[Features of the metabolism of nitric oxide in normal state and inflammation].

Brief analysis of the metabolism of nitric oxide in living cells in normal state and pathology and also the analysis of the causes, that hampered the progress of these studies, were carried out. It was established that most of physiological fluids, including blood, normally contain nitrite and non-thiolate nitroso compounds in concentration less than 100 nM. Literature data from different researchers on the normal range of nitrite concentration in plasma of healthy people from several hundreds of nM to several microM is probably the result of low selectivity of the methods used. But nitrite and non-thiolate nitroso compounds concentration in blood is dramatically increased in case of inflammatory diseases. It is proposed that the main mechanism for the production of these substances in blood is the nitrosyl iron complexes transformation by active oxygen species but not the activation of NO production as it was-considered previously.

[The early diagnostic of chorionic -amnionitic and intrauterine infection on content of nitrite and non-tiolate nitrosocompounds in plasma under premature discharge of amniotic fluid].

The article deals with the results of examination of 33 pregnant women with premature discharge of amniotic fluid with purpose to choose an optimal criterion of diagnostic of intrauterine infection. In blood plasma of all female patients the increased, from 0.5 to 2.5 mkmol/l, total content of nitrite and non-tiolate nitrosocompounds non-containing iron (NO2+RNO). In case of pregnant women without intrauterine infection the content of nitrite and netiolate nitrosocompounds did not exceed 0.1 mkmol/l as in all other subjects without inflammatory diseases. The antibacterial and anti-inflammatory therapy applied to female patients with premature discharge of amniotic fluid resulted in lowering of concentration of NO2+RNO up to 0.1 mmol/l and even lower. The study results permit to suppose that concentration of NO2+RNO in plasma is one of sensitive indicators of presence of inflammatory processes concomitant to premature discharge of amniotic fluid which by its sensitivity and specificity is superior to such indicators as number of leucocytes, ESR and concentration of C-reactive protein.

[The early diagnostic of somatic inflammatory diseases in patients with acute ischemic stroke].

We examined 29 patients with acute ischemic stroke. Measurement of plasma nitrite and N-nitroso-compounds (NO2- +RNNO) to test for the presence of acute inflammatory process allows to timely and definitely determine its onset before the development of clinical symptoms and to operatively control the treatment, the latter being particularly important due to the restricted communication ability of neurologic patients.

[The content of nitrite and N-nitroso compounds of plasma as a diagnostic test of nonspecific inflammation].

The authors' enzymatic sensor was applied to identify the content of nitrite and nitroso compounds of blood plasma in normal condition and under various inflammatory diseases. It is established that in normal conditions blood plasma contains nitrite, N-nitroso compounds (RNNO) and S-nitroso compounds (RSNO) in concentrations less than 100 nm. The plasma pool of nitroso compounds includes basically thiolferous nitrosate ferrum complex in concentration 3-20 microm. The concentration of nitrite in plasma is from 10 to 150 microm. The concentration (nitrite+RNNO) increases dramatically under inflammatory diseases. This indicator was 0.3-1.0 microm in examined patients with acute and chronic pancreatitis, cholecystitis, ENT diseases of inflammatory character and ARD. In the case of acute appendicitis the indicator reached 10 micro. In case of successful treatment the content (nitrite+RNNO) decreased to the concentration lower than 100 nm. The content of other nitrite and N-nitroso compounds had no reliable variations. Because of this, largely implemented evaluation of intensity of nitric oxide production by the aggregate indicator of nitrites content (NO(x)) in blood seems ambiguous. The reason is that in normal conditions nitrite is presented in trace amount and considerable quantity of nitrate can enter the organism in an exogenous way. Besides that the content of nitrite can depend on kidneys filterability. At the same time, based on the obtained data, the content (nitrite+RNNO) in plasma in concentrations higher than 150 nm are definitely to be considered as pathology.

[Detection of nitrite and nitrosocompounds in chemical systems and biological liquids by the calorimetric method].

The capacity of nitrite, S-nitrosothiols (RS-NO), dinitrosyl iron complexes (DNICs) with thiol-containing ligands, and nitrosoamines to inhibit catalase has been used for the selective determination of these compounds in purely chemical systems and biological liquids: cow milk and colostram. The limiting sensitivity of the method is 50 nM. A comparison of the results of the determinations of RS-NO, DNIC, and nitrite by the catalase method and the Greese method conventionally used for nitrite detection showed that, firstly, Greese reagents decompose DNIC and RS-NO to form nitrite. Therefore, the Greese method cannot be used for nitrite determination in solutions of these substances. Secondly, Greese reagents interact with complexes of mercury ions with RS-NO, inducing the release of nitrosonium ions from the complex followed by the hydrolysis of nitrosonium to nitrite. Thus, the proposition about the spontaneous decay of the complexes of mercury ions with RS-NO is incorrect. Keeping in mind a high sensitivity of the method, the use of catalase as an enzyme detector of nitrosocompounds allows one to detect these compounds in neutral medium without prior purification of the object, thereby preventing artificial effects due to noncontrolled modifications of the compounds under study.

[An enzymatic sensor for estimation of the content of nitro and nitroso compounds in biological objects].

Nitric oxide (NO) generation in the biological cells and tissues is a cause of the production of some nitro and nitroso compounds that differ in their physiological significance and toxicity. The resultant problem in the estimation of the content of all nitro and nitroso compounds totally and singly in complex systems, such as biological objects, remains to be very urgent since simple, highly sensitive and highly selective methods have not been proposed so far to determine all these NO derivatives. The method based on some specific biochemical property of NO metabolites, which manifests itself under physiological conditions and permits the fixation of all nitro and nitroso compounds without their prior modification fraught with unpredictable artifacts, seems to be optimal. The authors have designed an enzymatic sensor based on the previously established ability to inhibit the enzyme catalase in the presence of halide ions. The level of dinitrosyl iron complex (DNIC) was measured, by using its property to lose its ability to inhibit catalase on addition of a trap of NO and an iron chelator to a reaction medium. S-nitrosothiols are detected as substances that are able to produce DNIC after addition of ferrous iron and thiols, unlike nitrite that has not this property. The total content of nitro compounds was estimated, by reducing to nitroso compounds by vanadium (III) chloride. The nitro compounds showing the properties of NO donors (RNO2) were determined as the substances that acquire the properties of DNIC in the presence of ferrous iron and thiols. The content of nitrates was estimated as a difference between the total level of nitro compounds and the content of RNO2. The sensitivity of this method was as high as 50 nM. That is it is more than an order higher in sensitivity than the classical methods based on the Griess reaction. By keeping its high sensitivity in mind, the proposed catalase method as an enzymatic detector of nitro and nitroso compounds allows one to detect these compounds in the neutral medium, without pre-purifying the object, thereby preventing the influence of the factors that contribute to the uncontrolled modification of the compounds under study.

[Inhibitory effects of serotonin and sodium ascorbate on the oxidative aggregation of lipoproteins]. / Ingibiruiushchee deistvie serotonina i askorbinata natriia na okislitel'nuiu agregatsiiu lipoproteinov.

Milk lipoproteins (MLPs) are structurally and biochemically similar to blood lipoproteins, which allow the former to be used as model objects for studying the properties of the latter. The results of turbidimetric measurements showed a change in the light scattering from MLP suspensions upon contact with Fe3+ ions in the free from or in chelate complexes with o-phenanthroline and EDTA. No such effect was observed for Fe2+ ions. The effect of Cu2+ ions (in microscopic amounts) was similar to that of Fe3+, while Ca2+ and Mg2+ produce no effect. It was found that 1,1-azabicyclohexanecarbonitrile-2-methylpropionamidine dihydrochloride (an azoinitiator capable of spontaneous decomposition with the formation of peroxide radicals in an oxygen containing-medium) introduced into an MLP suspension produces the same effect as Fe3+ and Cu2+ ions. Study of the particle size distribution in a microcapillary by the method of impedance measurements showed that a change in the light scattering from the suspension is caused by the MLP aggregation. The action of aggregation factors upon the MLPs led to their oxidation, as indicated by accumulation of the TBA-active products. The ability of copper ions to oxidize MLPs agrees with the data reported on the copper-ion-induced oxidation of blood lipoproteins, which was observed in studying a relationship between this oxidation and clinical manifestations of atherosclerosis. Thus, pronounced oxidation of both milk an blood lipoproteins in the presence of microscopic amounts of copper ions is indicative of a similarity of these processes. The process of lipoprotein aggregation induced by various oxidizing agents is inhibited by sodium ascorbate and serotonin. At the same time, beta-naphthol (an antioxidant soluble in lipids) does not affect the aggregation process. It is suggested that the oxidative aggregation of lipoproteins mag be related to the problem of atherogenesis and thrombogenesis.

[Oxidative destruction of estradiol after treatment with hydrogen peroxide catalyzed by horseradish peroxidase and methemoglobin]. / Okislitel'naia destruktsiia éstradiola pod deistviem perekisi vodoroda, kataliziruemaia peroksidazoi khrena i metgemoglobinom.

It is shown that estradiol in the presence of horse radish peroxidase interacts with hydrogen peroxide, which is evidenced by an increase in its optical density at 280 nm. The photometering of samples containing estradiol and horse radish peroxidase upon their titration with hydrogen peroxide indicated that the increase in optical density stops after introducing hydrogen peroxide equimolar in concentration to estradiol. The stoichiometric ratio of estradiol consumed during oxidative destruction to hydrogen peroxide was 1:1. In the presence of ascorbate, the oxidative destruction of estradiol by the action of hydrogen peroxide, catalyzed by horse radish peroxidase, was observed only after a latent period and showed the same regularities as in the absence of ascorbate. It was found by calorimetry that, during the latent period, estradiol catalyzes the degradation of hydrogen peroxide and ascorbate without undergoing oxidative destruction. The substrates of the peroxidase reaction benzidine, 1-naphthol, and phenol interact with hydrogen peroxide in the presence of ascorbate and horse radish peroxidase in a similar way. Presumably, upon interaction with hydrogen peroxide in the presence of horse radish peroxidase, estradiol, like other substrates of this reaction, undergoes oxidative destruction by the mechanism of peroxidase reaction. It is shown that oxidative destruction of estradiol by the action of hydrogen peroxide can also be catalyzed by methemoglobin by the same mechanism. These data are important for understanding the role of estradiol in the organism and the pathways of its metabolic conversions.

[Chelating and oxidizing properties of tetracycline metabolites forming during its peroxidase or photoinduced oxidation]. / Khelatiruiushche-okisliaiushchie svoistva metabolitov tetratsiklina, poluchaemykh pri ego peroksidaznom ili fotoindutsirovannom okislenii.

Tetracycline metabolites forming on the antibiotic exposure to visible light or peroxidase as well as tetracycline as such showed the ability to bind iron cations. When the metabolites bound the cations of iron protoxide, they catalyzed its oxidation. Chelating agents such as o-phenanthroline and EDTA arrested the ions of iron protoxide and iron oxide in the respective iron/tetracycline complexes at a much lower rate than that with the use of the native tetracycline. This means that the affinity of the metabolites with the above mentioned iron ions was much higher than that of the native tetracycline. When the metabolites and tetracycline bound iron protoxide, they catalyzed its oxidation to the oxide. Tetracycline and its metabolites were shown as well to have the property of reversible regeneration of iron oxide to the protoxide.

[Metabolites of tetracycline obtained during its irradiation with visible light or peroxidase oxidation. Their toxic properties in relation to hemoglobin]. / Metabolity tetratsiklina, poluchennye pri ego obluchenii vidimym svetom ii peroksidaznom okislenii. Ikh toksicheskie svoistva v otnoshenii hemoglobina.

It was indicated in the literature that when exposed to visible light tetracycline induced phototoxic effects with respect to heme-containing proteins. The present study showed that when tetracycline was exposed to visible light it formed metabolites due to the photochemical transformations, the metabolites formation being slightly affected by the anti-radical drugs such as L-histidine, mannitol, ethanol and catalase. The investigation of the conditions of the metabolites formation as a result of the photochemical transformations revealed a specific role of ascorbate in the process. The comparative analysis of the physico-chemical properties of the metabolites resulting from the tetracycline exposure to visible light or peroxidase oxidation provided a conclusion that the nature of the metabolites was the same. It was shown that the metabolites were equal in their phototoxic capacity for the damage of hemoglobin by inducing its oxidative degradation.

[The property of tetracyclines to induce methemoglobin formation in erythrocytes and to inactivate catalase when exposed to radiation in the visible range]. / Svoistvo tetratsiklinov vyzyvat' metgemoglobinoobrazovanie v éritrotsitakh i inaktivirovat' katalazu pod deistviem izlucheniia.

When tetracycline and chlortetracycline were incubated an a dark room in the presence of erythrocytes with erythrocytic catalase completely inactivated by sodium azide, the antibiotics induced methemoglobin formation in them. If the catalase was not inactivated, no such phenomenon was observed. This meant that after the penetration into the erythrocytes the tetracyclines induced in them the generation of hydrogen peroxide which was the immediate cause of the methemoglobin formation. The effect of the methemoglobin formation on the erythrocytes was also induced by tetracycline without the catalase blocking when the erythrocytes were exposed to the antibiotic and visible light. The effect was not mediated by the hydrogen peroxide action on hemoglobin in the erythrocytes as it was in the previous case, since even when catalase was added exogenously to the suspension medium it induced no suppression of the methemoglobin formation in the erythrocytes. Additional introduction of exogenous catalase to the erythrocyte hemolysates prior to the exposure did not either influence the methemoglobin formation photoinduced in them by tetracycline. The effect manifestation was not practically influenced by L-histidine, mannitol or ethanol used as traps for the radicals which could form during the antibiotic exposure to visible light in the suspension medium. The calorimetric estimation of the catalase functional properties showed that when exposed to visible light in the presence of the enzyme (a commercial product) tetracycline induced its inactivation. It was indicated that the catalase photoinactivation by tetracycline was due not to a steady decrease of the activity of every molecule of the enzyme but to a dislodge of separate molecules among the active ones, i.e. a one-fold change of the enzyme molecule from the initial active state to the completely inactive one. The catalase photoinactivation by tetracycline was not eliminated by L-histidine or comparatively high concentrations of mannitol but was entirely eliminated by ethanol used in relatively low concentrations. When the erythrocytes were exposed to visible light in the presence of tetracycline, the effect of the catalase photoinactivation by the antibiotic was also observed. In this case the same as in the experiments with isolated catalase, ethanol as well protected the enzyme from the photoinactivation by tetracycline. The tetracycline photoeffects on hemoglobin, catalase and possibly other heme-containing proteins were likely realized in the immediate closeness of their hemes. The photoeffects of the tetracyclines associated with the heme-containing proteins possibly play a certain role in the phototoxicity of the antibiotics.

[Generation of active forms of oxygen by antibiotics of the tetracycline series during tetracycline catalysis of oxidation of ferrous iron]. / Generatsiia aktivnykh form kisloroda antibiotikami tetratsiklinovogo riada pri kataliziruemom imi okislenii zakisnogo zheleza.

During oxidation of Fe(2+) catalyzed by tetracyclines there was recorded lucigenin, activated chemiluminescence evident of generation of the oxygen radicals. It was also observed that during the Fe(2+) oxidation by the molecular oxygen catalyzed by tetracyclines there generated hydrogen peroxide which accelerated the Fe(2+) oxidation recorded photometrically by the formation of strongly absorbing tetracycline complexes with Fe(2+). In the presence of ascorbate reducing Fe(2+) in the complexes with tetracyclines and their subsequent oxidation there generated radicals modifying the antibiotic molecules evident from a change in their absorption spectra after the respective incubation. The results offered a pattern describing the mechanism of the tetracycline toxic effect on biological objects.

[Metabolic transformation of antibiotics of the tetracycline series in peroxidase reactions]. / Metabolicheskie prevrashcheniia antibiotikov tetratsiklinovogo riada v peroksidaznykh reaktsiiakh.

In was shown calorimetrically that in the presence of horse radish peroxidase tetracyclines induced degradation of hydrogen peroxide. Under such conditions changes in the tetracycline optical properties were detected photometrically. It was concluded that tetracyclines were metabolized in the peroxidase reactions catalyzed by horse radish peroxidase as their substrates. The tetracycline peroxidase oxidation was catalyzed not only by horse radish peroxidase but also by methemoglobin possessing the peroxidase activity. In the experiments with ascorbate there were detected characteristic peculiarities of the tetracycline peroxidase oxidation catalyzed by both horse radish peroxidase and methemoglobin. These peculiarities made it possible to classify the tetracyclines as the substrates of the peroxidase reaction belonging to the oxidogenic group. The fact that tetracyclines can be metabolized in peroxidase reactions is discussed in regard to its possible influence on their mechanism of antibacterial action and the development of tetracycline resistance.

[A new view of estradiol biotransformation in the body. Estradiol metabolism in the erythrocytes via the peroxidase reaction]. / Novyi vzgliad na biotransformatsiiu éstradiola v organizme. Metabolizm éstradiola v éritrotsitakh putem peroksidaznoi reaktsii.

The study has established that in the presence of horseradish peroxidase, estradiol was subject to oxidative destruction under the action of hydrogen peroxide via the peroxidase reaction. The stoichiometric ratio of the hydrogen peroxide consumption to estradiol oxidation is 1:1 in this peroxidase reaction. Estradiol peroxidation was found to be catalyzed by methemoglobin by the same order as in case of horseradish peroxidase. Based on these results and the fact that estradiol is bound to erythrocytes and penetrates inside, it is concluded that erythrocytic estradiol is metabolically converted via its peroxidation.

[Change in various properties of catalase in the small intestine of rats after vagotomy]. / Izmenenie nekotorykh svoistv katalazy tonkoi kishki krys pri vagotomii.

To evaluate the antioxidative system of the small intestine in disturbed parasympathetic innervation, the authors studied the activity of catalase, its resistance to substrate inactivation (catalytic capacity) and reduction potential (reduction of Fe3+ to Fe2+) in the enteric homogenate in different periods (on day 14 and 30) after bilateral subdiaphragmatic truncal vagotomy. Vagotomy did not change catalase activity but was attended by an increase of catalase catalytic capacity and reduction potential. It is suggested that the discovered changes of the studied parameters are manifestations of responses of the antioxidative protective system of the small intestine to the activation of lipid peroxidation.

[Mechanism of oxyhemoglobin oxidation induced by hydrogen peroxide]. / Mekhanism okisleniia oksigemoglobina, indutsirovannogo perekis'iu vodoroda.

The process of oxyhemoglobin oxidation initiated by hydrogen peroxide in low (10(-7) M) concentrations was investigated. It was found, that H2O2 in this concentration is able to induce the process of chain oxidation of oxyhemoglobin to methemoglobin. The following observations indicate that the process is essentially the chain reaction: 1) The amount of the methemoglobin in haem groups, produced in the reaction, exceed by 20 times the quantity of hydrogen, added initially, to induce the oxidation. 2) Catalase stopped this process at any stage of the reaction. This fact implies that the chain process involves generation of new molecules of H2O2 in the course of oxidation of oxyhemoglobin. The chain reaction proceeded only in the presence of oxygen. But if oxygen was introduced into hemoglobin solution, preincubated with H2O2 in vacuum, than again the oxidation of hemoglobin developed. Apparently, H2O2 in low concentrations appears, mainly, as an inductor of the oxyhemoglobin autooxidation.

[Mechanical properties of the small intestine of rats in the normal state and after disturbed parasympathetic innervation]. / Mekhanicheskie svoistva tonkoi kishki krys v norme i pri narushenii parasimpaticheskoi innervatsii.

In intact and vagotomized (in 14 and 30 days after the operation) rats by means of the dynamometric method values of maximal load and relative maximum elongation of the proximal and middle areas of the small intestine (SI) have been determined in vitro. Dependence of relative elongation of the SI fragments on the load applied has been investigated. The proximal part of the SI is the most firm to tearing in comparison to the middle one. Bilateral subdiaphragmatic+ truncal vagotomy results in an increased firmness to tearing and in relative maximal elongation of the SI proximal part in 14 days and in decrease of the former parameter in the same SI part in 30 days.