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It is shown that human serum albumin, previously treated with HOCl (HSA-Cl), enhances luminol-dependent chemiluminescence of neutrophils activated by phorbol-12-myristate-13-acetate (PMA). The enzyme-linked immunosorbent assay revealed that addition of HSA-Cl to neutrophils promotes exocytosis of myeloperoxidase. Inhibitor of myeloperoxidase--4-aminobenzoic acid hydrazide, without any effect on lucigenin-dependent chemiluminescence of neutrophils stimulated with PMA, effectively suppressed luminol-dependent chemiluminescence (IC50 = 20 microM) under the same conditions. The transfer of the cells from medium with HSA-Cl and myeloperoxidase to fresh medium abolished an increase in PMA-induced luminol-dependent chemiluminescence, but not the ability of neutrophils to respond to re-addition of HSA-Cl. A direct and significant (r = 0.75, p) correlation was observed between the intensity of PMA stimulated neutrophil chemiluminescence response and myeloperoxidase activity in the cell-free media after chemiluminescence measurements. These results suggest the involvement of myeloperoxidase in the increase of neutrophil PMA-stimulated chemiluminescence response in the presence of HSA-Cl. A significant positive correlation was found between myeloperoxidase activity in blood plasma of children with severe burns and the enhancing effects of albumin fraction of the same plasma on luminol-dependent chemiluminescence of PMA-stimulated donor neutrophils. These results support a hypothesis that proteins modified in reactions involving myeloperoxidase under oxidative/halogenative stress, stimulate neutrophils, leading to exocytosis of myeloperoxidase, a key element of halogenative stress, and to closing a "vicious circle" of neutrophil activation at the inflammatory site.

It is shown that in the presence of reduced glutathione at low concentrations (1-5 microM) the extent of platelet aggregation with neutrophils increases and the lag period of platelet aggregation induced by tumor cells decreases. At the same time in the presence of reduced glutathione at high concentration (3 mM) the extent of platelet aggregation with neutrophils decreases, and the lag period of platelet aggregation induced by tumor cells increases. It is established that glutathione-dependent regulation of the intercellular contact formation between platelets and neutrophils depends on the ratio of glutathione oxidized and reduced forms: at fixed total glutathione concentration of 5 microM, increase of glutathione redox potential from -175 mV to 0 mV led to reduction in platelet aggregation with neutrophils. Thus, it is shown for the first time, that GSH has priming effect on the platelet aggregation with neutrophils and tumor cells, which may contribute to the regulation of inflammatory diseases and cancer.

The chlorination activity of free myeloperoxidase and myeloperoxidase bound with ceruloplasmin or with both ceruloplasmin and lactoferrin has been studied by luminal-dependent chemiluminescence. It was shown that the addition of hydrogen peroxide to the "myeloperoxidase + Cl- + luminal" system is accompanied by a fast flash of light emission. In the absence of myeloperoxidase or Cl-, the flash intensity was considerably reduced. The inhibitor of myeloperoxidase NaN3, the HOCl scavengers taurine and methionine, and guaiacol, a substrate for peroxidation cycle of myeloperoxidase, prevented luminescence. These results suggest that the generation of luminescence was due to the halogenating activity of myeloperoxidase, and hence, the flash light sum may serve as a measure of chlorination activity of myeloperoxidase. The activity of myeloperoxidase was suppressed by ceruloplasmin. Lactoferrin exhibited no significant influence on the myeloperoxidase activity, nor did it prevent the inhibitory effect of ceruloplasmin when they both were combined with myeloperoxidase. These data were confirmed using alternative approaches for evaluating the myeloperoxidase activity, namely, the assessment of peroxidation activity and the taurine chlorination assay. It is noteworthy that the inhibitory effect of ceruloplasmin on chlorination and peroxidation activities of myeloperoxidase is seen with the latter, traditional approaches only if ceruloplasmin is present in a large excess relative to myeloperoxidase, whereas the chemiluminescence method allows the detection of the inhibitory effect of ceruloplasmin using lower proportions of the protein with respect to myeloperoxidase, which are close to the stoichiometry of the myeloperoxidase/ceruloplasmin and the myeloperoxidase'ceruloplasmin'lactoferrin complexes.

We have demonstrated that hypochlorite (HOCI/OCl-) and hypobromite (HOBr/OBr-) can react with tert-butyl hydroperoxide with close rate constants (k(HOCl) = 10,8 M(-1) x s(1); k(HOBr) = 8,9 M(-1) x (s(-1)). By means of the spin trap 4-pyridyl-1-oxide-N-tert-butyl nitron we have found that both reactions proceed through decomposition of tert-butyl hydroperoxide and generation of tert-butyl peroxyl (OOC(CH3)3) and tert-butoxyl (OC(CH3)3) radicals, the ratio of their the concentrations being dependent on the concentration of tert-butyl hydroperoxide. Thus, hypobromite, similar to hypochlorite, is a precursor of free radicals produced in the reaction with organic hydroperoxides. This reaction can be of great importance in the intensification of free radical processes, namely, in lipid peroxidation at the stage of chain branching.

Different approaches based on the spin probe method were used to compare the physical state of the surface lipid monolayer in subfractions of low-density lipoproteins: in native low-density lipoproteins constituting the bulk of human blood low-density lipoproteins and in circulating multiple-modified low-density lipoproteins whose portion is minor in healthy persons but significantly increases in atherosclerotic patients. The data obtained in in vitro experiments suggest that circulating multiple-modified low-density lipoproteins possess atherogenic properties. The order parameter S, rotational correlation time tau, and hydrophobicity parameter h were calculated from electron spin resonance spectra of a series of spin probes whose paramagnetic groups are located at different depths of the lipid monolayer. These parameters characterize the molecular packing, fluidity, and polarity in the microenvironment of paramagnetic groups. The kinetics of the reduction of paramagnetic groups by ascorbate and oxidation by hypochlorite were obtained for the spin probe whose paramagnetic group is located deeply in the lipid monolayer at the level of the terminal segments of phospholipid acyl chains. No difference between native low-density lipoproteins and circulating multiple-modified low-density lipoproteins was revealed in respect of the physical properties of the lipid domain of surface proteolipid layer, as sampled by spin probes.

Preparations of low-density lipoproteins from healthy donor blood contain lipoprotein particles with different capacity for aggregation: upon stirring, some particles form aggregates significantly more quick than others. After stirring, lipoprotein particles are separated by ultracentrifugation into two fractions: a fraction of large aggregates and a fraction of small particles without intermediate forms. It is known that lipoprotein aggregates can accelerate intracellular accumulation of lipids. Therefore, it is supposed that particles of high aggregation ability are more atherogenic.

The interaction of hypochlorite with linoleic acid hydroperoxides was studied by the coumarin C-525-enhanced chemiluminescence and ESR spin trapping techniques. Linoleic acid hydroperoxide was obtained in the reaction of lipoxygenase and linoleic acid. Alpha-(4-pyridyl-1-oxyl)-N-tert Butylnitron was used as a spin trap. It was shown that the addition of hypochlorite to the incubation media containing linoleic acid and lipoxygenase resulted in an intensive chemiluminescence flash. The intensity of this flash correlated with the hydroperoxide concentration. The analysis of ESR spectra of spin adducts produced in the reaction of hypochlorite with linoleic acid hydroperoxide showed the presence of O-centered, most likely peroxyl, radical with the splitting constants alphabetaH = 0.260 mT aN = 1.662 mT and C-centered penthyl radical with the splitting constants alphabetaH = 0.260 mT; aN = 1.662 mT. These data suggest that hypochlorite produced by phagocytes in vivo can induce the generation of free O- and C-centered radicals, promoters of free radical processes.

The interaction of hypochlorite (HOCl/OCl-) with tert-butyl hydroperoxide ((CH3)3COOH) was investigated by chemiluminescence. It was shown that the addition of HOCl/OCl- to (CH3)3COOH induces a fast chemiluminescent flash. The intensity of this flash increases with the increase in both HOCl/OCl- and (CH3)3COOH concentration. The chemiluminescence is quenched in a concentration-dependent manner in the presence of free radical spin traps N-tert-butyl nitrone and alpha-(4-pyridyl-1-oxyl)-N-tert-butyl nitrone. This fact proves that free radicals take part in the interaction of HOCl/OCl- and (CH3)3COOH. Hypochlorite yielded a very similar chemiluminescence spectrum in its reaction with (CH3)3COOH as Ce4+. It differed considerably from the spectrum in the system H2O2 and HOCl/OCl-. It is well known that the interaction of Ce4+ and (CH3)3COOH produces peroxyl radicals. These results confirm the hyothesis that the interaction of HOCl/OCl- and (CH3)3COOH is mediated by peroxyl radicals. Thus, organic hydroperoxides always present in unsaturated lipids can induce lipid peroxidation processes in the reaction with HOCl/OCl-.

In order to make a further sight into mechanism of hypochlorite (HOCl/OCl-)-induced lipid peroxidation we investigated the effect of pH of incubation medium on accumulation of lipid peroxidation products and also on kinetics of HOCl/OCl- decay and disappearance of double bonds during incubation of egg yolk phosphatidylcholine liposomes with hypochlorite. On the one hand, the increase in pH values led to decrease of the rate of a reaction of hypochlorite with double bonds of phosphatidylcholine acyl chains and, on the other hand, to increase in accumulation of primary (conjugated dienes and lipid peroxides) and secondary (TBA-reactive substances) products of lipid peroxidation. The present data and our previous results allow to suppose that besides the electrophilic addition of HOCl to double bonds another reaction of HOCl/OCl- with some lipid intermediate occurs that leads to initiation of free radical processes of lipid peroxidation. This reaction increases in velocity as pH rises at least in the range from 6.50 to 8.15. The role of intermediate may be performed by organic hydroperoxides which are known to be present in some proportion in lipid phase in vivo.

The ability of hemoglobin, modified by H2O2 or HOCl/OCl-, to induce lipid peroxidation (LPO) in low density lipoproteins (LDL) was studied, as well as the effects of haptoglobin. It was found that Hb modification by H2O2 or HOCl/OCl- increased generation of TBA-reactive substances in low density lipoproteins. Modified Hb was as double or more reactive compared to intact Hb. Free radical scavengers (ethanol and mannitol) gave no effect on LPO in LDL. On the other hand, ferric iron chelator desferrioxamine decreased LPO 5-6 times. Ferrous iron chelator- o-phenanthroline was effective only in the case of LPO, induced by H2O2 modified Hb. Haptoglobin (plasma protein forming complexes with Hb) decreased LPO induced by both intact or HOCl/OCl modified Hb. The results of the paper show that modification of Hb by H2O2 or HOCl/OCl- increase the ability of Hb to induce LPO in LDL, probably due to metHb, ferrylHb or free iron production.

The present study examined the kinetics of accumulation of lipid peroxidation products in phosphatidylcholine liposomes incubated with hypochlorite (HOCl/OCl-) or with FeSO4 in the presence of ascorbate. The incubation of liposomes with HOCl/OCl- led to the accumulation of primary (lipid hydroperoxides and conjugated dienes) and secondary (TBA-reactive substances) lipid peroxidation products identical to those generated in the presence of Fe2+/ascorbate. However the rate of the formation of lipid peroxidation products as well as the gain in their amount were sufficiently higher (4-150-fold and 3-6-fold, respectively) in the case of HOCl/OCl(-)-induced lipid peroxidation as compared to lipid peroxidation in the Fe2+/ascorbate system. The accumulation of lipid peroxidation products lasted until HOCl/OCl- was present in the reaction mixture. The data obtained support our hypothesis that hypochlorite produced by activated neutrophils and monocyte/macrophages may be reasonable for the initiation of lipid peroxidation in blood.

The chemiluminescence in the presence of luminol has been used to measure the amount of hypochlorite and its reduction during the interaction with oxidized and non-oxidized liposomes from egg yolk phosphatidylcholine as well as with organic peroxides (tert-butylhydroperoxide, cumene hydroperoxide, di-tert-butylperoxide, tert-butylperbenzoate, di-benzoylperoxide), and epoxides (cis- and trans-2,3-epoxy-butane, cholesterol-5 alpha,6 alpha-epoxide, and cis-9,10-epoxystearic acid). Since hypochlorite did not react with the saturated phospholipid, dimyristoylphosphatidylcholine (DMPC), and the reduction of double bonds in egg yolk phosphatidylcholine liposomes occurred at the same rate in both oxidized and non-oxidized liposomes, it may be suggested that hypochlorite interacted precisely with LPO products. None of the epoxides tested in this study, similar to di-tert-butylperoxide, tert-butylperbenzoate, di-benzoylperoxide, incorporated into liposomes reacted with hypochlorite. In contrast, tert-butylhydroperoxide and cumene hydroperoxide effectively reacted with it. The data obtained suggest that epoxides, dialkyl-, diacyl-, and alkyl-acyl-peroxides are not involved in hypochlorite-induced LPO. At the same time, organic hydroperoxides commonly present in certain amounts in the biomembrane lipid phase in vivo may play a role lf an intermediate; its interaction with HOCl/OCl- gives rise to free radical formation followed by accumulation of LPO products.

Surface potential of human blood lipoproteins was studied by the use of Mn(2+)-ions as a positively charged spin probe. The calculated values of surface potential of low density lipoproteins (LDL) appeared to be -17.7 +/- 1.2 mV. It was shown that hypochlorite (OCl-) induced an increase of negative surface potential of LDL. Agarose gel electrophoresis was used for the comparative analysis of electrophoretic mobility of human blood lipoproteins of different classes. It was shown that the interaction of hypochlorite with lipoproteins caused the partial degradation of lipoproteins and the increase in surface charge. High density lipoproteins appeared to be more resistant to the action of OCl- than LDL and especially very low density lipoproteins.

Changes of structural organization of liposomal phospholipid membranes, after their Fe2+-induced peroxidation were studied at different depth using nitroxyl derivatives of stearic acid. It was established that during Fe2+-induced lipid peroxidation a strong rise in lipid polarity accompanied with immobilization of acyl chains of phospholipids at the depth of 0.6-0.8 nm from the surface was measured. At the same time no significant changes in the structural organization were seen at the depth of 20-22 nm.

Surface potential of human plasma lipoproteins was studied by the use of positively charged spin probe. The calculated values of surface potential of high and low density lipoproteins appeared to be -29 +/- 1 and -16 +/- 1 respectively. It was shown that lipid peroxidation process induces an increase of surface potential of both high and low density lipoproteins. Probably, it is connected with the increase of the negative charge density on their surface. This fact can play an important role in pathogenesis of diseases with lipid metabolism and lipid peroxidation level disorders in plasma (atherosclerosis, ischemic heart disease etc.).

The thermo-induced structural changes in lipoproteins (LP) of human plasma were investigated by the electron paramagnetic resonance method, using 5-doxylstearate as a spin probe. LP were shown to undergo thermotropic transformations at temperatures specific for each LP type. Differences were found in the nature of temperature dependences of the order parameter for the probe localized in LP and for isolated lipids. The denser the LP, the more pronounced the differences. 5-Doxylstearate was shown to be localized not only in the lipid phase of LP, but also in the area of the protein--lipid contact. This indicates that 5-doxylstearate is a useful tool for the study of protein--lipid interactions in pathological processes coupled with their modifications.