Isolation of High Density Lipoprotein Subclasses by Electrofiltration and Their Chemical Components

Abstract

The exact role of high density lipoprotein in atheroprotection is not well understood yet due to its complex nature; it comprises more than ten subclasses that vary in size, composition, and function. Isolation and characterization of these subclasses is an important step for further studies addressing their functions in health and disease. In this work, we present a novel method that is relatively simple and efficient for isolation of high density lipoprotein subclasses. The method depends on fractional filtration of the subclasses through a preformed gel membrane system under the effect of an electric field, where the stepwise isolation of the subclasses depends on differences in their rates of migration in polyacrylamide gel. Using this design, we were able to isolate seven high density lipoprotein subclasses with relative molecular masses of 42,000–50,000; 71,000; 103,000; 124,000; 150,000; 182,000; and 219,000. All the subclasses contained apolipoprotein A-I, phosphatidylcholine, sphingomyelin, free cholesterol, esterified cholesterol, and triacylglycerols. Some fractions of some samples contained the apolipoproteins A-II, C-I, C-II, C-III, and E. A subclass of molecular mass of 106,000 was identified and isolated from a healthy young subject that contained albumin and apoA-I with some free and esterified cholesterol, but with no triacylglycerols. This electrofiltration technique offers a novel tool for isolating pure native high density lipoprotein subclasses in a concentrated form that can be used directly for detailed studies of their physicochemical and physiological properties.

Keywords:

• ApoA-I-Albumin lipoprotein
• Electroelution
• Electrofiltration
• Electroimmunoblotting
• Electrophoresis
• HDL lipids
• HDL subclasses isolation

ACKNOWLEDGMENT

This work was supported by the Deanship of Scientific Research at Jordan University of Science and Technology, Irbid, Jordan, projects Nos. 4/2002 and 132/2004. Part of this work was done during a Sabbatical leave for R.F. Atmeh (2006/2007).

Manuscript 7581

Notes

^a Detected in all samples.

^b Not detected in all samples.

^c Mean ± SD, n = 10.

^d Detected in some samples but not in others.