Determination of serum high- and low-density lipoprotein cholesterol by ultracentrifugation-high performance liquid chromatography--improvement and verification of β-quantification method

Abstract

Objective To propose and validate a reduced volume β-quantification method to measure serum high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C). Methods The reduced volume β-quantification method involved separation of LDL and HDL by ultracentrifugation and preparation of HDL by chemical precipitation. The sampling and reconstitution of the bottom fractions were performed gravimetrically and sample volume was thus decreased from 5 to 0.8 ml. High performance liquid chromatography was used to determine the cholesterol concentration of bottom fractions and HDL-C in the supernatant. Serum levels of LDL-C depended on a calculation of bottom fractions cholesterol minus HDL-C. Results The total CVs for HDL-C and LDL-C were 0.65%-1.75% and 0.63%-1.11%. The results of the developed method were consistent with the current reference method and well within the allowable bias for Cholesterol Reference Method Laboratory Network surveys. Conclusion A new method for the measurement of HDL-C and LDL-C has been established. This method requires a small amount of serum and is easy to operate, exhibiting a desirable precision and accuracy. It is reliable and can be used as a candidate reference method for HDL-C and LDL-C. Key words: Cholesterol,HDL; Cholesterol,LDL; Ultracentrifugation; Reference standards