#METABOLOMICS WORKBENCH michaelsa93_20170419_140637 DATATRACK_ID:889 STUDY_ID:ST000597 ANALYSIS_ID:AN000915 PROJECT_ID:PR000436
VERSION             	1
CREATED_ON             	April 23, 2017, 7:34 pm
#PROJECT
PR:PROJECT_TITLE                 	Dysfunctional lipid metabolism underlies the effect of the perinatal DDT
PR:PROJECT_TITLE                 	exposure on the development of metabolic syndrome
PR:PROJECT_SUMMARY               	This study aims to identify changes in lipid mediators in the hypothalamus with
PR:PROJECT_SUMMARY               	triphenyl phosphate (TPP) exposure. UC Davis type 2 diabetes mellitus (UCD-T2DM)
PR:PROJECT_SUMMARY               	rats were treated with TPP (n=8 per group) or not treated (n=8 per group). Each
PR:PROJECT_SUMMARY               	group was analyzed for oxylipin, nitro lipids, endocannabinoid, and
PR:PROJECT_SUMMARY               	endocannabinoid-like monoacylglycerol and N-acylethanolamide changes to
PR:PROJECT_SUMMARY               	investigate alterations in lipid mediator signaling due to TPP exposure.
PR:PROJECT_SUMMARY               	Targeted metabolomic analysis of lipid mediators in rat hypothalamus samples was
PR:PROJECT_SUMMARY               	performed by the Newman lab.
PR:INSTITUTE                     	UC Davis
PR:DEPARTMENT                    	Department of Environmental Toxicology
PR:LAST_NAME                     	La Merrill
PR:FIRST_NAME                    	Michele
PR:ADDRESS                       	1 Shields Ave., Davis, CA 95616
PR:EMAIL                         	mlamerrill@ucdavis.edu
PR:PHONE                         	(530) 754-7254
#STUDY
ST:STUDY_TITLE                   	Dysfunctional lipid metabolism underlies the effect of the perinatal DDT
ST:STUDY_TITLE                   	exposure on the development of metabolic syndrome
ST:STUDY_SUMMARY                 	This study aims to identify changes in lipid mediators in the hypothalamus with
ST:STUDY_SUMMARY                 	triphenyl phosphate (TPP) exposure. UC Davis type 2 diabetes mellitus (UCD-T2DM)
ST:STUDY_SUMMARY                 	rats were treated with TPP (n=8 per group) or not treated (n=8 per group). Each
ST:STUDY_SUMMARY                 	group was analyzed for oxylipin, nitro lipids, endocannabinoid, and
ST:STUDY_SUMMARY                 	endocannabinoid-like monoacylglycerol and N-acylethanolamide changes to
ST:STUDY_SUMMARY                 	investigate alterations in lipid mediator signaling due to TPP exposure.
ST:STUDY_SUMMARY                 	Targeted metabolomic analysis of lipid mediators in rat hypothalamus samples was
ST:STUDY_SUMMARY                 	performed by the Newman lab.
ST:INSTITUTE                     	U.S.D.A. Western Human Nutrition Research Center, University of California,
ST:INSTITUTE                     	Davis
ST:DEPARTMENT                    	Nutrition
ST:LABORATORY                    	Newman Lab
ST:LAST_NAME                     	Newman
ST:FIRST_NAME                    	John
ST:ADDRESS                       	430 W. Health Sciences Dr., Davis, CA 95616
ST:EMAIL                         	john.newman@ars.usda.gov
ST:PHONE                         	+1-530-752-1009
#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           	3	LAM-16	Treatment:E (vehicle control)	Organ=Hypothalamus; Species=Mouse
SUBJECT_SAMPLE_FACTORS           	36	LAM-01&09 avg.	Treatment:E (vehicle control)	Organ=Hypothalamus; Species=Mouse
SUBJECT_SAMPLE_FACTORS           	45	LAM-08	Treatment:E (vehicle control)	Organ=Hypothalamus; Species=Mouse
SUBJECT_SAMPLE_FACTORS           	50	LAM-07	Treatment:E (vehicle control)	Organ=Hypothalamus; Species=Mouse
SUBJECT_SAMPLE_FACTORS           	76	LAM-13	Treatment:E (vehicle control)	Organ=Hypothalamus; Species=Mouse
SUBJECT_SAMPLE_FACTORS           	108	LAM-15	Treatment:E (vehicle control)	Organ=Hypothalamus; Species=Mouse
SUBJECT_SAMPLE_FACTORS           	116	LAM-14	Treatment:E (vehicle control)	Organ=Hypothalamus; Species=Mouse
SUBJECT_SAMPLE_FACTORS           	132	LAM-03	Treatment:E (vehicle control)	Organ=Hypothalamus; Species=Mouse
SUBJECT_SAMPLE_FACTORS           	11	LAM-11&12 avg.	Treatment:T (treated with TPhP)	Organ=Hypothalamus; Species=Mouse
SUBJECT_SAMPLE_FACTORS           	20	LAM-10	Treatment:T (treated with TPhP)	Organ=Hypothalamus; Species=Mouse
SUBJECT_SAMPLE_FACTORS           	29	LAM-18	Treatment:T (treated with TPhP)	Organ=Hypothalamus; Species=Mouse
SUBJECT_SAMPLE_FACTORS           	59	LAM-04	Treatment:T (treated with TPhP)	Organ=Hypothalamus; Species=Mouse
SUBJECT_SAMPLE_FACTORS           	69	LAM-05	Treatment:T (treated with TPhP)	Organ=Hypothalamus; Species=Mouse
SUBJECT_SAMPLE_FACTORS           	90	LAM-06	Treatment:T (treated with TPhP)	Organ=Hypothalamus; Species=Mouse
SUBJECT_SAMPLE_FACTORS           	99	LAM-17	Treatment:T (treated with TPhP)	Organ=Hypothalamus; Species=Mouse
SUBJECT_SAMPLE_FACTORS           	126	LAM-02	Treatment:T (treated with TPhP)	Organ=Hypothalamus; Species=Mouse
#COLLECTION
CO:COLLECTION_SUMMARY            	"Prior to sacrifice, rats were fasted between 8 and12 h,theirbody weights
CO:COLLECTION_SUMMARY            	recorded, and blood was collected from the tail as above prior to euthanasia.
CO:COLLECTION_SUMMARY            	Rats were anesthetized with sodium pentobarbital and euthanized with a 1 mL/kg
CO:COLLECTION_SUMMARY            	intracardiac injection of saturated potassium chloride. Once cardiac movement
CO:COLLECTION_SUMMARY            	had stopped for 30 s the rat was decapitated and the hypothalamus, liver,
CO:COLLECTION_SUMMARY            	pancreas, heart, mesenteric adipose tissue, quadriceps, kidney, gonadal adipose
CO:COLLECTION_SUMMARY            	tissue, inguinal adipose tissue, and brown adipose tissue were collected. All
CO:COLLECTION_SUMMARY            	tissues were removed in the order listed above, wet weighed, and snap frozen in
CO:COLLECTION_SUMMARY            	liquid nitrogen"
CO:COLLECTION_PROTOCOL_FILENAME  	Green_et_al_2017-TPP_Exposure_Accelerat_T2DM_Rats.pdf
CO:SAMPLE_TYPE                   	Tissue
#TREATMENT
TR:TREATMENT_SUMMARY             	"Adult non-pregnant female UCD-T2DM rats (n = 16; 3 months old) were paired with
TR:TREATMENT_SUMMARY             	males (n = 10; 3–4 months old) for a 24 h period at which point males were
TR:TREATMENT_SUMMARY             	removed. This was defined as gestational day zero (G0) if a sperm plug was
TR:TREATMENT_SUMMARY             	observed or if the female rats gained at least 30 g of body weight over the next
TR:TREATMENT_SUMMARY             	7 days. The day of birth was designated postnatal day zero (P0). Pregnant dams
TR:TREATMENT_SUMMARY             	were randomly assigned to an exposure group (n = 8 per group), and received
TR:TREATMENT_SUMMARY             	daily oral TPhP or ethanol vehicle exposure from G8 through weaning (P21) as
TR:TREATMENT_SUMMARY             	described in Section 2.2 below. Gestational length and litter size were recorded
TR:TREATMENT_SUMMARY             	on P0 and the sex of pups was determined and recorded on P4. Body weights of all
TR:TREATMENT_SUMMARY             	pups in each litter were obtained periodically from P4–21. On P4 the litters
TR:TREATMENT_SUMMARY             	were culled to 8 pups ensuring up to 4males and 2 females in each litter by
TR:TREATMENT_SUMMARY             	random selection (Fig. 1A & B). This was done to ensure consistent exposure of
TR:TREATMENT_SUMMARY             	pups between litters [13,23]. The time ittakes to develop T2DM is accelerated
TR:TREATMENT_SUMMARY             	among UCD-T2DM rats with higher body weights on P21. Hence at weaning the
TR:TREATMENT_SUMMARY             	largest pups were housed in same sex littermate groups of two females and up to
TR:TREATMENT_SUMMARY             	four males as available (Fig. 1A & B). Urine was collected from the dams using
TR:TREATMENT_SUMMARY             	an adapted plastic wrap method outlined by Kurien [24], 60 mins after final
TR:TREATMENT_SUMMARY             	dose. Dams were placed in clean cages without bedding for at least 20 min then
TR:TREATMENT_SUMMARY             	using a pipette up to 500 L of urine was collected in ethanol rinsed glass vials
TR:TREATMENT_SUMMARY             	and placed on ice. At weaning all dams and remaining weanlings were sacrificed
TR:TREATMENT_SUMMARY             	(90–330 min post-exposure) by CO2 asphyxiation and rapid decapitation. Two
TR:TREATMENT_SUMMARY             	male rats weighing between 350–400 g on P61, from the TPhP group and the
TR:TREATMENT_SUMMARY             	vehicle group were weight-matched across treatments for the diabetes study to
TR:TREATMENT_SUMMARY             	eliminate confounding effects of body mass on the association between TPhP and
TR:TREATMENT_SUMMARY             	T2DM onset (Fig. 1B). This weight range was selected because male UCD-T2DM rats
TR:TREATMENT_SUMMARY             	that are between 350 and 400 g at 8 weeks of age develop T2DM at approximately
TR:TREATMENT_SUMMARY             	23 weeks of age [18]. Weight-matched rats were followed until 26 weeks or until
TR:TREATMENT_SUMMARY             	they developed T2DM, which was defined as two consecutive weekly non-fasting
TR:TREATMENT_SUMMARY             	glucose measurements of ≥200 mg/dL [18] in accordance with the American
TR:TREATMENT_SUMMARY             	Diabetes Association (ADA) guideline of diagnosing diabetes with a random plasma
TR:TREATMENT_SUMMARY             	glucose of 200 mg/dL or higher [19]. The remaining rats were not weight-matched
TR:TREATMENT_SUMMARY             	and followed for the 3.5 month obesity study (Fig. 1A).
TR:TREATMENT_PROTOCOL_FILENAME   	Green_et_al_2017-TPP_Exposure_Accelerat_T2DM_Rats.pdf
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Oxylipins and endocannabinoids were isolated using a Waters Ostro Sample
SP:SAMPLEPREP_SUMMARY            	Preparation Plate (Milford, MA). Hypothalamus samples were pulverized and
SP:SAMPLEPREP_SUMMARY            	aliquoted (~20-25mg) were added to 2mL polypropylene tubes and spiked with a 5
SP:SAMPLEPREP_SUMMARY            	µL anti-oxidant solution (0.2 mg/ml solution BHT/EDTA in 1:1 MeOH:water) and 10
SP:SAMPLEPREP_SUMMARY            	μL 1000nM analytical deuterated surrogates. A total of 100 µL methanol was
SP:SAMPLEPREP_SUMMARY            	added to the sample and vrtexed 90 sec. Next, 500 µL D.I. water and 1000 µL
SP:SAMPLEPREP_SUMMARY            	ethyl acetate was added and the tube was vortexed 3 minutes, before being
SP:SAMPLEPREP_SUMMARY            	centrifuged at 15,000g for 10 min at room temp. The supernate was then
SP:SAMPLEPREP_SUMMARY            	transferred into a clean 2 mL autosampler vial. The extraction with ethyl
SP:SAMPLEPREP_SUMMARY            	acetate was repeated and the eluent was dried by speed vacuum for 35 min at the
SP:SAMPLEPREP_SUMMARY            	medium BP setting. Once dry, samples were re-constituted with the internal
SP:SAMPLEPREP_SUMMARY            	standard 1-cyclohexyl ureido, 3-dodecanoic acid (CUDA) and 1-Phenyl
SP:SAMPLEPREP_SUMMARY            	3-Hexadecanoic Acid Urea (PHAU) at 100 nM (50:50 MeOH:CAN), vortexed 1 min,
SP:SAMPLEPREP_SUMMARY            	transferred to a spin filter (0.1 µm, Millipore, Billerica, MA), centrifuged
SP:SAMPLEPREP_SUMMARY            	for 3 min at 6ºC at <4500g (rcf), before being transferred to 2 mL LC-MS amber
SP:SAMPLEPREP_SUMMARY            	vials. Extracts were stored at -20ºC until analysis by UPLC-MS/MS. The internal
SP:SAMPLEPREP_SUMMARY            	standard was used to quantify the recovery of surrogate standards.
SP:SAMPLEPREP_PROTOCOL_FILENAME  	Hypothal_Newman_Data_Report.docx
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Waters Acquity
CH:COLUMN_NAME                   	Waters Acquity BEH C18 (150 x 2.1mm, 1.7um)
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:MS_COMMENTS                   	-
MS:INSTRUMENT_NAME               	ABI Sciex API 4000 QTrap
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS         	Concentration pmol/g
MS_METABOLITE_DATA_START
Samples	LAM-16	LAM-01&09 avg.	LAM-08	LAM-07	LAM-13	LAM-15	LAM-14	LAM-03	LAM-11&12 avg.	LAM-10	LAM-18	LAM-04	LAM-05	LAM-06	LAM-17	LAM-02
Factors	Treatment:E (vehicle control)	Treatment:E (vehicle control)	Treatment:E (vehicle control)	Treatment:E (vehicle control)	Treatment:E (vehicle control)	Treatment:E (vehicle control)	Treatment:E (vehicle control)	Treatment:E (vehicle control)	Treatment:T (treated with TPhP)	Treatment:T (treated with TPhP)	Treatment:T (treated with TPhP)	Treatment:T (treated with TPhP)	Treatment:T (treated with TPhP)	Treatment:T (treated with TPhP)	Treatment:T (treated with TPhP)	Treatment:T (treated with TPhP)
DHEA	57.2	65.15	44.1	44.6	48.1	66	50.3	70.8	42.75	35.4	49.9	45.5	48.4	22.6	37.5	77.1
AEA	24	30.05	17	15.7	18.7	28.7	16.6	32.7	18.15	13.8	17.9	20.7	17.5	13.5	16	37
LEA	15.9	23.1	11.6	11.7	11.5	20.6	12.3	21.8	14.3	8.68	14	15.7	14.5	11.3	13.9	25.9
2-AG	37300	28450	51600	32800	30500	39600	33800	51400	31100	26500	46400	54700	42300	33900	45800	43000
Dihomo GLA EA	0.895	2.085		0.878	0.809	1.5		2.1	0.9475	0.947	1.02	1.31	0.832	0.706	0.989	2.57
1-AG	2890	3045	4830	2320	2450	3770	3230	5560	2905	2270	4530	5250	3600	3000	4160	5140
2-LG	3280	3425	15000	11100	3420	4370	3540	10100	3410	6740	6170	11900	5760	6390	4930	4830
1-LG	931	994.5	2060	1600	1170	931	1040	1360	997	1220	1310	1120	1160	1150	1190	1070
PEA	587	554	438	502	481	641	445	873	590	495	541	464	538	511	494	675
OEA	126	169.5	79.1	100	114	166	89.6	227	132.5	104	111	95.8	103	88.1	107	249
2-OG	42300	39800	67200	54400	34900	51400	34600	123000	42100	33200	51400	55700	49900	37400	56800	91600
1-OG	6820	8515	9050	7260	6740	7570	6130	15700	5710	6000	7600	5410	6850	5920	6830	17600
SEA	149	203	105	105	111	137	90.8	249	121.5	114	123	92.4	109	93.1	105	314
DEA	8.05	13.05	6.1	6.67	9.38	12.2	7.81	21.6	8.865	7.96	8.34	8.04	9.17	6.73	10.6	21.9
NO-Gly	1.95	18.685	2.1	2.9	2.47	1.37	1.64	10.6	2.06	2.4	1.32	3.07	5.33	3.68	1.46	21
PGF2a EA																
PGE2 EA																
PGD2 EA																
PGF2a 1G																
PGE2 1G																
15-HETE EA																
11(12)-EpETre EA																
aLEA																
NA-Gly																
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	retention index	quantified m/z	PubChem ID	KEGG ID
DHEA			53245830
AEA			5281969	C11695
LEA			5283446
2-AG			5282280	C13856
Dihomo GLA EA			5282272	C13828
1-AG			16019980
2-LG			5365676
1-LG			6436630
PEA			4671	C16512
OEA			5283454
2-OG			5319879
1-OG			12178130
SEA			27902
DEA			5282273	C13829
NO-Gly			6436908
PGF2a EA			53481911
PGE2 EA
PGD2 EA
PGF2a 1G			24778485
PGE2 1G			52193688
15-HETE EA
11(12)-EpETre EA			16061183
aLEA			5283449
NA-Gly			5283389
METABOLITES_END
#END