[Oxidative stress in focal symptomatic and cryptogenic epilepsy in young patients]. / Okislitel'nyi stress pri fokal'noi simptomaticheskoi i kriptogennoi epilepsii v molodom vozraste.

OBJECTIVE: To study oxidative stress in young patients with focal symptomatic and cryptogenic epilepsy and after the new-onset epileptic seizures. MATERIAL AND METHODS: Patients, aged from 19 to 44 years, were distributed into three groups, 30 patients in each: patients after a few (first) epileptic seizures, patients in the clinical remission that has lasted at least one year, and patients with treatment-resistant epileptic seizures. The control group included 20 healthy people. Parameters of the pro-oxidant status (TBA-reactive products) and the antioxidant defense (total superoxide-scavenging activity, catalase, total antioxidant activity, and reduced thiols (SH groups)) were measured in the blood plasma. RESULTS: No significant differences in the concentrations of TBA-reactive products were identified in patients with epilepsy compared with healthy controls while concentrations of reduced SH groups, total superoxide-scavenging activity, catalase activity and the total antioxidant activity were significantly decreased in patients. In addition, some of the parameters displayed significant differences between different groups of patients. CONCLUSION: In patients with epilepsy, the changes in the free-radical processes are seen already after the first seizures and persist in the treatment-resistant epilepsy and in clinical remission. Since the parameters of the activity of the antioxidant defense are significantly different in different groups of patients, one can assume that different elements of the oxidative stress are present after the first epileptic seizures and in different courses of the disease.

Effect of Morphogenetic Protein BMP-2 on X-Ray Density of Bone Defect in the Experiment.

In experiments on Wistar rats, a simulated defect in the flat bones of the skull was filled with a collagen sponge of animal origin impregnated with BMP-2 or pure sponge; in control rats, the defect was left open. During follow-up, X-ray density of the collagen sponge in the experimental groups differed significantly. The results attest to the absence of spontaneous remodeling of the bone tissue under conditions modeled focal defect. Moreover, stimulation of reparative processes by the collagen matrix did not lead to positive dynamics. Saturation of the collagen sponge with BMP-2 in a concentration of 0.05 mg/ml allowed increasing Xray density of the bone starting from week 4.

Lipoic acid nanoforms based on phosphatidylcholine: production and characteristics.

We describe the dynamics of lipoic acid (LA) alone, incorporated in liposomes and as a part of nanoemulsions. Mass spectrometry shows that LA in water forms aggregates of two or three molecules in the form of a negatively charged ion and a neutral molecule. Phosphatidylcholine (PC)-based nanoforms of LA as liposomes and nanoemulsions with a particle size equal to 145 nm are characterized by a high degree of incorporation of LA into the nanoparticles and long-term stability during storage at room temperature. Dynamic light scattering (DLS) gives the polydispersity index of the nanoforms (> 0.3), characterizing the homogeneity of the obtained nanodispersions. We found that such emulsions can significantly (5 ×) increase the concentration of LA in the aqueous phase (5-7 mg/mL) when compared with an aqueous solution of LA (1 mg/mL) and by 40% when compared with PC liposomes (4 mg/mL). Moreover, the inclusion of LA in liposomes and nanoemulsions from PC did not change the neutral ζ-potential characteristic of PC nanoforms. CryoTEM established that the structural organization of the liposomes practically did not differ from nanoemulsions and both nanoforms contained both multilayer and single-layer vesicles. When studying the release kinetics of LA from phosphatidylcholine nanoforms, we found that at 22 h, 45-55% of LA was released from nanoparticles, but that at the initial stage of the process LA was slowly released from the nanoemulsions and rapidly from the liposomes. Conductance measurements indicate that LA delivered in all the three forms increase membrane permeability, though this result is most marked with the LA in PC liposomes.

[The study of the membrane-protective potential of the combination of 2-ethyl-6-methyl-3-hydroxypyridine-succinate and citicoline]. / Issledovanie membranoprotektivnogo potentsiala kombinatsii preparatov 2-étil-6-metil-3-oksipiridina-suktsinata i tsitikolina.

AIM: To assess the changes in the composition of plasma phospholipids in patients with chronic cerebrovascular disease treated with neuroprotectors 2-ethyl-6-methyl-3-hydroxypyridine succinate (neurox) and citicoline (neipilept), the natural metabolites involved in biochemical processes in the body, and their composition. MATERIAL AND METHODS: The study included 40 patients, 18 men and 22 women, aged from 54 to 72 years, with chronic cerebrovascular disease at the decompensation stage complicated with the hypertensive crisis and/or arrhythmia. RESULTS AND CONCLUSION: During extraction of phospholipids from blood cells, a significant decrease in the amount of total lipids was found to the end of treatment of patients who received neurox or neipilept or their combination. The study of quantitative composition of phospholipids showed no significant changes in patients treated with neurox, while the use of citicoline or combination of citicoline with 2-ethyl-6-methyl-3-hydroxypyridine succinate resulted in the increase of their total mass. There were no significant changes in the qualitative composition of phospholipid classes in blood plasma in patients treated with neurox. In patients treated with neipilept or with the combination of citicoline with 2-ethyl-6-methyl-3-hydroxypyridine succinate, plasma phosphatidylcholine was significantly increased. No significant changes in the content of phosphatidylinositol, phosphatidylserine and sphingomyelin were observed.

[Complex application 2-ethyl-6-methyl-3-hydroxypyridine-succinate and vinpocetine in cerebrovascular disorder.] / Kompleksnoe primenenie vinpotsetina i 2-étil-6-metil-3-oksipiridina-suktsinata pri tserebrovaskuliarnoi patologii.

Developing brain ischemia due to cerebral vascularization leads to disruption of brain metabolism. Chronic cerebral hypoperfusion leads to irreversible brain damage and plays an important role in the development of some types of dementia. Early use of antioxidants such as ethyl ether apovincamine acid (vinpocetine) and 2-ethyl-6-methyl-3-hydroxypyridine-succinate in the treatment of this pathology is seen as a real pathogenetically based method of correction of cerebral metabolism with cerebral vascular disorders, demonstrating the increase in cerebral blood flow and also neuroprotective effects. Clinical studies and studies on biological models show that the main mechanisms of action of vinpocetine and 2-ethyl-6-methyl-3-hydroxypyridine-succinate, although have a similar focus, but implementing neuroprotective and nootropic effects via various links in the pathogenesis of ischemic brain damage.

[Liposomal form of lipoic acid: preparation and determination of antiplatelet and antioxidant activity]. / Liposomal'naia forma lipoevoi kisloty: poluchenie i opredelenie antiagregatsionnoi i antioksidantnoi aktivnosti.

Optimal conditions for obtaining phosphatidylholine (PC) liposomes with lipoic acid (LA) are chosen that lead to the formation of nanoparticles with a size of 175¸284 nm with efficiency (extent) of inclusion of LA in liposomes equal 85% and characterized by a slow release of substance from the nanoparticles. The effect of "empty" liposomes and liposomal form of LA on platelet aggregation induced by arachidonic acid (AA) is established. It is found that liposomes with LА inhibit platelet aggregation, caused by AА, to 80%. In addition, it is shown that "empty" liposomes slightly (to 30%) suppress platelet aggregation, caused by AА. The amount of TBA-sensitive products in samples of platelet-rich plasma (PRP) incubated with liposomal LA is determined. It is shown that LA in the composition of liposomes retains its antioxidant properties, and the amount of products of lipid peroxidation in platelet-rich plasma decreases in a dose-dependent manner when arachidonic acid is used as an inductor of platelet aggregation. It is assumed that the antiplatelet action of the liposomal form of LА is induced by inhibition of the initiation of lipid peroxidation products caused by exogenous inducer AА. It is supposed that, after additional research, the liposomal form of LA can be considered as a new drug in complex treatment of cerebral ischemia.

[The individual and combined antioxidant effects of citicoline and ethylmethylhydroxypyridini succinas]. / Individual'nye i kombinirovannye antioksidantnye effekty tsitikolina i etilmetilgidroksipiridina suktsinata.

AIM: To study the antioxidant status of patients with chronic cerebral ischemia (CCI) during the individual treatment with 2-ethyl-6-methyl-3-hydroxypyridine-succinate (neurox) and in the combination with citicoline (neipilept). MATERIAL AND METHODS: A study included 40 patients, 18 men and 22 women, aged from 54 to 72 years, with CCI, stage 2, at the decompensation stage complicated with the hypertensive crisis and/or arrhythmia. RESULTS AND CONCLUSION: A significant increase in the serum superoxide dismutase activity after the complex therapy with neurox and neipilept was demonstrated compared to patients treated with neurox. A study of reduced sulfur-hydroxy groups in patients treated with 2-ethyl-6-methyl-3-hydroxypyridine-succinate and patients treated with the combination of 2-ethyl-6-methyl-3-hydroxypyridine-succinate and citicoline, revealed a significant increase in the number of reduced SH- groups after the treatment with neurox compared to the combined use of neurox and neipilept.

[Study of the influence of gold nanoparticles on activation of human blood neutrophils].

Activation of neutrophils in the presence of gold nanoparticles is accompanied by the formation of free radical peroxidation products recording the flash of chemiluminescence. The basis for the activation mechanism has its origins most likely in the influence of the gold particles on the surface membrane potential of neutrophils. Investigation of changes in the fluorescence intensity of the negatively charged ANS probe on the surface of model membranes by adding different concentrations of gold nanoparticles indicates the change in the membrane surface charge density that can cause cell activation.

[Multikinase inhibitor sorafenib and HDAC inhibitor suberoylanilide hydroxamic acid suppress confluent resistance of cancer cells to recombinant protein izTRAIL].

Suppression of human tumor cell resistance to TRAIL-induced apoptosis in confluent cultures, using molecular target drugs (sorafenib and SAHA) at non-toxic concentrations was studied. Sorafenib, a multikinase inhibitor, and SAHA, an inhibitor of histone deacetylase, effectively suppressed resistance of confluent human cells derived from the skin carcinoma (A431 cell line) and fibrosarcoma (HT-1080 cell line). The effectiveness of suppression of confluent resistance with these inhibitors for human carcinoma A431 cells was significantly higher than that for the human ovarian carcinoma OVCAR-3 cells. For all cell lines studied, suppression of confluent resistance with SAHA was more effective than when sorafenib was used. The possible reason for increasing tumor cell resistance in confluent cultures and the importance of this phenomenon for understanding drug resistance of cells in the tumor tissue are discussed.

[Increase in resistance of A431 cancer cells to TRAIL-induced apoptosis in confluent cultures].

It was shown that cancer cells acquired resistance to TRAIL-induced apoptosis in confluent cultures. Recombinant protein izTRAIL induced apoptosis of human carcinoma A431 cells in the first hours after cell plating at a concentration of 3-10 ng/ml, while in confluent cultures these cells acquire resistance to protein izTRAIL even at the concentration of 2 mkg/ml. Detachment and suspending of the cells of confluent cultures immediately suppressed the resistance to izTRAIL. The cells of confluent cultures, being resistant to TRAIL-induced apoptosis continue progression through the cell cycle, as evidenced by the DNA cytograms and the Ki67p-GFP reporter system. Thus, the results showed that tumor A431 cells can acquire resistance to TRAIL-induced apoptosis in confluent cultures, while continue progression through the cell cycle, keeping the proliferative potential.

[Myeloperoxidase-induced biodegradation of single-walled carbon nanotubes is mediated by hypochlorite].

Broad prospects for the use of single-walled carbon nanotubes (SWNTs) in medicine and biotechnology raise the concerns about both their toxicity, and the mechanisms of biodegradation and excretion from the body. SWNTs biodegradation as a result of catalytic activity of myeloperoxidase (MPO) was shown in the isolated MPO system as well as in the suspension of neutrophils [Kagan V.E., et al., 2010]. In the present study we analyzed the ability of different MPO-produced oxidants to participate in the modification and degradation of SWNTs. The comparison of the ability of various peroxidases to degrade SWNTs in vitro revealed that myeloperoxidase, due to its ability to produce hypochlorite, and lactoperoxidase, due to its ability to produce hypobromite, are extremely efficient in the degradation of carbon nanotubes. The biodegradation of SWNTs in the model system can also be caused by free radicals generated as a result of heme degradation and, to a lesser extent, by active oxoferryl intermediates of peroxidases. Our experiments showed that in the presence of blood plasma, peroxidase intermediates or free radical products of heme degradation were unable to initiate biodegradation of carbon nanotubes, only the generation of hypochlorite by MPO can cause the biodegradation of carbon nanotubes in vivo. Titration of SWNTs suspension containing plasma with hypochlorite at high concentrations resulted in the decrease in the optical absorbance of the suspension indicating the degradation of nanotubes. Our results clearly indicate that hypochlorite can serve as a main oxidizing agent which is able to modify and degrade nanotubes in the sites of inflammation and in the phagosomes.

[A search for new markers of oxidative stress in brain ischemia for the optimization of treatment approaches].

Data of literature on the role of inflammation factors in the pathogenesis of stroke are presented. The study of myeloperoxidase level in the early acute phase of ischemic stroke and dynamics of this parameter after the antioxidant treatment with the α-lipoic acid preparation berlition was carried out. It has been shown that the activation of systemic inflammation and related oxidative stress recorded in the early acute phase of brain infarction needs pharmacological treatment. Neuroprotective action of α-lipoic acid related to the prevention of damaging effect of free radicals on cell membranes and reduction of oxidative stress intensity is a pathogenetic explanation for using its preparations in ischemic brain lesions. The decrease in plasma myeloperoxidase content after the treatment with berlition may consider as a criterion of its efficacy.

[The role of recipient cells in the mechanism of pathological calcification of heart valve and vascular transplants].

It was found using the model of subcutaneous implantation in rats that the calcification of the aorta wall occurs by two mechanisms of which one is dependent on, and the other independent of the migration of recipient cells to the transplant.

[A study of the effect of ceruloplasmin and lactoferrin on the chlorination activity of leukocytic myeloperoxidase using the chemiluminescence method].

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.

[A comparative spin trapping study of hypobromous and hypochlorous acids interaction with tert-butyl hydroperoxide].

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.

Role of myeloperoxidase-mediated modification of human blood lipoproteins in atherosclerosis development.

The mechanism of interaction of hypochlorite and hypobromite formed in myeloperoxidase catalysis with lipids of human blood low-density lipoprotein is described. Both agents react with unsaturated lipids via two mechanisms: molecular (with the formation of mainly chloro- or bromohydrins and lysophospholipids) and free-radical (paralleled by lipid peroxidation). These reactions modify physicochemical properties of low-density lipoproteins and disorder their lipid-transporting function thus initiating early stages of atherosclerosis development.

Generation of free radicals during decomposition of hydroperoxide in the presence of myeloperoxidase or activated neutrophils.

It was shown with the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone that myeloperoxidase (MPO) in the presence of its substrates H2O2 and Cl- as well as activated neutrophils destroy tert-butyl hydroperoxide producing two adducts of O-centered radicals which were identified as peroxyl and alcoxyl radicals. Inhibitory analysis performed with traps of hypochlorite (taurine and methionine), free radical scavengers (2,6-di-tret-butyl-4-methylphenol and mannitol), and MPO inhibitors (salicylhydroxamic acid and 4-aminobenzoic acid hydrazide) revealed that the destruction of the hydroperoxide group in the presence of isolated MPO or activated neutrophils was directly caused by the activity of MPO: some radical intermediates appeared as a result of the chlorination cycle of MPO at the stage of hypochlorite generation, whereas the other radicals were produced independently of hypochlorite, presumably with involvement of the peroxidase cycle of MPO. The data suggest that the activated neutrophils located in the inflammatory foci and secreting MPO into the extracellular space can convert hydroperoxides into free radicals initiating lipid peroxidation and other free radical reactions and, thus, promoting destruction of protein-lipid complexes (biological membranes, blood lipoproteins, etc.).

[The interaction of hypochlorite with fatty acid hydroperoxides results in the generation of free radicals].

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.

[Interaction of tert-butyl hydroperoxide with hypochlorite induces peroxyl radicals. A chemiluminescence study]. / Vzaimodeistvie tret-butilgidroperoksida s gipokhloritom privodit k obrazovaniiu perekisnykh radikalov. Issledovanie metodom khemiliuminestsentsii.

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-.