A novel and precise method for simultaneous measurement of serum HDL and LDL subfractions and lipoprotein (a) cholesterol by ultracentrifugation and high-performance liquid chromatography

Abstract

Background: We developed an ultracentrifugation and high-performance liquid chromatography (HPLC) method for simultaneous measurement of cholesterol in serum high density lipoprotein (HDL) and low density lipoprotein (LDL) subfractions and lipoprotein (a) [Lp(a)].

Methods: Serum aliquots of 0.05 ml were centrifuged at background densities of 1.006, 1.044 kg/l, and in the presence of β-mercaptoethanol (ME) at background densities of 1.044, 1.063 and 1.125 kg/l for the separation of lipoprotein subfractions and Lp(a). Cholesterol levels in the ultracentrifugal bottom fractions were analyzed by HPLC.

Results: ME effectively dissociated Lp(a) into apolipoprotein (a) and Lp(a) remnant [Lp(a-)]. Lp(a-) showed a distinctive density distribution from that of the native Lp(a). Based on these data, a method was developed to separate lipoprotein into subfractions and Lp(a) by ultracentrifugation. The separated HDL and LDL subfractions were not contaminated with Lp(a). This method is highly precise with the total CVs for the measurement of HDL2-C, HDL3-C, LDLa-C, LDLb-C and Lp(a)-C 0.85%-2.66%, 0.87%-3.21%, 0.86%-1.11%, 2.59%-6.35% and 4.42%-12.29%, respectively.

Conclusion: A new method for the separation of HDL and LDL subfractions and Lp(a) and simultaneous measurement of cholesterol by ultracentrifugation and HPLC have been established. It is precise and sensitive and can be used in research or clinical laboratories for lipoprotein profiling.