A simple and precise method to detect sterol esterification activity of lecithin/cholesterol acyltransferase by high-performance liquid chromatography

• Published in: Analytical and Bioanalytical Chemistry Vol. 410; no. 6; pp. 1785 - 1792
• Main Authors: Wang, Yu, Wang, Siming, Zhang, Lijiao, Zeng, Jie, Yang, Ruiyue, Li, Hongxia, Tang, Yueming, Chen, Wenxiang, Dong, Jun
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Science and Business Media LLC 01.02.2018; Springer Berlin Heidelberg; Springer; Springer Nature B.V
• Subjects: 7-dehydrocholesterol; absorption; Acyltransferase; Adult; Aged; Analytical Chemistry; apolipoprotein A-I; Apolipoproteins; Biochemistry; blood serum; Cardiovascular diseases; Characterization and Evaluation of Materials; chemical bonding; Chemical properties; Chemical vapor deposition; Chemistry; Chemistry and Materials Science; Cholesterol; cholesterol acyltransferase; Chromatography; Chromatography, High Pressure Liquid; Coefficient of variation; Cofactors; correlation; Dehydrocholesterols; emulsifiers; Enzyme Assays; Esterification; Ethanol; Female; Food Science; Heart diseases; hexane; Hexanes; High density lipoprotein; high density lipoprotein cholesterol; High performance liquid chromatography; Humans; Laboratory Medicine; Lecithin; Linear functions; Lipid metabolism; Lipids; Liquid chromatography; Low density lipoprotein; Male; Metabolism; Methods; Middle Aged; Monitoring/Environmental Analysis; Observations; phosphatidylcholine-choline acyltransferase; Phosphatidylcholine-Sterol O-Acyltransferase; Phosphocholine; Reagents; Reproducibility; Reproducibility of Results; Research Paper; Risk assessment; Sterols; Substrate Specificity; Substrates; triacylglycerols; Triglycerides; Ultraviolet absorption; Young Adult; Lecithin: cholesterol acyltransferase; Sterol esterification; Cardiovascular disease; Enzyme activity; High-density lipoprotein; Chromatography
• ISSN: 1618-2642; 1618-2650; 1618-2650
• DOI: 10.1007/s00216-017-0834-4

The measurement of lecithin: cholesterol acyltransferase (LCAT, EC 2.3.1.43) activity is important in high-density lipoprotein (HDL) metabolism study and cardiovascular disease (CVD) risk assessment. However, current methods suffer from complex design and preparation of exogenous substrate, low reproducibility, and interference of cofactors. In this study, we developed a simple and precise high performance liquid chromatography (HPLC) method for the measurement of LCAT activity. By using 7-dehydrocholesterol (7-DHC) and 1,2-didecanoyl- sn -glycero-3-phosphocholine(10:0PC) as substrates, and an LCAT activating peptide (P642) as activator and emulsifier, the substrate reagent was easily made by vortex. The substrate reagent was mixed with serum samples (50:1, v / v ) and incubated at 37 °C for 1 h. After incubation, the lipid was extracted with hexane and ethanol. With a conjugated double bond and ultraviolet absorption, 7-DHC and its esterification product could be separated and analyzed by a single HPLC run without calibration. LCAT activity was a linear function of the serum sample volume and the intra- and total assay coefficients of variation (CV) less than 2.5% were obtained under the standardized conditions. The substrate reagent was stable, and assay result accurately reflected LCAT activity. LCAT activities in 120 healthy subjects were positively correlated with triglyceride ( P  < 0.05), fractional esterification rate of HDL cholesterol (FER HDL ) ( P  < 0.0001), and negatively correlated with apolipoprotein AI (apoAI) ( P  < 0.05) and HDL cholesterol (HDL-C) ( P  < 0.001). These results suggest that this method is sensitive, reproducible, and not greatly influenced by serum components and added substances, and will be a useful tool in the lipid metabolism study and the risk assessment of CVD.