Oxidative modification converts low-density lipoprotein (LDL) into its atherogenic form and appears to be a necessary precondition for LDL uptake by macrophages during foam cell formation. Cellular lipoxygenases have been implicated in this process. We studied the interaction of purified mammalian lipoxygenases with human LDL in v i m and found that the arachidonate 15-lipoxygenases of rabbit and man are capable of oxygenating lipoproteins as indicated by oxygen uptake and by the formation of thiobarbituric-acid-reactive substances. Furthermore, oxygenated polyenoic fatty acids, such as 13-hydro(pero)xy-9Z, 11 E-octadecadienoic acid and 15-hydro(pero)xy-5,8,11,13 (Z,Z, Z,E)-eicosatetraenoic acid were detected in the lipid compartment of various lipoproteins classes after lipoxygenase treatment. More than 90% of the oxygenated polyenoic fatty acids were found in the ester-lipid fraction, particularly in the cholesterol esters, whereas only small amounts of free hydro(pero)xy polyenoic fatty acids were detected. Lipoxygenase-catalyzed oxygenation of LDL is not restricted to the lipid compartment but also leads to a cooxidative modification of the apoproteins as indicated by changes in the electrophoretic mobility and by the formation of carbonyl derivatives of amino acid side chains. The possible biological significance of lipoxygenase-induced oxidative modification of lipoproteins in the pathogenesis of atherosclerosis is discussed.Atherosclerosis is a multifactoral disease, the pathogenesis of which is not fully understood [l-31. It has been shown by morphological studies that the accumulation of lipid-loaded foam cells in the subendothelial space leads to the formation of fatty streaks, which are generally accepted as the early atherosclerotic lesions [l -41. Foam cells develop from monocyte-derived macrophages [5, 61 or from smooth muscle cells [7] by taking up modified low-density lipoproteins (LDL) via scavenger-receptor(s)-mediated pathways. The occurrence of oxidatively modified proteins in atherosclerotic lesions [8, 91 and the fact that oxidatively modified LDL is rapidly taken up by macrophages [lo] suggested an involvement of oxidative processes in the pathogenesis of atherosclerosis. However, the mechanism of oxidative modification in vivo remains unclear. In-vitro studies with cell-free systems indicated that copper-mediated oxidation converts LDL into its atherogenic form [ll] The oxygenation of lipoproteins by mammalian lipoxygenases has not been studied so far. The soybean lipoxygenase which largely differs from mammalian lipoxygenases with respect to its protein chemical and enzymic properties is capable of oxidizing LDL only in the presence of phospholipase A, which provides free polyenoic fatty acids by cleaving lipoprotein phospholipids [20]. This result is not surprising since the soybean lipoxygenase has been shown to effectively oxygenate ester lipids only in the presence of detergents [21]. However, the rabbit reticulocyte lipoxygenase is capable of oxygenating complex ester lipids such ...

The arachidonate 15-lipoxygenase which is expressed in atherosclerotic lesions is implicated in the oxidative modification of low density lipoproteins during atherogenesis. To obtain experimental in vivo evidence for this hypothesis, we analyzed the structure of oxygenated lipids isolated from the aorta of rabbits fed with a cholesterol-rich diet for different time periods and compared the pattern of oxygenation products with that isolated from low density lipoproteins treated in vitro with the pure rabbit 15-lipoxygenase and with oxygenated lipids isolated from advanced human atherosclerotic lesions. In early atherosclerotic lesions (12-wk cholesterol feeding), specific lipoxygenase products were detected whose structure was similar to those isolated from lipoxygenase-treated low density lipoproteins. The appearance of these products did coincide with the lipid deposition in the vessel wall. In later stages of atherogenesis (26-wk cholesterol feeding) the degree of oxidative modification of the tissue lipids did increase but the share of specific lipoxygenase products was significantly lower, suggesting an increasing overlay of the specific lipoxygenase products by nonenzymatic lipid peroxidation. In advanced human atherosclerotic lesions, large amounts of oxygenation products were detected whose structure suggests a nonenzymatic origin. These data suggest that the arachidonate 15-lipoxygenase is of pathophysiological importance during the early stages of atherogenesis. In later stages of plaque development nonenzymatic lipid peroxidation becomes more relevant.

The oxidation of low density lipoprotein (LDL) by mammalian 15-lipoxygenases (15-LOX) was implicated in early atherogenesis. We investigated the molecular mechanism of 15-LOX/LDL interaction and found that during short term incubations, LDL cholesterol esters are oxygenated preferentially by the enzyme. Even when the LDL particle was loaded with free linoleic acid, cholesteryl linoleate constituted the major LOX substrate. In contrast, only small amounts of free oxygenated fatty acid isomers were detected, and re-esterification of oxidized fatty acids into the LDL ester lipid fraction was ruled out. When LDL was depleted from ␣-tocopherol, specific oxygenation of the cholesterol esters was not prevented, and the product pattern was not altered. Similar results were obtained at low (LDL/LOX ratio of 1:1) and high LOX loading (LDL/LOX ratio of 1:10) of the LDL particle. During long term incubations (up to 24 h), a less specific product pattern was observed. However, when the hydroperoxy lipids formed by the 15-LOX were immediately reduced by the phospholipid hydroperoxide glutathione peroxidase, when the reaction was carried out with vitamin E-depleted LDL, or when the assay sample was diluted, the specific pattern of oxygenation products was retained over a long period of time.These data suggest that mammalian 15-LOX preferentially oxidize LDL cholesterol esters, forming a specific pattern of oxygenation products. During long term incubations, free radical-mediated secondary reactions, which lead to a more unspecific product pattern, may become increasingly important. These secondary reactions appear to be suppressed when the hydroperoxy lipids formed are immediately reduced, when ␣-tocopherol-depleted LDL was used, or when the incubation sample was diluted. It may be concluded that 15-LOXinitiated LDL oxidation constitutes a dual-type oxygenase reaction with an initial enzymatic and a subsequent nonenzymatic phase. The biological relevance of this dual-type reaction for atherogenesis will be discussed. (15). (vi) A 15-LOX inhibitor that apparently lacks major antioxidative properties prevented lipid deposition in the aorta of cholesterolfed rabbits (16). These data are consistent with a proatherogenic activity of the enzyme. On the other hand, transgenic rabbits that overexpress the 15-LOX specifically in monocyte/ macrophages (17) develop significantly less atherosclerotic lesions when fed a cholesterol-rich diet or when cross-bred with LDL receptor-deficient Watanabe rabbits (18), suggesting an anti-atherogenic action of the enzyme.For a better understanding of the processes involved in LDL oxidation in vivo, the in vitro interaction of 15-LOXs with human LDL has been studied. Sparrow et al. (6) have shown that the 15-LOX from soybeans is capable of oxidizing LDL in the presence of phospholipase A 2 . Later on, we reported that the native rabbit (19) and the recombinant human 15-LOX (20) specifically oxidize LDL ester lipids in the absence of lipidhydrolyzing enzymes. Comparison of various LOX isoenzymes ind...

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The arachidonate 15‐lipoxygenase from rabbit reticulocytes oxygenates cholesterol esters containing polyenoic fatty acids. Cholesterol esterified with saturated fatty acids is not oxygenated. The structures of the oxygenation products formed from various cholesterol esters have been identified by high pressure liquid chromatography, UV‐spectroscopy and gas chromatography/mass spectroscopy. Oxygenated cholesterol esters have been detected in atherosclerotic plaques of human aortas.

Mitochondria1 membranes and plasma membranes of rabbit reticulocytes contain oxygenated polyenoic fatty acids such as (9Z,1 1E)-(l3S)-13-hydroxy-9,1l-octadecadienoic acid, 9 s and 9R isomers of (IOE,12Z)-9-hydroxy-10,12-octadecadienoic acid and their all-E isomers. Furthermore (5Z,8Z,112,13E)-(1 5S)-15-hydroxy-5,8,11,13-icosatetraenoic acid, 9-and 13-oxooctadecadienoic acid were detected as minor products. The chemical structure of these products has been identified by co-chromatography with authentic standards, by ultraviolet and infrared spectroscopy, and by gas chromatography/mass spectrometry of the native compounds and their hydrogenated derivatives. The oxygenated fatty acids originate most probably from the intracellular action of the erythroid arachidonate 15-lipoxygenase. In membranes of the mature erythrocyte only small amounts of hydroxy fatty acids were detected. Young peripheral reticulocytes contain more oxygenated polyenoic fatty acids in their membranes than older cells. In mixed cell populations, about 85% of the lipoxygenase products were found esterified to the membrane ester lipids, whereas 15% were associated as free hydroxy fatty acids with the membranes. The hydroxy fatty acid content of the mitochondrial membranes is more than threefold higher than that of the plasma membranes. The pattern of the products isolated from plasma membranes shows a high specificity with (92,llE)-(13S)-13-hydroxy-9,1 I-octadecadienoic acid as the main product. In contrast, the pattern found in the mitochondrial membranes was much more unspecific; a complex mixture of all positional and optical isomers was detected.The data presented indicate that the reticulocyte lipoxygenase in vivo acts on both plasma membranes and mitochondrial membranes. The results are discussed in the light of the involvement of the lipoxygenase in the breakdown of mitochondria and other organelles in reticulocytes during maturation.Reticulocytes of different species [l, 21 contain a lipoxygenase which is able to oxygenate polyenoic fatty acids esterified to membrane lipids [3]. This enzyme was proposed to play an important role in the breakdown of mitochondria during the maturation of red blood cells [4]. Free arachidonic acid was converted by the pure enzyme to a mixture of (52,8Z,lIZ,-13E)- (13S) In contrast to our knowledge of the properties of the isolated enzyme, little is known about the intracellular action of the lipoxygenase. Rabbit reticulocytes exhibit an antimycin-A-resistant oxygen uptake which was partly inhibited by various lipoxygenase inhibitors. This partial inhibition amounts to about 5% of the total oxygen uptake [9]. Recently the occurrence of oxygenated polyenoic fatty acids in the membranes of rabbit reticulocytes has been reported [lo], which indicates the intracelluIar action of the lipoxygenase on membrane lipids.In this study, we report the subcellular distribution of the oxygenated polyenoic fatty acids in various membrane fractions of rabbit reticulocytes and in reticulocytes at different stages of maturation.

Mammalian 15-lipoxygenases have been suggested to be involved in cell differentiation and atherogenesis because of their capability of oxygenating polyenoic fatty acids esterified to biomembranes and lipoproteins. We investigated the interaction of the lipid-peroxidizing 15-lipoxygenase and the hydroperoxy lipid-reducing phospholipid hydroperoxide glutathione peroxidase during their reaction with biomembranes and lipoproteins and obtained the following results. 1) Lipoxygenase treatment of submitochondrial membranes led to the formation of hydroperoxyphosphatidylethanolamine and hydroperoxyphosphatidylcholine as indicated by high performance liquid chromatography with chemiluminescence detection. In 15-lipoxygenase-treated low density lipoprotein cholesteryl hydroperoxylinoleate was the major oxygenation product. 2) Phospholipid hydroperoxide glutathione peroxidase was capable of reducing the hydroperoxy lipids formed by the 15-lipoxygenase to their corresponding alcohols. 3) Preincubation of low density lipoprotein and submitochondrial membranes with the phospholipid hydroperoxide glutathione peroxidase completely prevented the lipoxygenase reaction. However, addition of exogenous hydroperoxy lipids restored the oxygenase activity. 4) Short-term incubations of the complex substrates with the 15-lipoxygenase led to a specific pattern of oxidation products which was rendered more unspecific at long-term incubation or at high substrate concentrations. If the phosholipid hydroperoxide glutathione peroxidase was present during the reaction, the specific product pattern was preserved. These data indicate that the phospholipid hydroperoxide glutathione peroxidase is capable of reducing hydroperoxy ester lipids formed by a 15-lipoxygenase, and that it may down-regulate the 15-lipoxygenase pathways in mammalian cells. The specificity of 15-lipoxygenase-derived hydroperoxy lipids depends on their immediate reduction to the corresponding alcohols preventing postcatalytic isomerization.

Lipoxygenases (LOXs) form a heterogeneous family of lipid-peroxidizing enzymes, and several LOX-isoforms (12/15-LOX, 5-LOX) have been implicated in atherogenesis. However, the precise role of these enzymes is still a matter of discussion. 12/15-LOXs are capable of oxidizing lipoproteins (low-density lipoprotein (LDL), high-density lipoprotein (HDL)) to atherogenic forms, and functional inactivation of this enzyme in murine atherosclerosis models slows down lesion formation. In contrast, rabbits that overexpress this enzyme were protected from lesion formation when fed a lipid-rich diet. To contribute to this discussion, we recently investigated the impact of 12/15-LOX overexpression on in vitro foam cell formation. When 12/15-LOX-transfected J774 cells were incubated in culture with modified LDL, we found that intracellular lipid deposition was reduced in the transfected cells when compared with the corresponding control transfectants. This paper briefly summarizes the current status of knowledge on the biological activity of different LOX-isoforms in atherogenesis and will also provide novel experimental data characterizing the role of 12/15-LOX in cellular LDL modification and for in vitro foam cell formation.