#METABOLOMICS WORKBENCH nozomu_kono_20211017_174345 DATATRACK_ID:2892 STUDY_ID:ST001951 ANALYSIS_ID:AN003176 PROJECT_ID:PR001238
VERSION             	1
CREATED_ON             	October 23, 2021, 3:27 pm
#PROJECT
PR:PROJECT_TITLE                 	Analysis of ω-3 fatty acids and their derivatives in lungs from hypoxia-induced
PR:PROJECT_TITLE                 	pulmonary hypertension (PH) mice.
PR:PROJECT_SUMMARY               	Pulmonary arterial hypertension (PAH) is a rare, fatal disease that causes
PR:PROJECT_SUMMARY               	idiopathic pulmonary artery stenosis, which leads to increased pulmonary artery
PR:PROJECT_SUMMARY               	pressure and this chronic pressure-overload eventually results in right heart
PR:PROJECT_SUMMARY               	failure and death. Although the available therapies for PH have notably improved
PR:PROJECT_SUMMARY               	the survival of patients with PAH, there is still a significant portion of
PR:PROJECT_SUMMARY               	patients who do not achieve the expected efficacy. Here, in order to identify
PR:PROJECT_SUMMARY               	bioactive lipids which are protective against pulmonary hypertension (PH), we
PR:PROJECT_SUMMARY               	performed comprehensive lipidomic analysis of lung samples from hypoxia-induced
PR:PROJECT_SUMMARY               	PH mice.
PR:INSTITUTE                     	University of Tokyo
PR:LAST_NAME                     	Kono
PR:FIRST_NAME                    	Nozomu
PR:ADDRESS                       	7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
PR:EMAIL                         	nozomu@mol.f.u-tokyo.ac.jp
PR:PHONE                         	+81-3-5841-4723
#STUDY
ST:STUDY_TITLE                   	Quantification of ω-3 fatty acids and their derivatives in lungs from
ST:STUDY_TITLE                   	hypoxia-induced pulmonary hypertension (PH) mice.
ST:STUDY_SUMMARY                 	Using a liquid chromatography tandem mass spectrometry (LC-MS/MS) system, we
ST:STUDY_SUMMARY                 	quantified the ω-3 fatty acid (EPA and DHA) metabolites in the lung tissues of
ST:STUDY_SUMMARY                 	mice with PH induced by chronic hypoxia (10% oxygen concentration).
ST:INSTITUTE                     	University of Tokyo
ST:LAST_NAME                     	Kono
ST:FIRST_NAME                    	Nozomu
ST:ADDRESS                       	7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
ST:EMAIL                         	nozomu@mol.f.u-tokyo.ac.jp
ST:PHONE                         	+81-3-5841-4723
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
SU:GENDER                        	Male
#FACTORS
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	Day0-1	Time after hypoxia (days):-	RAW_FILE_NAME=Data180501 lung oxFFA-0d1
SUBJECT_SAMPLE_FACTORS           	-	Day0-2	Time after hypoxia (days):-	RAW_FILE_NAME=Data180501 lung oxFFA-0d2
SUBJECT_SAMPLE_FACTORS           	-	Day0-3	Time after hypoxia (days):-	RAW_FILE_NAME=Data180501 lung oxFFA-0d3
SUBJECT_SAMPLE_FACTORS           	-	Day4-1	Time after hypoxia (days):4	RAW_FILE_NAME=Data180501 lung oxFFA-4d1
SUBJECT_SAMPLE_FACTORS           	-	Day4-2	Time after hypoxia (days):4	RAW_FILE_NAME=Data180501 lung oxFFA-4d2
SUBJECT_SAMPLE_FACTORS           	-	Day4-3	Time after hypoxia (days):4	RAW_FILE_NAME=Data180501 lung oxFFA-4d3
SUBJECT_SAMPLE_FACTORS           	-	Day14-1	Time after hypoxia (days):14	RAW_FILE_NAME=Data180501 lung oxFFA-14d1
SUBJECT_SAMPLE_FACTORS           	-	Day14-2	Time after hypoxia (days):14	RAW_FILE_NAME=Data180501 lung oxFFA-14d2
SUBJECT_SAMPLE_FACTORS           	-	Day14-3	Time after hypoxia (days):14	RAW_FILE_NAME=Data180501 lung oxFFA-14d3
SUBJECT_SAMPLE_FACTORS           	-	Day28-1	Time after hypoxia (days):28	RAW_FILE_NAME=Data180501 lung oxFFA-28d1
SUBJECT_SAMPLE_FACTORS           	-	Day28-2	Time after hypoxia (days):28	RAW_FILE_NAME=Data180501 lung oxFFA-28d2
SUBJECT_SAMPLE_FACTORS           	-	Day28-3	Time after hypoxia (days):28	RAW_FILE_NAME=Data180501 lung oxFFA-28d3
#COLLECTION
CO:COLLECTION_SUMMARY            	Wild-type male mice (C57BL/6J) were anaesthetized and tissues were harvested.
CO:COLLECTION_SUMMARY            	Isolated mouse tissues were rinsed with cold PBS and immediately frozen in
CO:COLLECTION_SUMMARY            	liquid nitrogen.
CO:SAMPLE_TYPE                   	Lung
#TREATMENT
TR:TREATMENT_SUMMARY             	In chronic hypoxia mice model, the male mice were housed in a hypoxic chamber
TR:TREATMENT_SUMMARY             	(10 % O2) maintained using a hypoxic air generator (TEIJIN) and monitored with
TR:TREATMENT_SUMMARY             	an O2 analyzer (JIKO-255), for 4, 14 or 28 day. Animals were fed a standard
TR:TREATMENT_SUMMARY             	diet.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Lipids of tissues were extracted by the method of Bligh and Dyer. The extracted
SP:SAMPLEPREP_SUMMARY            	solutions were dried up with centrifugal evaporator, dissolved in methanol :
SP:SAMPLEPREP_SUMMARY            	isopropanol=1:1, and stored at −20°C. Fatty acid metabolites were further
SP:SAMPLEPREP_SUMMARY            	purified from tissues by solid-phase extraction using InertSep NH2 columns (GL
SP:SAMPLEPREP_SUMMARY            	Science) with deuterium-labelled internal standard (11(12)-EET-d11). Briefly,
SP:SAMPLEPREP_SUMMARY            	InertSep NH2 columns were precon- ditioned with 6mL of hexane and lipids
SP:SAMPLEPREP_SUMMARY            	extracted from tissues by the method of Bligh and Dyer were applied with 500μL
SP:SAMPLEPREP_SUMMARY            	of chloroform. Columns were then washed with 6mL of chloro- form/isopropanol
SP:SAMPLEPREP_SUMMARY            	(2/1, v/v), followed by the elution with diethyl ether/acetic acid (98/2,
SP:SAMPLEPREP_SUMMARY            	v/v).The extracted solutions were dried up with centrifugal evaporator,
SP:SAMPLEPREP_SUMMARY            	dissolved in methanol:isopro- panol=1:1, and stored at −20°C. For the
SP:SAMPLEPREP_SUMMARY            	detection of fatty acid metabolites, chromatographic separation was performed on
SP:SAMPLEPREP_SUMMARY            	a ACQUITY UPLC HSS T3 column (2.1×100 mm, 1.8 µm; Waters) maintained at
SP:SAMPLEPREP_SUMMARY            	40°C using mobile phase A (water/acetic acid (100/0.1, v/v) containing 10 mM
SP:SAMPLEPREP_SUMMARY            	ammonium acetate) and mobile phase B (acetonitrile/methanol (4/1, v/v)
SP:SAMPLEPREP_SUMMARY            	containing 10 mM ammonium acetate) in a gradient programme (0–2 min: 90%
SP:SAMPLEPREP_SUMMARY            	A; 2–10 min: 90% A →30% A; 10–24 min: 30% A →27% A; 24–27 min:
SP:SAMPLEPREP_SUMMARY            	1% A; 27–32 min: 90% A) with a flow rate of 0.2 mL/min(0–10 min),
SP:SAMPLEPREP_SUMMARY            	0.1 mL/min(10–15 min),0.2 mL/min(15–24 min) and
SP:SAMPLEPREP_SUMMARY            	0.5 mL/min(24–32 min).
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Shimadzu Nexera UC/s system (Shimadzu)
CH:COLUMN_NAME                   	ACQUITY UPLC HSS T3 column (2.1×100 mm, 1.8 µm; Waters)
CH:FLOW_GRADIENT                 	mobile phase A (water/acetic acid (100/0.1, v/v) containing 10 mM ammonium
CH:FLOW_GRADIENT                 	acetate) and mobile phase B (acetonitrile/methanol (4/1, v/v) containing 10 mM
CH:FLOW_GRADIENT                 	ammonium acetate) in a gradient programme (0–2 min: 90% A; 2–10 min: 90%
CH:FLOW_GRADIENT                 	A →30% A; 10–24 min: 30% A →27% A; 24–27 min: 1% A; 27–32 min:
CH:FLOW_GRADIENT                 	90% A) with a flow rate of 0.2 mL/min(0–10 min),
CH:FLOW_GRADIENT                 	0.1 mL/min(10–15 min),0.2 mL/min(15–24 min) and
CH:FLOW_GRADIENT                 	0.5 mL/min(24–32 min).
CH:FLOW_RATE                     	(0–2 min: 90% A; 2–10 min: 90% A →30% A; 10–24 min: 30% A →27%
CH:FLOW_RATE                     	A; 24–27 min: 1% A; 27–32 min: 90% A) with a flow rate of
CH:FLOW_RATE                     	0.2 mL/min(0–10 min), 0.1 mL/min(10–15 min),0.2
CH:FLOW_RATE                     	mL/min(15–24 min) and 0.5 mL/min(24–32 min)
CH:COLUMN_TEMPERATURE            	40
CH:SOLVENT_A                     	water/acetic acid (100/0.1, v/v) containing 10 mM ammonium acetate
CH:SOLVENT_B                     	acetonitrile/methanol (4/1, v/v) containing 10 mM ammonium acetate
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	ABI Sciex 4500 Qtrap
MS:INSTRUMENT_TYPE               	Ion trap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	The instrument parameters of QTRAP4500 for negative ion mode were as follows:
MS:MS_COMMENTS                   	curtain gas, 10 psi; ionspray voltage, −4500 V; temperature, 600°C; ion
MS:MS_COMMENTS                   	source gas 1, 70 psi; ion source gas 2, 80 psi. and quantification was performed
MS:MS_COMMENTS                   	using MultiQuant™ software (SCIEX).
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	ng/mg tissue
MS_METABOLITE_DATA_START
Samples	Day0-1	Day0-2	Day0-3	Day4-1	Day4-2	Day4-3	Day14-1	Day14-2	Day14-3	Day28-1	Day28-2	Day28-3
Factors	Time after hypoxia (days):-	Time after hypoxia (days):-	Time after hypoxia (days):-	Time after hypoxia (days):4	Time after hypoxia (days):4	Time after hypoxia (days):4	Time after hypoxia (days):14	Time after hypoxia (days):14	Time after hypoxia (days):14	Time after hypoxia (days):28	Time after hypoxia (days):28	Time after hypoxia (days):28
AA	1.332629635	0.691612138	1.420201592	0.87920638	1.158197869	1.625707737	1.35077187	1.096946827	0.823199611	0.816248741	0.876105027	1.030799112
EPA	0.150560071	0.054815096	0.113585988	0.0914062	0.101475505	0.091695302	0.121606737	0.088131893	0.080014934	0.090599818	0.073905628	0.07760927
DHA	1.309128582	0.679975966	0.837107885	0.888611198	0.941862326	1.082425218	0.835055096	0.665564738	0.571522553	0.882320074	0.546255092	0.737319721
5-HETE	0.196337935	0.163000925	0.231701085	0.149322731	0.133177392	0.1962096	0.126471413	0.183318386	0.156759444	0.218673419	0.176760459	0.221200588
8-HETE	0.001193885	0.002850373	0.001668369	0.001145624	0.001776613	0.00176225	0.001144255	0.001040577	0.001838212	0.001204792	0.001767122	0.001430269
9-HETE	0.010400255	0.009260183	0.009241666	0.006757653	0.009144106	0.011214914	0.011252245	0.011488776	0.007357704	0.008371222	0.009458246	0.010475905
11-HETE	0.006135379	0.003856802	0.004139105	0.00290529	0.003506085	0.017565401	0.007474203	0.01429298	0.002407256	0.006604251	0.005071509	0.014177479
12-HETE	0.026198439	0.008603209	0.058206228	0.011977688	0.007578779	0.046449489	0.012712611	0.019220612	0.005583195	0.012198707	0.008094884	0.019430236
15-HETE	0.20539058	0.128638245	0.193784071	0.103842759	0.143647033	0.42692801	0.223532806	0.351740347	0.10172974	0.162460806	0.134604623	0.330779486
16-HETE	0.000316032	0.000804494	0.000401673	0.000558456	0.00045047	0.00056043	0.000497206	0.000412363	0.000517881	0.000546159	0.000427448	0.000397052
17-HETE	0.000888794	0.000518758	0.000605069	0.000734795	0.000701998	0.000903858	0.000543285	0.000810252	0.000521084	0.000730981	0.00061419	0.000739482
18-HETE	0.001139068	0.000910515	0.001025649	0.001150089	0.000850997	0.000834178	0.000787392	0.000849545	0.000548019	0.001103448	0.000765411	0.000848986
19-HETE	0.004979492	0.00923135	0.011477702	0.009097368	0.009411751	0.006755135	0.007600561	0.00606174	0.006292206	0.006152217	0.006687295	0.007190154
20-HETE	0.005338916	0.002692067	0.002759803	0.002708477	0.003385459	0.002484171	0.001697161	0.002385226	0.00208261	0.002427638	0.001893613	0.00231502
5,6-EET	0.010792737	0.009603636	0.016191675	0.010428423	0.006006967	0.007519985	0.004347027	0.007083841	0.002223595	0.006967584	0.009248873	0.006547965
8,9-EET	0.003958708	0.004638982	0.005820906	0.005603249	0.004695024	0.005277554	0.006030303	0.00521503	0.004085029	0.003676768	0.004921212	0.003602496
11,12-EET	0.002094514	0.002732497	0.002310557	0.001986864	0.002257773	0.002248735	0.002022885	0.001690887	0.001693209	0.00174947	0.001761026	0.00188406
14,15-EET	0.003323461	0.003312913	0.003701922	0.003348135	0.003597316	0.003539823	0.002514956	0.002811755	0.002957505	0.002664161	0.003398566	0.00252877
5,6-DHET	0.001036644	0.000312633	0.000675006	0.00040921	0.000679564	0.000469119	0.000756082	0.000711309	0.000568696	0.00048208	0.000963547	0.000631886
8,9-DHET	0.000914492	0.000443639	0.000813278	0.000406313	0.000337838	0.000770325	0.000466707	0.000522119	0.000590457	0.000400302	0.000727654	0.000605758
11,12-DHET	0.000341166	0.000441905	0.000323266	0.000375622	0.000340215	0.000403358	0.000361373	0.000261501	0.00025319	0.000379297	0.000265099	0.000571349
14,15-DHET	0.000531924	0.000448375	0.000552092	0.000683728	0.000529682	0.000458196	0.000559588	0.000536267	0.000483348	0.00063985	0.000473683	0.001031458
5-HEPE	0.001447606	0.001710222	0.002347177	0.001673675	0.001109447	0.003385066	0.00208218	0.001990481	0.000877847	0.00127476	0.001173386	0.001521534
8-HEPE	0.002772061	0.002363133	0.002564892	0.002556335	0.002827671	0.001678206	0.002402156	0.002495337	0.001668195	0.001788211	0.002118089	0.0016306
9-HEPE	0.001352618	0.001092199	0.001304001	0.00111686	0.001808018	0.002184646	0.001188491	0.001815392	0.001574559	0.00115857	0.001229407	0.001509459
11-HEPE	0.001475127	0.001469387	0.001682758	0.001282885	0.001474392	0.00174132	0.001465607	0.001513288	0.001291095	0.001492179	0.001078286	0.001373264
12-HEPE	0.003572782	0.001838168	0.005180716	0.002188817	0.002050662	0.004363747	0.001739532	0.002563547	0.002014558	0.002265619	0.001786662	0.003169371
15-HEPE	0.000904339	0.00184965	0.00096501	0.001109136	0.001216333	0.001375312	0.000918271	0.001410702	0.000861182	0.000792707	0.00077545	0.002283696
18-HEPE	0.001444015	0.001443013	0.002335408	0.001635326	0.001451226	0.001632474	0.00141098	0.00161654	0.001150352	0.001008325	0.001248982	0.001525061
19-HEPE	0.029225129	0.083058922	0.119311218	0.07030342	0.056340871	0.047573469	0.040387259	0.035714024	0.029583977	0.028123318	0.02674681	0.027393244
20-HEPE	0.002691242	0.00300617	0.002675669	0.002079726	0.002257418	0.002766263	0.00104919	0.001750557	0.001792523	0.001415666	0.002601352	0.002726931
8,9-EpETE	0.001098563	0.000818829	0.001157783	0.001102431	0.00109103	0.000701908	0.00099135	0.000865649	0.000895431	0.000961715	0.000914629	0.00119699
11,12-EpETE	0.000324572	0.000390902	0.000329246	0.000267997	0.000234695	0.000222471	0.000411165	0.000440067	0.000262108	0.000394276	0.000418382	0.000248597
14,15-EpETE	0.000371648	0.000402217	0.000748776	0.000475064	0.000465161	0.00039245	0.000685373	0.000644303	0.000449973	0.000376875	0.000731234	0.000365145
17,18-EpETE	0.007099365	0.007656601	0.01136047	0.006129804	0.007776379	0.005777897	0.007831052	0.005249604	0.007029496	0.005512627	0.005666734	0.005516646
17,18-diHETE	0.029625564	0.021063093	0.022956876	0.018348716	0.014428852	0.0151627	0.01313576	0.014122211	0.010075713	0.012197281	0.010456614	0.015704342
4-HDoHE	0.003697283	0.002400365	0.002536101	0.001910938	0.002097336	0.001345377	0.001486951	0.001752228	0.001229813	0.001778994	0.001506239	0.001638356
7-HDoHE	0.001271772	0.001384278	0.001321313	0.001110957	0.00100417	0.001582427	0.001474802	0.001286324	0.000782137	0.001496957	0.000842445	0.001243897
8-HDoHE	0.003489205	0.002988734	0.004622431	0.004301688	0.004429032	0.003267222	0.003355959	0.003863018	0.003800602	0.004012358	0.003832049	0.00498631
10-HDoHE	0.002323813	0.002673189	0.001893541	0.001739818	0.003115421	0.001585152	0.001593392	0.001720838	0.002363037	0.001184357	0.001101074	0.001746215
11-HDoHE	0.001482286	0.001977382	0.001507211	0.001506963	0.001521635	0.001640305	0.001143453	0.001570896	0.001177879	0.001314854	0.001479621	0.001902711
13-HDoHE	0.010225919	0.010360634	0.011132579	0.012901842	0.010054564	0.011995851	0.010020194	0.010767141	0.009311602	0.009588136	0.009776729	0.009530636
14-HDoHE	0.0050382	0.006478542	0.003428571	0.004361316	0.006570555	0.008372524	0.002477679	0.003302095	0.003341862	0.005587302	0.003037363	0.016134454
17-HDoHE	0.002276999	0.003583841	0.002500376	0.002100465	0.003487827	0.003751488	0.002044896	0.002711566	0.001600338	0.002517703	0.001589058	0.007735919
16-HDoHE	0.001179098	0.002087149	0.001164256	0.00080584	0.001686015	0.001255911	0.000744496	0.000696261	0.000940262	0.000834817	0.000748817	0.00143511
20-HDoHE	0.001858111	0.00217719	0.002382648	0.001818066	0.001912851	0.001924471	0.001612007	0.001811437	0.001299915	0.001490395	0.001237665	0.001842213
21-HDoHE	0.006542104	0.016889719	0.017522215	0.020156516	0.02246036	0.017252919	0.015639589	0.020855275	0.019719331	0.016702977	0.019478561	0.021283023
22-HDoHE	0.009793878	0.002946921	0.003056561	0.004706156	0.00505883	0.004101045	0.003899629	0.003827119	0.003655452	0.003403282	0.002968275	0.002629611
16,17-EpDPE	0.00204749	0.002122042	0.001956219	0.001907502	0.001653411	0.001959662	0.002391405	0.002014048	0.001660827	0.001411114	0.001464524	0.001958519
19,20-EpDPE	0.003899079	0.003341083	0.004002406	0.003487831	0.003598926	0.003211723	0.003495037	0.004068782	0.002923312	0.003159957	0.00339395	0.003704739
19,20-diHDoPE	0.017859887	0.010200106	0.014041577	0.010659374	0.008694154	0.007764134	0.0084586	0.007498202	0.007048259	0.009341172	0.007683088	0.007332378
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Pubchem ID	Q1 Mass (Da)	Q3 Mass (Da)
AA	444899	303	259
EPA	446284 	301	257
DHA	445580	327.1	283
5-HETE	5280733	319	115
8-HETE	11976122	319.01	155
9-HETE	14123398	319.02	123
11-HETE	14123410	319.03	167
12-HETE	5353272	319.04	179
15-HETE	9966861	319.05	219
16-HETE	6442739	319.06	233
17-HETE	6442740 	319.07	247
18-HETE	6442778	319.08	261
19-HETE	 20839325  	319.09	231
20-HETE	5283157	319.1	245
5,6-EET	5283202	319.11	191
8,9-EET	 5283203 	319.12	155
11,12-EET	 5283204	319.13	167
14,15-EET	 5283205	319.14	219
5,6-DHET	5283142	337	145
8,9-DHET	5283144	337.01	127
11,12-DHET	 5283146	337.02	167
14,15-DHET	5283147  	337.03	207
5-HEPE	 6439678	317.2	115
8-HEPE	16061128 	317.21	155
9-HEPE	122164870  	317.21	149
11-HEPE	 16061130	317.21	167
12-HEPE	10041593 	317.2	179
15-HEPE	16061131	317.2	219
18-HEPE	16061132	317.21	259
19-HEPE	72551575	317.2	229
20-HEPE	72551579 	317.2	287
8,9-EpETE	16061086	317.2	155
11,12-EpETE	16061087	317.2	167
14,15-EpETE	16061088 	317.2	207
17,18-EpETE	 6439311 	317.2	259
17,18-diHETE	16061120 	335	247
4-HDoHE	14429109	343.1	101
7-HDoHE	16061142 	343.1	141
8-HDoHE	11976798 	343.1	109
10-HDoHE	11537494 	343.1	153
11-HDoHE	11631564 	343.1	149
13-HDoHE	11559259	343.1	221
14-HDoHE	11566378 	343.1	205
17-HDoHE	6439179	343.1	201
16-HDoHE	11595378 	343.11	233
20-HDoHE	16061143 	343.11	241
21-HDoHE	52922000	343.1	255
22-HDoHE	NA	343.1	269
16,17-EpDPE	14392758	343.1	233
19,20-EpDPE	11631565	343.1	241
19,20-diHDoPE	 16061148	361.1	273.1
METABOLITES_END
#END