Summary of Study ST003374
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 | ST003374 |
| Study Title | Incorporation of oleate-d9 and elaidate-d17 into complex sphingolipids in Huh7 cells while modulating SPT flux. |
| Study Summary | We analyzed complex sphingolipids (Lac-Cer, SM) in Huh7 cells treated with BSA-oleate-d9 or BSA-elaidate-d17 to determine their incorporation into the long-chain base (LCB) of intact sphingolipids. We validated their reduction in abundance through pharmacological inhibition of SPT with myriocin. 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 |
| 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: | SU003495 |
| 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 |
|---|---|---|---|
| SA366992 | Huh7_delip_SL2_Ed17_1 | Huh7 cells | BSA-elaidate-d17 + DMSO |
| SA366993 | Huh7_delip_SL2_Ed17_2 | Huh7 cells | BSA-elaidate-d17 + DMSO |
| SA366994 | Huh7_delip_SL2_Ed17_3 | Huh7 cells | BSA-elaidate-d17 + DMSO |
| SA366995 | Huh7_delip_SL2_Ed17myr_1 | Huh7 cells | BSA-elaidate-d17 + myriocin |
| SA366996 | Huh7_delip_SL2_Ed17myr_2 | Huh7 cells | BSA-elaidate-d17 + myriocin |
| SA366997 | Huh7_delip_SL2_Ed17myr_3 | Huh7 cells | BSA-elaidate-d17 + myriocin |
| SA366998 | Huh7_delip_SL2_Od9_1 | Huh7 cells | BSA-oleate-d9 + DMSO |
| SA366999 | Huh7_delip_SL2_Od9_2 | Huh7 cells | BSA-oleate-d9 + DMSO |
| SA367000 | Huh7_delip_SL2_Od9_3 | Huh7 cells | BSA-oleate-d9 + DMSO |
| SA367001 | Huh7_delip_SL2_Od9myr_1 | Huh7 cells | BSA-oleate-d9 + myriocin |
| SA367002 | Huh7_delip_SL2_Od9myr_2 | Huh7 cells | BSA-oleate-d9 + myriocin |
| SA367003 | Huh7_delip_SL2_Od9myr_3 | Huh7 cells | BSA-oleate-d9 + myriocin |
| Showing results 1 to 12 of 12 |
Collection:
| Collection ID: | CO003488 |
| Collection Summary: | Cells (~400,000 cells) were 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). Cells were scraped with 0.5 mL methanol and 0.5 mL H2O. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR003504 |
| 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: | SP003502 |
| Sampleprep Summary: | Cells (~400,000 cells) were 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). 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 plasma or 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 for cells were resuspended in 60 µl Buffer B, 5 µL of sample was injected. |
Combined analysis:
| Analysis ID | AN005526 |
|---|---|
| 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: | CH004202 |
| 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: | MS005251 |
| Analysis ID: | AN005526 |
| 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 |
