{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003038","ANALYSIS_ID":"AN004984","VERSION":"1","CREATED_ON":"January 8, 2024, 10:32 am"},

"PROJECT":{"PROJECT_TITLE":"Untargeted lipidomics of WT and Cyp2c44(-/-) mice liver.","PROJECT_TYPE":"Untargeted Lipidomics","PROJECT_SUMMARY":"Cytochrome P450 epoxygenase Cyp2c44 and their metabolite epoxyeicosatrienoic acids or EETs promotes insulin sensitivity. Mice lacking Cyp2c44 exhibits hepatic insulin resistance. Insulin resistance is also intricately related to increased hepatic lipid accumulation and hyperlipidemia. Interestingly, Cyp2c44(-/-) mice in standard chow diet had significantly increased hepatic and plasma lipid levels compared to wild-type mice. To identify the nature of these lipids, with a focus on fatty acids, we performed lipidomic analysis of liver homogenates from SD-fed WT and Cyp2c44(-/-) mice. We identified 2425 lipids (1152 in negative mode and 1273 in positive mode) that passed both quality control filters set as 25% for QC RSD and 10% for QC/blank ratio. Principal component analysis revealed two distinct lipid clusters in livers of WT and Cyp2c44(-/-) mice. Heatmap analysis revealed a hierarchical clustering of significant differences occurring in lipid species between WT and Cyp2c44(-/-) livers. Volcano plot analysis of the 1152 lipids identified in the negative mode (which contain fatty acids) revealed 160 lipid species upregulated, 61 downregulated, and 931 not significantly changed in Cyp2c44(-/-) livers compared to WT livers. Analysis of fatty acids classes in negative mode identified a total of 146 fatty acids, with 49 upregulated, 3 downregulated and 94 unchanged in Cyp2c44(-/-) compared to WT livers. Among the fatty acids that are significantly upregulated in the livers of Cyp2c44(-/-), we detected the saturated fatty acids palmitic acid; the monosaturated oleic acid; and the polyunsaturated arachidonic, linoleic, eicosapentaenoic and docosahexaenoic acids. Importantly, arachidonic acid is the major substrate of Cyp2c epoxygenases, although linoleic, eicosapentaenoic and docosahexaenoic acids are also efficient alternative substrates. We thus hypothesized that Cyp2c44 also governs hepatic lipid metabolism.","INSTITUTE":"Vanderbilt University Medical Center","DEPARTMENT":"Nephrology","LAST_NAME":"Ghoshal","FIRST_NAME":"Kakali","ADDRESS":"B-3106 Medical Center North, 1161 21st Ave South, Nashville, TN 37232","EMAIL":"kakali.ghoshal@vumc.org","PHONE":"615-322-7729"},

"STUDY":{"STUDY_TITLE":"Untargeted lipidomics of WT and Cyp2c44(-/-) mice liver.","STUDY_TYPE":"Untargeted Lipidomics","STUDY_SUMMARY":"Cytochrome P450 epoxygenase Cyp2c44 and their metabolite epoxyeicosatrienoic acids or EETs promotes insulin sensitivity. Mice lacking Cyp2c44 exhibits hepatic insulin resistance. Insulin resistance is also intricately related to increased hepatic lipid accumulation and hyperlipidemia. Interestingly, Cyp2c44(-/-) mice in standard chow diet had significantly increased hepatic and plasma lipid levels compared to wild-type mice. To identify the nature of these lipids, with a focus on fatty acids, we performed lipidomic analysis of liver homogenates from SD-fed WT and Cyp2c44(-/-) mice. We identified 2425 lipids (1152 in negative mode and 1273 in positive mode) that passed both quality control filters set as 25% for QC RSD and 10% for QC/blank ratio. Principal component analysis revealed two distinct lipid clusters in livers of WT and Cyp2c44(-/-) mice. Heatmap analysis revealed a hierarchical clustering of significant differences occurring in lipid species between WT and Cyp2c44(-/-) livers. Volcano plot analysis of the 1152 lipids identified in the negative mode (which contain fatty acids) revealed 160 lipid species upregulated, 61 downregulated, and 931 not significantly changed in Cyp2c44(-/-) livers compared to WT livers. Analysis of fatty acids classes in negative mode identified a total of 146 fatty acids, with 49 upregulated, 3 downregulated and 94 unchanged in Cyp2c44(-/-) compared to WT livers. Among the fatty acids that are significantly upregulated in the livers of Cyp2c44(-/-), we detected the saturated fatty acids palmitic acid; the monosaturated oleic acid; and the polyunsaturated arachidonic, linoleic, eicosapentaenoic and docosahexaenoic acids. Importantly, arachidonic acid is the major substrate of Cyp2c epoxygenases, although linoleic, eicosapentaenoic and docosahexaenoic acids are also efficient alternative substrates. We thus hypothesized that Cyp2c44 also governs hepatic lipid metabolism.","INSTITUTE":"Vanderbilt University Medical Center","LAST_NAME":"Ghoshal","FIRST_NAME":"Kakali","ADDRESS":"B-3106 Medical Center North, 1161 21st Ave South, Nashville, TN 37232","EMAIL":"kakali.ghoshal@vumc.org","PHONE":"615-3224635","NUM_GROUPS":"2","TOTAL_SUBJECTS":"9","NUM_MALES":"9"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","AGE_OR_AGE_RANGE":"12 weeks","WEIGHT_OR_WEIGHT_RANGE":"25 g","GENDER":"Male"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"POS_01",
"Factors":{"Factor":"WT"},
"Additional sample data":{"Ionization mode":"POS","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_POS_S01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"POS_02",
"Factors":{"Factor":"WT"},
"Additional sample data":{"Ionization mode":"POS","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_POS_S02.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"POS_03",
"Factors":{"Factor":"WT"},
"Additional sample data":{"Ionization mode":"POS","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_POS_S03.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"POS_04",
"Factors":{"Factor":"WT"},
"Additional sample data":{"Ionization mode":"POS","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_POS_S05.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"POS_05",
"Factors":{"Factor":"WT"},
"Additional sample data":{"Ionization mode":"POS","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_POS_S04.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"POS_06",
"Factors":{"Factor":"2c44KO"},
"Additional sample data":{"Ionization mode":"POS","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_POS_S06.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"POS_07",
"Factors":{"Factor":"2c44KO"},
"Additional sample data":{"Ionization mode":"POS","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_POS_S07.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"POS_08",
"Factors":{"Factor":"2c44KO"},
"Additional sample data":{"Ionization mode":"POS","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_POS_S09.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"POS_09",
"Factors":{"Factor":"2c44KO"},
"Additional sample data":{"Ionization mode":"POS","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_POS_S08.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"POS_10",
"Factors":{"Factor":"2c44KO"},
"Additional sample data":{"Ionization mode":"POS","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_POS_S10.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_01",
"Factors":{"Factor":"WT"},
"Additional sample data":{"Ionization mode":"NEG","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_NEG_S01.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_02",
"Factors":{"Factor":"WT"},
"Additional sample data":{"Ionization mode":"NEG","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_NEG_S02.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_03",
"Factors":{"Factor":"WT"},
"Additional sample data":{"Ionization mode":"NEG","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_NEG_S03.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_04",
"Factors":{"Factor":"WT"},
"Additional sample data":{"Ionization mode":"NEG","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_NEG_S04.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_05",
"Factors":{"Factor":"WT"},
"Additional sample data":{"Ionization mode":"NEG","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_NEG_S05.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_06",
"Factors":{"Factor":"2c44KO"},
"Additional sample data":{"Ionization mode":"NEG","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_NEG_S06.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_07",
"Factors":{"Factor":"2c44KO"},
"Additional sample data":{"Ionization mode":"NEG","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_NEG_S07.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_08",
"Factors":{"Factor":"2c44KO"},
"Additional sample data":{"Ionization mode":"NEG","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_NEG_S08.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_09",
"Factors":{"Factor":"2c44KO"},
"Additional sample data":{"Ionization mode":"NEG","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_NEG_S09.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_10",
"Factors":{"Factor":"2c44KO"},
"Additional sample data":{"Ionization mode":"NEG","RAW_FILE_NAME":"211210_SVC_Pozzi__Lipidomics_NEG_S10.mzXML"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"100 mg of mouse livers were collected from standard chow diet fed WT and Cyp2c44(-/-) mice.","SAMPLE_TYPE":"Liver"},

"TREATMENT":{"TREATMENT_SUMMARY":"Experiments conducted in mice were approved by the Vanderbilt Institutional Animal Care and Use Committee. Male 129/SvJ wild-type (WT) and Cyp2c44(-/-) mice littermates were housed in an Association for Assessment and Accreditation of Laboratory Animal Care–accredited facility. Mice were generated and maintained as described previously (Ghoshal K et al, Diabetes, 2021, doi: 10.2337/db21-0298). Mice were fed SD (~14% fat Purina Laboratory Rodent 5001) up to 12 weeks of age until euthanized to collect livers."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Mouse liver (100 mg) was homogenized in 500 µl PBS using FisherBrand BeadMill. Homogenates were spiked with 10 µl SPLASH-lipidomics internal standard mix (Avanti Polar Lipids/CRODA), transferred to glass tubes and extracted using the method of Bligh and Dyer. Lipid extracts were evaporated under a gentle stream of nitrogen gas and resuspended in 100 µl of HPLC-grade methanol and chloroform (9:1 ratio). Discovery lipidomics were performed using a Vanquish ultrahigh performance liquid chromatography (UHPLC) system interfaced to a Q Exactive HF quadrupole/orbitrap mass spectrometer (Thermo Fisher Scientific) operating in data-dependent mode. Lipid extracts (5 µl) were injected in both positive and negative ESI modes. Pooled QCs were injected to assess the performance of the LC and MS instruments at the beginning, middle and at the end of each sequence."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"MSRC Lipidomics LC method","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Waters ACQUITY UPLC BEH C18 (150 x 2.1mm,1.7um)","SOLVENT_A":"40% water/60% acetonitrile; 0.1% formic acid; 10 mM Ammonium formate, pH ~3","SOLVENT_B":"90% isopropanol/10% acetonitrile; 0.1% formic acid; 10 mM Ammonium formate, pH ~3","FLOW_GRADIENT":"Linear gradient from 20% B to 100% B over 7 minutes","FLOW_RATE":"0.25 mL/min","COLUMN_TEMPERATURE":"50"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive HF hybrid Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"MS was operating in data-dependent mode (Top7); Raw data data were processed with MS-DIAL version 4.70 in POS-lipidomics mode."},

"MS_METABOLITE_DATA":{
"Units":"peakarea",

"Data":[{"Metabolite":"CAR 4:0 (232.15382@1.505)","POS_01":"163401776","POS_02":"148844288","POS_03":"115605560","POS_04":"164865984","POS_05":"122016728","POS_06":"100837688","POS_07":"92799912","POS_08":"106382880","POS_09":"91922632","POS_10":"105400448"},{"Metabolite":"NAE 12:0 (244.22755@2.507)","POS_01":"65938196","POS_02":"104292600","POS_03":"43047108","POS_04":"64008820","POS_05":"85376856","POS_06":"148394832","POS_07":"161487888","POS_08":"136997104","POS_09":"187294560","POS_10":"117907648"},{"Metabolite":"CAR 5:0 (246.17033@1.68)","POS_01":"3435939328","POS_02":"4324818944","POS_03":"2664717568","POS_04":"3187928064","POS_05":"2897429760","POS_06":"2377873920","POS_07":"2747675904","POS_08":"2203023616","POS_09":"2482956544","POS_10":"2664140032"},{"Metabolite":"NAE 13:1 (256.22751@5.128)","POS_01":"61869216","POS_02":"63612496","POS_03":"63406800","POS_04":"64861924","POS_05":"66325256","POS_06":"60810088","POS_07":"61364432","POS_08":"61023324","POS_09":"61395232","POS_10":"57581892"},{"Metabolite":"CAR 6:0 (260.18619@1.946)","POS_01":"81224072","POS_02":"101946480","POS_03":"88376944","POS_04":"81554192","POS_05":"84983432","POS_06":"104666816","POS_07":"116364336","POS_08":"114287224","POS_09":"115846448","POS_10":"108206104"},{"Metabolite":"NAE 14:0 (272.2587@2.47)","POS_01":"101259168","POS_02":"84664832","POS_03":"199339504","POS_04":"110716400","POS_05":"12003112","POS_06":"73424136","POS_07":"111541824","POS_08":"97150600","POS_09":"70964808","POS_10":"13391640"},{"Metabolite":"CAR 7:0 (274.20102@1.848)","POS_01":"234206256","POS_02":"287041024","POS_03":"225609568","POS_04":"246343248","POS_05":"279855264","POS_06":"316324384","POS_07":"340383744","POS_08":"316680128","POS_09":"346147648","POS_10":"334819232"},{"Metabolite":"NAE 15:1 (284.258@6.136)","POS_01":"29129514","POS_02":"19059500","POS_03":"21273572","POS_04":"29243220","POS_05":"24922384","POS_06":"52412288","POS_07":"49610004","POS_08":"43674492","POS_09":"42814884","POS_10":"48759604"},{"Metabolite":"NAE 15:0 (286.2742@3.87)","POS_01":"355785152","POS_02":"383322272","POS_03":"326750272","POS_04":"351395168","POS_05":"365910240","POS_06":"372509408","POS_07":"457270176","POS_08":"381671232","POS_09":"388034656","POS_10":"368215392"},{"Metabolite":"CAR 8:0 (288.21732@1.792)","POS_01":"468264928","POS_02":"456329536","POS_03":"492364800","POS_04":"477379072","POS_05":"506865792","POS_06":"494152000","POS_07":"450880256","POS_08":"509272576","POS_09":"515952352","POS_10":"495275104"},{"Metabolite":"NAE 16:2 (296.25885@5.91)","POS_01":"9490365","POS_02":"18147484","POS_03":"5349412","POS_04":"13016619","POS_05":"18448344","POS_06":"29995832","POS_07":"40804284","POS_08":"24837926","POS_09":"30451644","POS_10":"56574096"},{"Metabolite":"NAE 16:1 (298.27466@6.504)","POS_01":"75239392","POS_02":"115068568","POS_03":"50204572","POS_04":"81014232","POS_05":"103493088","POS_06":"172732544","POS_07":"225579888","POS_08":"151087696","POS_09":"206565024","POS_10":"408555072"},{"Metabolite":"NAE 16:0 (300.28989@6.104)","POS_01":"153784384","POS_02":"112432096","POS_03":"66570500","POS_04":"201659600","POS_05":"88999656","POS_06":"245897952","POS_07":"632096576","POS_08":"166792112","POS_09":"1807598080","POS_10":"508891392"},{"Metabolite":"NAE 17:2 (310.27487@6.796)","POS_01":"18191556","POS_02":"32399482","POS_03":"18686178","POS_04":"18935430","POS_05":"22498574","POS_06":"2329561","POS_07":"2389170","POS_08":"2552694","POS_09":"1682484","POS_10":"1512317"},{"Metabolite":"NAE 17:1 (312.29013@3.116)","POS_01":"4098319","POS_02":"21025122","POS_03":"9427945","POS_04":"12778549","POS_05":"32148918","POS_06":"256333008","POS_07":"95067216","POS_08":"113618984","POS_09":"65271396","POS_10":"133509256"},{"Metabolite":"NAE 17:0 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(330.26416@2.331)","POS_01":"460860896","POS_02":"481084736","POS_03":"472817856","POS_04":"474060384","POS_05":"484569120","POS_06":"454548224","POS_07":"466014720","POS_08":"464138016","POS_09":"469040384","POS_10":"474664064"},{"Metabolite":"NAE 19:3 (336.2872@6.539)","POS_01":"4524596","POS_02":"2400839","POS_03":"1619399","POS_04":"8116888","POS_05":"2176783","POS_06":"3434741","POS_07":"10942309","POS_08":"2824477","POS_09":"31914954","POS_10":"2386147"},{"Metabolite":"NAE 19:2 (338.30597@3.568)","POS_01":"15809104","POS_02":"14977503","POS_03":"5796385","POS_04":"8646485","POS_05":"24310716","POS_06":"54441096","POS_07":"42897312","POS_08":"45557688","POS_09":"19508232","POS_10":"50880572"},{"Metabolite":"NAE 19:1 (340.32111@4.41)","POS_01":"227252240","POS_02":"253958624","POS_03":"138416608","POS_04":"188818080","POS_05":"313341152","POS_06":"1199992704","POS_07":"668196224","POS_08":"576212800","POS_09":"454816416","POS_10":"667555776"},{"Metabolite":"MG 16:3 (342.26395@4.476)","POS_01":"50921188","POS_02":"57145236","POS_03":"71986360","POS_04":"34325180","POS_05":"41690848","POS_06":"4497246","POS_07":"4534154","POS_08":"5099148","POS_09":"4443305","POS_10":"8467842"},{"Metabolite":"NAE 19:0 (342.3356@5.288)","POS_01":"1055487936","POS_02":"514654976","POS_03":"410692384","POS_04":"597963200","POS_05":"639798336","POS_06":"943319872","POS_07":"726736576","POS_08":"391923712","POS_09":"471500992","POS_10":"669098304"},{"Metabolite":"CAR 12:0 (344.28009@3.175)","POS_01":"87875672","POS_02":"91233064","POS_03":"95214184","POS_04":"86175536","POS_05":"87802752","POS_06":"87229184","POS_07":"87966128","POS_08":"85719256","POS_09":"89073152","POS_10":"132712824"},{"Metabolite":"NAE 20:5 (346.27322@6.124)","POS_01":"176315568","POS_02":"156679536","POS_03":"104740008","POS_04":"167466112","POS_05":"158411824","POS_06":"460985248","POS_07":"524443584","POS_08":"329545344","POS_09":"417369664","POS_10":"625857856"},{"Metabolite":"MG 16:0 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