KOSEN 한인과학기술자네트워크

>rat의 변을 수집하여 >담즙산의 배출 정도를 알아보려고하는데요 >논문에 자세한 method가 없어서요 >자세한 실험방법 있으시면 좀 알려주세요. > ============================= 변을 동결건조후 유기용매로 담즙산을 추출한후 HPLC로 검정합니다. HPLC느 JASCO 나 Hewlett Packard 등 어느기기도 가능합니다. 아래 간략한 method를 원문에서 인용합니다. Analysis of Fecal Bile Acids and Neutral Sterols. Hamsters were put in metabolic cages for 24 hrs to collect feces during the last 2 or 3 days of the feeding period. Quantitation of bile acids and neutral sterols in feces was measured by gas-liquid chromatography according to the method of Batta et al.(J Chromatogr B Analyt Technol Biomed Life Sci 775:153–161, 2002.) 방법: step 1. Two hundred ll n-butanol ontaining cholic acid and 5-alpha-cholestane (internal standards) and 50 ul of concentrated hydrochloric acid were added to 15 mg of freeze-dried fecal samples and to the standards (i.e., 10 ug and 20 ug of each bile acid and neutral sterol). step 2. The mixture was heated at 60'C for 4 hrs, and solvents were evaporated under nitrogen at 60'C. The butylesterified bile acids, neutral sterol standards, and fecal samples were reacted with 100 ul of Sil-prep (trimethyl silylation reagent; Alltech Associates Inc., Deerfield, IL) for 30 mins at 55'C, and solvents were evaporated under nitrogen at 55'C. Trimethyl silyl ether derivatives of samples and standards were resuspended in 200 ul of hexane and centrifuged to remove fecal debris, and 2 ul were injected onto the gas-liquid chromatography column. step 3. Analysis was performed using a Varian Chrompak CP-Sil 5 CB Low Bleed/MS fused silica capillary column, 25 M 3 0.25-mm ID 3 0.25-mm film thickness (Supelco Park, Bellefonte, PA) installed in a Hewlett Packard 6890 gas chromatograph equipped with a flame-ionization detector and autosampler. The carrier gas was helium at a flow rate of 1.5 ml/min, the injector temperature was 260'C, and the detector temperature was 290'C. Peaks were identified by comparing retention times with standards (i.e., hyodeoxycholic acid, deoxycholic acid, hyocholic acid, cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, ursocholic acid, lithocholic acid, coprostanol, campesterol, stigmasterol, stigmastanol, cholestanone, cholesterol, cholestenone, cholestane, b-sitosterol, lanosterol, lathosterol; Steraloids Inc., Newport, RI). The weight percentage of each bile acid and neutral sterol was determined by integration of the peak areas.