RU2455645C1 - Method of screening estimation of human functional body condition - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention describes a method of screening estimation of the humal functional body condition, involving the collection of exhaled moisture condensate (expirate), the preparation of a biosensor - luminescent lyophilised bacteria 'Ecolum', the addition of the expirate 0.5 cm³ to the biosensor 0.5 cm³, the 15-minute exposition, the measurement of luminescent emission of mixed bacterial suspension and condensate for 1000 sec, the recording of its maximum (Iop, imp/sec.), the comparison of said value with an analogue parameter Ik, imp/sec., derived by introducing distilled water in a bioluminometer tray instead of condensate in equal proportions, the determination of a coefficient K as the Iop/Ik relation which has a value of K=1 enables to state an optimal oxidative status; the value K>1 shows the prevailing pro-oxidative processes (a high level of formation of oxygen radicals, an insufficient level of protection against their damaging action), the value K<1 provides stating active antioxidant processes, sufficient reserves of protective mechanisms.

EFFECT: simplification, higher productivity and profitability of the method of the estimation of the functional body condition allowing detecting the oxidative status deviations.

3 ex

Description

The invention relates to medicine, more specifically to preventive medicine and laboratory diagnostics, and can be used to assess the functional state of the human body.

The aim of the invention is the simplification, high productivity and cost-effectiveness of the method for assessing the functional state of the body, which allows to identify deviations accompanied in its oxidative status. The advantage and novelty of the developed method consists in the transition from measuring the intensity of the induced chemiluminescence of the bioassay itself (expirate) to a bioassay based on establishing the level of bioluminescence of lyophilized bacteria due to the oxidative potential of the studied expirate. The use of a sensitive bacterial sensor to determine the capacity of the oxidizing system of the body while maintaining the information content of the method allows to evaluate the potential risk of oxidative stress for targeted correction in the screening mode for targeted correction, including in the early stages.

The method includes collecting exhaled moisture condensate (ekspirata) preparing the biosensor - lifilizirovannyh luminescent bacteria "Ekolyum", adding to 0.5 cm ³ biosensor ekspirata 0.5 cm ^3, 15 minute exposure, the luminescence intensity measurement of the bacterial suspension and the mixture of condensate within 1000 sec, fixing its maximum level (IOP, imp / sec), comparing this value with the same parameter Ic, imp / sec obtained when a bioluminometer was added to the cuvette instead of condensate of distilled water in an equal volume, setting to coefficient K as the ratio Iop / Ik, at values of which K = 1 state the optimal oxidative status, at K> 1 - the predominance of prooxidant processes (high level of formation of oxygen radicals, insufficient level of protection against their damaging effects), at K <1 - active antioxidant processes, sufficient reserves of protective mechanisms.

The technical result is a screening characteristic of the functional state of the body under the influence of endogenous and exogenous adverse factors .. A methodological approach to the use of luminescent bacteria to detect the oxidative properties of a biomedia - expirate was applied for the first time. The scientific fact of a change in the luminescence of bacteria in response to the introduction of the expirate has also been established for the first time.

The well-known "Method for assessing the functional state of the organism of miners", including the study of a non-invasive biosubstrate - expired air condensate by measuring the intensity of radical formation in it by the method of induced luminol-dependent chemiluminescence and superoxide-intercepting activity by spectrophotometry based on the inhibition of nitro blue tetrazolium in the generation system indicators and assessment of functional status rganizma like premorbid obtained with increasing ratio in comparison with standard [1]. The disadvantages of the method are the high cost, complexity and duration of execution, due to the need for two types of (biochemical and biophysical) studies of the sample.

There is a method for non-invasive diagnosis of the functional state of the organism of miners of the Far North [2], which involves collecting condensate of exhaled moisture, determining the intensity of radical formation (IR) in it by the light sum (S) of the luminol-dependent induced chemiluminescence signal of finding the total resistance coefficient S / SPA using the empirical dependence S SPA from S and diagnosis of deviations in the functional state of the body with an increase in the obtained coefficient in comparison with the norm, is determined Noah as an interval of 50-150%. The disadvantage of this method is the need for basing on empirical dependencies, as well as the registration of fast processes, which complicates the method and reduces its accuracy.

The closest in technical essence is the "Method for assessing the antioxidant balance of the human body" by collecting exhaled air condensate, sequentially adding 1 ml of phosphate buffer (pH 7.3-7.4) to a bioluminometer cuvette, 50 μl of a 0.006% luminol solution (pH 7.3-7.4), 0.2 ml and 0.2 ml of a freshly prepared 10% hydrogen peroxide solution, followed by immediate measurement of the glow intensity within 1-2 minutes, graphically plotting the CL intensity versus time, determining from her row show formations in the curve of induced luminol-dependent chemiluminescence, namely, the maximum values of spontaneous (Isp) and induced (I) chemiluminescence, the time to reach the maximum value (T1) and its decay time by half (T2), the calculation of the pair ratios A = I / Isp and T-T1 / T2, as well as the coefficient K = T / A, at a value of which is less than one, is judged about the violation of the antioxidant balance [3]. The disadvantage of this method is the complexity caused by the need to fix and obtain a graphic image of the CL formation curve — an extremely fast process and requiring in this connection either the availability of expensive devices or additional time and labor costs for constructing curves from observational data and establishing a large number of necessary parameters using computer programs.

The aim of the invention is to simplify the method and increase its productivity.

The goal is achieved by basing on the first established fact of the manifestation of changes in the bioluminescence of bacteria "Ekolyum" in contact with a sample of the expirate and interpretation of these changes for screening assessment of oxidative balance as one of the most important aspects of the functional state of the body.

Distinctive features of the method are that luminescence measurements are not carried out on the bioassay itself (expirate), but on the biosensor, thereby eliminating the need to study induced chemiluminescence, which greatly simplifies the method, while increasing its accuracy, since the fast-flowing process of induced chemiluminescence is always an artificial technique.

The method allows to establish the state of the functional reserves of the body for a targeted choice of preventive and correction measures, the formation of risk groups when examining various contingents of the adult and children's population.

Compared with the prototype method, the proposed method is more economical and affordable in execution. The method combines alternative (bacterial test) and non-invasive (expirate) methods, which makes it most suitable for laboratory practice.

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

EXAMPLES OF PERFORMING THE METHOD

Example 1

Patient P., 60 years old. He was observed at the clinic of occupational diseases with a diagnosis of suspected chronic bronchitis.

To assess the functional state of the body during the examination, an expirate (condensate of exhaled moisture) was collected for 20 minutes with unforced breathing. A sample was taken in a modernized Polezhaev absorber placed in a mixture of ice with sodium chloride for better condensation.

A suspension of the Ekolyum biosensor was prepared in accordance with the instructions [4].

0.5 ml of the biosensor and 0.5 ml of distilled water were added to one of the Biotoke-10 M bioluminometer ditches. After a 15-minute exposure, the maximum value of the chemiluminescence intensity was measured over a period of 1200 seconds (Ic), which amounted to 2002 pulse / sec. In another cuvette, 0.5 ml of biosensor and 0.5 ml of the test bioassay were added. Under similar measurement conditions, the maximum value of the intensity Iop = 833 imp / sec was recorded.

Coefficient K = 0.41 <1. A decrease in the intensity of bacterial bioluminescence indicates the predominance of antioxidant processes associated with a high level of protective reserves from the damaging effects of oxygen radicals.

At the same time, the study of the same bioassay was carried out using the prototype method [3]. For this purpose, 1 ml of phosphate buffer (pH 7.3–7.4), 50 μl of a 0.006% solution of luminol (pH 7.3–7.4), 0.2 ml were successively added to the cuvette of the PHL-01 bioluminometer. and 0.2 ml of a freshly prepared 10% hydrogen peroxide solution. Immediately after the introduction of hydrogen peroxide (initiator of peroxidation), the CL intensity readings were read for 2 minutes, and the time dependence of the CL intensity was constructed from the data obtained. Based on this dependence, the parameters of the intensity of spontaneous (Isp) and induced CL (I) were determined, the time to reach the maximum intensity and its decay time by half (T1 and T2, respectively), the values A = I / Isp and T = T1 / T2, and also coefficient K = T / A. The following data were obtained:

Use = 1.0

I = 3.53

T1 = 2.5 sec

T2 = 1.7 sec

A = 3.53

T = 1.51

K = 0.43

The value of the criterion K = 0.43 indicates the predominance of anti-radical defense processes characteristic of the course of chronic processes.

Thus, the conclusions made by the proposed method and the method of the prototype are comparable. However, the proposed method is more economical and simple to implement.

Example 2

To conduct a screening assessment of the functional state of the body of workers at a metallurgical plant, an equilibrium group of men aged 45-60 years was formed with at least 5 years of work with 12 people. According to the expirate of each subject, the oxidative status was evaluated in two ways - the proposed one and the prototype. The sequence of steps for each method is similar to that described in example 1.

The results obtained by the proposed method by measuring the intensity of the bioluminescence of the biosensor showed that 5 out of 12 examined (coefficient K> 1) may develop oxidative stress. In each case, the result of the study was obtained immediately after the analysis. It is recommended that these workers be included in the “risk group” for the correction.

The results obtained by the method of the prototype showed that out of the total number of 3 workers examined, a high level of radical formation was noted with a sufficient antioxidant defense capacity; 2 showed oxidative stress, expressed in changes both from the side of a high level of radical formation, and from the side of insufficient antiradical protection, that is, there are indications for mandatory correction. 3 cases of a decrease in the indicators characterizing the degree of antioxidant protection were revealed in the absence of deviations in the intensity of radical formation. These changes are also indications for inclusion in the “risk group”.

Thus, the number included in the "risk group" is similar to the assessment of the proposed method. However, it was additionally necessary to study induced chemiluminescence, as well as conduct a detailed analysis of changes in the state of oxidative status provided for by the prototype method, which together limits its use in screening studies.

Example 3

Patient K, 62 years old, electrolysis bath loader (MMC Norilsk). He has been living and working in the Arctic for 22 years. He was treated at the clinic of occupational diseases with a diagnosis of chronic bronchitis.

The functional state was assessed for this patient upon admission and after the prescribed course of treatment. Evaluation was carried out in parallel by the proposed method and by the prototype method. The collection of material for analysis, preparation and conduct are the same as in the previous examples.

As a result, the following data were obtained.

By the proposed method:

before treatment: Iop - 7926; Ik - 5825; K = 1.36;

after treatment: Iop - 3770; Ik - 5583; K = 0.675.

Thus, if, upon admission, the patient could have expected the development of oxidative stress, then according to the results of the treatment, activation of the anti-radical defense system and increased resistance of the body to the damaging effect of radicals were observed, as evidenced by a decrease in the K coefficient from 1.36 to 0.675. The results of the study according to the prototype method

Before treatment After treatment Spanish - 1.0 Spanish - 1.0 And - 1.29 And - 0.88 T1 - 1.25 sec T1 - 1.28 sec T2 - 1.5 sec T2 - 1.5 sec A = 1.29 A = 0.88 T = 0.84 T-0.85 K = 0.65 K = 0.96

Interpreting the obtained data, we can conclude that as a result of treatment, the patient showed a decrease in the level of radical formation (activation index decreased from 1.29 to 0.88), the antioxidant defense capacity remained practically unchanged (the T index remained unchanged), but in general, the oxidative balance was optimized (K = 0.96).

Therefore, research in one way or another proved the effectiveness of the treatment in terms of increasing the body's level of resistance to the action of damaging factors. However, laboratory studies of the proposed method are less time consuming while maintaining information content.

BIBLIOGRAPHY

1. Description of the invention to patent SU 1811608 A3 "Method for assessing the functional state of the organism of miners", published on 04/23/93. Authors: Yudina T.V., Egorova M.V., Borisenkova R.V., Lutsenko L.A., Halperin A.Sh., Larkina M.V., Fedorova N.E., Rushkevich O.P., Scriabin S.Yu.

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

3. Description of the invention to patent RU 2206891 C1 “Method for assessing the antioxidant balance of the human body”, published on 10.16.01. Authors: Yudina T.V., Rakitsky V.N., Egorova M.V.

4. The device of environmental control "Biotox-10M." Manual.

Claims (1)

Method skrinningovoy evaluating the functional status of the human body comprising a condensate collecting exhaled moisture (ekspirata) preparing the biosensor - lifilizirovannyh luminescent bacteria "Ekolyum", adding to 0.5 cm ³ biosensor ekspirata 0.5 cm ^3, 15 minute exposure, measurement of luminescence intensity a mixture of a suspension of bacteria and condensate for 1000 s, fixing its maximum level (IOP, imp / s), comparing this value with the same parameter Ic, imp / s, obtained by adding a bioluminometer to the cuvette instead of the condensate of distilled water in an equal volume, the establishment of the K coefficient as the Iop / Ik ratio, at values of K = 1, the optimal oxidative status is ascertained, at K> 1, the prevalence of prooxidant processes (high level of formation of oxygen radicals, insufficient level of protection against their damaging effects) , when K <1 - active antioxidant processes, sufficient reserves of protective mechanisms.