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 methanol to obtain oxidized phospholipids.
Lipid extracts were dried under a gentle stream of nitrogen, dissolved in 1mL of methanol, and stored at −80 ◦C until use.

メタノール 1mL で均質化し、氷上で抽出 1 h
遠心分離 (7000 rpm, 4 ◦C, 5min).
上清を pH 3.0, 0.1N HCl　の水10倍量で希釈
条件設定済み (20mL of methanol and 20mL of water) のC18 Sep-Pak カートリッジ (500mg, Waters, Millford, MA, USA)に設定
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
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

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