Separation of VLDL subfractions by density gradient ultracentrifugation

To assess the presence and composition of very-low-density lipoprotein (VLDL) in various types of hyperlipoproteinemia, a method of density gradient ultracentrifugation has been developed. After 2 hours of density gradient ultracentrifugation, human serum VLDL is separated into two distinct VLDL cholesterol peaks (VLDL1 and VLDL2). The two VLDL subfractions were detected in the serum samples from all subjects in the study, including subjects with normolipidemia (n = 10), familial dysbetalipoproteinemia (n = 12), and type IIa (n = 8), type IIb (n = 12), and type IV/V (n = 10) hyperlipoproteinemia. The cholesterol profiles obtained by the density gradient ultracentrifugation technique resembled the band patterns after electrophoresis of identical serum samples on 2% to 16% nondenaturing polyacrylamide gradient gel: VLDL1 represents relatively large VLDL particles (diameter of about 67 nm) and VLDL2 represents relatively small VLDL particles (diameter of about 38 nm). Recentrifugation of isolated VLDL1 and isolated VLDL2 did not result in any change in their density distribution. In all groups studied, the fluidity of VLDL1 was significantly higher than that of VLDL2, in accordance with the finding that VLDL1 particles were relatively rich in triglycerides and VLDL2 particles were relatively rich in cholesteryl esters. These results indicate that the two VLDL subfractions isolated represent distinct VLDL subclasses. The density gradient ultracentrifugation technique presented in this study allows the rapid isolation and characterization of VLDL subfractions from the serum samples of normolipidemic individuals and patients with hyperlipoproteinemia.