HPLC测定胆固醇代谢酶（CYP7A1）活性方法学的建立

发布时间：2018-05-23 16:34

本文选题：胆固醇7α-羟化酶 + HPLC　；参考：《山东大学》2010年硕士论文

【摘要】： 背景：胆汁酸是在肝细胞内由游离胆固醇生成的,第一步及限速反应是胆固醇在胆固醇7a-羟化酶的作用下进行的7a-羟化作用。胆固醇7a-羟化酶(CYP7A1)位于微粒体,属细胞色素P450酶系。据报道,它的活性受胆汁酸池体积、组成和疏水性,胆汁酸的肠肝循环,激素、肠道因子以及各种调节CYP7A1转录的核受体的表达的负反馈作用所影响。测定胆固醇7a-羟化酶活性的方法主要有三种：(1)用薄层色谱技术,采用同位素结合法将放射性标记的胆固醇与7a-羟胆固醇分离；(2)GLC-MS法；(3)用HLPC-分光光度法。用高效液相色谱法(HPLC)测定CYP7A1酶活性的基本原理是在肝微粒体的催化下,内源性胆固醇作为胆固醇7a-羟化酶的底物,适合的反应温度、时间等因素协同作用下,生成7a-羟胆固醇；我们通过加入胆固醇氧化酶后将7α-羟胆固醇转化为7α-羟-4-胆甾烯-3-酮(HCO),然后用HPLC对7a-羟胆固醇进行定量。以HCO产物量的多少来判断该CYP7A1活性的大小。本研究对前有的研究方法进行了改进,其一是采用了7β-羟胆固醇作为内标,其二是采用反相HPLC法对酶反应产物进行分离与定量。目的：对大鼠肝微粒体中胆固醇代谢酶(CYP7A1)活性测定方法的建立。在本研究中,我们采用反相液相色谱法(RP-HPLC)来测定胆固醇7a-羟化酶的活性。方法：采用XDB-C18 (4.6×250mm,5μm)色谱柱；柱温为室温；以乙腈-甲醇(95：5)为流动相,流速：0.8mL/min;检测器为可变波长扫描紫外检测器(VWD),VWD=240nm。以7β-羟胆固醇为内标与大鼠的肝微粒体进行体外孵育,以反相液相色谱法测定胆固醇的反应产物7α-羟-4-胆甾烯-3-酮(HCO)。结果： 1.以待测物浓度为横坐标,待测物与内标物的峰面积比值为纵坐标,求得线性回归方程,结果为：Y=1.31362786X+0.0268326, R2=0.99603。根据标准曲线,7a-羟胆固醇的线性范围为10-500ng/ml。当信噪比(S／N)为2.3时,方法最低检测限lOng/ml,回收率及精密度均符合检测要求。 2.在所建立的RP-HPLC条件下,胆固醇的产物(HCO)保留时间约为13.6min。杂质峰不干扰测定。 3.大鼠肝微粒体胆固醇7a-羟化酶的活性：1.214±1.153 nmol/h/mg protein。结论：该HPLC能够准确的检测出在大鼠肝微粒体中的胆固醇7a-羟化酶的活性。
[Abstract]:Background: bile acids are produced by free cholesterol in hepatocytes. The first step and rate-limiting reaction is the 7a-hydroxylation of cholesterol by cholesterol 7a-hydroxylase. Cholesterol 7a- hydroxylase CYP7A1) is located in microsomes and belongs to cytochrome P450 enzyme system. Its activity is reported to be affected by the volume, composition and hydrophobicity of bile acid pools, the intestinal and hepatic circulation of bile acids, hormones, intestinal factors and the negative feedback effects of various nuclear receptors regulating CYP7A1 transcription. Determination of cholesterol 7a-hydroxylase activity by TLC, radioisotope binding method was used to separate radioactive cholesterol from 7a-hydroxyl cholesterol by GLC-MS) HLPC-spectrophotometry was used to determine the activity of cholesterol 7a-hydroxylase. The basic principle of determining the activity of CYP7A1 by HPLC was that under the catalysis of liver microsomes, endogenous cholesterol was used as substrate of cholesterol 7a- hydroxylase, suitable reaction temperature, time and other factors to produce 7a-hydroxycholesterol. After the addition of cholesterol oxidase, 7 伪 -hydroxycholesterol was transformed into 7 伪 -hydroxy-4-cholestene -3-one HCO, and then 7a-hydroxycholesterol was quantified by HPLC. The activity of HCO was judged by the amount of the product. In this study, the former research methods were improved, one was to use 7 尾 -hydroxyl cholesterol as internal standard, the other was to separate and quantify the enzyme reaction products by reverse phase HPLC. Aim: to establish a method for the determination of cholesterol metabolism enzyme CYP7A1 in rat liver microsomes. In this study, RP-HPLC was used to determine the activity of cholesterol 7a-hydroxylase. Methods: a XDB-C18 column with a column temperature of 4.6 脳 250 mm ~ (5 渭 m) was used at room temperature, acetonitrile-methanol 95: 5) as mobile phase with a flow rate of 1: 0.8 mL / min and a variable wavelength scanning ultraviolet detector (VWDX) at 240 nm. Rat liver microsomes were incubated with 7 尾 -hydroxycholesterol as internal standard in vitro. The reaction product 7 伪 -hydroxy-4-cholesten-3-one was determined by reversed-phase liquid chromatography (RP-HPLC). Results: 1. The linear regression equation was obtained by using the concentration of the object under test as the horizontal coordinate and the ratio of the peak area of the object to the internal standard as the vertical coordinate. The results were as follows: 1. 31362786X 0.0268326, R2 + 0.99603. According to the standard curve, the linear range of 7 a-hydroxycholesterol is 10-500 ng / ml. When S / N = 2.3, the minimum detection limit of the method is lOng / ml, and the recovery rate and precision are in line with the detection requirements. 2. Under the conditions of RP-HPLC, the retention time of cholesterol product was about 13.6 mins. Impurity peaks do not interfere with the determination. 3. The activity of rat liver microsomal cholesterol 7a- hydroxylase: 1. 214 卤1.153 nmol/h/mg protein. Conclusion: the HPLC can accurately detect the activity of cholesterol 7a- hydroxylase in rat liver microsomes.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

【参考文献】