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.
ABSTRACT
Objective To summarize and review the comparative results of the isotope dilution mass spectrometry in Cholesterol Reference Method Laboratory Network (CRMLN)project of the Centers for Disease Control and Prevention (CDC)of the United States in order to provide quality controls for determination of blood lipid.Methods The isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS)methods for determination of serum total cholesterol (TC)and triglycerides (TG)were developed to participate in the comparison of CRMLN.The survey was conducted every three months before 2016 and every half year from 2016.Four kinds of reference materials with two parallel tubes for each material and each tube in duplicate were determined in every survey.At least two certified reference materials used as quality control samples were analyzed simultaneously in each determination.Results In the 15 comparisons the CV of the TC determination method in our laboratory was 0.43％ while the CV of all the participated laboratories in CRMLN was 0.42％.The bias from the overall mean value in our laboratory was 0.22％ while the bias from the CDC target values was 0.58％.The CV of the TG determination method in 15 tests of our laboratory was 0.62％ and the bias was-0.98％ from the overall mean value and-0.80％ from the target values of CDC.Among the 60 results for comparison,98％ (59/60)of CV in the TC determination met with CDC requirement for precision (CV≤ 1％),and 70％ of bias (42/60) of the results met with CDC requirement for accuracy (bias ≤ 1％).For the 60 results in comparison of TG determination,92％ (55/60)of bias of the results met with the accuracy requirement of CDC (bias ≤2.55％).Conclusion In CRMLN comparison the results of TC and TG determined by ID-LC/MS/MS method were consistent with the values which were certified by CDC and determined by other network laboratories.The comparative surveys may play an important role in the standardization of lipid determination,and should be expected to provide experiences and technical supports in the comparative plan for reference measurement in our country.