Inhibition of phospholipid hydrolysis by phospholipase A2 in microsomal and mitochondrial membranes subjected to lipid peroxidation

The rate of phospholipid hydrolysis in rat liver microsomal and mitochondrial membranes catalyzed by phospholipase A2 was shown to decrease after ascorbate + Fe2+-induced lipid peroxidation. The degree of inhibition was linearly dependent on the amount of lipid peroxidation products (malonyl dialdehyde) accumulated in the membrane. The decreased phospholipid hydrolysis rate in membranes after lipid peroxidation was registered using phospholipases A2 from two sources: porcine pancreas and bee venom. It was established that the inhibitory action of phospholipid peroxidation products was not linked with a direct effect on the enzyme and was not caused by depletion of phospholipase reaction substrates (as a result of lipid peroxidation). A possible role of lateral separation of oxidized and non-oxidized lipid phases in the mechanisms of inhibition of phospholipid hydrolysis by phospholipase A2 is discussed.     

A new synthetic route to 2-hydroxyl steroidal estrogens via acetylation and dakin oxidation

This paper describes a new two-step synthetic route to 2-hydroxy estrogens from either estrone or estradiol, via 2-acetylation followed by Dakin oxidation. This approach is characterized by its simplicity and excellence of yield.     

In vitro pro- and antioxidant properties of estrogens

The pro- and antioxidant properties of estrogens are subject of debate. The apparent discrepancy is largely caused by the chemical heterogeneity in the estrogen family and by their concentration and the environment in which they are found. To gain some insight into this debate, we determined whether estradiol (E(2)), estrone (E(1)), the 2-, 4- and 16alpha-hydroxyestrogens and also the 2- and 4-methoxyestrogens are: (1) good electron-donors; (2) capable of O(2) consumption and DNA strand break induction; (3) capable of inhibiting lipid peroxidation in vitro. E(2), E(1) and 16alpha-hydroxyestrone (16alpha-OHE(1)) were not pro-oxidants and were rather weak antioxidants, while the 2- and 4-hydroxyestrogens demonstrated both properties inducing DNA strand breaks damage as well as inhibiting lipid peroxidation. The 4-hydroxyestrogens consumed O(2) and induced DNA strand breaks to a level approximately 2.5-fold higher than the 2-hydroxyestrogens, but these hydroxyestrogens exhibited similar antioxidant capacity, as measured by inhibition of lipid peroxidation. The 4-methoxyestrogens cannot induce oxidative damage to DNA but can inhibit lipid peroxidation, although being less potent than the 2-methoxyestrogens and the 2- and 4-hydroxyestrogens. The 2-methoxyestrogens were both potent electron donors and inhibitors of lipid peroxidation. Although 2-methoxyestrogens cannot generate superoxide in vitro, they may also be considered pro-oxidants in vivo.     

Lipid peroxidation and phospholipase A2 activity in liposomes composed of unsaturated phospholipids: a structural basis for enzyme activation

The effect of lipid peroxidation on membrane structure and phospholipase A2 activity was studied using liposomes composed of bovine liver phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The phospholipids were mixed at set ratios and sonicated to yield small unilamellar vesicles. The liposome preparations were subjected to lipid peroxidation as induced by cumene hydroperoxide and hematin. Under these conditions, a sharp increase in lipid peroxidation was noted over a 30 min incubation period and was accompanied by loss of polyunsaturated fatty acids (PUFA). Liposomes enriched in PE were most extensively peroxidized with a preferred oxidation of this phospholipid. The extent of PC oxidation was also greater in liposomes containing the largest proportions of PE. Analysis of liposome anisotropy, via steady-state fluorescence polarization of diphenylhexatriene indicated that progressive increases in either PE content or the level of lipid peroxidation increased the apparent microviscosity of the vesicles. Moreover, lipid peroxidation increased anisotropy more effectively than variations in the ratios of PE vs. PC. Thus, peroxidation of 5-10% of the phospholipids produced the same anisotropy increase as a 20% increase in the ratio of PE vs. PC. Analysis of vesicle turbidity suggested that fusion was also more readily achieved through lipid peroxidation. When liposomes were incubated with 0.4 U/ml of snake venom phospholipase A2, a direct correlation was found between the degree of lipid peroxidation and the extent of phospholipid hydrolysis. The more unsaturated phospholipid, PE, was most extensively hydrolyzed following peroxidation. Increasing the proportion of PE also resulted in more extensive phospholipid hydrolysis. These findings indicate that lipid peroxidation produces a general increase in membrane viscosity which is associated with vesicle instability and enhanced phospholipase A2 attack. A structural basis for membrane phospholipase A2 activation as a consequence of lipid peroxidation is discussed in light of these findings.     

Formation of 9-hydroxy linoleic acid as a product of phospholipid peroxidation in diabetic erythrocyte membranes.

The increased production of oxygen-derived free radicals (OFR) and lipid peroxidation may contribute to vascular complications in diabetes. Some lipid peroxidation products have already been reported to be formed via glucose-induced oxidative stress. We have identified 9-hydroxy linoleic acid (9-OH-C18:2) in the red cell membrane phospholipid of diabetic subjects. We hypothesized that 9-OH-C18:2 would be formed in hydroxyl radical reactions to linoleic acid (C18:2) during glucose-induced oxidative stress, and confirmed that the formation of 9-OH-C18:2 was induced by ultraviolet (UV)-C irradiation to the synthetic C18:2. UV-C light generates highly reactive hydroxy radicals. C18:2 is confirmed to be the precursor of 9-OH-C18:2. To estimate the degree of oxidative damage to red cell membrane phospholipids, we developed a selective ion monitoring gas chromatography-mass spectrometric measurement for C18:2 and 9-OH-C18:2, following methanolysis of red cell membrane phospholipids. The relative peak height ratio of C18:2 to 9-OH-C18:2 (9-OH-C18:2/C18:2) was measured in phospholipid extracts of red cell membranes from healthy (n=29, 3.1+/-1.9%) and diabetic (n=27, 20. 9+/-16.1%) subjects. It was confirmed that 9-OH-C18:2/C18:2 is significantly (P<0.001) elevated in patients with diabetes. The measurement of 9-OH-C18:2/C18:2 in red cell membranes should be useful for assessing oxidative damage to membrane phospholipids in diabetes.     

Inhibitory effect of female hormones on lipid peroxidation

The female hormones estradiol, estrone, and estriol acted as antioxidants in the peroxidation of methyl linoleate by UV irradiation. All of them inhibited the peroxidation of microsomal lipids when they were added to the ADP-Fe3+ peroxidation system of rat liver microsomes. The efficiencies in the microsomal system were in the order of estradiol greater than estriol greater than estrone.     

Superoxide generation by NADPH-cytochrome P-450 reductase: the effect of iron chelators and the role of superoxide in microsomal lipid peroxidation

Superoxide generation, assessed as the rate of acetylated cytochrome c reduction inhibited by superoxide dismutase, by purified NADPH cytochrome P-450 reductase or intact rat liver microsomes was found to account for only a small fraction of their respective NADPH oxidase activities. DTPA-Fe3+ and EDTA-FE3+ greatly stimulated NADPH oxidation, acetylated cytochrome c reduction, and O(2) production by the reductase and intact microsomes. In contrast, all ferric chelates tested caused modest inhibition of acetylated cytochrome c reduction and O(2) generation by xanthine oxidase. Although both EDTA-Fe3+ and DTPA-Fe3+ were directly reduced by the reductase under anaerobic conditions, ADP-Fe3+ was not reduced by the reductase under aerobic or anaerobic conditions. Desferrioxamine-Fe3+ was unique among the chelates tested in that it was a relatively inert iron chelate in these assays, having only minor effects on NADPH oxidation and/or O(2) generation by the purified reductase, intact microsomes, or xanthine oxidase. Desferrioxamine inhibited microsomal lipid peroxidation promoted by ADP-Fe3+ in a concentration-dependent fashion, with complete inhibition occurring at a concentration equal to that of exogenously added ferric iron. The participation of O(2) generated by the reductase in NADPH-dependent lipid peroxidation was also investigated and compared with results obtained with a xanthine oxidase-dependent lipid peroxidation system. NADPH-dependent peroxidation of either phospholipid liposomes or rat liver microsomes in the presence of ADP-Fe3+ was demonstrated to be independent of O(2) generation by the reductase.     

Formation of 9-hydroxy linoleic acid as a product of phospholipid peroxidation in diabetic erythrocyte membranes

The increased production of oxygen-derived free radicals (OFR) and lipid peroxidation may contribute to vascular complications in diabetes. Some lipid peroxidation products have already been reported to be formed via glucose-induced oxidative stress. We have identified 9-hydroxy linoleic acid (9-OH-C18:2) in the red cell membrane phospholipid of diabetic subjects. We hypothesized that 9-OH-C18:2 would be formed in hydroxyl radical reactions to linoleic acid (C18:2) during glucose-induced oxidative stress, and confirmed that the formation of 9-OH-C18:2 was induced by ultraviolet (UV)-C irradiation to the synthetic C18:2. UV-C light generates highly reactive hydroxy radicals. C18:2 is confirmed to be the precursor of 9-OH-C18:2. To estimate the degree of oxidative damage to red cell membrane phospholipids, we developed a selective ion monitoring gas chromatography-mass spectrometric measurement for C18:2 and 9-OH-C18:2, following methanolysis of red cell membrane phospholipids. The relative peak height ratio of C18:2 to 9-OH-C18:2 (9-OH-C18:2/C18:2) was measured in phospholipid extracts of red cell membranes from healthy (n=29, 3.1±1.9%) and diabetic (n=27, 20.9±16.1%) subjects. It was confirmed that 9-OH-C18:2/C18:2 is significantly (P<0.001) elevated in patients with diabetes. The measurement of 9-OH-C18:2/C18:2 in red cell membranes should be useful for assessing oxidative damage to membrane phospholipids in diabetes.     

An investigation into the role of hydroxyl radical in xanthine oxidase-dependent lipid peroxidation

A model lipid peroxidation system dependent upon the hydroxyl radical, generated by Fenton's reagent, was compared to another model system dependent upon the enzymatic generation of superoxide by xanthine oxidase. Peroxidation was studied in detergent-dispersed linoleic acid and in phospholipid liposomes. Hydroxyl radical generation by Fenton's reagent (FeCl₂ + H₂O₂) in the presence of phospholipid liposomes resulted in lipid peroxidation as evidenced by malondialdehyde and lipid hydroperoxide formation. Catalase, mannitol, and Tris-Cl were capable of inhibiting activity. The addition of EDTA resulted in complete inhibition of activity when the concentration of EDTA exceeded the concentration of Fe²⁺. The addition of ADP resulted in slight inhibition of activity, however, the activity was less sensitive to inhibition by mannitol. At an ADP to Fe²⁺ molar ratio of 10 to 1, 10 mm mannitol caused 25% inhibition of activity. Lipid peroxidation dependent on the enzymatic generation of superoxide by xanthine oxidase was studied in liposomes and in detergent-dispersed linoleate. No activity was observed in the absence of added iron. Activity and the apparent mechanism of initiation was dependent upon iron chelation. The addition of EDTA-chelated iron to the detergent-dispersed linoleate system resulted in lipid peroxidation as evidenced by diene conjugation. This activity was inhibited by catalase and hydroxyl radical trapping agents. In contrast, no activity was observed with phospholipid liposomes when iron was chelated with EDTA. The peroxidation of liposomes required ADP-chelated iron and activity was stimulated upon the addition of EDTA-chelated iron. The peroxidation of detergent-dispersed linoleate was also enhanced by ADP-chelated iron. Again, this peroxidation in the presence of ADP-chelated iron was not sensitive to catalase or hydroxyl radical trapping agents. It is proposed that initiation of superoxide-dependent lipid peroxidation in the presence of EDTA-chelated iron occurs via the hydroxyl radical. However, in the presence of ADP-chelated iron, the participation of the free hydroxyl radical is minimal.     

The effect of oral contraceptive steroid therapy on the urinary metabolites of radioactive estrone and estradiol-17beta

Eight urinary metabolites of radioactive estrone and estradiol-17β (estrone, estradiol-17β, 2-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestrone 3-methyl ether, 16α-hydroxyestrone, 2-hydroxyestradiol and estriol) have been measured by reverse isotope dilution from young women on oral contraceptive therapy. There was a decrease in the sum of the 16α-hydroxy1ated metabolites in the Ketodase liberated fraction from the subjects taking ethynylestradiol containing preparations as compared to those taking preparations containing mestranol and those subjects who were taking no oral contraceptives. This report is also the first to document and measure 2-hydroxyestradiol as a urinary metabolite of radioactive estrone and estradiol.     

Lipid peroxidation and 4-hydroxy-2-nonenal formation by copper ion bound to amyloid-beta peptide

The lipid peroxidation product 4-hydroxy-2-nonenal (HNE) is proposed to be a toxic factor in the pathogenesis of Alzheimer disease. The primary products of lipid peroxidation are phospholipid hydroperoxides, and degraded reactive aldehydes, such as HNE, are considered secondary peroxidation products. In this study, we investigated the role of amyloid-beta peptide (A beta) in the formation of phospholipid hydroperoxides and HNE by copper ion bound to A beta. The A beta1-42-Cu2+ (1:1 molar ratio) complex showed an activity to form phospholipid hydroperoxides from a phospholipid, 1-palmitoyl-2-linoleoyl phosphatidylcholine, through Cu2+ reduction in the presence of ascorbic acid. The phospholipid hydroperoxides were considered to be a racemic mixture of 9-hydroperoxide and 13-hydroperoxide of the linoleoyl residue. When Cu2+ was bound to 2 molar equivalents of A beta(1-42) (2 A beta1-42-Cu2+), lipid peroxidation was inhibited. HNE was generated from one of the phospholipid hydroperoxides, 1-palmitoyl-2-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl) phosphatidylcholine (PLPC-OOH), by free Cu2+ in the presence of ascorbic acid through Cu2+ reduction and degradation of PLPC-OOH. HNE generation was markedly inhibited by equimolar concentrations of A beta(1-40) (92%) and A beta(1-42) (92%). However, A beta(1-42) binding 2 or 3 molar equivalents of Cu2+ (A beta1-42-2Cu2+, A beta1-42-3Cu2+) acted as a pro-oxidant to form HNE from PLPC-OOH. These findings suggest that, at moderate concentrations of copper, A beta acts primarily as an antioxidant to prevent Cu2+-catalyzed oxidation of biomolecules, but that, in the presence of excess copper, pro-oxidant complexes of A beta with Cu2+ are formed.     

Analysis and quantitation of free ceramide containing nonhydroxy and 2-hydroxy fatty acids, and phytosphingosine by high-performance liquid chromatography.

Reaction of ceramides containing nonhydroxy fatty acids with benzoyl chloride in pyridine at 70 degrees C for 1 hr resulted in N-benzoylation to form N,N-acyl,benzoyl derivatives; O-benzoylation also occurred. However with ceramides containing 2-hydroxy fatty acids and phytosphingosine only O-benzoylation occurred even on prolonged treatment. Only O-benzoylation occurred on reaction with benzoic an hydride. However, the benzoylation of ceramides with phytosphingosine could not be achieved with benzoic anhydride and this benzoylation was performed by reaction with benzoyl chloride at 70 degrees C for 4 hr. Because N,N-acyl,benzoyl derivatives of ceramides containing nonhydroxy fatty acids produced by treatment with benzoyl chloride overlap methyl benzoate on high-performance liquid chromatography, benzoic anhydride was preferable for benzoylation of ceramides with nonhydroxy and 2-hydroxy fatty acids. On the other hand, the reaction with benzoyl chloride at 70 degrees C for 4 hr was used for quantitation of benzoylated ceramides containing 2-hydroxy fatty acids and phytosphingosine. 3-(p-Phenylbenzoyl)estrone was used as an internal standard for both reactions and values for ceramides containing 2-hydroxy fatty acids obtained by the two reactions were in good agreement. This procedure was applied to measurement of the ceramide levels in the brain, liver, and kidney of rats during development. The levels of ceramides containing nonhydroxy and 2-hydroxy fatty acids in the brain, liver, and kidney increased to the adult levels and then remained unchanged. Ceramide with phytosphingosine was detected in the liver and kidney, where its concentration gradually increased with age, but it was not found in the brain. The composition of nonhydroxy fatty acids were also analyzed.     

Resolution and chromatographic configuration analysis of 2-hydroxy fatty acids

2-Hydroxyoctadecanoic acid was resolved into D and L isomers as salts of 1-phenylethylamine enantiomers The diastereomers of phenylethylamides of 2-hydroxy fatty acids and the corresponding derivatives with protected hydroxy group (acetyl, methyl, trifluoro-acetyl, trimethylsilyl) are well separated by thin-layer or gas-liquid chromatography. This allows a simple microanalysis of configuration and optical purity of 2-hydroxy fatty acids. With this method 2-hydroxy fatty acids from sphingomyelin of the honey-bee were shown to belong exclusively to the D series.     

Ethinyl estradiol: its interaction on blood-lipid

Lipophilicity (log P) of the drug plays an important role when drug reaches in the critical reaction site, i.e., active site cum receptors where the major constituent is lipid moieties. The drug molecule may be responsible for altering the lipid constituents, which is measured in terms of phosphorus content and can be explained by their fatty acid changes that are linked with biological effect of the drug. Having considered the lipophilicity of ethinyl estradiol (log P = 3.67), its interactions with the whole lipid of goat blood have been investigated along with fatty acid changes and lipid peroxidation phenomena. There was significant loss of phosphorus content of phospholipid and change of fatty acid constituents of whole lipid. This may be ascribed to binding affinity of ethinyl estradiol with lipid constituents in blood. Lipid binding potential of the drug may have role in its therapeutic effect. The peroxidation induced by drug has been quantitatively measured along with its suppression by using antioxidant. The results reveal that ethinyl estradiol caused significant extent of lipid peroxidation. Ascorbic acid, a promising antioxidant could significantly reduce drug induced lipid peroxidation.     

Phospholipid peroxidation induced by the catechol-Fe3+(Cu2+) complex: a possible mechanism of nigrostriatal cell damage

Ferric or cupric ions significantly promoted a peroxidative cleavage of unsaturated phospholipids in liposomes in vitro after coordinating with dopa and dopamine. Either alpha-tocopherol or desferrioxamine completely abolished the dopa-Fe3+ complex-induced phospholipid peroxidation, while superoxide dismutase, catalase, or sodium benzoate did not. A ferroxidase, ceruloplasmin, significantly inhibited the lipid peroxidation induced by the dopa-Fe3+ complex, indicating the importance of the reduction of the iron moiety in the complex for the lipid peroxidation. A possible mechanism of dopa-Fe3+ complex-induced phospholipid peroxidation is that oxene complexes, such as Fe(V) = O and Fe(IV) = O, produced abstract hydrogen atoms in unsaturated phospholipids to initiate lipid peroxidation.     

Biotransformation and bioconcentration of steroid estrogens by Chlorella vulgaris

The biotransformation and bioconcentration of natural and synthetic steroid estrogens by Chlorella vulgaris were investigated by using batch-shaking experiments with incubation for 48 h in the light or dark. Estradiol and estrone were interconvertible in both light and dark conditions; however, this biotransformation showed a preference for estrone. In the light, 50% estradiol was further metabolized to an unknown product. Apart from biotransformation, estrone, as well as hydroxyestrone, estriol, and ethinylestradiol, was relatively stable in the algal culture, whereas estradiol valerate was hydrolyzed to estradiol and then to estrone within 3 h of incubation. All of the tested estrogens exhibited a degree of partitioning to C. vulgaris; however, the concentrations of estriol, hydroxyestrone, ethinylestradiol, and estradiol valerate were always below the quantification limits. For estradiol and estrone, the partitioning of these estrogens in the algal extracts to the filtrates was <6% of the total amount present. The average concentration factor for estrone was ca. 27; however, the concentration factor for estradiol was not reported since no equilibrium was reached between the aqueous solution and that within the cells due to continuing biotransformation.     

Biotransformation and Bioconcentration of Steroid Estrogens by Chlorella vulgaris

The biotransformation and bioconcentration of natural and synthetic steroid estrogens by Chlorella vulgaris were investigated by using batch-shaking experiments with incubation for 48 h in the light or dark. Estradiol and estrone were interconvertible in both light and dark conditions; however, this biotransformation showed a preference for estrone. In the light, 50% estradiol was further metabolized to an unknown product. Apart from biotransformation, estrone, as well as hydroxyestrone, estriol, and ethinylestradiol, was relatively stable in the algal culture, whereas estradiol valerate was hydrolyzed to estradiol and then to estrone within 3 h of incubation. All of the tested estrogens exhibited a degree of partitioning to C. vulgaris; however, the concentrations of estriol, hydroxyestrone, ethinylestradiol, and estradiol valerate were always below the quantification limits. For estradiol and estrone, the partitioning of these estrogens in the algal extracts to the filtrates was <6% of the total amount present. The average concentration factor for estrone was ca. 27; however, the concentration factor for estradiol was not reported since no equilibrium was reached between the aqueous solution and that within the cells due to continuing biotransformation.     

Peroxidation and phospholipase A2 hydrolytic susceptibility of liposomes consisting of mixed species of phosphatidylcholine and phosphatidylethanolamine.

The relationship between lipid peroxidation and phospholipase A2 (PLA2) hydrolytic activity was studied using unilamellar vesicles (liposomes) as model membranes. Hydrolytic specificity was examined using vesicles prepared with pure bovine heart phosphatidylcholine (PC), bovine heart phosphatidylethanolamine (PE), or mixtures of these phospholipids, using two preparative procedures, i.e., sonication or extrusion. Lipid peroxidation was induced by incubating vesicles with cumene hydroperoxide and hematin at 37 degrees C. Determinations of the extent of peroxidation by means of diene conjugate content derived from second derivative spectra or by polarographic measurement of oxygen consumption rates provided a basis for comparing the extent of peroxidation of each phospholipid species to their subsequent hydrolysis by PLA2 (from Crotalus adamanteus). The extent of hydrolysis was determined through the release of arachidonic acid from either PC or PE. The PE distribution among the outer vs. inner leaflet of the membrane bilayer was nearly equal in sonicated vesicles, whereas most of the phospholipid was incorporated into the inner leaflet in extruded vesicles. The proportion of PE found in the inner leaflet progressively increased as the ratio of PE to PC increased in both sonicated and extruded vesicle preparations. Lipid peroxidation had no effect on PE distribution under the conditions examined. There was a clear preference for PC peroxidation for all vesicle compositions tested and PC was preferentially hydrolyzed by PLA2. This effect is proposed to result from a perturbation of membrane structure following peroxidation with assimilation of PC into PLA2-susceptible domains whereas PE peroxidation and hydrolysis is less affected in mixed PC/PE vesicles. Lipid peroxidation imposes an additional hydrolytic susceptibility over the effects exerted through the mixing of these phospholipids which is based on structural changes rather than formation of specific substrates for PLA2.     

Neutrality of amiodarone on the initiation and propagation of membrane lipid peroxidation.

Amiodarone is an iodinated benzofuran derivative largely used as an antiarrhythmic. Owing to the sensitivity of heart tissue to radicals, amiodarone was assayed for putative effects on lipid peroxidation studied in liposomes of soybean phosphatidylcholine and of bovine heart mitochondrial lipids used as model systems. Lipid peroxidations were initiated with Fe2+/ascorbic acid, and with peroxyl radicals generated from the azocompounds, AAPH and AMVN. These assays were carried out by following the quenching of the fluorescent probe cis-parinaric acid and by monitoring oxygen consumption. It has been ascertained that amiodarone does not protect or potentiate significantly the lipid peroxidation both lipidic systems. To fully ascertain the neutral behaviour of amiodarone in the lipid peroxidation process, the degradation of phospholipid acyl chains has been checked by GLC. These data confirm that amiodarone does not protect or potentiate lipid peroxidation to a significant extent. It is concluded that the limited effects of amiodarone might be related only indirectly with the lipid peroxidation. It is possible that the drug causes limited conformational and biophysical alterations in membrane phospholipid bilayers that can affect the process of peroxidation. Therefore, it is concluded that the therapeutic effects and benefits as a heart antiarrhythmic agent are independent of lipid peroxidation processes. Furthermore, the interaction of the drug with lipid bilayers does not induce significant conformational perturbations that could significantly favour or depress the peroxidation process.