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Cell-free Biochemical Fluorometric Enzymatic Assay for High-throughput Measurement of Lipid Peroxidation in High Density Lipoprotein
Published on: October 12, 2017
Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein:
M Navab1, S Y Hama, G M Anantharamaiah
1Atherosclerosis Research Unit, Division of Cardiology, Department of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
Insights
Normal high-density lipoprotein (HDL) prevents artery wall cells from oxidizing low-density lipoprotein (LDL). However, HDL from some coronary artery disease patients is defective, failing to inhibit LDL oxidation and its harmful effects.
Area of Science:
- Biochemistry
- Cardiovascular Biology
- Lipid Metabolism
Background:
- Artery wall cells can oxidize low-density lipoprotein (LDL).
- Oxidized LDL contributes to cardiovascular disease.
- The role of high-density lipoprotein (HDL) in preventing LDL oxidation by artery wall cells is not fully understood.
Purpose of the Study:
- To investigate the mechanisms of LDL oxidation by human artery wall cells.
- To determine the role of 12-lipoxygenase (12-LO) in LDL oxidation.
- To assess the ability of high-density lipoprotein (HDL) to inhibit LDL oxidation and the biological activity of oxidized phospholipids in patients with coronary artery disease (CAD).
Main Methods:
- Human aortic wall cells were treated with various agents, including apolipoprotein A-I (apoA-I), apoA-I peptide mimetic, HDL, and paraoxonase.
- Cells were transfected with antisense or sense oligonucleotides to 12-lipoxygenase (12-LO).
- The oxidation of phospholipids and cholesteryl linoleate was induced by hydroperoxides.
- The biological activity of oxidized phospholipids was assessed.
- HDL function was tested using LDL oxidation assays and oxidized phospholipid activity assays.
Main Results:
- Apolipoprotein A-I (apoA-I), HDL, and paraoxonase inhibited LDL oxidation by artery wall cells.
- Human aortic wall cells express 12-lipoxygenase (12-LO); inhibiting 12-LO prevented LDL-induced monocyte chemotactic activity.
- Hydroperoxides significantly enhanced the oxidation of phospholipids, forming biologically active oxidized phospholipids.
- Normal HDL inhibited LDL oxidation and the activity of oxidized phospholipids.
- HDL from CAD patients with normal lipid levels failed to inhibit LDL oxidation and oxidized phospholipid activity.
Conclusions:
- Mildly oxidized LDL is formed in a multi-step process involving 12-LO.
- Normal HDL effectively inhibits LDL oxidation and the biological activity of oxidized phospholipids.
- HDL from some coronary artery disease patients is functionally deficient, impairing its ability to prevent LDL oxidation and mitigate the effects of oxidized phospholipids.
Abstract:
Treatment of human artery wall cells with apolipoprotein A-I (apoA-I), but not apoA-II, with an apoA-I peptide mimetic, or with high density lipoprotein (HDL), or paraoxonase, rendered the cells unable to oxidize low density lipoprotein (LDL). Human aortic wall cells were found to contain 12-lipoxygenase (12-LO) protein. Transfection of the cells with antisense to 12-LO (but not sense) eliminated the 12-LO protein and prevented LDL-induced monocyte chemotactic activity. Addition of 13(S)-hydroperoxyoctadecadienoic acid [13(S)-HPODE] and 15(S)-hydroperoxyeicosatetraenoic acid [15(S)-HPETE] dramatically enhanced the nonenzymatic oxidation of both 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and cholesteryl linoleate. On a molar basis 13(S)-HPODE and 15(S)-HPETE were approximately two orders of magnitude greater in potency than hydrogen peroxide in causing the formation of biologically active oxidized phospholipids (m/z 594, 610, and 828) from PAPC. Purified paraoxonase inhibited the biologic activity of these oxidized phospholipids. HDL from 10 of 10 normolipidemic patients with coronary artery disease, who were neither diabetic nor receiving hypolipidemic medications, failed to inhibit LDL oxidation by artery wall cells and failed to inhibit the biologic activity of oxidized PAPC, whereas HDL from 10 of 10 age- and sex-matched control subjects did. We conclude that a) mildly oxidized LDL is formed in three steps, one of which involves 12-LO and each of which can be inhibited by normal HDL, and b) HDL from at least some coronary artery disease patients with normal blood lipid levels is defective both in its ability to prevent LDL oxidation by artery wall cells and in its ability to inhibit the biologic activity of oxidized PAPC.
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