高效液相色谱法测定血清卵磷脂胆固醇酰基转移酶活性的建立

Measurement of lecithin-cholesterol acyltransferase activity using high-performance liquid chromatography

目的建立高效液相色谱法(HPLC)测定血清卵磷脂胆固醇酰基转移酶(LCAT)活性的方法,观察LCAT活性与动脉粥样硬化性心血管病(CVD)传统危险因素的关系。方法选用7-脱氢胆固醇和1,2-二癸酰基甘油-3-磷酸(10∶0 PC)作为LCAT的反应底物,加入LCAT激活肽(LAP642),制备脂质体;在冰水浴中将500μL脂质体与10μL血清混合,37℃温育1 h;提取脂质,HPLC测定反应后7-脱氢胆固醇及其酯之比,计算LCAT活性。用所建方法测定120例健康志愿者血清的LCAT活性,分析其与CVD危险因素的关系。结果 7-脱氢胆固醇、10∶0 PC(物质的量的比为1∶8.5)与LAP642组成的脂质体制备简单、性质稳定。当温育时间0~8 h、血清量0~20μL时,LCAT活性与变量线性相关;平均批内不精密度(CV)和总CV分别小于1.76%、3.11%。方法比对结果显示,所建方法与ELISA测定的LCAT质量和内源底物法测定的LCAT活性显著正相关(P<0.01)。120例样本LCAT活性与体质量指数(BMI)、三酰甘油(TG)等呈正相关(P<0.05);与载脂蛋白AⅠ(apo AⅠ)(P<0.05)、高密度脂蛋白胆固醇(HDL-C)(P<0.01)等呈负相关。结论建立了HPLC测定LCAT活性的外源底物法,所建方法简便、精密、可靠,结果不受反应底物影响,为脂代谢机制研究和CVD危险分析提供了新的方法学基础。

Objective To develop a high-performance liquid chromatography （HPLC） method for the measurement of lecithin-cholesterol acyltransferase （LCAT） activity and analyze the relationships between LCAT activity and the traditional risk factors of atherosclerotic cardiovascular disease （CVD）.Methods The liposome which contained 7-dehydrocholesterol and 1,2-didecanoyl-sn-glycero-3-phosphocholine （10∶ 0 PC） as the substrate of LCAT and LCAT activating peptide （LAP642） as LCAT activator was mixed with 10 microliters of serum sample （50∶ 1,V/V） in ice-water bath and subsequently incubated at 37 ℃ for 1 h.After extracting with hexane,the lipid was analyzed by HPLC and the LCAT activity was calculated as the ratio of 7-dehydrocholesterol ester to free 7-dehydrocholesterol.LCAT activities of 120 health volunteers were measured and its relationship with traditional risk factors of CVD was analyzed.Results The liposome composed of substrates （7-dehydrocholesterol and 10∶ 0 PC with ratio of amount 1∶ 8.5） and LAP642 was stable,efficient and easy for preparation.LCAT activity was a linear correction during 8 hours of incubation and was independent of the volume of serum added in the range from 0 to 20 microliters.The averages of intra-and total coefficients of variation （CV） were less than 1.76%and 3.11% respectively.The comparison of two methods showed that the results of the HPLC method were highly correlated with LCAT mass measured by commercial ELISA method and LCAT activity measured by endogenous substrate fractional esterification of high density lipoprotein cholesterol （FERHDL） （P〈0.01）.LCAT activity positively correlated with body mass index （BMI）,triglyceride （TG） （P〈0.05） and negatively correlated with apolipoprotein AΙ （apo AΙ） （P〈0.05） and high density lipoprotein cholesterol （HDL-C） （P〈0.01） in the volunteers.Conclusion A simple,precise and reliable HPLC method for determination of LCAT activity using artificial substrate has been established,and the results were not influenced by endogenous cholesterol levels in serum.The newly developed method could be a useful tool in the study of lipid metabolism and the assessment for risk factors of CVD.