Optimized Lipid Extraction and NMR Analysis

The extraction of larger sample volumes for NMR was based on a method described previously^{22 (link),23 (link)}. Tissue homogenates were combined to give 5–10 mg of phospholipid per NMR sample. The samples of serum and prepared brain tissues from all groups were pooled and GCTU (250 µL) added to serum mixtures. Pooled solutions (5–8 mL) were diluted (DMT, 15 mL; Falcon tube) and made uniphasic (methanol, 15 mL). The mixture was agitated and diluted and made biphasic (dichloromethane, 10 mL) before centrifugation (3.2k × g, 2 min). The aqueous portion and any mesophasic solid was removed and discarded, and the organic solution dried under a flow of nitrogen. Samples were stored at −80 °C. Samples were dissolved in a modified^{22 (link)} form of the ‘CUBO’ solvent system^{43 (link)–46 (link)} (the amount of dueteriated dimethylformamide d₇-DMF was minimised). Stock solutions of the solvent consisted of dimethylformamide (3.5 mL), d₇-DMF (1.5 mL), triethylamine (1.5 mL) and guanidinium chloride (500 mg). Wilmad® 507PP tubes were used. Sample concentration was 5–10 mg lipids per sample (600 µL).