RU2629391C1 - Method for assessing body resistance to influence of prooxidant factors - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: value of the ratio of easily-accessible and hardly-accessible thiol groups of blood plasma, their oxidation by hydrogen peroxide are determined, the disbalance in the functioning of the thiol unit of the antioxidant system of the blood plasma is assessed by the integral coefficient (IC), and if the IC value is equal to or less than 1.5 relative units, the normal body resistance to the influence of prooxidant factors is determined, if the IC value is more than 1.5 relative units, the presence of conformational changes in proteins and the ratio of non-protein/protein thiol-containing blood plasma compounds and the functioning disruption of the thiol unit of antiradical protection are determined, accompanied by a reduced body resistance with the development of oxidative damage.

EFFECT: invention can be used in the diagnosis of diseases accompanied by oxidative stress, as well as for monitoring antioxidant therapy during the disbalance of the thiol unit of antiradical protection.

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Description

The present invention relates to medicine and can be used in the diagnosis of diseases accompanied by oxidative stress, as well as for monitoring antioxidant therapy with an imbalance of the thiol link of antiradical protection.

Oxidative stress is a common pathobiochemical condition that is widely encountered in various extreme physiological and pathological conditions [Menshchikova EB, Zenkov NK, Lankin V.Z., Bondar I.A., Trufakin V.A. Oxidative stress: pathological conditions and diseases. Novosibirsk .: Publishing house ARTA. 2008.284 p.].

Violation of the functioning of the thiol link of antioxidant protection is one of the significant indicators of the diagnosis of oxidative stress and control of antioxidant treatment for many diseases - diabetes, asthma, angina pectoris, myocardial infarction, cataracts, malignant neoplasms, rheumatoid arthritis and others. Moreover, often the key mechanism contributing to the formation of oxidative stress is the development of an imbalance in the work of thiol antioxidants, which leads to disruption of the processes of neutralization of free radicals, as well as to a violation of the regeneration of other classes of substances with antioxidant activity. Moreover, the thiol unit is the most sensitive component of the antioxidant system, which can not only neutralize free radicals, but also regenerate other antioxidants.

Timely diagnosis and correction of disorders in the work of the antioxidant system requires clearly formulated criteria, delimiting the adaptive tension of the thiol link of the antioxidant system, its regulatory multidirectional changes and the presence of an imbalance in the work of these compounds, leading to the formation of oxidative stress.

A known method for determining the antioxidant state of an organism, which can also be used to assess the body's response to external influences [Gorshkov ES, Ivanov VV, Sokolovsky VV Pat. 2431839 RU, IPC G01N 33/493. A method for determining the antioxidant state of an organism. - No. 2010122592/15; - declared. 06/02/2010; publ. 10/20/2011. - Bull. No. 29]. The essence of the method: during external exposure to the body repeatedly (7-8 times) during the day urine samples are taken. In each sample, thiol concentrations are determined using Ellman's reagent [Ellman G.L. Tissue sulfhydryl groups // Arch, bioch. biophys. - 1959. - Vol. 82. - P. 70-77], and urochrome, then calculate the thiolurochromic coefficient (TAC) as the ratio of the concentrations of thiols and urochrome. When the TUK value is 1.46 ± 0.2, the state of the body is assessed as satisfactory. When the TUK value is more than 1.66 or less than 1.26 - as satisfactory that went beyond the borders.

Disadvantages:

1) the biochemical composition of urine primarily reflects the functional state of the excretory system, but not the body as a whole, which significantly limits the possibility of using urine in non-invasive laboratory diagnostics;

2) the change in biochemical parameters in the urine is widely variable and depends, in addition to the state of the urinary system, on the time of day, the taken portion of urine, the nature of nutrition and water consumption, and ambient temperature, which introduces unreliability due to thermoregulatory effects;

3) the determination of thiol-containing compounds in urine, especially with the use of Ellman's reagent, is not widely used due to the high content in the urine of sulfur compounds in the form of inorganic sulfides and other compounds with intermediate values of the degree of oxidation of sulfur, which in turn actively react with Ellman's reagent Thus, a clearly overestimated result is obtained. Moreover, the described compounds do not reflect the state of the functional reserves of the body, but are the end products of the metabolism of sulfur-containing compounds;

4) the use of urochrome as a substance reflecting the intensity of oxidative metabolism also does not find wide application, since its content as, first of all, the final product of heme exchange depends on the intensity of protein metabolism, erythropoiesis, etc.

A known method (adopted for the closest analogue), which allows to determine the coefficient characterizing the content of reduced and oxidized thiol groups - thiol disulfide ratio [Sokolovsky V. The thioldisulfide ratio of blood as an indicator of the state of nonspecific resistance of the body: a Training manual. - SPb., 1996. - 30 S.], according to which the content of thiol (-SH) and disulfide (-SS-) groups in the test blood substrate is determined and their quantitative ratio is calculated. For its implementation, the content of SH-gr is determined. and -S-S-bonds using Ellman’s reagent [Verevkina I.V., Tochilkin A.I., Popova N.A. // Colorimetric definition of SH-gr. and -S-S bonds in proteins with 5.5 / - dithiobis (2-nitrobenzoic) acid. Modern methods in biochemistry, edited by Academician of the Academy of Medical Sciences of the USSR V.N. Orekhovich - Moscow, "Medicine" - 1977. - S. 223-231.] Or amperometrically [Sokolovsky V.V. Thiol antioxidants in the molecular mechanisms of the nonspecific reaction of the body to extreme effects (review) // Issues of medical chemistry. - 1988 - v. 34 - no. 6 - S. 2-11.] And determine their ratio. It is believed that the thiol disulfide ratio characterizes the state of antioxidant protection and redox processes of the body. The functions of this system include the regulation of enzymatic and antioxidant activity, immune responses, cell growth and permeability of biomembranes, etc.

The disadvantages of the analogue are:

1) large fluctuations in the thiol disulfide ratio over a short period of time;

2) a large spread in the indicators of the thiol disulfide ratio when using various laboratory research methods: spectrophotometric method with Ellman's reagent or other reagents, amperometric titration using various selective electrodes (mercury, silver, etc.);

3) when calculating this coefficient, the possibility of multidirectional changes in the content of thiol compounds for various pathologies due to various reasons is not taken into account, the concentration can vary both upward and downward in the content of both non-protein thiols and protein SH-groups, simultaneous increase in the content of disulfide groups. So, for example, the possibility of increasing the content of reduced thiols in the blood by washing them out of damaged cells has been shown.

4) the fact that the oxidation of SH groups does not always lead to the formation of disulfide groups is not always taken into account, sulfinic or sulfenic acids can also form. In this case, the pool of free thiol groups can decrease both against the background of the preservation of the disulfide content and against the background of their reduced concentration. Not all available -SS groups can be easily determined by common analytical methods; some of the total pool of disulfide groups are “hidden” inside the protein molecule and are not detected. During the oxidation of thiol groups, a conformational rearrangement of the protein molecule almost certainly occurs with a change in the ratio of functional groups available and inaccessible to reagents.

In view of this, it becomes difficult to interpret the results of calculating the thiol disulfide ratio.

The objective of the invention is the creation of a reliable and unambiguously interpreted method for the integral assessment of the functioning of the thiol link of antioxidant protection under conditions of oxidative stress, based on the use of a minimum number of additional special laboratory diagnostic methods, which allows one to determine the severity of the imbalance of thiol-containing compounds under conditions of oxidative stress, and to prescribe rational antioxidant therapy in due time volume and monitor its effectiveness in the process Behold treatment.

The main advantage of the proposed technology for the diagnosis of disorders of thiol metabolism in patients is that, in addition to quantitative colorimetric determination of thiol groups in blood plasma, it allows you to determine the ratio of fast-reacting (readily available, that is, reacting with Ellman's reagent in the first 3 minutes) and slow-reacting (hard to reach, reacting with Ellman's reagent for 3-30 minutes) of thiol groups, as well as the ability of native thiols to interact in vitro with hydrogen peroxide, which helped a differential diagnosis of metabolic disorders of sulfur-containing compounds.

The ratio of rapidly reacting (readily available) and slowly reacting (hard to reach) thiol groups is determined by the formula:

Where

SH _l - the content of readily available thiol groups, units of optical density;

SH _t - the content of inaccessible thiol groups, units of optical density;

Then, the percentage of oxidation of thiol groups by hydrogen peroxide (O _% ) is calculated, which reflects the ability of blood plasma to neutralize hydrogen peroxide, while maintaining a safe pool of reduced thiol groups, according to the formula:

Where

SH ₁ - total thiol groups of intact blood plasma, units of optical density;

SH ₂ - total thiol groups of blood plasma after exposure to hydrogen peroxide, optical density units.

After that, the integral coefficient (IR) is calculated, taking into account the antioxidant properties of blood plasma and, therefore, resistance to the effects of prooxidant factors on the body:

where 0.35 and 17.01 are the coefficients corresponding to the average values, respectively, To ₁ and O _{% of} healthy examined.

The use of the O _% factor in degree 2 is explained by the fact that the severity of the pathology is more dependent on the antioxidant properties of blood plasma.

The essence of the invention is a method for assessing the body's resistance to the effects of prooxidant factors, which provides that the complex using Ellman's reagent determines the ratio of readily available and inaccessible thiol groups of blood plasma, their oxidation by hydrogen peroxide, and the imbalance of the functioning of the antioxidant thiol unit is estimated by the integral coefficient (IR) blood plasma systems according to the formula:

IR = (K ₁ / 0.35) × (O _% / 17.01) ² , where

To ₁ - the ratio of easily and inaccessible thiol groups, determined by the formula:

K ₁ = SH _l / SH _t , where

SH _l - the content of readily available thiol groups, units of optical density;

SH _t - the content of inaccessible thiol groups, units of optical density;

About _% - the percentage of oxidized thiol groups with hydrogen peroxide, determined by the formula:

O _% = ((SH ₁ -SH ₂ ) / SH ₁ ) × 100, where

SH ₁ - total thiol groups of intact blood plasma, units of optical density;

SH ₂ - total thiol groups of blood plasma after exposure to hydrogen peroxide, optical density units,

and when the integral coefficient is equal to or less than 1.5 relative units, the normal resistance of the body to the action of prooxidant factors is determined, when the IR value is above 1.5 relative units, the presence of conformational changes in proteins and the ratio of non-protein / protein thiol-containing compounds of the blood plasma and the violation of the functioning of the thiol anti-radical defense link, accompanied by reduced body resistance during the development of oxidative damage.

The technical result of the invention is:

1) an integrated assessment of the functioning of the most sensitive link of antioxidant protection - thiol-containing compounds (protein and non-protein SH-groups), while determining their role in the development and formation of oxidative stress in the body of the subject;

2) determination of the imbalance of the thiol unit of the antioxidant system without taking into account the total content of SH-groups of the blood plasma of the subject, which allows to level the effect of increasing or decreasing the content of thiol groups and to determine the imbalance independently, and therefore more objectively;

3) all this allows us to abandon the determination of the concentration of blood plasma proteins, since there is no need to bring the content of thiol groups per 1 gram of protein due to the use of ratios of different fractions in the same subject.

4) to determine this integral indicator, an accessible biomaterial is used - blood plasma, which contains both protein and non-protein SH-groups in sufficient quantities, which increases the universality of the determination and use of this indicator for clinical studies in various diseases;

5) apply an approach to differentially assess the contribution of each of the components of the equation to the result when calculating the integral indicator of the functioning of the thiol unit of the antioxidant system by multiplying the ratio of readily available / inaccessible thiol groups by the percentage of oxidized thiol groups under the action of hydrogen peroxide, which allows to increase the sensitivity of the method, then there is an increase in the percentage of truly positive results in people suffering from diseases with oxidative stress, to and increase specificity, that is, reduce the frequency of false-positive results in healthy subjects, thus achieving the maximum possible diagnostic effectiveness for this method.

The method is as follows.

To assess the body's resistance to oxidative damage, an integral indicator is used, based on the determination of the functioning of the thiol link of antiradical protection. The object of the study is the blood plasma of the subjects stabilized with heparin sodium, in which the content of total, readily available and inaccessible SH-groups is determined using Ellman's reagent without prior exposure to hydrogen peroxide, as well as after exposure to hydrogen peroxide.

The content of SH-groups is determined in blood plasma by the colorimetric method using Ellman's reagent [Verevkina I.V., Tochilkin A.I., Popova N.A. // Colorimetric definition of SH-gr. and -SS bonds in proteins using 5.5 ^/ - dithiobis (2-nitrobenzoic) acid]. Modern methods in biochemistry, edited by Academician of the Academy of Medical Sciences of the USSR V.N. Orekhovich - Moscow, "Medicine" - 1977. - S. 223-231]. The technique is based on the ability of SH groups to react with 5,5'-dithiobis (2-nitrobenzoic) acid to form a thionitrophenyl anion. The amount of thionitrophenyl anion formed is directly proportional to the number of free SH groups. Determination process: 1.5 ml of 0.1 M phosphate buffer is added to the cuvette (pH = 7.4; the need for a reaction at physiological pH values is due to the need to preserve the native conformation of protein molecules), add 150 μl of blood plasma and 10 μl bidistilled water, after 5 minutes of incubation at 25 ° C, a spectrophotometer is removed from the prepared sample of the experimental solution in the cuvette, after which 25 μl of Ellman's ethanol reagent are added. After 3 and 30 minutes, the optical density of the samples was measured on a spectrophotometer at 412 nm (maximum absorption of TNFA), without stopping the reaction. Similarly, the extinction of control solution 1 (without adding Ellman's reagent) and control solution 2 (without adding plasma, but with Ellman's reagent) is determined relative to 0.1 M phosphate buffer. The result, expressed in units of optical density, minus the optical densities of control solutions 1 and 2, obtained after 3 minutes from the start of the reaction, corresponds to the content of readily available (rapidly reacting) SH groups, and after 30 minutes, minus the units of optical density obtained after 3 minutes corresponds to the content of inaccessible (slow-reacting) SH-groups.

The result obtained after 30 minutes of incubation, minus control solutions 1 and 2, corresponds to the sum of SH-groups.

The result obtained is left expressed in units of optical density, since the units of expression of the content of thiol groups will be unprincipled for further calculations. Recalculating the content of SH-groups in blood plasma per gram of protein is not required, since all calculations will be carried out with the ratios of different fractions of thiol groups in the blood plasma of one patient, without comparing the total content of SH-groups of different patients.

Then calculate the ratio of easily and inaccessible thiol groups according to the formula: K ₁ = SH _l / SH _t .

The determination of thiol groups is carried out with intact blood plasma and with blood plasma samples of the same patients, but after exposure to a solution of hydrogen peroxide. To do this, add 150 μl of blood plasma to the cuvette, then add 10 μl of a 0.03% solution of H ₂ O ₂ . After 5 minutes of incubation at 25 ° C, 1.5 ml of 0.1 M phosphate buffer (pH = 7.4) was added to the same cuvette and readily and inaccessible and the sum of thiol groups were determined, similarly to the method described above.

After that, the percentage of SH-groups oxidized by hydrogen peroxide is determined by the formula: O _% = ((SH ₁ -SH ₂ ) / SH ₁ ) × 100. And determine the integral coefficient by the formula: IR = (K ₁ / 0.35) × (O _% / 17.01) ² .

The results are interpreted as follows: if the integral index is equal to or less than 1.5 relative units, the absence of an imbalance in the functioning of the thiol unit of antioxidant protection is determined, if the ratio is higher than 1.5 relative units, the presence of conformational changes in proteins and the ratio of non-protein / protein thiol-containing blood plasma compounds is determined and impaired functioning of the thiol link of the anti-radical defense, accompanied by a reduced potential for response to oxidative Tress.

Justification of the results.

A feature of the proposed method is the assessment of conformational changes in proteins that occur during oxidative modification, including due to the oxidation of SH-groups of proteins, as well as other functional groups, with the partial “inversion” of the protein globule resulting in difficult to reach thiol groups outside the molecule, while the pool of hard-to-reach SH groups is correspondingly consumed, due to which the pool of readily available, more reactive, functionally active is partially replenished x, which is possible is the backup mechanism for adaptation during oxidative stress. This explains the increase in the ratio of readily available SH-groups to hard-to-reach ones. Also, an increase in the ratio of easily and inaccessible SH-groups can be partially due to the release of low molecular weight non-protein thiols from damaged cells into the blood plasma. In addition, with pathology, an increase in the number of thiol groups oxidized by hydrogen peroxide is observed, which is associated with a decrease in the antioxidant potential of blood plasma. This interpretation of the changes is consistent with in vitro model experiments on a solution of human serum albumin (see Appendix, - Fig. 1. Changes in the ratios of readily and inaccessible thiol groups, oxidation of thiol groups, and the integral index when modeling oxidative damage to proteins in albumin solution ) In the process of determining the ratio of readily and inaccessible SH-groups in an intact albumin solution under the same conditions under which we conducted a study of blood plasma, it was found that the coefficient K ₁ (according to formula 1) was 0.4; and after exposure to 0.003% hydrogen peroxide increased to values of 0.8-1.0 and to 1.8 when exposed to 0.03% H ₂ O ₂ , which indicates the correctness of the proposed approach. In addition, when evaluating the oxidizability of the thiol groups of albumin, it was found that hydrogen peroxide exerts an effect similar to the action of H ₂ O ₂ on blood plasma at an albumin solution at a concentration 10 times lower, which indicates the correctness of judgments about the antioxidant properties of blood plasma. So, when introducing into a model system containing 60 μl of a 20% albumin solution and 1.5 ml of sodium-potassium phosphate buffer solution (pH = 7.4), 10 μl of 0.003% (group A + 0.003% H ₂ O ₂ ) peroxide solution hydrogen, the total content of thiol groups decreased by 18%, and when 10 μl of 0.03% hydrogen peroxide was added to a similar system (group A + 0.03% H ₂ O ₂ ), the total content of SH groups decreased by 52%. The integral coefficient under the action of 0.003% H ₂ O ₂ amounted to 2.88 relative units, and under the action of 0.03% H ₂ O ₂ - 47.26 relative units. But it is worth noting that such a high concentration of hydrogen peroxide with a model albumin solution was not completely neutralized, and the remaining in the H ₂ O ₂ solution oxidized the thionitrophenyl anion formed during the interaction of thiol groups of proteins with Ellman's reagent, which distorted the result. However, this experiment clearly demonstrates that the introduced hydrogen peroxide in the blood plasma is neutralized not only by the SH-groups of proteins, but also by other components of the antioxidant system, therefore, the proposed experimental design adequately reflects the antiperoxidation activity of blood plasma.

Studies of the state of thiol metabolism in plasma in various categories of patients (n = 120) were carried out.

Analyzing the ratio of easily and inaccessible thiol groups in patients with chronic endometritis and in healthy women, there is a significant difference between them. Determination of the content of the sum of SH-groups showed that there were no significant differences between sick and healthy women. Thus, 0.69 units of optical density / 100 g of protein was determined in the blood plasma of healthy women, and 0.66 units of optical density / 100 g of protein in patients with chronic endometritis. In order to increase the information content of these categories of patients, an IR was calculated. In healthy women, it amounted to 1.33 relative units, and in patients with chronic endometritis - 26.29 relative units, which indicates that there is actually a significant imbalance in the functioning of the thiol unit of the nonspecific resistance system and a decrease in the body's resistance to oxidative damage.

Thus, the use of IR in comparison only with the assessment of the total content of SH-groups of blood plasma allows one to more accurately and efficiently determine the state of the thiol unit of the antioxidant system, which may be of importance when choosing the optimal method of treating a patient and monitoring the effectiveness of therapy.

The study was supported by the state task of the Ministry of Health of the Russian Federation (January 1, 2015, part 1, section 1) “Implementation of applied scientific research, including preclinical studies of drugs and clinical trials of drugs”.

Examples of specific performance.

Example No. 1. On June 10, 2016, patient F., aged 36 years old, was admitted to the gynecological department of the Basic Obstetric and Gynecological Clinic of the Federal State Budget Educational Institution of Higher Education at the KubSMU of the Ministry of Health of Russia. Diagnosis: "Chronic endometritis, bilateral salpingo-oophoritis." A survey was conducted to diagnose the possible presence of an imbalance in the thiol link of the antioxidant defense system (see Appendix, - Fig. 2. Changes in the coefficients of thiol-containing compounds in a patient with chronic endometritis and bilateral salpingo-oophoritis during treatment compared with the values of the control group): total SH content ₁ -groups of blood plasma was 0.49 units of optical density (e.p.), SH _l - 0.18 e.p., SH _t - 0.31 e.p., the indicator was calculated the ratio of easily and inaccessible thiol groups:

K ₁ = 0.18 / 0.31 = 0.58;

which significantly exceeded the control values. The content of SH ₂ groups of blood plasma (after exposure to hydrogen peroxide) was 0.21 e.p. Oxidation of thiol groups was 57.1%:

O _% = ((0.49-0.21) / 0.49) × 100 = 57.1;

which also significantly exceeds the indicator of a group of healthy women. The calculated IR was 18.71 relative units:

IR = (0.58 / 0.35) × (57.1 / 17.01) ² = 18.71 relative units;

Thus, the resistance to the action of prooxidant factors in patient F. was impaired.

Within 10 days, traditional drug treatment was carried out with the additional introduction of antioxidant drugs (sodium thiosulfate, vitamin E and C in standard daily dosages). After completion of treatment, a second examination was carried out to determine the dynamics of changes in the above indicators. After treatment, the total content of SH ₁ blood plasma groups was 0.50 e.p., SH _l - 0.16 e.p., SH _t - 0.34 e.p., the indicator was calculated the ratio of easily and inaccessible thiol groups:

K ₁ = 0.16 / 0.34 = 0.47;

The content of SH ₂ blood plasma groups (after exposure to hydrogen peroxide) was 0.36 e.p. Oxidation of thiol groups was 57.1%:

O _% = ((0.50-0.36) / 0.50) × 100 = 28.0;

which significantly exceeds the indicator of a group of healthy women. The calculated IR was 3.63 relative units:

IR = (0.47 / 0.35) × (28.0 / 17.01) ² = 3.63 relative units. Such results can be interpreted as the restoration of the native conformation of proteins and the functioning of the antioxidant system of the body, and the treatment carried out can be assessed as effective and adequate.

Example No. 2. On June 10, 2016, patient I., aged 35 years old, was admitted to the gynecological department of the Basic Obstetric and Gynecological Clinic of the Federal State Budget Educational Institution of Higher Education KubGMU of the Ministry of Health of Russia; Diagnosis: "Chronic endometritis." A survey was conducted to diagnose the possible presence of an imbalance in the thiol link of the antioxidant defense system (see Appendix, - Fig. 3. Change in the coefficients of thiol-containing compounds in a patient with chronic endometritis during treatment compared with the values of the control group): total content of SH ₁ groups blood plasma amounted to 0.55 e.p.p., SH _l - 0.19 e.p.p., SH _t - 0.36 e.p.p., the ratio of readily and inaccessible thiol groups was calculated:

K ₁ = 0.19 / 0.36 = 0.53;

which significantly exceeded the control values. The content of SH ₂ blood plasma groups (after exposure to hydrogen peroxide) was 0.35 e.p. The oxidation of thiol groups was:

O _% = ((0.55-0.35) / 0.55) × 100 = 36.4;

which also significantly exceeds the indicator of a group of healthy women. The calculated IR was:

IR = (0.53 / 0.35) × (36.4 / 17.01) ² = 6.91 relative units;

those. patient I.'s resistance to the effects of prooxidant factors was significantly reduced.

Within 10 days, a traditional drug treatment was carried out. After completion of treatment, a second examination was carried out in order to determine the dynamics of changes in the above indicators. After treatment, the total content of SH ₁ -groups of blood plasma was 0.55 e.o.p., SH _l - 0.15 e.o.p., SH _t - 0.30 e.o.p., the indicator was calculated the ratio of easily and inaccessible thiol groups:

K ₁ = 0.15 / 0.30 = 0.50.

The content of SH ₂ groups of blood plasma (after exposure to hydrogen peroxide) was 0.41 e.p. The oxidation of thiol groups was:

O _% = ((0.55-0.41) / 0.55) × 100 = 25.5;

which significantly exceeds the indicator of a group of healthy women. The calculated IR was:

IR = (0.50 / 0.35) × (25.5 / 17.01) ² = 3.21 relative units; Such results also indicate the effectiveness and adequacy of the therapy.

The proposed method allows the following.

- To assess the balance of readily available and inaccessible thiol groups as an indicator that demonstrates a violation of the functioning of the antioxidant defense of the body, as well as reflecting conformational disorders of protein molecules, which will allow the timely implementation of the necessary measures for their medical correction.

- To conduct a deeper assessment of the pathobiochemical significance of the imbalance of the thiol unit of antiradical protection due to an additional assessment of the sensitivity of this unit to simulate oxidative stress in the blood plasma of the studied patients by introducing a hydrogen peroxide solution with an assessment of the oxidation of thiol groups, which will allow to determine individual indications for prescribing drug antioxidant therapy and reduce dosages of used drugs with antioxidant activity, including containing thiol groups, for example lipoic acid, glutathione, thereby reducing the number of possible side effects, as well as reduce the cost of these drugs.

- This indicator allows you to reliably evaluate the effectiveness of restoring the balance of functioning of the thiol link of antiradical protection during treatment in order to predict possible complications and perform timely correction of therapy.

The practical result of the proposal is to determine the imbalance of the thiol link of the anti-radical defense in the examined patients and to assess the body's resistance to oxidative damage, which allows determining individual indications for drug correction, which means reducing the time spent by patients in hospitals and optimizing the amount of medication needed for treatment.

Claims (11)

A method for assessing the body's resistance to the effects of prooxidant factors, including the analysis of thiol groups in the patient's blood plasma, characterized in that the complex using the Ellman reagent determines the value of the ratio of readily available and inaccessible thiol groups of blood plasma, their oxidation by hydrogen peroxide and the integral coefficient (IR) assess the imbalance in the functioning of the thiol unit of the antioxidant system of blood plasma by the formula: IR = (K ₁ / 0.35) × (O _% / 17.01) ² , where K ₁ - the ratio of easily and inaccessible thiol groups, determined by the formula K ₁ = SH _l / SH _t where SH _l - the content of readily available thiol groups, units of optical density; SH _t - the content of inaccessible thiol groups, units of optical density; About _% - the percentage of oxidized thiol groups with hydrogen peroxide, determined by the formula O _% = ((SH ₁ -SH ₂ ) / SH ₁ ) × 100, where SH ₁ - total thiol groups of intact blood plasma, optical density units; SH ₂ - total thiol groups of blood plasma after exposure to hydrogen peroxide, optical density units, and when the integral coefficient is equal to or less than 1.5 relative units, the normal resistance of the body to the action of prooxidant factors is determined, if the IR value is above 1.5 relative units, the presence of conformational changes in proteins and the ratio of non-protein / protein thiol-containing compounds of blood plasma and the disruption of the functioning of the thiol link anti-radical protection, accompanied by reduced body resistance during the development of oxidative damage.

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