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A simple and precise method for direct measurement of fractional esterification rate of high density lipoprotein cholesterol by high performance liquid chromatography

Abstract Background: The relationship between fractional cholesterol esterification rate in plasma or serum high-density lipoprotein (HDL) (FERHDL) and lipoprotein subfractions and other cardiovascular disease (CVD) risk factors has been demonstrated. However, the current method for measuring FERHDL requires fresh serum samples and radioactive labeling of the samples, making it impractical for use in clinical laboratories. In this study, we developed a simple and precise HPLC method for the measurement of FERHDL. Correlations between FERHDL and CVD risk factors were evaluated in 119 healthy volunteers. Methods: Fasting blood samples were collected and serum was isolated within 2 h. Serum HDL was prepared by precipitation of apolipoprotein B (apoB)-containing lipoproteins with dextran sulfate and magnesium chloride. HDL fractions were divided into two aliquots and incubated at 0°C and 37°C, respectively, for 1 h. Free cholesterol in the HDL fractions was analyzed by HPLC. FERHDL was calculated as the percent decrease of free cholesterol during incubation. Results: The esterification reaction of HDL free cholesterol was not linear, but the measured FERHDL was stable when serum samples were stored at room temperature for <4 h, or at 4°C for <24 h. The intra-assay and total CVs for FERHDL measurements were 1.0%–2.1% and 1.6%–3.8%, respectively. Results of 119 healthy volunteers showed that FERHDL was positively correlated with age, BMI, blood pressure, total cholesterol (TC), triglyceride (TG) and small dense low-density lipoprotein-cholesterol (LDLb-C), and negatively correlated with HDL-C and HDL2-C. FERHDL has shown to be a predictor of HDL and LDL subfraction distributions. Conclusions: This method is simple, non-radioactive and precise and will be useful in prediction of lipoprotein subfraction distributions and in clinical assessment of CVD risks.