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Copper-induced oxidability of proteins was investigated in plasma and serum of blood of healthy donors. Incubation of plasma and serum samples with copper ions was accompanied by accumulation of carbonyl products of oxidized proteins. Quantity of the formed carbonyl products depended on both time of incubation, and dilution of plasma and serum. Under identical conditions of oxidation the accumulation of carbonyl products in serum of blood was higher than in plasma.

Autooxidation of low-density lipoproteins during incubation at 37 degrees C was accompanied by accumulation of LPO products, decrease in UV autofluorescence (FUV), and increase in autofluorescence in the visible band (FVIS). The degree of low-density lipoprotein modification was estimated by calculating the FVIS/FUV ratio. A positive correlation was revealed between this ratio and concentration of thiobarbituric acid-reactive LPO products (r=0.76, p<0.001). Autooxidation of low-density lipoproteins increased availability of tryptophanyls for fluorescence quenchers and inductive resonance energy transfer from tryptophanyls to adducts formed in the reaction of apoprotein and LPO products. These changes probably play a role in the decrease in FUV.

Oxidized forms of fibrinogen similarly to initial non-oxidized fibrinogen induced expression of P-selectin and ICAM-1 cell adhesion molecules in the cultured endothelial cells derived from human umbilical vein. The effect of oxidized fibrinogen on the expression of adhesion molecules was more pronounced. These data attest to more active participation of oxidized forms of fibrinogen into inflammation in the vascular wall, the first stage of atherogenesis.

A new approach for the evaluation of oxidizability of proteins and lipids in the same sample of blood plasma was proposed. We tested a method for evaluation of metal-catalyzed oxidation of fibrinogen by the formation of bityrosine cross-links during oxidation detected by the increase in fluorescence at 415 nm. A correlation was revealed between parameters of oxidizability estimated by this marker and carbonyl derivatives (dinitrophenylhydrazine method). Oxidizability of total proteins from whole plasma was compared with oxidizability of plasma lipids (marker malonic dialdehyde). Study of these parameters in patients with coronary heart disease showed that the proposed experimental approach allows us to divide the sample into several subgroups differing in the resistance to oxidative stress. These data can be used for diagnostic and prognostic purposes.

Aim: To investigate potentialities of a novel biochemical test assessing oxidative resistance of plasma as a marker of ischemic damage to the myocardium and to study this marker correlation with high-performance ECG parameters reflecting electrophysiological remodeling of the myocardium in patients with ischemic heart disease (IHD)., Material and Methods: A total of 145 IHD patients entered the trial: 32 patients free of effort angina, 67patients with stable effort angina of functional class (FC) II-III, 56 patients with acute coronary syndrome (ACS) and 32 healthy controls. ACS patients were examined according to the following protocol: stage 1--6-12 hours since the disease onset, stage 2--at the end of the first 24 hours, stage 3--day 5-7 of the disease. In ACS patients the following outcomes (end points) were evaluated: recurrent myocardial infarction (MI), hospitalization because of exacerbation of IHD, documented potentially harmful arrhythmias and death within 1 year. High-performance ECG and a novel biochemical test of plasma oxidizability were used. The latter was determined by accumulation of malonic dialdehyde after 24 hours of incubation with 20 mcM of copper sulphate., Results: A correlation was found between activity offree radical processes and electrophysiological remodeling of the myocardium by high-performance ECG. Amplitude and temporal characteristics of QRS complex and P wave can be used for follow-up of progression and severity of IHD in addition to standard electrocardiography., Conclusion: Unidirectional changes of high-performance ECG, plasma oxidizability and severity of IHD course allow using them as novel diagnostic tests of ischemic lesion and electrophysiological remodeling of the myocardium, for estimation of IHD severity.

Dynamics of lipoprotein oxidation in blood plasma was studied by Cu-induced plasma oxidation in 114 patients with atherosclerosis of lower extremities of various severity with and without ischemic heart disease. Preparedness of plasma lipoproteins to oxidation in patients was higher than in healthy subjects. Degree of oxidizeability increased with increase of severity and extent of atherosclerosis and was highest in patients with atherosclerosis of lower extremities and ischemic heart disease. There were no significant differences between groups of patients with various severity and extent of atherosclerosis in levels of total cholesterol and triglycerides as well as in other parameters of lipid spectrum. Correlation analysis revealed no relationship between age of patients and degree of plasma oxidizeability.

We studied the effect of UV-oxidized fibrinogen with oxidation degrees of 10 and 20% on rheological parameters of the blood. The effect of fibrinogen with 10% oxidation degree was moderate and variable, which attests to its partial compensation with the pool of natural antioxidants. The effect of fibrinogen with 20% oxidation degree was more pronounced. It dramatically decreased deformability of erythrocytes, delayed formation of linear aggregates, accelerated formation of 3D-aggregates, enhanced the total hydrodynamic strength of aggregates, but decreased stability of the largest aggregates. It did not increase plasma viscosity, but enhanced viscosity of the blood at all shear rates. At both degrees of oxidation, suspension stability of the blood decreased, the Caisson viscosity did not change, and the difference between the values of Caisson and asymptotic viscosities markedly increased. On the whole, oxidative fibrinogen produces negative changes in blood rheological parameters, and its effect depends on the degree of oxidation.

We studied the effect of UV-irradiated fibrinogen on blood coagulation. Fibrinogen with oxidation degree of 10% moderately activated the intrinsic pathway, but inhibited the extrinsic pathway of blood coagulation. Fibrinogen with oxidation degree of 20% inhibited both the extrinsic and intrinsic blood coagulation pathways. We revealed disturbances in the formation of fibrin clot with oxidized fibrinogen, suppression of platelet aggregation mediated by collagen receptors, and inhibition of aggregation associated with von Willebrand factor activity. ADP initiated platelet aggregation, which was in direct proportion to the degree of fibrinogen oxidation. Our results indicate that oxidized fibrinogen produces a dysregulatory effect on platelets.

Oxidized fibrinogen was more potent than native fibrinogen in inducing interleukin-8 production in primary culture of human endothelial cells. The optimal concentration of oxidized fibrinogen was 3 mg/ml. The optimal time of UV irradiation was 17 min. Secretion of interleukin-8 was maximum during culturing of endothelial cells in a serum-free medium.

For evaluation of the mechanisms underlying the effect of oxidized fibrinogen on platelet aggregation we studied ADP-induced platelet aggregation in the presence of UV-oxidized fibrinogen and inhibitors of major enzymes of platelet activation. Cyclooxygenase inhibitor acetylsalicylic acid, protein kinase C inhibitor H7, and to a lesser extent, protein tyrosine kinase inhibitor genistein suppressed ADP-induced platelet aggregation. In the presence of oxidized fibrinogen the degree of suppression was lower than in the presence of nonoxidized fibrinogen. Phospholipase C inhibitor U73122 markedly suppressed platelet aggregation in the presence of oxidized and nonoxidized fibrinogen. It can be hypothesized that oxidized fibrinogen activates platelets by modulating activity of the key signal component phospholipase C.

The simple way of quantitative evaluation of high density lipoproteins (HDL) capacity to absorb additive cholesterol quantity is proposed. It allows to evaluate indirectly intensity of the first, rate limiting stage of reverse cholesterol transport its accepting from the cells by means of HDL. The way includes the usage of stable artificial cholesterol donor--cholesterol covered inert polymer particles, which are than more convenient, than cell culture use. The total HDL rough fraction (i.e. serum after apoB lipoproteins removal) was shown to include more than 50% cholesterol in addition to yet presenting amount. But this ability is sharply reduced, or sometimes even is completely absent, in HDL of 63 coronary heart disease (CHD) patients (as compared with 41 healthy donors). This difference of potential cholesterol accepting capacity is revealed even at the same initial HDL concentrations. The negative correlation (r = - 0.32, p < 0.05) between this HDL property (delta HDL cholesterol) and their oxidability in the ions Cu2+ presence was observed. This underlines atherogenic role of HDL oxidability. The treatment of patients by phospholipids (as Lipostabyl) resulted to recovery of HDL cholesterol accepting capacity. The possible mechanisms of junction of this HDL activity with their oxidability are discussed, as well as necessity of evaluation of HDL properties and reverse cholesterol transport for the choice of care strategy of CHD patients.

Cholesterol (CH) acceptance ability of high density lipoproteins (HDL) was assessed in 43 ischemic heart disease (IHD) patients, including patients with post-infarction cardiosclerosis and class II-III effort angina. CH acceptance ability of HDL was measured as increment of HDL CH after incubation with artificial CH-containing system. Oxidabilities of HDL and total plasma were estimated by quantitation of lipid peroxidation products (hydroperoxides and thiobarbituric acid-reactive substances - TBARS) after incubation with Cu(2+) ions. HDL fraction (after apo B lipoproteins removal) of IHD patients appeared to include 2 times less additive CH compared with donor's HDL despite lower (-12%) HDL CH level. Negative correlation (r =-0.38, p<0.05) existed between formed TBARS in HDL and HDL CH acceptance. In total plasma of IHD patients elevation of both formed TBARS and particularly hydroperoxides was observed. Parallelism between decrease of CH acceptance by HDL, oxidability of HDL and of total plasma testifies on weakness not only of CH-accepting, but also of antioxidant HDL functions in IHD patients.

The aim of this study was to determine the concentration parameters and time-course of copper-induced oxidation in serum and whole blood plasma. We investigated the oxidizability of undiluted and (2-60-fold) diluted whole plasma (prepared with citrate as an anticoagulant) and serum by monitoring the change in the level of conjugated dienes, ketodienes and malondialdehyde in the samples. The kinetic curves of the accumulation of different lipid peroxidation (LPO) products had similar S-shape, but they differed by a number of quantitative parameters--lag-time, time of improvement and the level of maximal rate of oxidation, as well as time of improvement of the maximal accumulation and maximal amount of LPO products. When the LPO products were formed in undiluted plasma and serum, the lag phase of 2-2.5 h for conjugated dienes and ketodienes, and 11-12 h for malondialdehyde was observed. Oxidation profile showed a negligibly short period of inhibition followed by rapid oxidation for all investigated LPO products when plasma and serum were diluted at least 10-fold. The rate of accumulation and amount of LPO products decreased in the following order: conjugated dienes > ketodienes > malondialdehyde. Choice of blood plasma or serum and a dilution factor strongly influenced intensity of copper-induced LPO. Anticoagulant (sodium citrate), that was used by preparation of plasma, possessed an inhibitory action on LPO, due to formation of copper-citrate chelates. Dilution of plasma and serum increased LPO through lowering of influence of water-soluble antioxidants, but action of fat-soluble antioxidants (dissolved in the lipid phase of lipoprotein particles) by was unchanged. These results indicate that measurement of lipid oxidation induced in vitro in the whole plasma or serum might be more relevant model of the lipoprotein oxidation in the blood than the in vitro oxidation of single isolated lipoproteins.

In patients with coronary heart disease oxidizability of lipids during Cu(2+)-induced oxidation of blood plasma inversely correlated with fibrinogen content. A positive correlation was found between the amount of lipid peroxidation products in the plasma from these patients and fibrinogen content. The increase in fibrinogen content was associated with high levels of total lipids and triglycerides and low concentration of high-density lipoprotein cholesterol. In vitro experiments demonstrated that fibrinogen reduces oxidizability of blood plasma. Our results suggest that the decrease in lipid oxidizability at high concentration of fibrinogen in patients with coronary heart disease is related to predominant oxidation of fibrinogen and its competition with plasma lipids during Cu(2+)-induced oxidation.

The influence of oxidized fibrinogen on the intensity of luminol-dependent chemilumin escence of blood leukocytes, stimulated by opsonized zymosan was studied. It was shown that the introduction of fibrinogen modified by UV-irradiation in to a suspension of cells resulted in a significant increase in the intensity of the luminol-dependent chemiluminescence of leukocytes. It was suggested that oxidized fibrinogen can influence blood leukocytes, enhancing their functional activity.

Magnetic resonance spectroscopy revealed structural modifications of human plasma lipoproteins during peroxidation induced by copper sulfate in vitro. Decreased molecular mobility of fatty acid chains in lipoprotein lipids was demonstrated.

Using the method of peroxide-induced chemiluminescence we showed that incubation of the whole blood with oxidized LDL or oxidized blood plasma increased plasma hemoglobin concentration, which linearly depended on the degree of LDL oxidation. Similar effects were observed in erythrocyte suspension. Hemolytic activity of oxidized plasma 3-4-fold surpassed that of LDL isolated by ultracentrifugation. LDL capacity to oxidation in the presence of Cu(2+)increased by 50% and osmotic hemolysis of erythrocytes increased by 53% in coronary patients in comparison with healthy donors. These results indicate that oxidized LDL induce erythrocyte hemolysis.

The binding of bivalent metal ions Cu2+, Zn2+, Ca2+, Mg2+ to low-density lipoproteins (LDL) was investigated by the ESR technique. The monitoring of ESR spectra of paramagnetic Mn2+ ions in the presence of above-listed cations made it possible to evaluate the dissociation constants of their complexes with LDL. The effective dissociation constant of the complex Mn(2+)-LDL used for calculations was KD = (1.1 +/- 0.4) x 10(-4) M according to literature data. The investigated cations may be classified into two groups: 1) low dissociation constants were characteristic for Cu2+ ions [KD = (1.3 +/- 0.5) x 10(-4) M], which demonstrated a high oxidative ability, and for Zn2+ [KD = (0.95 +/- 0.45) x 10(-4) M] and Mn2+ ions, which could strongly influence the copper-induced LDL oxidation; 2) Ca2+ and Mg2+ were characterized by higher values of KD [(6 +/- 1) x 10(-4) M and (7.5 +/- 1.5) x 10(-4) M, accordingly] and slightly affected the Cu(2+)-induced oxidation of LDL. The results of the present work reinforced our earlier conjecture that cations may influence the process of lipid peroxidation, binding only to particular binding sites on the surface of LDL.

Fibrinogen intensified luminol-dependent chemiluminescence of blood leukocytes stimulated with opsonized zymosan. It is hypothesized that fibrinogen stimulates and prolongs functional activity of leukocytes during inflammation.

Oxidized UV-modified fibrinogen activates platelets in platelet-rich plasma. Kinetic turbidimetry showed that addition of oxidized fibrinogen to platelet-rich plasma led to platelet aggregation. Reversible aggregation is recorded starting from the 30th second and then constantly grows with the same rate. Nonoxidized fibrinogen produced no such effect. The relationship between aggregation intensity and rate and the degree of fibrinogen oxidation was described by a bell-shaped curve with a peak corresponding to 24% fibrinogen oxidation. The amplitude of aggregation increased with increasing the concentration of irradiated fibrinogen from 0.1 to 1.0 mg/ml and then plateaued. The rate of aggregation little depended on fibrinogen concentration.

The negative surface charge of low-density lipoproteins increased during their oxidative modification induced by autooxidation at 37 degrees C. The degree of changes depended on the time of autooxidation: the surface charge remained practically unchanged after short-term oxidation (6-h incubation), but then progressively increased and after 24-h oxidation it 2-fold surpassed the initial level. Long-term incubation of low-density lipoproteins in the presence of EDTA inhibiting lipid peroxidation did not change their surface charge. These changes probably contribute to atherogenic activity of oxidized low-density lipoproteins. The degree of oxidative modification of low-density lipoproteins was precisely estimated using fluorescence probes.

We evaluated antioxidant state of 62 patients with coronary heart disease and 47 patients with obliterating atherosclerosis of lower extremities by peroxide-depend chemiluminescence and inhibition of azo-initiated chemiluminescence and hydrogen peroxide-hemoglobin-luminol chemiluminescence system. The flash amplitude of peroxide-dependent chemiluminescence in the plasma from patients was 32% below the control. Antioxidant activity of the plasma from patients was higher than in healthy individuals by 33 and 27% depending on the type of free radical-generating systems. The increase in antioxidant activity was most pronounced in patients with combined pathology: coronary heart disease complicated by obliterating atherosclerosis. These results explain the decrease in peroxide-dependent chemiluminescence of the plasma and whole blood in patients with atherosclerosis compared to that in healthy individuals.

Relationship between serum oxidation of different degree and micro- and macrorheology of the blood and modification of this relationship in the presence of antioxidant alpha-tocopherol were studied. Lipid peroxidation affects blood rheology and erythrocyte osmotic resistance. Erythrocytes are the first to react to increased activity of free radical oxidation and to exhaust their compensatory potential. Plasma viscosity remains stable in serum oxidation of different degree, and therefore erythrocytes are responsible for changes in blood rheology during intensification of free radical oxidation. Moreover, erythrocytes are functionally resistant to oxidative stress in malonic dialdehyde concentrations under 3.62 +/- 0.41 nM/ml. alpha-Tocopherol increases functional resistance of erythrocytes and maybe of protein components of the plasma to damaging action of free radicals.

A comparative study of the effect of concomitant compensated diabetes mellitus (DM) on plasma oxidation in patients with ischemic heart disease (IHD) with stable angina of effort (functional class II-III) in 28 anginal patients with IHD, arterial hypertension and DM, 67 anginal patients with IHD and hypertension, 57 anginal patients with IHD and 28 donors. Lipoproteins oxidation was studied by enzymic methods. A significant effect of the type of DM on dynamics of plasma oxidation in IHD patients was found. This may be an additional prognostic criterion of IHD progression in DM patients.

In vitro experiments showed that copper-oxidized low-density lipoproteins activate factors of the prothrombin complex in the whole blood and inhibit fibrin generation in both blood and plasma. Moreover, oxidized low-density lipoproteins inhibit fibrinolysis and impair the structure of fibrin clot, which results in hypercoagulation.

The investigation of the effect of oxidized lipoproteins on platelet activity is important for the understanding of the plague formation under atherosclerosis. In the present work, we examined the influence of low density lipoproteins (LDL) on ADP-induced platelet aggregation in the platelet rich plasma. In was demonstrated that mixing of plasma and LDL was accompanied by the decrease of ADP-induced aggregation parameters as compared to control (mixing with buffer). After 1 h incubation, platelet ADP-aggregation in the sample containing oxidized LDL (oxLDL) exceeded the ADP-aggregation in the control sample. The dependence of the aggregation parameters on the incubation time and on the degree of LDL oxidation were obtained. No difference in the cholesterol and phospholipid content was observed between cells incubated with buffer, native or oxidized LDL. Therefore, the possible oxLDL-induced accumulation of cholesterol in platelet membranes is excluded as a reason for the increased cell aggregation.

The effect of metal cations on copper-catalyzed lipid peroxidation (LPO) of low density lipoproteins (LDL) was examined. The presence of metal cations in the incubation media containing LDL (0.8 mg protein/ml) and CuSO4 (0-80 microM) influenced on LPO of LDL as evident by the measurement of TBARS. With the concentrations of CuSO4 less than 10 microM, the metal cations caused an increase in LDL peroxidation. Zn2+ appeared to be the most effective inductor, Mn2+ was less effective, and the influence of Ca2+ and Mg2+ was insignificant. With greater CuSO4 concentrations Mg2+ showed no effect on TBARS formation in LDL while the addition of other nontransition metal cations to the incubation mixture led to the inhibition of LDL peroxidation. The capacity for inhibition decreased in the row Mn2+ > Zn2+ > Ca2+ > Mg2+. The possible mechanism explaining these results may be in the competition of metal ions for copper binding sites on LDL. Our results allow to suggest the existence of two types of copper binding sites on LDL, tight-binding sites which are non-effective in LPO and effective weak-binding sites.

Copper-catalyzed lipid peroxidation (LPO) in low density lipoprotein (LDL) and two subfractions of high density lipoprotein (HDL2 and HDL3) isolated from human serum was studied. The levels of LPO were estimated as thiobarbituric acid-reactive substances (TBARS). The amount of TBARS increased in a time-dependent manner when either LDL, HDL2 or HDL3 (0.4-1.2 mg protein/ml) was incubated at 37 degrees C during 4 h with phosphate-buffered saline containing CuSO4 (2-80 microM). The increase in CuSO4 concentration to some value therewith caused the enhancement of the rate of TBARS formation but the following increase in CuSO4 concentration beyond that value didn't already affect the kinetics of LPO in lipoproteins. It was also shown that the lesser was the concentration of lipoprotein the lesser CuSO4 concentration was needed to reach maximal rate of TBARS formation. The data obtained point to the versatility of the mechanism of copper-catalyzed oxidation of low and high density lipoproteins and suggest that the decisive role in the process belongs to copper ions bound to lipoprotein particle.

Oxidized low density lipoproteins (oxLDL) bounded to specific receptors on the platelet surface are able to activate platelets. However, the exact mechanism of signal transduction from LDL receptors into the cell still requires investigation. In the present paper inhibitors of the main enzymes of known platelet activation pathways were used to investigate the mechanism of the LDL-induced platelet aggregation. Our experiments were performed with autoxidized LDL (2-thiobarbituric acid reactive substances < 8 nmoles/mg). We demonstrated that the main enzymes of the arachidonate cycle do not play an important role in LDL-induced platelet aggregation, whereas inhibition of protein kinase C and phospholipase C--principal enzymes of the phosphoinositide cycle--resulted in the inhibition of LDL-induced platelet aggregation in a dose-dependent manner. It was also shown that transmembrane calcium transport was necessary for LDL-induced platelet activation. Thus, we conclude that the phosphoinositide cycle is the main mechanism of cellular signal transduction during LDL-induced platelet activation.

At neutral pH values 1,10-phenanthroline forms a colored complex with Fe(II), but it does not form such a complex with Fe(III). On the contrary, only Fe(III) forms with desferal a yellow complex with a g = 4.3 electron paramagnetic resonance (EPR) signal, but Fe(II) is rapidly oxidized by desferal to Fe(III), which gives then a yellow complex. On the basis of these facts, a method for simultaneous determination of both Fe(II) and Fe(III) ions was elaborated using a desferal-phenanthroline mixture. Two ways of detecting Fe(II) and Fe(III) have been suggested: (1) the spectrophotometric method for transparent water solutions, and (2) the EPR-spectrometric method for turbid solutions and tissue homogenates. The latter method was applied for determination of free and weakly bound iron in rat liver. The Fe(II) amount in intact liver was 22.2 +/- 7.6 nmol/g of wet tissue; free Fe(III) was not found.

Platelet aggregation induced by native and oxidized low-density lipoproteins (LDL) was studied. The minimal LDL concentration necessary for platelet activation was no more than 0.2 mg/ml. The aggregation rate increased when LDL concentration rose from 0 to 0.4-0.6 mg/ml for native LDL and to 0.2-0.4 mg/ml for oxidized LDL. The platelet aggregation induced by oxidized LDL appeared to be higher than that induced by the same concentration of native LDL. The dependence of aggregation rate on the degree of LDL peroxidation was linear up to MDA concentration equal to 3-4 mg/ml. Further LDL peroxidation leads only to the negligible increase in the induced aggregation rate.

Electron spin resonance spectroscopy was used for measurements of the surface potential and apoprotein structure of LDL and HDL in the presence of Ca2+ and dextran sulfate, heparin and chondroitin sulfate. A decrease in the absolute values of surface potential of LDL and HDL was observed after addition of Ca2+. In the presence of the negatively charged macromolecules the measured surface potential was less reduced. The spectral properties of a maleimide spin label covalently attached to the apoprotein were changed under conditions of aggregation of LDL induced by dextran sulfate, chondroitin sulfate or heparin in the presence of Ca2+. In the HDL system this effect was only observed for dextran sulfate. The influence of PEG on the spectral parameters of the spin label is dependent on the molecular weight of the polymer. PEG 400 decreased the mobility of the spin-labelled apoprotein region of LDL, whereas PEGs with higher molecular weight only slightly increased the maleimide mobility. On the other hand, the maleimide-labelled apoprotein region of HDL showed a higher sensibility to all PEGs used. Addition of PEG leads to immobilization of apoprotein A.

1. The addition of native cytochrome c to mitoplasts leads to a decrease of surface potential of the mitoplast membrane. However the surface potential is slightly decreased (approximately 3 mV) when PLP(Lys 86)-cytochrome c and PLP(Lys 79)-cytochrome c were added. 2. The native and PLP-modified cytochromes c do not influence the order parameters S and isotropic constant a when both spin probe I and probe II were used. It is shown that cytochrome c binding to the membrane does not affect the hydrophobic intermembrane area as well as the lipid arrangements of the mitoplast membrane. 3. At low ionic strength there was observed a significant difference in the membrane potential when PLP-cytochromes c were added to the mitoplasts. 4. At high ionic strength the addition of native or PLP-modified cytochromes c does not change the membrane potential.

Treatment of intact liver and liver homogenate with sodium nitrite, or desferal, brings about the appearance of g = 2.03 and g = 4.3 electron paramagnetic resonance spectroscopy (EPR) signals, respectively. The g = 2.03 signal is conditioned by the formation of dinitrosyl complexes of Fe(II); the g = 4.3 signal is related to the appearance of paramagnetic desferal-Fe(III) complexes. Desferal and sodium nitrite were administered successively into liver homogenate, resulting in only a g = 4.3 EPR signal. And, vice versa, if desferal was administered after sodium nitrite, there appeared only the signal with g = 2.03. These data testify to the fact that one and the same endogenous free iron is included in both paramagnetic centers. The concentration of iron ions was measured in intact tissue according to the formation of dinitrosyl-iron complexes and desferal-iron complexes. It was 33.2 +/- 4.6 and 20.3 +/- 4.0 nmol/g of tissue weight, respectively. The data obtained testify to the fact that free endogenous iron is present in intact tissue. Possibilities of the EPR method for estimation of the content of intracellular free iron are discussed.

Serum and red blood cell membrane lipid peroxidation (LPO), antioxidative activity (AOA) of the ceruloplasmin/transferrin (CP/TF) system, and biophysical parameters of the structure of a red blood cell membrane lipid bilayer were examined in fetuses experienced acute hypoxia at birth and in babies born to healthy mothers with uncomplicated pregnancy. The intensity of LPO product accumulation in mild asphyxia was ascertained to be proportional to the duration of hypoxic exposure of a fetus. In severe asphyxia accompanied by lower formation of primary LPO products, the levels of secondary LPO products showed a rise. A slight increase of AOA in the CP/TF system was unable to adequately compensate a high intensity of LPO processes, which provides strong evidence for altered structural parameters in the lipid bilayer. It was concluded that it was essential to correct hypoxic states in children with antioxidants immediately after birth.

The activation of freshly isolated human blood monocytes and neutrophils monitored by oxidized human plasma lipoproteins (LP) was measured by detecting luminol-amplified chemiluminescence. The activation was accompanied by production of superoxide radicals. This finding was confirmed by measuring superoxide dismutase-sensitive reduction of cytochrome c. Incubation of monocytes or neutrophils with low-density lipoproteins (LDL) resulted in the accumulation of lipid peroxidation (LPO) products which were assayed by the 2-thiobarbituric acid test. Data from inhibitory analysis suggest that the hydroxyl radical scavenger, mannitol, had no appreciable effect on the accumulation of LPO products during the incubation of LDL with either cell type. However, catalase, superoxide dismutase, the metal ion chelators desferrioxamine and EDTA, as well as the free radical scavenger, butylated hydroxytoluene, markedly decreased the accumulation of LPO products in the medium--by 88%, 67%, 38%, 52%, and 47%, respectively, after incubation of LDL with monocytes, and by 65%, 47%, 41%, 65%, and 100% after incubation of LDL with neutrophils. These results indicate that activation of monocytes and neutrophils by oxidized LP intensifies LPO which proceeds via a free-radical mechanism that is superoxide-dependent and is catalyzed by transition metals.

An electron spin probe study was made of the effect of lipid peroxidation (LPO) on the structure of surface proteolipid layer of human serum low-density lipoproteins (LDL). The results obtained with a positively charged spin label and stearic acid spin probes with doxyl labels at positions 5, 12, and 16 revealed that LPO caused a decrease in phospholipid molecule mobility both in the region of polar heads and in the region of acyl chains till the depth of at least 1.7 mm from water-lipid interface. Under relatively high levels of oxidation (more than 6 mumol MDA/g LDL phospholipid) the polarity of lipid phase increased. The decrease in efficiency of tryptophan fluorescence quenching by nitroxide fragments incorporated in hydrophobic regions at the depth of approximately 2 nm from water-lipid interface indicated that lipid-protein interaction was disturbed as a result of oxidation of LDL lipids. In addition, the LPO-induced modification of apo-B, the main protein of LDL, was examined with maleimide spin label. LPO led to increase in mobility of strongly immobilized maleimide labels and in the number of weakly immobilized ones. Oxidized LDL revealed decreased ability to incorporate spin-labeled steroid (androstane) as compared to native ones. LPO-induced structural changes of LDL surface are supposed to be a reason of enhanced accumulation of cholesterol in human monocytes during their incubation with oxidized LDL. The cholesterol content in red cells was shown to be directly correlated to MDA content in apo-B containing lipoproteins but not in whole serum. Our findings suggest that free radical modification of serum lipoproteins but not solely an increased level of LPO products in blood is one important cause for cholesterol accumulation in cells and, apparently, for their transformation into foam cells during atherosclerosis.

A spin-labelled fatty acid with an epr spectrum that is sensitive to the localization of the probe was used to show that albumin binds free fatty acids present in solution and also free fatty acids present in low density lipoproteins (LDL). Furthermore, albumin binds the thiobarbituric acid-reactive (TBA-reactive) products formed during the oxidation of linolenic acid, whereas the TBA-reactive substances formed during the oxidation of LDL are not bound by albumin. Linolenic acid bound to albumin essentially does not undergo peroxidation in the presence of ferrous ions, in contrast to a suspension of linolenic acid and LDL in which peroxidation occurs quite readily in the presence of ferrous ions. The highest rate of oxidation was found for linolenic acid alone. Albumin-bound spin-labelled fatty acid was essentially not reduced by ferrous ions, whereas free fatty acid or fatty acid incorporated into LDL was reduced quite rapidly, the highest rate of reduction being for free fatty acids. Thus the ability of fatty acids to undergo oxidation correlates with their accessibility to ferrous ions. The data obtained indicate that serum albumin is a relatively effective antioxidant in the blood and its mode of action is based on the immobilization of free fatty acids.

The tissue oxygen concentration, the serum antioxidant system state and the serum malondialdehyde (MDA) concentration were studied in patients with hepatitis B. The good correlations were studied in patients with hepatitis B. The good correlations between MDA concentration in patients serum and the oxygen concentration in tissues (R-0.79), and the cytoplasmic enzymes activity (R-0.75 for lactate dehydrogenase; R-0.75 for alanine transferase) were found. On the other hand, it was shown an antioxidant activity decrease of ceruloplasmin-transferrin system in patients serum. It is proposed, that the tissue hypoxia and the decrease of the serum antioxidant activity are the general factors leading to the MDA accumulation in the serum of patients with hepatitis B.

It was shown that thymectomy induces the injury of plasma membranes of mouse splenocytes. This membrane disorders completely inhibit T-cell response on Concanavalin. A. The incubation of splenocytes with Tactivin or injection of Tactivin or its subfractions to the animals restore the membrane structure and T-cell response.

An important index of neutrophil function is the production of superoxide radicals (O2-) upon activation. Thus a development of a new adequate assay of O2- generation measurement is of great interest for phagocyte researchers. The present article considers the quantitative determination of O2- generation based on the interaction of O2- with 1-oxy-2,2,6,6-tetramethyl-4-oxypiperidine producing 4-oxo-2,2,6,6-piperidine-1-oxyl, detected by ESR. The kinetic curve of nitroxyl radical (NR) formation has a linear character. The NR formation rate after a short induction period (appr. 2 min.) approaches 3.3 X 10(-3) M/s, where cell concentration was 4 X 10(5) per ml. Hydroxylamine (3.8 mM) auto-oxidation rate is negligible as compared with activated neutrophils and is equal to 2 X 10(-9) M/s. Sensitivity NR to the presence of superoxide dismutase (SOD) came as evidence that NR formation is due O2- radicals. SOD (10(-7) M) inhibits NR formation by 90%. Hydroxylamine oxidation by O2- is an irreversible reaction--20-min incubation of activated neutrophils with NR do not influence NR concentration. The NR generation rate dependence upon the neutrophil concentration is linear in the cell concentration range from 4 X 10(5 up to 6 X 10(6) per ml. In this range a quantitative measurement of O2- production is suitable. The sensitivity of hydroxylamine assay is close to the sensitivity of chemiluminescent method, but specificity is higher, as SOD inhibits chemiluminescence only by 50%.

It is shown that the dynamics of lipid peroxidation (LPO) in patients with purulent meningitis is unspecified in character and is encountered both in meningococcal and pneumococcal infection. The level of secondary LPO products and the degree of hyperfermentemia, which are determined according to the dynamics of changes in the activity of aspartate transaminase, are objective characteristics of the severity of the patient's condition and reach maximum on the 5th day of the disease. The correlation between the dynamics of the primary LPO products and the ceruloplasmin/transferin coefficient allows the severity of the disease to be prognosticated.

Rapid dissociated Fe(III)-ascorbic acid complexes are formed after the mixing of Fe(II) (100 mkM) with ascorbic acid (1 mM) at pH-7,4. As a result of these complexes formation Fe(II) concentration was decreased in solution, but o-phenantrolin addition or pH decreased up to 6.1 lead to the destruction of these complexes and reduction of iron up to Fe(II). Iron-ascorbic acid paramagnetic complexes were not observed in the perfused rat liver homogenate. Probably, these facts were due to the presence of other stronger reductants in the tissue.

The antioxidative system (AOS) ceruloplasmin-transferrin (Cp-Tr) was studied by means of electron paramagnetic resonance in 14 rabbits with experimental atherosclerosis and in 33 patients with ischemic heart disease (IHD). A correlation was found between the AOS Cp-Tr activity and the pathological process severity: mild disease was associated with high AOS activity, while in severe disease course, this activity was threefold lower. This regularity was detectable both in experimental animals and in human IHD patients. It was found that hemosorption (HS) exerted a positive effect only in the presence of low AOS Cp-Tr activity which increased after HS in these cases. In high AOS activity HS caused deterioration of the patients' condition and accumulation of lipid peroxidation products in the blood plasma; this was attended with lowering of the AOS Cp-Tr activity.

Using ESR spectroscopy of spin probes and spin labelling, the effects of lipid peroxidation (LPO) on the molecular organization of Ca-ATPase from rabbit skeletal muscle sarcoplasmic reticulum membranes were studied. Accumulation of LPO products caused an increase of the hydrophobicity and a decrease of the mobility of the Ca-ATPase polypeptide chain fragment immediately proximal to the enzyme active site. Induction of LPO in the membranes resulted in a decrease of the thermal stability of the lipoprotein Ca-ATPase complex which occurred as a biphasic process. At temperatures of 36-38 degrees C this thermodenaturation consisted in a shift of the Ca-transport ability towards a lower temperature region corresponding to the shift in the low temperature break on the Arrhenius plots for parameter 2A'zz of the maleimide spin label. Within the temperature range of 49-52 degrees C an earlier LPO-induced denaturation determines the low temperature shift of the point for a sharp decrease of the Ca-ATPase activity and of the corresponding break for parameter 2A'zz as well as the dissociation point for the lipoprotein Ca-ATPase complex and the corresponding breaks on the Arrhenius plots for the solubilization parameter, alpha, and the order parameter of spin probes, S.