Plasma lipid extraction

Biobank plasma samples were stored at -80 °C and thawed to 2 °C for 10 min before lipid extraction. The Folch method was used to extract plasma lipids (Folch et al., 1957). Briefly, in PYREX® culture tubes, 2 mL of chloroform: methanol (2:1, vol/vol), 8 µL of deuterated internal standard (SPLASH® LIPIDOMIX® mass spec standard, Avanti) were added to 100 µL of plasma, vortexed and incubated for 30 min. The internal standard contains deuterated lipids including phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidic acid (PA), lysophosphatidylcholine (LysoPC), lysophosphatidylethanolamine (LysoPE), cholesteryl ester (CE), monoglycerides (MG), diglycerides (DG), triglycerides (TG), and sphingomyelin (SM). To each sample, 400 µL of 0.9% NaCl was added, and the sample was centrifuged at 1200 RPM for 15 min. After centrifugation, the upper phase was removed, and the lower phase was collected into glass auto-sampler vials and evaporated under nitrogen flow. Finally, a 1:9 ratio of water and butanol-methanol (50:50) was added to the dried samples to resuspend the sample for analysis.