粪便中胆汁酸的提取方法

摘 要:

胆汁酸是胆汁的重要成分,在脂肪代谢中起着重要作用。 胆汁酸主要存在于肠肝循环系统并通过再循环起一定的保护作用。做代谢研究中有的会涉及到提取粪便中的胆汁酸。经过查询资料后,总结了一下粪便中胆汁酸的提取方法,供参考。

关键词: ,

胆汁酸是胆汁的重要成分,在脂肪代谢中起着重要作用。 胆汁酸主要存在于肠肝循环系统并通过再循环起一定的保护作用。做代谢研究中有的会涉及到提取粪便中的胆汁酸。经过查询资料后,总结了一下粪便中胆汁酸的提取方法,供大家参考:

(1)取粪便与水1:1匀浆化,取1g溶于20ml 1mol/L乙醇性氢氧化钠中,回流1h,加10ml水,用50ml石油醚除去中性甾类,向水层加2ml的10mol/L氢氧化钠,于热压灭菌器(202.65Kpa或2气压)中加热3h,用稀盐酸调PH值为2,用75ml氯仿-甲醇(1:1)及50ml氯仿提取2次。

(2)粪便中胆汁酸提取也可先干燥,然后用乙醇回流或用索氏提取器提取24~48h。也有用乙醇-丙酮(1:1)索氏提取器提取24h;氯仿-甲醇(1:1)提取48h;含有0.5%盐酸的丙酮提取48h。

(3)Fecal samples were lyophilized and weighed. For extraction of bile acids, an aliquot of dried feces (5 mg) was incubated in 1 mL alkaline methanol (methanol: 1 mol/L NaOH 3:1 [vol/vol]) for 2 hours at 80°C in screw-capped tubes using nor-hyodeoxycholate as an internal standard. Then 9 mL of distilled water was added, and the tubes were mixed and centrifuged. The supernatant was subsequently applied to a prepared Sep-Pak C18 solid-phase extraction cartridge. After a clean up by wash procedures, bile acids were eluted with 75% methanol. The solvent was evaporated to dryness, and the bile acids were derivatized by incubation with 50 μL trifluoroacetic anhydride and 30 μL 1,1,1,3,3,3-hexafluoro-2-propanol for 1 hour at 60°C. The bile acid derivatives were separated on a 25 m×0.25 mm capillary GC column (CP Sil 5B, Chrompack International) in a 3800 GC gas chromatograph (Varian) equipped with a flame ionization detector. The injector and the flame ionization detector were kept at 300°C. The column temperature was programmed from 230 to 280°C. Bile acid derivatives were introduced by split injection (split ratio, 20:1).

参考文献:

Post SM, de Crom R, van Haperen R, van Tol A, Princen HM. Increased fecal bile acid excretion in transgenic mice with elevated expression ofhuman phospholipid transfer protein. Arterioscler Thromb Vasc Biol. 2003,23(5):892-897.

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