#METABOLOMICS WORKBENCH hover_20170726_173354 DATATRACK_ID:1171 STUDY_ID:ST000901 ANALYSIS_ID:AN001467 PROJECT_ID:PR000461 VERSION 1 CREATED_ON November 15, 2017, 12:03 pm #PROJECT PR:PROJECT_TITLE Omega-6 and omega-3 oxylipins are implicated in soybean oil-induced obesity in PR:PROJECT_TITLE mice PR:PROJECT_SUMMARY Characterization and analysis of metabolomic, proteomic and metabolic profiles PR:PROJECT_SUMMARY of C57/Bl6N mice fed various high fat diets PR:INSTITUTE University of California, Riverside PR:LAST_NAME Sladek PR:FIRST_NAME Frances PR:ADDRESS 2115 Biological Sciences Building,University of California, Riverside, CA PR:ADDRESS 92521-0314 PR:EMAIL frances.sladek@ucr.edu PR:PHONE 951-827-2264 #STUDY ST:STUDY_TITLE Omega-6 and omega-3 oxylipins are implicated in soybean oil-induced obesity in ST:STUDY_TITLE mice - Plasma (part V) ST:STUDY_SUMMARY Characterization and analysis of metabolomic, proteomic and metabolic profiles ST:STUDY_SUMMARY of C57/Bl6N mice fed various high fat diets ST:INSTITUTE University of California, Davis ST:LAST_NAME Yang ST:FIRST_NAME Jun ST:ADDRESS 207 Everson Hall, Shields One Ave, Davis CA 95616 ST:EMAIL junyang@ucdavis.edu ST:PHONE 5307525109 #SUBJECT SU:SUBJECT_TYPE Animal 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 - Viv-1 Diet:Viv chow SUBJECT_SAMPLE_FACTORS - Viv-2 Diet:Viv chow SUBJECT_SAMPLE_FACTORS - Viv-4 Diet:Viv chow SUBJECT_SAMPLE_FACTORS - Viv-9 Diet:Viv chow SUBJECT_SAMPLE_FACTORS - PM D4-16 e1 Diet:HFD SUBJECT_SAMPLE_FACTORS - PM D4-16 e3 Diet:HFD SUBJECT_SAMPLE_FACTORS - PM D4-18 e1 Diet:HFD SUBJECT_SAMPLE_FACTORS - PM D4-18 e2 Diet:HFD SUBJECT_SAMPLE_FACTORS - PM D3-17 e2 Diet:LA-HFD SUBJECT_SAMPLE_FACTORS - PM D3-17 e3 Diet:LA-HFD SUBJECT_SAMPLE_FACTORS - PM D3-18 e1 Diet:LA-HFD SUBJECT_SAMPLE_FACTORS - PM D3-18 e2 Diet:LA-HFD SUBJECT_SAMPLE_FACTORS - PM PL-4 e3 Diet:PL-HFD SUBJECT_SAMPLE_FACTORS - PM PL-4 e4 Diet:PL-HFD SUBJECT_SAMPLE_FACTORS - PM PL-5 e1 Diet:PL-HFD SUBJECT_SAMPLE_FACTORS - PM PL-5 e2 Diet:PL-HFD #COLLECTION CO:COLLECTION_SUMMARY Liver tissue from mice on the diets for 24 weeks for metabolomic analysis was CO:COLLECTION_SUMMARY collected, rinsed in cold PBS, excess fluid was blotted with a kim-wipe and CO:COLLECTION_SUMMARY tissue was immediately snap frozen in liquid nitrogen before storage at -80°C. CO:COLLECTION_SUMMARY Blood was collected by cardiac puncture and centrifuged at 9 rcf for 5 min at CO:COLLECTION_SUMMARY 4°C. Plasma was stored immediately at -20°C. #TREATMENT TR:TREATMENT_SUMMARY Male C57/BL6N mice weaned at 3weeks of age were randomly assigned to one of the TR:TREATMENT_SUMMARY four diets-Viv chow, 40 kcal% coconut oil , 40 kcal% total fat soybean oil diet TR:TREATMENT_SUMMARY (21 kcal% from coconut oil and 19 kcal% from soybean oil) or 40 kcal% total fat TR:TREATMENT_SUMMARY Plenish oil diet (21 kcal% from coconut oil and 19 kcal% from Plenish oil) for TR:TREATMENT_SUMMARY 24 weeks. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Preparation of SPE on vacuum manifold: 1. Clean 60 mg Oasis HLB (Waters) spe SP:SAMPLEPREP_SUMMARY column with 1 column volume ethyl acetate followed by 2 column volumes MeOH SP:SAMPLEPREP_SUMMARY 2. Condition column with 2 column volume 0.1% acetic acid :5% MeOH solution SP:SAMPLEPREP_SUMMARY 3. Close valves tightly when meniscus reaches frit over sorbent bed. Sample SP:SAMPLEPREP_SUMMARY extraction by SPE on vacuum manifold: 4. If not in sample at collection, add 10 SP:SAMPLEPREP_SUMMARY µL of 0.2 mg/mL of EDTA and BHT in MeOH: H2O (1:1) to sorbent bed of all SP:SAMPLEPREP_SUMMARY columns 5. Vortex each sample a few seconds to homogenize just prior to loading. SP:SAMPLEPREP_SUMMARY 6. Load 250µL plasma sample into SPE column. 7. Spike each column with 10µl SP:SAMPLEPREP_SUMMARY 500nM surrogate solution 8. Load 1.5ml of 0.1% acetic acid: 5% MeOH solution to SP:SAMPLEPREP_SUMMARY each column and let sit a few minutes. 9. Open valves to extract by gravity (use SP:SAMPLEPREP_SUMMARY low vacuum only if necessary) 10. Wash SPE with 2 column volumes 0.1% acetic SP:SAMPLEPREP_SUMMARY acid :5% MeOH solution 11. Pull aqueous plug from SPE with a few seconds high SP:SAMPLEPREP_SUMMARY vacuum. 12. Further dry SPE with low vacuum. ~20min, -20psi. 13. For each SP:SAMPLEPREP_SUMMARY sample, spike 2ml eppendorf tube with 6µl 30% glycerol in MeOH prior to eluate SP:SAMPLEPREP_SUMMARY collection. 14. Elute spe into tube with 0.5 ml methanol followed by 1.5 mL of SP:SAMPLEPREP_SUMMARY ethyl acetate 15. Evaporate extract until 2 µl glycerol plug is left, by SP:SAMPLEPREP_SUMMARY Speed-vac. 16. Cap and freeze by liquid nitrogen (for shipping) or store at SP:SAMPLEPREP_SUMMARY -80ºC until analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1200SL CH:COLUMN_NAME Agilent Eclipse Plus C18 (150 x 2.1mm, 1.8um) CH:FLOW_RATE 0.35 mL/min CH:SAMPLE_INJECTION 10 uL #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Hammock Lab #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME ABI Sciex 4000 QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:ION_SOURCE_TEMPERATURE 600 C #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Concentration nmol/L MS_METABOLITE_DATA_START Samples PM PL-5 e1 PM PL-5 e2 PM PL-4 e3 PM PL-4 e4 Viv-4 Viv-9 Viv-1 Viv-2 PM D3-18 e1 PM D3-17 e2 PM D3-18 e2 PM D3-17 e3 PM D4-16 e1 PM D4-16 e3 PM D4-18 e1 PM D4-18 e2 Factors Diet:PL-HFD Diet:PL-HFD Diet:PL-HFD Diet:PL-HFD Diet:Viv chow Diet:Viv chow Diet:Viv chow Diet:Viv chow Diet:LA-HFD Diet:LA-HFD Diet:LA-HFD Diet:LA-HFD Diet:HFD Diet:HFD Diet:HFD Diet:HFD 9,12,13-TriHOME 59.45 25.52 62.19 32.40 303.89 1278.75 23.25 1706.00 69.41 41.30 302.25 977.75 303.15 1595.83 812.00 7590.00 9,10,13-TriHOME 30.00 14.39 36.24 11.60 162.33 861.25 13.23 1081.33 34.13 25.35 100.70 548.50 154.50 913.33 501.25 3990.00 15,16-DiHODE 1.56 0.85 1.10 0.88 11.71 15.36 16.77 17.93 2.96 5.85 4.13 5.45 4.40 6.40 3.15 2.75 8,15-DiHETE 2.89 2.14 0.10 0.25 1.22 0.37 0.64 0.43 3.39 3.71 1.00 7.09 2.41 6.15 3.88 5.90 9,10-DiHODE 0.35 0.21 0.02 0.17 2.41 1.88 5.04 4.62 0.51 1.29 0.69 1.51 0.72 1.32 0.56 0.36 12,13-DiHODE 0.42 0.16 0.01 0.05 2.28 2.54 3.85 4.26 0.21 0.62 0.37 0.35 0.48 0.71 0.54 0.30 5,15-DiHETE 0.20 0.16 0.32 0.12 0.23 0.06 0.13 0.13 0.11 0.36 0.04 0.41 0.08 0.66 0.14 0.61 17,18-DiHETE 1.44 0.94 0.85 2.56 3.09 3.36 7.28 4.33 1.67 7.36 1.61 3.72 0.59 2.28 1.12 0.79 14,15-DiHETE 0.38 0.21 0.19 0.61 1.10 1.08 1.82 1.81 0.60 2.08 0.45 0.92 0.06 0.52 0.38 0.06 11,12-DiHETE 0.22 0.21 0.09 0.23 0.67 0.52 0.99 0.60 0.27 0.59 0.29 0.30 0.12 0.26 0.19 0.14 12,13-DiHOME 7.84 4.75 8.96 2.70 100.69 80.68 131.75 169.32 11.29 20.28 12.86 13.07 13.30 16.73 12.28 6.55 9,10-DiHOME 3.82 2.92 3.01 1.72 41.23 30.99 47.97 64.13 6.59 11.80 7.23 8.60 7.65 9.99 6.94 5.86 19,20-DiHDPE 8.57 8.75 9.88 14.52 17.22 15.35 16.01 16.61 10.38 27.34 11.09 15.19 5.25 14.79 9.92 6.19 14,15-DiHETrE 1.39 1.41 0.68 1.40 2.01 1.65 2.38 2.35 2.74 4.17 2.67 1.86 0.90 1.90 2.12 0.92 16,17-DiHDPE 1.56 1.19 1.23 3.10 3.02 2.08 2.94 3.01 1.80 7.04 1.98 2.97 0.70 2.06 1.32 0.78 11,12-DiHETrE 0.78 0.70 0.50 0.55 1.23 1.09 1.31 1.02 1.36 1.62 1.50 0.73 0.55 0.91 0.99 0.54 13,14-DiHDPE 0.47 0.38 0.49 0.55 0.95 0.81 1.09 0.85 0.44 1.11 0.54 0.68 0.12 0.80 0.49 0.30 9-HOTrE 1.87 3.78 12.37 0.77 18.62 14.18 5.27 16.33 3.82 10.14 3.58 6.75 7.05 14.31 4.39 5.85 10,11-DiHDPE 0.38 0.22 0.19 0.14 0.63 1.09 1.18 0.62 0.25 0.45 0.44 0.31 0.16 0.41 0.29 0.30 8,9-DiHETrE 0.60 0.31 0.69 0.62 0.36 0.50 0.98 1.05 0.82 1.07 1.23 0.12 0.50 0.65 0.78 0.03 EKODE 40.73 51.38 88.80 17.28 432.50 1265.11 49.51 422.37 74.35 72.54 32.69 113.79 231.19 233.42 57.16 194.24 13-HOTrE 4.49 10.21 14.53 0.76 21.67 13.15 11.42 19.64 9.24 20.92 9.00 20.28 13.59 26.11 14.77 20.26 15-HEPE 10.23 6.18 1.73 0.97 38.38 9.49 21.24 7.48 8.49 42.73 5.22 36.90 12.51 85.66 8.08 20.17 8-HEPE 3.20 1.70 1.86 1.25 12.97 6.24 8.20 4.15 4.27 17.97 2.16 17.54 3.80 24.88 2.19 6.29 12-HEPE 17.04 11.38 5.34 4.34 167.74 113.11 119.56 91.20 15.92 54.97 28.97 52.67 12.15 99.29 15.80 18.71 5-HEPE 2.13 1.35 1.64 1.55 12.37 9.75 9.25 6.86 2.78 10.81 1.33 7.28 3.01 18.90 1.42 5.29 13-HODE 85.04 93.19 97.26 16.47 382.18 561.56 257.86 394.68 178.47 259.19 112.02 237.85 266.66 355.96 255.35 292.15 9-HODE 21.41 26.30 29.25 5.87 140.08 257.92 55.78 136.89 49.09 81.76 31.13 74.78 78.12 101.30 61.26 77.69 15(16)-EpODE 4.83 3.41 9.81 3.54 49.78 62.06 79.11 58.94 10.02 10.33 9.94 9.07 15.20 16.91 10.48 4.66 15-HETE 17.18 15.92 16.73 6.93 33.69 18.70 9.01 7.11 23.29 60.42 14.33 53.86 45.36 122.14 18.20 49.73 9(10)-EpODE 0.80 0.33 4.73 1.54 6.02 11.21 5.91 7.41 1.08 2.58 1.41 2.28 2.26 5.45 2.41 0.88 17(18)-EpETE 0.75 0.50 1.79 1.28 6.05 2.64 4.84 4.07 0.70 0.93 0.84 0.85 1.04 1.92 0.56 0.40 11-HETE 11.15 8.53 6.57 3.63 19.56 7.01 6.49 3.04 13.41 53.16 9.84 46.64 22.93 77.74 14.66 28.62 12(13)-EpODE 0.19 0.16 2.02 0.45 4.32 6.87 3.64 3.36 1.22 1.39 0.59 0.91 0.92 2.32 0.57 0.57 15-oxo-ETE 5.54 8.46 8.49 2.11 13.13 7.31 2.25 5.67 10.30 21.15 6.84 16.19 25.20 37.71 14.09 26.14 9-oxo-ODE 30.22 32.88 55.73 10.89 453.48 1325.87 86.22 714.56 69.03 99.31 34.49 89.15 151.62 178.34 85.41 129.78 14(15)-EpETE 8-HETE 9.54 7.48 6.62 3.21 13.14 9.12 6.76 4.42 13.51 43.49 9.42 36.30 18.49 73.72 11.64 24.18 12-HETE 60.75 68.53 31.24 36.06 370.03 205.01 136.16 147.38 83.59 138.76 297.15 139.95 60.62 266.86 61.42 67.50 9-HETE 6.92 6.35 4.91 4.28 13.23 5.07 5.12 1.22 9.68 46.02 5.78 41.65 13.68 61.59 9.46 15.41 15(S)-HETrE 8.81 5.93 4.04 2.55 7.56 6.20 3.09 3.02 7.64 19.83 4.48 15.97 8.81 26.93 9.21 16.94 5-HETE 7.84 5.26 10.00 5.51 12.64 11.04 3.26 4.88 11.62 27.52 6.43 16.25 18.64 53.86 8.28 24.91 19(20)-EpDPE 4.74 2.79 8.93 6.98 16.73 14.57 11.81 12.73 5.08 8.15 4.40 8.68 5.38 11.82 6.31 3.54 12(13)-EpOME 5.37 2.55 17.31 3.10 75.32 120.13 64.06 66.54 11.86 18.48 8.09 16.35 19.69 33.28 14.95 5.60 14(15)-EpETrE 0.66 0.39 1.33 0.31 1.57 2.15 1.41 0.88 1.19 0.99 0.64 0.88 1.44 3.20 1.56 0.61 9(10)-EpOME 3.90 1.72 14.75 1.89 64.59 119.02 48.99 49.74 10.25 17.92 6.51 12.96 20.45 34.45 12.42 6.80 16(17)-EpDPE 0.60 0.27 1.79 0.79 2.94 4.14 1.35 2.22 0.67 1.26 0.66 1.51 0.46 1.52 0.65 0.13 10(11)-EpDPE 1.59 0.29 1.87 1.02 4.33 6.77 2.37 1.87 1.65 0.76 0.57 1.30 1.05 3.50 1.25 0.89 11(12)-EpETrE 0.96 0.43 4.25 0.70 4.69 5.32 2.41 2.68 1.96 2.79 1.04 2.12 4.27 5.47 0.14 0.83 7(8)-EpDPE 19.03 10.47 10.56 16.29 99.31 131.68 44.68 46.53 34.39 18.45 16.18 39.27 34.75 64.06 13.37 16.54 8(9)-EpETrE 11.09 13.21 14.40 6.70 5.34 3.47 2.22 2.14 11.50 5.77 6.29 8.93 12.85 11.09 16.97 21.26 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name 9,12,13-TriHOME 9,10,13-TriHOME 15,16-DiHODE 8,15-DiHETE 9,10-DiHODE 12,13-DiHODE 5,15-DiHETE 17,18-DiHETE 14,15-DiHETE 11,12-DiHETE 12,13-DiHOME 9,10-DiHOME 19,20-DiHDPE 14,15-DiHETrE 16,17-DiHDPE 11,12-DiHETrE 13,14-DiHDPE 9-HOTrE 10,11-DiHDPE 8,9-DiHETrE EKODE 13-HOTrE 15-HEPE 8-HEPE 12-HEPE 5-HEPE 13-HODE 9-HODE 15(16)-EpODE 15-HETE 9(10)-EpODE 17(18)-EpETE 11-HETE 12(13)-EpODE 15-oxo-ETE 9-oxo-ODE 14(15)-EpETE 8-HETE 12-HETE 9-HETE 15(S)-HETrE 5-HETE 19(20)-EpDPE 12(13)-EpOME 14(15)-EpETrE 9(10)-EpOME 16(17)-EpDPE 10(11)-EpDPE 11(12)-EpETrE 7(8)-EpDPE 8(9)-EpETrE METABOLITES_END #END