#METABOLOMICS WORKBENCH Reeddom_20210522_192918_mwtab.txt DATATRACK_ID:2652 STUDY_ID:ST001911 ANALYSIS_ID:AN003108 PROJECT_ID:PR001205 VERSION 1 CREATED_ON August 20, 2021, 10:13 am #PROJECT PR:PROJECT_TITLE Metabolome changes induced by bitter melon (Momordica charantia)- intake in a PR:PROJECT_TITLE high-fat diet induced obesity model PR:PROJECT_TYPE LCMS quanitative analysis PR:PROJECT_SUMMARY Quanitative analysis of metabolomics on samples from murine samples (KEGG PR:PROJECT_SUMMARY Pathway MAPS, LIPIDMAPS, Human Metabolome Database) PR:INSTITUTE University of Colorado Anschutz Medical Campus PR:DEPARTMENT Pharmaceutical Sciences PR:LABORATORY Dr. Rajesh Agarwal's Laboratory and PR:LAST_NAME Agarwal PR:FIRST_NAME Rajesh PR:ADDRESS 12850 E. Montview Blvd, C238, Room V20-2118, Aurora, CO 80045, USA PR:EMAIL Rajesh.Agarwal@cuanschutz.edu PR:PHONE 303-724-4055 PR:FUNDING_SOURCE This work was supported by the National Institutes of Health/ National Cancer PR:FUNDING_SOURCE Institute grant R01CA195708, the National Institutes of Health Office of Dietary PR:FUNDING_SOURCE Supplements supplemental funds R01CA195708-02S1, and the pilot funding from the PR:FUNDING_SOURCE office of the Associate Dean for Research and Graduate Education (ADR), School PR:FUNDING_SOURCE of Pharmacy, UC AMC (to RA), and the National Cancer Institute diversity PR:FUNDING_SOURCE supplement grant R01CA195708-04S1 (to DR and RA). PR:CONTRIBUTORS Dominique Reed, Dileep Kumar, Sushil Kumar, Komal Raina, Reenu Punia, Charmion PR:CONTRIBUTORS Cruickshank-Quinn, Boris Tabakoff, Nichole Reisdorph, Michael Wempe, Chapla PR:CONTRIBUTORS Agarwal #STUDY ST:STUDY_TITLE A high-fat diet induced obesity murine model treated with bitter melon ST:STUDY_TITLE (Momordica charantia) ST:STUDY_TYPE A 40-day Intervention experiment ST:STUDY_SUMMARY Metabolic effects of lyophilized bitter melon juice (BMJ) extract (oral gavage ST:STUDY_SUMMARY 200mg/kg/body weight-daily for 40 days) intake were evaluated in diet-induced ST:STUDY_SUMMARY obese C57BL/6J male mice [fed-high fat diet (HFD), 60 kcal% fat]. Changes in ST:STUDY_SUMMARY metabolite abundance levels in lipid-phase plasma [liquid chromatography mass ST:STUDY_SUMMARY spectrometry (LC-MS)-based metabolomics] after BMJ intervention were assessed. ST:INSTITUTE University of Colorado Anschutz Medical Campus ST:DEPARTMENT Pharmaceutical Sciences ST:LABORATORY Dr. Rajesh Agarwal's Laboratory ST:LAST_NAME Agarwal ST:FIRST_NAME Rajesh ST:ADDRESS 12850 E. Montview Blvd, C238, Room V20-2118, Aurora, CO 80045, USA ST:EMAIL Rajesh.Agarwal@cuanschutz.edu ST:PHONE 303-724-4055 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 8 ST:NUM_MALES 8 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN wildtype SU:AGE_OR_AGE_RANGE 4 weeks SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER Jackson Laboratory SU:ANIMAL_HOUSING UC Denver SU:ANIMAL_FEED High fat diet (HFD), 60 kcal% fat from Research diets Inc., # D12492 SU:ANIMAL_WATER ad libitum #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 - OB_07(raw) Factor:High Fat RAW_FILE_NAME=OB_07 SUBJECT_SAMPLE_FACTORS - OB_11(raw) Factor:High Fat RAW_FILE_NAME=OB_11 SUBJECT_SAMPLE_FACTORS - OB_12_A(raw) Factor:High Fat RAW_FILE_NAME=OB_12_A SUBJECT_SAMPLE_FACTORS - OB+BMJ_03(raw) Factor:Treated RAW_FILE_NAME=OB+BMJ_03 SUBJECT_SAMPLE_FACTORS - OB+BMJ_04(raw) Factor:Treated RAW_FILE_NAME=OB+BMJ_04 SUBJECT_SAMPLE_FACTORS - OB+BMJ_05(raw) Factor:Treated RAW_FILE_NAME=OB+BMJ_05 #COLLECTION CO:COLLECTION_SUMMARY Blood was collected and plasma/serum flash frozen in liquid nitrogen and stored CO:COLLECTION_SUMMARY in -80 degrees Celsius for biochemical/metabolic assessments. CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY Sixteen-week-old diet-induced obese (DIO) C57BL/6J male mice [fed-high fat diet TR:TREATMENT_SUMMARY (HFD), 60 kcal% fat from Research diets Inc., # D12492) from 4 weeks of age] TR:TREATMENT_SUMMARY were purchased from Jackson labs (Bar Harbor, ME). Once received at the animal TR:TREATMENT_SUMMARY house facility in UC Denver, the mice were randomized into two groups (n=4/ TR:TREATMENT_SUMMARY group): a) Control-DIO group maintained on the 60 kcal% HFD for additional 40 TR:TREATMENT_SUMMARY days, and b) BMJ+DIO group maintained on 60 kcal% HFD fat for additional 40 days TR:TREATMENT_SUMMARY plus orally gavage with lyophilized bitter melon juice extract [(5mg per mouse; TR:TREATMENT_SUMMARY total volume of 100 μl, 200mg/kg body weight/ day), reconstituted in sterile TR:TREATMENT_SUMMARY deionized water immediately before use)] throughout the study. TR:TREATMENT Intervention TR:TREATMENT_COMPOUND Bitter melon juice TR:TREATMENT_ROUTE Oral gavage TR:TREATMENT_DOSE 200mg/kg body weight/ day TR:ANIMAL_FASTING On day 40 of study initiation, mice in control and treament groups were TR:ANIMAL_FASTING subjected to fasting for six hours prior to sacrifice. TR:ANIMAL_ENDP_EUTHANASIA On day 40, mice were sacrificed after CO2 asphyxiation and exsanguination. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Frozen plasma was stored at -80°C until sample preparation for untargeted SP:SAMPLEPREP_SUMMARY liquid-chromatography mass spectrometry (LC-MS)-based metabolomics. Briefly, SP:SAMPLEPREP_SUMMARY 100µL of sample underwent protein precipitation using methanol, followed by SP:SAMPLEPREP_SUMMARY liquid-liquid extraction using methyl-tert butyl ether as previously SP:SAMPLEPREP_SUMMARY described[1, 2] to obtain an aqueous fraction and a lipid fraction. The lipid SP:SAMPLEPREP_SUMMARY fraction was dried down and resuspended in 100% methanol for LC-MS analysis. SP:PROCESSING_STORAGE_CONDITIONS Described in summary #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The samples from the lipid fraction were analyzed an Agilent 1290 series pump CH:CHROMATOGRAPHY_SUMMARY with an Agilent Zorbax Rapid Resolution HD (RRHD) SB-C18, 1.8 micron (2.1 x 100 CH:CHROMATOGRAPHY_SUMMARY mm) analytical column and an Agilent Zorbax SB-C18, 1.8 micron (2.1 x 5 mm) CH:CHROMATOGRAPHY_SUMMARY guard column. The autosampler tray temperature was set at 4°C, column CH:CHROMATOGRAPHY_SUMMARY temperature was set at 60°C, and the sample injection volume was 4µL. The flow CH:CHROMATOGRAPHY_SUMMARY rate was 0.7 mL/min with the following mobile phases: mobile phase A was water CH:CHROMATOGRAPHY_SUMMARY with 0.1% formic acid, and mobile phase B was 60:36:4 isopropyl CH:CHROMATOGRAPHY_SUMMARY alcohol:acetonitrile:water with 0.1% formic acid. Gradient elution was as CH:CHROMATOGRAPHY_SUMMARY follows: 0-1 minute 30-70% B, 1-7.92 minutes 70-100% B, 7.92-10.4 minutes 100% CH:CHROMATOGRAPHY_SUMMARY B, 10.4-10.5 minutes 100-30% B, followed by column re-equilibration with 30% B CH:CHROMATOGRAPHY_SUMMARY from 10.5-15.1 minutes. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 CH:COLUMN_NAME Agilent Zorbax RRHD SB-C18 (100 x 2.1mm, 1.8um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6210 TOF MS:INSTRUMENT_TYPE TOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The mass spectrometry conditions were as follows: Agilent 6210 Time-of-Flight MS:MS_COMMENTS mass spectrometer (TOF-MS) in positive ionization mode with dual electrospray MS:MS_COMMENTS (ESI) source, mass range 60-1600m/z, scan rate 2.03, gas temperature 300°C, gas MS:MS_COMMENTS flow 12.0L/min, nebulizer 30psi, skimmer 60V, capillary voltage 4000V, MS:MS_COMMENTS fragmentor 120V, reference masses 121.050873 and 922.009798 (Agilent reference MS:MS_COMMENTS mix). Proprietary analytical software for integration and peak picking MS:MS_RESULTS_FILE ST001911_AN003108_Results.txt UNITS:Abundance (mass spectral counts) Has m/z:Neutral masses Has RT:Yes RT units:Minutes #END