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第二次實驗課程- 食物中之膽固醇定量
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實驗原理 利用酵素反應法使樣品中的 cholesterol ester 經 cholesterol esterase 及cholesterol oxidase 反應後變成4-cholesten-3-one 及 H2O2,而所生成之 H2O2 會與4-amino-antipyrine 藉由 peroxidase 的作用生成red quinone,可於500nm下測其生成量。其反應式如下: cholesterol ester+H2O > cholesterol+fatty acid cholesterol esterase cholesterol oxidase cholesterol+O >4 quinone+4 H2O-cholesten-3-one+H2O2 H2O2+phenol+4-amino-antipyrine >red peroxidase
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實驗藥品 Calibrator (standard) 200mg/dl Working reagents:
PIPES buffer pH6.9 40 mmol/l Phenol 24 mmol/l Peroxidase > 100 U/l Cholesterol esterase > 250 U/l Cholesterol oxidase > 200 U/l 4-amino-antipyrine(4-AA) 0.05 mmol/l
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實驗步驟 將A、B及C於水浴37℃下5分鐘,再分別於500 nm下測量其吸光值。
所得之個別吸光值帶入公式計算後,可得知樣品之膽固醇含量(mg/dl) A B C Reagent blank (μl) Standard (μl) Sample DH2O 10 - Reagent 1000
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計算公式 OD of unknown Conc. of unknown= × Conc. standard OD of standard
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