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.

Effect of gold nanoparticles coated with plasma components on ADP-induced platelet aggregation.

We studied the effect of gold nanospheres coated with components of autologous blood plasma on ADP-induced platelet aggregation. Gold nanoparticles in the chosen concentration range (5-40 µM) and particle size (5-30 nm) with or without coating produced no activating effect on platelet aggregation caused by aggregation inductor ADP in all applied doses (1.6, 2.0, and 5.0 µM). Nanoparticles with a diameter of >60 nm inhibited platelet aggregation. These findings and published data confirm the biological safety of gold nanoparticles for targeted delivery of drugs and phototherapy.

[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.

Studies of oxidant-induced changes in albumin transport function with a fluorescent probe K-35. Metal-catalyzed oxidation.

The dynamics of albumin transport function was studied during metal-catalyzed oxidation of albumin in diluted blood plasma from healthy donors and in the solution of purified albumin using fluorescent probe K-35. The changes were compared with the dynamics of free radical oxidation markers. For oxidation, different concentrations of Cu(2+), Fe(2+), Fe(3+) ions as well as EDTA and H(2)O(2) were used. Oxidative modification of proteins was assessed by carbonyl and bityrosine fluorescent products. Oxidation of plasma lipids was assessed by the levels of TBA-reactive products. It was found that oxidation markedly decreased effective concentration of albumin characterizing albumin binding capacity, and leads to accumulation of carbonyl products of protein oxidation, bityrosine fluorescent products in proteins, and TBA-active products of lipid oxidation. It was hypothesized that reduced effective concentration of albumin is related to impairment of its binding sites and/or accumulation of free-radical oxidation products filling the binding sites of albumin.

Studies of oxidant-induced changes in albumin transport function with a fluorescent probe k-35. Effect of hypochlorite.

The dynamics of changes in albumin transport function during hypochlorite-induced oxidation of isolated albumin in blood plasma and serum was studied with a fluorescent probe K-35. Binding of the probe K-35 to albumin was characterized by effective concentration of albumin. Oxidative modification of proteins was evaluated by the content of carbonyl products of protein oxidation and bityrosine fluorescent products. Oxidation with hypochlorite was accompanied by a decrease in the effective concentration of albumin in albumin, diluted plasma, and serum and accumulation of carbonyl products of protein oxidation and bityrosine fluorescent products. The decrease in the effective concentration of albumin during oxidation with hypochlorite can be explained by oxidative damage to albumin binding sites. Oxidative modification of probe K-35 binding sites with hypochlorite contributes to a decrease in effective concentration of albumin under pathological conditions.

[Association of a polymorphic marker Trp719Arg of KIF6 gene with effects of atorvastatin and simvastatin in patients with early ischemic heart disease].

Action of statins is characterized by pronounced variability what is caused by effects of a multitude of factors. Main of these factors appears to be genetic peculiarity of patients. We studied influence of polymorphic marker Trp719Arg of KIF6 gene on lipid and nonlipid effects of atorvastatin and simvastatin. The studied genetic marker is associated with risk of development of ischemic heart disease and myocardial infarction as well as efficacy of therapy with statins according to data of a number of large multicenter studies. We examined 60 men with ischemic heart disease which had manifested in young age when genetic factors were most expressed and had special significance. Efficacy of 40 mg/day simvastatin did not depend on genotypes of polymorphic marker Trp719Arg of KIF6. Therapy with 10 mg/day atorvastatin was more effective in carriers of polymorphic marker Trp719Arg of KIF6 gene by action on dynamics of changes of high sensitivity C-reactive protein and dispersion of high density lipoprotein response. Increase of atorvastatin dose to 80 mg/day abolished influence of genotypes. Thus for the first time we discovered influence of polymorphic marker Trp719Arg of KIF6 gene on individual response to therapy with 10 mg/day of atorvastatin, while and apoA1, structural protein of high density lipoproteins can be considered as a marker of "fast response".

[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.

[Relationship between clinical characteristics and primary and secondary products of lipid and protein peroxidation].

Recent studies showed that activation of free oxygen radicals and the resulting stress play a key role in the development of brain vascular lesions related to hypertensive disease and atherosclerosis leading to chronic cerebral ischemia. To recall, activation of oxidative stress precedes chronic cerebral ischemia and stroke. Therefore, detection of stress markers may be a step toward the development of a new method for the identification of groups at high risk of stroke among patients with the unfavourable course of cerebral ischemia. This study was focused on the relationship between lipid and protein peroxidation products and clinical manifestations of the disease.

Oxidative modification of fibrinogen inhibits its transformation into fibrin under the effect of thrombin.

Changes in the capacity of fibrinogen subjected to oxidative modification to transform into fibrin under the effect of thrombin and to form a fibrin clot were studied. The effects of oxidized fibrinogen preparations on the clot formation by citrate-treated donor plasma were evaluated by the thrombin time test. Oxidation impaired the capacity of isolated fibrinogen to form a fibrin clot under the effect of thrombin. Addition of oxidized fibrinogen solutions to donor plasma led to prolongation of the plasma clotting time. Maximum addition (33% volume) of oxidized fibrinogen led to a 10-26% prolongation of clotting time in comparison with addition of the same volume of the same solution without fibrinogen.

Effect of oxidized fibrinogen on aggregation of activated platelets and neutrophils.

The effect of oxidized fibrinogen on platelet-neutrophil complex formation was evaluated by studying the platelet aggregation (changes in light transmission and turbidimetric assay). Activation of cells by thrombin (0.015 U/ml) in the presence of oxidized fibrinogen was accompanied by the formation of larger intermolecular aggregates of platelets and leukocytes as compared to those detected in experiments with non-oxidized fibrinogen. Addition of thrombin (0.2 U/ml) in the presence of oxidized fibrinogen was followed by the formation of more stable complexes of platelets and leukocytes as compared to those revealed in experiments with non-oxidized fibrinogen. An increase in the width of aggregation curves was most pronounced in the system of 10(-4) M Fe(2+) and 10(-4) M H(2)O(2) with oxidized fibrinogen. Our results indicate that oxidized fibrinogen contributes to the "floating" or suspension of platelet-leukocyte complexes.

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.

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.

[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.

[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).

Oxidative resistance of the plasma and electrophysiological remodeling of the myocardium in coronary patients.

The diagnostic and prognostic potentialities of the parameters of free-radical processes and high resolution ECG in coronary disease were evaluated. A relationship between oxidative resistance of the plasma (MDA concentration after 24-h copper-induced oxidation) and clinical characteristics of coronary disease (functional class of angina and high-resolution ECG parameters) was detected. Changes in ECG parameters directly correlated with MDA levels, the frequency of their registration reflects the severity of coronary disease. The absolute values of ECG, MDA, and their dynamics correlated with the severity of coronary disease and outcome of acute coronary syndrome, the prognosis was unfavorable for patients with MDA level >100 nmol/ml. The level of MDA increased by days 5-7 of observation in all patients with acute coronary syndrome, indicating exhaustion of the plasma antioxidant system during exacerbation of the coronary syndrome. Hence, evaluation of the plasma oxidative resistance and high resolution ECG can serve as a new diagnostic complex approach for detecting coronary patients with an unfavorable prognosis.

[Correlations between electrophysiological myocardial remodeling and parameters of free radical oxidation in patients with various forms of ischemic heart disease].

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.