Revision as of 04:42, 22 October 2010

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時間放置し脂質を抽出
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
水20mL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
脂質を窒素ガス下で乾固、メタノール1mLに溶解、 −80 ◦C で保管

酸化脂肪酸解析のための分子特異的測定方法

MRMの特異性と逆相LCによる酸化脂肪酸の分離

酸化リン脂質のMS/MS解析と測定方法

逆相LCによるリン脂質・酸化リン脂質の分離

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@@ Line 9: / Line 9: @@
 # 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時間脂質を抽出
+# 組織をメタノール 1mL でホモジナイズ、氷上で1時間放置し脂質を抽出
 # 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