#METABOLOMICS WORKBENCH michaelsa93_20170419_143343 DATATRACK_ID:890 STUDY_ID:ST000598 ANALYSIS_ID:AN000916 PROJECT_ID:PR000436
VERSION             	1
CREATED_ON             	April 23, 2017, 7:51 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                      	NEGATIVE
#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)
6-keto PGF1a	12.3	3.66	8.65	10.4	4.26	8.71	3.72	10	12.135	8.99	4.07	9.76	6.8	1	3.1	1.62
TXB2	23	12.9	17.4	17.8	16.8	19.4	16.7	27.4	22.9	27.9	18.9	23.5	12.9	14	18.9	9.79
9,12,13-TriHOME	21.9	3.99	14.6						17.65							
PGF2a	25.5	8.315	11.7	16.5		14.1		12.4	12.85	19.1	9.42	14.3	12			
PGE2	13	6.235	9.18	10.1	6.46	7.47	5.1	6.93	8.99	11.7	5.53	8.62	7.16	3.1	3.5	3.95
PGD2	77.9	34.75	55.4	57.7	38.4	48.8	42.9	55.5	62.3	86.2	43.4	46	45	14	18.7	19.7
9,10-DiHOME	0.968	0.977	1.41	1.09	0.867	0.415		0.535	0.713	0.866	0.851	0.791	0.891	0.7		
19,20-DiHDoPA	0.804	0.809	0.484	0.955	0.494	0.588	0.7	1.18	0.664	0.918	0.408	0.895	1.08	0.734	0.657	1
14,15-DiHETrE		0.31	0.23	0.38	0.17		0.32	0.19	0.32	0.15		0.36	0.33		0.2	
11,12-DiHETrE	0.36	0.41	0.17	0.22	0.24	0.17	0.17	0.32	0.31	0.28	0.29	0.38	0.2	0.26		0.23
9-HOTE		1.1	1.31		0.779	0.679	0.95		0.819	1.08	1.06	1.03	0.949	2.97	1.2	
13-HOTE		4.515	1.07	0.959	13.5			1.54	1.19	2.95	5.4	2	9.94	12.6		
12-HEPE	0.391	3.385	1.78	2.27	3.31	2.07	2.2	2.1	3.175	2.11	3.91	1.6	2.12	3.84	1.36	1.13
13-HODE		52.4	50.9	41.9	80					38.7	43.4	35.7	48.1	119		
9-HODE		28.8	34.9									19.2		60.2		
15(16)-EpODE	2.27	1.585	3.93	2.84	2.09	0.348	0.8	0.945	0.611	3.82	2.55	2.82	2.38	4.8	0.495	0.63
17-HDoHE	14.2	24.55	10.6	12.4	18.6	10.6	9.6	10.1	14.4	23.3	16.5	25.8	25.4	13.8	18.3	8.3
12(13)-EpODE	0.174	0.2715	0.487	0.361			0.0639	0.279	0.484	0.863	0.256	0.329	0.724	0.658		0.131
14-HDoHE		19.5	14.5	17.7	20.5		11.9	11.5	17.7	14.5	21.5	30.5	18.9	14.6		10.6
11-HETE	49.7	28.7	38.5	39.6	19.3	34	26.5	27.8	43.2	43.9	25.3	38.6	30.8	18.1	27.9	19.7
12-HETE	47.8	106.45	108	118	151	122	123	109	159	116	126	129	106	109	92.1	77.6
8-HETE	7.35	6.07	7.37	4.21	3.83	2.86	3.47	1.72	6.24	6.63	3.81	7.28	3.54	4.09	5.11	3.58
5-HETE	21.4	12.5	15.8	16.4	8.59	11.1	11.4	11.1	16	17.2	10.1	16	13.8	11.6	14.7	12.1
12(13)-EpOME			20.6									11		15.6		
14(15)-EpETrE	4.22	3.91	3.1	4.94	2.08	1.91	3.72	2.45	3.89	3.79	3.49	2.88	2.05	3.04	4.42	3.62
5-KETE	3.46	2.93	2.23	1.99	1.57	1.11	1.3	1.51	2.38	1.67	1.94	2	2.01	1.23	2.08	1.97
11(12)-EpETrE	1.14	2.125	1.35	1.67	1.28	1.22	2.1	1.21	1.815	1.54	1.54	1.81	1.57	1.31	1.34	1.84
8(9)-EpETrE	2.51	1.85	1.43	1.73	0.516		1.05		1.405	1.93	1.81	1.89		0.871		
9,10-EpO			84.9						62.5							
10-Nitrooleate			6.43	6.49	8.99		8.26		11.9				12	7.25		17.4
12(13)-Ep-9-KODE																
15-HEPE																
15-HETE																
15-HpETE																
9-KODE																
9-HETE																
9(10)-EpOME																
9,10-DiHHex																
PGF3a																
PGE3																
PGE1																
15-Keto PGE2																
Resolvin D1																
Lipoxin A4																
LTB5																
15,16-DiHODE																
12,13-DiHODE																
8,15-DiHETE																
9,10-DiHODE																
17,18-DiHETE																
5,15-DiHETE																
6-trans-LTB4																
14,15-DiHETE																
LTB4																
9,10-e-DiHO																
8,9-DiHETrE																
15-deoxy PGJ2																
20-HETE																
5,6-DiHETrE																
9-HEPE																
5-HEPE																
17(18)-EpETE																
13-KODE																
9(10)-EpODE																
13-HpODE																
9-HpODE																
15-KETE																
14(15)-EpETE																
11(12)-EpETE																
12-HpETE																
5-HpETE																
19(20)-EpDPE																
4-HDoHE																
16(17)-EpDPE																
10-Nitrolinoleate																
9-Nitrooleate																
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	retention index	quantified m/z	PubChem ID	KEGG ID
6-keto PGF1a			5280888
TXB2			5283137	C05963
9,12,13-TriHOME			9858729	C14833
PGF2a
PGE2			5280360	C00584
PGD2			448457	C00696
9,10-DiHOME			9966640
19,20-DiHDoPA			16061148
14,15-DiHETrE			5283147	C14775
11,12-DiHETrE			5283146	C14774
9-HOTE			53480359
13-HOTE			10469728
12-HEPE			10041593
13-HODE			5282948	C14762
9-HODE			5282944	C14767
15(16)-EpODE			16061056
17-HDoHE			6439179
12(13)-EpODE			16061061
14-HDoHE			11566378
11-HETE			5312982
12-HETE			5312983
8-HETE			5312975
5-HETE			5280733
12(13)-EpOME			5356421
14(15)-EpETrE			5283205
5-KETE			5353355
11(12)-EpETrE			53480479
8(9)-EpETrE			5283203
9,10-EpO
10-Nitrooleate			53394576
12(13)-Ep-9-KODE			5283007
15-HEPE			16061131
15-HETE			9966861
15-HpETE			5280893
9-KODE			9839084
9-HETE			14123398
9(10)-EpOME			12097313
9,10-DiHHex			193113
PGF3a			5229256
PGE3			5280937
PGE1			5280723
15-Keto PGE2			5280719
Resolvin D1			44251266
Lipoxin A4			5280914
LTB5			5283125
15,16-DiHODE			16061068
12,13-DiHODE			16061067
8,15-DiHETE			53480358
9,10-DiHODE			16061066
17,18-DiHETE			16061120
5,15-DiHETE			5283158
6-trans-LTB4			5283128
14,15-DiHETE			16061119
LTB4			5280492
9,10-e-DiHO
8,9-DiHETrE			5283144
15-deoxy PGJ2			5311211
20-HETE			5283157
5,6-DiHETrE			5283142
9-HEPE			16061129
5-HEPE			6439678
17(18)-EpETE			6439311
13-KODE			6446027
9(10)-EpODE			16061051
13-HpODE			5280720
9-HpODE			5282856
15-KETE			5280701
14(15)-EpETE			71433622
11(12)-EpETE
12-HpETE
5-HpETE
19(20)-EpDPE
4-HDoHE
16(17)-EpDPE
10-Nitrolinoleate
9-Nitrooleate
METABOLITES_END
#END