A cell-free assay for detecting HDL that is dysfunctional in preventing the formation of or inactivating oxidized phospholipids.
We have developed a novel and rapid cell-free assay of the ability of HDL to prevent the formation of or inactivate oxidized phospholipids. HDL was tested for its ability to inhibit the oxidation of LDL, or inhibit the oxidation of l-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) by hydroperoxyoctadecadienoic acid (HPODE), or inactivate oxidized PAPC (Ox-PAPC). In each case the fluorescent signal generated in the presence of the test substances and the test HDL was determined. As little as 2.5 microg of normal human HDL cholesterol significantly inhibited the fluorescent signal generated by Ox-PAPC; results did not differ regardless of whether the HDL was prepared by gel electrophoresis, fast protein liquid chromatography, or dextran sulfate precipitation. HDL from each of 27 patients with coronary atherosclerosis failed to inhibit the fluorescent signal generated by a control LDL, whereas HDL from each of 31 matched normal subjects with the same levels of HDL cholesterol significantly inhibited the signal. Results from an established cell-based assay (Navab, M., S. Hama, J. Cooke, G. M. Anantharamaiah, M. Chaddha, L. Jin, G. Subbanagounder, K. F. Faull, S. T. Reddy, N. E. Miller, and A. M. Fogelman. 2000. J. Lipid Res. 41: 1481-1494) were identical. HDL from the patients also failed to inhibit the fluorescent signal generated from PAPC plus HPODE (10 of 10 patients) whereas HDL from matched controls (8 of 8 patients) significantly inhibited the fluorescent signal. We conclude that this new assay has the potential to allow widespread testing of the hypothesis that HDL that is dysfunctional in preventing the formation or inactivating oxidized phospholipids may play an important role in the development of atherosclerosis.
Navab
M
M
Atherosclerosis Research Unit, Division of Cardiology, Department of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA. mnavab@mednet.ucla.edu
Hama
S Y
SY
Hough
G P
GP
Subbanagounder
G
G
Reddy
S T
ST
Fogelman
A M
AM
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001011:Aorta; D002474:Cell-Free System; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D051152:Clusterin; D018920:Coculture Techniques; D003324:Coronary Artery Disease / Q000097:blood; D004730:Endothelium, Vascular; D005260:Female; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006023:Glycoproteins / Q000494:pharmacology; D006801:Humans; D008041:Linoleic Acids / Q000494:pharmacology; D015227:Lipid Peroxidation / Q000187:drug effects; D008054:Lipid Peroxides / Q000037:antagonists & inhibitors*; D008075:Lipoproteins, HDL / Q000097:blood* / Q000494:pharmacology*; D008077:Lipoproteins, LDL / Q000097:blood; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D018832:Molecular Chaperones / Q000494:pharmacology; D009000:Monocytes / Q000502:physiology; D009131:Muscle, Smooth, Vascular; D010084:Oxidation-Reduction; D010742:Phospholipid Ethers / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence
C400184:1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine; D000975:Antioxidants; C495242:CLU protein, human; C495243:Clu protein, mouse; D051152:Clusterin; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006023:Glycoproteins; D008041:Linoleic Acids; D008054:Lipid Peroxides; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D018832:Molecular Chaperones; D010742:Phospholipid Ethers; C037631:2',7'-dichlorofluorescein