Oxygen-mediated heterogeneity of apo-low-density lipoprotein.

Abstract

Mild oxidation of human serum low-density lipoprotein (LDL) converts the apoprotein from a nearly homogeneous component of high apparent molecular weight to a mixture of apparently lower mo ecular weight polypeptide components, as characterized by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis. This protein alteration, which correlates temporally with increases in the formation of lipid oxidation products and in the fluorescence of the apoprotein, is markedly reduced when oxygen is excluded or when EDTA or the free-radical-scavenging antioxidants, butylated hydroxytoluene or propyl gallate, are added. The conversion thus appears to be due to a reaction between the protein moiety and auto-oxidizing lipid. The presence of the antibacterial agent sodium azide markedly accelerates the oxidation, suggesting that it should only be used with caution in lipid-containing solutions. Structural and functional characterization of serum lipoproteins has received attention because molecular or metabolic abnormalities associated with this class of molecules might be significant in the development of atherosclerosis (1). The lowdensity lipoprotein fraction (LDL) has been of particular interest because of its apparent role in atherogenesis. Such a role is indicated by a correlation between high serum levels of LDL and clinical atherosclerosis (2), as well as the accelerated atherosclerosis in patients who have an inherited abnormality in LDL catabolism (familial hypercholesterolemia) (3). LDL is catabolized after it is bound through its protein moiety (apo-B or apo-LDL) to a cell-surface receptor (4).Although characterization of the apoprotein of LDL has been difficult (5), studies on the lipid moiety appear to be less controversial. LDL contains a large proportion of unsaturated lipids (somewhat variable on diet) (6) and is unique among the serum lipoproteins in its susceptibility for undergoing auto-oxidation in vitro (7-9). This finding has led some authors to speculate that such auto-oxidation plays a role in atherogenesis (7,10,11). Although structural (7-13), spectrophotometric (9,13,14), and lipid compositional (8, 11) changes in LDL have been observed upon oxidation, its polypeptide chain has not been characterized. Model studies with lipid-protein mixtures have shown that oxidized lipid can lead to major modification of protein components; such modifications include crosslinking (15), polypeptide scission (16), and loss of amino acids (17, 18). It thus seems possible that some of the difficulties encountered in characterizing apo-B are associated with partial oxidation of LDL during purification and/or storage. The observation that plasma lipids from human subjects contain significant diene conjugation (an early manifestation of lipid auto-oxidation) suggests that oxidation of LDL can be initiated in vivo (19).We show here that mild oxidation of LDL in vitro results in altering the sodium dodecyl sulfate (NaDodSO4)-apoprotein electrophoretic pattern from a single major band of low mobility into nume...

Lipoprotein oxidation and lipoprotein-induced cytotoxicity.

“…Our measure of lipoprotein oxidation is in terms of the equivalents of 215 216 ARTERIOSCLEROSIS VOL 3, No 3, MAY/JUNE 1983 malondialdehyde (MDA) as measured by reactivity to thiobarbituric acid. 16 Our results show that lipoprotein toxicity strongly correlates with the presence of thiobarbituric acid-reacting substances (TBARS). Although MDA has been shown to be injurious to mammalian cells in culture 17 " 19 and to be carcinogenic in experimental animals, 20 it appears from our results that MDA, itself, is not the agent responsible for the cytoxic effects induced by oxidized LDL.…”

Native and Oxidized Low Density Lipoprotein Induction of Tissue Factor Gene Expression in Smooth Muscle Cells Is Mediated by Both Egr-1 and Sp1

“…The range of the endotoxin level of all preparations was 0.000 to 0.013 pg/g. Electrophoretic mobility (Corning) and thiobarbituric acid reactivity (25,26) were tested in all lipoprotein preparations. Thiobarbituric acid reactivities for LDL were 0.1 to 0.9 nmol of malondialdehyde (as standard)/mg LDL cholesterol, and for oxLDL were 3.9 to 8.1.…”

Endothelial and smooth muscle cells alter low density lipoprotein in vitro by free radical oxidation.

“…A modification of the spectrophotometric assay described by Schuh et al 20 was used to determine the malondialdehyde-like content of the lipoprotein samples and supernatants. Lipoprotein samples were assayed by adding 1 ml of 20% trichloroacetic acid (TCA) to 100 y.\ of lipoprotein of known protein, cholesterol, and triglyceride content.…”

“…Bovine aortic EC were subcultured in 5% bovine plasma-derived serum in Dulbecco-Vogt modified Eagle's medium (Dulbecco-Vogt medium) supplemented with sodium bicarbonate, penicillin, streptomycin, nonessential amino acids, and sodium pyruvate as described by Glenn et a\. u Bovine aortic EC were subcultured in 75 cm 2 flasks (Falcon, Cockeysville, Maryland) with trypsin at a split ratio of 1:3 and used in this study in passage [15][16][17][18][19][20]. Human umbilical vein EC were isolated by published methods 15 and used for experiments when primary cultures were confluent (5 days).…”