Lipid peroxidation in membranes and low-density lipoproteins: similarities and differences

Lipid peroxidation has been a central aspect of studies of the nature of free radical species and their origin in biological systems. Moreover, there has been a growing interest in lipid peroxidation based on evidence that biologically active products are formed that influence cell function and the course of major human diseases. A review of the work in this area is contributed by Lars Ernster is presented with an emphasis on the mechanisms by which lipid peroxidation is initiated in biological lipid systems. Based on what was described for metal catalyzed oxidation of cell membranes, and the seminal studies on cytochrome P-450-mediated lipid peroxidation, several parallel and distinct aspects of lipid peroxidation are described. A key distinction between lipid peroxidation in cell membranes and lipoproteins reveals aspects of free radical initiated reactions involving proteins and lipids that determine pro- vs. anti-oxidant outcomes, and the role of lipid structure and order in delineating the progress of oxidation.     

4-Hydroxynonenal,an end-product of lipid peroxidation,induces apoptosis in human leukemic T- and B-cell lines

4-Hydroxynonenal (HNE) is the major aldehydic product resulting from lipid peroxidation and has been implicated as involved in several pathological conditions. In our continuing studies on the role of membranes and lipid peroxidation in the induction of apoptosis, we investigated the effect of HNE on cultured human malignant immune system cells. Two cell lines were utilized; MOLT-4, a human T-cell leukemia cell line, and Reh, a human B-cell lymphoma cell line. A 10 min treatment with 0.01 mM HNE resulted in the apoptotic death, as determined by flow cytometric and morphological analyses, of both cell lines within 24 h. MOLT-4 cells exhibited the manifestations of impending apoptotic death much sooner than did Reh cells, indicating that MOLT-4 cells were more sensitive or not as efficient at detoxifying HNE than were Reh cells. These results suggest that peroxidative damage to cellular membranes resulting in the production of HNE may be a trigger for the induction of apoptosis in immune system cells.     

4-Hydroxynonenal, an end-product of lipid peroxidation, induces apoptosis in human leukemic T- and B-cell lines

4-Hydroxynonenal (HNE) is the major aldehydic product resulting from lipid peroxidation and has been implicated as involved in several pathological conditions. In our continuing studies on the role of membranes and lipid peroxidation in the induction of apoptosis, we investigated the effect of HNE on cultured human malignant immune system cells. Two cell lines were utilized; MOLT-4, a human T-cell leukemia cell line, and Reh, a human B-cell lymphoma cell line. A 10 min treatment with 0.01 mM HNE resulted in the apoptotic death, as determined by flow cytometric and morphological analyses, of both cell lines within 24 h. MOLT-4 cells exhibited the manifestations of impending apoptotic death much sooner than did Reh cells, indicating that MOLT-4 cells were more sensitive or not as efficient at detoxifying HNE than were Reh cells. These results suggest that peroxidative damage to cellular membranes resulting in the production of HNE may be a trigger for the induction of apoptosis in immune system cells.     

The mechanism of action of 4-hydroxynonenal in cell injury

The effect of the C9 ketoaldehyde, 4-hydroxynonenal (HNE), a cytotoxic product of lipid peroxidation, on DNA, RNA and protein synthesis has been investigated in cells in culture. Macromolecular synthesis is powerfully inhibited by this agent which readily enters the lipid-rich membranes and is considerably more toxic than the polar ketoaldehyde, methyl glyoxal (MG). The entry of HNE into membranes lowers their glutathione GSH content. This is associated with an increased lipid peroxidation measured in vitro which is blocked by added GSH or alpha-tocopherol. It is proposed that this latter sequence of events is the underlying cause of the cytopathic effect of HNE in cells in culture.     

Ferric(III) ions inhibits copper(II)/hydrogen peroxide-catalyzing lipid peroxidation in human erythrocyte membranes.

1. Effect of ferric ions (Fe3+) on the lipid peroxidation catalyzed by copper ions (Cu2+) and hydrogen peroxide (H2O2) was studied in human erythrocyte membranes. 2. The formation of thiobarbituric acid-reactive products elicited by CuCl2/H2O2 was inhibited by FeCl3 in a concentration-dependent manner; 0.25 mM FeCl3 were enough to cause 50% inhibition of the formation of peroxides. 3. The inhibitory effect of FeCl3 is not due to competition against Cu2+. 4. FeCl3 inhibited the initiation, but did not inhibit the propagation of Cu2+/H2O2-catalyzing lipid peroxidation. 5. In the heat- or trypsin-treated erythrocyte membranes, FeCl3 had no inhibitory effect on Cu2+/H2O2-catalyzing lipid peroxidation. 6. Sodium azide, an inhibitor of catalase, had no effect on the inhibitory effect of FeCl3. 7. These results suggest that a protein factor(s), which is not catalase, is involved in the inhibition of Cu2+/H2O2-catalyzing lipid peroxidation by Fe3+.     

Changes in the fluorescence parameters of bound N-(1-pyrene) maleimide by lipid peroxidation of intestinal brush-border membranes

Using a fluorogenic thiol reagent, N-(1-pyrene)maleimide (NPM), we have examined of lipid peroxidation on the microenvironment around SH groups of the membrane proteins in porcine intestinal brush-border membrane vesicles. The lipid peroxidation of the membranes was performed with various concentrations of t-butylhydroperoxide (t-BuOOH) in the presence of 100 microM ascorbic acid and 10 microM Fe2+. Treatment of NPM-labeled membranes with these oxidizing agents resulted in a decrease of the fluorescence lifetime, suggesting modification of the environmental properties around the bound dye. Measurement of the steady-state fluorescence anisotropy of the labeled membranes indicated restriction of the motion of the bound dye by the lipid peroxidation membranes. This interpretation was further supported by an elevation of the transition temperature of the anisotropy, a decrease in the quenching rate constant of the fluorescence with acrylamide and a decrease in the SH reactivity of the membrane proteins for NPM by lipid peroxidation. Based on these results, the possibility of conformation changes in the vicinity of SH groups in the membrane proteins associated with lipid peroxidation has been discussed.     

Cytochrome P450 inactivation in microsomal membranes damaged by Fe2+-ascorbate-dependent lipid peroxidation

It has been established that Fe2+-ascorbate-dependent lipid peroxidation in rat liver microsome membranes is followed by the decrease of microsome cytochrome P450 content and the increase of the reduced haemoprotein inactivation rate. These changes are proportional to the amount of lipid peroxidation products (malonic dialdehyde) accumulating in the membranes.     

Phosphorescent analysis of lipid peroxidation products in isolated human erythrocyte membranes

The room temperature phosphorescence of lipid peroxidation products in the composition of isolated human erythrocyte membranes was registered, and its kinetic parameters were determined. The excitation and emission spectra of phosphorescence of lipid peroxidation products in the composition of erythrocyte membranes at 0 degree C measured. The nature of lipid peroxidation products possessing the phosphorescencing capacity was discussed. Based on the analysis of temperature dependences of the intensity and lifetimes of phosphorescence of lipid peroxidation products in the range -2 divided by 26 degrees C, it is concluded that the deactivation of excited triplet states of lipid chromophores was realized by the dynamic type.     

Sensitivity of ATPase-ADPase activities from synaptic plasma membranes of rat forebrain to lipid peroxidation in vitro and the protective effect of vitamin E

The in vitro effects of membrane lipid peroxidation on ATPase-ADPase activities in synaptic plasma membranes from rat forebrain were investigated. Treatment of synaptic plasma membranes with an oxidant generating system (H₂O₂/Fe²⁺/ascorbate) resulted in lipid peroxidation and inhibition of the enzyme activity. Besides, trolox as a water soluble vitamin E analogue totally prevented lipid peroxidation and the inhibition of enzyme activity. These results demonstrate the susceptibility of ATPase-ADPase activities of synaptic plasma membranes to free radicals and suggest that the protective effect against lipid peroxidation by trolox prevents the inhibition of enzyme activity. Thus, inhibition of ATPase-ADPase activities of synaptic plasma membranes in cerebral oxidative stress probably is related to lipid peroxidation in the brain.     

Radiation induced peroxidative damage: mechanism and significance

An interest has been generated in free radicals after the discovery of superoxide dismutase. These free radicals cause a number of diseases and are involved in the detrimental effect of ionizing radiation. Efforts have been made to understand their role in damage and death of the cell using lipid peroxidation process. Lipid peroxidation is an important effect of radiation on membranes, which apart from DNA, are critical targets of radiation action. This paper addresses the basic mechanism of radiation induced lipid peroxidation. Various factors, which determine the mode and magnitude of lipid peroxidation, are also discussed. Lipid peroxidation is shown to have importance in understanding the modifications of radiation effects. Efforts are made to show similarities between radiolytic and non-radiolytic lipid peroxidation. Recent findings related to the close link between radiation-induced lipid peroxidation and apoptosis are likely to open new avenues for future research and to develop new approaches for radiomodification of biological effects.     

Phosphorescent analysis of lipid peroxidation products in liposomes]

It was found that lipid peroxidation products incorporated into liposomes prepared from oxidized preparations of bovine heart phosphatidylcholine and the total lipid fraction of human erythrocyte membranes are able to phosphoresce at room temperature was studied. The temperature dependences of kinetic and spectral parameters of phosphorescence were measured. It is shown that mechanism of phosphorescence quenching of lipid chromophores has a dynamic nature. It is proposed to use endogenic molecules of the lipid peroxidation products capable of phosphorescence as intrinsic phosphorescence probes for studying the slow molecular dynamics of lipids in artificial and biological membranes in a millisecond range.     

Carotenoids are degraded by free radicals but do not affect lipid peroxidation in unilamellar liposomes under different oxygen tensions

It has been questioned whether carotenoids can act as antioxidants in biological membranes. Biological membranes can be modeled for studies of lipid peroxidation using unilamellar liposomes. Both carotenoid depletion and lipid peroxidation were increased with increasing oxygen tension in unilamellar liposomes. Carotenoids in such liposomes were found to be very sensitive to degradation by free radicals generated from iron and 2,2'-azobis(2-amidinopropane) dihydrochloride, but they were not protective against lipid peroxidation. Lycopene and beta-carotene were more sensitive to free radical attack than lutein, zeaxanthin, and beta-cryptoxanthin.     

Effects of membrane-stabilizing agents,cholesterol and cepharanthin,on radiation-induced lipid peroxidation and permeability in liposomes

Effects of two membrane-stabilizing agents, cholesterol and cepharanthin, on radiation-induced lipid peroxidation and membrane permeability were examined. Radiation-induced lipid peroxidation caused an increase in membrane permeability in phosphatidylcholine liposomes. The presence of cholesterol in liposomal membranes caused a decrease in the degree of membrane permeability in spite of an increased lipid peroxidation. On the other hand, cepharanthin suppressed both lipid peroxidation and the changes in permeability induced by radiation. The membrane-stabilizing effect of cholesterol against radiation-induced changes in permeability seemed to depend on the rigidification of membranes, which was estimated by ESR studies. Cepharanthin suppressed the degree of membrane permeability mainly by inhibiting the radiation-induced lipid peroxidation. However, cepharanthin did not exhibit a radical-trapping ability.     

Dynamics of lipid peroxidation of membranes in cells and mitochondrial fraction of neocortex in non- and preconditioned rats after severe hypobaric hypoxia

There was studied effect of severe hypobaric hypoxia and subsequent reoxygenation on level and dynamics of lipid peroxidation in membranes of neocortex cells and in mitochondriaenriched neocortex fraction of non-preconditioned rats and of rats preconditioned thrice with a moderate hypobaric hypoxia. The threefold hypoxic preconditioning increasing brain resistance has been shown to significantly prevent disturbance of lipid peroxidation processes in neocortex—one of the most hypoxia-sensitive brain structures—and to modify development of these processes in mitochondria.     

Reactive oxygen species and sperm cells

There is a dynamic interplay between pro- and anti-oxidant substances in human ejaculate. Excessive reactive oxygen species (ROS) generation can overwhelm protective mechanism and initiate changes in lipid and/or protein layers of sperm plasma membranes. Additionally, changes in DNA can be induced. The essential steps of lipid peroxidation have been listed as well as antioxidant substances of semen. A variety of detection techniques of lipid peroxidation have been summarized together with the lipid components of sperm membranes that can be subjected to stress. It is unsolved, a threshold for ROS levels that may induce functional sperm ability or may lead to male infertility.     

Induction of lipid peroxidation in the erythrocytes during cholesterol oxidation catalysed by cholesterol oxidase

Using the chemiluminescence technique to assay the activity of cholesterol oxidase it has been shown that enzymic oxidation of cholesterol to cholest-4-en-3-one red cell membranes is accompanied by accumulation of lipid peroxidation products--malonyl dialdehyde (MDA). The amount of MDA formed was dependent on the amount of cholesterol oxidized. The free radical scavenger 4-methyl-2,6-ditretbutylphenol, the transition metal chelator EDTA and catalase inhibited lipid peroxidation in red blood cells. The participation of OH radicals in the initiation of lipid peroxidation in red cell membranes in the course of cholesterol oxidation is discussed.     

The role of lipid peroxidation products in the effect of He-Ne laser on human blood leukocytes

The role of lipid peroxidation products formed in membranes of human blood leukocytes after irradiation with He-Ne laser was studied. It was found that low-intensity laser irradiation (0.3-1.6 J/cm2) leads to both cell activation and an increase in the content of lipid peroxidation products. The intensity of lipid peroxidation was analyzed by estimating the amount of TBA reactive products and lipid diene conjugates. Irradiation in the presence of an exogenous photosensitizer (protoporphyrin IX) enhanced the phenomena observed. The use of antioxidants (tocopherol and ionol) completely eliminated the laser-induced effects (changes in leukocyte activity and accumulation of lipid peroxidation products). These results can be explained by the fact that laser irradiation leads to the activation of lipid peroxidation in leukocyte membranes, which in turn enhances the response of cells to the stimulus (priming).     

Ganglioside-dependent factor inhibiting lipid peroxidation in synaptosomal membranes

The inhibitory effect of exogenous monosialoganglioside GM1 on lipid peroxidation was studied in synaptosomal membranes from rat brain. When this effect was studied over a wide GM1 concentration range, the biphasic kinetics was observed, the highest per cent of inhibition (70%) was found at GM1 concentration of 10(-9)- 10(-8) M. In liposomes made from lipids isolated from rat synaptosomal membranes the inhibition of lipid peroxidation by exogenous GM1 was much less pronounced (25% at maximum) it reached the saturation at ganglioside concentration of 10(-8)-10(-6) M. The thermal denaturation (90 degrees C), storage at 0 degrees C, addition of polymyxin B result in considerable decrease of inhibitory effect of GM1 on lipid peroxidation in synaptosomal membranes. On the contrary phorbol-12-myristate-13-acetate (10(-6)M) or Ca2+ (2.10(-3)M) inhibit lipid peroxidation in synaptosomal membranes, the presence of exogenous GM1 in incubation medium having additional inhibitory effect. Possible mechanisms of ganglioside participation in regulation of functional activity of excitatory membranes are discussed.     

Cytochrome P-450 involvement in the NADPH-dependent lipid peroxidation in human placental mitochondria

The NADPH-dependent lipid peroxidation in human placental mitochondria has been found to be inhibited strongly by amphenone B, aminoglutethimide and carbon monoxide, inhibitors of cytochrome P-450-mediated reactions, but was hardly affected by respiratory chain inhibitors. Cytochrome c, an exogenous electron acceptor which is known to compete with cytochrome P-450 for the reducing equivalents, showed an inhibitory effect on NADPH-dependent lipid peroxidation. The observed NADPH-dependent superoxide generation was also strongly inhibited by amphenone B and aminoglutethimide. Moreover, the lipid peroxidation in placental mitochondria was demonstrated to be stimulated by xanthine/xanthine oxidase added as superoxide generating system. This peroxidation was not affected by amphenone B and aminoglutethimide. On the other hand, the superoxide dismutase was found to inhibit both the xanthine oxidase- and NADPH-dependent lipid peroxidation. These data provide evidence that cytochrome P-450 is involved in NADPH-dependent mitochondrial lipid peroxidation. It is suggested that superoxide liberated from cytochrome P-450, in combination with iron, may be responsible for initiation of NADPH-dependent lipid peroxidation in human placental mitochondria.     

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.