Separation of the principal HDL subclasses by iodixanol ultracentrifugation

Abstract

HDL subclasses detection, in cardiovascular risk, has been limited due to the time-consuming nature of current techniques. We have developed a time-saving and reliable separation of the principal HDL subclasses employing iodixanol density gradient ultracentrifugation (IxDGUC) combined with digital photography. HDL subclasses were separated in 2.5 h from prestained plasma on a three-step iodixanol gradient. HDL subclass profiles were generated by digital photography and gel scan software. Plasma samples (n = 46) were used to optimize the gradient for the resolution of HDL heterogeneity and to compare profiles generated by IxDGUC with gradient gel electrophoresis (GGE); further characterization from participants (n = 548) with a range of lipid profiles was also performed. HDL subclass profiles generated by IxDGUC were comparable to those separated by GGE as indicated by a significant association between areas under the curve for both HDL 2 and HDL 3 (HDL 2 , r = 0.896, P < 0.01; HDL 3 , r = 0.894, P < 0.01). The method was highly reproducible, with intra- and interassay coefficient of variation percentage < 5 for percentage area under the curve HDL 2 and HDL 3 , and < 1% for peak Rf and peak density. The method provides time-saving and cost-effective detection and preparation of the principal HDL subclasses. Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc.