1 2 dinonadecanoyl sn glycero 3 phosphocholine

All the restriction enzymes were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Microcystin-LR was purchased from Taiwan Algal Science Inc (Taoyuan, Taiwan, China). Lipid internal standards of 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC 38:0); 1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine (PE 34:0); 1-nonadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC 19:0); N-lauroyl-D-erythro-sphingosylphosphorylcholine (SM 12:0); and N-heptadecanoyl-D-erythro-sphingosine (Ceramide 17:0) were from Avanti Polar Lipids, Inc. (Alabaster, Alabama, USA). Palmitic acid-16,16,16-d3 (FFA 16:0_d3), stearic acid-18,18,18-d3 (FFA 18:0_d3) and glyceryl tripentadecanoate (TAG 45:0) were purchased from Sigma-Aldrich (Munich, Germany). All other chemicals were from Sigma-Aldrich (St. Louis, Missouri, USA) or Sangon Biotech (Shanghai, China).

The plasma level of PL fatty acids was analyzed by GC with a flame ionization detector (FID)¹⁸ . First, the lipids were isolated by Folch extraction using chloroform/methanol mixture (2:1, v/v) in the presence of 0.01% butylated hydroxytoluene (BHT). TLC separated PL fatty acids with the mobile chase heptane—diisoprophyl ether—acetic acid (60:40:3, v/v/v) and transmetylated to fatty acid methyl ester (FAME) with boron trifluoride in methanol reagent under nitrogen atmosphere at 100 °C for 30 min for PLs. Two standards were used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) (Avanti Polar Lipids, Alabaster, AL, USA) to assess the overall recovery of the extraction method, and methyl nonadecanoate (MeC19:0) (Supelco, North Harrison Road, Bellefonte, PA, USA) as an internal standard (ISTD) for the GC analysis. The FAMES were analyzed by gas chromatography with a flame ionization detector (GC-FID; Clarus 500, PerkinElmer, Shelton, CT, USA) using a capillary column coated with CP-Sil 88 stationary phase (50 m × 0.25 mm × 0.20 μm; Agilent, Palo Alto, CA, USA). The injector and FID detector temperatures were kept at 260 °C. The column temperature was programmed from 150 °C (2 min)–230 °C (10 min) at 4 °C/min. Identification of FAMEs was made by comparison with their retention time with standards.