Glc7890a

Cases and controls from both cohorts were processed within the same batch, and laboratory personnel were blinded to participant information. To determine fatty acid concentration, 10 µg C15 phosphatidylcholine internal standard (Nuchek Prep, Inc.) was added to 200 µL plasma and phospholipids were extracted using a Folch method as previously described (43 (link), 44 (link)). Briefly, lipids were extracted from the plasma sample and total phospholipids were separated by spotting the samples on a heat-activated silica gel “G” TLC plate (Analtech) and developing plate in a chamber with solvent containing 80:20:1 petroleum ether:diethyl ether:acetic acid. Methyl ester bands were prepared by a mixture of BF₃(boron trifluoride) and hexane at 100°C. Total phospholipid fatty acids were separated by automated GLC 7890A (Agilent Technologies) on a CP-Sil 88 column (100 m × 0.25 mm; Agilent) (45 (link)). To control for variations between batches of samples, control measures were used in addition to the internal standard (concentration = 20 μg/mL), and individual GC peaks were identified and validated against phospholipid standards (GLC-502 and GLC-643) from NuChek Prep, Inc., which were run for each batch to verify retention time and quantification for each individual fatty acid.