Isolation, characterization, and functional assessment of oxidatively modified subfractions of circulating low-density lipoproteins.
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.


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 approximately 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.
Yang
Chao-yuh
CY
Department of Medicine, Baylor College of Medicine, 6565 Fannin, MS A.601, Houston, Tex 77030, USA. cyang@bcm.tmc.edu
Raya
Joe L
JL
Chen
Hsin-Hung
HH
Chen
Chu-Huang
CH
Abe
Yasunori
Y
Pownall
Henry J
HJ
Taylor
Addison A
AA
Smith
Charles V
CV
D000293:Adolescent; D000328:Adult; D000818:Animals; D001161:Arteriosclerosis / Q000097:blood; D002417:Cattle; D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects; D002648:Child; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004261:DNA Replication / Q000187:drug effects; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D042783:Endothelial Cells / Q000166:cytology; D004730:Endothelium, Vascular / Q000166:cytology; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000097:blood*; D008077:Lipoproteins, LDL / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D008297:Male; D009000:Monocytes / Q000166:cytology; D010084:Oxidation-Reduction; D012212:Rheology
D008077:Lipoproteins, LDL; C099252:oxidized low density lipoprotein; D016222:Fibroblast Growth Factor 2