#METABOLOMICS WORKBENCH wenchaowei_20230317_221807 DATATRACK_ID:3794 STUDY_ID:ST002514 ANALYSIS_ID:AN004140 VERSION 1 CREATED_ON 06-30-2023 #PROJECT PR:PROJECT_TITLE The investigation of the role of dietary inulin in Non-alcoholic Steatohepatits PR:PROJECT_TITLE NASH progression PR:PROJECT_TYPE non-targeted LC-MS analysis PR:PROJECT_SUMMARY To investigate the role of dietary fiber in non-alcoholic steatohepatitis (NASH) PR:PROJECT_SUMMARY progression, male C57 mice was randomly assigned into four groups that received PR:PROJECT_SUMMARY normal chow diet (NCD), choline deficient high fat diet (CDHFD), CDHFD + 10% PR:PROJECT_SUMMARY inulin (CDHFD-I), CDHFD + 10% Cellulose (CDHFD-C). Mice stool and portal vein PR:PROJECT_SUMMARY serum was collected. Additionally, some mice received inulin or cellulose was PR:PROJECT_SUMMARY treated with 13C labelled fiber for 36 hours. Metabolites were analyzed by PR:PROJECT_SUMMARY non-targeted metabolomics. PR:INSTITUTE The Chinese University of Hong Kong PR:DEPARTMENT Medicine PR:LAST_NAME Wei PR:FIRST_NAME Wenchao PR:ADDRESS Prince of Wales Hospital PR:EMAIL 1155118538@link.cuhk.edu.hk PR:PHONE 56001913 PR:PUBLICATIONS https://www.nature.com/articles/s41564-023-01418-7 PR:DOI http://dx.doi.org/10.21228/M81T5C #STUDY ST:STUDY_TITLE The investigation of the role of dietary inulin in NASH progression with mouse ST:STUDY_TITLE fecal metabolites ST:STUDY_TYPE non-targeted LC-Mass analysis ST:STUDY_SUMMARY To investigate the role of dietary fiber in non-alcoholic steatohepatitis (NASH) ST:STUDY_SUMMARY progression, male C57 mice was randomly assigned into four groups that received ST:STUDY_SUMMARY normal chow diet (NCD), choline deficient high fat diet (CDHFD), CDHFD + 10% ST:STUDY_SUMMARY inulin (CDHFD-I), CDHFD + 10% Cellulose (CDHFD-C). Additionally, some mice ST:STUDY_SUMMARY received inulin or cellulose was treated with 13C labelled fiber for 36 hours. ST:STUDY_SUMMARY Fecal metabolites were analyzed by non-targeted metabolomics.Results shown that ST:STUDY_SUMMARY the fecal metabolites from Inulin treatment group signficantly distinguished ST:STUDY_SUMMARY from that from CDHFD only group, while only negligible alterations was induced ST:STUDY_SUMMARY by cellulose treatment, indicating that inulin not cellulose could be well ST:STUDY_SUMMARY fermented by gut microbiota. The 13C tracing results shown that nineteen ST:STUDY_SUMMARY 13C-inulin labelled metabolites were also captured including pantothenate, ST:STUDY_SUMMARY phosphoethanolamine and adenosine. These metabolites are reported to play a ST:STUDY_SUMMARY protective role in reducing fat accumulation and ameliorating cellular oxidative ST:STUDY_SUMMARY stress and inflammation, indicating that the mechanism inulin suppresses NASH ST:STUDY_SUMMARY may through mediating modulating gut metabolites. ST:INSTITUTE The Chinese University of Hong Kong ST:LAST_NAME Wei ST:FIRST_NAME Wenchao ST:ADDRESS Prince of Wales Hospital ST:EMAIL 1155118538@link.cuhk.edu.hk ST:PHONE 56001913 ST:SUBMIT_DATE 2023-03-17 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57 BL/6 SU:AGE_OR_AGE_RANGE 23-24 weeks SU:WEIGHT_OR_WEIGHT_RANGE 30-60g SU:HEIGHT_OR_HEIGHT_RANGE N/A SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER The Laboratory Animal Services Centre in the Chinese univeristy of Hong Kong SU:ANIMAL_HOUSING The Laboratory Animal Services Centre in the Chinese univeristy of Hong Kong SU:ANIMAL_LIGHT_CYCLE 12/12 SU:ANIMAL_FEED libitum SU:ANIMAL_WATER libitum #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - F1125_neg Samplet_Type:Mice Stool | Treatment:CDHFD RAW_FILE_NAME=F1125_neg-F1125.mzML SUBJECT_SAMPLE_FACTORS - F1125_pos Samplet_Type:Mice Stool | Treatment:CDHFD RAW_FILE_NAME=F1125_pos-F1125.mzML SUBJECT_SAMPLE_FACTORS - F1236_neg Samplet_Type:Mice Stool | Treatment:CDHFD RAW_FILE_NAME=F1236_neg-F1236.mzML SUBJECT_SAMPLE_FACTORS - F1236_pos Samplet_Type:Mice Stool | Treatment:CDHFD RAW_FILE_NAME=F1236_pos-F1236.mzML SUBJECT_SAMPLE_FACTORS - F1241_neg Samplet_Type:Mice Stool | Treatment:CDHFD RAW_FILE_NAME=F1241_neg-F1241.mzML SUBJECT_SAMPLE_FACTORS - F1241_pos Samplet_Type:Mice Stool | Treatment:CDHFD RAW_FILE_NAME=F1241_pos-F1241.mzML SUBJECT_SAMPLE_FACTORS - F1242_neg Samplet_Type:Mice Stool | Treatment:CDHFD RAW_FILE_NAME=F1242_neg-F1242.mzML SUBJECT_SAMPLE_FACTORS - F1242_pos Samplet_Type:Mice Stool | Treatment:CDHFD RAW_FILE_NAME=F1242_pos-F1242.mzML SUBJECT_SAMPLE_FACTORS - F1246_neg Samplet_Type:Mice Stool | Treatment:CDHFD RAW_FILE_NAME=F1246_neg-F1246.mzML SUBJECT_SAMPLE_FACTORS - F1246_pos Samplet_Type:Mice Stool | Treatment:CDHFD RAW_FILE_NAME=F1246_pos-F1246.mzML SUBJECT_SAMPLE_FACTORS - F1203_neg Samplet_Type:Mice Stool | Treatment:CDHFD-13C-C RAW_FILE_NAME=F1203_neg-F1203.mzML SUBJECT_SAMPLE_FACTORS - F1203_pos Samplet_Type:Mice Stool | Treatment:CDHFD-13C-C RAW_FILE_NAME=F1203_pos-F1203.mzML SUBJECT_SAMPLE_FACTORS - F1249_neg Samplet_Type:Mice Stool | Treatment:CDHFD-13C-C RAW_FILE_NAME=F1249_neg-F1249.mzML SUBJECT_SAMPLE_FACTORS - F1249_pos Samplet_Type:Mice Stool | Treatment:CDHFD-13C-C RAW_FILE_NAME=F1249_pos-F1249.mzML SUBJECT_SAMPLE_FACTORS - F1250_neg Samplet_Type:Mice Stool | Treatment:CDHFD-13C-C RAW_FILE_NAME=F1250_neg-F1250.mzML SUBJECT_SAMPLE_FACTORS - F1250_pos Samplet_Type:Mice Stool | Treatment:CDHFD-13C-C RAW_FILE_NAME=F1250_pos-F1250.mzML SUBJECT_SAMPLE_FACTORS - F1251_neg Samplet_Type:Mice Stool | Treatment:CDHFD-13C-C RAW_FILE_NAME=F1251_neg-F1251.mzML SUBJECT_SAMPLE_FACTORS - F1251_pos Samplet_Type:Mice Stool | Treatment:CDHFD-13C-C RAW_FILE_NAME=F1251_pos-F1251.mzML SUBJECT_SAMPLE_FACTORS - F1257_neg Samplet_Type:Mice Stool | Treatment:CDHFD-13C-C RAW_FILE_NAME=F1257_neg-F1257.mzML SUBJECT_SAMPLE_FACTORS - F1257_pos Samplet_Type:Mice Stool | Treatment:CDHFD-13C-C RAW_FILE_NAME=F1257_pos-F1257.mzML SUBJECT_SAMPLE_FACTORS - F1259_neg Samplet_Type:Mice Stool | Treatment:CDHFD-13C-C RAW_FILE_NAME=F1259_neg-F1259.mzML SUBJECT_SAMPLE_FACTORS - F1259_pos Samplet_Type:Mice Stool | Treatment:CDHFD-13C-C RAW_FILE_NAME=F1259_pos-F1259.mzML SUBJECT_SAMPLE_FACTORS - F1218_neg Samplet_Type:Mice Stool | Treatment:CDHFD-13C-I RAW_FILE_NAME=F1218_neg-F1218.mzML SUBJECT_SAMPLE_FACTORS - F1218_pos Samplet_Type:Mice Stool | Treatment:CDHFD-13C-I RAW_FILE_NAME=F1218_pos-F1218.mzML SUBJECT_SAMPLE_FACTORS - F1225_neg Samplet_Type:Mice Stool | Treatment:CDHFD-13C-I RAW_FILE_NAME=F1225_neg-F1225.mzML SUBJECT_SAMPLE_FACTORS - F1225_pos Samplet_Type:Mice Stool | Treatment:CDHFD-13C-I RAW_FILE_NAME=F1225_pos-F1225.mzML SUBJECT_SAMPLE_FACTORS - F1227_neg Samplet_Type:Mice Stool | Treatment:CDHFD-13C-I RAW_FILE_NAME=F1227_neg-F1227.mzML SUBJECT_SAMPLE_FACTORS - F1227_pos Samplet_Type:Mice Stool | Treatment:CDHFD-13C-I RAW_FILE_NAME=F1227_pos-F1227.mzML SUBJECT_SAMPLE_FACTORS - F1228_neg Samplet_Type:Mice Stool | Treatment:CDHFD-13C-I RAW_FILE_NAME=F1228_neg-F1228.mzML SUBJECT_SAMPLE_FACTORS - F1228_pos Samplet_Type:Mice Stool | Treatment:CDHFD-13C-I RAW_FILE_NAME=F1228_pos-F1228.mzML SUBJECT_SAMPLE_FACTORS - F1280_neg Samplet_Type:Mice Stool | Treatment:CDHFD-13C-I RAW_FILE_NAME=F1280_neg-F1280.mzML SUBJECT_SAMPLE_FACTORS - F1280_pos Samplet_Type:Mice Stool | Treatment:CDHFD-13C-I RAW_FILE_NAME=F1280_pos-F1280.mzML SUBJECT_SAMPLE_FACTORS - F1247_neg Samplet_Type:Mice Stool | Treatment:CDHFD-C RAW_FILE_NAME=F1247_neg-F1247.mzML SUBJECT_SAMPLE_FACTORS - F1247_pos Samplet_Type:Mice Stool | Treatment:CDHFD-C RAW_FILE_NAME=F1247_pos-F1247.mzML SUBJECT_SAMPLE_FACTORS - F1253_neg Samplet_Type:Mice Stool | Treatment:CDHFD-C RAW_FILE_NAME=F1253_neg-F1253.mzML SUBJECT_SAMPLE_FACTORS - F1253_pos Samplet_Type:Mice Stool | Treatment:CDHFD-C RAW_FILE_NAME=F1253_pos-F1253.mzML SUBJECT_SAMPLE_FACTORS - F1254_neg Samplet_Type:Mice Stool | Treatment:CDHFD-C RAW_FILE_NAME=F1254_neg-F1254.mzML SUBJECT_SAMPLE_FACTORS - F1254_pos Samplet_Type:Mice Stool | Treatment:CDHFD-C RAW_FILE_NAME=F1254_pos-F1254.mzML SUBJECT_SAMPLE_FACTORS - F1255_neg Samplet_Type:Mice Stool | Treatment:CDHFD-C RAW_FILE_NAME=F1255_neg-F1255.mzML SUBJECT_SAMPLE_FACTORS - F1255_pos Samplet_Type:Mice Stool | Treatment:CDHFD-C RAW_FILE_NAME=F1255_pos-F1255.mzML SUBJECT_SAMPLE_FACTORS - F1216_neg Samplet_Type:Mice Stool | Treatment:CDHFD-I RAW_FILE_NAME=F1216_neg-F1216.mzML SUBJECT_SAMPLE_FACTORS - F1216_pos Samplet_Type:Mice Stool | Treatment:CDHFD-I RAW_FILE_NAME=F1216_pos-F1216.mzML SUBJECT_SAMPLE_FACTORS - F1217_neg Samplet_Type:Mice Stool | Treatment:CDHFD-I RAW_FILE_NAME=F1217_neg-F1217.mzML SUBJECT_SAMPLE_FACTORS - F1217_pos Samplet_Type:Mice Stool | Treatment:CDHFD-I RAW_FILE_NAME=F1217_pos-F1217.mzML SUBJECT_SAMPLE_FACTORS - F1222_neg Samplet_Type:Mice Stool | Treatment:CDHFD-I RAW_FILE_NAME=F1222_neg-F1222.mzML SUBJECT_SAMPLE_FACTORS - F1222_pos Samplet_Type:Mice Stool | Treatment:CDHFD-I RAW_FILE_NAME=F1222_pos-F1222.mzML SUBJECT_SAMPLE_FACTORS - F1223_neg Samplet_Type:Mice Stool | Treatment:CDHFD-I RAW_FILE_NAME=F1223_neg-F1223.mzML SUBJECT_SAMPLE_FACTORS - F1223_pos Samplet_Type:Mice Stool | Treatment:CDHFD-I RAW_FILE_NAME=F1223_pos-F1223.mzML SUBJECT_SAMPLE_FACTORS - F1230_neg Samplet_Type:Mice Stool | Treatment:CDHFD-I RAW_FILE_NAME=F1230_neg-F1230.mzML SUBJECT_SAMPLE_FACTORS - F1230_pos Samplet_Type:Mice Stool | Treatment:CDHFD-I RAW_FILE_NAME=F1230_pos-F1230.mzML SUBJECT_SAMPLE_FACTORS - F1261_neg Samplet_Type:Mice Stool | Treatment:NCD RAW_FILE_NAME=F1261_neg-F1261.mzML SUBJECT_SAMPLE_FACTORS - F1261_pos Samplet_Type:Mice Stool | Treatment:NCD RAW_FILE_NAME=F1261_pos-F1261.mzML SUBJECT_SAMPLE_FACTORS - F1262_neg Samplet_Type:Mice Stool | Treatment:NCD RAW_FILE_NAME=F1262_neg-F1262.mzML SUBJECT_SAMPLE_FACTORS - F1262_pos Samplet_Type:Mice Stool | Treatment:NCD RAW_FILE_NAME=F1262_pos-F1262.mzML SUBJECT_SAMPLE_FACTORS - F1263_neg Samplet_Type:Mice Stool | Treatment:NCD RAW_FILE_NAME=F1263_neg-F1263.mzML SUBJECT_SAMPLE_FACTORS - F1263_pos Samplet_Type:Mice Stool | Treatment:NCD RAW_FILE_NAME=F1263_pos-F1263.mzML SUBJECT_SAMPLE_FACTORS - F1264_neg Samplet_Type:Mice Stool | Treatment:NCD RAW_FILE_NAME=F1264_neg-F1264.mzML SUBJECT_SAMPLE_FACTORS - F1264_pos Samplet_Type:Mice Stool | Treatment:NCD RAW_FILE_NAME=F1264_pos-F1264.mzML SUBJECT_SAMPLE_FACTORS - F1267_neg Samplet_Type:Mice Stool | Treatment:NCD RAW_FILE_NAME=F1267_neg-F1267.mzML SUBJECT_SAMPLE_FACTORS - F1267_pos Samplet_Type:Mice Stool | Treatment:NCD RAW_FILE_NAME=F1267_pos-F1267.mzML SUBJECT_SAMPLE_FACTORS - QC01_neg Samplet_Type:Mice Stool | Treatment:QC RAW_FILE_NAME=QC01_neg-QC01.mzML SUBJECT_SAMPLE_FACTORS - QC01_pos Samplet_Type:Mice Stool | Treatment:QC RAW_FILE_NAME=QC01_pos-QC01.mzML SUBJECT_SAMPLE_FACTORS - QC02_neg Samplet_Type:Mice Stool | Treatment:QC RAW_FILE_NAME=QC02_neg-QC02.mzML SUBJECT_SAMPLE_FACTORS - QC02_pos Samplet_Type:Mice Stool | Treatment:QC RAW_FILE_NAME=QC02_pos-QC02.mzML SUBJECT_SAMPLE_FACTORS - QC03_neg Samplet_Type:Mice Stool | Treatment:QC RAW_FILE_NAME=QC03_neg-QC03.mzML SUBJECT_SAMPLE_FACTORS - QC03_pos Samplet_Type:Mice Stool | Treatment:QC RAW_FILE_NAME=QC03_pos-QC03.mzML SUBJECT_SAMPLE_FACTORS - QC04_neg Samplet_Type:Mice Stool | Treatment:QC RAW_FILE_NAME=QC04_neg-QC04.mzML SUBJECT_SAMPLE_FACTORS - QC04_pos Samplet_Type:Mice Stool | Treatment:QC RAW_FILE_NAME=QC04_pos-QC04.mzML SUBJECT_SAMPLE_FACTORS - QC05_neg Samplet_Type:Mice Stool | Treatment:QC RAW_FILE_NAME=QC05_neg-QC05.mzML SUBJECT_SAMPLE_FACTORS - QC05_pos Samplet_Type:Mice Stool | Treatment:QC RAW_FILE_NAME=QC05_pos-QC05.mzML SUBJECT_SAMPLE_FACTORS - QC06_neg Samplet_Type:Mice Stool | Treatment:QC RAW_FILE_NAME=QC06_neg-QC06.mzML SUBJECT_SAMPLE_FACTORS - QC06_pos Samplet_Type:Mice Stool | Treatment:QC RAW_FILE_NAME=QC06_pos-QC06.mzML #COLLECTION CO:COLLECTION_SUMMARY Collected mouse stool was stored in liquid nitrogen immediately and stored in CO:COLLECTION_SUMMARY -80℃ for long term storage. CO:COLLECTION_PROTOCOL_FILENAME Protocol_for_The_role_of_fiber_in_NASH_progression_stool.docx CO:SAMPLE_TYPE Feces CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Male C57 mice was randomly assigned into 4 groups that received Normal chow diet TR:TREATMENT_SUMMARY (NCD), Choline deficient high fat diet (CDHFD), CDHFD + 10% inulin (CDHFD-I), TR:TREATMENT_SUMMARY CDHFD + 10% Cellulose (CDHFD-C). Among the mice received inulin or cellulose, TR:TREATMENT_SUMMARY some of them received 13C labelled inulin or cellulose for 36 hours. Mouse stool TR:TREATMENT_SUMMARY were collected. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 25 mg of sample was weighted to an EP tube, and 500 μL extract solution SP:SAMPLEPREP_SUMMARY (acetonitrile: methanol: water = 2: 2: 1) containing internal standard SP:SAMPLEPREP_SUMMARY (L-2-Chlorophenylalanine, 4 μg/mL) was added. After 30 s vortex, the samples SP:SAMPLEPREP_SUMMARY were homogenized at 35 Hz for 4 min and sonicated for 5 min in ice-water bath. SP:SAMPLEPREP_SUMMARY The homogenization and sonication cycle was repeated for 2 times. Then the SP:SAMPLEPREP_SUMMARY samples were incubated at -40 ℃ for 1 h and centrifuged at 12000 rpm for 15 SP:SAMPLEPREP_SUMMARY min at 4 ℃. 400 μL of supernatant was transferred to a fresh tube and dried SP:SAMPLEPREP_SUMMARY in a vacuum concentrator at 37 ℃. Then, the dried samples were reconstituted SP:SAMPLEPREP_SUMMARY in 200 μL of 50% acetonitrile by sonication on ice for 10 min. The constitution SP:SAMPLEPREP_SUMMARY was then centrifuged at 13000 rpm for 15 min at 4 ℃, and 75 μL of supernatant SP:SAMPLEPREP_SUMMARY was transferred to a fresh glass vial for LC/MS analysis. The quality control SP:SAMPLEPREP_SUMMARY (QC) sample was prepared by mixing an equal aliquot of the supernatants from all SP:SAMPLEPREP_SUMMARY of the samples. SP:SAMPLEPREP_PROTOCOL_FILENAME Protocol_for_The_role_of_fiber_in_NASH_progression_stool.docx #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Non-targeted metabolomic analysis was performed on a 1290 Infinity series UHPLC CH:CHROMATOGRAPHY_SUMMARY System (Agilent Technologies) coupled to a Triple TOF 6600 mass spectrometer (AB CH:CHROMATOGRAPHY_SUMMARY Sciex ) equipped with an electrospray ionization (ESI) source by Biotree company CH:CHROMATOGRAPHY_SUMMARY (Shanghai, China). Briefly, chromatographic separation was achieved by using a CH:CHROMATOGRAPHY_SUMMARY Waters BEH Amide column (2.1 * 100 mm, 1.7 µm) under 25 ℃. The mobile phase CH:CHROMATOGRAPHY_SUMMARY consisted of water with 25mM ammonium acetate and 25 mM ammonium hydroxide (A) CH:CHROMATOGRAPHY_SUMMARY and acetonitrile (B). The flow rate was 0.5 ml/min. CH:METHODS_FILENAME Protocol_for_The_role_of_fiber_in_NASH_progression_stool.docx CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) CH:COLUMN_TEMPERATURE 25 ℃ CH:FLOW_GRADIENT 0~0.5 min, 95% B; 0.5~7 min, 95%~65% B; 7~8 min, 65%~40% B; 8~9 min, 40% B; CH:FLOW_GRADIENT 9~9.1 min, 40%~95% B; 9.1~12 min, 95% B CH:FLOW_RATE 0.5 ml/min. CH:SOLVENT_A water with 25mM ammonium acetate and 25 mM ammonium hydroxide CH:SOLVENT_B acetonitrile CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE Protocol_for_The_role_of_fiber_in_NASH_progression_stool.docx #MS MS:INSTRUMENT_NAME ABI Sciex 6600 TripleTOF MS:INSTRUMENT_TYPE Triple TOF MS:MS_TYPE ESI MS:MS_COMMENTS MS analysis was operated in information-dependent basis (IDA) mode under MS:MS_COMMENTS positive. The 12 most abundant ions were selected, fragmented and sent to TOF MS:MS_COMMENTS mass analyzer to acquire the MS2 information. MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST002514_AN004140_Results.txt UNITS:Peak ion intensity Has m/z:Yes Has RT:Yes RT units:Seconds #END