Oxidized phospholipids

Samples were homogenized with 1mL methanol and extracted for 1 h at on ice.
Homogenates were centrifuged (7000 rpm, 4 ◦C, 5min).
Supernatant was diluted with 10 volumes of water adjusted to pH 3.0 with 0.1N HCl.
Samples were applied to preconditioned (20mL of methanol and 20mL of water) C18 Sep-Pak cartridges (500mg, Waters, Millford, MA, USA).
Cartridge was washed with 20mL of water to exclude nonvolatile ions followed by 10mL of hexane to exclude cholesterols and neutral lipids.
Samples were eluted with 10mL of methyl formate to obtain oxidized fatty acids and 10mL of methanol to obtain oxidized phospholipids, successively.
Lipid extracts were dried under a gentle stream of nitrogen, dissolved in 1mL of methanol, and stored at −80 ◦C until use.

組織をメタノール 1mL でホモジナイズ、氷上で1時間抽出
遠心分離 (7000 rpm, 4 ◦C, 5min)
上清を 0.1N HClでpH 3.0に調整した10倍量の水で希釈
メタノール20mL 次いで水20mL で洗浄したC18 Sep-Pak カートリッジ (500mg, Waters, Millford, MA, USA)にサンプルを注入
20mL の水でカートリッジに残るイオンを洗浄、ヘキサン 10mL でコレステロールと中性脂質を除く
ギ酸メチル　10mLで酸化脂肪酸を溶出、メタノール 10mL で酸化リン脂質を溶出
脂質をゆるやかな窒素ガスで乾燥、1mL メタノールに溶解、 −80 ◦C で保管