The method of determining the activity and functional status of the synthetase of nitric oxide

 

The invention relates to medicine, in particular for histochemistry. The method provides a more accurate assessment of functional status and activity of the synthetase of nitric oxide in specific cells to solve diagnostic and research tasks. Conduct assessments of NADPH2-diaphorase activity of the enzyme by treatment with the investigational product solutions of salts of tetrazole in the presence of NADPH2after exposure of the drug for 10 min in 4% aqueous solution of neutral formalin, thus provide for complex histochemical reactions, including additional assessment mates functioning of the catalytic center of the enzyme during NBT-test by staining of the drug for 30 min in 0.2% solution narasinga of tetrazole in buffered saline solution (pH 7.4) and nitrite by exposure of the drug prepared Griss reagent for 30 min at 37oWith, and the activity and functional status synthetase of nitric oxide determined photometrically according to the content in the cells of the drug products cytochemical reaction for nitrite and deformazione. 7 Il.

The invention relates to about the Vano for diagnostic processes of tissue damage, associated with activation of free radical oxidation.

Studies in recent decades have led synthase-nitric oxide among the most studied enzymes [1, 2]. This is due to the variety of functions and high biological activity of the final product of the enzymatic reaction of nitric oxide is one of the five known intracellular messengers [3]. Nitric oxide can enhance the defense mechanisms and increase the resistance of cells to the effects. However, interacting with superoxidedismutase, nitric oxide forms peroxynitrite, which in the acidic environment dissociates with the formation of hydroxyl radical. These radicals are dlitelnoye and have a strong damaging effect on the cell structure. Their education provides antimicrobial protection of the body, but under pathological conditions (stroke, heart attacks, colourvue inflammation and others) can contribute to areas of damage and the loss of highly specialized cells with critical violations of the functions of the organs. Accordingly, the formation of nitric oxide, performs, in addition, regulatory functions, subject to a detailed analysis for prediction of disease outcomes is C which performs a specific catalytic function [4].

The known method histochemical evaluation of the activity of the enzyme in the final product of the enzymatic reaction. In the formation of a portion of the nitric oxide binds to thiols, forming nitrosothiol. The content of the latter in the tissues assess immunohistochemically number of nitrotyrosine [5]. The advantages of this method are the ability to assess the activity of the enzyme by the number of the final product of the enzymatic reaction, which increases the specificity of the results. Among the disadvantages of this method, which limited its use are the need of the use of serums, production and purification which are associated with significant financial costs, and the possibility of estimating the content of nitric oxide associated with only one amino acid, while the other more numerous nitrosothiol not analyzed.

The known method histochemical determination of enzyme activity by means of a colour reaction with nitrosonium tetrazolium (BAT), recovering in painted formazan the oxidation restored nicotinamide dinucleotide phosphate (NADPH2) on the reductase domain of the enzyme [5, 6]. This histochemical reaction was successfully used to identify NO-ergy nitrogen in various organs and tissues [7-9].

However, the use of this method did not allow to obtain accurate results due to the fact that the same enzymatic reaction is carried out at least another five enzymes, to allocate among which the component synthetase of nitric oxide is not possible. To overcome this drawback, a method of pre-processing experiencing material to inhibit the activity of NADPH2-diaphorase not associated with the metabolism of nitric oxide. This processing is reduced prior to immersing the slices in a solution of formaldehyde or potassium permanganate [5, 10, 11].

The use of pre-treatment of sections allowed us to increase the specificity of the method, which is confirmed by immunohistochemical detection of protein NO-synthetase. It was shown that the modified identification diaphorase activity with a high degree coincides with the distribution of immunologic labels and marks the localization of the enzyme [12, 13].

Describes the histochemical method for determining the activity of NO-synthetase adopted as a prototype as the most widespread and close in order actions.

Its advantages are the simplicity and the ability to assess samostatnou, consisting of: - assessment of enzyme activity is not on the end-products of the enzymatic reaction, which eliminates the possibility of obtaining information about the work of the enzyme deficiency, tetrahydrobiopterin when dissociation centers metabolism of L-arginine and one-electron oxygen reduction together with produces NO superoxidedismutase (Fig.1); - inability to inactivation by formalin or potassium permanganate all side of NADPH2-diaphorase. Excessive processing coagulating agents are able to inhibit the activity of the synthetase of nitric oxide, and in case of insufficient pre-treatment can be identified additional activities not related to the formation of nitric oxide.

Arguments determine the necessity of carrying multiple histochemical reactions characterizing the processes oxidase and reductase domains of the enzyme and determining the degree of their agreement.

The aim of the invention is the finding method for determining the activity of the synthetase of nitric oxide, allowing more accurate assessment of functional status and activity of this enzyme in specific cells to solve diagnostic and scientific-IP is located in along with detection of NADPH2-diaphorase activity of the enzyme assess mates functioning of the catalytic centers in the course of the NBT-test and histochemical detection of nitrite reagent Griss.

The possibility of achieving the goal of the invention is proved by the following examples.

Example 1. The method of determining the activity of the synthetase of nitric oxide is performing the next procedure. The tissue sample is extracted from a biological object and frozen in liquid nitrogen after the correction of the surface tension of the last talc. For the manufacture of frozen sections used microtome with a table TOZ or cryostat, making slices in which is carried out at a temperature of -18oC. a Sample of tissue is removed from liquid nitrogen and placed in a marked drop of water primarily to preparatorily. By micropolicy a tissue sample progressive raise of 10 μm, cutting raised above the plane of the knife section.

In the manufacture cryostatic slices carry them with knife and brush on the pre-chilled glass and through local heating of the opposite edge surfaces of the glass reaches the unfolding of the drug. The best is s is transferred into chilled to 4oWith buffered saline (pH 7.4). In the implementation of this method using three tissue section mounted on three glass or placed in three containers with saline solution. For control reactions using three more of the drug.

In the case of using the method to evaluate the activity of the synthetase of nitric oxide in cell cultures and tissues by staining cultures on glass do not perform the previous manipulation. Incubation medium is drained and drugs crops washed three times in test tubes cold buffered saline solution (pH 7.4, temperature 4oC).

The first drug is transferred from saline in a freshly prepared solution of reagent Griss. When using cryostatic slices solutions of coloring reagents dotted on the glass. Staining of tissue culture slides and drugs entirely immersed in the dye solution by pouring the latter into tubes containing culture. Staining performed for 30 min at a temperature of 37oC. Preparation of reagent Griss carried out by dissolving 350 mg officinal preparation (manufacture of NGOs "Reagent") in 50 ml of distilled water. Control are drugs in the and L-nitroarginine at a concentration of 10-5M

The second drug is placed in a 0.2% solution narasinga of tetrazole in buffered saline solution (pH 7.4) for NBT-test. Staining performed within 30 minutes Control are drugs, incubated without the addition of nst.

A third drug for 10 min, treated by the method prototype, putting in 4% aqueous solution of neutral formalin for inactivation of NADPH2-diaphorase not associated with the formation of nitric oxide. After treatment with formalin preparations incubated in substrate buffer mixture containing CNT and NADPH2by noting loida [14]. Control are drugs, pre-treated in a solution of salts of diphenylethane at a concentration of 10-5M

After dyeing preparations dried with a Hairdryer and without additional dehydration illuminate xylene and conclude in canadian balsam or syrup of Apathy.

Content of products cytochemical reactions in the cells of the tissues of the drug is evaluated quantitatively on a light microscope using photometric consoles. The intensity loss of deformazione appreciate at a wavelength of 530 nm, and the photometry products histochemical reaction for nitrite detected at a wavelength of 480 nm. Activity farm the rats Extracted tissue sample of the sciatic nerve of rats were treated in accordance with the claimed method. The results of staining of the slices shown in Fig. 2, 3 and 4.

It is obvious that the greatest accumulation of deformazione in the detection of NADPH2-diaphorase occurs in the cells of the vascular wall and glorith, whereas nerve fibers remain maloobrazovannimi. Similarly distributed product diazoacetate, marking the accumulation of nitrite. When conducting NBT-test patterns nerve stained weakly, indicating that the pair of processes oxidase and reductase domains of the enzyme.

In this example, the distribution of the products of the reaction for nitrite and coincided with the results of detection of NADPH2-diaphorase (the prototype method), however, additional application of NBT-test allowed us to assess the correlation functioning domains of the enzyme.

Thus, the inventive method compared with the method of the prototype allows to obtain additional information about the enzymatic process.

Example 3. Evaluation of the activity of NO-synthetase in cell cultures of renal epithelial Preparations cultures of renal epithelial cells adhered on top of the glass was treated by the claimed method.

Microscopic examination of the cells revealed granules products histochemical reactions. The show is located and intensity were significantly large in comparison with the results of the histochemical reaction in the detection of nitrite and the results of the NBT-test. This, apparently, is due to the fact that the cells of this differen Express several enzymes with formalistically of NADPH2-diaphorase activity.

In this example, the distribution of the products of the reaction for nitrite and the NBT-test did not coincide with the results of detection of NADPH2-diaphorase (the prototype method), which is probably due to the additional staining of tissue not associated with the processes of synthesis of NO.

Thus, the inventive method is superior to the prototype based on specificity and provides more accurate information about the activity of the enzyme.

Example 4. Evaluation of the activity of NO-synthetase in the sciatic nerve after limb ischemia and reperfusion Outbred white rats weighing 220 g were narcoticyou the thiopental dose of 100 mg/kg intraperitoneally and were fixed on the machine in position on the back with outstretched limbs. On the right foot in the groin area transversely applied pressure turnstile 1 hour after the time the obstruction to flow is eliminated and after 10 min of reperfusion of the limb anesthetized animal is taken out of the experience. The rat was deceptional and dissected sciatic nerve ischemic limb. Contralateral to the Ali by the claimed method. The results of staining of sections of intact nerve did not differ from that given in example 2. The results of staining nerve ischemic limb shown in Fig.5, 6 and 7.

Staining of the tissues of the sciatic nerve is damaged limbs differed from those with the contralateral side. Found a significantly greater intensity distributions of the color reaction for nitrite and a high level of NBT-test, however, NADPH2-diaporama activity was not changed.

In this example, the distribution of the reaction products diazoketone exceeded the results diaphorase test of the prototype method). Intense staining of sections deformation when neindutsirovannom recovery nst suggest dissociation functioning domains and leakage of electrons without changing diaphorase activity.

Thus, the inventive method is superior to the prototype to analyze the processes of synthesis of oxide of nitrogen under pathological conditions. Intense reaction in the determination of nitrite in combination with intensive staining of tissues when performing NBT-test allow you to install the formation of peroxynitrite and to predict the extent of tissue damage.

PR is Quatro functioning synthetase of nitric oxide in normal, and in pathological conditions, when a sandboxed application of NADPH2-diaphorase test of the prototype method) gives distorted results.

The claimed invention satisfies the criterion of "novelty", as first proposed effective way to estimate the activity and functional status of the synthetase of nitric oxide, comprising a complex of histochemical reactions and allows to identify pathological changes in specific cell populations, but not in the tissue as a whole, which significantly increases the value of the information received. Histochemical determination of nitrite previously not described in literature.

The claimed invention satisfies the criterion of "inventive step", because it does not use the known technical solutions. In the proposed method to evaluate the activity and functional status of NO-synthetase applied complex histochemical reactions, allowing to evaluate the functioning of the enzyme and conjugation reactions metabolizirovannom L-arginine and one-electron oxygen reduction in the formation of the final products of the enzymatic reaction.

The criterion of "suitability for industrial application we prove the results of Imeretia when conducting scientific research and laboratory diagnostic practice to obtain reliable results in the identification of the biochemical mechanisms of development of pathological conditions.

Sources of information 1. Goren A. K. F., Mayer B. Universal and comprehensive Enzymology synthase nitric oxide // Biochemistry. - 1998. - T. 63, vol. 7. - S. 870-880.

2. Taylor, B. C., Larson L. H., Billiar, T. D. Inducible synthase nitric oxide in the liver: regulation and functions // Biochemistry. - 1998. - T. 63, vol. 7. - S. 905-923.

3. Reutov B. N., Sorokin, E., NO-sintsyana and nitratreduktaznaya cycle components of nitric oxide // Biochemistry. - 1998. - T. 63, vol. 7. - S. 1029-1040.

4. Costa E. T., do-Nascimento J. L., Picanco-Diniz, C. W., J. Quaresma, A., Silva-Filho M. Histochemical characterization ofNADPH-diaphorase activity in area 17 of diurnal and nocturnal primates and rodents // Braz. J. Med. Biol. Res. - 1996. - Vol. 29, 10. - P. 1355-1362.

5. Mirza M. H. , Oliver J. L., Seaborn T. L., Hosgood g, Moore R. M. Detection and comparison of nitric oxide in clinically normal horses and those with naturally acquired small intestinal strangulation obstruction // Can. J. Vet. Res. - 1999. - Vol. 63, 4. - P. 230-240.

6. Alonso J. R., Porteros, A., Crespo C., Arevalo, R., Brinon J. G., Weruaga e , J. Aijon Chemical anatomy of the macaque monkey humans bulb: NADPH-diaphorase/nitric oxide synthase activity // J. Tech. Neurol. - 1998. - Vol. 402, 3. - P .419-434.

7. Matakin P. A., Okhotin, C. E., Sulimov, Y. Cholinergic pyramidal neurons in the motor areas of neocortex person // Morphology. - 1990. , 99, vol. 8. - S. 34-39.

8. Liu L., Liu G. L., L. Barajas Distribution of nitric oxide synthase-containing ganglionic neuronal somata and postganglionic fibers in the rat kidney // J. Tech. Neurol. - 1996. - Vol. 369. 1. - P. 16-30.

9. Nakajima T. , Sakaue, M., Kato, M., Saito, S., Ogawa K., Taniguchi K. R. Immunohistoche Nitric oxide synthase I immunoreactivity and NOS-associated NADPHd histochemistry in the visceral epithelial cells of the intraplacental mouse yolk sac // Acta Histochem. - 1996. - Vol. 98, 2. - P. 173-183.

11. Nahar N. S., Chowdhury, J. U., Tokuno, H., Tomita T., Torihashi S., during closing , S., Kobayashi S. Nitrinergic nerves controlling pacemaker activities of the inner sublayer(P-layer) in the canine proximal colon circular muscles // Arch. Histol. Cytol. - 1996. - Vol. 59, 1. -P. 37-46.

12. Blute T. A., Mayer C., Eldred W. D. Immunocytochemical and histochemical localization of nitric oxide synthase in the turtle retina // Vis. Neurosci. - 1997. - Vol. 14, 4. - P. 717-729.

13. Lamanna, S., Costagliola, A., A. Vittoria, C. Mayer, Assisi L., V. Botte , Cecio A. NADPH-diaphorase and NOS enzymatic activities in some neurons of reptilian gut and their relationships with two neuropeptides // Anat. Embryol. (Beri). - 1999. - Vol. 199, 5. - P. 397-405.

14. Loyd H., Gossrau R., Kibler So the Histochemistry of enzymes: laboratory methods. TRANS. from English. - M.: Mir, 1982. - 272 S.

Claims

The method of determining the activity and functional status of the synthetase of nitric oxide, which includes the assessment of NADPH2-diaphorase activity of the enzyme by treatment with the investigational product solutions of salts of tetrazole in the presence of NADPH2after exposure of the drug for 10 min in 4% aqueous solution of neutral formalin, characterized in that it includes a complex of histochemical reactions, including additional assessment mates functioning catalytic zentropia in buffered saline solution (pH 7.4) and nitrite by exposure of the drug prepared Griss reagent for 30 min at 37oWith, and the activity and functional status synthetase of nitric oxide determined photometrically according to the content in the cells of the drug products cytochemical reaction for nitrite and deformazione.

 

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FIELD: medicine, analytical biochemistry.

SUBSTANCE: invention relates to laboratory methods of investigations. Method involves sampling specimen from patient to be inspected, extraction of serotonin and histamine from a specimen, chromatography of extract and determination of concentration of serotonin and histamine by the fluorescence intensity value. Saliva is used as biological fluid. Saliva by volume 1 ml is extracted with 4 ml of 1 N hydrochloric acid solution, 2 g of anhydrous potassium carbonate and 5 ml of mixture of butanol and chloroform in the ratio 3:2 are added, extract is shaken up and centrifuged. Organic phase (4 ml) is sucked off from extract and passed through chromatography column (diameter is 3 mm, height is 16 mm) filled with ion-exchange resin KB-4 or KB-4P-2 or Bio Rex-70 in H+-form, size of granules is 0.1 ± 0.02 mm. Histamine is eluted with 4 ml of 0.1 N hydrochloric acid at the rate of eluting solution 0.4 ml/min. Histamine concentration is determined by reaction with ortho-phthalic aldehyde dissolved in ethanol. Serotonin concentration is determined by reaction with ninhydrin in organic passed through column. Method provides assaying the saliva concentration of serotonin and histamine with high precision.

EFFECT: improved assay method.

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