Summary of Study ST003372

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	ST003372
Study Title	Incorporation of oleate-d9 and elaidate-d17 into neutral lipids of Huh7 cells.
Study Summary	We analyzed neutral lipid classes including DG and TG in Huh7 cells treated with BSA-oleate-d9 or BSA-elaidate-d17 to determine their incorporation into the broader lipidome. 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 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
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

Select appropriate tab below to view additional metadata details:

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:	SU003493
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	Treatment
SA366974	Huh7_C30Lip2_elaidicd17_1	Huh7 cells	BSA-elaidate-d17
SA366975	Huh7_C30Lip2_elaidicd17_2	Huh7 cells	BSA-elaidate-d17
SA366976	Huh7_C30Lip2_elaidicd17_3	Huh7 cells	BSA-elaidate-d17
SA366977	Huh7_C30Lip2_oleicd9_1	Huh7 cells	BSA-oleate-d9
SA366978	Huh7_C30Lip2_oleicd9_2	Huh7 cells	BSA-oleate-d9
SA366979	Huh7_C30Lip2_oleicd9_3	Huh7 cells	BSA-oleate-d9

Showing results 1 to 6 of 6

Showing results 1 to 6 of 6

Collection:

Collection ID:	CO003486
Collection Summary:	Cells (~400,000 cells)were spiked with 1 µg of each of the following internal standards: 15:0-18:1(d7) phosphatidylcholine (Avanti Polar Lipids, Cat #791637), 15:0-18:1(d7) phosphatidylethanolamine (Avanti Polar Lipids, Cat #791638), 18:1(d7) lysophosphatidylcholine (Avanti Polar Lipids, Cat#791643), 18:1(d7) lysophosphatidylethanolamine (Avanti Polar Lipids, Cat #791644), 15:0-18:1(d7) diacylglycerol (Avanti Polar Lipids, Cat #791647), 15:0-18:1(d7)-15:0 triacylglycerol (Avanti Polar Lipids, Cat #791648). Cells were scraped with 0.5 mL methanol and 0.5 mL H2O.
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR003502
Treatment Summary:	Huh7 cells were treated with 1) 100 µM BSA-oleate-d9 or 2) 100 µM BSA-elaidate-d17 for 48 hours in delipidated media.

Sample Preparation:

Sampleprep ID:	SP003500
Sampleprep Summary:	Cells (~400,000 cells) were spiked with 1 µg of each of the following internal standards: 15:0-18:1(d7) phosphatidylcholine (Avanti Polar Lipids, Cat #791637), 15:0-18:1(d7) phosphatidylethanolamine (Avanti Polar Lipids, Cat #791638), 18:1(d7) lysophosphatidylcholine (Avanti Polar Lipids, Cat#791643), 18:1(d7) lysophosphatidylethanolamine (Avanti Polar Lipids, Cat #791644), 15:0-18:1(d7) diacylglycerol (Avanti Polar Lipids, Cat #791647), 15:0-18:1(d7)-15:0 triacylglycerol (Avanti Polar Lipids, Cat #791648). Cells were scraped with 0.5 mL methanol and 0.5 mL H2O. Homogenate aliquot of 100 µL was taken to determine protein content using the BCA protein assay (Thermo Fisher Scientific). The remaining homogenate was transferred to a new Eppendorf tube and 1 mL chloroform was added. For media, 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 were resuspended in 60 µl 65:30:5 ACN: IPA:H2O, 5 µL of sample was injected.

Sampleprep ID:

SP003500

Sampleprep Summary:

Cells (~400,000 cells) were spiked with 1 µg of each of the following internal standards: 15:0-18:1(d7) phosphatidylcholine (Avanti Polar Lipids, Cat #791637), 15:0-18:1(d7) phosphatidylethanolamine (Avanti Polar Lipids, Cat #791638), 18:1(d7) lysophosphatidylcholine (Avanti Polar Lipids, Cat#791643), 18:1(d7) lysophosphatidylethanolamine (Avanti Polar Lipids, Cat #791644), 15:0-18:1(d7) diacylglycerol (Avanti Polar Lipids, Cat #791647), 15:0-18:1(d7)-15:0 triacylglycerol (Avanti Polar Lipids, Cat #791648). Cells were scraped with 0.5 mL methanol and 0.5 mL H2O. Homogenate aliquot of 100 µL was taken to determine protein content using the BCA protein assay (Thermo Fisher Scientific). The remaining homogenate was transferred to a new Eppendorf tube and 1 mL chloroform was added. For media, 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 were resuspended in 60 µl 65:30:5 ACN: IPA:H2O, 5 µL of sample was injected.

Combined analysis:

Analysis ID	AN005524
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Agilent 1260 Infinity II
Column	Thermo Accucore C30 (150 x 2.1mm,2.6µm)
MS Type	ESI
MS instrument type	Triple quadrupole
MS instrument name	Agilent 6460 QQQ
Ion Mode	POSITIVE
Units	Peak area

Chromatography:

Chromatography ID:	CH004200
Instrument Name:	Agilent 1260 Infinity II
Column Name:	Thermo Accucore C30 (150 x 2.1mm,2.6µm)
Column Temperature:	40˚C
Flow Gradient:	0 min, 30% B; 3 min, 30% B; 8 min, 43% B; 9 min, 50% B; 18 min, 90% B; 26 min, 99% B; 30 min, 99%B; 36 min, 30% B
Flow Rate:	0 min, 0.1 mL/min; 8 min, 0.2 mL/min
Solvent A:	60% Acetonitrile/40% water; 0.1% formic acid; 10 mM ammonium formate
Solvent B:	90% Isopropanol/10% acetonitrile; 0.1% formic acid; 10 mM ammonium formate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS005249
Analysis ID:	AN005524
Instrument Name:	Agilent 6460 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Lipid 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