Conformational changes in oxidized LDL recognized by mouse peritoneal macrophages.

Mouse peritoneal macrophages have been considered to recognize and take up oxidized LDL by a scavenger receptor. However, it is still unknown what conformational changes in oxidized LDL contribute to recognition by the macrophage scavenger receptor. In the present study, it was shown that the amount of oxidized LDL taken up by macrophages correlated well with the fluorescence intensity formed in oxidized LDL. The autofluorescent products generated in oxidized LDL were characterized by Ex:365 nm Em:430 nm, and the intensity of the fluorescence was reduced at base pH, and restored by adjusting the pH to neutral. The characteristics of the fluorescent products indicate that a Schiff base structure was formed in oxidized LDL. Oxidized LDLs were fractionated into native size and aggregated large particles with HPLC by monitoring fluorescence. It was demonstrated that macrophages ingest selectively or preferentially aggregated oxidized LDL, but not native size oxidized LDL. The incorporation of aggregated oxidized LDL was remarkably suppressed by heparin and cytochalasin B. These results suggest that mouse peritoneal macrophages recognize the conformational changes in oxidized LDL related to the formation of a Schiff base structure with increasing autofluorescence, and ingest selectively aggregated large particles in oxidized LDL in a phagocytic process.

Oxidation of lipids. 7. Oxidation of phosphatidylcholines in homogeneous solution and in water dispersion.

The rates and products of the oxidations of phosphatidylcholines (PCs) in organic homogeneous solutions and as multilamellar liposomes in an aqueous dispersion were studied at 50 degrees C initiated by oil-soluble and water-soluble azo compounds and with the aim primarily of elucidating the effect of substrate and the reaction medium. Polyunsaturated fatty acids were oxidized exclusively among the fatty acids in PC. The product distribution was determined by the number of double bonds: in the oxidation of methyl linoleate and dilinoleoyl PC, the conjugated diene hydroperoxides were formed almost quantitatively, whereas in the oxidation of PC and polyunsaturated fatty acids having three or more double-bonds, cyclic peroxides were also formed. The oxidizability of PC was dependent on the content of doubly allylic hydrogens and their reactivities were suggested to be similar, independent of parent substrate and reaction medium, although the oxidizability of PC in nonpolar organic solvents is markedly enhanced by the formation of reverse-phase micelles.

Accumulation of ceroid-like pigments in macrophages cultured with phosphatidylcholine liposomes in vitro.

When mouse peritoneal macrophages as well as P388D1 cells, an established macrophage-like cell line, were cultured with liposomes composed of rat liver phosphatidylcholine and phosphatidylserine, storage of fluorescent products, ceroid-like pigments, within those cells was observed with light and fluorescence microscopy, and fluorescence spectrophotometry. The amounts of thiobarbituric acid-reactive substances and fluorescent products in macrophages were increased gradually to reach a maximal level to between 6 and 8 days of culture. The involvement of peroxidation of liposomal lipids in the formation of the pigments was further suggested by the 6 days that incorporation of alpha-tocopherol into liposomes decreased the storage of the pigments. No appreciable formation of the pigments was observed in macrophages cultured with liposomes containing dipalmitoylphosphatidylcholine instead of rat liver phosphatidylcholine. The fluorescent products formed in cultured cells were found in lipid-soluble and -insoluble fractions. Lipid-insoluble fluorescent products had an excitation maximum at 360 nm and a fluorescence maximum at 430 nm in SDS-aqueous solution (pH 7.4) and the intensity of the fluorescence was quenched at base pH, but it was not changed in acidic media. These findings indicate that the macrophages can store Schiff base fluorescent substances formed by the reaction between peroxidation products of exogenous lipids and amino compounds in the cells, under some pathological conditions.

Formation of age pigment-like fluorescent substances during peroxidation of lipids in model membranes.

The formation of age pigment-like fluorescent substances during the lipid peroxidation of model membranes has been studied. Ferrous ion and ascorbate-induced lipid peroxidation of liposomal membranes containing phosphatidylethanolamine led to the formation of fluorescent substances which have characteristics similar to those of compounds derived from the reaction of phosphatidylethanolamine with purified fatty acid hydroperoxides. The fluorescent substances were accumulated in liposomal membranes, whereas thiobarbituric acid-reactive substances formed during lipid preoxidation were immediately released from the liposomal membranes. The thiobarbituric acid-reactive substances free from the membranes were not reactive with amino compounds such as phosphatidylethanolamine in liposomes or glycine in aqueous phase. It was suggested that the products reacting with amino compounds are short-lived, and may be rapidly inactivated after released into aqueous phase. The formation of fluorescent products was inefficient when phosphatidylethanolamine incorporated into the liposomes insensitive to lipid preoxidation was incubated with ferrous ion and ascorbate in the presence of liposomes sensitive to the peroxidation. The results suggest that some products generated from peroxidation-sensitive lipids react with the amino group of phosphatidylethanolamine molecules which are located on the same membranes, forming fluorescent substances. The presence of phosphatidylethanolamine in the membrane suppressed the formation of thiobarbituric acid-reactive substances, suggesting that phosphatidylethanolamine may react with radicals formed and terminate the propagation.

Oxidation of biological membranes and its inhibition. Free radical chain oxidation of erythrocyte ghost membranes by oxygen.

The oxidation of human and rat erythrocyte ghost membranes by molecular oxygen has been performed in an aqueous suspension at 37 degrees C. A constant rate of oxygen uptake was observed in the presence of radical initiator. alpha-Tocopherol in the membrane suppressed the oxidation and the induction period was clearly observed. alpha-Tocopherol decreased linearly during the induction period and when it was depleted the induction period was over and a rapid oxidation started. The rate of oxidation was proportional to the square root of the rate of initial radical generation. The kinetic chain length, the ratio of the rate of propagation to that of initiation, was long, ranging from 7 to 100. These results indicate that the erythrocyte ghost membranes are oxidized by a free radical chain mechanism by molecular oxygen. Among the fatty acids of membrane lipids, polyunsaturated fatty acids were oxidized exclusively. Proteins as well as polyunsaturated fatty acids were oxidized and the formation of the high- and low-molecular-weight proteins and the decrease of protein bands were observed on gel electrophoresis.

Oxidized low-density lipoprotein induces the production of superoxide by neutrophils.

Exposure of guinea pig peritoneal neutrophils to ox-LDL led to the production of superoxide, which was measured by the formation of superoxide-dependent chemiluminescence. The cells exposed to unoxidized LDL, e.g. native LDL, acetyl-LDL, and self-aggregates of LDL showed no production of superoxide. The superoxide production was correlated with the levels of oxidative modification of LDL and reached a maximum between 10 and 30 min during incubation, but preincubating the cells with cytochalasin B decreased the superoxide production. These findings indicate that neutrophils rapidly take up ox-LDL by phagocytosis and generate superoxide which may cause superoxide-mediated lipid peroxidation in vivo.

Studies of effects of trans fatty acids in the diet on lipid metabolism in essential fatty acid deficient rats.

Effects of diets containing mixtures of safflower oil, hydrogenated coconut oil with elaidate of linolelaidate on growth, fatty acid composition, serum lecithin: cholesterol acyl transferase (LCAT) and postheparin plasma lipoprotein lipase activities in essential fatty acid (EFA) deficient rats were determined. Addition of trans fatty acids to the diet lowered the growth response to linoleic acid. Both elaidate and linolelaidate accumulated in the serum and liver, imparied the conversion of oleic acid to eicosatrienoic acid and linoleic acid to arachidonic acid, and the incorporation of eicosatrienoic acid into cholesteryl esters. Trans fatty acids also influenced the fatty acid composition of testicular lipids, but much lower amounts of these acids accumlated in tests than in liver or serum. Serum lecithin:cholesterol acyl transferase activity was elevated by an EFA deficiency, was unaffected by dietary elaidate, but was significantly decreased by linolelaidate. These effects were nullified by the addition of safflower oil to the diet. Postheparin plasma extrahepatic and hepatic lipase activities were also affected by an EFA deficiency, and by the addition of elaidate or linolelaidate alone or in combination with safflower oil to the diets of EFA deficient rats. It is suggested that trans fatty acids exhibit particular effects on the metabolism of lipids in addition to aggravation of an EFA deficiency.

Age and organ difference in amount and distribution of autofluorescent granules in rats.

The amount and distribution of autofluorescent granules in various organs and tissues of rats of different ages (2-, 11-, and 29.5-month-old) were compared by morphometrical analysis. The age-related increase of the granules was observed in the cardiac muscle and hepatic cells, but the amount of granules was little even in 29.5-month-old rats. In some other organs, the granules appeared early, at 2 months of age, increased up to 11 months, but thereafter no significant increase was observed. The autofluorescent granules are not merely considered to be an age-related pigment, but seems to be influenced by relationship to the cell metabolism and other functions.

Morphometrical and biochemical analysis on autofluorescent granules in various tissues and cells of the rats under several nutritional conditions.

The effects of age and nutritional conditions on accumulation of autofluorescent granules in various organs and tissues of male Sprague-Dawley rats were compared morphometrically. The relative intensity of the specific fluorescence of these autofluorescent granules was similar in all tissues and cells examined. In almost all cases, there were more autofluorescent granules in the 12-month experiment than in the 4-month one. Multiple necrotic foci of myofibrils with an accumulation of autofluorescent granules were seen in striated muscles in the rats on vitamin E-deficient diets for 12 months. In splenocytes, renal proximal convoluted tubules and hepatic cells, autofluorescent granules quantitatively increased significantly with an increase of the corn oil contents in the diets. The increase was rather marked in the splenocytes and renal epithelia of vitamin E-deficient rats. In the Purkinje cells and bronchial epithelial cells, no significant differences were noted according to the difference in the vitamin E and corn oil contents in diets. The accumulation of autofluorescent granules was not merely considered to be an age-related change, but to be influenced by a relationship to the cell metabolism and functional activity in various organs.

Comparison of fluorescence characteristics of products of peroxidation of membrane phospholipids with those of products derived from reaction of malonaldehyde with glycine as a model of lipofuscin fluorescent substances.

The fluorescence characteristics of product (I), formed during the lipid peroxidation of rat liver phosphatidylcholine liposomes containing glycine, and fluorescent product (II), derived from the reaction of malonaldehyde with glycine, were examined to elucidate the mechanism of fluorescent chromophore formation. Fluorescent product (I) had a fluorescence emission maximum at 430 nm when excited at 360 nm; its fluorescence intensity decreases in alkaline medium, but is restored by readjustment of pH to neutrality. In contrast, fluorescent product (II) exhibited an emission maximum at 458 nm, and the fluorescence was quenched at acidic pH. The fluorescent substances formed during the lipid peroxidation of hemoglobin-free human erythrocyte ghost membranes had similar fluorescence characteristics to product (I). Gel filtration experiments showed that molecular size of fluorescent product (I) was larger than that of fluorescent product (II). The thiobarbituric acid-reactive substances released from peroxidizing liposomal phospholipids had a larger molecular size than malonaldehyde, and produced little or no fluorescence with glycine. It is concluded that the precursor of the fluorescent product formed during the lipid peroxidation of membrane phospholipids differs from malonaldehyde. The mechanism of the formation of blue emitting fluorescent material, believed to be a component of lipofuscin, seems to involve peroxidized phospholipids of the membrane.

Mild hypothermia reduces the rate of metabolism of arachidonic acid following postischemic reperfusion.

Free fatty acid (FFA) accumulation during cerebral ischemia has been described as an indicator of ischemic damage. Furthermore arachidonic acid (AA) metabolites, liberated from glycerophospholipids, have been confirmed to induce disturbances of membrane functions. Are there differences in AA levels in the hippocampus of normo- and hypothermic gerbils following ischemia-reperfusion? In an attempt to answer this question, we first studied the time course of changes in the amount of AA liberated from glycerophospholipids using gerbils subjected to 5 min of ischemia-reperfusion under normo- and mild hypothermia. FFAs (including AA) were separated from total lipids by Bond Elut (NH2) column chromatography and analyzed by gas-liquid chromatography. Mild intra-ischemic hypothermia (MIH) did not affect the ischemia-induced AA accumulation following of 5 min of forebrain ischemia. The accumulated AA amounts under MIH tend to decrease more slowly to baseline levels from 15 to 30 min of reperfusion than do the levels under normothermia. These results suggested that MIH reduced the rate of metabolism of AA after reperfusion and might suppress the generation of free radical, eicosanoids and other bioactive metabolites.

Lethal forebrain ischemia stimulates sphingomyelin hydrolysis and ceramide generation in the gerbil hippocampus.

Ceramide, a hydrolyzed product of sphingomyelin, is reported to play an important role in apoptosis. In this study, we measured the sphingomyelin and ceramide levels in the hippocampus of the gerbil after transient forebrain ischemia for 5 min (lethal) or 2 min (sublethal). The aim was to examine alterations in the sphingomyelin cycle during delayed neuronal death, which we considered could be due to apoptosis. Sphingolipids were separated on high-performance thin-layer chromatography plates and analyzed by gas-liquid chromatography. At 30 min and 24 h after lethal ischemia, sphingomyelin levels were decreased and ceramide levels were increased compared with control levels. No significant changes were observed after sublethal ischemia. These results suggest that the sphingomyelin cycle may have a role in neuronal death.

Regional distribution of ethanolamine plasmalogen in the hippocampal CA1 and CA3 regions and cerebral cortex of the gerbil.

Although ethanolamine plasmalogens (EtnPm) are the predominant phospholipids in neural tissue, their physiological role has not been clarified. The biophysical conformation of EtnPm in the proteoliposome enhances the activity of the sodium-calcium exchanger, which has been proposed to induce intracellular calcium ion accumulation during ischemia and early reperfusion. The levels of EtnPm in the areas of the gerbil brain selectively vulnerable to ischemia, namely the hippocampal CA1 and CA3 regions and the cerebral cortex, were measured by high-performance thin-layer chromatography and gas-liquid chromatography. The concentration of EtnPm in the CA1 region, which is the most vulnerable to ischemic and anoxic stress, was 2.6- and 2.7-fold higher than that in the CA3 region and cerebral cortex, respectively. The significantly higher concentration of EtnPm in the hippocampal CA1 region may enhance sodium-calcium exchanger activity and play an important role in the vulnerability of this region to ischemia.

Inhibition of oxidation of low density lipoprotein by vitamin E and related compounds.

The oxidation of low density lipoprotein (LDL) was carried out aiming specifically at elucidating the anti-oxidant action of alpha-tocopherol. Lipophilic and hydrophilic azo compounds and copper induced the oxidation of LDL similarly to give cholesterol ester and phosphatidylcholine hydroperoxides as major products. The antioxidant potency of alpha-tocopherol in LDL was much poorer than in homogeneous solution. Doxyl stearic acids were used as spin probe and incorporated in LDL. The rate of reduction of doxyl nitroxide in LDL by ascorbate decreased with increasing distance from the LDL surface. From the competition between the spin probe and alpha-tocopherol in scavenging radical, it was found that the efficacy of radical scavenging by alpha-tocopherol became smaller as the radical went deeper into the interior of LDL. On the other hand, 2,2,5,7,8-pentamethyl-6-chromal spared the spin label regardless of the position of nitroxide. The antioxidant activity of chromanols against LDL oxidation increased with decreasing length of isoprenoid side chain at the 2-position. All these results were interpreted by location and low mobility of alpha-tocopherol in LDL. The tocopherol mediated propagation was observed notably at low rate of radical flux, but this was suppressed by reductant such as ascorbic acid and ubiquinol.

Sphingomyelin changes in rat cerebral cortex during focal ischemia.

To better define the sphingolipid metabolism during focal brain ischemia, levels of ceramide, sphingomyelin, cerebroside and gangliosides were determined in rat cerebral cortex during focal ischemia produced by occlusion of the middle cerebral artery. Sphingomyelin began to decrease at 2 hours of ischemia and continued to decrease for 96 hours. In contrast, ceramide increased at 6 hours and increased to 4.2-fold at 96 hours after ischemia, and the fatty acid composition of ceramide was solely nonhydroxylated fatty acid similar to sphingomyelin. Hydroxylated fatty acid-linked cerebroside decreased at 6 hours of ischemia, whereas any significant decrease of nonhydroxylated fatty acid-linked cerebroside didn't occur for 96 hours of ischemia. There were no measurable changes in the levels of gangliosides. These results suggested that ceramide was produced in the cerebral cortex by the breakdown of sphingomyelin during early ischemia.

Therapeutic time window in the penumbra during permanent focal ischemia in rats: changes of free fatty acids and glycerophospholipids.

To better define a therapeutic time window for reducing the extent of damage in ischemic penumbra, the time courses of changes in the glycerophospholipid and free fatty acid (FFA) levels were determined in the rat cerebral cortex following induction of the permanent focal ischemia. Focal ischemia induced a biphasic increase in FFA levels in the cerebral cortex, which had been recognized as the ischemic penumbra during the early stages after permanent occlusion of the middle cerebral artery (MCA). The first increase in FFA levels, in which the polyunsaturated fatty acid (PUFA) contained a large number of arachidonic acid (C20:4) molecules, began at 30 min and reached a peak at 1 h, followed by transient return to each sham level 2-6 h after the onset of MCA occlusion. Thereafter, the delayed increase in FFA levels, showing more increases of docosahexaenoic acid (C22:6) molecules than the C20:4 in PUFA compositions, occurred at 24 h. In contrast, the levels of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) decreased rapidly at 30 min of ischemia and returned transiently to each sham level at 1-6 h. The levels of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), including polyphosphoinositides (PIPs), began to decrease significantly during the late stages, i.e., 24 h after induction of ischemia. These results suggest that the time-dependent changes in FFA and PIPs levels during the early stages of ischemia (until 6 h after induction) might be an important determinant of the subsequent neuronal death in the ischemic penumbra and that the breakdown of glycerophospholipids in the later stages after the induction of focal ischemia was associated with the development of infarction in the cerebral cortex.

Isolation of isoflavonoids possessing antioxidant activity from the fermentation broth of Streptomyces sp.

Three antioxidant isoflavonoids characterized as 4',7,8-trihydroxyisoflavone (1), 3',4',7-trihydroxyisoflavone (2) and 8-chloro-3',4',5,7-tetrahydroxyisoflavone (3) were isolated from the cultured broth of Streptomyces sp. OH-1049. Among them, 3 is a novel isoflavonoid possessing a chlorine atom in the molecule. In in vitro studies, these antibiotics were found to possess antioxidant activity whereas showed almost no cytocidal activities against HeLa S3 cells.

Isolation and analysis of age-related fluorescent substances in rat testes.

Fluorescent substances were extracted from rat testicular tissue with 2,2-dimethoxypropane (DMP) and analyzed by 2-dimensional thin layer chromatography (TLC). One substance that accumulated with increasing age of the animals was isolated and analyzed quantitatively by spectrophotofluorometry using quinine sulfate as a standard. This substance, which was designed as an age-related fluorescent substance (ARFS), exhibited an excitation maximum at 355 nm and an emission maximum at 490 nm. Its fluorescence was quenched by metal chelators and at alkaline pH, indicating it contained a conjugated Schiff base structure. Quantitative analysis of this substance in the testes of rats 1, 2, 11 and 20 months of age showed that it increased linearly with age. The relation of this substance to aging also was indicated by its detection in animals of different ages fed diets of both low and high unsaturation.

Covalent binding of peroxidized linoleic acid to protein and amino acids as models for lipofuscin formation.

The fluorescent substances produced by the reaction of linoleic acid hydroperoxides (LOOH) with ca. 20 different amino acids and bovine serum albumin (BSA) were studied. Only the amino acids, lysine, glycine, arginine, histidine and phenylalanine, gave products with strong fluorescent properties. Products of lysine had a fluorescence intensity of ca. 10 times those of glycine and 100 times those of phenylalanine. The N-acylation of amino acids greatly reduced the fluorescence of the products of the reaction except lysine and arginine. The fluorescence of the products of the reaction of LOOH with N-acetyl BSA was only ca. 25% of the control BSA under the same conditions. It appeared that the substances formed from the reaction of LOOH with BSA were crosslinked polymers as evidenced by column chromatography and polyacrylamide gel electrophoresis. These products were insoluble in common organic solvents and their fluorescent intensities correlated well with the thiobarbituric acid (TBA) test. These observations appear to be highly important in the formation of lipofuscin substances, particularly those associated with the aging pigments which accumulate during aging in mammalian tissues.

Antioxidative activity of 3,4-dihydroxyphenylacetic acid and caffeic acid in rat plasma.

The purpose of the present paper is to study and compare in vitro the inhibitory effect of 3,4-dihydroxyphenylacetic acid (DOPAC) and caffeic acid (CA) on lipid peroxidation in rat plasma. Rat plasma was oxidized at 37 degrees C by the radical initiators 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) or 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (MeO-AMVN). The consumption of endogenous alpha-tocopherol (alpha-TOH) and the accumulation of conjugated diene hydroperoxides were measured by high-performance liquid chromatography and by ultraviolet spectroscopy, respectively. Alpha-TOH was consumed at the same rate in the presence of 20 mM AAPH or 2 mM MeO-AMVN. DOPAC and CA suppressed the alpha-TOH consumption in a dose-dependent manner. A concentration of 50 microM of both phenolic acids was sufficient to induce a lag phase and to delay the rate of alpha-TOH consumption. The effect was more pronounced in rat plasma oxidation by AAPH than by MeO-AMVN. CA spared vitamin E more effectively than DOPAC in both oxidations. DOPAC and CA suppressed the formation of conjugated diene hydroperoxides. DOPAC and CA at concentration 50 microM suppressed alpha-TOH consumption during oxidation of soybean phosphatidylcholine (2.8 mM) multilamellar vesicles containing 15 microM alpha-TOH, in which the lipophilic initiator 2,2'-azobis (2,4-dimethylvaleronitrile) (6 mM) was incorporated. In conclusion, we demonstrated that DOPAC and CA in micromolar concentrations have antioxidant activity in rat plasma, a medium very close to the conditions in vivo, suggesting that supplementation with the phenolic acids will provide significant antioxidant protection.