Chemiluminescence of lipid vesicles supplemented with cytochrome c and hydroperoxide.

The increase in light emission of hydroperoxide-supplemented cytochrome c observed on addition of lipid vesicles was related to the degree of unsaturation of the fatty acids of the phospholipids: dipalmitoyl phosphatidylcholine was without effect, whereas dioleoyl phosphatidylcholine and soya-bean phosphatidylcholine enhanced chemiluminescence 2- and 3-fold respectively. Effects on light-emission were similar to those on O2 uptake. The chemiluminescence of the present system was sensitive to cyanide and to the radical trap 2,5-di-t-butylquinol, indicating a catlytic activity of cytochrome c and the presence of free-radical species respectively. Lipid-vesicle enhanced chemiluminescence showed different kinetic behaviours, apparently depending on unsaturation: three phases are described for soya-bean phosphatidylcholine, whereas only one phase was present in mixtures containing dipalmitoyl and dioleoyl phospholipids. Chemiluminescence of lipid vesicles supplemented with cytochrome c and hydroperoxide showed similar kinetic patterns with H2O2 and primary (ethyl) and tertiary (t-butyl and cumene) hydroperoxides. Participation of singlet molecular oxygen, mainly on the phase III of chemiluminescence, is suggested by the increase of light-emission by 1,4-diazabicyclo[2.2.2]-octane as well as by data from spectral analysis.     

Formation of Keto and Hydroxy Compounds of Linoleic Acid in Submitochondrial Particles of Bovine Heart

To observe lipid peroxidation of additive-free submitochondrial particles, we incubated submitochondrial particles in the absence of exogenous irons and t-butyl hydroperoxide. After the incubation, the phospholipids were hydrolyzed by phopholipase A₂, and the fatty acid constituents were analyzed by high-performance liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry. Contrary to a commonly accepted theory, lipid peroxidation in the submitochondrial particles did not need the addition of NADH. In the phospholipid constituent fatty acids of the oxidized submitochondrial particles, derivatives of hydroperoxides of linoleic acid such as keto, hydroxy, trihydroxy, and hydroxyepoxy compounds were generated. Lipid peroxidation in the submitochondrial particles was not inhibited by the addition of catalase, superoxide dismutase, hydroxyl radical scavengers, or ethylenediaminetetraacetic acid, but was inhibited by the addition of KCN, antimycin-A, NADH, ubiquinol, deferoxamine mesylate, ascorbic acid, and -tocopherol. The cardiolipin–cytochrome c lipid peroxidation system could mimic the lipid peroxidation of the submitochondrial particles, in terms of linoleic acid products and the inhibitory patterns of radical scavengers and electron transfer chain inhibitors. Thus, lipid peroxidation in the submitochondrial particles seems to be due to phospholipid–hemoprotein lipid peroxidation systems such as the cardiolipin–cytochrome c system.     

Hydroperoxide-induced chemiluminescence of the perfused lung.

Light-emission of the perfused lung is induced by t-butyl hydroperoxide, giving chemiluminescence yields that oscillate between 800 and 1500 counts/s depending on the site and position of the lung. The response of the perfused lung to infusion with different hydroperoxides gives a pattern similar to that observed with the liver microsomal fraction; ethyl hydroperoxide shows a much higher chemiluminescence yield than the tertiary (t-butyl and cumene)hydroperoxides. Alveolar oedema affected the light-emission of the perfused lung depending on the time at which oedema developed, decreasing light emission on infusion of hydroperoxide in the oedematous lung and increasing it when oedema appeared after the maximal chemiluminescence yield was already achieved. Paraquat, administered in vivo, augmented light-emission by approximately 2-fold. The effect of paraquat was a time-dependent process. Lung chemiluminescence, compared with liver chemiluminescence, needed higher hydroperoxide concentration to induce light-emission.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis

Red cells exposed to t-butyl hydroperoxide undergo lipid peroxidation, haemoglobin degradation and hexose monophosphate-shunt stimulation. By using the lipid-soluble antioxidant 2,6-di-t-butyl-p-cresol, the relative contributions of t-butyl hydroperoxide and membrane lipid hydroperoxides to oxidative haemoglobin changes and hexose monophosphate-shunt stimulation were determined. About 90% of the haemoglobin changes and all of the hexose monophosphate-shunt stimulation were caused by t-butyl hydroperoxide. The remainder of the haemoglobin changes appeared to be due to reactions between haemoglobin and lipid hydroperoxides generated during membrane peroxidation. After exposure of red cells to t-butyl hydroperoxide, no lipid hydroperoxides were detected iodimetrically, whether or not glucose was present in the incubation. Concentrations of 2,6-di-t-butyl-p-cresol, which almost totally suppressed lipid peroxidation, significantly inhibited haemoglobin binding to the membrane but had no significant effect on hexose monophosphate shunt stimulation, suggesting that lipid hydroperoxides had been decomposed by a reaction with haem or haem-protein and not enzymically via glutathione peroxidase. The mechanisms of lipid peroxidation and haemoglobin oxidation and the protective role of glucose were also investigated. In time-course studies of red cells containing oxyhaemoglobin, methaemoglobin or carbonmono-oxyhaemoglobin incubated without glucose and exposed to t-butyl hydroperoxide, haemoglobin oxidation paralleled both lipid peroxidation and t-butyl hydroperoxide consumption. Lipid peroxidation ceased when all t-butyl hydroperoxide was consumed, indicating that it was not autocatalytic and was driven by initiation events followed by rapid propagation and termination of chain reactions and rapid non-enzymic decomposition of lipid hydroperoxides. Carbonmono-oxyhaemoglobin and oxyhaemoglobin were good promoters of peroxidation, whereas methaemoglobin relatively spared the membrane from peroxidation. The protective influence of glucose metabolism on the time course of t-butyl hydroperoxide-induced changes was greatest in carbonmono-oxyhaemoglobin-containing red cells followed in order by oxyhaemoglobin- and methaemoglobin-containing red cells. This is the reverse order of the reactivity of the hydroperoxide with haemoglobin, which is greatest with methaemoglobin. In studies exposing red cells to a wide range of t-butyl hydroperoxide concentrations, haemoglobin oxidation and lipid peroxidation did not occur until the cellular glutathione had been oxidized. The amount of lipid peroxidation per increment in added t-butyl hydroperoxide was greatest in red cells containing carbonmono-oxyhaemoglobin, followed in order by oxyhaemoglobin and methaemoglobin. Red cells containing oxyhaemoglobin and carbonmono-oxyhaemoglobin and exposed to increasing concentrations of t-butyl hydroperoxide became increasingly resistant to lipid peroxidation as methaemoglobin accumulated, supporting a relatively protective role for methaemoglobin. In the presence of glucose, higher levels of t-butyl hydroperoxide were required to induce lipid peroxidation and haemoglobin oxidation compared with incubations without glucose. Carbonmono-oxyhaemoglobin-containing red cells exposed to the highest levels of t-butyl hydroperoxide underwent haemolysis after a critical level of lipid peroxidation was reached. Inhibition of lipid peroxidation by 2,6-di-t-butyl-p-cresol below this critical level prevented haemolysis. Oxidative membrane damage appeared to be a more important determinant of haemolysis in vitro than haemoglobin degradation. The effects of various antioxidants and free-radical scavengers on lipid peroxidation in red cells or in ghosts plus methaemoglobin exposed to t-butyl hydroperoxide suggested that red-cell haemoglobin decomposed the hydroperoxide by a homolytic scission mechanism to t-butoxyl radicals.     

Prevention of lipid peroxidation by NAD(P)H in rat liver submitochondrial particles

In rat liver submitochondrial particles both NADH and NADPH inhibit lipid peroxidation induced by cumene hydroperoxide. Concomitantly with the inhibition of lipid peroxidation, NADH and NADPH strongly stimulate the peroxidase activity of rat liver submitochondrial particles. Rotenone slightly prevents both the protective effect on malondialdehyde formation and peroxidase activity. The peroxidase activity of rat liver submitochondrial particles was attributed to the NAD(P)H-mediated reduction of mitochondrial cytochrome P-450 which can act upon hydroperoxides, by decomposing them to alcohols.     

Low-level chemiluminescence of hydroperoxide-supplemented cytochrome c

Ferricytochrome c showed low-level chemiluminescence, with a light-emission measured of about 1×10³–3×10³ counts/s, when supplemented with organic hydroperoxides. Tertiary hydroperoxides (cumene hydroperoxide and t-butyl hydroperoxide) showed a saturation behaviour at about 5mm-hydroperoxide, whereas primary hydroperoxides showed a quadratic dependence on the hydroperoxide concentration. Chemiluminescence depended linearly on cytochrome c concentration, and optimal light-emission was observed at [t-butyl hydroperoxide]/[ferricytochrome c] ratios of 160–500. Hydroperoxide-supplemented ferricytochrome c consumed O₂ at a rate of 1.0μmol/min per μmol of cytochrome c; the rate of O₂ uptake was linearly related to the concentration of cytochrome c. The Soret absorption band of ferricytochrome c decreased about 64% after incubation with t-butyl hydroperoxide, whereas the 530nm band was almost totally abolished. Light-emission was (a) inhibited competitively by cyanide. (b) inhibited by singlet-oxygen quenchers (e.g. β-carotene), scavengers (e.g. dimethylfuran) and traps (e.g. histidine and tryptophan) and (c) increased by singlet-oxygen-chemiluminescence enhancer 1,4-diazabicyclo[2.2.2]-octane. Superoxide dismutase had no effect on the present system. The participation of free radicals is suggested by the effect of the radical trap 2,5-di-t-butylquinol. Singlet-oxygen dimol emission seems to be mainly responsible for the observed light-emission; a mechanism that can account for the major part of the present experimental observations is proposed.     

Independent biosynthesis of soluble and membrane-bound alkaline phosphatases in the suckling rat ileum

Oxygenation of anaerobically isolated brain and liver homogenates is associated with chemiluminescence and formation of lipid hydroperoxides, the latter determined by the thiobarbituric acid assay. Light emission and formation of malonaldehyde are 20-fold higher in the brain than in liver; chemiluminescence of both decays when accumulation of malonaldehyde ceases. Exogenous organic peroxides, such as t-butyl hydroperoxide, inhibit the light-emission response to oxygenation by brain homogenate, whereas they enhance that of liver homogenate. t-Butyl hydroperoxide-induced photoemission of liver homogenate shows a polyphasic kinetic pattern that is O2-dependent. The spectral analysis of chemiluminescence arising from brain and liver homogenates on oxygenation shows a spectrum with five emission bands at 420-450, 475-485, 510-540, 560-580 and 625-640 nm. These bands are subjected to intensity changes or shifts of the wavelength whenever t-butyl hydroperoxide is present, either inhibiting or stimulating light emission. The blue-band chemiluminescence, around 435 nm, is possibly due to the weak light emission arising from excited carbonyl compounds [Lloyd (1965) J. Chem. Soc. Faraday Trans. 61, 2182-2193; Vassil'ev (1965) Opt. Spectrosc. (USSR) 18, 131-135], whereas the presence of other bands suggests generation of singlet molecular oxygen either in the process triggered on oxygenation (lipid oxygenation) or after supplementation with organic hydroperoxides. We offer several explanations for the spectral analysis presented here.     

Peroxidation stimulated by lipid hydroperoxides on bovine retinal pigment epithelium mitochondria: effect of cellular retinol-binding protein

This study analyzes the effect of cellular retinol-binding protein (CRBP), partially purified from retinal pigment epithelium (RPE) cytosol, on the non-enzymatic lipid peroxidation induced by fatty acid hydroperoxides of mitochondrial membranes isolated from bovine RPE. The effect of different amounts (50, 75 and 100 nmol) of linoleic acid hydroperoxide (LHP), arachidonic acid hydroperoxide (AHP) and docosahexaenoic acid hydroperoxide (DHP) on the lipid peroxidation of RPE mitochondria was studied; RPE mitochondria deprived of exogenously added hydroperoxide was utilized as control. The process was measured simultaneously by determining chemiluminescence as well as polyunsaturated fatty acid (PUFA) degradation of total lipids isolated from RPE mitochondria. The addition of hydroperoxides to RPE mitochondria produces a marked increase in light emission that was hydroperoxide concentration dependent. The highest value of activation was produced by LHP. The major difference in the fatty acid composition of total lipids isolated from native and peroxidized RPE mitochondria incubated with and without hydroperoxides was found in the docosahexaenoic acid content, this decreased 40.90+/-3.01% in the peroxidized group compared to native RPE mitochondria. The decrease was significantly high: 86.32+/-2.57% when the lipid peroxidation was stimulated by 100 nmol of LHP. Inhibition of lipid peroxidation (decrease of chemiluminescence) was observed with the addition of increasing amounts (100-600 microg) of CRBP to RPE mitochondria. The inhibitory effect reaches the highest values in the presence of LHP.     

Interaction of PGBx and peroxides with cytochrome c and inhibition of lipid peroxidation

PGBx, a derivative of prostaglandin B1, stimulated the oxidation of cytochrome c in the presence of H2O2. Although the reaction was nonenzymatic, the apparent activation energies of 12 and 4.9 kcal above and below the transition at 21.5 degrees C were similar to those for oxidation by cytochrome oxidase. Depletion of H2O2 and oxidation of cytochrome c followed similar time courses, suggesting that H2O2 was consumed in the reaction. PGBx was a specific requirement, but organic hydroperoxides (ethyl and T-butyl) could replace H2O2. Low concentrations of ethyl or t-butyl hydroperoxide initially stimulated the oxidation of cytochrome c; this stimulation disappeared before completion of the oxidation, but was restored when the hydroperoxide concentration was renewed, suggesting that these hydroperoxides were probably also consumed in the reaction. The concentration of PGBx (8.9 microM) required for half-maximum stimulation of the oxidation was similar to the apparent Kd for its dissociation from oxidized cytochrome c (6.8 microM). Binding data and CD spectra suggested that a 1:1 complex between cytochrome c and PGBx was formed, altering the conformation of the heme region. This conformational change caused a shift of the Soret absorption peak from 410 to 406 nm and may be responsible for the enhanced oxidizability of the cytochrome c by H2O2. Cytochrome c inhibited lipid peroxidation in microsomes, an effect enhanced by the addition of PGBx. In the absence of lipid peroxidation, cytochrome c and PGBx stimulated NADPH oxidation via NADPH-cytochrome c reductase. Thus the inhibition of lipid peroxidation by cytochrome c and PGBx may involve either the removal of hydroperoxides or deviation of electron transfer away from the pathway for lipid peroxidation.     

Detection of Picomole Levels in Lipid Hydroperoxides by a Chemiluminescence Assay

A highly sensitive and simple chemiluminescent method for the quantitation of lipid hydroperoxides at the picomole level is described. The method is based on detecting the chemiluminescence generated during the oxidation of luminol by the reaction with hydroperoxide and cytochrome c under mild conditions. A semilogarithmic relationship was observed between the hydroperoxide added and the chemiluminescence produced. For lipid hydroperoxides, cytochrome c was a most favorable catalyst for generating the chemiluminescence, rather than cytochrome c heme peptide and horseradish peroxidase. This method had high sensitivity to methyl linoleate hydroperoxide, arachidonic acid hydroperoxide and cholesterol hydroperoxide, but low to /-butyl hydroperoxide, J-butyl perbenzoate, diacyl peroxides (lauroyl peroxode and benzoyl peroxide) and dialkyl peroxides (di-/-butyl peroxide and dicumyl peroxide).     

Retinal fatty acid binding protein reduce lipid peroxidation stimulated by long-chain fatty acid hydroperoxides on rod outer segments

In the present study we have investigated the effect of partially purified retinal fatty acid binding protein (FABP) against nonenzymatic lipid peroxidation stimulated by hydroperoxides derived from fatty acids on rod outer segment (ROS) membranes. Linoleic acid hydroperoxide (LHP), arachidonic acid hydroperoxide (AHP) and docosahexaenoic acid hydroperoxide (DHP) were prepared from linoleic acid, arachidonic acid and docosahexaenoic acid, respectively, by means of lipoxidase. ROS membranes were peroxidized using an ascorbate-Fe(+2) experimental system. The effect on the peroxidation of ROS containing different amounts of lipid hydroperoxides (LOOH) was studied; ROS deprived of exogenously added LOOH was utilized as control. The degradative process was measured simultaneously by determining chemiluminescence and fatty acid composition of total lipids isolated from ROS. The addition of hydroperoxides to ROS produced a marked increase in light emission. This increase was hydroperoxide concentration-dependent. The highest value of activation was produced by DHP. The decrease percentage of the more polyunsaturated fatty acids (PUFAs) (20:4 n6 and 22:6 n3) was used to evaluate the fatty acid alterations observed during the process. We have compared the fatty acid composition of total lipids isolated from native ROS and peroxidized ROS that were incubated with and without hydroperoxides. The major difference in the fatty acid composition was found in the docosahexaenoic acid content, which decreased by 45.51+/-1.07% in the peroxidized group compared to native ROS; the decrease was even higher, 81.38+/-1.11%, when the lipid peroxidation was stimulated by DHP. Retinal FABP was partially purified from retinal cytosol. Afterwards, we measured its effect on the reaction of lipid peroxidation induced by LOOH. As a result, we observed a decrease of chemiluminescence (inhibition of lipid peroxidation) when adding increasing amounts (0.2 to 0.6 mg) of retinal FABP to ROS. The inhibitory effect reaches its highest value in the presence of DHP (41.81+/-10.18%). Under these conditions, bovine serum albumin (BSA) produces a smaller inhibitory effect (20.2+/-7.06%) than FABP.     

Visible chemiluminescence induced by t-butyl hydroperoxide in red blood cell suspensions

Red blood cells from Wistar rats were exposed to milimolar concentrations of t-butyl hydroperoxide. Extensive hemoglobin oxidation (methemoglobin formation), t-butyl hydroperoxide cleavage (t-butanol formation) and peroxidation (measured by oxygen consumption and thiobarbituric acid reactive substances) was observed. Significant chemiluminescence was emitted by the system. Hemoglobin oxidation and t-butanol production were independent of oxygen pressure and free radical scavengers, however, luminescence was enhanced as oxygen pressure increased and it was reduced by addition of free radical scavengers. The spectral distribution of the light emitted suggests that the luminescence detected is not due to singlet oxygen dimol emission. The results are in agreement with a lipid peroxidative mechanism initiated by t-butoxy radicals produced in the interaction of hemoglobin and t-butyl hydroperoxide.     

Detection of chemiluminescence in lipid peroxidation of biological systems and its application to HPLC

A combined system of chemiluminescence detection and high performance liquid chromatography (CL–HPLC) was developed to determine primary peroxidation products in biological tissues, such as phosphatidylcholine hydroperoxide (PCOOH). The CL–HPLC assay consists of separation of lipid classes with HPLC and detection of hydroperoxide-specific chemiluminescence. Hydroperoxides react with heme compounds to produce oxidants as suggested by our early studies on tissue low-level chemiluminescence in which singlet molecular oxygen is generated as one of the excited species in several biological systems involving free radical events. In the CL–HPLC method, a cytochrome c–luminol mixture was used as a hydroperoxide-specific luminescent reagent, and the quantification of hydroperoxide was performed by detecting chemiluminescence due to the luminol oxidation caused by the oxidant produced during the lipid hydroperoxides with heme. The detection limit of PCOOH was 10 pmole hydroperoxide–O₂. PCOOH in normal human blood was found to be 10–500 pmol/ml plasma and significantly higher levels of PCOOH were observed in some hospitalized patients.     

Ferrous ion-mediated cytochrome P-450 degradation and lipid peroxidation in adrenal cortex mitochondria.

The relationship between the degradation reaction of cytochrome P-450 and lipid peroxidation was studied utilizing bovine adrenal cortex mitochondria. The two reactions were found to be closely correlated in terms of their response to storage of the mitochondrial preparation, stimulation by Fe2+, inhibition by EDTA and their initiation by cumene hydroperoxide. Both reactions were also found not to be inhibited by catalase, superoxide dismutase, 1,4-diazabicyclo-(2,2,2)-octane and alcohols, indicating that H2O2, superoxide, singlet oxygen and hydroxyl radicals do not participate in these reactions. Yet, diphenylamine proved to be a powerful inhibitor for both reactions, suggesting the involvement of a radical species. Cumene hydroperoxide could induce these two reactions at below 0.1 mM concentrations in the presence of molecular oxygen. The chemiluminescence observed during the Fe2+-mediated lipid peroxidation reaction which was not inhibited by either superoxide dismutase or 1,4-diazabicyclo-(2,2,2)-octane, was biphasic: one was a rapid burst; and the other was a slowly increasing emission. The latter portion of the emission of light coincided with the formation of malondialdehyde. These results indicate that in adrenal cortex mitochondria the degradation of cytochrome P-450 is closely related to lipid peroxidation.     

Hydroperoxide-metabolizing systems in rat liver.

1. Metabolism of added hydroperoxides was studied in hemoglobin-free perfused rat liver and in isolated rat hepatocytes as well as microsomal and mitochondrial fractions. 2. Perfused liver is capable of removing organic hydroperoxides [cumene and tert-butyl hydroperoxide] at rates up to 3--4 mumol X min-1 X gram liver-1. Concomitantly, there is a release of glutathione disulfide (GSSG) into the extracellular space in a relationship approx. linear with hydroperoxide infusion rates. About 30 nmol GSSG are released per mumol hydroperoxide added per min per gram liver. GSSG release is interpreted to indicate GSH peroxidase activity. 3. GSSG release is observed also with added H2O2. At rates of H2O2 infusion of about 1.5 mumol X min-1 X gram liver-1 a maximum of GSSG release is attained which, however, can be increased by inhibition of catalase with 3-amino-1,2,4-aminotriazole. 4. A contribution of the endoplasmic reticulum in addition to glutathione peroxidase in organic hydroperoxide removal is demonstrated (a) by comparison of perfused livers from untreated and phenobarbital-pretreated rats and (b) in isolated microsomal fractions, and a possible involvement of reactive iron species (e.g. cytochrome P-450-linked peroxidase activity) is discussed. 5. Hydroperoxide addition to microsomes leads to rapid and substantial lipid peroxidation as evidenced by formation of thiobarbituric-acid-reactive material (presumably malondialdehyde) and by O2 uptake. Like in other types of induction of lipid peroxidation, malondialdehyde/O2 ratios of 1/20 are observed. Cumene hydroperoxide (0.6 mM) gives rise to 4-fold higher rates of malondialdehyde formation than tert-butyl hydroperoxide (1 mM). Ethylenediamine tetraacetate does not inhibit this type of lipid peroxidation. 6. Lipid peroxidation in isolated hepatocytes upon hydroperoxide addition is much lower than in isolated microsomes or mitochondria, consistent with the presence of effective hydroperoxide-reducing systems. However, when NADPH is oxidized to the maximal extent as evidenced by dual-wavelength spectrophotometry, lipid peroxidation occurs at large amounts. 7. A dependence of hydroperoxide removal rates upon flux through the pentose phosphate pathway is suggested by a stimulatory effect of glucose in hepatocytes from fasted rats and by an increased rate of 14CO2 release from [1-14C]glucose during hydroperoxide metabolism in perfused liver.     

Arachidonic acid hydroperoxide stimulates lipid peroxidation in rat liver nuclei and chromatin fractions

Arachidonic acid, the most abundant polyunsaturated fatty acid in rat liver nuclei phospholipids is a major target of free radical attack, which induces lipid peroxidation. The non-enzymatic lipid peroxidation process in intact rat liver nuclei and in several chromatin fractions indicated that the most sensitive fatty acid for peroxidation is arachidonic acid C20:4 n-6. In this study, the effect of different amounts of arachidonic acid hydroperoxide on the lipid peroxidation of rat liver nuclei and chromatin fractions was studied; rat liver nuclei and chromatin fractions deprived of exogenous added hydroperoxide were utilized as control. The addition of arachidonic acid hydroperoxide to liver nuclei produces a marked increase in light emission that was hydroperoxide concentration dependent. The maximal peak of chemiluminescence displayed by the different chromatin fractions analyzed was observed between 20 and 80 min of incubation. The highest value of light emission was displayed by the high-density chromatin fractions, the 27.5 K fraction showed intermediate values of light emission, whereas the lowest density fraction produced very low chemiluminescence. A high correlation between arachidonic acid hydroperoxide concentration and chemiluminescence in the different chromatin fractions was observed. AC is Members of Carrera del Investigador Científico, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.     

Cytochrome P-450 deficiency and resistance to t-butyl hydroperoxide of hepatoma microsomal lipid peroxidation

Lipid peroxidation of microsomes from rat liver and Morris hepatoma 9618A was induced by means of tert-butyl hydroperoxide (t-BuOOH). In rat liver microsomes t-BuOOH stimulated an early formation of lipid hydroperoxides (LOOH) and an increasing accumulation of malondialdehyde; t-BuOOH was completely consumed and cytochrome P-450 was rapidly destroyed. In hepatoma microsomes (60% deficiency of cytochrome P-450) a remarkable inhibition of both malondialdehyde and LOOH was observed; t-BuOOH was consumed only partially and cytochrome P-450 was destroyed slowly. In the presence of aminopyrine, malondialdehyde production was inhibited to the same extent (about 70%) in normal and tumour microsomes. The concentration of t-BuOOH required to achieve half-maximal velocity of malondialdehyde accumulation was comparable in the two microsome types. It is proposed that the deficiency of cytochrome P-450 limits the activation of t-BuOOH to the free radical species which initiate lipid peroxidation. Low cytochrome P-450 content would also affect the LOOH-dependent propagation of lipid peroxidation.     

Hematin- and peroxide-catalyzed peroxidation of phospholipid liposomes

The effect of hydroperoxides on hematin-catalyzed initiation and propagation of lipid peroxidation was examined utilizing soybean phosphatidylcholine liposomes as model membranes. Polarographic and spectrophotometric methods revealed a bimodal pseudocatalytic activity for hematin. A slow initiation phase of peroxidation was observed in the presence of low peroxide concentrations, whereas a fast propagative phase was observed at higher peroxide levels. Peroxide levels were manipulated enzymatically by the combination of phospholipase A2 and lipoxidase or by the direct addition of linoleic acid hydroperoxide, cumene hydroperoxide, or hydrogen peroxide. In addition, the effect of two different techniques for liposome preparation, i.e., sonication and extrusion, were compared on the basis of peroxidation kinetics. High pressure liquid chromatography analysis showed that sonicated liposomes contained higher levels of endogenous peroxides than the extruded ones. These sonicated liposomes also exhibited more rapid peroxidation following hematin addition. Extruded liposomes were more resistant to hematin-catalyzed peroxidation but became better substrates when exogenous hydroperoxides were added. All three peroxides reacted with hematin during which decomposition of peroxide and irreversible oxidation of hematin took place. Spectral analysis of hematin indicated that a higher oxidation state of hematin iron may be transiently formed during reaction with hydroperoxides and accounts for the propagation of lipid peroxidation when reactions proceed in the presence of soybean phosphatidylcholine liposomes. Of the three peroxides studied, linoleic acid hydroperoxide was most efficient in supporting hematin-catalyzed lipid peroxidation. The relevance of our findings is discussed in terms of the concentration dependence for lipid peroxides in determining the rate and extent of radical propagation chain reactions catalyzed by heme-iron catalysts such as hematin. Variation of hematin and linoleic hydroperoxide concentrations may provide an efficient and reproducible method for inducing and manipulating the rates and extent of lipid peroxidation through facilitation of the propagative phase of lipid peroxidation. In addition, we address a problem inherent to in vitro studies of heme-catalyzed lipid peroxidation where preparations of peroxide-free membranes should be of concern.     

The role of selenium peroxidases in the protection against oxidative damage of membranes

The present review deals with the chemical properties of selenium in relation to its antioxidant properties and its reactivity in biological systems. The interaction of selenite with thiols and glutathione and the reactivity of selenocompounds with hydroperoxides are described. After a short survey on distribution, metabolism and organification of selenium, the role of this element as a component of the two seleno-dependent glutathione peroxidases is described. The main features of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are also reviewed. Both enzymes reduce different hydroperoxides to the corresponding alcohols and the major difference is the reduction of lipid hydroperoxides in membrane matrix catalyzed only by the phospholipid hydroperoxide glutathione peroxidase. However, in spite of the different specificity for the peroxidic substrates, the kinetic mechanism of both glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase seems identical and proceeds through a tert-uni ping pong mechanism. In the reaction cycle, indeed, as supported by the kinetic data, the oxidation of the ionized selenol by the hydroperoxide yields a selenenic acid that in turn is reduced back by two reactions with reduced glutathione. Special emphasis has been given to the role of selenium-dependent glutathione peroxidases in the prevention of membrane lipid peroxidation. While glutathione peroxidase is able to reduce hydrogen peroxide and other hydroperoxides possibly present in the soluble compartment of the cell, this enzyme fails to inhibit microsomal lipid peroxidation induced by NADPH or ascorbate and iron complexes. On the other hand, phospholipid hydroperoxide glutathione peroxidase, by reducing the phospholipid hydroperoxides in the membranes, actively prevents lipid peroxidation, provided a normal content of vitamin E is present in the membranes. In fact, by preventing the free radical generation from lipid hydroperoxides, phospholipid hydroperoxide glutathione peroxidase decreases the vitamin E requirement necessary to inhibit lipid peroxidation. Finally, the possible regulatory role of the selenoperoxidases on the arachidonic acid cascade enzymes (cyclooxygenase and lipoxygenase) is discussed.