RU2547790C1 - Method of evaluating tolerance to physical loading by measurement of autofluorescent properties of skin - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to functional diagnostics, and can be applied for the evaluation of tolerance to physical loading. Skin is illuminated by exciting radiation. Signals, generated by the secondary skin irradiation, are measured by a spectrometer in two spectral areas. The first spectral area is selected from the field of excitation, the second - in the area of fluorescent NADH emission. Evaluation of the autofluorescent AF parameter is carried out in accordance with formula: AF=F(λ_m)/R(λ_x)^0.85, where R(λ_x) is a signal, obtained from the first spectral area, F(λ_m) is a signal, obtained from the second spectral area. Measurement of the signals is carried out in symmetric zones of the right and left parts of the body in an initial condition of the human organism. Then, the human organism is influenced by a loading test, and the measurement of signals is performed in the same areas of the skin. The index of the functional status IFS is calculated by formula: IFS=AF_o/AF_p, where AF_o is the parameter of autofluorescence before the influence with the loading test, AF_p is the parameter of autofluorescence after the influence with the loading test. The value of AF_p, maximally different from AF_o is used_. Tolerance to physical load is evaluated by the obtained value of IFS.

EFFECT: method makes it possible to increase the reliability of estimation of tolerance to physical loading due to the differential principle of measurement, in which only changes of the skin autofluorescence, associated with NADH changes, are traced, as well as to reduce the evaluation time.

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Description

The invention relates to medicine, namely to functional diagnostics, and can be used for rapid assessment and monitoring of a person’s functional state under normal conditions, in pathological conditions and under extreme conditions of activity, to determine the degree of fatigue, latent pathology, and establishment of reserve capabilities of the body, choosing the optimal treatment, rehabilitation and activation tactics.

The method is based on measuring autofluorescence of the skin and therefore is non-invasive.

Autofluorescence (AF) or intrinsic fluorescence of biological tissues (i.e. their ability to glow without introducing any external agent into the tissue) is determined by the presence of endogenous fluorophores, including pyridine nucleotide (NAD), flavoproteins (Fp) ), glycation end products (AGE), etc.

The glow of NAD and Fp depends on their being in an oxidized or reduced state. To assess the redox processes in organs and tissues, fluorescence of the reduced pyridine NADH nucleotide is most often used, which emits in the visible spectral range in the range 420-480 nm, when excited in the ultraviolet region of the spectrum in the absorption band of 320-380 nm, which is absent in the oxidized state this respiratory enzyme (Mayevsky A, Rogatsky GG Mitochondrial function in vivo evaluated by NADH fluorescence: from animal models to human studies. Am. J. Physiol. Cell 292: C615-C640, 2007).

The basis of the known method is the phenomenon that, when there is a lack of oxygen in the tissue, an accumulation of excess amounts of reduced pyridine nucleotides (NADH) occurs as a result of a violation of its oxidation in NAD ⁺ through the mitochondrial electron transport chain. The intensity of autofluorescence depends on the ratio of reduced and oxidized pyridine nucleotides, increasing with hypoxia. Thus, the phenomenon of tissue autofluorescence can be used to record ischemic changes in organs and tissues.

A study using multiphoton microscopy is known to evaluate the intensity of NADH autofluorescence in human epidermal cells during occlusion of the brachial artery reperfusion [Balu Μ, Mazhar A, Hayakawa SK, Mittal R, Krasieva TV, Konig K, Venugopalan V, Tromberg BJ. In vivo multiphoton NADH fluorescence reveals depth-dependent keratinocyte metabolism in human skin. Biophys J. 2013 Jan 8; 104 (l): 258-67].

The result of assessing the level of fluorescence was compared with the level of oxy- and deoxyhemoglobin in the tissue. It was shown that during a 3-minute occlusion of the artery there is a significant increase in the level of NADH fluorescence, which the authors attribute to a decrease in the intensity of oxidative metabolism in keratinocytes of the basal layer of the epidermis.

The phenomenon of autofluorescence is widely used in medical diagnostics, including by measuring the glow of the skin under the influence of ultraviolet radiation.

A known method and device described in the patent (US 7966060. Method and apparatus for determining autofluorescence of skin tissue), which are designed to evaluate the final products of glycation of AGE by measuring the AF of the skin areas located in the forearm of the patient. A source of exciting radiation is a fluorescent lamp emitting in the UV region in the range of 300-420 nm. The collection and registration of radiation is carried out using a spectrometer in the range of 420-600 nm. To reduce the effect of skin pigmentation, a parameter is introduced that represents the ratio of fluorescence radiation to reflected exciting radiation (285-420 nm). Measurements are taken on skin areas with a size of 0.4 cm ² . This method allows for screening studies of the population to identify patients with diabetes mellitus, to predict complications in diseases of the cardiovascular system and in renal failure.

The disadvantage of this method is the limited scope of its application.

Also known is a method and device (patent US 6697657 Method and devices for laser induced fluorescence attenuation spectroscopy), which are designed to determine ischemia and hypoxia of biological tissue. The operation of this system is based on measuring NAD fluorescence spectra in the range from 470 to 490 nm with two-photon laser excitation. The complexity of this technology does not allow its use in the routine practice of clinical research.

The disadvantage of this method is also the limited scope.

There is a method of measuring autofluorescence properties of the skin to assess the functional state of the body, implemented by the Skin-AGE spectrometer designed to conduct fluorescence-reflective biomedical research (Kang Uk; Papayan GV, Berezin VB, Petrishchev N.N. ., Galaguza MM Spectrometer for fluorescence-reflective biomedical research. Optical journal. 2013. V. 80 .. No. 1. P. 32-38).

This method is the closest to the claimed invention and can serve as a prototype. It consists in irradiating the skin with excitation radiation from a illuminator generating radiation in the near ultraviolet region with a central wavelength of 365 nm, measuring the signals caused by secondary skin radiation in a spectrometer in two spectral regions, of which the first region is selected in the excitation region, and the second in areas of fluorescence emission of the skin, and the subsequent calculation of the autofluorescence parameter, which is a function of the two indicated signals. The method is implemented with regular, for a long time, episodic measurement of autofluorescence properties of the skin of a certain location.

The disadvantage of this method is that such an assessment is possible only when performing lengthy measurements, when you can set the values of the individual norm. It can vary significantly among different people, because the fluorescence signal is affected not only by NADH, but also by the luminescence of other substances. When examining the skin, these are, first of all, AGE, keratin, collagen, the excitation and emission spectra of which are in the same wavelength regions as NADH. This overlap makes it difficult to unambiguously interpret the results when assessing the functional status by known methods. For example, a higher AF value in a given person compared to average normal values can be associated with both the state of hypoxia at a given time and the increased content of AGE, which can be caused by long-term exposure to various adverse factors.

The objective of the present invention is to increase the reliability of assessing exercise tolerance and the possibility of monitoring it by measuring the NADH fluorescence of the skin, which reflects the oxidation-reduction state of the body in normal conditions for various pathologies and various types of extreme human activity, as well as reducing the time to assess physical tolerance load due to the application of the differential principle of measurement and the expansion of functionality.

The achievement of this task is achieved by the fact that in the method of assessing exercise tolerance by measuring the autofluorescence properties of the skin, including illumination of the skin with exciting radiation, measuring signals caused by secondary skin radiation with a spectrometer in two spectral regions, from which the first region is selected in excitation region, and the second - in the field of NADH fluorescence emission, the calculation of the autofluorescence parameter AF in accordance with the formula:

AF = F (λ _m ) / R (λ _x ) ^0.85 ,

where R (λ _x ) is the signal obtained from the first spectral section,

F (λ _m ) is the signal obtained from the second spectral region,

according to the invention, said measurement of signals is carried out in symmetrical zones of the right and left parts of the body in the initial state of the human body, after which the human body is exposed to a load breakdown and the signals are measured in the same skin areas, the functional status index (IFS) is determined by the formula:

IFS = AF _{0 /} AF _p ,

where AF _o - autofluorescence parameter before exposure to a load sample,

AF _p - autofluorescence parameter after exposure to a stress test,

moreover, the value of AF _{p is used} , which differs as much as possible from AF _o , and exercise tolerance is estimated from the obtained IFS value.

The inventive method for assessing exercise tolerance by measuring the autofluorescence properties of the skin differs from the prototype in that it is based on the measurement of skin AF (ROS) independent of the variation of fluorophores that are not involved in the biological oxidation process. This is achieved through the use of a differential measurement principle, in which only AF changes associated with a change in NADH concentration are monitored. Changes can be stimulated by various stress tests.

It is known that the state of the oxidation-reduction metabolism of the body can be influenced by various factors: physical activity, orthostatic breakdown, respiratory breakdown, electromagnetic radiation, exposure at acupuncture points, hydraulic effects, temperature effects, drug effects, etc. The effect of some of them on AF has been verified experimentally. The following examples show this effect.

As a result of studies of a method for assessing exercise tolerance by measuring the autofluorescence properties of the skin, it can be concluded that the invention provides the achievement of the task, which is expressed in increasing the reliability of assessing exercise tolerance and the possibility of monitoring it, as well as in reducing assessment time exercise tolerance and enhanced functionality.

The claimed invention is new, not known in the practice of biomedical research, and the combination of distinctive features does not follow from the prior art. The invention is applicable due to the simplicity of the measurement method and. the possibility of using existing devices.

The invention is illustrated by drawings, where

in FIG. 1- presents a graph of IFS in athletes at different stages of the experiment on the first day of testing;

in FIG. 2 is a graph of IFS in athletes at different stages of the experiment a week after the first test;

in FIG. 3 is a graph of IFS during exercise in healthy subjects and in patients with heart failure (dashed curves);

in FIG. 4 is a graph of the relationship between the IFS and the amount of oxygen absorbed during a load test;

The method is as follows.

As a device for carrying out fluorescence-reflective measurements, for example, a Skin-AGE fiber spectrometer can be used, in which a wavelength of 365 nm is used to excite fluorescence, which is suitable for excitation of NADH. Due to the presence of a fiber probe in the device, the place of skin measurements can be arbitrary. It is selected on the basis of the research task, ease of measurement and skin uniformity. In particular, to assess exercise tolerance, measurements can be performed on the medial surface of the skin of the shoulder, which is convenient for the doctor and patient, which has minimal hairiness. The signal R (λ _x ) is measured from the first spectral portion λ _x located in the fluorescence excitation region and the signal F (λ _m ) from the second spectral portion λ _m located in the NADH fluorescence emission region. To eliminate the influence of the scatter of readings at various points of the selected skin area, the signals are averaged when it is scanned with a probe. Based on the received signals, the autofluorescence parameter AF is calculated by the formula:

AF = F (λ _m ) / R (λ _x ) ^0.85 (1)

where R (λ _x ) is the signal obtained from the first spectral section,

F (λ _m ) is the signal obtained from the second spectral region,

and to take into account the possibility of an asymmetric reaction of the body to the load, paired measurements are taken in areas that are selected in the symmetric zones of the right and left parts of the body. To account for the effect of signal changes over time, multiple measurements are taken in the same skin areas.

After the measurements in the initial state and determination by the formula (1) of the initial autofluorescence parameter AF _o , the human body is subjected to a stress test and the signals are measured in the same areas of the skin. The autofluorescence parameter is calculated using the same formula (1) after a stress test AF _p. As a result of exposure, different values of AF _p can be obtained on the right and left. When calculating IFS use the value of AF _p , the most different from AF _o . According to the obtained values of AF _o and AF _p according to the formula:

IFS = AF ₀ / Af _p (2)

where AF _o - autofluorescence parameter before exposure to a load sample,

AF _p - autofluorescence parameter after exposure to a stress test,

determine the index of functional status (IFS), the value of which assesses exercise tolerance.

The method is illustrated by examples of studies on the effects of various loads on subjects with different physical conditions. The studies were carried out using a Skin-AGE fiber spectrometer. The measurement site is a section with a diameter of 20-30 mm on the medial surface of the skin of the shoulder. The number of measurements is 10 (5 on the left hand, 5 on the right hand), in strictly fixed sections that were selected, in the symmetric zones of the right and left body regions. The duration of one measurement is 0.5 seconds. The output parameter AF was calculated by formula (2) at wavelengths λ _x = 380 nm and λ _m , = 440 nm and averaged over all five measurements on each side.

Example 1. Assessment of the functional status of athletes during exercise

The contingent of the examined was represented by two groups of athletes: 1) athletes-wrestlers (n = 5), 2) athletes-athletes (n = 5). The average age of the subjects was 21 ± 1 year. Measurements of the functional state of athletes were performed in the initial state (February 6-7, 2013), and also after a week (February 14, 2013).

The load execution protocol included the following time periods:

1) Test 1 (velo). It is performed on a bicycle ergometer for 30 seconds at the maximum possible intensity for a given person.

2) Rest 1. It is carried out within 30 minutes after the end of the load "led".

3) Rest 2. It is carried out within 2-4 hours.

4) Test 2 (treadmill). It is carried out on a treadmill for 20-25 minutes with a stepwise increase in speed from about 5 km / h to 15 km / h. The last 10 step lasts as long as the athlete withstands.

5) Rest 3. It is carried out within 30 minutes after the end of the load on the treadmill.

The results of AF measurements in the initial state and at various points in the test tests are presented in FIG. 1. When analyzing the data obtained, the following is noteworthy. After the end of the short-term ultimate load, the effect of “hypercompensation” is observed on the bicycle ergometer, at which the IFS averages 103%. Rest 1 for 30 minutes after the bicycle ergometer leads to the effect of "fatigue", in which there is a drop in the IFS to an average of 96%. Rest 2 for 2-4 hours after the bicycle ergometer is accompanied by almost complete compensation for the effect of "fatigue". Long-term load on the treadmill, which is accompanied by a transition from the aerobic nature of the load to anaerobic, gives a more significant effect of "hypercompensation" (IFS increases to an average of 109% )

Rest 3 for 30 minutes after the treadmill does not lead to the effect of "fatigue" - AF returns to the level that occurred before the load on the treadmill. The individual differences in the effect of "hypercompensation" under the load on the treadmill in this study reached 30%. So, the greatest effect (increase in IFS up to 128%) took place in subject A. (sambo) and was completely absent (IFS 98%) in subject K. (skiing).

The results of measuring IFS one week after the initial testing according to the same scheme are presented in FIG. 2. In this case, the same patterns were observed as in the initial testing.

However, in one of the subjects the reaction was atypical, namely, there was a significant continuous drop in the IFS at all stages of the experiment, especially immediately after performing the bicycle ergometry test. However, his health was poor.

Thus, the results of measurements of IFS with various types of physical activity can be summarized as follows. Firstly, the previously discovered effect of the correlation of skin AF parameters with the physiological state of the body was confirmed on the group of examined athletes. The change in the skin AF during exercise is explained by a change in the degree of its oxygenation. A new phenomenon has been discovered - the effect of "hypercompensation" - which consists in a short-term increase in the degree of oxygenation during the period of the most intense load. It can be assumed that this effect occurs only in well-trained people. To confirm this hypothesis, the response to prolonged exercise in untrained individuals and in people with reduced exercise tolerance should be evaluated. The previously observed effect of "fatigue" is also manifested in athletes, but only after a short-term maximum load.

Example 2. Evaluation of the functional status in healthy subjects and in patients with heart failure during exercise

The load was performed by the test subjects on a treadmill and included movement with an initial track speed of 5 km / h at a lift level of 0 units. (in units of treadmill) for the first 2 minutes. Every next 2 minutes, an increase in load was carried out (speed increases by 1.6 km / h). Work is performed until submaximal heart rate is reached. The following parameters were recorded:

1. VO ₂ peak (ml / min)

2. VO ₂ peak (ml / kg / min)

3. VO ₂ peak (% of maximum values due)

4. The ratio of O ₂ / HRpeak

5. The ratio of O ₂ / HRpeak (% of due maximum values)

6. VE at peak load (l / min)

7. Respiratory reserve (BR),%

8. VE / VCO ₂ rest

9. VE / VCO ₂ at peak load

10. The amount of oxygen absorbed (% of MDA)

The subjects were divided into two groups: 1) practically healthy young people (n = 6) aged 20 to 34 years, 2) patients with cardiomyopathy and heart failure of the II-III functional class (n = 2).

The results of measurements of parameters characterizing the tolerance of the subjects of both groups to physical activity are given in the table. From the table it follows that patients with heart failure were significantly less resistant to stress, which is confirmed by a significantly lower value of peak oxygen consumption.

Table. Exercise tolerance indicators when performing a treadmill test in subjects

The results of measuring IFS in subjects of both groups are presented in FIG. 3.

The response to the load was different in norm and pathology. The degree of change after exercise is different for the right and left hand. In the vast majority, extreme changes were recorded on the right hand and only in one of the cardiological patients on the left hand.

In cardiological patients (dashed curves in Fig. 3), the IFS was 91-97% (cf.), 90-93% (extr.), And in a patient preparing for surgery, the degree of decline was the greatest. In practically healthy subjects, IFS, on the contrary, increases - 103-120% (cf.), 106-125% (extr.).

The correlation between IFS and the amount of oxygen absorbed is shown in FIG. 4. It follows from it that the functional status correlates well with such an indicator as the amount of oxygen absorbed, which makes it possible to consider IFS as a sensitive marker that determines the amount of reserve capacity of the body and, therefore, the maximum achievable sports result.

The above examples of experimental studies show that the dynamics of the skin AF during exercise testing clearly correlates with the reserve capabilities of the body and its tolerance to physical activity. A correlation is observed between the level of AF and most indicators characterizing the body's resistance to physical work. In individuals with reduced exercise tolerance (heart failure), a decrease in IFS was observed, while in practically healthy people and athletes, an increase in IFS was noted. The degree of increase in IFS when performing a standard load test can be a screening method for selecting athletes who have at the given time the maximum indicators of physical disability.

The method of assessing exercise tolerance can be used to monitor not only under normal and extreme conditions of human activity, but also to identify hidden pathology and during its treatment, in order to monitor the effectiveness of various procedures aimed at improving well-being and increasing physical performance. In addition, the method can also be used for rapid assessment of the effectiveness of correction tools. So, for example, repeated load testing with a standard load power before and after application of the functional status correction means can indicate the effectiveness of the correction method used and its safety. Based on these data, an accelerated selection of individual methods, means and doses of correction of the athlete's functional state can be made.

Thus, the claimed invention ensures the achievement of the task, namely, it increases the reliability and reduces the time to assess the functional state of the human body, and also extends the functionality of the method.

Claims (1)

A method for assessing exercise tolerance by measuring the autofluorescence properties of the skin, which includes irradiating the skin with excitation radiation, measuring signals caused by secondary skin radiation using a spectrometer in two spectral regions, of which the first region is selected in the field of excitation and the second in the region NADH fluorescence emission, calculation of the autofluorescence parameter (AF) in accordance with the formula:
AF = F (λ _m ) / R (λ _x ) ^0.85 ,
Where:
R (λ _x ) is the signal obtained from the first spectral region,
F (λ _m ) is a signal obtained from the second spectral region, characterized in that the said measurement of signals is carried out in symmetric zones of the right and left parts of the body in the initial state of the human body, after which a load breakdown is applied to the human body and signal measurements are carried out in those the same areas of the skin, determine the index of functional status (IFS) by the formula:
IFS = AF ₀ / AF _p ,
Where:
AF _{o -} autofluorescence parameter before exposure to stress test,
AF _p is the autofluorescence parameter after exposure to a stress test, and an AF _p value is used that is as different as possible from AF _o , and exercise tolerance is estimated from the obtained IFS value.

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