Measurement of lecithin-cholesterol acyltransferase activity using high-performance liquid chromatography
10.13602/j.cnki.jcls.2017.09.02
- VernacularTitle: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
- Author:
Yu WANG
1
;
ming Si WANG
;
Jie ZENG
;
yue Rui YANG
;
xia Hong LI
;
Jun DONG
;
xiang Wen CHEN
Author Information
1. 北京大学第五临床医学院(北京医院)
- Keywords:
lecithin-cholesterol acyltransferase;
cardiovascular disease;
high-performance liquid chromatography;
7-dehydrocholesterol
- From:
Chinese Journal of Clinical Laboratory Science
2017;35(9):644-648
- CountryChina
- Language:Chinese
-
Abstract:
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-dehydroeholesterol.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 AI (apoAI) (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.
