Serum lipoprotein subclass and lipoprotein (a) cholesterol determined by ultracentrifugation and HPLC

Objective To develop a reliable method for the measurement of serum high density lipoprotein(HDL)and low density lipoprotein(LDL)subclass and lipoprotein(a)[Lp(a)] cholesterol.Methods Serum aliquots were centrifuged in the presence of proline and in the presence or absence of mercaptoethanol at densities of 1.125,1.063,1.044 and 1.006 g/ml for the separation of lipoprotein fractions.Cholesterol levels in the ultracentrifugation bottom fractions were analyzed by high- performance liquid chromatography(HPLC).HDL subclass(HDL_2 and HDL_3),LDL subclass(LDLa and LDL_b)and Lp(a)cholesterol was calculated by subtractions.Results Mercaptoethanol effectively dissociated Lp(a)into apolipoprotein(a)and LDL-like lipoproteins[Lp(a-)].Lp(a-)showed a distinctive density distribution from that of the native Lp(a).This finding enabled reliable separation of lipoprotein subclasses and Lp(a)by ultracentrifugation.The total CVs for the measurement of HDL_2、HDL_3、 LDL_a、LDL_b and Lp(a)cholesterol were 0.85%-2.66%,0.87%-3.21%,0.86%-1.11%,2.59% -6.35% and 4.42%-12.29%.Conclusions A new method for the measurement of HDL and LDL subclass cholesterol and Lp(a)cholesterol by ultracentrifugation and HPLC has been established.It is simple,reliable and precise and may be used as a candidate reference method.