The method of evaluation of antioxidant balance of the human body

 

(57) Abstract:

The invention relates to medicine, more specifically to clinical biochemistry, hygiene, and can be used to assess the antioxidant status of the organism in different populations. The method includes collecting condensate alveolar moisture, then determine the maximum magnitude of the H2O2induced chemiluminescence (And), the maximum magnitude of spontaneous chemiluminescence (COI), calculate the index of activation And=And/COI, set the time to achieve the maximum chemiluminescence T1and the time of its decay half - T2calculate the ratio T=T1/ T2and the coefficient K=T/A, and when set To<1 determine the violation of antioxidant balance. The technical result is a more accurate assessment of antioxidant balance for the General characteristics of the functional state of the organism.

Antioxidant balance is an important part of functional homeostasis of the human body, characterized by indicators of the level of radiculopathy and the ability to inactivate their damaging effects (superoxidedismutase activity - SPA).

Izobreteny of status (AOS) of the organism in different segments of the population.

The aim of the invention is to improve the accuracy of evaluation of antioxidant balance for the General characteristics of the functional state of the organism.

The method includes collecting condensate alveolar moisture (expirate) in modernized absorber Polezhaeva, determine the maximum values of chemiluminescence of the sample - spontaneous (SP) and H2O2induced (And), the index calculation activation And=And/COI characterizing the intensity of processes of free radical oxidation (CPO), determining the time of maximum signal PI ( T1and a time of recession it in half (T2), calculate the relations T=T1/T2reflecting the degree of antiradical protection, the establishment of a K-factor= T/And whose values less than 1 indicate a violation of antioxidant balance and deviations in the overall functional state of the organism.

A known method for determining superoxide activity exhaled air of patients and healthy people by collecting the test person's breath of air in a sealed plastic bag, standard incubation volume of the tested air from nicrosini tetrazolium in the presence of superoxide is to interact superoxide bioradicals and narasinga of tetrazole - formisano, spectrometric determination of their optical density, calculate the concentration of formisano on the standard volume of the test exhaled air, calculation and evaluation of the difference between the content of superoxidation in the studied air of the patient and a healthy person, which characterizes superoxide activity in the tested air /1/.

The disadvantage of this method is the possibility of estimating only one side of the AOC - superoxide activity, which does not allow to make a full conclusion on the status of antioxidant balance.

There is a method of assessing the functional state of the organism miners on the basis of the study, two components of the AOC by selecting condensate alveolar moisture in the modernized absorber Polezhaeva, determine the sample superoxidedismutase activity by spectrophotometry based on the inhibition narasinga tetrazolium in the system generation of superoxide anion radicals in biological material by sequentially depositing in the cuvette of a spectrophotometer 1.3 ml of buffer a mixture of one-deputizing potassium phosphate and sodium hydroxide, 0.4 ml of the investigated samples, 0.5 ml of a solution of nicotinamide-adenine-dinucleotide vos is asin of metasulfite, as well as determining the intensity of the H2O2induced chemiluminescence in sitesome signal (S) using bioluminometer when posledovatelna making his cuvette 1 ml of phosphate buffer (pH 7.3 to 7.4), 50 μl of 0,006% solution lyuminola (pH 7.3 to 7.4), investigated condensate in a volume of 0.2 ml, the introduction of 0.2 ml of freshly prepared 10% solution of hydrogen peroxide and immediate measurement readings of the light intensity within 1-2 minutes of finding the ratio S/SPA and evaluation of the functional state of the organism as premorbid with increasing coefficient obtained in comparison with the norm /2/.

The disadvantages of the method are its complexity and duration, due to the need to perform two diverse research assay (biochemical and biophysical) to characterize the two sides of free radical oxidation - radiculopathy and antiradical protection.

The closest in technical essence is a method of non-invasive diagnostics of functional state of organism miners of the Far North under the influence of complex factors /3/ by collection of expirate in modernized absorber Polezhayev, is swesome (S) signal Lomonosovskiy H2O2-induced chemiluminescence in sequential introduction into the cuvette 1 ml of phosphate buffer (pH 7.3 to 7.4), 50 μl of 0,006% solution lyuminola (pH 7.3 to 7.4), condensation in the volume of 0.2 ml, the introduction of 0.2 ml of freshly prepared 10% solution of hydrogen peroxide and immediate measurement readings of the light intensity within 1-2 minutes of finding the coefficient of total resistance S/SPA on the basis of empirical dependences S/SPA from S and diagnosing abnormalities in a functional condition of an organism with increasing coefficient obtained in comparison with the norm (50-150%).

The disadvantage of this method is its inaccuracy related to the fact that this really is determined by the rate of only one side SRO - intensity radiculopathy, while the other side is superoxidedismutase activity, is estimated using the empirical relationship between these two indicators.

The aim of the invention is to improve the accuracy of evaluation of antioxidant balance for the General characteristics of the functional state of the organism.

The objective is achieved by collecting 1 ml of expirate in modernized absorber Polezhaeva, placed in a container with a mixture of ice and NaC ml of phosphate buffer (pH 7.3 to 7.4), 50 μl of 0,006% solution lyuminola (pH 7.3 to 7.4), sample assay volume of 0.2 ml, the introduction of 0.2 ml of freshly prepared 10% solution of hydrogen peroxide and immediate measurement readings of the light intensity within 1-2 minutes, check the maximum intensity of the spontaneous (SP) and H2O2induced PI (S), calculation of the index activation And=And/COI, determine the time of maximum signal PI (T1and its decline to one-half (T2), calculate the relations T= T1/T2, setting the K-factor=T/A, the output of which is beyond the limits of physiological equilibrium (1,0) indicates disturbances in antioxidant balance and deviations in the overall functional state of the organism.

Distinctive features of the method are that the two sides SRO - radiculopathy and antiradical protection, assessed by one - holdem-study of biological samples - expirati for patterns radiculopathy determine the index of activation (A), for the characteristics of antiradical protection is the time of maximum signal PI to the time of its decline to one-half (T), and violation of antioxidant balance state ividually variations in antioxidant balance, can be applied in the examination of the various contingents of the adult and child population.

In comparison with the method of the prototype of the proposed method is more accurate as the estimate of the ratio of two sides of the SRO is based on real specific data about the formation of a curve induced chemiluminescence in expirate which is essential for the monitoring and screening.

The application of the method is illustrated by the following examples.

EXAMPLES OF THE WAY

Example 1

As a result of hygienic analysis of power structures in one of the kindergartens were observed failure rate of consumption of a number of important produktov that allowed us to assess the nature of power as unbalanced.

To assess the AOC in a child 6 years attending this kindergarten for 20 minutes at deforsirovannom degenii was assembled 1.0 ml of expirate in modernized absorber Polezhaeva, placed in a mixture of ice / sodium chloride to condense.

In selected bioassays conducted by the definition of the parameters of spontaneous and induced chemiluminescence using chemiluminometer PHL-01. To do this, in thermally Cam the Oba, we measured the magnitude of spontaneous chemiluminescence ( with continuous stirring) - COI, then injected 0.2 ml of hydrogen peroxide solution as the activating agent and the registered intensity changes induced chemiluminescence in 2-3 minutes. When the idle measurements instead of the investigated sample in the cuvette was made the same amount of buffer solution.

Reportedly built the dependence of the intensity signal (S) from time to time, which are defined by the following parameters in relative units compared to the same parameters for the blank sample:

- the intensity of spontaneous chemiluminescence (COI) - 1,05;

- maximum intensity of the stimulated luminescence (S) - 5,73;

the time of maximum intensity PI (T1) - 1,0;

the time of decrease in the intensity to half of the maximum value (T2) - 0,87.

Then calculate the index of activation And=And/COI, which was 5,46, regarding the timing of the maximum signal to the time of recession T=T1/T2, which amounted to 1.15 and the coefficient K=T/a, which in this case was equal to 0.21, which is less than 1.0. Thus, the child was found violating AO balance.

For comparison with the way the situation in the cuvette of chemiluminometer 1 ml of buffer solution, 50 μl of a solution lyuminola, 0.2 ml of a measured sample, 0.2 ml of hydrogen peroxide solution as the activating agent and the registration of the change of the intensity of induced chemiluminescence in 2-3 minutes. Sutasoma was defined as the area under the curve PI. When the idle measurements instead of the investigated sample in the cuvette was made the same amount of buffer solution. Reading values setosum was calculated in percentage to sutasoma signal induced chemiluminescence for the blank sample.

Coefficient of resistance taken out in accordance with empirical formulas /2/ depending on the values of sutasoma. In this example, the value of setosum (S) was 265,98%, coefficient of total resistance - 292,16%, which is beyond the limits of physiological norm, installed at intervals of 50-150% and allows you to mark a violation of the functional state of the organism in the examined child, installed on the presence of deviations in AOC.

According to the results of the examination, the child was assigned and carried out preventive measures, including anti-oxidant therapy, then the study of the antioxidant balance were repeated and showed that the intensity of the radical (K=1,03). The scores setosum CHL showed that its value also decreased to 97.7% and defined by the empirical formula of the coefficient of total resistance was 98%, which is within the physiological norm.

Thus, evaluation of antioxidant balance in the child, carried out in two ways by exploring expirate allowed to come to the same conclusion, but in the proposed method, the findings are based on real data obtained about the state of free radical oxidation in the study of the biological media at the time of the study, while the way the prototype uses a previously established empirical relationship obtained by summarizing the data, a large contingent of patients, which reduces the accuracy and reliability of results.

Example 2

To assess the functional state of the organism workers metallurgical enterprises selected homogeneous group of men aged 20-28 years old with work experience not less than 5 years consisting of 16 persons.

In the air of working zone was established, among other factors, the presence of complex heavy metals in concentrations close to the MPC.

In selected each of the surveyed expirate about the obtained results to the whole group. Studies conducted in the same way as example 1, showed that the intensity of radiculopathy (A) was 1.22, superoxidedismutase activity, characterized by the index T is 0.41 and the K-factor is 0.42. A low value To indicate a violation of antioxidant balance and is the basis for the course antioxidant prevention. Evaluation according to the method of the prototype showed that the average value of the total resistance amounted to 352%, which coincides with the evaluation of the proposed method and confirms the conclusions.

After the course antioxidant therapy in the same group conducted a second survey, which show that antioxidant levelled off balance: A=1,26; T=1,36; K=1,08. The intensity of radiculopathy as assessed by sitesome signal, amounted to 115.5% and the total resistance of 1.15, which is within the physiological norm.

Thus, as in example 1, the proposed method allows for the quick and simple to carry out an adequate assessment of antioxidant balance for the General characteristics of the functional state without resorting to the use of empirical regularities.

References

1. SPO is ptx2">

2. The method of estimation of the functional state of the organism miners. Patent SU 1811608 A3.

3. Non-invasive diagnostics of the functional state of the organism miners of the Far North under the influence of complex factors, preventive measures. Guidelines.-M.,1992.-28 C.

The method of evaluation of antioxidant balance of the human body, including the collection of condensate alveolar moisture, determination of the intensity values of H2ABOUT2induced chemiluminescence of the sample, characterized in that to determine the maximum magnitude of the H2ABOUT2induced chemiluminescence (And), the maximum magnitude of spontaneous chemiluminescence (COI), calculate the index of activation And= And/COI, set the time to achieve the maximum chemiluminescence T1and the time of its decay half - T2calculate the ratio T= T1/T2and the coefficient K=T/A, and when set To<1 determine the violation of antioxidant balance.

 

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