Biofactors - Volume 6, issue 2

Authors: Flohé, Leopold | Schaur, Jörg | Poli, Giuseppe

Article Type: Other

Citation: Biofactors, vol. 6, no. 2, pp. 89-89, 1997

Authors: Bellomo, Giorgio | Maggi, Elena | Palladini, Giuseppina | Perugini, Cristina | Seccia, Milfred

Article Type: Research Article

Citation: Biofactors, vol. 6, no. 2, pp. 91-98, 1997

Authors: Kontush, Anatol | Spranger, Torsten | Reich, Axel | Djahansouzi, Sirus | Karten, Barbara | Braesen, Jan Hinrich | Finckh, Barbara | Kohlschütter, Alfried | Beisiegel, Ulrike

Article Type: Research Article

Abstract: Lipoprotein oxidation induced in vitro in whole plasma is expected to be a more relevant model of the lipoprotein oxidation in the arterial wall than the in vitro oxidation of single isolated lipoproteins, e.g., low density lipoprotein (LDL). However, it is unclear, whether the oxidizability of whole plasma may serve as an adequate measure of the oxidizability of plasma lipoproteins. We measured the oxidizability of whole plasma diluted 150‐fold as an absorbance increase at 234 nm known to reflect the level of conjugated dienes in the samples. Plasma oxidation was induced by Cu(II), 2,2' ‐azobis‐(2‐amidinopropane) hydrochloride (AAPH), lipoxygenase or …myeloperoxidase{}+{} H_{2} O_{2} . Oxidizability of human plasma measured in the presence of Cu(II) was found to correlate with the oxidizability of LDL measured in the common Cu(II)‐based LDL oxidation assay. The plasma oxidizability also correlated positively with plasma oxidizable fatty acid and negatively with plasma antioxidant content. Supplementation of human plasma with different antioxidants (albumin, urate, ascorbate, bilirubin, \alpha ‐tocopherol and ubiquinol‐10) in vitro decreased its oxidizability. Supplementation of Watanabe heritable hyperlipidaemic rabbits with different antioxidants (vitamin E, ubiquinone‐10, probucol, carvedilol) in vivo lowered the oxidizability of rabbit plasma in comparison with rabbits fed standard diet. When plasma from hyperlipidaemic patients with or without coronary heart disease and from age‐matched healthy controls was studied, the plasma oxidizability was found to be highest in the patients with coronary heart disease and lowest in the controls. Taken together, these data indicate that the plasma oxidation assay (i) provides information similar to that obtained using the common LDL oxidation assay, (ii) upgrades the latter, taking into account the effect of hydrophilic antioxidants on lipoprotein oxidation and characterizing the oxidizability of all plasma lipoproteins, and (iii) offers important practical advantages, such as fast and simple sample processing, low amount of plasma required and avoidance of artefactual oxidation during lipoprotein isolation. We propose the measurement of plasma oxidizability at 234 nm as an adequate practical index of the oxidizability of plasma lipoproteins. Show more

Keywords: Blood plasma, low density lipoprotein, antioxidants, lipid peroxidation, atherosclerosis, hyperlipidaemia, Watanabe heritable hyperlipidaemic rabbits

Citation: Biofactors, vol. 6, no. 2, pp. 99-109, 1997

Authors: Menzel, E.J. | Sobal, G. | Staudinger, A.

Article Type: Research Article

Abstract: Advanced glycation is a major pathway for the posttranslational modification of plasma and tissue proteins. The initiating reaction is the nonenzymatic addition of sugars such as glucose to the primary amino groups of proteins, i.e., mainly to lysine residues. These “early” Schiff base and Amadori products then undergo a series of inter‐ and intramolecular rearrangements to produce the “late” products termed advanced glycation end products (AGEs). Incubation of LDL with glucose or glucose‐6‐phosphate produces AGE moieties on both the lipid and apolipoprotein B components. In addition, we tried to generate AGE‐LDL by reaction with AGE‐peptides (<10 kD) obtained …by enzymatic digestion of long‐term glycated fibronectin as a model for connective tissue AGE‐peptides. AGE‐formation can be assessed by monitoring of fluorescence (370/440 nm) which is easily differentiated from the much lower autofluorescence of oxidized low density lipoproteins (oxLDL). Alternatively, AGE formation was detected by an AGE‐specific ELISA using antibodies elicited in rabbits against bovine AGE‐RNAse. In the present study we investigated the influence of oxidative stress on the long‐term glycation of LDL and the modulation of LDL‐oxidation by AGE‐modification. We observed (a) that the rate of AGE formation is reduced by BHT/EDTA both on LDL and serum albumin (glycation vs.glycoxidation), (b) long‐term glycated LDL is more readily oxidized than unglycated LDL, (c) oxLDL is more prone to AGE‐modification, (d) AGE‐modification of LDL strongly alters its epitope spectrum and (e) that aminoguanidine at higher concentrations (1–10 mM) inhibits copper‐catalyzed LDL oxidation in the way of a classical antioxidant. Show more

Keywords: LDL oxidation, advanced glycation, diabetes

Citation: Biofactors, vol. 6, no. 2, pp. 111-124, 1997

Authors: Tatzber, F. | Wonisch, W. | Schmidl, E. | Esterbauer, H.

Article Type: Research Article

Abstract: It is generally accepted, that lipid peroxidation plays a pathogenic role in atherosclerosis. Furthermore, recent studies indicate that antibodies directed against oxidative modifications of Low Density Lipoprotein (oLAb) contribute to atherosclerotic processes and may have some function in other disorders. These antibodies have been determined predominantly in humans, because assays for oLAb measurement use species specific anti IgG conjugates. From such assay designs it is not possible to get directly comparable data from various animal species. Main advantages of comparable data between animal species are that results of animal experiments can be interpreted using human calibrators and that results of …immunisations and production of monoclonal antibodies are directly comparable not only within, but also between animal species. The aim of this study was to find a modification for ELISAs for oLAb determination, which allows to measure sera of various animal species simultaneously. Microtitration plates were coated with oxidised LDL and blocked with bovine serum albumine. Human and animal sera were then pipetted into the plate in logarithmic serial dilutions and incubated for 2 h at 37^\circ C. After washing, a protein A horse‐radish peroxidase conjugate (Biomakor, Israel) was added to each well in a dilution of 1\, :\, 20,000. The incubation conditions had to be optimized to achieve reliable results. After another washing step, the assay was developed with TMB. Absorptions were read at 450 nm in a microplate photometer. Following the manufacturers incubation instructions, which recommended a duration of 1 h at room temperature, the system did not work optimally. No binding of protein A to IgG molecules bound to oxidised LDL could be observed, if the system was incubated at 37^\circ C. In our hands, best results were achieved for several animal species, if the conjugate was incubated for two hours at 2–4^\circ C in a refrigerator. Under these conditions, assay sensitivity was the same as in the standard method, which uses anti‐species IgG conjugates. The protein A modification of oLAb allows direct reading of animal oLAb titres from human calibrators. With this method, results of animal experiments can be interpreted on the basis of the situation in humans. Preliminary results obtained show that immunisation experiments with oxidised LDL give serum titres in animals, which are in the same order of magnitude as human sera with high oLAb concentrations. The results of this study, in accordance with findings of other authors, give further indications that atherosclerotic processes are influenced by the specific immune system. Show more

Keywords: Oxidized LDL, antibodies, lipid peroxidation, animals, nutrition, protein A

Citation: Biofactors, vol. 6, no. 2, pp. 125-130, 1997

Authors: Chen, Qi | Wei, Enhui | Chen, Xiuying | Wang, Nan | Jürgens, Günther

Article Type: Research Article

Abstract: In order to investigate the influence of collagen on the interactions between macrophages and oxidatively modified low density lipoprotein (ox‐LDL), type I collagen was isolated from rat tail tendon and prepared as a gel. The binding of ^{125} I‐ox‐LDL, ^{125} I‐malondialdehyde (MDA)‐LDL and ^{125} I‐acetyl‐LDL to collagen was higher but the binding of ^{125} I‐4‐hydroxynonenal (HNE)‐LDL was lower than that of native ^{125} I‐LDL. When mouse peritoneal macrophages were cultivated on this collagen gel, most of the modified LDL was bound to the collagen gel rather than taken up by macrophages. The amount of modified …^{125} I‐LDL degraded by the macrophages decreased in the presence of the collagen gel. In the absence of gel a similar degree of reduction in degradation of modified ^{125} I‐LDL by macrophages was obtained when the cells were treated with cytochalasin D, an inhibitor of non‐specific phagocytosis. However, the treatment of the macrophages cultivated on the collagen gel with cytochalasin D did not influence the degradation of ^{125} I‐ox‐LDL and ^{125} I‐HNE‐LDL. These results suggest that the uptake of ox‐LDL by macrophages grown on collagen gels is primarily mediated via the scavenger receptors pathway, whereas in the absence of collagen also other mechanisms of uptake are operating. Show more

Keywords: Collagen, macrophages, oxidized low density lipoprotein

Citation: Biofactors, vol. 6, no. 2, pp. 131-138, 1997

Authors: Tertov, Vladimir V. | Kaplun, Victor V. | Orekhov, Alexander N.

Article Type: Research Article

Abstract: We have recently found that adducts of lipids, particularly cholesterol, with apolipoprotein B (apoB) are stable markers of human plasma low density lipoprotein (LDL) oxidation [V.V. Tertov, V.V. Kaplun, S.N. Dvoryantsev and A.N. Orekhov, Biochem. Biophys. Res. Commun. 214 (1995), 608–613]. In this study we attempt to assess the relationship between the degree of plasma LDL oxidation, evaluated by the content of apoB‐bound cholesterol and the ability of LDL to induce cholesterol accumulation in cultured human aortic intima smooth muscle cells, i.e., LDL atherogenic potential. LDL samples of 32 out of 39 healthy subjects did not increase cholesterol content in …cells cultured from grossly normal intima of human aorta. Most of LDL preparations isolated from coronary atherosclerosis patients with (34 out of 43) or without (35 out of 45) hypercholesterolemia stimulated intracellular cholesterol acumulation by 32–302%. The ability of human LDL to induce cholesterol accumulation in aortic smooth muscle cells did not correlate with the degree of in vivo LDL oxidation (r=0.10 , n=127 ). These results suggest that atherogenicity of LDL circulating in human plasma does not depend on the degree of lipid peroxidation in LDL particles. Show more

Keywords: Human blood plasma, low density lipoprotein, apolipoprotein B‐bound cholesterol, human intimal smooth muscle cells, intracellular cholesterol accumulation

Citation: Biofactors, vol. 6, no. 2, pp. 139-143, 1997

Authors: Heinecke, Jay W.

Article Type: Research Article

Abstract: Many lines of evidence implicate oxidation of low density lipoprotein (LDL) in the pathogenesis of atherosclerosis, a chronic inflammatory disease. The physiologically relevant mechanisms have not been identified, but phagocytic white cells may play an important role because macrophage‐rich lesions characterize the disorder. Recent studies have shown that myeloperoxidase, a heme enzyme secreted only by phagocytes, is present in human atherosclerotic tissue. The enzyme is a potent catalyst of LDL oxidation in vitro, it co‐localizes with macrophages in lesions, and it generates products that are detectable in atherosclerotic plaque. These findings suggest that myeloperoxidase may promote LDL oxidation in …the artery wall. This article reviews the enzyme’s ability to generate a range of oxidants, including tyrosyl radical, reactive aldehydes, hypochlorous acid and molecular chlorine. These products have the potential to damage host molecules as well as microbes, suggesting a mechanism that may contribute to atherosclerotic vascular disease. Show more

Keywords: 3‐Chlorotyrosine, dityrosine, chlorine gas, atherosclerosis, phagocyte

Citation: Biofactors, vol. 6, no. 2, pp. 145-155, 1997

Authors: Couderc, Rémy | Bonneau, Christine | Tissot, Michèle | Bailleul, Sophie | Roch‐Arveiller, Monique | Giroud, Jean Paul

Article Type: Research Article

Abstract: Polymorphonuclear leukocytes (PMN) generate highly reactive oxygen derived free radicals that may cause lipoprotein lipid oxidation and so contribute to the pathogenesis of atherosclerosis. On the other hand it has been shown that lipoproteins can alter cell functions in vitro. We therefore studied the effects of atherogenic lipoproteins, VLDL and LDL, on the production of superoxide anion by human PMN in the presence or absence of formyl‐methionyl‐leucyl‐phenylalanine (fMLP). VLDL and LDL stimulate PMN superoxide production and potentialize PMN stimulation by fMLP. The lipid moiety of the lipoproteins might be mainly involved in these effects. The binding of radio‐labelled fMLP to …its specific membrane receptor was significantly enhanced in the presence of VLDL and only slightly in the presence of LDL. The study of the signal transduction suggests that modulation of phospholipase D and A2 activities could be involved in the modification by LDL of PMN response to fMLP. Show more

Keywords: Lipoproteins, polymorphonuclear leukocyte, superoxide, atherogenesis

Citation: Biofactors, vol. 6, no. 2, pp. 157-163, 1997

Authors: Grune, T. | Davies, K.J.A.

Article Type: Research Article

Abstract: The degradation of oxidized proteins is an essential part of antioxidant defenses against free radical attack. Selective degradation of oxidatively damaged proteins allows proteolytic systems to function directly in the removal of useless cellular debris and therefore prevent the accumulation of potentially toxic fragments or large aggregates of cross‐linked proteins. The degradation of oxidized proteins in dividing mammalian cells after hydrogen peroxide treatment has been demonstrated. Cells are able to increase proteolysis rates after treatment with moderate levels of oxidants. The role of proteasome in the removal of oxidized proteins has been demonstrated by treatment of cells with an …anti‐sense oligodesoxynucleotide to the proteasome C2 subunit gene. This treatment decreases the proteasome content of the cells and prevents increased proteolysis rates after hydrogen peroxide treatment. Thus proteasome clearly plays a role in the removal of oxidized proteins. As part of antioxidant defenses the proteasome provides a second line of defense against the numerous radicals and oxidants which contact cells during their lifetime. The degradation of oxidatively damaged proteins enables cells to recover from a moderate oxidant attack. Show more

Keywords: Oxidized proteins, protein degradation, proteasome, antioxidative defense

Citation: Biofactors, vol. 6, no. 2, pp. 165-172, 1997