Macrophage cholesteryl ester hydrolases and hormone-sensitive lipase prefer specifically oxidized cholesteryl esters as substrates over their non-oxidized counterparts.

The oxidative modification of low-density lipoprotein (LDL) has been implicated as a pro-atherogenic process in the pathogenesis of atherosclerosis. Macrophages rapidly take up oxidized LDL via scavenger-receptor-mediated pathways and thereby develop into lipid-laden foam cells. The uptake mechanism has been studied extensively and several types of scavenger receptors have been identified. In contrast, the intracellular fate of oxidized LDL lipids is less well investigated. We studied the degradation of specifically oxidized cholesteryl esters by murine macrophages using an HPLC-based assay, and found that oxidized substrates are hydrolysed preferentially from a 1:1 molar mixture of oxidized and non-oxidized cholesteryl esters. This effect was observed at both neutral and acidic pH. Similar results were obtained with lysates of human monocytes and with pure recombinant human hormone-sensitive lipase. These data suggest that the intracellular oxidation of cholesteryl esters may facilitate intracellular cholesteryl ester hydrolysis, and thus may represent an anti-atherogenic process.

The rabbit 15-lipoxygenase preferentially oxygenates LDL cholesterol esters, and this reaction does not require vitamin E.

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 alpha-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 alpha-tocopherol-depleted LDL was used, or when the incubation sample was diluted. It may be concluded that 15-LOX-initiated 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.

Lipoxygenase treatment render low-density lipoprotein susceptible to Cu2+-catalysed oxidation.

Oxidative modification of low-density lipoprotein (LDL) has been implicated in foam-cell formation at all stages of atherosclerosis. Since transition metals and mammalian 15-lipoxygenases are capable of oxidizing LDL to its atherogenic form, a concerted action of these two catalysts in atherogenesis has been suggested. Cu2+-catalysed LDL oxidation is characterized by a kinetic lag period in which the lipophilic antioxidants are decomposed and by a complex mixture of unspecific oxidation products. We investigated the kinetics of the 15-lipoxygenase-catalysed oxygenation of LDL and found that the enzyme is capable of oxidizing LDL in the presence of the endogenous lipophilic antioxidants. In contrast with the Cu2+-catalysed reaction, no kinetic lag phase was detected. The pattern of products formed during short-term incubations was highly specific, with cholesterol-esterified (13S)-hydroperoxy-(9Z,11E)-octadecadinoic acid being the major product. However, after long-term incubations the product pattern was less specific. Preincubation with 15-lipoxygenase rendered human LDL more susceptible to Cu2+-catalysed oxidation as indicated by a dramatic shortening of the lag period. Addition of Cu2+ to lipoxygenase-treated LDL led to a steep decline in its antioxidant content and to a greatly reduced lag period. Interestingly, if normalized to a comparable hydroperoxide content, autoxidation and addition of exogenous hydroperoxy fatty acids both failed to overcome the lag period. The local peroxide concentrations in various LDL subcompartments will be discussed as a possible reason for this unexpected behaviour.

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase controls the activity of the 15-lipoxygenase with complex substrates and preserves the specificity of the oxygenation products.

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.

Oxidative modification of human lipoproteins by lipoxygenases of different positional specificities.

Cellular lipoxygenases have been implicated in foam cell formation during the early stages of atherogenesis. We studied the interaction of lipoxygenases of different positional specificities with human lipoproteins and found that the arachidonate 15-lipoxygenases of rabbit and humans and the arachidonate 12-lipoxygenase of porcine leukocytes oxygenate lipoproteins as indicated by the formation of oxygenated lipids and changes in electrophoretic mobility of low density lipoprotein. The arachidonate 12-lipoxygenase of human platelets, the recombinant arachidonate 5-lipoxygenase of human leukocyte, and the soybean lipoxygenase I were less effective in oxidizing human LDL. As a major oxygenation product, esterified 13S-hydro(pero)xy-9Z,11E-octadecadienoic acid was identified for both the rabbit reticulocyte 15- and the porcine leukocyte 12-lipoxygenase. In addition, esterified 15S-hydro(pero)xy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid (for the rabbit 15-lipoxygenase) and 12S-hydro(pero)xy-5,8,10,14(Z,Z,E,Z)-eicosatetraenoic acid (for the porcine 12-lipoxygenase) as well as small amounts of racemic 9-hydro(pero)xy-10,12-octadecadienoic acid isomers were detected. More than 90% of the oxygenated polyenoic fatty acids were found in the ester lipid fraction, particularly in the cholesteryl esters and in various phospholipid classes (phosphatidylcholine and phosphatidylethanolamine). The possible biological significance of lipoxygenase-induced oxidative modification of lipoproteins in the pathogenesis of atherosclerosis is discussed.

Involvement of 15-lipoxygenase in early stages of atherogenesis.

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.

Oxygenation of lipoproteins by mammalian lipoxygenases.

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 vitro 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,11E-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.

Structure elucidation of oxygenated lipids in human atherosclerotic lesions.

Oxidative modification of low density lipoproteins and tissue lipids has been proposed to be involved in the pathogenesis of atherosclerosis. We examined human atherosclerotic lesions of various stages from fifteen victims of acute heart failure and detected substantial amounts of oxygenated fatty acids in the tissue ester lipids. The degree of lipid oxygenation correlated with the stage of advancement of the lesion. More than 85% of the oxygenated fatty acids were localized in the cholesterol esters, whereas phospholipids contained only small amounts. Structure elucidation of the oxygenation products indicated a nonspecific product pattern of various isomers of keto- and hydroxy-octadecadienoic acid. The data presented suggest an involvement of lipid peroxidation in the pathogenesis of atherosclerosis and indicate that the majority of the oxygenation products are formed via nonspecific, non-enzymatic reactions possibly initiated by the action of a 15-lipoxygenase.

Identification of oxidatively modified lipids in atherosclerotic lesions of human aortas.

The oxygenated lipids of human aortas with atherosclerotic lesions were analysed by RP-, SP- and Chiral Phase-HPLC and compared with those obtained after treatment of human LDL with pure 15-lipoxygenase (LOX) of rabbit reticulocytes. The data suggest an indirect role of 15-LOX in the atherogenesis in aortas.

The oxygenation of cholesterol esters by the reticulocyte lipoxygenase.

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.

Oxygenation of biological membranes by the pure reticulocyte lipoxygenase.

We find that the reticulocyte lipoxygenase can oxygenate rat liver mitochondrial membranes, beef heart submitochondrial particles, rat liver endoplasmic membranes, and erythrocyte plasma membranes (inside-out and right side-out ghosts) without prior action of a phospholipase. After alkaline hydrolysis of the ester lipids, the main products were identified as 15S-hydro(pero)xy-5Z,8Z,11Z,13E-eicosatetr aenoic acid, 17S-hydro(pero)xy-4Z,7Z,10Z,13Z,15E, 19Z,-docosahexaenoic acid, 13S-hydro(pero)xy-9Z,11E-octadecadienoic acid, 9(S/R)-hydro(pero)xy-10E,12Z-octadecadienoic acid as well as the two all-E hydro(pero)xy octadecadienoic acid isomers. At low membrane concentrations (1 mg of protein/ml), the enzyme maintains a high stereospecificity for the S-configuration, but at higher concentrations (20 mg/ml), the products were virtually racemic. Addition of the antioxidant 2,6-ditert-butyl-p-cresol counteracted this tendency to lose stereospecificity. During these enzyme-catalyzed reactions, substantially more oxygen is consumed than can be accounted for as the hydro(pero)xy products. This discrepancy is due to secondary reactions which lead to the decomposition of the primary oxygenation products, the hydroperoxy lipids, and to oxidative modifications of membrane proteins. These data indicate that the reticulocyte lipoxygenase can oxygenate polyenoic fatty acids in various types of biological membrane and that the oxidative modifications are not restricted to the membrane lipids. The results are discussed in terms of the proposed role of the enzyme in the breakdown of mitochondria and other intracellular organelles during the maturation of red blood cells.

Subcellular distribution of lipoxygenase products in rabbit reticulocyte membranes.

Mitochondrial membranes and plasma membranes of rabbit reticulocytes contain oxygenated polyenoic fatty acids such as (9Z,11E)-(13S)-13-hydroxy-9,11-octadecadienoic acid, 9S and 9R isomers of (10E,12Z)-9-hydroxy-10,12-octadecadienoic acid and their all-E isomers. Furthermore (5Z,8Z,11Z,13E)-(15S)-15-hydroxy-5,8,11,13-icosa tetraenoic 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 (9Z,11E)-(13S)-13-hydroxy-9,11-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.

Occurrence of 9- and 13-keto-octadecadienoic acid in biological membranes oxygenated by the reticulocyte lipoxygenase.

Membranes of intact rabbit reticulocytes and rat liver mitochondrial membranes oxygenated by the pure reticulocyte lipoxygenase contain 13-keto-9Z,11E-octadecadienoic acid and 9-keto-10E,12Z-octadecadienoic acid. In mitochondrial membranes not treated with lipoxygenase and in rabbit erythrocyte membranes these products were not detected. The chemical structure of the compounds has been identified by cochromatography with authentic standards on various types of HPLC columns, by uv and ir spectroscopy and GC/MS. In the membranes of rabbit reticulocytes up to 2% of the linoleate residues are present as its 9- and 13-keto derivatives. Most of the keto compounds (up to 90%) are esterified in the membrane ester lipids, only about 10% were found in the free fatty acid fraction. It is proposed that the keto dienoic fatty acids are formed via decomposition of hydroperoxy polyenoic fatty acids originating from the oxygenation of the membrane lipids by the reticulocyte lipoxygenase.

New evidence for endoplasmic reticulum and Golgi apparatus in rabbit reticulocytes.

The incorporation of 14C-galactose and 14C-fucose in rabbit reticulocytes obtained by bleeding anaemia and separated according to their stage of maturation was studied. Only the youngest reticulocytes were shown to incorporate the two sugars with galactosylation being three times higher. The glycosylation behaved different from the synthesis of membrane proteins with respect to the dependence on temperature and time. Among the newly synthesized membrane proteins the signal sequence receptor (SSR), a compound of the system addressing protein translocation, was detected immunologically.

Metabolism of polyenoic fatty acids by rabbit reticulocytes. Intracellular action of the erythroid lipoxygenase on membrane lipids.

Rabbit reticulocytes metabolize exogenous polyenoic fatty acids via three different pathways. (i) incorporation into the cellular ester lipids, predominantly into phospholipids, (ii) beta-oxidation forming CO2 and (iii) lipoxygenase reaction. The lipoxygenase pathway contributes to about 30% to the metabolism of exogenously added linoleic acid. The endogenous substrates of the lipoxygenase pathway are not only the free polyenoic fatty acids bound to the cellular membranes but also the membrane phospholipids. The lipoxygenase products detected in the membrane lipids have been isolated and their complete chemical structure has been identified as 13-hydroxy-9Z,11E-octadecadienoic acid. 9-hydroxy-10E,12Z-octadecadienoic acid, their all E isomers and 15-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid. Subcellular fractionation of different cellular membranes indicated that these products occur in both the mitochondrial membranes and the plasma membrane. By quantitative HPLC analysis it has been shown that the mitochondrial membranes contain about 3 times more oxygenated fatty acids than the plasma membranes; one out of ten linoleic acid residues in the mitochondrial membranes is present as hydroxylated derivative. The pattern of lipoxygenase products detected in the mitochondrial membranes was much more unspecific than that of plasma membranes. These data are discussed in the light of the involvement of the lipoxygenase pathway in the degradation of mitochondria during the maturation of red blood cells.

Oxygenation of biological membranes by the reticulocyte lipoxygenase. Lack of stoichiometry between oxygen uptake and product formation.

The oxygenation of different types of biological membranes (rat liver mitochondria, rat liver endoplasmic membranes, inside-out erythrocyte ghosts, right side-out erythrocyte ghosts) was studied with respect to products formed during the reaction. In all cases a very similar product pattern was observed with 15S-hydroperoxy-5Z.8Z.11Z.13E-eicosatetraenoic acid (15-HETE) and 13S-hydroperoxy-9Z,11E-octadecadienoic acid, (13-HODE) being the major products. Comparison of the amount of lipoxygenase products formed with the oxygen uptake measured during the reaction indicated an excessive oxygen uptake. With mitochondrial membranes the oxygen consumption was almost one order of magnitude higher than the amount of the products detected. The origin of the excessive oxygen uptake remains unclear. These data, however, indicate that the oxygen consumption with complex substrates is not a reliable measure for the lipoxygenase activity.

Approaches to characterize by density-fractionation age-dependent properties of reticulocyte mitochondria.

Mitochondria from rabbit reticulocytes of varying maturity were prepared by means of two different methods and separated in sucrose and Percoll density gradients. A mechanical method of cell disintegration under isotonic conditions followed by centrifugation in a Percoll density gradient proved to be adequate to reflect the physiological conditions. "Young" mitochondria exhibit in this system greater density and higher specific activity of cytochrome oxidase.

[In vitro maturation of reticulocytes. Behavior of RNA and inorganic pyrophosphatase]. / In vitro-Reifung von Retikulozyten. Verhalten von RNS und anorganischer Pyrophosphatase.

A simple system of incubation of reticulocytes with a small degree of haemolysis has been developed. It was possible to test the influence of inorganic phosphate, pH-values in the range of 7.0--9.0 and anaerobic conditions on the in vitro maturation of reticulocytes. As main criteria of the maturation the RNA-content and the activity of PPase has been tested. The decrease of the RNA-content and the activity of the PPase is highly significant during maturation. The decrease of RNA and the reduction of reticulocytes are stimulated by inorganic phosphate.