Effect of oxidatively modified and non-modified human serum albumin on luminol-dependent chemiluminescence of human peripheral blood leukocytes stimulated with opsonized zymosan.

We studied the effects of native and oxidized human serum albumin on luminol-dependent chemiluminescence of human peripheral blood leukocytes stimulated with opsonized zymosan. Human serum albumin was added simultaneously with opsonized zymosan at the beginning of the chemiluminescent reaction. Otherwise, leukocytes were incubated with human serum albumin at 37°C for various periods before addition of opsonized zymosan. Oxidized human serum albumin was obtained by the method of metal-catalyzed oxidation. In control to non-modified albumin, oxidized albumin produced an inhibitory effect on luminol-dependent chemiluminescence of leukocytes. These changes were observed in experiments with addition of oxidized albumin at the beginning of a chemiluminescent reaction and after incubation of study agent with cells.

Effects of gold nanoparticles on erythrocyte hemolysis.

We studied hemolytic activity of gold nanoparticles added to the whole blood (ex vivo) and of nanoparticles coated and not coated with plasma components on erythrocytes in hypotonic medium (osmotic hemolysis) in vitro. Gold nanoparticles did not stimulate erythrocyte hemolysis after 4-h incubation with the whole blood ex vivo. Hemolysis tended to increase in the presence of small gold nanoparticles (5, 10, 20 nm) at the maximum concentration of 20 µM (by gold content) used in our study in comparison with the control. This tendency was detected during the 1st hour of the nanoparticles incubation with blood. Gold nanoparticles in the used concentrations (up to 20 µM of gold) coated with plasma components after preincubation with autologous plasma and nanoparticles without coating caused no osmotic hemolysis of erythrocytes in vitro.

Effect of gold nanoparticles on production of reactive oxygen species by human peripheral blood leukocytes stimulated with opsonized zymosan.

We studied the effect of gold nanoparticles on ROS production by leukocytes. ROS production was detected by luminol-dependent chemiluminescence (LDCL) of human peripheral blood leukocytes stimulated with opsonized zymosan. Nanoparticle size was 5, 10 and 30 nm. Simultaneous addition of nanoparticles and opsonized zymosan showed that 5-nm nanoparticles inhibited LDCL intensity in comparison with the control, when LDCL recording was conducted in the presence of opsonized zymosan. Increasing nanoparticle size from 5 up to 30 nm enhanced LDCL intensity. Preincubation of gold nanoparticles with autologous blood plasma increased LDCL intensity. In the control (without gold nanoparticles), blood plasma produced no activating effect on LDCL. We found that the effect of gold nanoparticles on leukocyte LDCL depended on nanoparticle size: 10- and 30-nm nanoparticles inhibited LDCL intensity in comparison with the control (incubation in the absence of nanoparticles) irrespective of the duration of incubation, while 5-nm gold nanoparticles had no effect on LDCL intensity. Incubation of gold nanoparticles with autologous plasma increased LDCL intensity if nanoparticle size was 30 and 10 nm.

[The association between oxidative stress markers and clinical course of chronic cerebral ischemia].

Authors studied 360 patients with different stages of chronic cerebral ischemia (CBI), including 180 patients followed-up for 12 months after the first examination, who were stratified into two groups with regard to disease course - favorable (stable) and unfavorable (progressive or with acute episodes of cerebral blood circulation disturbance). Oxidative stress markers were evaluated by the level of lipid- (malonic dialdehyde) and protein - (carbon products of protein oxidation, the level of plasma SH-groups, the accumulation of the products of deep oxidation of proteins) oxidation. Along with indicators of oxidative stress, we evaluated the binding capacity of albumin using fluorescent probe K-35. Initial level of these markers and their concentrations after the copper ion induced oxidation of the plasma were determined. The highest increase in oxidative stress indicators was seen in patients with acute episodes. Authors identified significant differences in these indicators in the groups of patients with different clinical variants of CBI course as well as qualitative and quantitative diagnostic criteria of unfavorable course and risk of stroke. Our findings suggest that the imbalance of oxidative-antioxidative system contributes to the course of CBI. Prediction of unfavorable course of CBI determines the timeliness of adequate treatment.

[The influence of oxidized fibrinogen on apoptosis of endothelial cells].

Oxidative stress plays an important role in cardio-vascular diseases and atherosclerosis. Fibrinogen (FB), plasma coagulation protein, is a risk factor of atherosclerosis. Importantly, it can be readily oxidized during oxidative stress and in pathological conditions. FB can promote angiogenesis by supporting migration and proliferation of endothelial cells. On the other hand, recent reports demonstrated cytotoxicity of oxidized fibrinogen (oxFB). Endothelial dysfunction plays a critical role in the atherosclerosis development, therefore it is important to understand the effect of oxFB on human endothelial cells (hEC), and the mechanism of the cell death. Here, we studied influence of oxFB on hEC during 24 h incubation in two conditions: (1) at low serum level (0.1%) and in the absence of growth factors ("starvation"); (2) in full medium (5% FBS) with growth factor supplement. Apoptosis was evaluated using analysis of nuclear morphology, phosphatidylserine externalization on hEC surface and caspase-3 activation. In starvation, we observed significant cell death via apoptosis. FB prevented starvation-induced cell death and caspase activation. Caspase activity in the presence of oxFB was 1.5 times higher as compared to FB, yet oxFB demonstrated significant cell protection during stress. Similarly, in optimal cultivation conditions FB decreased the rate of apoptosis by three times, while oxFB supported cell viability to the lesser extent. Thus, FB can protect hEC in stress conditions (in starvation); oxidative modification of FB diminishes its antiapoptotic properties.

[Comparative analysis of antioxidant activity of nebivolol in patients with chronic heart failure with and without concomitant type 2 diabetes].

UNLABELLED: Consistent neurohormonal activation of sympatho-adrenal system in patients with chronic heart failure (CHF) and hyperglycemia contributes to development of oxidative stress--one of the most important pathogenetic mechanisms of endothelial dysfunction. PURPOSE: To study the impact of nebivolol concerning modification of clinical and hemodynamic indicators and parameters of oxidative stress in patients with CHF and with or without concomitant diabetes mellitus type 2 (DM2). MATERIAL: Nebivolol was used in complex therapy of CHF in 82 patients, suffering from NYHA class I - III CHF (EF < 50%) of ischemic genesis with or without comorbid DM2, average age 63.2 +/- 8.2 years. RESULTS: After 8 months of therapy significant improvement of clinical status was observed in both groups, tolerance to physical activity increased (significant reduction of average class of CHF in the group with DM2 from 2.5 +/- 0.58 to 2.125 +/- 0.71, p = 0.001, and in the second group from 2.3 +/- 0.5 to 1.9 +/- 0.4, p = 0.01). We also noted in both groups increase of plasma oxidative resistance (reduction of intensity of fast flash in lipid peroxidation h from 7 to 6 mm, p = 0.016, and from 8 to 6 mm, p = 0.03, respectively) and increase of antioxidant plasma protection (increase of SH-groups from 154.19 to 182.4 mmol/1, p = 0.00035, and from 176 to 205, p = 0.004, respectively). CONCLUSION: Nebivolol is a modern neurohormonal modulator, which contributes to reverse evolution of oxidative changes in patients with CHF and hyperglycemia.

[Clinical and prognostic significance of free radical processes in patients with coronary heart disease].

The aim of the work was to evaluate the diagnostic and prognostic value of the characteristics of processes involving free radicals and of high-performance ECG (hpECG) in patients with coronary heart disease (CHD). A new method for early assessment of the severity of myocardial ischemia and its electric remodelling was developed. Malonic dialdehyde levels (MDA) following Cu-induced oxidation during 24 hr were shown to correlate with clinical features of CHD (functional class of angina, hpECG patterns). hpECG characteristics changed parallel to MDA levels, their frequency was related to the severity of CHD. Long-term prognosis of unstable angina (12 month follow-up) was aggravated in patients with MDA level of 100 nmol/ml. MDA concentration and its dynamics correlated with CHD severity and outcome of acute coronary syndrome (ACS): non-Q wave myocardial infarction and angina. All patients with ACS underwent its exacerbation within 5-7 days and had depleted plasma antioxidative system. MDA and hpECG dynamics can be used to evaluate ACS severity and prognosis as a new diagnostic approach to identifying CHD patients with the expected unfavourable outcome of the disease.

Effect of oxidation-modified fibrinogen on the formation and lysis of fibrin clot in the plasma.

Turbidimetry studies showed that after addition of thrombin to fresh donor plasma light scatter in the sample increases and slowly attains a plateau. The process of fibrin formation was less intensive in the presence of oxidized fibrinogen. The formation of fibrin clot in lyophilized plasma was characterized by a biphasic kinetic of light scatter, oxidized fibrinogen inhibited both phases of the process. In the presence of streptokinase, oxidized fibrinogen did not modify the kinetics of fibrin clot lysis. Addition of oxidized fibrinogen to plasma reduced optical density of fibrin clot the more intensely the higher was the degree of oxidative modification of fibrinogen.

Effect of iron ions on functional activity of thrombin.

The kinetics of thrombin inhibition by irons ions was studied in the thrombin time test with normal plasma. The kinetic and concentration characteristics for recovery of thrombin activity by desferal were evaluated at various periods of thrombin incubation with iron ions. The thrombin time test showed that incubation of thrombin with iron sulfate in a final concentration of 200 microM for 25-35 min is followed by the loss of thrombin activity. Pretreatment of iron-containing incubation system with desferal was shown to decelerate the process of thrombin inactivation. The kinetic characteristics for recovery of thrombin activity by 2 mM desferal were estimated at various periods after addition of iron sulfate in the inhibitory dose. The effect of reversibility was shown to depend on the time of thrombin preincubation with iron. Incomplete recovery of thrombin activity after increasing the time of incubation with iron (more than 30 min) was probably related to oxidative modification of thrombin.

[The free-radical processes and antioxidant therapy in brain ischemia].

A role of the free-radical processes and disturbances of oxidative-restorative blood homeostasis and nervous tissue in the pathogenesis of brain ischemic pathology and other diseases are reviewed. Attention is focused on the search for optimal ways of pharmacological correction of oxidative stress in the schemes of complex treatment of chronic blood circulation insufficiency and on the necessity of combined application of several antioxidants with different mechanisms of action which reciprocally potentiate each other. Experimental and clinical suppositions of the use of a-lipoic acid as one of the most studied antioxidant in the treatment of brain ischemia as well as the results of own studies on the preparation berlition which contains a-lipoic acid used in the neuroprotective therapy of chronic brain ischemia for correction of free-radical processes are discussed.

[The method for detection of protein oxidability in serum and plasma].

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.

Effects of oxidized fibrinogen on the functions of blood cells, blood clotting, and rheology.

Oxidatively-modified fibrinogen induces platelet aggregation and potentiates ADP-induced platelet aggregation and production of active oxygen forms in zymosan-stimulated leukocytes. Fibrinogen induces IL-8 production in primary culture of endothelial cells from human umbilical vein; the oxidized form of fibrinogen is more active, similarly as during induction of the expression cell adhesion molecules (P-selectin and ICAM-1). Oxidized fibrinogen (10 and 20% oxidation degree) impairs microrheological properties of the blood, sharply reduces erythrocyte deformability, modifies blood viscosity, and reduces suspension stability of the blood. Oxidized fibrinogen modified blood clotting parameters and ADP-, ristocetin-, and collagen-induced platelet aggregation in whole blood. Oxidized fibrinogen disordered the formation of fibrin clot and blood clotting process. Platelet aggregation was activated in response to ADP, but not to ristocetin and collagen, the degree of activation increased in direct proportion to the degree of fibrinogen oxidation. This indicates the "dysregulatory" effect of oxidized fibrinogen on platelets. The formation of platelet complexes with polymorphonuclear leukocytes was intensified in the presence of oxidized fibrinogen; polymorphonuclear leukocyte luminol-dependent fluorescence intensity in the presence of platelets increased after incubation with oxidized fibrinogen in comparison with native fibrinogen. Hence, oxidized fibrinogen plays an important role in the development of atherosclerosis and its complications (thromboses).

Oxidized forms of fibrinogen induce expression of cell adhesion molecules by cultured endothelial cells from human blood vessels.

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.

Fibrinogen and its oxidized form induce interleukin-8 [correction of interleukin-2] production in cultured endothelial cells of human vessels.

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.

Mechanism of activation of ADP-induced platelet aggregation under the influence of oxidatively modified fibrinogen.

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.

Oxidatively modified fibrinogen modulates blood rheological parameters.

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.

Effect of oxidized fibrinogens on blood coagulation.

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.

Effects of fibrinogen on lipid peroxidation in patients with coronary heart disease and in vitro.

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.

Effect of UV-modified fibrinogen on platelet aggregation in platelet-rich plasma.

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 effect of an antioxidant preparation based on bioflavonoids and vitamin C on the antioxidant activity of blood plasma]. / Vliianie antioksidantnogo preparata na osnove bioflavonoidov i vitamina C na antioksidantnuiu aktivnost' plazmy krovi.

The effect of single and long-term reception of the antioxidant preparation Magnum C (MC) on blood plasma antioxidant activity (AOA) of volunteers has been investigated. AOA was measured with chemiluminescence test using the hemoglobin-hydrogen peroxide-luminol system. MC contains ethereal form of vitamin C and bioflavonoids as the basic antioxidant components. It was determined that blood plasma AOA was increased on the average by 36% (p < 0.01) 3 hours after single intake of 4 capsules of MC. In case of long-term reception of MC (1 capsule twice in day during 4 weeks) the greatest increase in the blood plasma AOA was about 60% (p < 0.001) and was observed on the 14th day. On the 7th day after completion of MC reception the level of blood plasma AOA was, approximately, 20% above its initial value (p < 0.05). This is probably due to prolonged action of MC.