Lipid Peroxidation. Definition of Experimental Conditions for Selective Study of the Propagation and Termination Phases

To find experimental conditions to selectively study the propagation phase of lipoperoxidation we studied the lipoperoxidation, catalyzed by FeCl₂, of liposomes in a buffering condition where Fe²⁺ autoxidation and oxygen active species generation does not occur. Liposomes from egg yolk phosphatidylcholine. prepared by vortex mixing, do not oxidize Fe²⁺: on the contrary they oxidize Fe²⁺ when prepared by ultrasonic irradiation. Dimyristoyl phosphatidylcholine liposomes prepared by ultrasonic irradiation do not oxidize Fe²⁺. During sonication polyunsaturated fatty acid residues autoxidize and lipid hydroperoxides (LOOH) are generated. Only when LOOH are present in the liposimes Fe²⁺ oxidizes and its rate of oxidation depends on the amount of LOOH in the assay. The reaction results in the generation of both LOOH and thiobarbituric acid reactive material (TBAR): it is inhibited by butylated hydroxytoluene and has a acidic pH optimum; it is not inhibited by catalase and OH' scavengers. The reaction studied. thus, appears to be the chain branching and propagation phase of lipoperoxidation. When we studied the dependence of Fe²⁺ oxidation, LOOH and TBAR generation on FeCl₂ concentration, we observed that at high FeCl₂ concentrations the termination phase of lipoperoxidation was prevalent. Thus. by selecting the appropriate FeCl₂ concentration the proposed experimental system allows study of either the propagation or the termination phase of lipoperoxidation.     

Lipid Peroxidation. Definition of Experimental Conditions for Selective Study of the Propagation and Termination Phases

To find experimental conditions to selectively study the propagation phase of lipoperoxidation we studied the lipoperoxidation, catalyzed by FeCl₂, of liposomes in a buffering condition where Fe²⁺ autoxidation and oxygen active species generation does not occur. Liposomes from egg yolk phosphatidylcholine. prepared by vortex mixing, do not oxidize Fe²⁺: on the contrary they oxidize Fe²⁺ when prepared by ultrasonic irradiation. Dimyristoyl phosphatidylcholine liposomes prepared by ultrasonic irradiation do not oxidize Fe²⁺. During sonication polyunsaturated fatty acid residues autoxidize and lipid hydroperoxides (LOOH) are generated. Only when LOOH are present in the liposimes Fe²⁺ oxidizes and its rate of oxidation depends on the amount of LOOH in the assay. The reaction results in the generation of both LOOH and thiobarbituric acid reactive material (TBAR): it is inhibited by butylated hydroxytoluene and has a acidic pH optimum; it is not inhibited by catalase and OH' scavengers. The reaction studied. thus, appears to be the chain branching and propagation phase of lipoperoxidation. When we studied the dependence of Fe²⁺ oxidation, LOOH and TBAR generation on FeCl₂ concentration, we observed that at high FeCl₂ concentrations the termination phase of lipoperoxidation was prevalent. Thus. by selecting the appropriate FeCl₂ concentration the proposed experimental system allows study of either the propagation or the termination phase of lipoperoxidation.     

Protective effect of a new anti-oxidant on the rat brain exposed to ischemia-reperfusion injury: inhibition of free radical formation and lipid peroxidation.

A new oligomeric derivative was synthesized from prostaglandin B2 and ascorbic acid, and its effect on rat brain ischemia-reperfusion injury was studied. Brain ischemia was produced in the rat by the combination of bilateral common carotid artery occlusion and hemorrhagic hypotension (30 mmHg, 20 min). The cerebral cortex was homogenized in the presence of the spin trap agent, N-tert-butyl-alpha-phenyl-nitrone (PBN). Spin-adducts were detected using an electron spin resonance spectrometer (EPR). Lipid peroxidation was estimated from the amounts of both thiobarbituric acid reactive substances (TBAR) and conjugated diene. In control experiments, reperfusion induced a burst of free radical formation which peaked at 5 min reperfusion time (238 +/- 41%). Lipid peroxidation increased significantly after 20 min of reperfusion (TBAR, 161 +/- 50%; conjugated diene, 160 +/- 29%). When the oligomeric derivative was administered (9 mg/kg i.p. 30 min before ischemic insult), it significantly reduced both spin adduct formation (103 +/- 13%) and lipid peroxidation (TBAR, 109 +/- 14%; conjugated diene, 97 +/- 33%).     

Vitamin A inhibits lipoperoxidation ascorbate-Fe++ dependent of rat kidney microsomes and mitochondria

In the present study it was investigated if Vitamin A supplementation could protect rat kidney microsomes and mitochondria from in vitro lipoperoxidation. After incubation of rat kidney microsomes and mitochondria in an ascorbate-Fe⁺⁺ system, at 37°C during 60 min, it was observed that the total cpm/mg protein originated from light emission (chemiluminescence) was lower in those organelles obtained from the control group when compared with the vitamin A supplemented group. The fatty acid composition of microsomes and mitochondria from control group was profoundly modified when subjected to nonenzymatic lipoperoxidation with a considerable decrease of arachidonic acid, C20:4 (n-6) and docosapentaenoic acid, C22:5 (n–3) in mitochondria and docosahexaenoic acid C22:6 (n-3) in microsomes.As a consequence the peroxidizability index, a parameter based on the maximal rate of oxidation of specific fatty acids was higher in the supplemented animals than in those used as control. These results indicate that Vitamin A may act as antioxidant protecting rat kidney microsomes and mitochondria from deleterious effect.     

Erythrocyte defects precede the onset of CCl4-induced liver cirrhosis. Protection by silymarin.

The time-course of some alterations produced in erythrocytes during the onset of CCl4-induced liver cirrhosis was studied in rats. Erythrocyte membranes were isolated to measure Na+, K+ and Ca+2-ATPase activities. Membrane lipid composition was determined to calculate the cholesterol/phospholipid ratio and serum samples were used to measure lipoperoxidation. The results demonstrated that as CCl4 treatment progressed, serum lipoperoxidation and membrane cholesterol/phospholipid ratio increased while ATPase activities decreased. ATPase activities in red blood cells of cirrhotic rats were 50% below normal values but those determined in cells of animals treated simultaneously with CCl4 + silymarin were significantly improved. Silymarin co-treatment also preserved the normal cholesterol/phospholipid ratio in the membranes. Our results suggest that the measure of ATPase activities in erythrocytes membranes could be a simple, safe and useful early marker of liver damage and also valuable to test the effectiveness of a given drug therapy.     

Is chemiluminescence an index of hepatic lipoperoxidation accompanying chloroform anesthesia?

Hepatic lipoperoxidation by highly reactive metabolites produced during biodegradation of chloroform is believed to cause delayed hepatic necrosis. Chemiluminescence occurs during interaction of these metabolites with a lipid membrane. We have made continuous in vivo measurements of hepatic light output in the phenobarbital-induced rat breathing either air or chloroform vaporized in air. The data permitted direct estimation of the time course of chloroform-induced lipoperoxidation. These potentially toxic events began 15 min after initiation of anesthesia and continued for the duration of the study. Chemiluminescence did not occur with inhalation of isoflurane, an anesthetic undergoing minimal biodegradation.     

Lipoperoxidation of rod outer segments of bovine retina is inhibited by soluble binding proteins for fatty acids

In the present study it was investigated if soluble-binding proteins for fatty acids (FABPs) present in neural retina show protection from in vitro lipoperoxidation of rod outer segment membranes (ROS). After incubation of ROS in an ascorbate-Fe⁺⁺ system, at 37°C during 90-120 min, the total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of soluble binding proteins for fatty acids. The fatty acid composition of rod outer segment membranes was substantially modified when subjected to non-enzymatic lipoperoxidation with a considerable decrease of docosahexaenoic acid (22:6 n-3) and arachidonic acid (20:4 n-6). As a result of this, the unsaturation index, a parameter based on the maximal rate of oxidation of specific fatty acids was higher in the native and control membranes when compared with peroxidized ones. A similar decrease of chemiluminescence was observed with the addition of increasing concentrations of native or delipidated FABP retinal containing fractions to rod outer segment membranes. These results indicate that soluble proteins with fatty acid binding properties may act as antioxidant protecting rod outer segment membranes from deleterious effect.     

High resolution dual laser flow cytometry.

Flow cytometers based on optical sensing utilize external light sources and fluorescent dyes to measure one or more specific components or properties of individual cells or subcellular particles in liquid suspension. To provide for independent excitation of two dyes used in double staining experiments we have constructed a high resolution flow cytometer that uses two laser beams to provide two wavelengths of excitation. These beams are separated spatially so that cells flow through them sequentially, with a time separation of about 20 musec. Since the dyes are excited sequentially their emission occurs at different times and their emission spectra may overlap without causing any difficulty in analysis. We have developed new light collection optics that permit up to four measurements to be made on each cell. This approach greatly increases the number of dye combinations that can be used in flow cytometry, thus removing a significant limitation of single illumination instruments.     

Daily Oscillation of Fatty Acids and Malondialdehyde in the Dinoflagellate Lingulodinium polyedrum

The levels of the fatty acids, cis eicosahexenoic acid and cis linolenic acid, as well as the extent of lipoperoxidation, measured as malondialdehyde (MDA), were analyzed in the photosynthetic marine dinoflagellate Lingulodinium polyedrum at different times during the light : dark (L : D) cycle. Levels of MDA were twice as high during the day phase than during the night phase. This may be related to the circadian rhythm in photosynthesis as during photosynthetic electron flux, electrons can 'leak' and react with molecular oxygen producing reactive oxygen species (ROS) which in turn react with lipids, proteins, and DNA among other biomolecules. Fatty acid levels were highest during the day phase. Our findings indicate that unsaturated fatty acids are more susceptible to attack and degradation when L. polyedrum is exposed to light and that the cells compensate for this by an increased fatty acid content during the day. Excessive lipoperoxidation during the light phase could result in a higher level of chloroplast or plasma membrane disruption leading to cell death.     

Diabetes impairs the enzymatic disposal of 4-hydroxynonenal in rat liver

This study assesses whether the HNE accumulation we formerly observed in liver microsomes and mitochondria of BB/Wor diabetic rats depends on an increased rate of lipoperoxidation or on impairment of enzymatic removal. There are three main HNE metabolizing enzymes: glutathione-S-transferase (GST), aldehyde dehydrogenase (ALDH), and alcohol dehydrogenase (ADH). In this study we show that GST and ALDH activities are reduced in liver microsomes and mitochondria of diabetic rats; in contrast, ADH activity remains unchanged. The role of each enzyme in HNE removal was evaluated by using enzymatic inhibitors. The roles of both GST and ALDH were markedly reduced in diabetic rats, while ADH-mediated consumption was significantly increased. However, the higher level of lipohydroperoxides in diabetic liver indicated more marked lipoperoxidation. We therefore think that HNE accumulation in diabetic liver may depend on both mechanisms: increased lipoperoxidation and decreased enzymatic removal. We suggest that glycoxidation and/or hyperglycemic pseudohypoxia may be involved in the enzymatic impairment observed. Moreover, since HNE exerts toxic effects on enzymes, HNE accumulation, deficiency of HNE removal, and production of reactive oxygen species can generate vicious circles able to amplify the damage.     

Visible chemiluminescence from rat brain homogenates undergoing autoxidation. II. Kinetics of the luminescence decay

The visible luminescence emitted in the autoxidation of brain homogenates is only partially quenched when antioxidants are added at concentrations such that further oxidation is prevented. From the time course of the emission after antioxidant addition, it can be estimated that nearly 50% of the light arises from an intermediate that decays with a first order kinetics and with a lifetime of ca. 40 s at 32 degrees C. The remaining light arises from the decomposition of one or several intermediates, and show a kinetics that is independent of the incubation time. From the data obtained it is concluded that bimolecular free radical processes, such as the recombination of peroxy radicals, do not significantly contribute to the observed luminescence.     

Effect of hydroxyl radical on Na(+)-K(+)-ATPase activity of the brain microsomal membranes.

Sphingomyelin liposomes and brain microsomes were oxidized by exposure to hydrogen peroxide and ferrous ion. Lipid peroxidation were measured by the formation of thiobarbituric acid- reactive substances (TBAR). Hydroxyl radical was detected using the spin- trapping technique. Incubation of sphingomyelin liposomes with H2O2-Fe2+ resulted in an increase in the formation of TBAR. Na(+)-K(+)-ATPase activity was markedly inhibited and the SH group content decreased during incubation of microsomes in the presence of H2O2-Fe2+. Sodium ferulate effectively inhibited TBAR formation, protected Na(+)-K(+)-ATPase activity and prevented the oxidative modification of SH groups. Spin-trapping experiments showed that sodium ferulate effectively scavenged the hydroxyl radicals.     

A polychromator-based microspectrophotometer

A microspectrophotometer is a digital microscope used to measure absorption and fluorescence spectra. In this paper we describe a polychromator-based microspectrophotometer that performs in vivo absorption or emission measurements at the same time on different subcellular compartments such as photoreceptive and photosynthetic structures of algal cells. In this system, a flat field imaging concave grating polychromator is connected to the slit-shaped exit pupil of a light-guide probe mounted onto a microscope equipped with an epifluorescence module. The subcellular components, on which the spectra will be measured, are placed in the microscope field and finely adjusted. The outer bundle of the probe is used for centering the objects, while the central bundle of the probe, containing 19 light guides, is used for acquiring either transmitted or emitted light (i.e. fluorescence). The light transmitted or emitted by the subcellular components is collected by the probe mounted in the back focal plane of the ocular. The exit pupil of this probe, connected to a flat field imaging concave grating polychromator, produces a dispersion image that in turn is focused onto a digital slow scan cooled CCD camera. Absorption and emission spectra of algal subcellular compartments are presented.     

Regulation of lipid peroxidation induced by cumene hydroperoxide in the liver mitochondria of irradiated rats

A study was made of the accumulation of lipoperoxidation products and O2- generation induced by cumene hydroperoxide in mitochondria of irradiated rat liver. O2- generation and formation of lipoperoxidation products were found to be connected with the function of mitochondrial P-450 cytochrome. During the first 24 h following X-irradiation of rats with a dose of 10 Gy, the rate of O2- generation sharply increased and mitochondria could not regulate the intensity of lipoperoxidation with incubation medium tonicity being altered.     

Time-resolved delayed luminescence image microscopy using an europium ion chelate complex.

Improvements and extended applications of time-resolved delayed luminescence imaging microscopy (TR-DLIM) in cell biology are described. The emission properties of europium ion complexed to a fluorescent chelating group capable of labeling proteins are exploited to provide high contrast images of biotin labeled ligands through detection of the delayed emission. The streptavidin-based macromolecular complex (SBMC) employs streptavidin cross-linked to thyroglobulin multiply labeled with the europium-fluorescent chelate. The fluorescent chelate is efficiently excited with 340-nm light, after which it sensitizes europium ion emission at 612 nm hundreds of microseconds later. The SBMC complex has a high quantum yield orders of magnitude higher than that of eosin, a commonly used delayed luminescent probe, and can be readily seen by the naked eye, even in specimens double-labeled with prompt fluorescent probes. Unlike triplet-state phosphorescent probes, sensitized europium ion emission is insensitive to photobleaching and quenching by molecular oxygen; these properties have been exploited to obtain delayed luminescence images of living cells in aerated medium thus complementing imaging studies using prompt fluorescent probes. Since TR-DLIM has the unique property of rejecting enormous signals that originate from scattered light, autofluorescence, and prompt fluorescence it has been possible to resolve double emission images of living amoeba cells containing an intensely stained lucifer yellow in pinocytosed vesicles and membrane surface-bound SBMC-labeled biotinylated concanavalin A. Images of fixed cells represented in terms of the time decay of the sensitized emission show the lifetime of the europium ion emission is sensitive to the environment in which it is found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid peroxidation induced by cercosporin as a possible determinant of its toxicity

The photodynamic action of cercosporin was assayed in various kinds of natural and artificial membranes. Cerosporin induces lipoperoxidation of liposomes, rat liver and pea internode mitochondria and microsomes, estimated both as malondialdehyde (MDA) formation and O2 consumption. Cercosporin-induced lipoperoxidation is inhibited by either singlet oxygen quenchers, free radical trapping agents or EDTA. Superoxide anion (O2-), hydrogen peroxide and hydroxyl radicals (.OH) are not involved in the activity of cercosporin. In addition cercosporin, by chelating iron, lowers the lipoperoxidation induced by such a metal. Therefore cercosporin stimulates, through singlet oxygen production, the hydroperoxide formation but, at the same time, it inhibits the continuation of the iron-mediated free radical chain. The present results suggest that cellular lipid peroxidation has a certain relevance to toxic activity of cercosporin.     

Hyperglycemic Damage to Mitochondrial Membranes During Cerebral Ischemia: Amelioration by Platelet-Activating Factor Antagonist BN 50739

Abstract: The Pulsinelli-Brierley four-vessel occlusion model was used to study the consequences of hyperglycemic ischemia and reperfusion. Rats were subjected to either 30 min of normo- or hyperglycemic ischemia or 30 min of normo- or hyperglycemic ischemia followed by 60 min of reperfusion. In some animals, 2 mg/kg BN 50739, a platelet-activating factor receptor antagonist, was administered intraarterially either before or after the ischemic insult. The changes in mitochondrial membrane free fatty acid levels, phosphatidylcholine fatty acyl composition, and thiobarbituric acid-reactive material (TBAR) content plus the mitochondrial respiratory control ratio (RCR) were monitored. When the platelet-activating factor antagonist was present during normoglycemia, (a) the mitochondrial free fatty acid release both during and after ischemia was slowed, (b) reacylation of phosphatidylcholine following ischemia was promoted, and (c) TBAR accumulation during and following ischemia was decreased. The detrimental effects of hyperglycemia were muted when BN 50739 was present during ischemia. The RCR was preserved and phosphatidylcholine hydrolysis during ischemia was decreased. TBAR levels were consistently higher in hyperglycemic brain mitochondria both during and after ischemia. The RCR correlated directly with mitochondrial phosphatidylcholine polyunsaturated fatty acid content during ischemia and reperfusion. BN 50739 protection of mitochondrial membranes in brain may be influenced by tissue pH.     

Use of synchronously excited fluorescence to assess the accumulation of membrane potential probes in yeast cells

Evaluation of emission spectra of fluorescent probes used for the monitoring of membrane potential in microbial cells can be greatly facilitated by using synchronously excited spectroscopy (SES). This method permits the suppression of undesirable spectrum components (contributions due to scattered light or cell autofluorescence) and leads to considerable increase in monitored emission intensity and to narrowing of spectral peaks. It allows an efficient fractional decomposition of the probe fluorescence spectra into their free and bound dye fluorescence components. The usefulness of the method was tested by monitoring the accumulation of the fluorescent membrane potential probe diS-C3(3) in yeast cells, which serves as a qualitative measure of the membrane potential.     

Hematin catalysed autooxidation of hydroquinone or 1,2,4-benzenetriol

Autooxidation of hydroquinone (HQ) or 1,2,4-benzenetriol (BT), catalysed by hemin in the presence of dithiothreitol was studied in phosphate buffered saline. Inclusion of glutamate in the above reaction mixture resulted in the formation of thiobarbituric acid reactive products (TBAR) only in an aerobic atmosphere and was linear up to 2 h. Oxygen consumption was noticed during the reaction process. The formation of TBAR was linear with the increase in concentration of heme (1 – 4 μM), dithiothreitol (0.2 – 2 mM) or BT (0.17 – 0.85 mM). Linearity of TBAR formation from glutamate for up to 2 h was observed during the autooxidation of BT in the presence of heme. Besides glutamate, heme concentration dependent formation of TBAR from deoxyuridine or DNA was also observed. Almost complete inhibition of TBAR formation from glutamate, deoxyuridine or DNA was observed in the presence of catalase or superoxide dismutase (SOD). The presence of thiourea or mannitol in the reaction mixture caused substantial diminution of TBAR formation. Albumin or dimethyl sulfoxide also caused partial inhibition. Complete to partial inhibition observed in the presence of oxyradical scavengers in this study indicates that hemin catalysed autooxidation of BT results in the formation of reactive oxygen radicals.     

Inhibition of lipid peroxidation by prostaglandin oligomeric derivatives.

The inhibition of lipid peroxidation by oligomeric derivatives synthesized from prostaglandin E1 (PGE1) and PGB2 was studied using two rat models. In an in vitro model, the brain was exposed to decapitation-ischemia, the cortex was removed and homogenized, and the formation of thiobarbituric acid reactive substances (TBAR) was measured after exposing the homogenate to in vitro reoxygenation either in the presence or absence of oligomers. It was found that these oligomers could inhibit lipid peroxidation, and that their activities were higher than that of superoxide dismutase (SOD). In an in vivo administration model, either the oligomer or the vehicle was injected i.p. 30 min before decapitation. The brain was exposed to decapitation-ischemia, the cortex was homogenized and exposed to 'in vitro' reoxygenation, after which TBAR value was determined. Ester-type compounds had a greater activity than free-acid type compounds in inhibiting lipid peroxidation. A possible mechanism of the protective effect of these oligomers in ischemia/reperfusion injury may be to scavenge oxygen free radicals.