title: 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 HDL2 and HDL3 (HDL2, r = 0.896, P < 0.01; HDL3, 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 HDL2 and HDL3, and < 1% for peak Rf and peak density. The method provides time-saving and cost-effective detection and preparation of the principal HDL subclasses.
authors.affiliation: Healing Foundation Cleft and Craniofacial Clinical Research Centre, University of Manchester, Manchester M13 9WL, United Kingdom.
authors.forename: Nicola L
authors.initials: NL
authors.lastname: Harman
authors.affiliation: Department of Nutritional Science, Faculty of Health and Medical Sciences, University of Surrey, Guildford GU2 7XH, United Kingdom; and.
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Griffin
authors.affiliation: Faculty of Education, Community and Leisure, Liverpool John Moores University, Liverpool L17 6BD, United Kingdom. Electronic address: i.g.davies@ljmu.ac.uk.
authors.forename: Ian G
authors.initials: IG
authors.lastname: Davies
mesh_terms: D008075:Lipoproteins, HDL / Q000145:classification* / Q000302:isolation & purification*; D014283:Triiodobenzoic Acids / Q000737:chemistry*; D014461:Ultracentrifugation
chemical_list: D008075:Lipoproteins, HDL; D014283:Triiodobenzoic Acids; C044834:iodixanol
keywords: HDL subfractions; HDL2; HDL3; LDL subclasses; atherogenic lipoprotein phenotype; cardiovascular disease; high density lipoprotein; plasma lipoproteins; plasma triacylglycerol; self-generating gradients