J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
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Originally published In Press as doi:10.1194/jlr.D700044-JLR200 on March 12, 2008

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Journal of Lipid Research, Vol. 49, 1364-1371, June 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Methods

Lipoprotein separation in a novel iodixanol density gradient, for composition, density, and phenotype analysis

Michael S. Yee1, Darrell V. Pavitt, Tira Tan, Soundararajan Venkatesan, Ian F. Godsland, William Richmond and Desmond G. Johnston

Section of Endocrinology and Metabolic Medicine, Faculty of Medicine, Imperial College London, St Mary's Hospital, London W2 1NY, United Kingdom

This research was supported by a grant from GlaxoSmithKline.

Published, JLR Papers in Press, March 12, 2008.

1 To whom correspondence should be addressed. e-mail: m.yee{at}imperial.ac.uk

Separation of lipoproteins by traditional sequential salt density floatation is a prolonged process (~72 h) with variable recovery, whereas iodixanol-based, self-generating density gradients provide a rapid (~4 h) alternative. A novel, three-layered iodixanol gradient was evaluated for its ability to separate lipoprotein fractions in 63 subjects with varying degrees of dyslipidemia. Lipoprotein cholesterol, triglycerides, and apolipoproteins were measured in 21 successive iodixanol density fractions. Iodixanol fractionation was compared with sequential floatation ultracentrifugation. Iodixanol gradient formation showed a coefficient of variation of 0.29% and total lipid recovery from the gradient of 95.4% for cholesterol and 84.7% for triglyceride. Recoveries for VLDL-, LDL-, and HDL-cholesterol, triglycerides, and apolipoproteins were approximately 10% higher with iodixanol compared with sequential floatation. The iodixanol gradient effectively discriminated classic lipoproteins and their subfractions, and there was evidence for improved resolution of lipoproteins with the iodixanol gradient. LDL particles subfractionated by the gradient showed good correlation between density and particle size with small, dense LDL (<25.5 nm) separated in fractions with density >1.028 g/dl. The new iodixanol density gradient enabled rapid separation with improved resolution and recovery of all lipoproteins and their subfractions, providing important information with regard to LDL phenotype from a single centrifugation step with minimal in-vitro modification of lipoproteins.

Supplementary key words ultracentrifugation • apolipoprotein B • very low density lipoprotein • low density lipoprotein • high density lipoprotein • lipoprotein (a)

Abbreviations: ApoA-I, apolipoprotein A-I; HMW, high molecular weight; IDGU, iodixanol density gradient ultracentrifugation; SFU, sequential floatation ultracentrifugation; TBE, Tris borate EDTA


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