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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:1083.)
© 2003 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Isolation, Characterization, and Functional Assessment of Oxidatively Modified Subfractions of Circulating Low-Density Lipoproteins

Chao-yuh Yang; Joe L. Raya; Hsin-Hung Chen; Chu-Huang Chen; Yasunori Abe; Henry J. Pownall; Addison A. Taylor; Charles V. Smith

From the Departments of Medicine (C.y.Y., J.L.R., H.-H.C., C.-H.C., Y.A., H.J.P., A.A.T.) and Biochemistry (C.y.Y.), Baylor College of Medicine, Houston, Tex, and Center for Developmental Pharmacology and Toxicology (C.V.S.), Children’s Research Institute, Ohio State University, Columbus, Ohio.

Correspondence to Chao-yuh Yang, Department of Medicine, Baylor College of Medicine, 6565 Fannin, MS A.601, Houston, TX 77030. E-mail cyang{at}bcm.tmc.edu

Objective— Current evidence suggests that oxidatively modified human plasma low-density lipoproteins (ox-LDLs) are proatherogenic and cytotoxic to endothelial and vascular smooth muscle cells. The present study describes a method using ion-exchange chromatography that is capable of large-scale subfractionation of LDL for adequate analyses of composition or bioactivities.

Methods and Results— LDLs from normolipidemic (N-LDL) and homozygous familial hypercholesterolemic (FH-LDL) subjects were separated into 5 subfractions (L1 through L5) by high-capacity ion-exchange chromatography. The most strongly retained fraction from FH subjects, FH-L5, suppressed DNA synthesis in cultured bovine aortic endothelial cells and stimulated mononuclear cell adhesion to cultured endothelial cells under flow conditions in vitro. L5, which represented 1.1±0.2% and 3.7±1.7% of the LDL from N-LDL and FH-LDL, respectively, was more triglyceride-rich (17% versus 5%) and cholesteryl ester-poor (23% versus 33%) than were L1 through L4. Electrophoretic mobilities on agarose gels increased from L1 to L5. According to SDS-PAGE, apolipoprotein B-100 in N-LDL fractions L1 through L5 appeared as a single 500-kDa band. In contrast, the fractions isolated from FH-LDL showed substantial fragmentation of the apolipoprotein B-100, including bands between 200 and 116 kDa. Competitive ELISA analyses using a malondialdehyde-specific monoclonal antibody against Cu²⁺ ox-LDL suggest that FH-L5 is malondialdehyde-modified.

Conclusions— Relative to N-LDL, FH-LDL contains higher concentrations of a fraction, L5, that exhibits distinctive physicochemical properties and biological activities that may contribute to initiation and progression of atherogenesis in vivo.

Key Words: oxidized LDL • atherosclerosis • hypercholesterolemia • DNA synthesis • adhesion molecule induction

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