高效液相色譜法測定血清卵磷脂膽固醇酰基轉移酶活性的建立*

王玉，王思明，曾潔,3，楊睿悅，李紅霞，董軍,，陳文祥,3

(1.北京大學第五臨床醫學院(北京醫院)，北京 100730；2.北京醫院 國家老年醫學中心，北京 100730；3.衛生部臨床檢驗中心，北京 100730)

·臨床檢驗技術研究·

高效液相色譜法測定血清卵磷脂膽固醇酰基轉移酶活性的建立*

王玉1，王思明2，曾潔2,3，楊睿悅2，李紅霞2，董軍1,2，陳文祥2,3

(1.北京大學第五臨床醫學院(北京醫院)，北京 100730；2.北京醫院 國家老年醫學中心，北京 100730；3.衛生部臨床檢驗中心，北京 100730)

目的建立高效液相色譜法(HPLC)測定血清卵磷脂膽固醇酰基轉移酶(LCAT)活性的方法，觀察LCAT活性與動脈粥樣硬化性心血管病(CVD)傳統危險因素的關系。方法選用7-脫氫膽固醇和1,2-二癸酰基甘油-3-磷酸(10∶0 PC)作為LCAT的反應底物，加入LCAT激活肽(LAP642)，制備脂質體；在冰水浴中將500 μL脂質體與10 μL血清混合，37 ℃溫育1 h；提取脂質，HPLC測定反應后7-脫氫膽固醇及其酯之比，計算LCAT活性。用所建方法測定120例健康志愿者血清的LCAT活性，分析其與CVD危險因素的關系。結果7-脫氫膽固醇、10∶0 PC(物質的量的比為1∶8.5)與LAP642組成的脂質體制備簡單、性質穩定。當溫育時間0～8 h、血清量0～20 μL時，LCAT活性與變量線性相關；平均批內不精密度(CV)和總CV分別小于1.76%、3.11%。方法比對結果顯示，所建方法與ELISA測定的LCAT質量和內源底物法測定的LCAT活性顯著正相關(P<0.01)。120例樣本LCAT活性與體質量指數(BMI)、三酰甘油(TG)等呈正相關(P<0.05)；與載脂蛋白AⅠ(apoAⅠ)(P<0.05)、高密度脂蛋白膽固醇(HDL-C)(P<0.01)等呈負相關。結論建立了HPLC測定LCAT活性的外源底物法，所建方法簡便、精密、可靠，結果不受反應底物影響，為脂代謝機制研究和CVD危險分析提供了新的方法學基礎。

卵磷脂膽固醇酰基轉移酶；心血管病；高效液相色譜；7-脫氫膽固醇

Abstract：ObjectiveTo 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).MethodsThe 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.ResultsThe 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(apoAΙ)(P<0.05)and high density lipoprotein cholesterol(HDL-C)(P<0.01)in the volunteers.ConclusionA 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.

Keywords: lecithin-cholesterol acyltransferase; cardiovascular disease; high-performance liquid chromatography; 7-dehydrocholesterol

高密度脂蛋白膽固醇(HDL-C)降低是動脈粥樣硬化(atherosclerosis，As)性心血管病(cardiovascular diseases，CVD)的重要危險因素[1]，這與HDL在膽固醇逆轉運(reverse cholesterol transport，RCT)中發揮的作用密不可分。RCT指外周組織過量的游離膽固醇酯化后被運回肝臟進而排出體外的過程；……