#METABOLOMICS WORKBENCH dongf01_20200326_184856 DATATRACK_ID:1957 STUDY_ID:ST001337 ANALYSIS_ID:AN002231 PROJECT_ID:PR000913 VERSION 1 CREATED_ON April 1, 2020, 8:59 am #PROJECT PR:PROJECT_TITLE aryl hydrocarbon receptor-related compounds studies PR:PROJECT_SUMMARY The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor PR:PROJECT_SUMMARY that responds to a variety of structurally diverse exogenous and endogenous PR:PROJECT_SUMMARY small molecules. Gut microbiota utilizing tryptophan and indole metabolism as a PR:PROJECT_SUMMARY reservoir, has been demonstrated to provide an abundant source of AHR ligands. PR:PROJECT_SUMMARY So untargeted global profiling was performed to find the potential candidates of PR:PROJECT_SUMMARY AHR activator in human feces. PR:INSTITUTE The Pennsylvania State University PR:LAST_NAME DONG PR:FIRST_NAME FANGCONG PR:ADDRESS 314 Life Sciences Building, University Park, PA 16802 PR:EMAIL fxd93@psu.edu PR:PHONE 8148637610 #STUDY ST:STUDY_TITLE Global profiling for human feces ST:STUDY_SUMMARY The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor ST:STUDY_SUMMARY that responds to a variety of structurally diverse exogenous and endogenous ST:STUDY_SUMMARY small molecules. Gut microbiota utilizing tryptophan and indole metabolism as a ST:STUDY_SUMMARY reservoir, has been demonstrated to provide an abundant source of AHR ligands. ST:STUDY_SUMMARY So untargeted global profiling was performed to find the potential candidates of ST:STUDY_SUMMARY AHR activator in human feces. ST:INSTITUTE The Pennsylvania State University ST:LAST_NAME DONG ST:FIRST_NAME FANGCONG ST:ADDRESS 314 Life Sciences Building, University Park, PA, 16802 ST:EMAIL fxd93@psu.edu ST:PHONE 8148637610 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 - Human feces_ALA007 Treatment:defined diet RAW_FILE_NAME=Human feces_ALA007.raw SUBJECT_SAMPLE_FACTORS - Human feces_ALA011 Treatment:defined diet RAW_FILE_NAME=Human feces_ALA011.raw #COLLECTION CO:COLLECTION_SUMMARY Feces were collected from individuals at risk for cardiovascular disease CO:COLLECTION_SUMMARY involved in a randomized, controlled, 3‐period, crossover, feeding trial. CO:COLLECTION_SUMMARY Following a 2‐week standard Western diet run‐in (12% saturated FAs [SFA], 7% CO:COLLECTION_SUMMARY polyunsaturated FAs, 12% monounsaturated FAs), participants consumed 3 CO:COLLECTION_SUMMARY isocaloric weight‐maintenance diets for 6 weeks each: a walnut diet (.D 7% CO:COLLECTION_SUMMARY SFA, 16% polyunsaturated FAs, 3% ALA, 9% monounsaturated FAs); a walnut CO:COLLECTION_SUMMARY FA‐matched diet; and an oleic acid–replaced‐ALA diet (7% SFA, 14% CO:COLLECTION_SUMMARY polyunsaturated FAs, 0.5% ALA, 12% monounsaturated FAs), which substituted the CO:COLLECTION_SUMMARY amount of ALA from walnuts in the WD with oleic acid. CO:SAMPLE_TYPE Feces #TREATMENT TR:TREATMENT_SUMMARY Feces were collected from individuals at risk for cardiovascular disease TR:TREATMENT_SUMMARY involved in a randomized, controlled, 3‐period, crossover, feeding trial. TR:TREATMENT_SUMMARY Following a 2‐week standard Western diet run‐in (12% saturated FAs [SFA], 7% TR:TREATMENT_SUMMARY polyunsaturated FAs, 12% monounsaturated FAs), participants consumed 3 TR:TREATMENT_SUMMARY isocaloric weight‐maintenance diets for 6 weeks each: a walnut diet (.D 7% TR:TREATMENT_SUMMARY SFA, 16% polyunsaturated FAs, 3% ALA, 9% monounsaturated FAs); a walnut TR:TREATMENT_SUMMARY FA‐matched diet; and an oleic acid–replaced‐ALA diet (7% SFA, 14% TR:TREATMENT_SUMMARY polyunsaturated FAs, 0.5% ALA, 12% monounsaturated FAs), which substituted the TR:TREATMENT_SUMMARY amount of ALA from walnuts in the WD with oleic acid. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Freeze dried human stool (~ 30 mg) were mixed with 1 mL of ice cold 80% methanol SP:SAMPLEPREP_SUMMARY (v/v) containing 0.1% formic acid (v/v). Each mixture was homogenized with 1 mm SP:SAMPLEPREP_SUMMARY zirconium beads using a BeadBlasterTM 24 (Benchmark Scientific, Edison, NJ, USA) SP:SAMPLEPREP_SUMMARY homogenizer. All samples were homogenized according to the program parameters: SP:SAMPLEPREP_SUMMARY 6500 - 1×30 - 005 (×3). After vortexing, samples were sonicated for 20 min in SP:SAMPLEPREP_SUMMARY an ice water bath, prior to centrifugation at 20,000 × g for 20 min at 4 ℃. SP:SAMPLEPREP_SUMMARY The supernatants were collected, dried in a Savant SpeedVac (Thermo Scientific, SP:SAMPLEPREP_SUMMARY Waltham, MA, USA), and reconstituted in 100 μL of 3% methanol (v/v) containing SP:SAMPLEPREP_SUMMARY 1 µM chlorpropamide (internal standard). SP:SAMPLEPREP_PROTOCOL_ID MS_protocol_for_global_profiling SP:SAMPLEPREP_PROTOCOL_FILENAME MS_protocol_for_global_profiling.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Vanquish UHPLC system CH:COLUMN_NAME BEH C18 column (2.1 × 100 mm, 1.7 µm particle size; Waters) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Fusion Tribrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Solvent A was HPLC-grade water with 0.1% formic acid, and solvent B was MS:MS_COMMENTS HPLC-grade acetonitrile with 0.1% formic acid. The initial condition was 97% A MS:MS_COMMENTS and 3% B, increasing to 45% B at 10 min and 75% B at 12 min, where it was held MS:MS_COMMENTS at 75% B until 17.5 min before returning to the initial conditions. MS:MS_RESULTS_FILE ST001337_AN002231_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END