Oxidative stress in patients with beta-thalassemia major.

The aim of this work is to study the level of oxidative stress in blood of beta-thalassemia major patients with transfusional iron overload and chelation therapy as a central pathological process. Beta-thalassemia major results in an increase in the concentration of lipid peroxidation products in blood plasma of more than 100% and in the intensity of chemiluminescence - about 20% in comparison to healthy controls. The activity of the antioxidant enzyme superoxide dismutase in the blood of beta-thalassemia major patients is decreased by more than 30% and the total antioxidant activity is diminished by about 70% compared to controls. Experimental data confirm the progression of oxidative stress in patients with beta-thalassemia major: activation of free radical processes and lipid peroxidation, decreased antioxidant capacity. Strong oxidative damage and essential alternations define these parameters as sensitive markers of oxidative stress in patients with beta-thalassemia major. The combination of effective iron-chelatory agents with natural or synthetic antioxidants can be extremely helpful in clinical practice in the regulation of the antioxidant status of patients with beta-thalassemia major.

Chemiluminescent in vitro estimation of the inhibitory constants of antioxidants ascorbic and uric acids in Fenton's reaction in urine.

The goal of this research was to measure in vitro the inhibitory constants of the antioxidants ascorbic and uric acid in urine, with lucigenin enhanced chemiluminescence (CL) in Fenton's system. Maximum CL emission is registered in urine containing H2O2 (5.10(-4) M), Fe2+ (5.10(-5) M), EDTA (5.10(-5) M), and chemical enhancer lucigenin (10(-4) M) at pH 5.5 and 36 degrees C. Ascorbic acid exhibits up to 4-fold stronger antioxidant effect than uric acid. The constants of antioxidant inhibition in urine were measured at concentrations 10(-3) and 10(-4) M: for ascorbic acid, 5.92 +/- 0.04 and 24.05 +/- 1.82 micromol.sec(-1); for uric acid, 1.60 +/- 0.02 and 21.45 +/- 0.97 micromol.sec(-1), respectively. Three phases of CL kinetics of urine are well observed: spontaneous CL (0-10 sec), fast flash of CL (10-50 sec), and latent period (50-300 sec). The antioxidant efficiency of ascorbic and uric acids in the final stage of catabolic processes in the body is discussed.

Effect of vitamin E and vitamin C combination on experimental influenza virus infection.

Successful antioxidant treatment of the so-called "free radical diseases" has been reported in the literature. In this study we examined the preventive effect of vitamin E and vitamin C, alone and in combination, on the damage caused by influenza virus infection (IVI). Male mice (ICR), infected with influenza virus A/2/68/(H3N2) (1.5 of LD(50)), were administered single once-daily doses of vitamin E (60 mg/kg b.w.) and vitamin C (80 mg/kg b.w.) intraperitoneally (3 days before virus inoculation). On the 5th and 7th day, respectively, after virus inoculation, animals were decapitated. Monooxygenase enzyme activity (ethylmorphine N-demethylase, amidopyrin N-demethylase, analgin N-demethylase, aniline hydroxylase, cytochrome P-450 content and NADPH-cytochrome C reductase [CCR]) was determined in liver 9000 x g supernatant. Primary and secondary products of lipid peroxidation (LPO; conjugated dienes [CD] and TBA-reactive substances) were measured in blood plasma, lung and liver 9000 x g supernatant. Vitamin E effectively restored LPO-levels increased by IVI. The effect of vitamin C was similar, but slighter. The combination (vitamin E + C) had greater effect on LPO levels than their separate administration. P-450-dependent monooxygenase activity was significantly restored and more pronounced cytochrome P-450 content and NADPH-CCR activity was noted. The preventive effect of vitamin E was stronger than the effect of vitamin C, but the combination (vitamin E + C) had the strongest effect. The superior protective effect of the combination is probably due to vitamin C's repairing effect on vitamin E's tocopheroxyl radical.

[Mechanisms of the effects of Ca2+ ions on lipid peroxidation]. / Mekhanizmy deistviia ionov Ca2+ na protsess perekisnogo okisleniia lipidov.

Mechanisms underlying Ca2+ effects on lipid peroxidation (LPO) induced in liposomes (from egg yolk lecithin) and UFsomes (from linolenic acid, methyl linolenate) with the aid of O2- -system (Fe2+ + ascorbate) were studied. It was shown that stimulation of lipid peroxidation by low Ca2+ concentrations (10(-6)-10(-5) M) was due to its ability to release Fe2+-ions bound to negatively charged (phosphate, carboxylic) lipid groups (of licethin, linolenic acid), thus increasing the concentration of catalytically active Fe2+. The inhibitory effect of high Ca2+ concentrations was caused by its interaction with superoxide anion-radicals and was not observed in LPO-systems, independent of O2- generation (e. g. Fe2+ + cumol hydroperoxide).

[Study of the mechanism of initiation of enzymatic NADPH-dependent lipid peroxidation in membranes of the endoplasmic reticulum]. / Izuchenie mekhanizma initsiirovaniia fermentativnogo NADPH-zavisimogo perekisnogo okisleniia lipidov v membranakh éndoplazmaticheskogo retikuluma.

The localization and mechanism of generation of active oxygen species in the enzymatic NADPH-dependent lipid peroxidation system in liver microsomes were studied. Using the spin-trapping method, the key role of active oxygen species in the initiation of NADPH-dependent enzymatic lipid peroxidation was confirmed. It was shown that active oxygen species are generated via consecutive one-electron reduction of the oxygen molecule by NADPH-cytochrome P-450 reductase.

[Initiation of nonenzymatic lipid peroxidation in a Fe2+-ascorbate-linolenate system]. / Initsiirovanie nefermentativnogo perekisnogo okisleniialipidov v sisteme Fe2+-askorbat-linolenat.

Using spin trapping method there were discovered and identified the radicals of linolenic acid formed when initiating its peroxidation by the system Fe2+-ascorbate. Mechanism of formation of linolenic acid radicals and their role in initiation of peroxidation were studied. A scheme of reactions of peroxidation initiation in the system Fe2+-ascorbate. linolenic acid is proposed.

[Lipid peroxidation in the myocardium in experimental infarct]. / Perekisnoe okislenie lipidov v miokarde pri éksperimental'nom infarkte.

Changes in the content of lipid peroxidation (LP) products and activities of antioxidant enzymes--superoxide dismutase, glutathione peroxidase and catalase in myocardium of rats after experimental infarction as well as after pretreatment with antioxidant ionol, beta-adrenoblocker inderal and verapamil, an inhibitor of slow Ca2+-channels have been studied. In the left ventricles of the control animals decreased levels of LP-products (Schiff bases and lipid hydroperoxides) have been registered as compared with right ventricles, accompanied by increased activity of antioxidant enzymes in the left ventricles. In experimental infarction the level of LP products increases and activity of antioxidant enzymes decreases both in ischemic and nonischemic regions of the heart. In nonischemic zone these changes can be prevented by pretreatment with inderal and ionol but not with verapamil.

[Lipid peroxidation in experimental myocardial infarct: the action of hyperbaric oxygenation]. / Perekisnoe okislenie lipidov pri éksperimental'nom infarkte miokarda: deistvie giperbaricheskoi oksigenatsii.

Rats with experimental myocardial infarction demonstrated decrease in the activity of superoxide dismutase and catalase and increase in the content of lipid peroxidation (LPO) products and Schiff bases both in and outside the area of necrosis. The combined ischemic damage and hyperbaric oxygenation resulted in the over additive effect of accumulation of LPO products in and outside the area of infarction. The data suggest that it is desirable to use antioxidants during hyperbaric oxygenation.

[Study of radicals formed in the interaction of organic hydroperoxides with ferric ions using the spin trap method]. / Issledovanie radikalov, obrazuiushchikhsia pri vzaimodeistvii organicheskikh gidroperekisei s ionami zheleza, metodom spinovykh lovushek.

The mechanism of free radical generation in the reaction of ferrous ion with t-butyl and linolenic acid hydroperoxide was investigated by spin trapping method. The t-butoxyl, methyl, linolenic acid alkoxyl and alkyl radical spin adducts EPR spectra were observed and identified.

[Role of lipid peroxidation in the pathogenesis of arrhythmias and the anti-arrhythmogenic action of antioxidants]. / Rol' perekisnogo okisleniia lipidov v patogeneze aritmii i antiaritmogennoe deistvie antioksidantov.

In the initial phase of its action on the contracting myocardium the inductor of lipid peroxidation (LPO) H2O2 displays marked positive ino- and chronotropic as well as relaxant effects which are, therefore, close to catecholamine effects. Since catecholamines activate LPO it suggests that such activation may be involved in the mechanism of their physiologic action. The prolongation of H2O2 action inevitably leads to the development of bradycardia and bradyarrhythmic arrhythmia which may ultimately end in cardiac arrest. The atrial resistance to H2O2 in animals exposed to stress is considerably diminished: in response to this inductor of LPO such animals develop more pronounced bradyarrythmic arrhythmia and cardiac arrest without the stage of the initially positive inotropic effect. The preincubation of the contracting atrium by HP-6, a LPO inhibitor of the hydroxypyridine class, checks the development of bradyarrhythmic arrhythmia and in many cases prevents cardiac arrest. Taken as a whole these data suggest that LPO activation may play an important role in the pathogenesis of cardiac rhythm disorders which may serve as substantiation for the use of antioxidants in the treatment and prevention of arrhythmias.

[Mechanisms of mixed-function oxidase system breakdown in the hepatic endoplasmic reticulum. The role of membrane phospholipid peroxidation]. / Mekhanizmy razborki sistemy oksigenaz so smeshannoi funktsiei v éndoplazmaticheskom retikulume pecheni. Rol' perekisnogo okisleniia membrannykh fosfolipidov.

It has been shown that endogenous lipid peroxidation (LPO) is an effective mechanism participating in the destruction of endoplasmic reticulum membranes (cytochrome P450) in liver. Antioxidants are able to control the rate of degradation of cytochrome P450 in vivo. Stock of the constitutive cytochrome P450 as compared with induced P450 is more resistive to LPO in vivo and in vitro. Spontaneous as well as induced by Fe2+--ADP+ +NADPH system destruction of cytochrome P450 due to accumulation of LPO products malonic dialdehyde (MDA) occurs during incubation of isolated rats hepatocytes. The LPO inhibitors (4-methyl-2,6- ditretbutilphenol , pyrogallol) stabilize cytochrome P450 preventing accumulation MDA hepatocytes. Degradation of cytochrome P450 in microsomes during trypsin proteolysis has been found to be enhanced by PLO induction. Efficiency of proteolysis depends on the way of induction and decreases in such an order: NADPH-- HNDH --ascorbate-dependent LPO. LPO may be considered as a trigger mechanism that makes some forms of cytochrome P450 available for endogenous proteases.

Mechanisms of disassembly of a mixed function oxygenase system in liver endoplasmic reticulum: I. The role of peroxidation of membrane phospholipids.

It was demonstrated that endogenous lipid peroxidation (LPO) is an effective mechanism of disassembly of endoplasmic reticulum membranes and cytochrome P-450 (P-448) in the liver. The rate of cytochrome P-450 (P-448) degradation in vivo can be regulated by free radical scavengers. The constitutive forms of cytochrome P-450 (P-448) are less sensitive to LPO induced in vivo or in vitro than the inducible ones.

Mechanisms of disassembly of a mixed function oxygenase system in liver endoplasmic reticulum: II. The interrelation between lipid peroxidation and proteolytic degradation of cytochrome P-450.

The degradation of cytochrome P-450 as a result of proteolytic action of trypsin is a biphasic process. Lipid peroxidation (LPO) increases the rate of the fast phase of cytochrome P-450 degradation and its accessibility to protease. The efficiency of this process depends on the mode of LPO induction and decreases in the following order: NADPH----NADH----ascorbate-dependent LPO. The induction of the monooxygenase system increases the efficiency of proteolysis. LPO and proteolysis seem to be mutually enhancing processes which provide for a high efficiency of cytochrome P-450 degradation. LPO can be regarded as a triggering mechanism which makes various forms of cytochrome P-450 accessible to endogenous proteases.

Formation of inhibitors of lipid peroxidation during oxidative metabolism of hydrophobic xenobiotics catalyzed by mixed function oxygenases.

It was shown that benz (alpha) pyrene inhibits the NADPH-dependent lipid peroxidation (LPO) in rat liver microsomes in vitro. The degree of LPO inhibition is correlated with the accumulation of hydroxylated derivatives of benz (alpha) pyrene in the presence of NADPH. Benz (alpha) pyrene protects cytochrome P-450 against conversion into its inactive form, P-420, induced by LPO. Another inhibitor of the NADPH-dependent LPO in rat liver microsomes is chlorpromazine. Inhibition of LPO is due to the antioxidant effect of hydroxylated derivatives of chlorpromazine formed in the course of its metabolism by NADPH-dependent microsomal oxygenase. NADPH-dependent formation of hydroxylated metabolites of chlorpromazine, possessing antioxidant properties, was also estimated in brain cortex microsomes from rats and men. It is shown that chlorpromazine when preliminarily injected to rats, protects against LPO activation in brain tissue in vivo induced by exposure of the animals to hyperbaric oxygenation.

[Test systems for biomonitoring based on membrane-bound enzyme complexes. IV. Assessment of genotoxic effects in an Ames test system with metabolic activation by the microsomal mono-oxygenases from fish livers]. / Test-sistemy dlia biomonitoringa na osnove membranno-sviazannykh fermentnykh kompleksov. IV. Otsenka genotokischeskikh éffektov v testsisteme Eimsa s metabolicheskoi aktivatsiei mikrosomal'nymi monooksigenazami iz pecheni ryb.

The study of mutagenic effect of 2-aminoantracene and benz(alpha)pyrene on Salmonella triphimurium TA 100 in the Ames test-system in the presence of postmitochondrial fractions S-9 from carp liver with 3-methylcholantrene induced by microsomal oxidation system has been carried out. The metabolic activity and cytochrome P450 contence in carp liver microsomes have been shown to concede considerably those in rats liver. But these characteristics are sufficient for the use of fraction S-9 from carp liver for the study of genotoxic effect of these xenobiotics in the Ames test-system. Several regimes of storage of S-9 preparations from carp liver have been compared. S-9 preparations frozen immediately after isolation preserve their metabolic activity with respect to 2-aminoantracene and benz(alpha)pyrene well.

[Peroxidation of lipids in mitochondrial membranes, induced by enzymatic deamination of biogenic amines]. / Perekisnoe okislenie lipidov v mitokhondrial'nykh membranakh, indutsiruemoe fermentativnym dezaminirovaniem biogennykh aminov.

In presence of ferrous cations and ascorbate lipid peroxidation in mitochondrial membranes has been induced by incubation of fragments of the membrane devoid of catalase activity with amines which are substrates of monoamine oxidases of the B type (2-phenyl ethylamine, benzylamine) or transformed monoamine oxidases of type A (cadaverine). In the samples containing both cadaverine and benzylamine the highest stimulation of lipid peroxidation was noted. To the contrary, a substrate of the monoamine oxidases of the type A (serotonin) caused under the same conditions an antioxidative effect. The following conditions are obligatory to induce lipid peroxidation in mitochondria by incubation with amines: I. absence of catalase activity in the biomembranes; 2. presence of physiological concentrations of Fe2+. Physiological concentrations of ascorbate or alterations of pH in the samples caused additional stimulation of the lipid peroxidation.

[Role of lipid peroxidation in damage to serotonin receptors and development of epileptiform seizures during hyperoxia]. / Rol' perekisnogo okisleniia lipidov v povrezhdenii retseptorov serotonina i vozniknovenii épileptoformnykh sudorog pri giperoksii.

Hyperoxia brought about substantial accumulation of primary and end products of lipid peroxidation (LPO) and a significant lowering of alpha-tocopherol content in rat brain tissues. Preinjection of animals with synthetic and natural antioxidants (4-methyl-2,6-ditretbutylphenol and alpha-tocopherol) prevented LPO activation and decreased the frequency of epileptiform seizures induced by hyperoxia. Administration of a mixture of unsaturated fatty acids led to an opposite effect. The changes in the properties of serotonin receptors were found to be dependent on the hyperoxia-induced LPO. These changes were marked by the reduced specific binding of serotonin with neuronal membranes of the rat brain cortex. The data obtained allowed the conclusion about the key role played by LPO activation in toxic action of hyperbaric activation on the brain.

[Accumulation of gaseous products of lipid peroxidation in the expired air of human subjects during hyperbaric oxygenation]. / Nakoplenie gazoobraznykh produktov perekisnogo okisleniia lipidov v vydykhaemom vozdukhe u liudei pri giperbaricheskoi oksigenatsii.

Hyperbaric oxygenation (1 atm. of pure oxygen, 60 min. exposure) resulted in a sharp increase of the endogenous lipoperoxidation level in humans which was evaluated by the pentane content in the exhaled air. That activation of endogenous lipid peroxidation was a short-term process: 2-3-fold increase of pentane content 10 min after exposure to hyperbaric oxygenation and levelling off to control values in 1 hour. It is recommended to use determination of endogenous lipid peroxidation by the pentane content in the exhaled air in order to find optimal regimens of hyperbaric oxygenation.