DOI: 10.5281/zenodo.17775129
title: PUFA supplementation-dependent incorporation of ω-3 and ω-6 fatty acids into phospholipids and triglycerides in the intestinal epithelium of Gpx4⁺/⁻IEC mice on a Western diet

description: Transgenic Gpx4+/−IEC mice exhibit diminished GPX4 activity specifically in the intestinal epithelium and develop features of small-intestinal Crohn’s-like disease when challenged with a Western diet and exposed to excess polyunsaturated fatty acids (PUFAs) [1,2]. To investigate the incorporation and distribution of PUFA species in intestinal epithelial phospholipid and neutral lipid classes, we profiled phosphatidylcholines, phosphatidylethanolamines, and triglycerides using a lipidomics approach based on ultraperformance chromatography-ESI-tandem mass spectrometry. The analysis focused on changes in the epithelial proportion of ω-3 and ω-6 PUFAs in these lipid classes.

For this purpose, 8- to 10-week-old Gpx4+/−IEC mice were fed either a Western diet (ssniff, TD88137) or a PUFA-enriched Western diet (TD88137 + 10% fish oil) ad libitum for three months. Lipids were extracted from tissue homogenates of small intestinal scrapings from Gpx4⁺/⁻IEC mice fed the respective diets. Our data reveal substantial enrichment of ω-3 and ω-6 PUFAs across lipid classes upon PUFA supplementation, accompanied by rearrangements in overall lipid composition.

The uploaded files contain the following parts: 

1) A sample list describes the sample id, mice genotype (wt and Gpx4+/−IEC), diet (WD and PUFA-WD), intestine tissue section (Duodenum, Jejunum or Ileum), tissue weight, volume of PBS for homogenate preparation as well as the homogenate concentration.

2) A batch list describes the lipid species (phosphatidylcholine (PC), phosphatidylethanolamine (PE) and triglycerides (TG)), batch number, sample number as well as the corresponding Analyst wiff file name.

3) A zip file including the individual raw data files of PC, PE and TG after the lipidomics measurement.

 

The data has been shown in the following original article: 

Meyer et al., Metabolic stress sensing by epithelial RXRα links Westernization of diet with Crohn’s disease. Cell Metab. 2025, 10.1016/j.cmet.2025.11.008.
description: Extraction and analysis of phospholipids and triglycerides by UPLC-MS/MS

Lipids were extracted from tissue homogenates of small intestinal scrapings of Gpx4+/−IEC mice fed a PUFA-WD or a WD for 3 months, by the sequential addition of PBS pH 7.4, methanol (spiked with internal standards), chloroform, and saline (final ratio 14:34:35:17). The chloroform layer was recovered and evaporated using an Eppendorf Concentrator Plus system (Hamburg, Germany; non-polar phase: high vapor pressure application mode), stored at -20°C, and dissolved in methanol prior to ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) analysis. 1-pentadecanoyl-2-oleoyl(d7)-sn-glycero-3-phosphocholine [PC(15:0/18:1-d7), Avanti Polar Lipids, Alabaster, AL/Sigma-Aldrich, Darmstadt, Germany], 1-pentadecanoyl-2-oleoyl(d7)-sn-glycero-3-phosphoethanolamine [PE(15:0/18:1-d7), Avanti Polar Lipids/Sigma-Aldrich] and 1,3-dipentadecanoyl-2-oleyol(d7)-glycerol [TG(15:0/18:1-d7/15:0), Avanti Polar Lipids/Sigma-Aldrich] were used as internal standards.

Lipids were separated on an Acquity UPLC BEH C8 column (130 Å, 1.7 μm, 2.1×100 mm, Waters, Milford, MA) using an ExionLC AD UHPLC system (Sciex, Framingham, MA). For the analysis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the gradient containing mobile phase A (water/acetonitrile 90/10, 2 mM ammonium acetate) and B (water/acetonitrile 5/95, 2 mM ammonium acetate) was ramped from 75 to 85% B over 5 minutes and then further increased to 100% B within 2 minutes, which was maintained isocratically for another 2 minutes. For the analysis of triglycerides (TG), mobile phase A (isopropanol) and mobile phase B (acetonitrile/water 95/5, 10 mM ammonium acetate) were ramped from 90% to 70% B over 6 min followed by isocratic elution for 4 min. The flow rate was set to 0.75 ml/minute and the column temperature to 45°C.

Lipids were detected by a QTRAP6500+ mass spectrometer (Sciex) equipped with an IonDrive Turbo V Ion Source and a TurboIonSpray probe for electrospray ionization. The QTRAP6500+ system was operated in negative (for PC, PE) or positive (for TG) ionization mode using scheduled multiple reaction monitoring (MRM). For the detection of PE and PC species, the transitions from [M-H]- to both fatty acid anions were monitored. For the detection of TG species, the transitions from [M + NH4]+ adduct ions to [M - fatty acid anion]+ ions were monitored, without differentiating between fatty acid positional isomers. Table 1 shows the previously reported optimized mass spectrometry parameters for PC, PE and TG.

The instruments were operated by Analyst 1.7.1 (Sciex), and the mass spectra were processed by Analyst 1.6.3 (Sciex). For the quantitative analysis of PC, PE and TG species, combined peak areas from the transitions to the two fatty acid anions (for PC, PE) or the three [M - fatty acid anion]+ ions (for TG) were calculated. Fatty acid compositions are given as percentage of all species detected in the corresponding PE, PC or TG subclass (= 100%).

 

Table 1 Mass spectrometry parameters of the QTRAP6500+ system.






parameters



PC



PE



TG





Curtain gas (CUR)



40 psi



40 psi



40 psi





Collision gas (CAD)



medium



medium



medium





Ion spray voltage (IS)



-4,500 V



-4,500 V



5,500 V





Heated capillary temperature (TEM)



350°C



650°C



400°C





Sheath gas pressure (GS1)



55 psi



55 psi



60 psi





Auxiliary gas (GS2)



75 psi



75 psi



70 psi





Declustering potential (DP)



-44 V



-50 V



120 V





Entrance potential (EP)



-10 V



-10 V



10 V





Collision energy (CE)



-46 eV



-38 eV



35 eV





Collision cell exit potential (CXP)



-11 V



-12 V



26 V