#METABOLOMICS WORKBENCH nchorna_20231015_045517 DATATRACK_ID:4402 STUDY_ID:ST002932 ANALYSIS_ID:AN004810 PROJECT_ID:PR001823 VERSION 1 CREATED_ON October 16, 2023, 11:33 am #PROJECT PR:PROJECT_TITLE Analyzing Metabolic Alterations in the Gut, Hippocampus and Brainstem of Mice PR:PROJECT_TITLE Induced by Running Exercise Through Liquid Chromatography Mass Spectrometry PR:PROJECT_SUMMARY Metabolic analysis of the impact of running exercise on the gut, hippocampus and PR:PROJECT_SUMMARY brainstem is crucial as it provides insights into how exercise affects memory, PR:PROJECT_SUMMARY mood, and vital physiological functions. In our six-week study, we utilized a PR:PROJECT_SUMMARY mouse model (C57BL/6 genotype) to investigate these metabolic changes. Employing PR:PROJECT_SUMMARY liquid chromatography coupled with mass spectrometry followed by metabolomics PR:PROJECT_SUMMARY for a comprehensive analysis, our approach offers insights into how exercise PR:PROJECT_SUMMARY influences metabolic processes, including brain function. Our findings hold the PR:PROJECT_SUMMARY potential to shape more effective exercise strategies for enhancing overall PR:PROJECT_SUMMARY health and cognitive function. PR:INSTITUTE University of Puerto Rico, School of Medicine PR:DEPARTMENT Biochemistry PR:LAST_NAME Chorna PR:FIRST_NAME Nataliya PR:ADDRESS University of Puerto Rico, Medical Sciences Campus, San Juan, PR 00935 PR:EMAIL nataliya.chorna@upr.edu PR:PHONE 7877582525 ext 1640 PR:FUNDING_SOURCE NIGMS-NIH-PRINBRE-P20GM103475 #STUDY ST:STUDY_TITLE Analyzing Metabolic Alterations in the Gut, Hippocampus and Brainstem of Mice ST:STUDY_TITLE Induced by Running Exercise Through Liquid Chromatography Mass Spectrometry ST:STUDY_SUMMARY Metabolic analysis of the impact of running exercise on the gut, hippocampus and ST:STUDY_SUMMARY brainstem is crucial as it provides insights into how exercise affects memory, ST:STUDY_SUMMARY mood, and vital physiological functions. In our six-week study, we utilized a ST:STUDY_SUMMARY mouse model (C57BL/6 genotype) to investigate these metabolic changes. Employing ST:STUDY_SUMMARY liquid chromatography coupled with mass spectrometry followed by metabolomics ST:STUDY_SUMMARY for a comprehensive analysis, our approach offers insights into how exercise ST:STUDY_SUMMARY influences metabolic processes, including brain function. Our findings hold the ST:STUDY_SUMMARY potential to shape more effective exercise strategies for enhancing overall ST:STUDY_SUMMARY health and cognitive function. ST:INSTITUTE University of Puerto Rico, School of Medicine ST:DEPARTMENT Biochemistry ST:LAST_NAME Chorna ST:FIRST_NAME Nataliya ST:ADDRESS University of Puerto Rico, Medical Sciences Campus, San Juan, PR 00935 ST:EMAIL nataliya.chorna@upr.edu ST:PHONE 7877582525 ext 1640 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 24 ST:NUM_MALES 24 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6J SU:AGE_OR_AGE_RANGE 20 wks SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER Jackson Lab #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 bs_lc_r1 bs_r1 Factor:brainstem Treatment=Running; RAW_FILE_NAME=bs_lc_r1.mzML SUBJECT_SAMPLE_FACTORS bs_lc_r2 bs_r2 Factor:brainstem Treatment=Running; RAW_FILE_NAME=bs_lc_r2.mzML SUBJECT_SAMPLE_FACTORS bs_lc_r3 bs_r3 Factor:brainstem Treatment=Running; RAW_FILE_NAME=bs_lc_r3.mzML SUBJECT_SAMPLE_FACTORS bs_lc_r4 bs_r4 Factor:brainstem Treatment=Running; RAW_FILE_NAME=bs_lc_r4.mzML SUBJECT_SAMPLE_FACTORS bs_lc_r5 bs_r5 Factor:brainstem Treatment=Running; RAW_FILE_NAME=bs_lc_r5.mzML SUBJECT_SAMPLE_FACTORS bs_lc_r6 bs_r6 Factor:brainstem Treatment=Running; RAW_FILE_NAME=bs_lc_r6.mzML SUBJECT_SAMPLE_FACTORS bs_lc_r7 bs_r7 Factor:brainstem Treatment=Running; RAW_FILE_NAME=bs_lc_r7.mzML SUBJECT_SAMPLE_FACTORS bs_lc_r8 bs_r8 Factor:brainstem Treatment=Running; RAW_FILE_NAME=bs_lc_r8.mzML SUBJECT_SAMPLE_FACTORS bs_lc_r9 bs_r9 Factor:brainstem Treatment=Running; RAW_FILE_NAME=bs_lc_r9.mzML SUBJECT_SAMPLE_FACTORS bs_lc_r10 bs_r10 Factor:brainstem Treatment=Running; RAW_FILE_NAME=bs_lc_r10.mzML SUBJECT_SAMPLE_FACTORS bs_lc_r11 bs_r11 Factor:brainstem Treatment=Running; RAW_FILE_NAME=bs_lc_r11.mzML SUBJECT_SAMPLE_FACTORS bs_lc_r12 bs_r12 Factor:brainstem Treatment=Running; RAW_FILE_NAME=bs_lc_r12.mzML SUBJECT_SAMPLE_FACTORS bs_lc_s1 bs_s1 Factor:brainstem Treatment=Sedentary; RAW_FILE_NAME=bs_lc_s1.mzML SUBJECT_SAMPLE_FACTORS bs_lc_s2 bs_s2 Factor:brainstem Treatment=Sedentary; RAW_FILE_NAME=bs_lc_s2.mzML SUBJECT_SAMPLE_FACTORS bs_lc_s3 bs_s3 Factor:brainstem Treatment=Sedentary; RAW_FILE_NAME=bs_lc_s3.mzML SUBJECT_SAMPLE_FACTORS bs_lc_s4 bs_s4 Factor:brainstem Treatment=Sedentary; RAW_FILE_NAME=bs_lc_s4.mzML SUBJECT_SAMPLE_FACTORS bs_lc_s5 bs_s5 Factor:brainstem Treatment=Sedentary; RAW_FILE_NAME=bs_lc_s5.mzML SUBJECT_SAMPLE_FACTORS bs_lc_s6 bs_s6 Factor:brainstem Treatment=Sedentary; RAW_FILE_NAME=bs_lc_s6.mzML SUBJECT_SAMPLE_FACTORS bs_lc_s7 bs_s7 Factor:brainstem Treatment=Sedentary; RAW_FILE_NAME=bs_lc_s7.mzML SUBJECT_SAMPLE_FACTORS bs_lc_s8 bs_s8 Factor:brainstem Treatment=Sedentary; RAW_FILE_NAME=bs_lc_s8.mzML SUBJECT_SAMPLE_FACTORS bs_lc_s9 bs_s9 Factor:brainstem Treatment=Sedentary; RAW_FILE_NAME=bs_lc_s9.mzML SUBJECT_SAMPLE_FACTORS bs_lc_s10 bs_s10 Factor:brainstem Treatment=Sedentary; RAW_FILE_NAME=bs_lc_s10.mzML SUBJECT_SAMPLE_FACTORS bs_lc_s11 bs_s11 Factor:brainstem Treatment=Sedentary; RAW_FILE_NAME=bs_lc_s11.mzML SUBJECT_SAMPLE_FACTORS bs_lc_s12 bs_s12 Factor:brainstem Treatment=Sedentary; RAW_FILE_NAME=bs_lc_s12.mzML SUBJECT_SAMPLE_FACTORS fec_lc_r1 fec_r1 Factor:feces Treatment=Running; RAW_FILE_NAME=fec_lc_r1.mzML SUBJECT_SAMPLE_FACTORS fec_lc_r2 fec_r2 Factor:feces Treatment=Running; RAW_FILE_NAME=fec_lc_r2.mzML SUBJECT_SAMPLE_FACTORS fec_lc_r3 fec_r3 Factor:feces Treatment=Running; RAW_FILE_NAME=fec_lc_r3.mzML SUBJECT_SAMPLE_FACTORS fec_lc_r4 fec_r4 Factor:feces Treatment=Running; RAW_FILE_NAME=fec_lc_r4.mzML SUBJECT_SAMPLE_FACTORS fec_lc_r5 fec_r5 Factor:feces Treatment=Running; RAW_FILE_NAME=fec_lc_r5.mzML SUBJECT_SAMPLE_FACTORS fec_lc_r6 fec_r6 Factor:feces Treatment=Running; RAW_FILE_NAME=fec_lc_r6.mzML SUBJECT_SAMPLE_FACTORS fec_lc_r7 fec_r7 Factor:feces Treatment=Running; RAW_FILE_NAME=fec_lc_r7.mzML SUBJECT_SAMPLE_FACTORS fec_lc_r8 fec_r8 Factor:feces Treatment=Running; RAW_FILE_NAME=fec_lc_r8.mzML SUBJECT_SAMPLE_FACTORS fec_lc_r9 fec_r9 Factor:feces Treatment=Running; RAW_FILE_NAME=fec_lc_r9.mzML SUBJECT_SAMPLE_FACTORS fec_lc_r10 fec_r10 Factor:feces Treatment=Running; RAW_FILE_NAME=fec_lc_r10.mzML SUBJECT_SAMPLE_FACTORS fec_lc_r11 fec_r11 Factor:feces Treatment=Running; RAW_FILE_NAME=fec_lc_r11.mzML SUBJECT_SAMPLE_FACTORS fec_lc_r12 fec_r12 Factor:feces Treatment=Running; RAW_FILE_NAME=fec_lc_r12.mzML SUBJECT_SAMPLE_FACTORS fec_lc_s1 fec_s1 Factor:feces Treatment=Sedentary; RAW_FILE_NAME=fec_lc_s1.mzML SUBJECT_SAMPLE_FACTORS fec_lc_s2 fec_s2 Factor:feces Treatment=Sedentary; RAW_FILE_NAME=fec_lc_s2.mzML SUBJECT_SAMPLE_FACTORS fec_lc_s3 fec_s3 Factor:feces Treatment=Sedentary; RAW_FILE_NAME=fec_lc_s3.mzML SUBJECT_SAMPLE_FACTORS fec_lc_s4 fec_s4 Factor:feces Treatment=Sedentary; RAW_FILE_NAME=fec_lc_s4.mzML SUBJECT_SAMPLE_FACTORS fec_lc_s5 fec_s5 Factor:feces Treatment=Sedentary; RAW_FILE_NAME=fec_lc_s5.mzML SUBJECT_SAMPLE_FACTORS fec_lc_s6 fec_s6 Factor:feces Treatment=Sedentary; RAW_FILE_NAME=fec_lc_s6.mzML SUBJECT_SAMPLE_FACTORS fec_lc_s7 fec_s7 Factor:feces Treatment=Sedentary; RAW_FILE_NAME=fec_lc_s7.mzML SUBJECT_SAMPLE_FACTORS fec_lc_s8 fec_s8 Factor:feces Treatment=Sedentary; RAW_FILE_NAME=fec_lc_s8.mzML SUBJECT_SAMPLE_FACTORS fec_lc_s9 fec_s9 Factor:feces Treatment=Sedentary; RAW_FILE_NAME=fec_lc_s9.mzML SUBJECT_SAMPLE_FACTORS fec_lc_s10 fec_s10 Factor:feces Treatment=Sedentary; RAW_FILE_NAME=fec_lc_s10.mzML SUBJECT_SAMPLE_FACTORS fec_lc_s11 fec_s11 Factor:feces Treatment=Sedentary; RAW_FILE_NAME=fec_lc_s11.mzML SUBJECT_SAMPLE_FACTORS fec_lc_s12 fec_s12 Factor:feces Treatment=Sedentary; RAW_FILE_NAME=fec_lc_s12.mzML SUBJECT_SAMPLE_FACTORS hp_LC_r1 hp_r1 Factor:hippocampus Treatment=Running; RAW_FILE_NAME=hp_LC_r1.mzML SUBJECT_SAMPLE_FACTORS hp_LC_r2 hp_r2 Factor:hippocampus Treatment=Running; RAW_FILE_NAME=hp_LC_r2.mzML SUBJECT_SAMPLE_FACTORS hp_LC_r3 hp_r3 Factor:hippocampus Treatment=Running; RAW_FILE_NAME=hp_LC_r3.mzML SUBJECT_SAMPLE_FACTORS hp_LC_r4 hp_r4 Factor:hippocampus Treatment=Running; RAW_FILE_NAME=hp_LC_r4.mzML SUBJECT_SAMPLE_FACTORS hp_LC_r5 hp_r5 Factor:hippocampus Treatment=Running; RAW_FILE_NAME=hp_LC_r5.mzML SUBJECT_SAMPLE_FACTORS hp_LC_r6 hp_r6 Factor:hippocampus Treatment=Running; RAW_FILE_NAME=hp_LC_r6.mzML SUBJECT_SAMPLE_FACTORS hp_LC_r7 hp_r7 Factor:hippocampus Treatment=Running; RAW_FILE_NAME=hp_LC_r7.mzML SUBJECT_SAMPLE_FACTORS hp_LC_r8 hp_r8 Factor:hippocampus Treatment=Running; RAW_FILE_NAME=hp_LC_r8.mzML SUBJECT_SAMPLE_FACTORS hp_LC_r9 hp_r9 Factor:hippocampus Treatment=Running; RAW_FILE_NAME=hp_LC_r9.mzML SUBJECT_SAMPLE_FACTORS hp_LC_r10 hp_r10 Factor:hippocampus Treatment=Running; RAW_FILE_NAME=hp_LC_r10.mzML SUBJECT_SAMPLE_FACTORS hp_LC_r11 hp_r11 Factor:hippocampus Treatment=Running; RAW_FILE_NAME=hp_LC_r11.mzML SUBJECT_SAMPLE_FACTORS hp_LC_r12 hp_r12 Factor:hippocampus Treatment=Running; RAW_FILE_NAME=hp_LC_r12.mzML SUBJECT_SAMPLE_FACTORS hp_LC_s1 hp_s1 Factor:hippocampus Treatment=Sedentary; RAW_FILE_NAME=hp_LC_s1.mzML SUBJECT_SAMPLE_FACTORS hp_LC_s2 hp_s2 Factor:hippocampus Treatment=Sedentary; RAW_FILE_NAME=hp_LC_s2.mzML SUBJECT_SAMPLE_FACTORS hp_LC_s3 hp_s3 Factor:hippocampus Treatment=Sedentary; RAW_FILE_NAME=hp_LC_s3.mzML SUBJECT_SAMPLE_FACTORS hp_LC_s4 hp_s4 Factor:hippocampus Treatment=Sedentary; RAW_FILE_NAME=hp_LC_s4.mzML SUBJECT_SAMPLE_FACTORS hp_LC_s5 hp_s5 Factor:hippocampus Treatment=Sedentary; RAW_FILE_NAME=hp_LC_s5.mzML SUBJECT_SAMPLE_FACTORS hp_LC_s6 hp_s6 Factor:hippocampus Treatment=Sedentary; RAW_FILE_NAME=hp_LC_s6.mzML SUBJECT_SAMPLE_FACTORS hp_LC_s7 hp_s7 Factor:hippocampus Treatment=Sedentary; RAW_FILE_NAME=hp_LC_s7.mzML SUBJECT_SAMPLE_FACTORS hp_LC_s8 hp_s8 Factor:hippocampus Treatment=Sedentary; RAW_FILE_NAME=hp_LC_s8.mzML SUBJECT_SAMPLE_FACTORS hp_LC_s9 hp_s9 Factor:hippocampus Treatment=Sedentary; RAW_FILE_NAME=hp_LC_s9.mzML SUBJECT_SAMPLE_FACTORS hp_LC_s10 hp_s10 Factor:hippocampus Treatment=Sedentary; RAW_FILE_NAME=hp_LC_s10.mzML SUBJECT_SAMPLE_FACTORS hp_LC_s11 hp_s11 Factor:hippocampus Treatment=Sedentary; RAW_FILE_NAME=hp_LC_s11.mzML SUBJECT_SAMPLE_FACTORS hp_LC_s12 hp_s12 Factor:hippocampus Treatment=Sedentary; RAW_FILE_NAME=hp_LC_s12.mzML #COLLECTION CO:COLLECTION_SUMMARY Extraction of fecal metabolites: 50 mg of fresh feces were collected from each CO:COLLECTION_SUMMARY mouse and frozen in liquid nitrogen. Samples were spiked with 0.5 mL water, CO:COLLECTION_SUMMARY homogenized for 5 min followed by 10 s of sonication, and centrifuged at 14K rpm CO:COLLECTION_SUMMARY X 10 min at 4°C. Collected supernatants were evaporated to dryness under a CO:COLLECTION_SUMMARY nitrogen gas stream and stored at −80°C. Extraction of brain metabolites: CO:COLLECTION_SUMMARY Approximately 20-40 mg of frozen tissues was resuspended in 4 volumes of MPER CO:COLLECTION_SUMMARY and homogenized on ice. 130 µL of tissue homogenates were spin filtered, as CO:COLLECTION_SUMMARY above, and the flow through was diluted 1:1 with deionized water, followed by CO:COLLECTION_SUMMARY sonication for 10 sec using a Sonic Dismembrator on ice, vortexed for 10 s, and CO:COLLECTION_SUMMARY centrifuged at 14K rpm X 15 min at 4°C. 100 uL of eluate was evaporated to CO:COLLECTION_SUMMARY dryness under a nitrogen gas stream and stored at −80°C. CO:SAMPLE_TYPE feces, hippocampus, brainstem CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY To evaluate our hypothesis that running exercise reshapes gut microbiota TR:TREATMENT_SUMMARY diversity that balances TRP metabolism in the gut, we used an established model TR:TREATMENT_SUMMARY of voluntary running exercise in 20-week-old male mice (C57BL/6J, Jackson Lab) TR:TREATMENT_SUMMARY housed individually in a standard cage in temperature-controlled (21°C) TR:TREATMENT_SUMMARY quarters with a 12-h light/12-h dark cycle. Animals were given water and food TR:TREATMENT_SUMMARY (Purina Chow) ad libitum as previously described (Chorna et al., 2013). Briefly, TR:TREATMENT_SUMMARY mice were randomized into two groups, sedentary control (n=12) and running TR:TREATMENT_SUMMARY experimental (n=12), housed with free access to a wireless running wheel (Med TR:TREATMENT_SUMMARY Associates) for six weeks. For the sedentary group, the wheels were locked in TR:TREATMENT_SUMMARY the experiment. Therefore, this group of mice could not perform running TR:TREATMENT_SUMMARY exercises. Exercise activities of the running group were recorded for each TR:TREATMENT_SUMMARY animal for the investigation to ensure that each mouse was physically active. TR:TREATMENT_SUMMARY The recording was conducted using an automatic counter and Med Associates TR:TREATMENT_SUMMARY software. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Extraction of fecal metabolites: 50 mg of fresh feces were collected from each SP:SAMPLEPREP_SUMMARY mouse and frozen in liquid nitrogen. Samples were spiked with 0.5 mL water, SP:SAMPLEPREP_SUMMARY homogenized for 5 min followed by 10 s of sonication, and centrifuged at 14K rpm SP:SAMPLEPREP_SUMMARY X 10 min at 4°C. Collected supernatants were evaporated to dryness under a SP:SAMPLEPREP_SUMMARY nitrogen gas stream and stored at −80°C. Extraction of brain metabolites: SP:SAMPLEPREP_SUMMARY Approximately 20-40 mg of frozen tissues was resuspended in 4 volumes of MPER SP:SAMPLEPREP_SUMMARY and homogenized on ice. 130 µL of tissue homogenates were spin filtered, as SP:SAMPLEPREP_SUMMARY above, and the flow through was diluted 1:1 with deionized water, followed by SP:SAMPLEPREP_SUMMARY sonication for 10 sec using a Sonic Dismembrator on ice, vortexed for 10 s, and SP:SAMPLEPREP_SUMMARY centrifuged at 14K rpm X 15 min at 4°C. 100 uL of eluate was evaporated to SP:SAMPLEPREP_SUMMARY dryness under a nitrogen gas stream and stored at −80°C. SP:PROCESSING_STORAGE_CONDITIONS Described in summary #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The LC/MS experiments were conducted using an i-Class LC system coupled to a CH:CHROMATOGRAPHY_SUMMARY Synapt-XS mass spectrometer (Waters). Liquid chromatography was performed on CH:CHROMATOGRAPHY_SUMMARY fecal, hippocampus, and brainstem samples collected from both the SED and RUN CH:CHROMATOGRAPHY_SUMMARY groups. The chromatographic separation was conducted using a BEH HILIC column CH:CHROMATOGRAPHY_SUMMARY with 1.7 µm particle size dimensions and 2.1 x 100 mm (Waters). The column CH:CHROMATOGRAPHY_SUMMARY temperature was maintained at 50ºC, and the flow rate was set at 0.4 mL/min. CH:CHROMATOGRAPHY_SUMMARY The mobile phase consisted of 0.1% formic acid in water as solvent A and 0.1% CH:CHROMATOGRAPHY_SUMMARY formic acid in acetonitrile as solvent B. The initial mobile phase composition CH:CHROMATOGRAPHY_SUMMARY was 5% A and 95% B, which was held constant for 2 minutes. Subsequently, a CH:CHROMATOGRAPHY_SUMMARY linear gradient from 5% to 50% A was applied over 8 minutes, followed by a CH:CHROMATOGRAPHY_SUMMARY 2-minute hold at 50% B before returning to the initial condition for a 5-minute CH:CHROMATOGRAPHY_SUMMARY re-equilibration. The total run time for each analysis was 19 minutes. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Synapt-XS Waters CH:COLUMN_NAME Waters ACQUITY UPLC BEH HILIC (100 x 2.1mm,1.7um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 5% to 50% A was applied over 8 minutes, followed by a 2-minute hold at 50% B CH:FLOW_GRADIENT before returning to the initial condition for a 5-minute re-equilibration. CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 50ºC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Synapt-XS MS:INSTRUMENT_TYPE Ion Mobility TOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The mass spectrometer operated at a frequency of 5 Hz, covering a mass range of MS:MS_COMMENTS 50-1200 m/z. MS:MS_RESULTS_FILE ST002932_AN004810_Results.txt UNITS:peak intensities Has m/z:Yes Has RT:Yes RT units:Minutes #END