#METABOLOMICS WORKBENCH Juneyoung_20250501_040408 DATATRACK_ID:5876 STUDY_ID:ST003909 ANALYSIS_ID:AN006416 PROJECT_ID:PR002446 VERSION 1 CREATED_ON May 9, 2025, 10:04 am #PROJECT PR:PROJECT_TITLE Fecal and serum untargeted metabolomics analysis in MDR2 WT, KO mouse PR:PROJECT_SUMMARY Colesevelam treatment induces significant change of serum and fecal metabolite PR:PROJECT_SUMMARY profile in Primary sclerosing cholangitis mouse model We aimed to identify PR:PROJECT_SUMMARY metabolites differing between Mdr2 WT and KO mice through fecal and serum PR:PROJECT_SUMMARY metabolomic analyses, and to determine whether changes in specific metabolites PR:PROJECT_SUMMARY induced by the bile acid sequestrant colesevelam contribute to the amelioration PR:PROJECT_SUMMARY of liver pathology. PR:INSTITUTE Kyung Hee University PR:LAST_NAME June-Young PR:FIRST_NAME Lee PR:ADDRESS Kyungheedae-ro 26, Seoul, South Korea PR:EMAIL hgod2356@khu.ac.kr PR:PHONE +8210-7720-0118 #STUDY ST:STUDY_TITLE Colesevelam induced serum and fecal metabolite profile in Mdr2 WT and KO mice - ST:STUDY_TITLE Serum ST:STUDY_SUMMARY Multidrug resistance protein 2 (MDR2) is essential for proper bile acid ST:STUDY_SUMMARY transport, and its deficiency leads to spontaneous development of chronic ST:STUDY_SUMMARY cholestatic liver disease resembling primary sclerosing cholangitis (PSC). To ST:STUDY_SUMMARY investigate the metabolic consequences of MDR2 loss and the therapeutic effects ST:STUDY_SUMMARY of bile acid sequestration, we utilized an Mdr2-knockout mouse model and ST:STUDY_SUMMARY performed fecal and serum metabolomic profiling. Comparative analyses between ST:STUDY_SUMMARY Mdr2 wildtype and knockout mice revealed significant alterations in bile ST:STUDY_SUMMARY acid–related metabolites and inflammatory metabolic signatures. Upon treatment ST:STUDY_SUMMARY with colesevelam, a clinically approved bile acid sequestrant, we observed a ST:STUDY_SUMMARY marked attenuation of hepatic inflammation and fibrosis. Metabolomic shifts ST:STUDY_SUMMARY following colesevelam administration highlighted specific metabolite changes ST:STUDY_SUMMARY potentially responsible for the observed hepatic improvement. These findings ST:STUDY_SUMMARY suggest a mechanistic link between bile acid modulation, systemic metabolism, ST:STUDY_SUMMARY and hepatic pathology, offering insights into the therapeutic potential of ST:STUDY_SUMMARY metabolic intervention in PSC. Serum metabolite showes significant change in ST:STUDY_SUMMARY bile acid metabolism associated metabolites by treatment of colesevelam ST:INSTITUTE Kyung Hee University ST:LAST_NAME June-Young ST:FIRST_NAME Lee ST:ADDRESS Kyungheedae-ro 26, Seoul, South Korea ST:EMAIL hgod2356@khu.ac.kr ST:PHONE +8210-7720-0118 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN FVB.129P2-Abcb4tm1Bor/J SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER Jackson SU:ANIMAL_HOUSING Kyung Hee University #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 KO-COL10 KO-COL10_Serum_positive Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=KOCOL10_serum_pos.mzML SUBJECT_SAMPLE_FACTORS KO-COL11 KO-COL11_Serum_positive Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=KOCOL11_serum_pos.mzML SUBJECT_SAMPLE_FACTORS KO-COL15 KO-COL15_Serum_positive Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=KOCOL15_serum_pos.mzML SUBJECT_SAMPLE_FACTORS KO-COL5 KO-COL5_Serum_positive Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=KOCOL5_serum_pos.mzML SUBJECT_SAMPLE_FACTORS KO-COL6 KO-COL6_Serum_positive Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=KOCOL6_serum_pos.mzML SUBJECT_SAMPLE_FACTORS KO-COL7 KO-COL7_Serum_positive Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=KOCOL7_serum_pos.mzML SUBJECT_SAMPLE_FACTORS KO-COL8 KO-COL8_Serum_positive Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=KOCOL8_serum_pos.mzML SUBJECT_SAMPLE_FACTORS KO-COL9 KO-COL9_Serum_positive Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=KOCOL9_serum_pos.mzML SUBJECT_SAMPLE_FACTORS KO-VEH10 KO-VEH10_Serum_positive Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=KOVEH11_serum_pos.mzML SUBJECT_SAMPLE_FACTORS KO-VEH11 KO-VEH11_Serum_positive Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=KOVEH13_serum_pos.mzML SUBJECT_SAMPLE_FACTORS KO-VEH14 KO-VEH14_Serum_positive Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=KOVEH14_serum_pos.mzML SUBJECT_SAMPLE_FACTORS KO-VEH3 KO-VEH3_Serum_positive Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=KOVEH3_serum_pos.mzML SUBJECT_SAMPLE_FACTORS KO-VEH4 KO-VEH4_Serum_positive Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=KOVEH4_serum_pos.mzML SUBJECT_SAMPLE_FACTORS KO-VEH5 KO-VEH5_Serum_positive Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=KOVEH5_serum_pos.mzML SUBJECT_SAMPLE_FACTORS KO-VEH9 KO-VEH9_Serum_positive Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=KOVEH9_serum_pos.mzML SUBJECT_SAMPLE_FACTORS WT-COL10 WT-COL10_Serum_positive Sample source:Serum | Genotype:Wild-type | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=WTCOL10_serum_pos.mzML SUBJECT_SAMPLE_FACTORS WT-COL12 WT-COL12_Serum_positive Sample source:Serum | Genotype:Wild-type | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=WTCOL12_serum_pos.mzML SUBJECT_SAMPLE_FACTORS WT-COL1 WT-COL1_Serum_positive Sample source:Serum | Genotype:Wild-type | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=WTCOL1_serum_pos.mzML SUBJECT_SAMPLE_FACTORS WT-COL2 WT-COL2_Serum_positive Sample source:Serum | Genotype:Wild-type | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=WTCOL2_serum_pos.mzML SUBJECT_SAMPLE_FACTORS WT-COL3 WT-COL3_Serum_positive Sample source:Serum | Genotype:Wild-type | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=WTCOL3_serum_pos.mzML SUBJECT_SAMPLE_FACTORS WT-COL6 WT-COL6_Serum_positive Sample source:Serum | Genotype:Wild-type | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=WTCOL6_serum_pos.mzML SUBJECT_SAMPLE_FACTORS WT-COL8 WT-COL8_Serum_positive Sample source:Serum | Genotype:Wild-type | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=WTCOL8_serum_pos.mzML SUBJECT_SAMPLE_FACTORS WT-VEH11 WT-VEH11_Serum_positive Sample source:Serum | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=WTVEH11_serum_pos.mzML SUBJECT_SAMPLE_FACTORS WT-VEH14 WT-VEH14_Serum_positive Sample source:Serum | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=WTVEH14_serum_pos.mzML SUBJECT_SAMPLE_FACTORS WT-VEH1 WT-VEH1_Serum_positive Sample source:Serum | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=WTVEH1_serum_pos.mzML SUBJECT_SAMPLE_FACTORS WT-VEH2 WT-VEH2_Serum_positive Sample source:Serum | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=WTVEH2_serum_pos.mzML SUBJECT_SAMPLE_FACTORS WT-VEH4 WT-VEH4_Serum_positive Sample source:Serum | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=WTVEH4_serum_pos.mzML SUBJECT_SAMPLE_FACTORS WT-VEH7 WT-VEH7_Serum_positive Sample source:Serum | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=WTVEH7_serum_pos.mzML SUBJECT_SAMPLE_FACTORS WT-VEH8 WT-VEH8_Serum_positive Sample source:Serum | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=WTVEH8_serum_pos.mzML SUBJECT_SAMPLE_FACTORS KO-COL10 KO-COL10_Serum_negative Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=KOCOL10_serum_neg.mzML SUBJECT_SAMPLE_FACTORS KO-COL11 KO-COL11_Serum_negative Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=KOCOL11_serum_neg.mzML SUBJECT_SAMPLE_FACTORS KO-COL15 KO-COL15_Serum_negative Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=KOCOL15_serum_neg.mzML SUBJECT_SAMPLE_FACTORS KO-COL5 KO-COL5_Serum_negative Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=KOCOL5_serum_neg.mzML SUBJECT_SAMPLE_FACTORS KO-COL6 KO-COL6_Serum_negative Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=KOCOL6_serum_neg.mzML SUBJECT_SAMPLE_FACTORS KO-COL7 KO-COL7_Serum_negative Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=KOCOL7_serum_neg.mzML SUBJECT_SAMPLE_FACTORS KO-COL8 KO-COL8_Serum_negative Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=KOCOL8_serum_neg.mzML SUBJECT_SAMPLE_FACTORS KO-COL9 KO-COL9_Serum_negative Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=KOCOL9_serum_neg.mzML SUBJECT_SAMPLE_FACTORS KO-VEH10 KO-VEH10_Serum_negative Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=KOVEH11_serum_neg.mzML SUBJECT_SAMPLE_FACTORS KO-VEH11 KO-VEH11_Serum_negative Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=KOVEH13_serum_neg.mzML SUBJECT_SAMPLE_FACTORS KO-VEH14 KO-VEH14_Serum_negative Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=KOVEH14_serum_neg.mzML SUBJECT_SAMPLE_FACTORS KO-VEH3 KO-VEH3_Serum_negative Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=KOVEH3_serum_neg.mzML SUBJECT_SAMPLE_FACTORS KO-VEH4 KO-VEH4_Serum_negative Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=KOVEH4_serum_neg.mzML SUBJECT_SAMPLE_FACTORS KO-VEH5 KO-VEH5_Serum_negative Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=KOVEH5_serum_neg.mzML SUBJECT_SAMPLE_FACTORS KO-VEH9 KO-VEH9_Serum_negative Sample source:Serum | Genotype:Mdr2-knockout | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=KOVEH9_serum_neg.mzML SUBJECT_SAMPLE_FACTORS WT-COL10 WT-COL10_Serum_negative Sample source:Serum | Genotype:Wild-type | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=WTCOL10_serum_neg.mzML SUBJECT_SAMPLE_FACTORS WT-COL12 WT-COL12_Serum_negative Sample source:Serum | Genotype:Wild-type | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=WTCOL12_serum_neg.mzML SUBJECT_SAMPLE_FACTORS WT-COL1 WT-COL1_Serum_negative Sample source:Serum | Genotype:Wild-type | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=WTCOL1_serum_neg.mzML SUBJECT_SAMPLE_FACTORS WT-COL2 WT-COL2_Serum_negative Sample source:Serum | Genotype:Wild-type | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=WTCOL2_serum_neg.mzML SUBJECT_SAMPLE_FACTORS WT-COL3 WT-COL3_Serum_negative Sample source:Serum | Genotype:Wild-type | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=WTCOL3_serum_neg.mzML SUBJECT_SAMPLE_FACTORS WT-COL6 WT-COL6_Serum_negative Sample source:Serum | Genotype:Wild-type | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=WTCOL6_serum_neg.mzML SUBJECT_SAMPLE_FACTORS WT-COL8 WT-COL8_Serum_negative Sample source:Serum | Genotype:Wild-type | Treatment:Colesevelam RAW_FILE_NAME(Raw file name)=WTCOL8_serum_neg.mzML SUBJECT_SAMPLE_FACTORS WT-VEH11 WT-VEH11_Serum_negative Sample source:Serum | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=WTVEH11_serum_neg.mzML SUBJECT_SAMPLE_FACTORS WT-VEH14 WT-VEH14_Serum_negative Sample source:Serum | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=WTVEH14_serum_neg.mzML SUBJECT_SAMPLE_FACTORS WT-VEH1 WT-VEH1_Serum_negative Sample source:Serum | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=WTVEH1_serum_neg.mzML SUBJECT_SAMPLE_FACTORS WT-VEH2 WT-VEH2_Serum_negative Sample source:Serum | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=WTVEH2_serum_neg.mzML SUBJECT_SAMPLE_FACTORS WT-VEH4 WT-VEH4_Serum_negative Sample source:Serum | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=WTVEH4_serum_neg.mzML SUBJECT_SAMPLE_FACTORS WT-VEH7 WT-VEH7_Serum_negative Sample source:Serum | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=WTVEH7_serum_neg.mzML SUBJECT_SAMPLE_FACTORS WT-VEH8 WT-VEH8_Serum_negative Sample source:Serum | Genotype:Wild-type | Treatment:Vehicle RAW_FILE_NAME(Raw file name)=WTVEH8_serum_neg.mzML #COLLECTION CO:COLLECTION_SUMMARY Fecal and serum samples were collected from Mdr2-knockout and wildtype mice CO:COLLECTION_SUMMARY following an 8-week treatment period with either colesevelam or vehicle control. CO:COLLECTION_SUMMARY Fecal samples were directly collected at the end of the treatment period, while CO:COLLECTION_SUMMARY serum samples were obtained via cardiac puncture immediately prior to liver CO:COLLECTION_SUMMARY tissue harvest. For metabolomic analysis, 0.04 g of fecal material and CO:COLLECTION_SUMMARY 100 μL of serum were used per sample. All samples were snap-frozen and stored CO:COLLECTION_SUMMARY at –80 °C until extraction. No in vitro cell lines were used in this study. CO:SAMPLE_TYPE Blood (serum) CO:COLLECTION_LOCATION Serum CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY FVB/N wild-type (WT, Mdr2+/+) and Mdr2−/− (knock-out, KO) littermate mice TR:TREATMENT_SUMMARY were obtained by breeding heterozygous pairs bought from the Jackson Laboratory TR:TREATMENT_SUMMARY (Bar Habor, Maine, USA). WT and KO littermates were housed under a 12-hour TR:TREATMENT_SUMMARY light/dark cycle, and allowed access to water and a standard chow diet (2918C; TR:TREATMENT_SUMMARY Envigo) ad libitum. Both WT and KO mice were randomly assigned to cages for each TR:TREATMENT_SUMMARY group (vehicle- and colesevelam-treated) at 3–4 weeks of age. Colesevelam (a TR:TREATMENT_SUMMARY BA sequestrant, cas#182815-44-7) was obtained from BOC Sciences (Ramsey Road TR:TREATMENT_SUMMARY Shirley, NY, USA). From 7–8 weeks of age, male Mdr2+/+ and Mdr2−/− TR:TREATMENT_SUMMARY littermate mice received either a control chow diet or a diet supplemented with TR:TREATMENT_SUMMARY 2% (w/w) colesevelam for 8 weeks. TR:TREATMENT_COMPOUND normal chow diet or Colesevelam included diet TR:TREATMENT_ROUTE oral route TR:TREATMENT_DOSE 2% (w/w) colesevelam TR:TREATMENT_DOSEDURATION 8 weeks TR:TREATMENT_VEHICLE normal chow diet #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Serum and fecal samples were thawed at 4°C before extraction. Briefly, 100 μL SP:SAMPLEPREP_SUMMARY of serum and 0.04 g of feces was added to 900 μL of a solution comprising SP:SAMPLEPREP_SUMMARY MeOH:acetonitrile:water (1:1:2, v/v) containing reserpine 10 μM and 0.1% (v/v) SP:SAMPLEPREP_SUMMARY formic acid. The serum and fecal solutions were then homogenized by vortexing SP:SAMPLEPREP_SUMMARY for 5 min, followed by centrifugation at 13,000 × g at 4°C for 10 min. After SP:SAMPLEPREP_SUMMARY centrifugation, the supernatant was filtered through a 0.2 μm pore SP:SAMPLEPREP_SUMMARY polytetrafluoroethylene polymer filter and transferred to a new glass vial. SP:SAMPLEPREP_SUMMARY Quality control (QC) samples were prepared by mixing 50 μL of filtered samples SP:SAMPLEPREP_SUMMARY (serum and feces). QC samples were analyzed together and inserted into every SP:SAMPLEPREP_SUMMARY five samples regularly before and after the operation. SP:SAMPLEPREP_PROTOCOL_FILENAME JYL_protocol.pdf SP:EXTRACT_STORAGE On ice #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY see protocol file CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Agilent ZORBAX Eclipse Plus C18 (100 x 2.1mm,3.5um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT The gradient started with 98% A, decreasing to 10% A in 13 min, holding at 10% CH:FLOW_GRADIENT A for 3 min, moving immediately to 98% A, and then holding at 98% A for CH:FLOW_GRADIENT 4 min. CH:FLOW_RATE 0.5 mL/min CH:COLUMN_TEMPERATURE 45 CH:METHODS_FILENAME JYL_protocol.pdf #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Seoul Biohub AN:SOFTWARE_VERSION Xcalibur, version 4.1.31.9 AN:ACQUISITION_DATE 2023.08 AN:ANALYSIS_PROTOCOL_FILE JYL_protocol.pdf AN:DATA_FORMAT RAW #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS positive mode: see protocol file MS:ION_SPRAY_VOLTAGE 3.5 kV MS:IONIZATION heated electrospray ionization source MS:MS_RESULTS_FILE ST003909_AN006416_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Seconds #END