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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.

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 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.

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.

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.

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.

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.

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).

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.

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.