The way to determine the status of free radical oxidation and antioxidant systems in areas of the skin lesions in patients with vitiligo

 

The invention relates to medicine, namely to dermatology. The essence of the method: produce samples of affected and unmodified skin, crushed, homogenized, separate aliquots of the supernatant, define them in spectrophotometric activity of enzymes of antioxidant system, choose system induction of free radical oxidation and conduct appropriate response induction. In the resulting supernatant record the absorption spectrum of thiobarbituric-active products in the range 480-590 nm, find its maximum, which determine the concentration of products induced free radical oxidation. Then build a graph of the dependence of their accumulation on the time of incubation, which determine the rate of their accumulation, and when it is exceeded by more than 30% in the affected skin in comparison with unmodified and simultaneously reduced or unchanged activity of enzymes determine the state of activation of free radical oxidation and decreased activity of antioxidant systems and set according to the free radical damage to the skin. The method allows to determine the true value of free radical processes and antiradical protection aquatoy this skin condition therapy. 2 Il.

The invention relates to medicine, namely to dermatology, and can be used in the diagnosis and choice of treatment in patients with vitiligo, as well as in biochemistry to study pathogenetic mechanisms of development of this disease.

Vitiligo is a disease with a poorly understood etiology and pathogenesis, therefore, the application of adequate treatment of this disease difficult. In the last 5 years has been actively discussed the activation of free radical oxidation in the skin, as well as the decreased activity of antioxidant systems as the main links of the pathogenesis of vitiligo. In this regard, the important is the choice of adequate, accessible and informative way of assessing free radical reactions and antioxidant systems in the skin in this disease with the aim of choice pathogenetically reasonable method of diagnosis and treatment.

There is a method of evaluation of free radical oxidation in the skin using spectroscopy electron paramagnetic resonance (Fuchs J., Herrling Th., Groth N. Detection of free radicals in skin: a review of the literature and new developments. / In: Oxidants and antioxidants in cutaneous biology. Eds: Thiele j, Elsner P. Current Problems in De the oxidation by resonant absorption of microwave radiation in samples of unmodified skin, having paramagnetic centers. Due to the short lifetime of the free radicals registration is possible either in the deep-frozen samples, or by using a specific spin traps, which catch radicals and translate them into a more stable form, which allows you to register endogenous free radical state. The study evaluated the amplitude spectrum of electron paramagnetic resonance. This method is used in the last 5-7 years to study the pathogenesis of skin diseases.

The disadvantage of this method is the possibility of registering only intermediate products of free radical oxidation, which are usually found in low concentrations, which requires high sensitivity of the method, i.e. the use of special, expensive equipment, allowing multiple accumulate signals and their mathematical treatment. The complexity, the complexity, the need for specially trained qualified personnel and high cost limit the functionality of the method.

There is a method of evaluation of free radical oxidation in the skin of measuring the level of one of the products of this process is the responsibility of the m progesterone on lipid peroxidation and the redox system of glutathione in skin tissues of rats in experimental dermatitis / bul. the experimental. biology and medicine, 2000, vol 129, No. 1, pp. 186-188). The method consists in carrying out the reaction between malonic dialdehyde and its derivatives contained in the tissue, and thiobarbituric acid. As a result of this reaction is a colored compound - crimminy complex, in which one molecule of malondialdehyde have two molecules thiobarbituric acid (Ohkawa H., Ohishi n, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction / Analyt Biochem, 1979, v.95, R. 351-358). The intensity of staining trimethylboron complex is then registered on the spectrophotometer, which allows quantitative determination of the level of malondialdehyde in the tissue sample.

Despite the availability of this method uninformative, since it allows the skin to measure only the initial, the initial level of oxidation products. However, the sensitivity of the skin to damaging agents can be assessed only by the reaction of tissues to the induction of free radical oxidation. It is the rate of product accumulation induced free radical oxidation, but not their initial level, directly correlates with the state of the tissue (Arkhipenko Yu.V., Sazontova T. G., Rice-Evans C. Hypertrophy and regression of the rat heart: free radical ralated metabolic systems. / Pathophysiology, 1997, v.4, No. Trigo, and used only in experimental work on animals in the study of the pathogenesis of psoriasis and contact dermatitis.

As the closest analogue accepted way to determine the status of free radical oxidation in peripheral blood of patients with vitiligo (Dell'Anna M. L., L. V., S. Briganti et al. Mitochondrial his or her in peripheral blood mononuclear cells during the active phase of vitiligo / J. Invest Dermatol, 2001, v.117(4), p.908-913). The method is based on the study of damage induced by reactive oxygen species in erythrocytes and monocytes in peripheral blood of patients with vitiligo. The state of free radical oxidation determined by the ability of reactive oxygen species damage the membrane and disrupt the membrane potential of mitochondria, changes which register with special electrodes.

However, in addition to the complexity and intensity of registration this method is indirect, since it is not measured level of free radical products, and membrane damage, which in addition to the action of reactive oxygen species depends on several factors, such as maintenance of membrane unsaturated fatty acids, the level of metals of variable valency, and other factors. In addition, estimated in this way, the level Smolnogo process in the skin and thus, it is not a reliable diagnostic parameter.

The objective of the invention is to create an efficient, simple and accurate method to determine the status of free radical oxidation and antioxidant systems in areas of the skin lesions in patients with vitiligo.

The essence of the invention is that the method of determining the status of free radical oxidation and antioxidant systems in areas of the skin lesions in patients with vitiligo is characterized by the fact that produce a collection of samples of affected and unmodified skin, frozen in liquid nitrogen, pulverized, and homogenized in two stages, followed by the separation of aliquot of the supernatant, define them in spectrophotometric activity of antioxidant enzymes - catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase and establish the nature of its changes in the direction of increase, decrease or lack of it in the affected skin compared to the unmodified, in accordance with which selects the induction system of free radical oxidation, then aliquots of homogenate diluted with medium to homogenization, is placed into the microwells, preincubated, then add the previously selected system induction and p is you, add 50 ál to 150 ál containing 0.9-1.2% of 2-thiobarbituric acid, 0.54% of sodium dodecyl sulphate and 10.8-15.3% of acetic acid, the samples preincubated, then heated to 95° C and incubated for 40 minutes after the appearance of staining settled coagulated protein is separated and the resulting supernatant determine the degree of staining on the spectrophotometer by recording absorption spectrum of thiobarbituric-active products in the range 480-590 nm, find its maximum, which determine the concentration of products induced free radical oxidation at different time intervals of incubation, samples, build a dependency graph of their accumulation on the time of incubation, which determine the rate of their accumulation, and when it is exceeded by more than 30% in the affected skin in comparison with unmodified and simultaneously reduced or unchanged activity of enzymes determine the state of activation of free radical oxidation and decreased activity of antioxidant systems and set according to the free radical damage to the skin.

The use of the invention allows to obtain the following technical result.

The method allows the school to protection of damaged skin in vitiligo lesions, to assess these indicators of the severity of the pathological process and the sensitivity of the skin in vitiligo lesions to the action of exogenous damaging factors associated with active forms of oxygen, and to find adequate this skin condition treatment.

The entire diagnostic process, including tissue sampling, takes a few hours. The method is simple to perform, does not require sophisticated equipment or expensive consumables, vasocontraction.

The method can be effectively used not only biochemical but also in any clinical laboratory, allowing you to decide on an adequate assessment of Pro - and antioxidant systems in skin samples in vitiligo, which allows the most objectively choose the treatment method and methodology in each case. Special attention to this method can be manifested in cases of complex pathology when vitiligo burdened allergic symptoms, cancer changes and so on, i.e., in cases where there are no objective criteria for the decision on the appointment of certain medicines that can change the balance between oxidants and antioxidants in the cell.

Technical financial p is satou directly from pathological lesion of the skin of the patient vitiligo and not modified on the type of skin of the same patient. First proposed and accurate, adequate to the state of the tissue method evaluation of free radical reactions and antioxidant systems in the skin of patients with vitiligo, which is based on determining the sensitivity of the modified skin tissues to the induction of free radical oxidation at existing in this area of the skin level antiradical protection.

By changing the ratio between prooxidant and antioxidant systems in the hearth of the modified skin with vitiligo compared to the normal skin from the same patient was first asked to evaluate the shift of the equilibrium towards oxidative processes in the hearth of vitiligo. For the first time the efficiency of determining the correlation between the altered sensitivity of tissues to the induction of free radical oxidation in vitro (change rate of product accumulation induced free radical oxidation and antioxidant protection for pathogenetically proved diagnosis of free radical damage to melanocytes in vitiligo and treatment options for a patient.

The method is as follows.

Produce bioptates of the skin of the patient from the affected vitiligo (depigmented what about the punch dissect the sample skin. Samples of affected and unmodified skin quickly frozen in liquid nitrogen until the determination of biochemical parameters. The time from the commencement of the bioptates before freezing should not exceed 15 seconds. The samples are weighed and crushed in a mortar using inert Teflon or quartz granules for 50 seconds at a temperature of 4° C, then continue grinding in the homogenizer Teflon-glass environment homogenization containing 20 mm Tris-HCl (pH of 7.4) and 100 mm NaCl in the ratio of tissue-Wednesday, equal to 1:5. Then repeat the previous step homogenization. The total time of homogenization does not exceed 3.5 minutes and homogenization is carried out at a temperature of 4° C. the samples centrifuged in micro test tubes at 1500 rpm for 10 minutes and take an aliquot of supernatant for carrying out free-radical oxidation and determination of activity of enzymes of antioxidant protection.

Determine the activity of antioxidant enzymes - catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase - known for these enzymes spectrophotometric methods: the catalase activity by the method of Luck N. (Luck And. Catalase. / In: Methods of enzymatic analyses. Editor: Bergmeyer H. U., improved assay and an assay applicable to acrilamide gels. / Analit Biochem, 1971, v.44, p.276-287); the activity of glutathione peroxidase by the method of Paglia D. (Paglia D., Valentin W. Studies of a quantitative and qualitative characterization of erythrocyte glutation buffer. / J. Lab Clin Methods, 1967, v.70, p.158-169); glutathione reductase activity by the method of Beutler E. (Beutler E. Red cell metabolism. A manual of biochemical methods. / In: Orlando, Fl. Gune and Stratton inc., 1984, 188 p.), using microcube 0.1-0.2 ml and a path length of beam 1 cm, when rigid fixation microcube in the cell holder.

Establish the nature of changes in the activity of enzymes in the direction of increase, decrease or lack of it in the affected skin compared to the unmodified, which is judged on the presence of the activation of the antioxidant system, decrease or no change.

Then the reaction induced free radical oxidation choose a system based on induction of free radical oxidation in vitro, depending on the degree of changes in the activity of antioxidant enzymes in the hearth vitiligo compared with a plot of apparently unaltered skin. When increasing the activity of antioxidant enzymes in the hearth vitiligo compared to similar indicators in the sample plot apparently not modified skin of the same patient, choose the oxidation system, containing 0.25 mm ascorbic) the antioxidant defense in the hearth vitiligo compared with the sample plot, apparently unaltered skin apply system containing 0.1-0.75 mm ascorbic acid.

Aliquots of the homogenate is diluted 5 times environment homogenization (containing 20 mm Tris-HCl (pH 7.4) and 100 mm NaCl) and placed into the microwells. Then preincubated received the sample in each well for 5 minutes at 37° C and constant stirring. The protein concentration in the mixture in each well should not exceed 1.5 mg/ml Solutions of ascorbic acid and FeSO4to induce free radical oxidation cook for 15 minutes prior to the inactivated and added to the microwells previously selected system induction of free radical oxidation in the respective concentrations.

Conduct the reaction induction of free radical oxidation at 37° C with constant stirring for 20-40 minutes depending on the selected system oxidation. Then separate aliquots for determination of free radical oxidation products. In parallel, put the control on the auto-oxidation under the same conditions, but without addition of inductors from the oxidation of ascorbic acid and FeSO4.

In the selected aliquot determine the concentration of free radical oxidation products, which is appreciated by the reaction with 2-thiobarbituric acid. For this to 150 ál of re is add 50 ál of the selected aliquot.

The samples preincubated at 4° C for 60 min to improve the stability of the obtained results (stability of the color characterizing the concentration of free radical oxidation products). Then these samples are heated to 95° C and incubated for 40 min using a reverse refrigerators for each sample in order to avoid evaporation of the mixture. After the appearance of staining settled, coagulated protein is separated from the sample by centrifugation in microcentrifuge tubes at 1400g for 10 minutes In the obtained transparent supernatant determine the degree of staining on the spectrophotometer by recording absorption spectrum of thiobarbituric-active products in the range 480-590 nm and find the maximum of this absorption spectrum, which determines the concentration of the products induced free radical oxidation at different time intervals of incubation of the samples in the selected oxidation system and Express them in nmol/mg of protein/ml

Then build the graph of the accumulation of free radical oxidation products from the incubation time. The rate of product accumulation induced free radical oxidation is determined according to the received schedule, using compo oxidation in the affected skin (focus vitiligo) compared to unmodified more than 30% and simultaneously reduced or unchanged activity of one or more enzymes of the antioxidant system determine the state of activation of free radical oxidation, the decreased activity of antioxidant systems and set according to the free radical damage to the skin. This allows you to choose pathogenetically-based therapy, including anti-oxidant drugs. The dose and the destination schema antioxidant drugs chosen individually depending on the rate of change of speed of free radical oxidation and the rate of change of the activity of antioxidant enzymes.

The method is tested on the basis of the Russian medical Academy of postgraduate education and Institute of General pathophysiology and morphology of the RAMS 17 of vitiligo patients, 10 men and 7 women aged from 16 to 53 years, disease duration from 1 to 12 years. All patients were identified free-radical nature of the skin lesions in vitiligo lesions, suggesting his leading role in the development of vitiligo.

Example 1. Patient K., 27 years old, suffers from widespread vitiligo in the last 3 years. Under local anesthesia the patient from the humeral region took a biopsy of the affected and apparently unaltered skin. Samples of affected and unmodified skin immediately frozen in liquid nitrogen.

Samples were grind and homogenized. Then primetime. In damaged skin showed a reduction of catalase activity by 27% compared to the same indicators in an unmodified skin, decreased activity of superoxide dismutase by 23%, a decrease in activity of glutathione peroxidase by 14%, however, the activity of glutathione reductase was not changed. Considering the obtained data, chose the system for the induction of free radical oxidation, containing 0.1 mm ascorbic acid.

Aliquots of the homogenate were taken biopsies were diluted 5-fold environment homogenization containing 20 mm Tris-HCl (pH 7.4) and 100 mm NaCl, plaincourault at 37° C for 5 minutes with constant stirring. Then the resulting mixture was added previously selected and prepared immediately before carrying out the reaction of the ascorbic acid solution.

The reaction induction of free radical oxidation was carried out at 37° C for 40 minutes. Then selected aliquots, of which 50 μl was added to 150 ál containing 0.9-1.2% of 2-thiobarbituric acid, 0.54% of sodium dodecyl sulphate and 10.8-15.3% of acetic acid (pH 3.5). The obtained samples were plaincourault at 4° C for 60 min to improve the stability of the obtained results (stability of the color characterizing the coin, using a reverse refrigerators for each sample in order to avoid evaporation of the mixture. After the appearance of staining settled, coagulated protein was separated from the samples by centrifugation in microcentrifuge tubes at 1400g for 10 minutes In the obtained transparent supernatant on the spectrophotometer was determined by the degree of staining by recording the absorption spectrum of thiobarbituric-active products in the range 480-590 nm and found the maximum of this absorption spectrum. Then build graphs of the accumulation of free radical oxidation products from the incubation time. The rate of product accumulation induced free radical oxidation is calculated based on the obtained graphs (see Fig.1 and 2).

As a result of the reactions and calculations have determined that the suspect in the area of damaged skin initial level of free radical oxidation products was not changed in comparison with the same indicators in the area of unmodified skin, while the rate of product accumulation induced free radical oxidation was increased by 45%. These data indicate that free-radical damage to the skin lesion of vitiligo, for the correction to the m;">Claims

The way to determine the status of free radical oxidation and antioxidant systems in areas of the skin lesions in patients with vitiligo, characterized by the fact that produce a collection of samples of affected and unmodified skin of the patient, frozen in liquid nitrogen, and then at 4°crushed, homogenized twice in a medium containing 20 mm Tris-HCl and 100 mm NaCl, then centrifuged, separated aliquots of the supernatant, define them in spectrophotometric activity of antioxidant enzymes - catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase and establish the nature of its changes in the affected skin compared to the unmodified, in accordance with choosing the induction system of free radical oxidation, while enhancing the activity of enzymes containing 0.25 mm ascorbic acid and 1,2-10 μm FeSO4·10H2O, in the absence of changes or reduce the activity of enzymes containing 0.1-0.75 mm ascorbic acid, and then an aliquot of homogenate diluted with medium to homogenization, is placed into the microwells, preincubated at 37°C, and then add the selected induction system and carry out the reaction of inducing free radical oxide is 50 ál to 150 ál, containing 0.9-1.2% of 2-thiobarbituric acid, 0.54% of sodium dodecyl sulfate and 10.8-15.3% of acetic acid, the samples preincubated at 4°C, then heated to 95°C and incubated for 40 min, after the appearance of staining settled coagulated protein is separated by cetrifugation, the resulting supernatant determine the degree of staining on the spectrophotometer by recording absorption spectrum of thiobarbituric-active products in the range 480-590 nm, find its maximum, which determine the concentration of products induced free radical oxidation, build a graph of the dependence of their accumulation on the time of incubation, which determine the rate of their accumulation, and when it is exceeded by more than 30% in the affected skin in comparison with unmodified and simultaneously reduced or unchanged activity of enzymes determine the state of activation of free radical oxidation and decreased activity of antioxidant systems and set according to the free radical damage to the skin.



 

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