Lipidomic Analysis of LPA and PA

The total lipid extract was obtained by the classical Bligh-Dyer method [31 (link)] and purified through PTFE filters (Agilent, 5190–5265). To isolate lysophosphatidic (LPA) and phosphatidic (PA) acids, the total extract was separated by TLC (Fluka Silica gel TLC Al foils 10 × 10 cm) in hexane:acetone:acetic acid (40:50:2). Phospholipids remained in situ and were detected by staining with primulin (0.05% in acetone:water 80:20); a mixture of 1-oleoyl LPA (http://www.gzsopo.com/product/PNOALD-O353308.html. CAS: 655528-98-5; accessed on 31 December 2023), PA sodium salt (http://www.gzsopo.com/product/PNOMACKLIN-L864045.html. CAS 383907-53-7; accessed on 31 December 2023), and phosphatidylcholine (PC) was used as a standard. The appropriate silica gel sections were taken from the plate and transferred to Eppendorf tubes, where they were washed with a chloroform–methanol (1:1) mixture followed by an equivalent volume of water. The bottom fraction containing phospholipids was chosen after centrifugation. To separate phospholipids, the chloroform:methanol:acetone:water:acetic acid (6:2:8:1:1:1) system was utilized. The LPA and PA standards had R_fs of 0.4 and 0.7, respectively. To detect lipid groups, primulin staining was applied. Lipids from all four detected groups were isolated, extracted similarly to phospholipids, and treated in methanol with 1% H₂SO₄ for 30 min at 55 °C [32 (link)]. The FAMEs were then extracted with n-hexane and analyzed using GC-MS as described above.