#METABOLOMICS WORKBENCH kmercier_20160831_073314 DATATRACK_ID:711 VERSION 1 CREATED_ON November 17, 2016, 3:13 pm #PROJECT PR:PROJECT_TITLE Metabolomic analysis of oxytocin effects on social deficits in mice PR:PROJECT_SUMMARY The goal of this study was to determine the effects of the neuropeptide oxytocin PR:PROJECT_SUMMARY (OT) on metabolomic profiles, using a mouse model of autism-like behavior, the PR:PROJECT_SUMMARY BALB/cByJ inbred strain. We have previously reported that subchronic treatment PR:PROJECT_SUMMARY with OT can lead to persistent reversal of social deficits in BALB/cByJ and PR:PROJECT_SUMMARY other models of autism spectrum disorder (ASD). In this study, mice were given a PR:PROJECT_SUMMARY subchronic regimen with either vehicle or OT, and then evaluated for social PR:PROJECT_SUMMARY approach. At the end of the study, brain and blood were collected for PR:PROJECT_SUMMARY metabolomic analysis. In addition, fecal samples were taken at different time PR:PROJECT_SUMMARY points during the treatment and testing regimen. The results from this project PR:PROJECT_SUMMARY could elucidate mechanisms underlying the prosocial effects of oxytocin, and PR:PROJECT_SUMMARY identify new targets for the development of highly specific oxytocin-related PR:PROJECT_SUMMARY drugs. PR:INSTITUTE University of North Carolina PR:DEPARTMENT Department of Psychiatry PR:LAST_NAME Moy PR:FIRST_NAME Sheryl PR:ADDRESS CB# 7146, Chapel Hill, NC 277599 PR:EMAIL ssmoy@med.unc.edu PR:PHONE 919-966-3082 #STUDY ST:STUDY_TITLE Metabolomic analysis of oxytocin effects on social deficits in mice (part IV) ST:STUDY_SUMMARY This metabolomics pilot study evaluated serum from mice treated with oxytocin ST:STUDY_SUMMARY and vehicle to understand how these factors influence the metabotype. ST:INSTITUTE RTI International ST:LAST_NAME Sumner ST:FIRST_NAME Susan ST:ADDRESS 3040 E. Cornwallis Road ST:EMAIL ssumner@rti.org ST:PHONE 919-541-7479 #SUBJECT SU:SUBJECT_TYPE Murine SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS C3 S_11 Treatment:Veh .raw data file=MOY_LIPIDS_S_11_POS.raw SUBJECT_SAMPLE_FACTORS B6 S_9 Treatment:Veh .raw data file=MOY_LIPIDS_S_9_POS.raw SUBJECT_SAMPLE_FACTORS B3 S_6 Treatment:Oxytocin .raw data file=MOY_LIPIDS_S_6_POS.raw SUBJECT_SAMPLE_FACTORS C2 S_10 Treatment:Oxytocin .raw data file=MOY_LIPIDS_S_10_POS.raw SUBJECT_SAMPLE_FACTORS D4 S_18 Treatment:Veh .raw data file=MOY_LIPIDS_S_18_POS.raw SUBJECT_SAMPLE_FACTORS C4 S_12 Treatment:Oxytocin .raw data file=MOY_LIPIDS_S_12_POS.raw SUBJECT_SAMPLE_FACTORS D1 S_15 Treatment:Oxytocin .raw data file=MOY_LIPIDS_S_15_POS.raw SUBJECT_SAMPLE_FACTORS B1 S_4 Treatment:Veh .raw data file=MOY_LIPIDS_S_4_POS.raw SUBJECT_SAMPLE_FACTORS A5 S_3 Treatment:Oxytocin .raw data file=MOY_LIPIDS_S_3_POS.raw SUBJECT_SAMPLE_FACTORS D2 S_16 Treatment:Veh .raw data file=MOY_LIPIDS_S_16_POS.raw SUBJECT_SAMPLE_FACTORS A1 S_1 Treatment:Oxytocin .raw data file=MOY_LIPIDS_S_1_POS.raw SUBJECT_SAMPLE_FACTORS B4 S_7 Treatment:Veh .raw data file=MOY_LIPIDS_S_7_POS.raw SUBJECT_SAMPLE_FACTORS D6 S_20 Treatment:Veh .raw data file=MOY_LIPIDS_S_20_POS.raw SUBJECT_SAMPLE_FACTORS A4 S_2 Treatment:Oxytocin .raw data file=MOY_LIPIDS_S_2_POS.raw SUBJECT_SAMPLE_FACTORS B5 S_8 Treatment:Oxytocin .raw data file=MOY_LIPIDS_S_8_POS.raw SUBJECT_SAMPLE_FACTORS C6 S_14 Treatment:Oxytocin .raw data file=MOY_LIPIDS_S_14_POS.raw SUBJECT_SAMPLE_FACTORS D3 S_17 Treatment:Oxytocin .raw data file=MOY_LIPIDS_S_17_POS.raw SUBJECT_SAMPLE_FACTORS B2 S_5 Treatment:Oxytocin .raw data file=MOY_LIPIDS_S_5_POS.raw SUBJECT_SAMPLE_FACTORS D5 S_19 Treatment:Oxytocin .raw data file=MOY_LIPIDS_S_19_POS.raw SUBJECT_SAMPLE_FACTORS C5 S_13 Treatment:Veh .raw data file=MOY_LIPIDS_S_13_POS.raw SUBJECT_SAMPLE_FACTORS Pool PS_1 Treatment:Pool .raw data file=MOY_LIPIDS_PS_1_POS.raw SUBJECT_SAMPLE_FACTORS Pool PS_2 Treatment:Pool .raw data file=MOY_LIPIDS_PS_2_POS.raw #COLLECTION CO:COLLECTION_SUMMARY Trunk blood was collected into centrifuge tubes and centrifuged for 15 min at CO:COLLECTION_SUMMARY ~1,000 x g at 4o C. Supernatant (serum) was extracted, frozen on dry ice, and CO:COLLECTION_SUMMARY stored at -80o C. #TREATMENT TR:TREATMENT_SUMMARY BALB/cByJ male mice (4-5 weeks in age; Jackson Laboratory, Bar Harbor, ME) were TR:TREATMENT_SUMMARY given four treatments of either vehicle or OT (1.0 mg/kg; IP) across 8 days, TR:TREATMENT_SUMMARY with at least 48 hours between each injection. Mice were evaluated in a TR:TREATMENT_SUMMARY 3-chamber choice test for social preference 24 hr after the final injection. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 30 µL of each serum sample was transferred to a new, pre-labeled 1.5 mL LoBind SP:SAMPLEPREP_SUMMARY eppendorf tube to make the individual study samples, and 12 µL was transferred SP:SAMPLEPREP_SUMMARY to a new, pre-labeled 1.5 mL LoBind Eppendorf to make the total pool. The total SP:SAMPLEPREP_SUMMARY pool was aliquoted into 3 Total Pool QC Samples and 4 Column Equilibrium samples SP:SAMPLEPREP_SUMMARY with volumes of 30 µL in 1.5 mL LoBind Eppendorf tubes. 600 uL of Lipid SP:SAMPLEPREP_SUMMARY Extraction Solvent (PC(17:0/17:0) 50 µg/mL in 2:1 dichloromethane:methanol) was SP:SAMPLEPREP_SUMMARY added to all samples, and samples were vortexed at 4,000 rpm for 2mins on SP:SAMPLEPREP_SUMMARY multiple-tube vortexer. 120 µL of H2O was added to each tube and the samples SP:SAMPLEPREP_SUMMARY were vortexed at 4,000 rpm for 1min. Samples sat at room temperature for 10 SP:SAMPLEPREP_SUMMARY minutes, and were centrifuged at 16,000 rcf for 10 min at 10°C. 240 µL of the SP:SAMPLEPREP_SUMMARY lower lipid-rich DCM layer was transferred into new pre-labeled 1.5 mL LoBind SP:SAMPLEPREP_SUMMARY Eppendorf tubes, and rubber stoppers were inserted. Samples were placed at SP:SAMPLEPREP_SUMMARY -80˚C for 60 mins, and then lyophilized for 19.5 hrs. 240 µL of ACN/IPA/H2O SP:SAMPLEPREP_SUMMARY (65:30:5 v/v/v) was added to each sample, and the samples were vortexed at 4,000 SP:SAMPLEPREP_SUMMARY rpm for 2mins and centrifuged at 16,000 rcf for 4 min. 225 µL of the SP:SAMPLEPREP_SUMMARY supernatant was transferred to pre-labeled autosampler vials and 10 µL was SP:SAMPLEPREP_SUMMARY injected into OrbiTrap Velos. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity CSH C18 (100 x 2.1mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS MS MS_METABOLITE_DATA_START Samples PS_1 PS_2 S_11 S_9 S_16 S_18 S_4 S_13 S_20 S_7 S_19 S_12 S_15 S_1 S_17 S_2 S_6 S_10 S_14 S_3 S_8 S_5 Factors Treatment:Pool Treatment:Pool Treatment:Veh 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10261.23853 10193.5629 10883.06769 7708.399528 12217.69999 11117.94639 13764.31831 12980.90742 13129.85723 10231.76793 11916.04489 10210.52827 9688.12545 11290.90166 6860.274855 8670.874335 10708.24489 13178.44027 10326.30131 10087.99032 16.99_1581.2469m/z 52521.96377 41709.99716 70453.48464 54649.94968 39136.6939 34162.42096 38196.66522 63901.35328 48433.68708 48127.49589 68947.83889 53766.56644 39213.26855 54768.91655 45803.64214 53987.46332 61391.59229 74470.33229 50897.77049 52757.20494 43646.81251 61005.83014 4.55_521.3451n 3420699.082 3104695.955 3449101.745 4335264.838 3508830.127 3336545.504 2557567.042 2597131.02 2744892.686 2961175.47 4938484.065 3327107.341 3619965.188 3425631.751 3744784.848 3327147.509 4721307.641 3683881.301 1106463.933 3204065.267 3346608.636 3184658.818 10.65_829.5811m/z 6615.702464 5326.05245 4265.865409 2665.937763 5294.406201 9963.716407 5054.819734 4896.426621 5587.467921 5172.055717 6289.621496 6314.817027 6032.019589 4728.746648 5401.189952 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