#METABOLOMICS WORKBENCH Microbial Metabolites in Liver Disease METADATA.xlsx DATATRACK_ID:385 STUDY_ID:ST000254 ANALYSIS_ID:AN000402 VERSION 1 CREATED_ON 02-08-2024 #PROJECT PR:PROJECT_TITLE The role of microbial metabolites in experimental liver disease PR:PROJECT_TYPE Targeted Metabolomics PR:PROJECT_SUMMARY Liver fibrosis is the result of chronic liver damage from various etiologies PR:PROJECT_SUMMARY including toxins, alcohol abuse, obesity, or viral hepatitis. Chronic liver PR:PROJECT_SUMMARY disease may progress to cirrhosis, an end stage organ disease, and liver cancer. PR:PROJECT_SUMMARY Patients with chronic liver disease show intestinal bacterial overgrowth and PR:PROJECT_SUMMARY dysbiosis. They also demonstrate increased intestinal permeability, and disease PR:PROJECT_SUMMARY severity correlates with systemic levels of bacterial products. Although PR:PROJECT_SUMMARY experimental liver fibrosis is dependent on gut derived bacterial products, the PR:PROJECT_SUMMARY exact contribution of the commensal microflora to chronic liver disease in PR:PROJECT_SUMMARY unknown. Since the interaction of bacterial products with the innate immune PR:PROJECT_SUMMARY system can also confer protection to the host, we subjected germfree mice to PR:PROJECT_SUMMARY experimental models of liver fibrosis. Results from our laboratory demonstrate PR:PROJECT_SUMMARY that germfree mice show exacerbated liver fibrosis as compared to conventional PR:PROJECT_SUMMARY mice. Our previous studies also have indicated that a bacterial metabolite of PR:PROJECT_SUMMARY tryptophan, Indole-3-propionic Acid (IPA), is absent in germ free mice. In this PR:PROJECT_SUMMARY study we are investigating the role of IPA in acute and chronic models of liver PR:PROJECT_SUMMARY fibrosis. PR:INSTITUTE University of California, San Diego PR:LAST_NAME Schnabl PR:FIRST_NAME Bernd PR:ADDRESS 9500 Gilman Drive, MC0063 La Jolla, CA 92093 PR:EMAIL beschnabl@ucsd.edu PR:PHONE 858-822-5311 PR:DOI http://dx.doi.org/10.21228/M8RW2F #STUDY ST:STUDY_TITLE The role of microbial metabolites in experimental liver disease ST:STUDY_TYPE Targeted Metabolomic Analysis of plasma samples ST:STUDY_SUMMARY Aim 1: Our experimental approach is to understand the effect of drinking water ST:STUDY_SUMMARY supplemented bacterial metabolite, Indole-3-propionic Acid (IPA), in liver ST:STUDY_SUMMARY disease in an acute alcohol model. Aim 2: Determine the levels of IPA in plasma ST:STUDY_SUMMARY of conventional mice in a chronic alcohol model. Aim 3: Determine the levels of ST:STUDY_SUMMARY IPA in plasma of conventional WT (C57BL/6) and mutant SL (sublytic, which is a ST:STUDY_SUMMARY mouse with a point mutation in ATP4a) mice. ST:INSTITUTE University of North Carolina ST:DEPARTMENT Discovery Science Technology ST:LABORATORY Sumner Lab ST:LAST_NAME Sumner ST:FIRST_NAME Susan ST:ADDRESS Eastern Regional Comprehensive Metabolomics Resource Core, UNC Nutrition ST:ADDRESS Research Institute, 500 Laureate Way, Kannapolis, NC, 28081 ST:EMAIL susan_sumner @unc.edu ST:PHONE 704-250-5066 ST:SUBMIT_DATE 2015-09-29 #SUBJECT SU:SUBJECT_TYPE Animal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6 SU:SPECIES_GROUP Mammal #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - P7 Groups:Acute Alcohol Model | Treatment:0 mM IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P8 Groups:Acute Alcohol Model | Treatment:0 mM IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P13 Groups:Acute Alcohol Model | Treatment:10 mM IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P14 Groups:Acute Alcohol Model | Treatment:10 mM IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P15 Groups:Acute Alcohol Model | Treatment:10 mM IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P10 Groups:Acute Alcohol Model | Treatment:1 mM IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P11 Groups:Acute Alcohol Model | Treatment:1 mM IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P12 Groups:Acute Alcohol Model | Treatment:1 mM IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P9 Groups:Acute Alcohol Model | Treatment:1 mM IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - STD10 Groups:Calibration Standard 10 | Treatment:2000 ng/ml of IPA | Strain:N/A SUBJECT_SAMPLE_FACTORS - STD11 Groups:Calibration Standard 11 | Treatment:2500 ng/ml of IPA | Strain:N/A SUBJECT_SAMPLE_FACTORS - STD12 Groups:Calibration Standard 12 | Treatment:3000 ng/ml of IPA | Strain:N/A SUBJECT_SAMPLE_FACTORS - STD1 Groups:Calibration Standard 1 | Treatment:10 ng/ml of IPA | Strain:N/A SUBJECT_SAMPLE_FACTORS - STD2 Groups:Calibration Standard 2 | Treatment:20 ng/ml of IPA | Strain:N/A SUBJECT_SAMPLE_FACTORS - STD3 Groups:Calibration Standard 3 | Treatment:40 ng/ml of IPA | Strain:N/A SUBJECT_SAMPLE_FACTORS - STD4 Groups:Calibration Standard 4 | Treatment:80 ng/ml of IPA | Strain:N/A SUBJECT_SAMPLE_FACTORS - STD5 Groups:Calibration Standard 5 | Treatment:160 ng/ml of IPA | Strain:N/A SUBJECT_SAMPLE_FACTORS - STD6 Groups:Calibration Standard 6 | Treatment:320 ng/ml of IPA | Strain:N/A SUBJECT_SAMPLE_FACTORS - STD7 Groups:Calibration Standard 7 | Treatment:650 ng/ml of IPA | Strain:N/A SUBJECT_SAMPLE_FACTORS - STD8 Groups:Calibration Standard 8 | Treatment:1000 ng/ml of IPA | Strain:N/A SUBJECT_SAMPLE_FACTORS - STD9 Groups:Calibration Standard 9 | Treatment:1500 ng/ml of IPA | Strain:N/A SUBJECT_SAMPLE_FACTORS - P28 Groups:Chronic Alcohol Model | Treatment:No IPA | Strain:SL SUBJECT_SAMPLE_FACTORS - P29 Groups:Chronic Alcohol Model | Treatment:No IPA | Strain:SL SUBJECT_SAMPLE_FACTORS - P30 Groups:Chronic Alcohol Model | Treatment:No IPA | Strain:SL SUBJECT_SAMPLE_FACTORS - P31 Groups:Chronic Alcohol Model | Treatment:No IPA | Strain:SL SUBJECT_SAMPLE_FACTORS - P20 Groups:Chronic Alcohol Model | Treatment:No IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P21 Groups:Chronic Alcohol Model | Treatment:No IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P22 Groups:Chronic Alcohol Model | Treatment:No IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P23 Groups:Chronic Alcohol Model | Treatment:No IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P24 Groups:Chronic Alcohol Model | Treatment:No IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P25 Groups:Chronic Alcohol Model | Treatment:No IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P26 Groups:Chronic Alcohol Model | Treatment:No IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P27 Groups:Chronic Alcohol Model | Treatment:No IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P1 Groups:Control | Treatment:0 mM IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P2 Groups:Control | Treatment:0 mM IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P5 Groups:Control | Treatment:10 mM IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P6 Groups:Control | Treatment:10 mM IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P3 Groups:Control | Treatment:1 mM IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P4 Groups:Control | Treatment:1 mM IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P16 Groups:Control | Treatment:No IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P17 Groups:Control | Treatment:No IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P18 Groups:Control | Treatment:No IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - P19 Groups:Control | Treatment:No IPA | Strain:WT SUBJECT_SAMPLE_FACTORS - QC1 Groups:Quality Control Sample 1 | Treatment:30 ng/ml of IPA | Strain:N/A SUBJECT_SAMPLE_FACTORS - QC2 Groups:Quality Control Sample 2 | Treatment:200 ng/ml of IPA | Strain:N/A SUBJECT_SAMPLE_FACTORS - QC3 Groups:Quality Control Sample 3 | Treatment:600 ng/ml of IPA | Strain:N/A SUBJECT_SAMPLE_FACTORS - QC4 Groups:Quality Control Sample 4 | Treatment:1800 ng/ml of IPA | Strain:N/A SUBJECT_SAMPLE_FACTORS - QC5 Groups:Quality Control Sample 5 | Treatment:2400 ng/ml of IPA | Strain:N/A #COLLECTION CO:COLLECTION_SUMMARY - CO:SAMPLE_TYPE Blood #TREATMENT TR:TREATMENT_SUMMARY Control vs IPA Supplemented Wild Type and SL Mice TR:TREATMENT Indole-3-propionic Acid (IPA) Supplemention TR:TREATMENT_COMPOUND Indole-3-propionic Acid (IPA) TR:TREATMENT_ROUTE Drinking Water TR:TREATMENT_DOSE 0, 1, and 10 mM IPA #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Aliquots of plasma samples (20 µL) were transferred into pre-labeled 2.0 mL SP:SAMPLEPREP_SUMMARY low-bind Eppendorf tubes for experimental samples. For all samples 100 µL SP:SAMPLEPREP_SUMMARY Protein Precipitation Buffer with internal standard [2.5 ng/ml of SP:SAMPLEPREP_SUMMARY 2-methyl-4-chlorophenyl-acetic acid (MCPA) in methanol] was added to each tube SP:SAMPLEPREP_SUMMARY and were vortexed for 2 min at 5000 rpm. The samples were centrifuged at 16,000 SP:SAMPLEPREP_SUMMARY rcf for 4 min. A 90 µL of the supernatant wastransferred into pre-labeled SP:SAMPLEPREP_SUMMARY auto-sampler vials and diluted by adding 90 µL of water with 10% Formic Acid. SP:SAMPLEPREP_SUMMARY Samples were then mixed on a multiple tube vortexer for 5 min at 5000 rpm. 10 SP:SAMPLEPREP_SUMMARY µL of samples were injected onto API-4000 Q-trap. SP:PROCESSING_STORAGE_CONDITIONS 4°C SP:EXTRACT_STORAGE -80C SP:SAMPLE_RESUSPENSION Methanol, H2O, formic acid SP:SAMPLE_SPIKING 2-methyl-4-chlorophenyl-acetic acid (MCPA) #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Reversed-Phase Gradient Seperation CH:METHODS_ID RTI-RCMRC-IPA-Analysis CH:METHODS_FILENAME RTI-RCMRC-IPA-Analysis CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity BEH C18 (50 x 2.1mm,1.7um) CH:COLUMN_PRESSURE 6000-10000 CH:COLUMN_TEMPERATURE 50 C CH:FLOW_GRADIENT Time(min) Flow Rate %A %B Curve ;Initial 0.600 99.0 1.0 6;0.75 0.600 CH:FLOW_GRADIENT 99.0 1.0 6 ;2.75 0.600 1.0 99.0 6 ;3.00 0.600 1.0 99.0 6 ;3.10 CH:FLOW_GRADIENT 0.600 99.0 1.0 6 ;3.50 0.600 99.0 1.0 6; CH:FLOW_RATE 0.6 ml/min CH:INJECTION_TEMPERATURE 8 C CH:INTERNAL_STANDARD 2-methyl-4-chlorophenyl-acetic acid (MCPA) CH:SOLVENT_A 95% water/5% acetonitrile; 0.15% acetic acid CH:SOLVENT_B 95% acetonitrile/5% water; 0.15% acetic acid CH:ANALYTICAL_TIME 3.5 mins CH:WEAK_WASH_SOLVENT_NAME 95:5 Water:ACN with 0.15% Acetic Acid CH:WEAK_WASH_VOLUME 600 uL CH:STRONG_WASH_SOLVENT_NAME 95:5 ACN:Water with 0.15% Acetic Acid CH:STRONG_WASH_VOLUME 600 uL CH:TARGET_SAMPLE_TEMPERATURE 8 C CH:SAMPLE_LOOP_SIZE 10 uL CH:SAMPLE_SYRINGE_SIZE 100 uL CH:RANDOMIZATION_ORDER Yes CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:LABORATORY_NAME RTI AN:ANALYSIS_TYPE MS AN:ACQUISITION_DATE 42166 AN:ACQUISITION_PARAMETERS_FILE RTI-RCMRC-IPA-Analysis AN:SOFTWARE_VERSION Analyst 1.6.2 AN:OPERATOR_NAME Suraj Dhungana AN:PROCESSING_PARAMETERS_FILE RTI-RCMRC-IPA-Analysis AN:DETECTOR_TYPE TOF #MS MS:INSTRUMENT_NAME ABI Sciex API 4000 QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:COLLISION_ENERGY -20 MS:COLLISION_GAS 6 MS:ION_SOURCE_TEMPERATURE 450 C MS:ION_SPRAY_VOLTAGE -4500 volts #END