Linolenic acid d4

Linolenic acid-d₄ and linoleic acid-d₄ (Cayman chemical), 4 μM, were incubated with 0.8 mg/mL fatty acid free BSA in MV2 medium for 2 h at 37°C. HAOEC were treated for 24 h with T0901317 (10 μM) or vehicle (DMSO) then incubated with the deuterated fatty acid-BSA mix. Total lipids were extracted using the Folch method, and then analyzed and quantified by gas chromatography-mass spectrometry (GC-MS) negative chemical ionization (NCI) with a specific determination of corresponding synthetized n-6 deuterated fatty acids (C18:2 n-6 d4, C18:3 n-6 d4, C20:3 n-6 d4, C20:4 n-6 d4, C22:4 n-6 d4, C24:4 n-6 d4, C24:5 n-6 d4, C22:5 n-6 d4) and n-3 deuterated fatty acids (C18:3 n-3 d14, C18:4 n-3 d12, C20:4 n-3 d12, C20:5 n-3 d10, C22:5 n-3 d10, C24:5 n-3 d10, C24:6 n-3 d10, C22:6 n-3 d10) (Cayman chemical). Data were normalized to cell protein concentration.

Docosahexaenoic acid, Docosahexaenoic acid—d5, Eicosapentaenoic acid, Eicosapentaenoic acid—d5, Arachidonic acid, Arachidonic—d8, Linoleic acid, and α-Linolenic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Linolenic Acid—d4 and α-Linolenic acid—d14 were purchased from Cayman Chemical Company (Ann Arbor, MI, USA).
Methanol and Hexane were purchased from Honeywell (Seelze, Germany), potassium hydroxide from Chimreactiv (Bucharest, Romania), acetonitrile from AppliChem GmbH (Darmstadt, Germany), isopropanol, formic acid, and ammonium acetate from Merck (Darmstadt, Germany), and water from VWR International (Radnor, Pennsylvania, USA). All reagents were HPLC-grade.