Way to accelerate the oxidation of nitric oxide (no) in a heterogeneous environment

 

(57) Abstract:

The invention relates to acceleration of the reaction of nitric oxide in heterogeneous environments, including blood plasma, the addition of hydrophobic fluorine-containing compounds and may find application in medicine for correction of conditions associated with impaired metabolism of nitric oxide. The invention consists in that the reaction mixture is injected component(s) causing the formation of one or more new phases, so that the value of the expression

< / BR>
where N is the acceleration of the reaction; kithe reaction rate constant in the i-th phase, QNO,i, is the equilibrium distribution coefficients of NO and O2in the i-th phase; xi- the share of the i-th phase in the total volume has increased, with the added component(s) contains(a)t fluorocarbon, or halogen-substituted derivative fluorocarbon, or perforaciones, or protein solution, solyubilizirovannye fluoride organic matter with a value of QNOand/or in the two-phase system with water, higher than the maximum of the values of QNOand/or for arbitrary pairs of phases of the reaction mixture before adding, and heterogeneous environment is blood plasma. The invention allows to accelerate the reaction of the one-electron akaltara and pressure in the system. 5 C.p. f-crystals, 6 ill., table 2.

The invention relates to the field of chemistry, biochemistry and medicine, in particular to the regulation of NO-dependent processes in living organisms.

Nitric oxide (NO) is the major metabolite of inorganic all higher animals [1-5] . He is involved in the regulation of the diameter of blood vessels [3] and septic shock [6], in the transmission and storage of information [4]; is the starting product for the synthesis of peroxynitrite, which kills pathogenic bacteria and cancer cells [7, 8]; and acts as a scavenger of free radicals [9, 10]. In mammals, NO is formed by the oxidation of arginine by oxygen under the action of NO-synthase [4, 11] and disintegrates upon further oxidation. There are two main competing ways oxidation NO: complexes of transition metals, for example, Hb-O2quickly and irreversibly oxidizes NO to nitrate (1)-trachelectomy oxidation; in the absence of transition metals free O2oxidizes NO to nitrite or nitrosoguanidine (2) - one-electron process [1, 5, 12]

NO + Hb+2- O2---> NO-3+ Hb+3; (1)

4NO + O2+ 2H2O ---> 4NO2-+ 4H+; 4NO + O2+ 2RSH ---> 2RSNO + 2NO2-+ 2H+(2)

In fact, reaction (2) optimista processes (1) and (2) regulates the pool of NO and NO-equivalents, and through them the most important functions of an organism in norm and pathology [5, 10, 16].

Known methods of acceleration of chemical reactions, including (2), are increasing the concentrations of reacting substances (reaction (2) total third order), achieved by various methods, and fever. For biochemical and medical applications, this approach is often unacceptable because the concentration of NO and oxygen are set by physiology. In many instances (e.g., septic shock) in order to accelerate the reaction (2) is the need of the local decrease of the NO concentration with a simultaneous increase in the concentrations of oxidation products (e.g., nitrosothiols performing various physiological functions, including the transport of NO in the form of NO-equivalents).

There is also known a technique for accelerating the oxidation of NO in the micellar catalysis [1, 2], while the hydrophobic phase acts as a sponge, concentrating reagents in a small volume. In [1] the method is implemented experimentally in addition to homogeneous aqueous solution of emulsions containing hydrophobic components, for example, liposomes. Thus the reaction rate observed decrease of the NO concentration was increased, i.e., it was shown that the oxidation rate in hetero acceleration from the share of added hydrophobic phase monotone (increasing the share of the hydrophobic phase, the reaction rate is constantly growing).

The closest analogue of the proposed technical solutions can be considered the work of [2], in which it was theoretically shown that this dependence has a maximum at a relatively small fractions of the hydrophobic phase, with further increase in the share of the hydrophobic phase, the reaction rate falls, the proposed formula for calculating acceleration of the oxidation of NO in multicomponent systems. This work can be taken as a prototype. However, in [2] was not offered a practical way to accelerate the reaction of the one-electron oxidation of NO in a heterogeneous environment, such as in the blood. The paper also contained an algebraic error in the formula.

In the paper [17] N. Trouble, and So Suncool (the authors of this application in accordance with article 4-1 of the Patent law of the Russian Federation application is submitted earlier than six months from the date of publication of the article (18.06.99)) error in previously published studies were analyzed.

In the prior art it is unknown as to accelerate the reaction of the one-electron oxidation of NO (2) in the initially heterogeneous system (practically important case is in the blood), without changing any of the quantities of the reacting substances or temperature and pressure in the system. The proposed solution consists in changing the reaction credc, to the value of the expression,

< / BR>
where H is the acceleration of the reaction, kithe reaction rate constant in the i-th phase, QNO,i, is the equilibrium distribution coefficient of NO and O2in the i-th phase, xi- the share of the i-th phase in the total volume.

describing the overall acceleration of the reaction throughout the system (total for all phases) has increased. This can be achieved by incorporating in the reaction environment more hydrophobic component (e.g., fluorocarbon) with a high value of QNOand/or

The proposed solution involves an inventive step (i.e., not obvious from the prior art) is indeed issued by the U.S. patent [19] (1998), where the claimed emulsions of fluorosurfactants for increasing the concentration of NO in the body of the patient, the authors suggest that NO will dissolve in the hydrophobic phase and the oxidation neglected.

The proposed solution is feasible, for example, when using a mixture of fluorosurfactants "perftoran" (dosage form is permitted for clinical use). Fluorosurfactants are allowed to use as blood substitutes, while in the bloodstream impose significantly more fluorocarbon, which illustrate the invention the following examples. Effect of micellar catalysis was proven initial study of the kinetics of the oxidation of nitric oxide by oxygen in the water in the blood plasma, in the solution of the emulsion of fluorosurfactants in water and in blood plasma after solubilization of fluorosurfactants. As the emulsion of the fluorosurfactants used emulsion performanceline and emulsion performancebased, stable surface - active agent (Pluronic), and the pharmaceutical form emulsion "perftoran" (content performanceline 13 g, the content of performancecriteria 6.5 g per 100 ml). Solubilization of fluorosurfactants plasma proteins was performed under the action of ultrasound by mixing plasma with a solution of an emulsion of fluorosurfactants. Resolubilization part of the emulsion was separated from the solution of the plasma by centrifugation. The water, after removal of oxygen, saturated with gaseous nitric oxide. Using tight glass syringe water V = 1 ml, saturated with nitric oxide, was added to the reaction mixture under air. Sampling was performed every 10 seconds, the volume of the sample was 50 µl. For the kinetics of oxidation of nitric oxide followed by reaction product is nitrite ion.

4NO + O2+ 2H2

The ratio of the initial rates of oxidation of NO in the presence of the emulsion of fluorosurfactants and in distilled water (see table.1).

The ratio of the initial rates of oxidation of NO in the presence of PFC emulsion, solubilizing in plasma and in plasma without a PFC emulsion WPFC0/Wplasma0(see tab. 2).

In Fig. 1 shows a typical kinetics of NO oxidation in the water and emulsion PFCs.

Example 6. Micellar catalysis of the oxidation of NO in vivo.

The experiments were performed on 40 rats male normotensive Wistar weight 180-400, the day before the experiment the animals under ketamine anesthesia (5 mg/100 g administered intraperitoneally) was implanted polyethylene catheters (PE-10). The femoral artery was katerinovka for further connection to the sensor is registering HELL, and femoral vein for intravenous administration of drugs. Blood pressure and heart rate were measured in the abdominal aorta using a pressure sensor connected to the input of the amplifier. The signal from such operation is received into two groups, each of which consisted of control and experimental subgroups. Experienced subgroup was introduced perftoran (NGOs "Perftoran") at a rate of 5 ml/kg, control the same number of modified Krebs solution-Henseleit (KG). With the help of perfusate solutions were presented in venous catheter at a constant speed (0.2 ml/min). Monitoring of blood pressure and heart rate was continued for one and a half hours after intravenous injections.

After registration of the original level of HELL the first group of rats (group L-NAME) was administered selective inhibitor of NO-synthase - methyl ester of N- nitro-L-arginine (Sigma Chemical Co.) at a dose of 50 mg/kg (1 ml physiologic saline) intraperitoneally. An hour and a half, when the HELL was set at a constant level, animals were injected with sodium nitrite (1 mg/kg in 1 ml of physiologic saline intraperitoneally). After persistent reduction in HELL, the animals were injected Perftoran (or KG).

The second group of rats (FC-group) Perftoran (or KG) was administered immediately after registration of the original level of HELL.

Before each experiment, after the HELL the infusion of L-NAME and sodium nitrite were set at a constant level, and immediately after the completion of monitoring, from arterial the plasma.

The amount of nitrite and nitrate in the blood was determined photometrically using a standard set of reagents firms "R&D Systems". For analysis it was necessary to 0.1 ml of plasma 50 ál for each anion. The procedure was as follows.

Determination of nitrite:

1) in a test tube of 1.5 ml (measured in 50 µl reaction buffer;

2) was added to 50 μl of the sample (zero in the solution were added 50 μl of reaction buffer);

3) was added to the sample 50 ál reagent Gris I (was intensively shaken);

4) after 10 min was added 50 μl of reagent Gris II;

5) the protein precipitate was separated by centrifugation for 10 min (spindle speed 10000 rpm);

6) the solution above the sediment was transferred into an optical cuvette with an optical path length of 1 cm and a volume of 1 ml, and then was adding 0.8 ml of distilled H2O;

7) was detected optical density of the solution at a wavelength of 540 nm;

8) the amount of nitrite was calculated from the calibration graph.

Determination of nitrate:

1) in an Eppendorf tube was added 50 μl of the sample (zero in the solution were added 50 μl of reaction buffer);

2) was added 25 μl of NADH solution (intensively mixed);

3) was added 25 μl of a solution of nitrate reductase;

4>6) the amount of nitrate was calculated by the difference between the second and the first determination of nitrite.

In Fig. 2 shows a calibration graph for the determination of nitrite.

In Fig. 2A presents the calibration graph for the determination of nitrate.

Group L-NAME.

The infusion of the NOS inhibitor content of nitrite and nitrate in plasma experimental and control subgroups unreliable decreased. After injection of sodium nitrite content of both ions in the plasma increased dramatically, (the amount of nitrite increased by 344 68 microns in experience and 362 73 microns in control and nitrate - 87 15 μm experience and 76 16 in the control). The introduction of perftoran experienced subgroup resulted in significant change in the nitrite-nitrate balance of the blood. Thus, the content of nitrite decreased by 220 40 μm, and the content of nitrate increased by 157 12 μm. In the control subgroup infusion solution KG resulted in a slight (33 13 μm) to decrease the content of nitrite and nitrate in plasma.

In Fig. 3 shows the change in the concentration of nitrite and nitrate in the course of the experiment in the experimental group L-NAME.

In Fig. 4 shows the change in the concentration of nitrite and nitrate in the course of the experiment in the control group L-NAME.

Group FC
< increase the content of nitrite 49 6 μm, the concentration of nitrate decreased by 23 8 μm. As a result, total content of products of NO oxidation was increased. In the control subgroup introduction KG resulted in even a slight decrease in the concentration of both ions to 74 microns. Therefore, the total amount of nitrite and nitrate in the blood slightly reduced.

Thus, in both series of experiments introduction into the blood of the new phase with a higher distribution coefficients Q led to the acceleration of the oxidation of NO. In the group NAME own NO synthesis under the action of NO-synthase was zingiberales and NO could be formed only when restoring entered nitrite and tionaries obtained by one-electron oxidation of NO in the presence of thiols blood. Activation of the one-electron oxidation of NO under the influence of perftoran resulted in increasing the content of tionaries and, consequently, to accelerate the synthesis of NO during their recovery. Formed thus NO competitive oxidized complex of the hemoglobin-oxygen and other Fe-containing complexes to nitrate, so the nitrite concentration decreased, and the concentration of nitrate increased. In the absence of the inhibitor of NO-synthase and exogenous nitrite introduction of perftoran led to uskorenie the competitive reaction, fell. The example shows the possibility of practical realization of the invention to accelerate the oxidation of NO in the blood.

In Fig. 5 shows the change in the concentration of nitrite and nitrate in the course of the experiment in the experimental group FC.

In Fig. 6 shows the change in the concentration of nitrite and nitrate in the course of the experiment in the control group FC.

Literature

1. Liu, X., Miller, M. J. S., Joshi, M. S., Thomas, D. D. and Lancaster, J. R., Jr. (1998) Proc. Natl. Acad. Sci. USA 95, 2175 - 2179.

2. Gordin, V. A. and Nedospasov, A. A. (1998) FEBS Letters 424, 239-242.

3. Gow, A. J. and Stamler, J. S. (1998) Nature 391, 169-173.

4. Mayer, C. and Hemmens, B. N. (1997) Trends Biochem. Sci. 22, 477-481.

5. Nedospasov, A. A. (1998) Biochemistry (Moscow), 63, 881-904.

6. Kuhl, S. J. and Rosen, H. (1998) West. J. Med. 168, 176-181.

7. Pryor, W. A. and Squadrito, G. L. (1995) Am. J. Physiol. 268, L699-L722.

8. Xie, K., and Fidler, I. J. (1998) Cancer Metastasis Rev 17, 55-75.

9. Gorbunov, N. V. Tyurina, Y. Y., Salama, G., Day, B. W., Claycamp, H. G., Argyros, G. , Elsayed, N. M. and Kagan, V. E. (1998) Biochem. Biophys. Res. Comman. 244, 647 - 651.

10. Darley-Usmar, V. , Wiseman, H. and Halliwell, B. (1995) FEBS Lett 369, 131-135.

11. Stuehr, D. J. (1997) Annu. Rev. Pharmacol. Toxicol 37, 339-359.

12. Liu, X., Miller, M. J. S., Joshi, M. S., Sadowska-Krowicka, H., lark, D. A. and Lancaster, J. R., Jr. (1998) J. Biol. Chem. 273, 18709-18713.

13. Reutov, V. P., Sorokina, E. G. and Kaiushin, L. P. (1994) Vopr. Med. Khim. 40(6), 31-35.

14. Singh, R. J. , Hogg, N., Joseph, J. and Kalyanaraman, B. (1996) J. LASS="ptx2">

16. Whittle, B. J. (1995) Histochem. J. 27, 727-737.

17. Beda, N. V., Suntsova, T. P. (1999) FEBS Letters 453, 229-235.

18. Patent USA 5726209 (1998) "Liquid Fluorcarbon Emulsion as a Vascular Nitric Oxide Reservoir.

1. The way to accelerate a one-electron oxidation of nitric oxide (NO) with oxygen in a heterogeneous environment by changing the composition of the reaction mixture, namely, that in the reaction mixture is injected component(s) causing the formation of one or more new phases, so that the value of the expression

< / BR>
where H is the acceleration of the reaction;

kithe reaction rate constant in the i-th phase;

QNO,i, is the equilibrium distribution coefficients of NO and O2in the i-th phase;

xi- the share of the i-th phase in the total volume,

increased.

2. The method according to p. 1, wherein the input component(s) contain fluorocarbon.

3. The method according to p. 1, wherein the input component(s) contain halogen-substituted derivative of fluorocarbon.

4. The method according to p. 1, wherein the input component(s) contain perforaciones.

5. The method according to p. 1, wherein the input component(s) contain the protein solution, solyubilizirovannye fluoride organic matter with a value of QNOand/or two-phase Noy mixture prior to the introduction.

6. The method according to p. 1, characterized in that the heterogeneous environment is blood plasma.

 

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