7 beta-hydroperoxycholest-5-en-3 beta-ol, a component of human atherosclerotic lesions, is the primary cytotoxin of oxidized human low density lipoprotein.

Abstract

Modification of low density lipoprotein (LDL) by free radical oidat renders this molecular complex cytotoxic. Oxidized lipoproteins exist in vivo In atherosclerotic lesions and in the plasma of diabetic i , suggesting that Upoprotein-induced tissue damage may occur in cen diseases. We undertook purifiation and Identicati of the major cytotoxin In oxidized LDL. The lipid extract from oxidized LDL was subjected to multiple HPLC separations, and the fractions were assayed for cytotoxicity. Mass spectrometry and nuclear magnetic resonance Identified the purified toxin as 7p-hydroperoxycholest-5-en-3I3-ol Vascular cells are susceptible to the toxic effects of low density lipoprotein (LDL) or very low density lipoprotein (VLDL) after modification of the lipoproteins by free radical oxidation (1-4). Oxidation of LDL leads to cytotoxin formation whether oxidation is mediated by lipoxygenases (5), metal ions (6), or ultraviolet irradiation (7) in cell-free systems or by the action of free radicals from cultured endothelial cells (8), vascular smooth muscle cells (8), neutrophils (9), or stimulated human monocytes (10). The cytotoxic moiety of oxidized LDL (oxLDL) is extractable with organic solvents (4), and numerous candidate substances could be proposed to explain its toxic action (11).Oxidized lipoproteins occur in vivo in vascular lesions (12)(13)(14) and in plasma of certain diabetic subjects (15). OxLDL has been proposed to play a causal role in atherosclerosis (11,(16)(17)(18) is an important step toward testing evolving theories of atherogenesis that include lipoprotein oxidation (11,(16)(17)(18) and vascular injury (20,21) as putative early events.by dialysis against 2-6 /uM cupric sulfate for various times (23). OxLDL preparations were dialyzed against 0.15 M NaCl/0.5 mM EDTA, pH 8.5, to remove cupric ions. Relative oxidation, measured as thiobarbituric acid reactivity (6,24), was equivalent to 4-6 nmol of malondialdehyde (MDA) per mg of LDL cholesterol.Human foreskin fibroblasts were plated in a 1:1 (vol/vol) mixture of Dulbecco's minimal essential medium and Ham's F-12 medium (DME/F-12) supplemented with 5% (vol/vol) fetal bovine serum (6) Aliquots of5 mg ofnative LDL or oxLDL were lyophilized overnight, and the lipid was extracted with 5 ml of acetone. The mixture was sonicated for 10-20 sec, mixed for 1 min, and allowed to stand for 20 min. After centrifugation at 1000x g for 10-20 min, the residue was reextracted twice, as above. Preliminary experiments revealed that the recovery of the cytotoxic activity by this extraction procedure was equivalent or superior to other means, including chloroform/ methanol extraction ofaqueous lipoprotein solutions. Pooled extracts were dried under nitrogen and redissolved in isopropanol/acetonitrile, 1:1 (vol/vol). After centrifugation for 5 min at 1000 x g, the supernatant was analyzed by reversephase HPLC (Waters 1LBondapak C1s preparative column).The solvent gradient for elution consisted of water/ acetonitrile, 1:1 (vol/vol), which was increased over 5 ...

Lipoprotein-like Phospholipid Particles Inhibit the Smooth Muscle Cell Cytotoxicity of Lysophosphatidycholine and Platelet-Activating Factor

“…28 Incubation of SMCs with 7␤-hydroxycholesterol in concen- trations up to 25 g/mL decreased cell viability by 20.1Ϯ12.3% (PϽ.01), as assessed by the MTT assay. Addition of 50 g/mL apoA-I-containing phospholipid particles and apoA-I Milano -containing phospholipid particles completely inhibited this cytotoxicity.…”

Circulating Oxidized Low Density Lipoprotein Levels

“…10 -16 Furthermore, cytotoxicity of OxLDL from cultured endothelial cells has been clearly demonstrated to be atherogenic. 17,18 In contrast to our increasing knowledge of the atherogenic mechanisms of oxidation of LDL in the arterial wall, the clinical importance of circulating OxLDL is poorly understood, mainly because of a lack of a sensitive method to specifically detect circulating OxLDL levels. Although a number of investigators have measured thiobarbituric acidreactive substances in plasma, such as lipid hydroperoxides and isoprostanes, 19,20 none have directly measured the formation of oxidatively modified LDL.…”

Involvement of Oxysterols and Lysophosphatidylcholine in the Oxidized LDL–Induced Impairment of Serum Albumin Synthesis by HEPG2 Cells

“…Cytotoxicity and chemotactic activity have been reported for oxysterols, LPC, and 4-hydroxynonenal. 14,40,41 Not surprisingly, many of the various proatherogenic and cell-injuring capacities of Ox-LDL have been attributed to a vast array of these products.…”