284016 7.035293987 7.925049965 7.826993303 6.678212786 9.054767095 7.377557603 7.315330737 7.727171692 7.413458688 6.557042415 7.678001459 6.621319301 5.784503983 5.050066298 5.799605422 7.466708681 8.453805629 6.604219619 7.480992104 6.102238194 7.067380578 6.710806434 6.238022518 7.725809309 7.892694591 7.996953601 6.487196907 6.656639374 8.613052738 5.269407241 6.845364594 6.688739868 8.193673195 7.691743519 6.666614587 6.116031993 Cer(d18:1/24:1) 6.804389199 6.680465018 6.788555215 5.844988157 7.017365205 5.712320641 6.866413481 6.653060017 5.079804755 7.618091794 5.925999419 5.702380502 6.336640446 6.350497247 5.833396134 6.124534929 5.267535798 5.377470843 4.915042791 4.819157224 6.503507754 7.209550842 5.268284667 6.056149961 4.878725341 4.979110755 5.607034346 5.332707934 6.738632747 6.728464765 6.666472569 5.395405605 5.391630262 6.972118218 4.083213368 5.123086751 5.985955755 6.540244302 5.440952198 5.032100843 5.356848079 Cer(d18:1/25:0) 3.486714373 3.896271849 3.847996907 2.361768359 4.295723025 2.596935142 4.377123749 3.813524689 1.646162657 4.915998852 3.021479727 4.213347282 3.771885579 3.821710215 3.22342255 3.331991778 2.454175893 2.66448284 2.140778656 2.788685711 3.934516502 4.855491443 2.86393845 4.237257771 2.965322548 2.757023247 3.353323291 2.671293372 4.004501392 4.348374075 2.89917563 1.941106311 3.59812696 4.412104045 1.790772038 2.937344392 2.275007047 4.277984747 3.185866545 2.189033824 2.189033824 Cer(d35:1) 9.062882631 9.056908618 8.863814587 9.003686556 9.005456131 9.052812555 9.00739175 9.02017436 9.025361072 9.037135089 8.983991141 9.037382222 8.960494334 8.986922842 9.024447124 9.015415052 8.928281484 9.036008724 8.997744026 8.888134388 8.945209709 9.065254555 9.016028242 9.003686556 9.024474828 8.923327485 8.90529674 8.871812853 9.09816371 8.979653836 8.952974156 9.052078919 8.939579214 8.94976836 8.978624667 9.012568674 9.039988272 9.073606041 8.836744702 8.978967805 8.996303999 Cer(d42:1) 7.189330401 6.455491621 6.0725346 5.962549023 6.71918344 6.00966069 6.794025896 6.847245307 5.765534746 8.014913158 6.197314999 6.319401167 6.726831217 6.384395238 5.548128324 6.755688777 5.496014873 4.991408467 4.00270252 4.605257263 6.463360886 7.553437306 5.499208091 6.442777659 5.13996057 5.890689878 5.675815931 5.13422094 6.904965719 6.873567152 6.868760834 5.540399306 5.630230716 7.460414939 4.231893162 5.815063017 5.635754391 7.279378431 6.322829498 5.581953751 5.168321116 Cholecalciferol (D3) 2.584962501 1.516015147 1.739848103 1.438292852 2.307428525 1.769771739 1.575312331 1.855989697 1.786596362 1.855989697 1.526068812 1.448900951 1.851998837 2.61117238 1.604071324 1.709290636 1.244887059 2.163498732 2.411426246 1.86393845 1.443606651 2.673556424 0.855989697 1.933572638 1.097610797 1.485426827 1.49057013 2.100977648 1.851998837 2.336283388 2.189033824 1.500802053 0.855989697 1.655351829 0.084064265 1.608809243 1.137503524 2 1.495695163 1.941106311 1.682573297 GlcCer(d18:1/20:0) 3.367371066 2.456806149 1.735522177 2.513490746 3.220329955 2.244887059 4.339850003 2.528571319 2.327687364 3.702657543 2.289834465 0.90303827 2.295723025 2.176322773 0.790772038 2.64385619 2.09085343 1.765534746 1.50589093 0.411426246 2.704871964 2.901108243 3.478971805 2.163498732 1.084064265 0.98550043 2.709290636 0.765534746 3.212569339 3.404630684 2.86393845 1.761285273 2.659924558 2.587364991 1.748461233 1.735522177 2.384049807 4.080657663 1.769771739 1.459431619 2.392317423 GlcCer(d18:1/24:0) 5.475409193 2.217230716 2.922197848 2.998195503 3.836934011 3.160274831 3.528571319 5.102658131 3.350497247 4.397802962 3.054848477 2.95419631 3.320484678 4.197708158 3.033863452 4.768713657 2.392317423 3.357552005 3.424922088 2.942983598 4.052241981 3.874796966 1.831877241 5.578032972 2.361768359 3.752748591 3.481557281 3.23572706 3.333423734 4.667892125 2.759155834 3.155425432 2.403267722 3.480265122 1.10433666 3.700439718 2.843983844 3.958842675 0.632268215 4.224966365 2.066950244 HexCer(d18:1/22:0) 5.470536865 3.23878686 5.037821465 4.860962798 5.08491466 4.529196268 4.841469999 4.818646083 4.286881148 5.115615931 4.151371776 3.617063344 4.35473424 5.021923839 4.412104045 4.693208149 3.956056652 3.23572706 3.220329955 3.646162657 4.540399306 5.348019909 3.399171094 5.154210531 3.434294618 4.397118409 4.838447593 3.908812908 4.622344723 4.646738698 4.920769673 3.109360559 4.54225805 4.269781238 2.757023247 4.847996907 3.181102551 5.593652558 3.670160514 4.017921908 3.798050515 HexCer(d18:1/23:0) 4.131754091 2.580145484 4.574101508 3.22342255 4.070389328 2.639232163 4.052241981 3.037382222 2.533563348 3.679198571 2.893362211 2.613531653 3.016139703 3.295723025 2.901108243 3.552131108 2.056583528 2.272023189 1.761285273 2.22650853 3.351910961 4.107687869 2.364572432 3.970853654 2.358958826 2.722466024 3.565597176 2.472487771 3.484138131 3.727920455 3.03562391 2.066950244 3.407352751 3.078951341 2.070389328 3.305970521 2.17951105 3.791814071 1.782408565 2.257010618 2.748461233 HexCer(d18:1/24:0) 5.456149035 3.655351829 5.836934011 5.049630768 5.252854626 4.524189078 5.484138131 5.14974712 4.702103407 5.706530553 4.603477988 3.725741157 4.72955277 4.927422429 4.510961919 4.880685525 4.063502942 3.073820233 3.514753498 3.917431755 4.7382274 5.476705707 3.851998837 5.14974712 3.720278465 4.442943496 4.885574364 3.710393192 4.850999395 5.262282806 4.828326926 3.016139703 4.995032192 4.655924213 3.117695043 5.01658546 3.196921734 5.64038956 3.702657543 4.203201156 4.040892431 HexCer(d18:1/24:1) 8.258283356 6.554128344 7.72900887 6.805937293 7.823813037 8.1237593 8.264348763 7.724582064 7.725127635 8.004220466 7.302684614 7.85124932 7.998082647 8.474395481 7.117695043 8.062423969 7.670018844 7.207209596 5.58345891 7.163196797 8.006859013 8.439249119 6.493295071 8.670762456 7.040673293 7.619633096 8.153248008 7.365360097 7.62388149 8.243459637 8.208185577 7.778734244 7.307610672 8.059777069 5.481557281 8.368899275 6.882520782 7.759022639 6.967514481 6.66831772 6.63531908 Retinyl Palmitate 0.545968369 0.189033824 -2.184424571 0.918386234 1.344828497 0.584962501 1.176322773 0.545968369 1.207892852 2.358958826 0.344828497 2.09085343 1.411426246 1.891419187 1.250961574 2.14404637 0.669026766 1.438292852 -1.152003093 0.464668267 0.739848103 2.629939409 0.042644337 1.182692298 -1.089267338 -0.074000581 0.650764559 1.03562391 2.592158002 2.669026766 2.066950244 -0.514573173 0.815575429 1.618238656 -1.358453971 0.454175893 0.028569152 2.153805336 0.910732662 0.333423734 0.042644337 Retinol 1.752748591 -1.089267338 1.316145742 2.35614381 1.03562391 1.286881148 2.807354922 2.531069493 1.124328135 2.584962501 1.769771739 1.86393845 1.89917563 2.007195501 1.316145742 1.851998837 1.480265122 0.321928095 -0.251538767 1.042644337 1.244887059 1.811471031 1.500802053 2.166715445 0.545968369 1.169925001 1.372952098 1.169925001 1.23878686 1.906890596 1.201633861 1.014355293 1.40599236 2.682573297 0.831877241 1.389566812 1.361768359 2.025028794 1.063502942 1.63691458 0.40053793 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m/z retention time Catacerebroside A TBC TBC Catacerebroside B TBC TBC Cer(d40:1) TBC TBC Cer(d18:1/16:0) TBC TBC Cer(d18:1/22:0) TBC TBC Cer(d18:1/23:0) TBC TBC Cer(m42:0) TBC TBC Cer(d18:1/24:0) TBC TBC Cer(d18:1/24:1) TBC TBC Cer(d18:1/25:0) TBC TBC Cer(d35:1) TBC TBC Cer(d42:1) TBC TBC Cholecalciferol (D3) TBC TBC GlcCer(d18:1/20:0) TBC TBC GlcCer(d18:1/24:0) TBC TBC HexCer(d18:1/22:0) TBC TBC HexCer(d18:1/23:0) TBC TBC HexCer(d18:1/24:0) TBC TBC HexCer(d18:1/24:1) TBC TBC Retinyl Palmitate TBC TBC Retinol TBC TBC METABOLITES_END #END