#METABOLOMICS WORKBENCH michaelsa93_20170623_104303 DATATRACK_ID:1079 STUDY_ID:ST000656 ANALYSIS_ID:AN001002 PROJECT_ID:PR000461
VERSION             	1
CREATED_ON             	June 26, 2017, 10:32 am
#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               	In this study we have compared the metabolic effects of conventional soybean oil
PR:PROJECT_SUMMARY               	to those of genetically modified Plenish soybean oil, that is low in linoleic
PR:PROJECT_SUMMARY               	acid and high in oleic acid. This work builds on our previous study showing that
PR:PROJECT_SUMMARY               	soybean oil, rich in polyunsaturated fats, is more obesogenic and diabetogenic
PR:PROJECT_SUMMARY               	than coconut oil, rich in saturated fats (PMID: 26200659). Here, in order to
PR:PROJECT_SUMMARY               	elucidate the mechanisms responsible for soybean oil induced obesity, we have
PR:PROJECT_SUMMARY               	performed the first ever metabolomics (in plasma and liver) and proteomics on
PR:PROJECT_SUMMARY               	the livers of mice fed the two soybean oil diets (plus those fed a high coconut
PR:PROJECT_SUMMARY               	oil and Viv chow diet). Our results show that the new high oleic soybean oil
PR:PROJECT_SUMMARY               	induces less obesity and adiposity than conventional soybean oil, but can cause
PR:PROJECT_SUMMARY               	hepatomegaly and liver dysfunction. Metabolomic analysis reveals that the
PR:PROJECT_SUMMARY               	hepatic and plasma metabolic profiles differ considerably between the two
PR:PROJECT_SUMMARY               	soybean oils. Hepatic C18 oxylipin metabolites of omega-6 (ω6) and omega-3
PR:PROJECT_SUMMARY               	(ω3) fatty acids (linoleic and α-linolenic acid, respectively) in the
PR:PROJECT_SUMMARY               	cytochrome P450/soluble epoxide hydrolase pathway were found to correlate
PR:PROJECT_SUMMARY               	positively with obesity.
PR:INSTITUTE                     	University of California, Riverside
PR:DEPARTMENT                    	Cell Biology and Neuroscience
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 (part III)
ST:STUDY_SUMMARY                 	In this study we have compared the metabolic effects of conventional soybean oil
ST:STUDY_SUMMARY                 	to those of genetically modified Plenish soybean oil, that is low in linoleic
ST:STUDY_SUMMARY                 	acid and high in oleic acid. This work builds on our previous study showing that
ST:STUDY_SUMMARY                 	soybean oil, rich in polyunsaturated fats, is more obesogenic and diabetogenic
ST:STUDY_SUMMARY                 	than coconut oil, rich in saturated fats (PMID: 26200659). Here, in order to
ST:STUDY_SUMMARY                 	elucidate the mechanisms responsible for soybean oil induced obesity, we have
ST:STUDY_SUMMARY                 	performed the first ever metabolomics (in plasma and liver) and proteomics on
ST:STUDY_SUMMARY                 	the livers of mice fed the two soybean oil diets (plus those fed a high coconut
ST:STUDY_SUMMARY                 	oil and Viv chow diet). Our results show that the new high oleic soybean oil
ST:STUDY_SUMMARY                 	induces less obesity and adiposity than conventional soybean oil, but can cause
ST:STUDY_SUMMARY                 	hepatomegaly and liver dysfunction. Metabolomic analysis reveals that the
ST:STUDY_SUMMARY                 	hepatic and plasma metabolic profiles differ considerably between the two
ST:STUDY_SUMMARY                 	soybean oils. Hepatic C18 oxylipin metabolites of omega-6 (ω6) and omega-3
ST:STUDY_SUMMARY                 	(ω3) fatty acids (linoleic and α-linolenic acid, respectively) in the
ST:STUDY_SUMMARY                 	cytochrome P450/soluble epoxide hydrolase pathway were found to correlate
ST:STUDY_SUMMARY                 	positively with obesity.
ST:INSTITUTE                     	University of California, Davis
ST:DEPARTMENT                    	Genome and Biomedical Sciences Facility
ST:LABORATORY                    	WCMC Metabolomics Core
ST:LAST_NAME                     	Fiehn
ST:FIRST_NAME                    	Oliver
ST:ADDRESS                       	1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis,
ST:ADDRESS                       	CA 95616
ST:EMAIL                         	ofiehn@ucdavis.edu
ST:PHONE                         	(530) 754-8258
#SUBJECT
SU:SUBJECT_TYPE                  	Animal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
SU:GENOTYPE_STRAIN               	C57/BL6N
SU:GENDER                        	Male
SU:ANIMAL_HOUSING                	SPF facility
SU:ANIMAL_LIGHT_CYCLE            	12:12 h light-dark cycle
SU:ANIMAL_FEED                   	Ad libitum
SU:ANIMAL_WATER                  	Ad libitum
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	-	CSH_BioRec_01_pos mode_MS_07212014.d	GROUP_DESCRIPTION:N/A | TIME_POINT:N/A | Genotype:N/A	
SUBJECT_SAMPLE_FACTORS           	-	CSH_BioRec_02_pos mode_MS_07212014.d	GROUP_DESCRIPTION:N/A | TIME_POINT:N/A | Genotype:N/A	
SUBJECT_SAMPLE_FACTORS           	-	CSH_BioRec_03_pos mode_MS_07212014.d	GROUP_DESCRIPTION:N/A | TIME_POINT:N/A | Genotype:N/A	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM D3-17 e1_pos mode_MS_07212014.d	GROUP_DESCRIPTION:LA-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM D3-17 e2_pos mode_MS_07212014.d	GROUP_DESCRIPTION:LA-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM D3-17 e3reinject_pos mode_MS_07212014.d	GROUP_DESCRIPTION:LA-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM D3-17 e4_pos mode_MS_07212014.d	GROUP_DESCRIPTION:LA-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM D3-18 e1_pos mode_MS_07212014.d	GROUP_DESCRIPTION:LA-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM D3-18 e2reinject_pos mode_MS_07212014.d	GROUP_DESCRIPTION:LA-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM D3-18 e3_pos mode_MS_07212014.d	GROUP_DESCRIPTION:LA-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM D3-18 e4_pos mode_MS_07212014.d	GROUP_DESCRIPTION:LA-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM D4-16 e1_pos mode_MS_07212014.d	GROUP_DESCRIPTION:HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM D4-16 e2_pos mode_MS_07212014.d	GROUP_DESCRIPTION:HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM D4-16 e3_pos mode_MS_07212014.d	GROUP_DESCRIPTION:HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM D4-16 e4_pos mode_MS_07212014.d	GROUP_DESCRIPTION:HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM D4-17 e1_pos mode_MS_07212014.d	GROUP_DESCRIPTION:HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM D4-17 e2_pos mode_MS_07212014.d	GROUP_DESCRIPTION:HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM D4-18 e1_pos mode_MS_07212014.d	GROUP_DESCRIPTION:HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM D4-18 e4_pos mode_MS_07212014.d	GROUP_DESCRIPTION:HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM PL-4 e1_pos mode_MS_07212014.d	GROUP_DESCRIPTION:PL-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM PL-4 e3_pos mode_MS_07212014.d	GROUP_DESCRIPTION:PL-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM PL-4 e4_pos mode_MS_07212014.d	GROUP_DESCRIPTION:PL-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM PL-5 e1_pos mode_MS_07212014.d	GROUP_DESCRIPTION:PL-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM PL-5 e2_pos mode_MS_07212014.d	GROUP_DESCRIPTION:PL-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM PL-5 e3_pos mode_MS_07212014.d	GROUP_DESCRIPTION:PL-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM PL-6 e1_pos mode_MS_07212014.d	GROUP_DESCRIPTION:PL-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_PM PL-6 e4_pos mode_MS_07212014.d	GROUP_DESCRIPTION:PL-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_Viv-1_pos mode_MS_07212014.d	GROUP_DESCRIPTION:Viv chow | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_Viv-2_pos mode_MS_07212014.d	GROUP_DESCRIPTION:Viv chow | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_Viv-4_pos mode_MS_07212014.d	GROUP_DESCRIPTION:Viv chow | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_Viv-5_pos mode_MS_07212014.d	GROUP_DESCRIPTION:Viv chow | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_Viv-6_pos mode_MS_07212014.d	GROUP_DESCRIPTION:Viv chow | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_Viv-7_pos mode_MS_07212014.d	GROUP_DESCRIPTION:Viv chow | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_Viv-8_pos mode_MS_07212014.d	GROUP_DESCRIPTION:Viv chow | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
SUBJECT_SAMPLE_FACTORS           	-	CSH_Viv-9_pos mode_MS_07212014.d	GROUP_DESCRIPTION:Viv chow | TIME_POINT:24 weeks | Genotype:WT C57-Bl6	
#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: 1) VIV chow- normal rodent chow , low in fat and high in fiber 2)
TR:TREATMENT_SUMMARY             	HFD (referred to as CO in the manuscript) -40 kcal% high fat diet with 36 kcal%
TR:TREATMENT_SUMMARY             	from coconut oil and 4 kcal% from conventional soybean oil 3) LA-HFD (referred
TR:TREATMENT_SUMMARY             	to as SO+CO in the manuscript) - 40 kcal% high fat diet with 21 kcal% fat
TR:TREATMENT_SUMMARY             	calories from coconut oil and 19 kcal% from conventional soybean oil, of which
TR:TREATMENT_SUMMARY             	10 kcal% were from LA 4) PL-HFD (referred to as PL+CO in the manuscript)
TR:TREATMENT_SUMMARY             	-40kcal% high fat diet in which conventional soybean oil in LA-HFD was replaced
TR:TREATMENT_SUMMARY             	on a per gram basis with the genetically modified (GM) High Oleic Soybean Oil ,
TR:TREATMENT_SUMMARY             	Plenish
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	1) Keep Specimen on Dry Ice 2) Transfer Tissue Contents into a new 1.5mL labeled
SP:SAMPLEPREP_SUMMARY            	eppendorf tube; keep on dry ice at all times 3) Add three (3) 3mm metal grinding
SP:SAMPLEPREP_SUMMARY            	balls to each sample; store in -80C for 10minutes 4) Homogenize the entire
SP:SAMPLEPREP_SUMMARY            	tissue to fine powder using genogrinder; make sure that the metal grinding balls
SP:SAMPLEPREP_SUMMARY            	are ice-cold prior to homogenization (step 3) 5) Upon completion of
SP:SAMPLEPREP_SUMMARY            	homogenization, keep samples on dry ice 6) Weight out two (2) aliquots: a ~5mg
SP:SAMPLEPREP_SUMMARY            	aliquot for CSH_lipidomics and a ~4mg aliquot for Primary Metabolites by GCTOF
SP:SAMPLEPREP_SUMMARY            	a. Record the exact weight weighed out for each sample b. Keep all samples on
SP:SAMPLEPREP_SUMMARY            	dry ice 7) KEEP remaining tissue specimen (>90mg) for analysis of Oxylipins
SP:SAMPLEPREP_SUMMARY            	(store in -80C) 8) Analysis of Primary Metabolites (GCTOFMS) a. Add 1mL of
SP:SAMPLEPREP_SUMMARY            	ice-cold “degassed” 3:3:2 ACN/IPA/H2O b. Vortex for 10seconds c. Shake on
SP:SAMPLEPREP_SUMMARY            	shaker for 20min at -4C d. Centrifuge the samples for 2min at 14,000 rcf e.
SP:SAMPLEPREP_SUMMARY            	Transfer two (2) 500μL aliquots to new 1.5mL eppendorf tubes; one for backup
SP:SAMPLEPREP_SUMMARY            	the other to be dried to dryness using the SpeedVac f. IMPORTANT: The
SP:SAMPLEPREP_SUMMARY            	precipitated protein will be used for analysis of the proteome, DO NOT DISCARD
SP:SAMPLEPREP_SUMMARY            	THESE; Place these in a separate labeled box and store in -20C g. Keep all
SP:SAMPLEPREP_SUMMARY            	samples on ice during extraction period h. Dry down one (1) 500μL aliquot to
SP:SAMPLEPREP_SUMMARY            	complete dryness i. Perform cleanup on dried aliquot using 500μL of 50/50 v/v
SP:SAMPLEPREP_SUMMARY            	ACN/H2O j. Transfer supernatant and dry to completeness k. Submit for
SP:SAMPLEPREP_SUMMARY            	Derivatization 9) Analysis of Complex Lipids (LCQTOF) a. Add 225μL of ice-cold
SP:SAMPLEPREP_SUMMARY            	“degassed” MeOH containing “ISTD mixture” to homogenized 5mg aliquot b.
SP:SAMPLEPREP_SUMMARY            	Vortex for 10 seconds c. Add 750μL of ice-cold “degassed” MTBE containing
SP:SAMPLEPREP_SUMMARY            	22:1 CE ISTD d. Vortex for 10 seconds e. Shake on Orbital Mixer for 6min at 4C
SP:SAMPLEPREP_SUMMARY            	f. Add 188μL of room temperature H2O g. Vortex for 20 seconds h. Centrifuge for
SP:SAMPLEPREP_SUMMARY            	2min at 14,000 rcf i. Transfer two (2) aliquots of 350μL of top layer, one for
SP:SAMPLEPREP_SUMMARY            	backup stored in -20C, the other for analysis j. Keep bottom layer and store in
SP:SAMPLEPREP_SUMMARY            	-20C k. Dry down one (1) 350μL aliquot to dryness using the Speedvac l.
SP:SAMPLEPREP_SUMMARY            	Resuspend samples in 108.6μL of 50ng/mL CUDA m. Vortex and sonicate for
SP:SAMPLEPREP_SUMMARY            	5minutes n. Centrifuge for 2min at 14,000 rcf o. Transfer 90μL to an amber vial
SP:SAMPLEPREP_SUMMARY            	with micro-insert (non-diluted) p. Transfer 10μL to a new 1.5mL eppendorf tube,
SP:SAMPLEPREP_SUMMARY            	dilute 20X with 50ng/mL CUDA in 90:10 MeOH:Toluene (10μL + 190μL CUDA) and
SP:SAMPLEPREP_SUMMARY            	transfer 100μL to amber vial with micro-insert (diluted for TGs) i. The
SP:SAMPLEPREP_SUMMARY            	dilution is based off previous experiences with liver samples
SP:SAMPLEPREP_PROTOCOL_FILENAME  	SP_Extraction_Protocol_for_liver_multi-omic.pdf
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Agilent 6550
CH:COLUMN_NAME                   	Waters Acquity CSH C18 (100 x 2.1mm, 1.7um)
CH:COLUMN_NAME                   	1.7um Pre-Column
CH:FLOW_GRADIENT                 	15% B to 99% B
CH:FLOW_RATE                     	0.6 mL/min
CH:COLUMN_TEMPERATURE            	65 C
CH:METHODS_FILENAME              	Data_Dictionary_Fiehn_laboratory_GCTOF_MS_primary_metabolism_10-15-2013_general.pdf
CH:SOLVENT_A                     	60:40 Acetonitrile:Water +10mM Ammonium Acetate +10mM Acetic Acid
CH:SOLVENT_B                     	9:1 Isopropanol:Acetonitrile +10mM Ammonium Acetate +10mM Acetic Acid
CH:COLUMN_PRESSURE               	450-850 bar
CH:INJECTION_TEMPERATURE         	4 C
CH:INTERNAL_STANDARD             	See data dictionary
CH:RETENTION_TIME                	See data dictionary
CH:SAMPLE_INJECTION              	5.0 uL
CH:ANALYTICAL_TIME               	13 min
CH:CAPILLARY_VOLTAGE             	3500 eV
CH:TIME_PROGRAM                  	15 min
CH:WEAK_WASH_SOLVENT_NAME        	Isopropanol
CH:STRONG_WASH_SOLVENT_NAME      	Isopropanol
CH:TARGET_SAMPLE_TEMPERATURE     	Autosampler temp 4 C
CH:RANDOMIZATION_ORDER           	Excel
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:DETECTOR_TYPE                 	TOF MCP
AN:SOFTWARE_VERSION              	Masshunter
AN:DATA_FORMAT                   	.d
#MS
MS:MS_COMMENTS                   	-
MS:INSTRUMENT_NAME               	Agilent 6550 QTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:CAPILLARY_VOLTAGE             	3500 eV
MS:COLLISION_ENERGY              	25 eV
MS:COLLISION_GAS                 	Nitrogen
MS:DRY_GAS_FLOW                  	13L/min
MS:DRY_GAS_TEMP                  	200 C
MS:FRAGMENT_VOLTAGE              	175 eV
MS:FRAGMENTATION_METHOD          	Auto MS/MS
MS:ION_SOURCE_TEMPERATURE        	325 C
MS:ION_SPRAY_VOLTAGE             	1000
MS:IONIZATION                    	Neg
MS:PRECURSOR_TYPE                	Intact Molecule
MS:REAGENT_GAS                   	Nitrogen
MS:SOURCE_TEMPERATURE            	325 C
MS:DATAFORMAT                    	.d
MS:DESOLVATION_GAS_FLOW          	11 L/min
MS:DESOLVATION_TEMPERATURE       	350 C
MS:NEBULIZER                     	35 psig
MS:OCTPOLE_VOLTAGE               	750
MS:RESOLUTION_SETTING            	Exteded Dyamic Range
MS:SCAN_RANGE_MOVERZ             	60-1700 Da
MS:SCANNING_CYCLE                	2 Hz
MS:SCANNING_RANGE                	60-1700 Da
MS:SKIMMER_VOLTAGE               	65
MS:MS_RESULTS_FILE               	ST000656_AN001002_Results.txt	UNITS:Counts
#END