Summary of Study ST003360

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 PR002085. The data can be accessed directly via it's Project DOI: 10.21228/M83R6P This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003360
Study Title	Secretion of sphingomyelin from Huh7 cells treated with oleate-d9 or elaidate-d17 while modulating SPT flux.
Study Summary	We analyzed SM in the fresh and spent media from Huh7 cells treated with BSA-oleate-d9 or BSA-elaidate-d17. We validated their reduction in abundance through pharmacological inhibition of SPT with myriocin.
Institute	Salk Institute for Biological Studies
Last Name	Gengatharan
First Name	Jivani
Address	10010 N Torrey Pines Rd, La Jolla, CA, 92037, USA
Email	jivani14@gmail.com
Phone	(858) 453-4100
Submit Date	2024-07-30
Raw Data Available	Yes
Raw Data File Type(s)	d
Analysis Type Detail	LC-MS
Release Date	2024-08-12
Release Version	1

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Project:

Project ID:	PR002085
Project DOI:	doi: 10.21228/M83R6P
Project Title:	Altered sphingolipid biosynthetic flux and lipoprotein trafficking contribute to trans fat-induced atherosclerosis
Project Summary:	The goal of the project is to determine the role of sphingolipid metabolism in atherosclerosis induced by dietary trans fat. We analyzed lipid metabolites in Huh7 cells following various fatty acid treatments, with specific focus on cis and trans unsaturated fatty acids. Additionally, we analyzed lipid metabolites in plasma and liver of Ldlr-/- mice fed high-fat diets enriched in cis or trans fatty acids in the presence or absence of myriocin, a pharmacological inhibitor of Serine palmitoyltransferase (SPT), the initial rate-limiting enzyme of sphingolipid biosynthesis.
Institute:	Salk Institute for Biological Studies
Last Name:	Gengatharan
First Name:	Jivani
Address:	10010 N Torrey Pines Rd, La Jolla, CA, 92037, USA
Email:	jivani14@gmail.com
Phone:	(858) 453-4100

Subject:

Subject ID:	SU003481
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Cell Strain Details:	Huh7

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Condition
SA365850	H7spentmed_delip_Ed17_2	Huh7 media	BSA-elaidate-d17 + DMSO spent media
SA365851	H7spentmed_delip_Ed17_1	Huh7 media	BSA-elaidate-d17 + DMSO spent media
SA365852	H7spentmed_delip_Ed17_3	Huh7 media	BSA-elaidate-d17 + DMSO spent media
SA365853	H7spentmed_delip_Ed17myr_1	Huh7 media	BSA-elaidate-d17 + myriocin spent media
SA365854	H7spentmed_delip_Ed17myr_2	Huh7 media	BSA-elaidate-d17 + myriocin spent media
SA365855	H7spentmed_delip_Ed17myr_3	Huh7 media	BSA-elaidate-d17 + myriocin spent media
SA365856	H7spentmed_delip_Od9_1	Huh7 media	BSA-oleate-d9 + DMSO spent media
SA365857	H7spentmed_delip_Od9_2	Huh7 media	BSA-oleate-d9 + DMSO spent media
SA365858	H7spentmed_delip_Od9_3	Huh7 media	BSA-oleate-d9 + DMSO spent media
SA365859	H7spentmed_delip_Od9myr_1	Huh7 media	BSA-oleate-d9 + myriocin spent media
SA365860	H7spentmed_delip_Od9myr_2	Huh7 media	BSA-oleate-d9 + myriocin spent media
SA365861	H7spentmed_delip_Od9myr_3	Huh7 media	BSA-oleate-d9 + myriocin spent media
SA365862	H7freshmed_delip_1	Huh7 media	Fresh media
SA365863	H7freshmed_delip_2	Huh7 media	Fresh media
SA365864	H7freshmed_delip_3	Huh7 media	Fresh media

Showing results 1 to 15 of 15

Showing results 1 to 15 of 15

Collection:

Collection ID:	CO003474
Collection Summary:	0.75 mL of fresh media or 1.5 mL of spent media was evaporated under vacuum at 4°C and resuspended into 0.1 mL of H2O.
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR003490
Treatment Summary:	Huh7 cells were treated with (1) 100 µM BSA-oleate-d9 with DMSO, (2) 100 µM BSA-100 µM oleate-d9 with 100 nM myriocin, (3) 100 µM BSA-elaidate-d17 with DMSO, or (4) 100 µM BSA-elaidate-d17 with 100 nM myriocin for 48 hours in delipidated media.

Sample Preparation:

Sampleprep ID:	SP003488
Sampleprep Summary:	Media was spiked with the following internal standards: 20 pmol sphinganine-d7 (Avanti Polar Lipids, Cat# 860658), deoxysphinganine-d3 (Avanti Polar Lipids, Cat# 860474), 100 pmol d18:0-d7/13:0 dihydroceramide (Avanti Polar Lipids, Cat# 330726), 200 pmol d18:1-d7/15:0 ceramide (Avanti Polar Lipids, Cat# 860681), 100 pmol d18:1-d7/15:0 glucosylceramide (Avanti Polar Lipids, Cat# 330729), 100 pmol d18:1-d7/15:0 lactosylceramide (Avanti Polar Lipids, Cat# 330727), 200 pmol sphingosine-d7 (Avanti Polar Lipids, Cat# 860657), 200 pmol sphingosine-d7 (Avanti Polar Lipids, Cat# 860657), and 200 pmol sphingomyelin (d18:1/18:1)-d9 (Avanti Polar Lipids, Cat#860740). 0.5 mL methanol, 0.5 mL H2O, and 1 mL chloroform were added directly. Samples were vortexed for 5 min and centrifuged for 5 min at 4°C at 15,000g. The organic phase was collected and 2 μL of formic acid was added to the remaining polar phase which was re-extracted with 1 mL of chloroform. Combined organic phases were dried under nitrogen. After dried extracts for cells were resuspended in 60 µl Buffer B, 5 µL of sample was injected.

Sampleprep ID:

SP003488

Sampleprep Summary:

Media was spiked with the following internal standards: 20 pmol sphinganine-d7 (Avanti Polar Lipids, Cat# 860658), deoxysphinganine-d3 (Avanti Polar Lipids, Cat# 860474), 100 pmol d18:0-d7/13:0 dihydroceramide (Avanti Polar Lipids, Cat# 330726), 200 pmol d18:1-d7/15:0 ceramide (Avanti Polar Lipids, Cat# 860681), 100 pmol d18:1-d7/15:0 glucosylceramide (Avanti Polar Lipids, Cat# 330729), 100 pmol d18:1-d7/15:0 lactosylceramide (Avanti Polar Lipids, Cat# 330727), 200 pmol sphingosine-d7 (Avanti Polar Lipids, Cat# 860657), 200 pmol sphingosine-d7 (Avanti Polar Lipids, Cat# 860657), and 200 pmol sphingomyelin (d18:1/18:1)-d9 (Avanti Polar Lipids, Cat#860740). 0.5 mL methanol, 0.5 mL H2O, and 1 mL chloroform were added directly. Samples were vortexed for 5 min and centrifuged for 5 min at 4°C at 15,000g. The organic phase was collected and 2 μL of formic acid was added to the remaining polar phase which was re-extracted with 1 mL of chloroform. Combined organic phases were dried under nitrogen. After dried extracts for cells were resuspended in 60 µl Buffer B, 5 µL of sample was injected.

Combined analysis:

Analysis ID	AN005502
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Agilent 1260 Infinity II
Column	Peeke Scientific Spectra C8SR (150 x 3.0 mm, 3μm)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	Agilent 6460 QQQ
Ion Mode	POSITIVE
Units	Peak area

Chromatography:

Chromatography ID:	CH004184
Instrument Name:	Agilent 1260 Infinity II
Column Name:	Peeke Scientific Spectra C8SR (150 x 3.0 mm, 3μm)
Column Temperature:	40°C
Flow Gradient:	0 min, 82% B; 3 min, 82% B; 4 min, 90% B; 18 min, 99% B; 25 min, 99% B; 27 min, 82% B; 30 min, 82% B
Flow Rate:	0.5 mL/min
Solvent A:	100% water; 0.2% formic acid; 2 mM ammonium formate
Solvent B:	100% methanol; 0.2% formic acid; 1 mM ammonium formate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS005228
Analysis ID:	AN005502
Instrument Name:	Agilent 6460 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Sphingolipid species were analyzed by multiple reaction monitoring of the transition from precursor to product ions at associated optimized collision energies, and fragmentor voltages using Agilent Masshunter. The m/z values of the precursor and product ions are provided in the metabolite metadata section.
Ion Mode:	POSITIVE