RU2613910C1 - Method for skeletal muscle and myocardium tissue hypoxia determination during physical inactivity - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for skeletal muscle and myocardium tissue hypoxia determination in case of physical inactivity comprises determination of acetol (hydroxiacetone C₃H₆O₂ GAS116-09-6) in exhaled air by chromatography mass spectrometry, prior to inactivity and during exposure, and in case of authentic reduction of acetol in exhaled air skeletal muscle and myocardium tissue hypoxia is diagnosed during physical inactivity.

EFFECT: method avoids invasive procedures and provides its implementation under different conditions for unlimited number of times and with any duration, that allows to carry out preventive measures against skeletal muscle and myocardium tissue hypoxia development during inactivity.

2 tbl, 2 dwg

Description

The invention relates to medicine, more specifically to space medicine, and can be used to monitor the state of hypoxia of skeletal muscles and myocardium of persons who have been under conditions of physical inactivity for a long time.

Reducing the load on skeletal muscles and myocardium during hypodynamia is accompanied by the development of metabolic changes in the human body and, above all, changes in the energy and plastic support of muscle tissue. The limitation of periods of stress and decay of macroergs in muscle tissue and myocardium during hypodynamia is accompanied by a violation of the neuroendocrine regulation of energy processes of biological oxidation (decrease in the rate of synthesis of adenosine triphosphate (ATP)), due to a decrease in the degree of conjugation of oxidative phosphorylation, structural disorders, as well as a decrease in the activity of enzymes that regulate synthetic processes in the organs [1, 2]. Studies of gas exchange at rest and during the exercise of physical activity during prolonged (520 days in the MARS-500 program) limitation of motor activity showed that ventilation and gas exchange efficiency, during the entire exposure period, were within the physiological norm of a healthy person [3] . Apparently, with a long-term restriction of motor activity, energy expenditure in the body is covered mainly by catabolic processes in tissues due to microcirculation disorders [1, 2]. So, in [4, 5], a decrease in the intensity of cellular respiration during gravitational unloading (up to 14 days) with a disruption of the processes in the respiratory chain of mitochondria and a decrease in the activity of cytochrome oxidase in the soleus muscle [4], as well as the intensity of cellular respiration in the cardiomyocytes of the left ventricle of the heart, was shown rats on the 3rd day of rehabilitation [5]. A decrease in muscle mass with a prolonged limitation of human motor activity in model experiments (370 days) and in astronauts with different durations of flights was accompanied by the development of negative nitrogen balance, a slowdown in nucleic acid metabolism, increased excretion of urea, uric acid with urine, due to a slowdown in the synthesis of energy-rich phosphorus compounds and tissue respiration [1, 2, 6].

Moreover, a decrease in the muscle mass quota in humans with prolonged (370 days) inactivity occurred mainly in the most functionally active groups of skeletal muscles and in the myocardium [1, 2]. Carbohydrate metabolism in modeling long-term inactivity was characterized by inhibition of aerobic glycolysis processes with a decrease in ATP production and an accumulation of lactic and pyruvic acids. Confirmation of the predominance of the anaerobic phase of glycolysis is an increase in the activity of lactate dehydrogenase (LDH) and the conversion of pyruvic acid into lactate for reoxidation of reduced nicotinamide adenine dinucleotide (NADH) and the oxidized form of NAD ⁺ nicotinamide adenine dinucleotide with decreased activity of muscular tissue and muscular tissue (muscle) enzymes hypodynamia [1, 2]. The process of stressful mobilization of the body during adaptation to long (370 days) inactivity was accompanied by a decrease in the function of enzymatic systems, a weakening of the processes of glycolysis and enzymatic catalysis in cells, and a uniform decrease in the activity of enzymes in energy homeostasis [1]. The dynamics of creatine phosphokinase (CPK) reflected a decrease in the activity of muscle creatine phosphokinase (CPK-MM) and, to a lesser extent, myocardial creatine phosphokinase (CPK MB) [1]. At the same time, a study of the enzyme spectrum during long-term (370 days) inactivity showed that as the duration of exposure increases, there is an increase in changes in the myocardial energy exchange [1]. Based on the literature data characterizing metabolic manifestations during prolonged physical inactivity [1, 2, 6], in order to verify the information content of acetol as a biomarker of hypoxia in skeletal muscles and myocardium, the following biochemical parameters were observed: the activity of the enzyme energy homeostasis (CPK) and its isoenzymes: muscle MM-KFK and myocardial MV-KFK, the activity of dehydrogenase glycolysis of lactate dehydrogenase (LDH), glucose and an indicator of a decrease in the functional load on skeletal muscle - creatinine [1, 7, 8]. When choosing volatile organic compounds (VOCs) for analysis, the data presented in [9, 10, 11, 12, 13] were used as biomarkers of hypoxia.

As a prototype of the claimed method for determining tissue hypoxia of skeletal muscles and myocardium in humans with physical inactivity, the method described in [1] was selected, in which the determination of tissue hypoxia was carried out by modeling long-term (370 days) inactivity in ground-based model experiments and in astronauts under zero gravity . In the prototype, hypoxia in skeletal muscles and myocardium was determined by the dynamics of biochemical parameters characterizing aerobic oxidation of carbohydrates, the activity of energy enzymes specific for the analysis of the aerobic type of energy metabolism and the activity of enzymes of energy homeostasis: total creatine kinase (CPK) and its muscle (MM KK) and myocardial (MB CC) isoenzymes, the decrease in activity of which is observed with a decrease in muscle mass in humans and a sedentary lifestyle (inactivity).

The disadvantage of the selected prototype is the need for venous blood sampling from subjects for analysis, i.e. the need for invasive intervention.

The technical result of the claimed method is that the proposed method eliminates invasive intervention, which allows the method to be carried out in a variety of conditions, in unlimited dynamics, with the frequency necessary to assess the adverse effects of physical inactivity and the effectiveness of preventive measures.

This technical result is achieved by the fact that in the known method for determining tissue hypoxia of skeletal muscles and myocardium in case of physical inactivity, including analysis of molecular metabolites of tissue hypoxia in a test subject, acetol (hydroxyacetone C3H6O2) is determined as a molecular metabolite in the air exhaled by the test subject. GAS116-09-6), before hypodynamic onset and during its exposure and with a significant decrease in acetol in exhaled air, tissue hypoxia of skeletal muscles and myocardium is diagnosed inactivity.

The method is as follows.

1. Sampling

Samples of exhaled air from the subjects were taken in bags of neutral polymeric material, a volume of 5 l under the control of the CO ₂ "straw" method. CO ₂ control was performed on an Oxycon Pro complex from Jaeger-VITASYS (Germany), which has a high-speed CO ₂ infrared analyzer [4]. The exhaled air was taken without inhalation with an extended exhalation; therefore, the obtained air samples contained mainly alveolar gas corresponding to FET _CO2 — the content of CO ₂ (%) in the final portion of exhaled air. The samples were concentrated in sorption tubes containing 180 mg of Tepah TA sorbent, pumping 1 liter of exhaled air through the sorbent using a Supelco pump. Samples were introduced into a gas chromatograph-mass spectrometer (GC-MS) through a TDS3 thermal desorber (Gerstel, Germany). During desorption, the carrier gas flow was 50 ml / min, the temperature rose from an initial temperature of 25 ° C to 250 ° C at a speed of 60 ° C / min (the final temperature was kept for 4 minutes in the non-dividing mode). The desorbed substances were concentrated by cryofocusing at -30 ° C with liquid nitrogen in a CIS-4 injector containing a liner filled with Tenax TA. The introduction of the sample directly into the column began by heating the CIS-4 injector at a rate of 12 ° C / second to 250 ° C (the final temperature was delayed for 3 minutes in the non-dividing mode). GC-MS analysis was performed on a 6890N gas chromatograph with a 5973N mass-selective detector (Agilent Technologies) operating at an ionization energy of 70 eV. Chromatographic separation was performed on a DB-VRX 60 m × 0.25 mm × 1.4 μm capillary column (Agilent Technologies) at a temperature program: the initial temperature was maintained at 45 ° C for 15 minutes, then heated to 190 ° C at a rate of 8 ° C / min, holding 2 minutes, then heating to 250 ° C at a speed of 8 ° C / min and holding 1 min at the final temperature. The flow of helium carrier gas was constant at 1.0 ml / min. The mass spectrometer was in TIC mode with a range of m / z from 20 to 350. Chromatographic data were processed through the Agilent Chemstation Software.

2. Identification of detected compounds

Identification of the detected compounds was performed using the NIST mass spectra library. In addition, retention times obtained from the analysis of calibration mixtures were used for identification. Standard gas mixtures were prepared by evaporation of liquid substances in a 5 L teflon bag. The bag was previously purged with pure nitrogen (99.999%), followed by evacuation and repeating the operation 10 times. Then the bag was filled with 3 liters of nitrogen. Liquid standards (a few microliters depending on the desired concentration) were introduced into the bag using a liquid chromatography syringe (Agilent Technologies). After evaporation of the standards, the bag was additionally filled with 2 liters of nitrogen. Standard samples were taken and analyzed in accordance with the procedure described above.

3. Detected compounds

Biochemical studies included: determination of the activity of enzymes of energy homeostasis (CPK) and its isoenzymes: muscle CPK MM and myocardial CPK MB, glycolysis activity was evaluated by the dynamics of lactate dehydrogenase activity (LDH), as well as glucose concentration. The functional activity of skeletal muscles was evaluated by the dynamics of creatinine concentration in the blood. Biochemical studies were carried out using commercial kits of reagents manufactured by DiaSys, Germany. The measurements were carried out on a Targa 3000 biochemical analyzer, manufactured by Biotecnica Instruments SPA, Italy. Venous blood was taken in the morning, on an empty stomach 7 days before the start of the tests, when the volunteers were isolated in an airtight room and on the 7th day during the recovery period (VP). When exposed to “dry” immersion, blood samples were taken before exposure on days 3 and 7 of exposure, and on day 7 of RP.

4. Statistical data processing was performed in the Statistica 8.0 package (StatSoft, Inc). Due to the small number of subjects, the dynamics of VOCs in exhaled air (EX) and biochemical blood parameters during the experiment were investigated using nonparametric methods. Comparisons between series of measurements were performed using the Wilcoxon test. The differences were considered statistically significant at p <0.05. [14, 15].

Research results

To conduct research with prolonged isolation of a person in sealed rooms, permission was obtained from the bioethical commission of the Institute of Biomedical Problems of the Russian Academy of Sciences.

The study involved 11 healthy men aged 21-38 years, who underwent clinical examination and admitted to the research by the medical commission. The studies were conducted at the experimental base of the Institute of Biomedical Problems of the Russian Academy of Sciences. The modeling of physical inactivity included 2 series of studies: the 1st series (11 people, duration 105 days) and the 2nd 520-day series ("Mars-500"). The isolation of the examined was carried out in an airtight room with a volume of 550 m, equipped with autonomous life support systems. The daily routine during prolonged physical inactivity included an 8-hour night's sleep, three meals a day (standard diet with a calorie content of 2600-2800 kcal), medical monitoring and controlled optimal environmental conditions (O ₂ - 20-22.5%, CO ₂ - 0.03 %, temperature - 21-23 ° С, humidity 60-70%).

Exhaled air sampling (1 series) was carried out 1-3 days before planting in the heat chamber, during the isolation period monthly (1 time) and 3 days after the subjects examined from the heat chamber — the rehabilitation period (RP). Exhaled air samples were taken before exercise (at rest), immediately after exercise, and 1 hour after dosed work on a bicycle ergometer in RP. During physical activity, the examined performed pedaling at an average pace (60 ± 5 rpm), increasing the load power by 15 W every minute (initial load - 30 W). The load power of the last stage for each tester was selected individually in accordance with training and ranged from 150 to 210 watts. At the same time, air samples were taken in the pressure chamber and the room (immersion stand) and their analysis according to ISO 16000-6: 2004. Up to 120 volatile organic compounds, including saturated normal and branched hydrocarbons, their oxygen-containing derivatives (alcohols) were identified in the exhaled air of practically healthy patients. , ketones, aldehydes, acids), unsaturated hydrocarbons (mainly isoprene), aromatic hydrocarbons.

VOCs were selected from the list of detected compounds, in the concentration of which the dynamics was determined as the duration of inactivity increased (Table 1).

Acetol, isoprene, and acetone were chosen as potential biomarkers of hypoxia. The basis for choosing VOCs was their formation as intermediate metabolites of lipid and carbohydrate metabolism, providing energy consumption of skeletal muscles and myocardium at rest and under other physiological conditions [8]. It was found that a change in the concentration of isoprene and acetone is uninformative. The decrease in the acetol content in the exhaled breath of the examined was significant (P = 0.0002) during all periods of exposure to physical inactivity with a maximum effect of 500 days of exposure (Fig. 1). It is important to note that, in contrast to the dynamics of isoprene and acetone, the concentration of exhaled acetol in RP remained significantly lower (P = 0.002) than the value observed before exposure to physical inactivity. This significant decrease in the content of exhaled acetol with an increase in the duration of inactivity allowed us to choose acetol as the most likely biomarker of tissue hypoxia and to conduct a comparative analysis with changes in the biochemical parameters of energy metabolism.

Acetol as a metabolite of methylglyoxal in an alternative pathway of carbohydrate metabolism is a substrate for the synthesis of lactate and allows you to bypass the stages of glycolysis, which go with a low yield of ATP.

When hypoxia of various genesis occurs, including during intense physical work, the pool of restored NAD in the body is depleted, the cofactor is consumed only in mitochondria, and the electron flux into transhydrase reactions is inhibited. Accordingly, the formation of acetol from methylglyoxal in the methylglyoxal metabolic pathway is inhibited [7, 16].

The results of biochemical blood parameters examined with a prolonged restriction of motor activity are presented in table. 2.

Thus, the intensification of catabolic processes during skeletal muscle “underloading” was accompanied by a decrease in the activity of enzymes of energy homeostasis (CPK), more pronounced in skeletal muscle (CPK MM), starting from 168 days of exposure (Table 2). A decrease in acetol (P = 0.0002) in the exhaled breath of the examined was observed from 44 days of inactivity to 500 days, the marker content, compared with the value before the exposure to the factor, decreased significantly (Fig. 1). The activity of myocardial creatine phosphokinase (CPK MB) changed in a wave-like fashion with alternating periods of increase and decrease in enzyme activity (Table 2). An increase in the activity of the myocardial enzyme KFK MB during the period of the body's adaptation stress to physical inactivity (up to 60 days) may be due to both the biological activation of the adaptation processes in the myocardium (stabilization or increase in cellular respiration), and with the processes of energy production in skeletal muscles, because approximately 10% of creatine phosphokinase activity in muscle tissue is represented by KFK MB (7, 8). Confirmation of the information content of acetol as a biomarker of the intensity of the process of oxidative phosphorylation with a decrease in the functional load on skeletal muscles and myocardium is the unidirectional changes in acetol and creatinine in the blood as the duration of exposure to physical inactivity increases (Fig. 2).

An increase in the activity of bioenergetic processes in skeletal muscles and in the myocardium during the rehabilitation period (Table 2) was not accompanied by a significant increase in acetol in the exhaled breath of the examined patients, indicating the preservation of reduced energy production in muscle tissue, which is consistent with the lack of creatinine dynamics in the blood (Fig. 2).

To clarify the information content of acetol, as a marker of tissue hypoxia, tests were performed with physical activity, modifying the metabolism of carbohydrates in conditions of physical inactivity. An analysis of the data showed that physical work, restoring the processes of energy exchange, reduces the manifestations of tissue hypoxia, increasing the content of acetol in exhaled air. However, the general direction of reducing exhaled acetol with increasing duration of inactivity both at rest and during physical work does not fundamentally change. So, as the effect of muscle “underloading” increases, the increase in the acetol content, in response to physical work, gradually lengthens and significantly decreases by 500 days compared with the value before exposure. Activation of carbohydrate metabolism and an increase in energy production in skeletal muscles and myocardium on the 3rd day after the end of exposure to physical inactivity (Table 2) was accompanied by a tendency to an increase in the concentration of exhaled acetol at rest and during exercise in the exhaled air of the examined before, after and during exercise conditions of prolonged restriction of motor activity). However, the increase in the marker content in the exhaled air of those examined during exercise in PR did not reach the values observed before exposure to physical inactivity.

Apparently, an alternative way of energy production is not able to compensate for a long time the insufficiency of aerobic and anaerobic oxidation in the energy supply of tissues with a long-term restriction of human motor activity.

Thus, the research results showed that the dynamics of the acetol content in exhaled air during prolonged physical inactivity is consistent with a change in the activity of energy metabolism enzymes and can be considered as an indicator of tissue hypoxia. The unidirectional increase in acetol in exhaled air and the activity of CPK MB and MM in the blood after the end of the action of physical inactivity in RP suggests that the dynamics of acetol can be a reflection of metabolic changes in both skeletal muscle and myocardium.

Brief description of tables and drawings

Tab. 1. The main VOC metabolites identified in the exhaled air of a healthy person with a prolonged restriction of motor activity.

Tab. 2. A change in the enzyme spectrum of blood serum while limiting motor activity was characterized by periods of increase and decrease in the activity of energy enzymes and an indicator of the functional load on skeletal muscle - creatinine.

In the table. 2 the following notation is used

- * Background - before exposure to physical inactivity;

- ** from 274 days - prevention (physical activity on a bicycle ergometer according to an individual scheme for volunteers)

- RP - rehabilitation period

FIG. 1. Dynamics of the acetol biomarker in the expired air of the examined with a prolonged restriction of motor activity.

FIG. 2. Changes in acetol and creatinine in the blood as the duration of exposure to physical inactivity increases.

In FIG. 2, dashed line indicates acetol, the solid line is creatinine.

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Claims (1)

A method for determining tissue hypoxia of skeletal muscles and myocardium in case of physical inactivity, including analysis of tissue hypoxia in the subject’s molecular metabolites, characterized in that acetol (hydroxyacetone C3H6O2 GAS116-09-6 is determined by chromatography-mass spectrometry) ), before the onset of hypodynamia and during its exposure and with a significant decrease in acetol in the exhaled air, tissue hypoxia of skeletal muscles and myocardium is diagnosed with hypodynamia.

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