Lipid Profiling in Tissues

The total lipids were extracted from the tissues (liver, brain cortex, and retina) using a 2:1 mixture of methanol/chloroform. As phospholipids are dominant in the brain and neurons, the phospholipid fraction was isolated further from the total lipid of the brain cortex in Exp. II using an SPE column (CHROMABOND NH₂) (Macherey-Nagel GmbH & Co. KG, Düren, Germany). This was not feasible for retina lipids because there were only trace amounts of samples, even when pooled from four mice within a group. Extracts were taken to dryness and weighed to estimate the total lipid content. The total lipids or phospholipid fraction was subjected to transesterification by methanol/dichloromethane = 3/1, as described elsewhere [26 (link)], and the resulting fatty acid methyl esters were dissolved in n-hexane for fatty acid analysis in a Hewlett-Packard 5890 gas chromatograph with flame ionization detection on a SP-2380 fused silica capillary column (30 m × 0.25 mm× 0.2 µm; Supelco, Bellefonte, PA, USA) using nitrogen as the carrier gas (1.5 mL/min). The oven temperature program was set to 110℃ for 5 min, which was then increased at 10℃/min to 170℃, then at 3℃/min to 230℃, and held at 230℃ for 5 min. The fatty acid peaks were identified by a comparison of the retention times with authentic standards.