Abstract 288: An Improved Method for the Isolation of HDL from Human Serum

In an effort to reduce the time and labor resources required for the standard HDL isolation protocol using sequential ultracentrifugation, our laboratory has investigated a modified process for isolation of HDL from human serum. Our method employs an alternative step for removing VLDL and LDL from human serum that requires less than one hour and replaces the first 18 hour ultracentrifugation step. This alternative process is based on the well documented ability of polyethylene glycol (PEG) to selectively and irreversibly precipitate apolipoprotein B-containing lipoproteins. Subsequently, both methods then use a 21 hour ultracentrifugation spin to separate HDL from all other non-lipoprotein components of human serum. For this study comparing the methods, 1.5 ml of human serum was used. The human serum starting material had a total cholesterol value of 262 mg/dL with the following lipoprotein cholesterol profile: 178 mg/dL LDL-C (68%), 34 mg/dL VLDL-C (13%) and HDL-C 48 mg/dL (18%). Comparison of the two methods demonstrates similar recovery of HDL-C from human serum, 35% vs 37% for the standard method versus modified method, respectively. The total cholesterol recovered using the standard protocol was slightly higher than the modified method. However, as shown by lipoprotein gel electrophoresis, HDL isolated by the standard method was only 80% pure, with 20% of the cholesterol present as VLDL and LDL contaminants remaining in the preparation. In contrast, using our modified process, isolated HDL was >99% pure with no detectable VLDL or LDL cholesterol present. In summary, our laboratory has developed a methodology for the isolation of HDL from human serum that: 1) requires substantially less time and resources, 2) provides the same recovery of HDL-C from serum as the standard method and 3) provides a higher purity HDL than is achieved from the standard sequential ultracentrifugation method.