A simple and precise method to detect sterol esterification activity of lecithin/cholesterol acyltransferase by high-performance liquid chromatography
The measurement of lecithin: cholesterol acyltransferase (LCAT, EC 22.214.171.124) 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 (FERHDL) (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.
KeywordsHigh-density lipoprotein Lecithin: cholesterol acyltransferase Enzyme activity Sterol esterification Chromatography Cardiovascular disease
Cholesterol esterification rate
Fractional esterification rate of HDL cholesterol
Reverse cholesterol transport
This study was supported by research grants from the National Natural Science Foundation of China (81472035, 81171647, 81501842).
Compliance with ethical standards
This study was reviewed and approved by the Beijing Hospital Ethics Committee. All studied individuals were informed in writing of the intended use of their samples and each provided written consent.
Conflict of interest
No authors declared any potential conflicts of interest. The funding organizations played no role in the design of the study, review and interpretation of data, or preparation or approval of the manuscript.
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