Gerhard Ledinski

Delay of copper-catalyzed oxidation of low density lipoprotein by in vitro enrichment with choline or ethanolamine plasmalogens

Low density lipoprotein (LDL) isolated from human serum of different donors was enriched with plasmalogens and their diacyl analogs in order to investigate a possible effect of these phospholipids on the rate of lipid peroxidation in this lipoprotein. LDL was incubated with either vesicles of choline plasmalogen or phosphatidylcholine in presence of lipoprotein- deficient serum, or with liposomes of ethanolamine plasmalogen or phosphatidylethanolamine together with the non-specific phospholipid transfer protein isolated from beef liver. After re-isolation of LDL by ultracentrifugation, a dose-dependent incorporation of the exogenous phospholipids was obtained. Enrichment of LDL with choline plasmalogen resulted in a delay of the copper-catalyzed oxidation of LDL from five different donors. LDL from two donors was also enriched with diacylglycerophosphocholine which led to a delay of oxidation, but the protective effect was smaller than with choline plasmalogen. Enrichment of LDL from two additional donors with ethanolamine plasmalogen resulted in the strongest protection against oxidation, whereas, diacylglycerophospho-ethanolamine had little effect. The delay of the copper-catalyzed LDL oxidation may be due to a direct antioxidative action of the plasmalogens, which are partially degraded during the lag phase of oxidation, or to an indirect effect caused by alteration of the LDL surface in the presence of an excess of glycerophospholipids.

Neopterin and 7,8-dihydroneopterin interfere with low density lipoprotein oxidation mediated by peroxynitrite and/or copper.

Low density lipoprotein (LDL) oxidation within the artery wall likely represents a key event in the formation of atherosclerotic lesions. Oxidatively modified LDL particles exert chemotactic properties on macrophages, and the uncontrolled uptake of modified LDL by macrophages leads to the formation of lipid-loaded foam cells, a hallmark of early stage atherosclerosis. Human macrophages stimulated by interferon- n generate reactive oxygen species (ROS), neopterin, and 7,8-dihydroneopterin. Higher concentrations of neopterin were found in atherosclerosis, and earlier studies have provided evidence that these neopterin derivatives are able to interfere with reactive species. We therefore investigated whether they also modulate LDL oxidation mediated by Cu(II) and/or peroxynitrite (ONOO m ). By means of UV-absorption recording the formation of conjugated dienes in the course of lipid oxidation as well as by measuring the relative electrophoretic mobility of oxidized LDL, we found that neopterin is capable of enhancing ONOO m - as well as Cu(II)-mediated LDL oxidation, whereas 7,8-dihydroneopterin mainly protects LDL from oxidation. However, in case of Cu(II)-mediated LDL oxidation, an initial prooxidative effect of 7,8-dihydroneopterin could be observed. We hypothesize that 7,8-dihydroneopterin may chemically reduce Cu(II) to Cu(I) thereby increasing its oxidative capacity. After total reduction of Cu(II), excess 7,8-dihydroneopterin may block the oxidative potential of Cu(I) and thus decrease the oxidation of LDL. These findings confirm the general behavior of pteridines in redox processes and suggest an in vivo contribution to the process of LDL oxidation.

Comparison of laboratory parameters as risk factors for the presence and the extent of coronary or carotid atherosclerosis : the significance of apolipoprotein B to apolipoprotein all ratio

Abstract We compared several “new” risk factors (autoantibodies to oxidatively modified low density lipoprotein (LDL), sialic acid content of LDL, bilirubin and C-reactive protein) with “conventional” risk factors (apolipoprotein (apo) AI, AII and B, lipoprotein(a), triglycerides, and total, LDL and high density lipoprotein (HDL) cholesterol) for the presence and the extent of coronary or carotid atherosclerosis. Forty male patients with angiographically proven coronary atherosclerosis and 31 male patients with ultrasound-proven extracranial carotid atherosclerosis were compared to 40 age matched (53 ± 5 years) healthy males as control subjects, with negative parental history of atherosclerosis, no clinical signs of systemic or organ-related ischemic disease and normal extracranial carotid arteries. The apo B/apo AII ratio most powerfully indicated the presence and the extent of coronary or carotid atherosclerosis. Elevated lipoprotein(a) contributed significant additional information in the assessment of the atherosclerotic risk. Increase in Creactive protein indicated the presence (but not the extent) of coronary or carotid atherosclerosis with a similar power as lipoprotein(a). Decreased values of total bilirubin indicated the presence of atherosclerosis only in smokers. Autoantibodies to oxidatively modified LDL additionally described the atherosclerotic process, but were less important than apolipoproteins, lipoprotein(a), C-reactive protein or bilirubin. Sialic acid content of LDL added no information to the parameters discussed above. We demonstrated that in male patients apolipoproteins, especially the apo B/apo AII ratio, were better indicators of the presence and the extent of coronary or carotid atherosclerosis than C-reactive protein, bilirubin, autoantibodies to oxidatively modified LDL or sialic acid content of LDL.

Binding and uptake of differently oxidized low density lipoprotein in mouse peritoneal macrophages and THP-1 macrophages: involvement of negative charges as well as oxidation-specific epitopes.

Oxidatively modified low‐density lipoprotein (LDL) has been found in vivo, and oxidized LDL (oxLDL) could bind to scavenger receptors, leading to foam cell formation. Macrophages bear a number of different scavenger receptors for oxLDL, and macrophages of different origins may have a different scavenger receptor repertoire. In addition, LDL oxidized to different degrees may differ in the ability to bind macrophage scavenger receptors. In this study, we characterized the patterns of the binding and uptake of differently oxidized LDL in mouse peritoneal macrophages (MPM) and human THP‐1 macrophages, and the influence of negative charge and oxidation‐specific epitopes in oxLDL on these processes. Thresholds of increased binding and uptake in MPM were found when LDL was oxidized to the degrees with a relative electrophoretic mobility (REM) of 2.6 (minor threshold) and 3.0 (major threshold), corresponding to 49 and 57%, respectively, of the loss of free amino groups in these oxLDL. There was no threshold for the binding of oxLDL to THP‐1 macrophages, while for uptake, a major threshold with REM of 3.0 (57% free amino groups lost) was found. The presence of the F(ab′)2 fragments of the monoclonal antibody OB/04, which was raised against copper‐oxidized LDL, led to the reduction of the binding and uptake, respectively, of Eu3+‐oxLDL (REM:3.6) in MPM by 31 and 29%, and by 19 and 22% in THP‐1 macrophages. It is concluded that LDL oxidized to different degrees binds differently to macrophages, and the patterns of binding and uptake are different for MPM and human THP‐1 macrophages. Both, the negative charge and the oxidation‐specific epitopes of oxLDL are involved in these processes. J. Cell. Biochem. 81:557–569, 2001.

Relationship between the sialic acid content of low-density lipoprotein (LDL) and autoantibodies to oxidized LDL in the plasma of healthy subjects and patients with atherosclerosis.

Abstract To determine whether the sialic acid (SA) content of the low-density lipoprotein (LDL) is related to the plasma concentration of autoantibodies to oxidized LDL (oxLDL), we measured the SA content of LDL and the concentrations of oxLDL and autoantibodies to oxLDL in plasma of 20 apparently healthy subjects and 20 patients with advanced coronary atherosclerosis. In the healthy subjects the SA content of LDL correlated positively with plasma concentration of autoantibodies to oxLDL. In agreement with the literature the decreased SA content of LDL was associated with an increased fraction of oxLDL; a decreased fraction of oxLDL was associated with an increased plasma concentration of autoantibodies to oxLDL. In the patients the SA content of LDL and plasma concentrations of oxLDL and autoantibodies to oxLDL were not related. We conclude that the SA content of LDL correlates positively with plasma concentration of autoantibodies to oxLDL in healthy subjects. However, this association may vary depending on the stage of atherogenesis. Although our results suggest dependence of LDL SA content on the clearance of oxidatively modified (desialylated and oxidized) LDL from blood by autoantibodies to oxLDL, the mechanisms regulating the SA content of LDL await further studies.

Presence of aldehydic epitopes on a minor low-density lipoprotein fraction.

A more negatively charged low-density lipoprotein (LDL), named minor LDL (mi-LDL), was separated by ionic exchange chromatography and further characterized. This mi-LDL contained lower amounts of polyunsaturated fatty acids, alpha- or gamma- tocopherol, but higher amounts of lipid hydroperoxides than the major LDL fraction (ma-LDL). We show here for the first time that apoB of mi-LDL is modified by lipid peroxidation products, such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). Using polyclonal antibodies, generated against 4-HNE- or MDA-LDL and apoB, the ratio of 4-HNE- or MDA-derived epitopes to apoB of mi-LDL and ma-LDL was estimated by means of a solid phase fluorescence immunoassay. The ratio of 4-HNE-derived epitopes to apoB on mi-LDL was fourfold higher, while the ratio of MDA-derived epitopes to apoB was twofold higher, compared with the ratios obtained with ma-LDL. In a competition assay with mi- and ma-LDL, only mi-LDL was an effective competitor to inhibit the immunoreaction of anti-4-HNE-LDL with 4-HNE-LDL (by 24%) and of anti-MDA-LDL with MDA-LDL (by 10%).

Low-density lipoprotein modification by normal, myeloperoxidase-deficient and NADPH oxidase-deficient granulocytes and the impact of redox active transition metal ions

Abstract The modification of low-density lipoprotein (LDL) by normal, myeloperoxidase (MPO)-deficient and NADPH oxidase-deficient granulocytes was investigated using the monoclonal antibody (mAb) OB/04, which was originally generated against copper-oxidized LDL. Incubation of LDL with normal granulocytes increased the reactivity of LDL with mAb OB/04. These effects were even more pronounced using MPO-deficient granulocytes. Inhibitors of oxidative reactions (the NADPH oxidase inhibitor diphenyleneiodonium chloride [DPI], catalase, superoxide dismutase [SOD]) did not significantly reduce LDL oxidation by normal granulocytes. Furthermore, granulocytes of a patient with NADPH oxidase deficiency were almost equally effective as normal granulocytes, indicating that oxidative burst-derived reactive oxygen species are of only minor importance in the generation of mAb OB/04-detectable new epitopes on LDL in vitro. In contrast, incubation of LDL with iron and copper prior to and during incubation with normal granulocytes markedly enhanced the generation of OB/04-detectable epitopes. It is supposed that, besides superoxide (in normal and MPO-deficient granulocytes) or instead of superoxide (in NADPH oxidase-deficient granulocytes), lytic enzymes released by activated granulocytes may enhance the availability of transition metals for oxidation of LDL. Our results support the concept that transition-metal-dependent pathways of LDL oxidation in combination with degranulation products of granulocytes are important.