Summary of Study ST000890

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000619. The data can be accessed directly via it's Project DOI: 10.21228/M8NT1F This work is supported by NIH grant, U2C- DK119886.

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Study ID	ST000890
Study Title	Identification of RXR Ligands
Study Type	Idenfication of Ligands by HPLC-MS
Study Summary	Free fatty acids in mouse plasma were identified and quantified by LC-MS. Through differential feeding and PHZ (phnylhydrazine) dosing, coupled with mass spectrometry, we identified the long chain fatty acid C24:5 as a natural RXRA ligand, which was dynamically increased in concentration in response to hematopoietic stress. Collectively, these data demonstrate that natural RXRA ligands are present and are dynamically regulated in vivo in mouse hematopoietic cells.
Institute	Washington University in St. Louis
Department	Diabetic Cardiovascular Disease Center, School of Medicine
Laboratory	Metabolomics Core
Last Name	Fujiwara
First Name	Hideji
Address	660 South Euclid Ave, St. Louis MO 63110
Email	hfujiwar@wustl.edu
Phone	314-747-0494
Submit Date	2017-09-22
Study Comments	Units of measurement:peak area ratio: analyte peak area/peak area of internal standard
Analysis Type Detail	LC-MS
Release Date	2017-10-22
Release Version	1

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Project:

Project ID:	PR000619
Project DOI:	doi: 10.21228/M8NT1F
Project Title:	Endogenous Retinoid X Receptor Ligands in Mouse Hemotopoietic Cells
Project Type:	MS Identification and Quantification of Ligands
Project Summary:	The retinoid X receptor α (RXRA) has been implicated in diverse hematological processes. However, it is unknown whether natural ligands of RXRA are present or regulated in hematopoietic cells. We quantified lipids in the serum of mice treated with a vitamin A deficient diet, a fatty acid deficient diet, and following phenylhydrazine treatment. In parallel, these serum samples were applied to RXRA reporter cells to identify conditions which contained increased concentrations of natural RXRA ligands. Mass spectrometry quantification of serum lipids was correlated with data from the RXRA reporter cells to identify serum lipids that increased in conditions associated with augmented RXRA ligand concentrations in the RXRA reporter assay.
Institute:	Washington University in St. Louis
Department:	Diabetic Cardiovascular Disease Center
Laboratory:	Metabolomics Core
Last Name:	Fujiwara
First Name:	Hideji
Address:	660 South Euclide Avenue, St. Lois, MO 63110
Email:	hfujiwar@wustl.edu
Phone:	314-747-0494
Funding Source:	NIH R01 HL128447, NIH P30 DK020579
Publications:	Science Signaling 2018, being accepted for publication
Contributors:	Haixia Niu, Hideji Fujiwara, Orsola di Martino, Gayla Hadwiger, Thomas E. Frederick, María P. Menéndez-Gutiérrez, Mercedes Ricote, Gregory R. Bowman, John S. Welch

Subject:

Subject ID:	SU000927
Subject Type:	Animals
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57/B6
Age Or Age Range:	8-16 weeks
Species Group:	Mammal

Factors:

Subject type: Animals; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Diet of subject mice	Feeding period (wk)	PHZ dose (mg)
SA052449	4630 BM2-PHZ	control chow	12	1
SA052450	VADC-PHZ-4	control chow	12	1
SA052451	4629 BM2-PHZ	control chow	12	1
SA052452	4629 BM1-PHZ	control chow	12	1
SA052453	VADC-PHZ-3	control chow	12	1
SA052454	4630 PB1-PHZ	control chow	12	1
SA052455	4630 BM1-PHZ	control chow	12	1
SA052456	4629 PB1-PHZ	control chow	12	1
SA052457	4630 PB2-PHZ	control chow	12	1
SA052458	4629 PB2-PHZ	control chow	12	1
SA052459	VADC-PHZ-1	control chow	12	1
SA052460	VADC-PHZ-2	control chow	12	1
SA052461	4633 PB1	control chow	12	mock
SA052462	VDAC41	control chow	12	mock
SA052463	VADC-23	control chow	12	mock
SA052464	VADC-13	control chow	12	mock
SA052465	VADC-33	control chow	12	mock
SA052466	VADC-43	control chow	12	mock
SA052467	VADC-53	control chow	12	mock
SA052468	VDAC52	control chow	12	mock
SA052469	VDAC42	control chow	12	mock
SA052470	VDAC21	control chow	12	mock
SA052471	VDAC51	control chow	12	mock
SA052472	VDAC22	control chow	12	mock
SA052473	VDAC32	control chow	12	mock
SA052474	4632 PB1	control chow	12	mock
SA052475	VDAC31	control chow	12	mock
SA052476	VADC-2	control chow	12	mock
SA052477	VADC-3	control chow	12	mock
SA052478	VADC-1	control chow	12	mock
SA052479	4632 PB2	control chow	12	mock
SA052480	4632 BM2	control chow	12	mock
SA052481	4633 PB2	control chow	12	mock
SA052482	VADC-4	control chow	12	mock
SA052483	4633 BM2	control chow	12	mock
SA052484	4632 BM1	control chow	12	mock
SA052485	4633 BM1	control chow	12	mock
SA052486	VADC-PHZ-5	control chow	12	mock
SA052487	NFC-PHZ-22	control chow	4	1
SA052488	NFC-PHZ-23	control chow	4	1
SA052489	NFC-PHZ-12	control chow	4	1
SA052490	NFC-PHZ-21	control chow	4	1
SA052491	NFC-PHZ-13	control chow	4	1
SA052492	NFC-PHZ-11	control chow	4	1
SA052493	FC-31	control chow	4	mock
SA052494	FC-21	control chow	4	mock
SA052495	FC-41	control chow	4	mock
SA052496	FC-12	control chow	4	mock
SA052497	FC-11	control chow	4	mock
SA052498	FC-42	control chow	4	mock
SA052499	FC-22	control chow	4	mock
SA052500	FC-32	control chow	4	mock
SA052418	NF-PHZ-41	Non-fat-diet	4	1
SA052419	NF-PHZ-31	Non-fat-diet	4	1
SA052420	NF-PHZ-32	Non-fat-diet	4	1
SA052421	NF-PHZ-23	Non-fat-diet	4	1
SA052422	NF-PHZ-43	Non-fat-diet	4	1
SA052423	NF-PHZ-21	Non-fat-diet	4	1
SA052424	NF-PHZ-42	Non-fat-diet	4	1
SA052425	NF-PHZ-33	Non-fat-diet	4	1
SA052426	F-41	Non-fat-diet	4	mock
SA052427	F-31	Non-fat-diet	4	mock
SA052428	F-51	Non-fat-diet	4	mock
SA052429	NF-11	Non-fat-diet	4	mock
SA052430	NF-23	Non-fat-diet	4	mock
SA052431	NF-33	Non-fat-diet	4	mock
SA052432	F-21	Non-fat-diet	4	mock
SA052433	F-11	Non-fat-diet	4	mock
SA052434	NF-2	Non-fat-diet	4	mock
SA052435	NF-3	Non-fat-diet	4	mock
SA052436	NF-1	Non-fat-diet	4	mock
SA052437	NF-31	Non-fat-diet	4	mock
SA052438	NF-13	Non-fat-diet	4	mock
SA052439	NF-12	Non-fat-diet	4	mock
SA052440	NF-21	Non-fat-diet	4	mock
SA052441	NF-22	Non-fat-diet	4	mock
SA052442	NF-PHZ-22	Non-fat-diet	4	mock
SA052443	F-12	Non-fat-diet	4	mock
SA052444	F-52	Non-fat-diet	4	mock
SA052445	NF-32	Non-fat-diet	4	mock
SA052446	F-42	Non-fat-diet	4	mock
SA052447	F-32	Non-fat-diet	4	mock
SA052448	F-22	Non-fat-diet	4	mock
SA052501	VAD-PHZ2-32	vitamin A deficient diet	12	1
SA052502	VAD-PHZ2-12	vitamin A deficient diet	12	1
SA052503	VAD-PHZ2-42	vitamin A deficient diet	12	1
SA052504	VAD-PHZ2-22	vitamin A deficient diet	12	1
SA052505	VAD-PHZ1-11	vitamin A deficient diet	12	1
SA052506	VAD-PHZ1-31	vitamin A deficient diet	12	1
SA052507	VAD-PHZ1-21	vitamin A deficient diet	12	1
SA052508	VAD-PHZ2-52	vitamin A deficient diet	12	1
SA052509	VAD-PHZ1-41	vitamin A deficient diet	12	1
SA052510	VAD-PHZ2-51	vitamin A deficient diet	12	1
SA052511	VAD-PHZ1-42	vitamin A deficient diet	12	1
SA052512	VAD-PHZ1-22	vitamin A deficient diet	12	1
SA052513	VAD-PHZ2-41	vitamin A deficient diet	12	1
SA052514	VAD-PHZ2-31	vitamin A deficient diet	12	1
SA052515	VAD-PHZ2-11	vitamin A deficient diet	12	1
SA052516	VAD-PHZ2-21	vitamin A deficient diet	12	1
SA052517	VAD-PHZ1-12	vitamin A deficient diet	12	1

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Collection:

Collection ID:	CO000921
Collection Summary:	Mouse plasma was collected by obtaining peripheral blood via peri-orbital bleeding. Approximately 100 ul blood is placed in a microvette vial with pre-loaded KEDTA and then centrifuged at 14,000 rpm for 10 minutes at 4 degrees. Plasma is removed and stored at -80 C until analysis.
Sample Type:	Blood

Treatment:

Treatment ID:	TR000941
Treatment Summary:	Phenylhydrazine was administered at 1mg once on day 1 and plasma was collected on day 4. Vitamin A deficient diet: Teklad, TD.86143. Control diet: Teklad, TD.91280. Fatty acid deficient diet: Teklad, TD.03314. Control diet: Teklad, TD.130321.

Sample Preparation:

Sampleprep ID:	SP000934
Sampleprep Summary:	For free fatty acids analysis, 100 μL of plasma extraction sample or 400 μL pull-down samples was used for DMAPA (dimethylaminopropylamine) derivatization for improving MS sensitivity of free fatty acids. Prior to the derivatization, 50 ng of AA-d8 (deuterated arachidonic acid –d8) as the internal standard was added to each sample. The solvents in the sample were dried under a stream of nitrogen. To derivatize the sample, 50 μL of 50 mM EDC (N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide hydrochloride) and 50 mM DMAPA and 50 mM DMAP (4-dimethylaminopyridine) were added to the dried fatty acid samples and heated at 50 ◦C for 30 minutes. The samples were dried under nitrogen and then dissolved in 1 mL of ethanol : water for MS.

Sampleprep ID:

SP000934

Sampleprep Summary:

For free fatty acids analysis, 100 μL of plasma extraction sample or 400 μL pull-down samples was used for DMAPA (dimethylaminopropylamine) derivatization for improving MS sensitivity of free fatty acids. Prior to the derivatization, 50 ng of AA-d8 (deuterated arachidonic acid –d8) as the internal standard was added to each sample. The solvents in the sample were dried under a stream of nitrogen. To derivatize the sample, 50 μL of 50 mM EDC (N-ethyl-N-(3-dimethylaminopropyl)-carbodiimide hydrochloride) and 50 mM DMAPA and 50 mM DMAP (4-dimethylaminopyridine) were added to the dried fatty acid samples and heated at 50 ◦C for 30 minutes. The samples were dried under nitrogen and then dissolved in 1 mL of ethanol : water for MS.

Combined analysis:

Analysis ID	AN001452
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Shimadzu 20AD
Column	Agilent Eclipse XDB-C18 (100 x 3.0mm,3.5um)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	ABI Sciex API 4000 QTrap
Ion Mode	POSITIVE
Units	peak area ratio

Chromatography:

Chromatography ID:	CH001021
Chromatography Summary:	For DMAPA derivatized Fatty acid analysis. Shimadzu 20AD two pump system was used. Solvent A: 0.1% formic acid in water. Solvent B: 0.1% in Acetonitrile. The initial LC condition was 30% B. The solvent gradient was programmed from 30% B to 99% B in 4 min and was hold for 1 min at the flow rate of 1 mL/min. Then, it was immediately brought back to 30 % B and was hold for 2 min. An Agilent Eclipse XDB-C18 column (3.0 x100 mm x 3.5 µm) was used for this analysis.
Instrument Name:	Shimadzu 20AD
Column Name:	Agilent Eclipse XDB-C18 (100 x 3.0mm,3.5um)
Column Temperature:	60 C
Flow Gradient:	30% B to 99% B in 4 min
Flow Rate:	1 mL/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Analytical Time:	7 min
Chromatography Type:	Reversed phase

MS:

MS ID:	MS001342
Analysis ID:	AN001452
Instrument Name:	ABI Sciex API 4000 QTrap
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Units of measurement:peak area ratio: analyte peak area/peak area of internal standard
Ion Mode:	POSITIVE