Summary of Study ST001252

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench,, where it has been assigned Project ID PR000839. The data can be accessed directly via it's Project DOI: 10.21228/M87H7W This work is supported by NIH grant, U2C- DK119886.


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Study IDST001252
Study TitleEicosanoid profiles of dermal fibroblasts (part-I)
Study SummaryThe sphingolipid, ceramide-1-phosphate (C1P), directly binds and activates Group IVA cytosolic phospholipase A2 (cPLA2) to generate eicosanoids. Due to the role of eicosanoids in wound healing, we choose to use our novel genetic mouse model expressing cPLA2 with an ablated C1P interaction site (KI) to examine the cPLA2/C1P interaction in wound healing. Wound closure rate was not affected, but wound maturation was enhanced by loss of the C1P/cPLA2α interaction based on the following findings. Wounds in KI mice displayed: i) increased infiltration of dermal fibroblasts into the wound environment; ii) increased wound tensile strength; and iii) higher Type I/Type III collagen ratios. These findings were recapitulated in vitro as primary dermal fibroblasts (pDFs) from KI mice showed significantly increased collagen deposition and migration velocity compared to WT and KO pDFs. Additionally, the KI showed an altered eicosanoid profile of reduced pro-inflammatory prostaglandins (PGE2) and increased levels of specific HETE species (5-HETE). Elevated 5-HETE levels promoted increased dermal fibroblast migration and collagen deposition. This “gain of function” role for the mutant cPLA2 was also linked to differential cellular localization of cPLA2α and 5-HETE biosynthetic factors. These studies demonstrate regulation of key biological mechanisms by a defined protein:lipid interaction in vivo and provide new insights into cPLA2 function.
University of South Florida
Last NameChalfant
First NameCharles
Address4202 E Fowler Ave, Tampa, FL 33620
Submit Date2019-09-22
Raw Data AvailableYes
Raw Data File Type(s)wiff
Analysis Type DetailMALDI-MS
Release Date2019-10-11
Release Version1
Charles Chalfant Charles Chalfant application/zip

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Sample Preparation:

Sampleprep ID:SP001328
Sampleprep Summary:Samples were stored at − 80 °C until the time of analysis. Lipids were extracted for eicosanoids. Eicosanoid Preparation Eicosanoids were extracted using a modified extraction process from previously described. (1, 2). 4 mL of media was combined with an IS mixture comprised of comprised of 10% methanol (400 μl), glacial acetic acid (20 μl), and internal standard (20 μl) containing the following deuterated eicosanoids (1.5 pmol/μl, 30 pmol total) (All standards purchased from Cayman Chemicals): (d4) 6keto-Prostaglandin F1α, (d4) Prostaglandin F2α, (d4) Prostaglandin E2, (d4) Prostaglandin D2, (d8) 5-Hydroxyeicosa-tetranoic acid (5-HETE), (d8) 12-Hydroxyeicosa-tetranoic acid (12-HETE) (d8) 15-Hydroxyeicosa-tetranoic acid (15-HETE), (d6) 20-Hydroxyeicosa-tetranoic acid (20-HETE), (d11) 8,9 Epoxyeicosa-trienoic acid, (d8) 14,15 Epoxyeicosa-trienoic acid, (d8) Arachidonic acid, (d5) Eicosapentaenoic acid, (d5) Docosahexaenoic acid, (d4) Prostaglandin A2, (d4) Leukotriene B4, (d4) Leukotriene C4, (d4) Leukotriene D4, (d4) Leukotriene E4, (d5) 5(S),6(R)-Lipoxin A4, (d11) 5-iPF2α-VI, (d4) 8-iso Prostaglandin F2α, (d11) (±)14,15-DHET, (d11) (±)8,9-DHET, (d11) (±)11,12-DHET, (d4) Prostaglandin E1, (d4) Thromboxane B2, (d6) dihmo gamma linoleic acid, (d5) Resolvin D2, (d5) Resolvin D1, (d5) Maresin 2, and (d5) Resolvin D3. Samples and vial rinses (5% MeOH; 2 ml) were applied to Strata-X SPE columns (Phenomenex), previously washed with methanol (2 ml) and then dH2O (2 ml). Eicosanoids eluted with isopropanol (2 ml), were dried in vacuuo and reconstituted in EtOH:dH2O (50:50;100 μl) prior to UPLC ESI-MS/MS analysis.