Relationship between the hemolytic action of heavy metals and lipid peroxidation.

It is well known that some of the heavy metals have a hemolytic action, but the mechanisms responsible for this effect are not well established. In order to elucidate whether or not the hemolytic action of heavy metal ions is associated with the peroxidation of membrane lipids, the relationship between metal-induced hemolysis and the generation of malonaldehyde has been studied. The results obtained show that the metal-induced hemolysis is associated with the development of peroxidative processes in erythrocyte membranes. The peroxidation is caused by metals with and without pro-oxidant catalytic action. The level of the malonaldehyde products rises before the appearance of hemolysis which proves that the development of peroxidative processes precedes but does not result from hemolysis. The suggestion has been made that the peroxidation of membrane lipids is a possible mechanism of damage to the red cell membrane in metal-induced hemolysis.

The effect of lead on hemoglobin-catalyzed lipid peroxidation.

Lead significantly increases the rate of hemoglobin-catalyzed lipid peroxidation. The inhibition of this effect by superoxide dismutase and catalase suggests that superoxide radicals and H2O2 are somehow involved. Furthermore, lead catalyzes methemoglobin formation both in pure hemoglobin solutions and in hemolysates in which all protecting systems are present. It is speculated that a superoxide radical released in lead-catalyzed hemoglobin autoxidation may initiate the peroxidation of unsaturated fatty acids in red cell membrane. This hypothesis is supported by the fact that the preliminary conversion of oxyhemoglobin to methemoglobin decreases the rate of peroxidation. A conclusion is drawn that in native erythrocyte Pb2+ may exert a pro-oxidant effect, possibly by interacting with hemoglobin.

HgCl2 increases the methemoglobin prooxidant activity. Possible mechanism of Hg2+-induced lipid peroxidation in erythrocytes.

In an attempt to elucidate the mechanism of initiation of peroxidation in HgCl2-treated erythrocytes, the effect of HgCl2 on methemoglobin-catalyzed lipid peroxidation was studied. It was found that HgCl2 reinforces the prooxidant action of methemoglobin. This effect seems not to be due to dissociation or degradation of the hemoglobin molecule to heme-containing fragments or iron-containing products of low molecular weight. The results obtained indicate that Hg2+ increases the binding of oxy- and methemoglobin to liposomes. A suggestion is made that the acceleration of methemoglobin-catalyzed peroxidation by HgCl2 is mainly due to increased binding of methemoglobin to liposomes. On the basis of these results and the results obtained previously the possible mechanism of initiation of peroxidation in Hg2+-treated erythrocytes is discussed.

Hemoglobin-catalyzed lipid peroxidation in the presence of mercuric chloride.

In order to elucidate the possible mechanism of initiation of peroxidative processes in Hg2+-treated erythrocytes, the effect of HgCl2 on hemoglobin-catalyzed peroxidation of phospholipid liposomes was studied. It was demonstrated that HgCl2 significantly increases the rate of hemoglobin-catalyzed peroxidation. The addition of superoxide dismutase and catalase partially inhibits this effect. Furthermore, it was found that HgCl2 potentiates the hemoglobin oxidation. A suggestion was made that the acceleration of hemoglobin-catalyzed peroxidation by HgCl2 is associated at least in part with the increased production of superoxide anion radicals from hemoglobin.

Effect of vitamin E on lipid peroxidation and liver monooxigenase activity in experimental influenza virus infection.

Influenza virus infection was associated with development of oxidative stress in liver of mice, viz. increase in amount of lipid peroxidation products, decrease in cytochrome P-450 and NADP. H-cytochrome c-reductase activity, and inhibition of liver monooxygenases (aniline hydroxylase, ethylmorphine-N-demethylase, amidopyrine-N-demethylase and analgin-N-demethylase). These effects were most pronounced on the 7th day after virus inoculation as compared to the 5th one. Supplementation of mice with vitamin E before virus inoculation leads to liver protection against oxidative stress and toxicosis. A marked decrease of lipid peroxidation products and an increase of cytochrome P-450 and activities of monooxygenases was established. The stabilizing effect of vitamin E was dose-dependent and was most pronounced on the 5th day after virus inoculation as compared to the 7th one.

Effect of vitamin E supplementation on lipid peroxidation in blood and lung of influenza virus infected mice.

The influenza virus infection (A/Aichi/2/68) was associated with development of oxidative stress in lung and blood of mice, accompanied by an increase in levels of lipid peroxidation products (conjugated dienes and total malondialdehyde) and a decrease in endogenous amounts of natural antioxidant vitamin E. These effects were most pronounced on the 5th day after virus inoculation, in comparison with those on the 7th. Supplementation of mice with exogenous vitamin E before virus inoculation lead to lung and blood protection against lipid peroxidation. A marked decrease in lipid peroxidation products and an increase in vitamin E content was established in blood and lung on the 5th and 7th day after virus inoculation. The stabilizing effect of vitamin E is dose-dependent in blood and dose-independent in lung, and was most pronounced on the 5th day after virus inoculation in comparison with the 7th day.

A chemiluminescent method for measurement of activated oxygen forms in biological fluids and homogenates.

A chemiluminescent method for measuring the concentration of activated oxygen species (O-2 and H2O2) is described. Its main features are: high sensitivity (10(-9) M H2O2), its applicability to systems with high optical absorbance in the visible spectral region, a wide linear dynamic range, and the possibility for recording the kinetics of the processes, in which activated oxygen species are involved.

Pulmonary xanthine oxidase activity of rats exposed to prolonged immobilization stress.

This study was designed to study xanthine oxidase (XO) and xanthine dehydrogenase (XD) activity in the lung of rats exposed to prolonged restraining immobilization stress. Immobilization caused more than twofold increase of xanthine oxidase activity in the rat lung. The activity of xanthine oxidase decreased in lung homogenates incubated at -20 degrees C for 24 h. The same incubation of homogenates from control rats caused a non-significant increase of the activity. No measurable NAD(+)-dependent xanthine dehydrogenase activity could be established in the lungs of both control rats and rats subjected to immobilization. All rats revealed methylene blue-dependent xanthine dehydrogenase activity which was more than two-times higher in the immobilized animals. Incubation at -20 degrees C for 24 h increased the methylene blue-dependent xanthine dehydrogenase activity in homogenates from control rats and decreased the enzyme activity in homogenates from immobilized rats. A working hypothesis was proposed for the sequence of events explaining the results obtained: XO-catalyzed generation of activated oxygen species may take place in the initiation of lipid peroxidation in the lung of rats immobilized for prolonged periods of time.

The interaction of copper chloride with erythrocyte membrane as a source of activated oxygen species. A chemiluminescent study.

The possibility for the generation of activated oxygen species during the interaction between copper chloride and erythrocyte membranes was investigated. A chemiluminescent method for detecting superoxide radicals and hydrogen peroxide was used. It was found that the interaction of CuCl2 with erythrocyte membrane is accompanied with O-2 and H2O2 generation. On the base of this result it is proposed that the activated oxygen species generated by CuCl2-membrane interaction may be able to initiate peroxidative breakdown processes in erythrocytes eventually leading to haemolysis.

Relationships between serum levels of autoantibodies against oxidized low density lipoproteins, lipid-soluble antioxidants and apolipoprotein B in patients with coronary heart disease.

High affinity IgG autoantibodies against oxidized low density lipoproteins (oxLDLs), apolipoprotein B and lipid-soluble antioxidants--alpha-tocopherol and beta-carotene, were tested in patients with coronary heart disease. Correlation relationships between these parameters were analysed. Fifty one patients with coronary heart disease (37 males/14 females) defined as Q-wave myocardial infarction and/or stenosis of more than 50%, and 51 healthy blood donors (34 males/17 females) as controls participated in this study. LDLs were isolated by density gradient ultracentrifugation and oxidized with Cu2+. OxLDLs or native LDLs (nLDLs) were used as antigens in enzyme immunoassay (ELISA) to detect IgG autoantibodies in the serum. The contents of alpha-tocopherol and beta-carotene were measured by HPLC. Apolipoprotein B was determined by immunoturbidimetry. Correlation analysis of the parameters was carried out by Spearmann's test. Alpha-tocopherol was decreased significantly in the serum of patients with coronary heart disease (2.96+/-1.63 nmol/mg serum protein vs 6.23+/-2.28 nmol/mg serum protein in Control group) (p < 0.01). Also, the serum level of beta-carotene was decreased in patients with coronary heart disease (174.0+/-95.7 pmol/mg serum protein vs 313.2+/-141.5 pmol/mg serum protein in Control group) (p < 0.01), while apolipoprotein B was increased significantly (1.20+/-0.34 g/l in patients with coronary heart disease vs 0.86+/-0.23 g/l in Control group) (p < 0.001). In a previous study we established that the mean serum level of IgG autoantibodies against oxLDLs (expressed in optical density units) was about 2.5 times higher in patients with coronary heart disease as compared to control subjects (p < 0.001). A good positive linear correlation was observed between alpha-tocopherol and apolipoprotein B levels in Control group (r = 0.78, p < 0.001), as well as in the group of patients with coronary heart disease (r = 0.42, p < 0.001). Poor nonsignificant correlations were established between all another measured parameters. In conclusion, the lipid-soluble antioxidants--alpha-tocopherol and beta-carotene, are not informative with respect to the susceptibility of the serum to oxidative modifications and as to the extent of the subsequent humoral immune response. Presumably, the reduction of the correlation coefficient between apolipoprotein-B and alpha-tocopherol in patients with coronary heart disease in comparison with control subjects could provide indirect information on modifications of apolipoprotein-B and on a decrease of its susceptibility to interact with this major lipid-soluble antioxidant in atherogenesis.

Effect of phenothiazines on activated macrophage-induced luminol-dependent chemiluminescence.

The inhibitory effect of some phenothiazine neuroleptics (chlorpromazine, levomepromazine, thioridazine, promethazine and trifluoperazine) on the ability of rat peritoneal macrophages to produce O2- during phagocytosis was investigated. The superoxide radical release was estimated by measuring the luminol-dependent chemiluminescence (CL). The effect of drugs was studied in the concentration range of 0.1-100 mumol/l. Additional experiments to determine the ability of the drugs to scavenge O2- were carried out. They included measuring the effect of phenothiazines on the luminol-dependent CL in systems with enzymatically (xanthine-xanthine oxidase) and non-enzymatically (KO2) generated O2-. The ability of phenothiazines to scavenge O2- was additionally tested by a "non-luminescence" method in which the superoxide concentration was determined spectrophotometrically by the reduction of nitro blue tetrazolium to formazan. All drugs tested decreased significantly CL of stimulated macrophages at concentrations greater than 1 mumol/l. The C50 values were between 0.45 and 1.74 mumol/l. Also phenothiazines were found to act as scavengers of O2-. However, this effect occurred at significantly higher drug concentrations. The C50 values for 50% scavenging of O2- in systems with different sources of O2- were in the concentration range of 5-160 mumol/l. These results suggested that phenothiazines predominantly affected the ability of macrophages to produce O2- during phagocytosis. The findings may provide some insight into the untoward effects of the drugs tested.

Serum level of IgG autoantibodies against oxidized low density lipoproteins and lag-phase of serum oxidation in coronary heart disease--inverse correlation.

High affinity IgG autoantibodies (ABs) against oxLDLs and lag-phase of serum oxidation were tested in patients with coronary heart disease (CHD). Fifty one (37 M/14 F) patients with CHD defined as Q-wave myocardial infarction and/or stenosis of more than 50% and 51 (34 M/17 F) healthy blood donors as controls participated in this study. LDLs were isolated by gradient ultracentrifugation and oxidized with CuSO4. The modified LDLs (oxLDLs) or native LDLs (nLDLs) were used as antigens in an enzyme immunoassay (ELISA) to detect IgG ABs in both groups. The serum was oxidized by CuSO4 and the oxidation was monitored spectrophotometrically at lambda = 234 nm to follow the formation of conjugated diens. The lag-phase (in minutes) is the interval between the addition of CuSO4 to the serum and the beginning of extensive oxidation (increasing absorbance at 234 nm). The concentrations of total cholesterol, triglycerides, HDL-cholesterol, apo-A and apo-B were measured as well. The mean level of ABs against oxLDLs (expressed as optical density units) was 0.590 +/- 0.330 in CHD-patients vs 0.244 +/- 0.200 in controls (p < 0.001). The lag-phase in minutes was 47.00 +/- 27.19 in CHD-patients and 80.23 +/- 26.30 in controls (p < 0.001). A negative correlation between ABs levels and lag-phase was established in CHD-patients (r = -0.69, p < 0.001) and controls (r = -0.62, p < 0.001). A poor correlation was established between ABs levels or lag-phase, on one hand, and other measured parameters. In conclusion, the lag-phase of serum oxidation by Cu2+ could be informative for LDL susceptibility to modification and the extent of consequent humoral immune response.

Study of activated oxygen production by some thiols using chemiluminescence.

2-3-dimercapto-1-propane sulfonic acid, D-penicillamine and meso-dimercapto succinic acid, drugs widely applied as antidota against metal poisoning, and cysteine and glutathione were studied with respect to their ability to generate and to scavenge superoxide anion radical. Superoxide production and scavenging were tested by means of luminol-dependent chemiluminescence. In presence of 1 mumol/l ADP-Fe3+ only cysteine and meso-dimercapto succinic acid induced chemiluminescence which could be inhibited by superoxide dismutase. 2,3-dimercapto-1-propane sulfonic acid, D-penicillamine and glutathione acted as O2- scavengers. These thiols inhibited O2(-)-dependent lipid peroxidation thus acting as antioxidants, whereas cysteine and meso-dimercapto succinic acid accelerated peroxidation. It is suggested that the toxic side effects of thiols may be due to their ability to generate or to scavenge free radicals.

Oxidative damage of the membrane lipids after electroporation.

Electric field pulses used for cell manipulation can cause irreversible cell damage. The mechanisms of the processes leading to such cell damage are very complicated. Our work demonstrated that exponential electric pulses with intensity of 2-7.5 kV/cm and duration of 5.2 ms were able to initiate peroxidation of fatty acid emulsions, liposomal membranes, red blood and Ehrlich ascite tumor cells. Electric pulses-induced peroxidation of erythrocyte membranes was followed by hemolysis. The electric treatment caused damage of E. coli membrane lipids which was accompanied by decreased cell survival. All these effects depended on field intensity. A relatively good correlation between pulse-induced peroxidation of erythrocyte membranes and hemolysis was observed. These results suggest that free radical mediated processes as lipid peroxidation and/or lipid degradation or fragmentation may be possible causes for electric pulses-induced irreversible cell damage.

Polyamines as modulators of lipoperoxidation.

The polyamines spermine and spermidine and the diamine putrescine inhibit lipid peroxidation in phospholipid liposome suspensions and rat liver homogenates. Using the chemiluminescence technique the antioxidant activity of polyamines was found to be due to reactions with the free radical intermediates of lipid peroxidation and/or superoxide radicals. Also, the antioxidant action of polyamines correlated with the amount of their amino groups: the antioxidant activity increases from putrescine to spermine.

Effect of oxidized phospholipids on the chemiluminescence of zymosan-activated leukocytes.

The effect of liposomes with different degree of oxidation on the zymosan-induced chemiluminescence (CL) of leukocytes was investigated. Non-oxidized liposomes did not influence significantly the CL response of leukocytes. In contrast previously oxidized liposomes increased CL even if liposomes and cells were separated by a dialysis membrane. Based on the observed increase of luminol-activated CL by oxidized liposomes, lipid peroxidation (LPO) products may be suggested to enhance cell activation. Zymosan-activated leukocytes did not affect the amount of malondialdehyde (MDA) in non-oxidized liposomes unless iron salts were added. Fe3+ + ADP added to non-oxidized liposomes triggered LPO. Both catalase and superoxide dismutase (SOD) prevented the effect. In experiments with previously oxidized liposomes the activated oxygen species produced by leukocytes did not increase the amount of MDA; on the contrary, they decreased it both in the presence and in the absence of chelated iron in the liposome suspension. The reaction between lipid hydroperoxide and O2- widely accompanied by CL. SOD decreased CL in this system by a factor of 1.7. On the other hand, peroxidized lipids may "opsonize" initially inactive particles: oxidized liposomes increased CL response of leukocytes similarly as opsonized zymosan routinely used as a phagocyte activator.

Oxidation of low density lipoproteins leads to disturbance of their binding with alpha-tocopherol.

The dynamics of binding of exogenous alpha-tocopherol (alpha-T) added to native or oxidatively modified LDLs (LDLs or oxLDLs) were investigated. Venous blood from 31 clinically healthy blood donors (15 males and 16 females) was used. LDLs were isolated by density gradient ultracentrifugation. LDLs were oxidized in vitro by CuSO4. LDLs or oxLDLs were enriched with exogenous alpha-T (initial concentrations: 0; 10; 20; 50; or 100 nmol per mg protein). The contents of alpha-T in LDLs or in oxLDLs were measured by HPLC. Lag-phase of LDL oxidation before or after saturation with alpha-T was recorded. Correlation analysis of the lag-phase of LDL oxidation and alpha-T content in LDLs was carried out by the method of Esterbauer et al. The experimental results demonstrated that: (i) alpha-T was incorporated into native LDLs to a higher extent as compared to oxLDLs. (ii) A saturation of LDLs and oxLDLs with alpha-T was observed. (iii) A positive correlation was observed between the duration of the lag-phase of LDL oxidation in vitro and the content of alpha-T in LDLs. (iv) Based on LDL saturation with alpha-T, the persons could be classified in two groups: LDLs from group I of 26 persons were found to incorporate exogenous alpha-T to the extent of 1.8 to 3 times its initial concentration; LDLs from group II of 5 persons incorporated little or no exogenous alpha-T. In the first group, oxidation of LDLs lead to a considerable decrease in alpha-T dependent variable k and to a moderate reduction of alpha-T-independent variable alpha in the equation of Esterbauer et al.: lag-phase = k.[alpha-tocopherol]+alpha. In the second group, oxidation of LDLs lead to insignificant changes in k, as well as in a. (v) According to the levels of k and a the native LDLs from the second group of 5 persons were very close to oxLDLs from the first group of 26 persons. Presumably, native LDLs from the second group of persons were initially oxidatively modified, and probably this will be a risk group in relation to atherogenic disorders.

Determination of malondialdehyde in biological samples by solid-phase extraction and high-performance liquid chromatography.

An analytical procedure for determination of malondialdehyde in tissue homogenates and blood serum was developed. A reaction with 2,4-dinitrophenylhydrazine is used followed by cleaning up of the derivative by solid-phase extraction. The samples were analyzed by isocratic high-performance liquid chromatography (HPLC) using a narrow-bore HPLC-column. A good separation of the 1-pyrazole peak from that of 2,4-dinitrophenylhydrazine was observed. A high linear dependence was established by the concentration of 1-pyrazole in the range of 10-5000 ng/ml. The detection limit of the method applied for tissue homogenates and blood serum was approximately 10 ng/ml or lower, and RSD of the method was 9% (n = 8). The peak of 1-pyrazole for these samples was well separated from the other matrix peaks. Experiments carried out evaluated that the solid-phase extraction might be an effective step of the sample preparation, significantly increasing the selectivity of the analysis and the life-time of the column. The method seems to be applicable for determination of malondialdehyde in different biological samples.

Antioxidant properties of rimantadine in influenza virus infected mice and in some model systems.

Influenza virus infection is associated with development of oxidative stress in lung and blood plasma, viz. increase of primary and secondary lipid peroxidation products. It was established that rimantadine treatment led to a decrease of the products of lipid peroxidation in tissues of mice experimentally infected with influenza virus A/Aichi/2/68 (H3N2). The effect is strongest in blood plasma (a decrease of about 50%) and weaker in the lung (about 20%). To elucidate the mechanism of this action of rimantadine, experiments were carried out with some model systems. The capability of rimantadine to scavenge superoxide radicals (scavenging properties) was studied in a system of xanthine-xanthine oxidase to generate superoxide. The amount of superoxide was measured spectrophotometrically by the NBT-test and chemiluminesce. Rimantadine does not show scavenging properties and its antioxidant effect observed in vivo, is not a result of its direct action on the processes of lipid peroxidation and/or interaction with antioxidant enzymes. The antioxidant properties of rimantadine were investigated by measurement of induced lipid peroxidation in a Fe2+ and (Fe2+ - EDTA) system with an egg liposomal suspension. Our findings with model systems do not prove an antioxidant or prooxidant effect of the drug on the processes of lipid peroxidation. Apparently, the observed antioxidant effect of rimantadine in vivo is not connected directly with free radical processes in the organism.

[Study of monoamine oxidase from human placenta mitochondria by the chemoluminescence method]. / Issledovanie monoaminoksidazy mitokhondrii platsenty cheloveka metodom khemiliuminestsentsii.

The activity of monoamine oxidase from human placenta mitochondria was determined with 2-phenylethylamine and benzylamine as substrates by the generation of hydrogen peroxide in a conjugated luminol-peroxidase system, using the chemiluminescence method. The monoamine oxidase was found to oxidize at a high rate MAO substrates and revealed high sensitivity to clorgyline, a specific inhibitor of monoamine oxidase type A. It was shown that the use of the chemiluminescence technique for determining the monoamine oxidase activity gives the results that are fully consistent with those obtained by other methods.