RU2503003C1 - Method for prediction of progression of bronchial obstruction in patients suffering chronic obstructive pulmonary disease - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for prediction of the development of chronic obstructive pulmonary disease involves pre- and post-exercise blood sampling to determine a mitochondrial membrane potential index that is a COPD progression risk factor. If the mitochondrial membrane potential index is 1.40 standard units or more, a favourable course of COPD is predicted, while the mitochondrial membrane potential index of 1.20 standard units or less shows an unfavourable course of COPD.

EFFECT: diagnostic technique is reliable, informative and quick; it promotes both assessing the severity, and predicting the potential progression of chronic obstructive pulmonary disease that is necessary for giving the differentiated, forehanded and high-quality treatment.

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Description

The invention relates to medicine, namely to pulmonology, and can be used to accelerate the prognosis of the progression of disorders of bronchial conduction in terms of the membrane potential of mitochondria in platelets.

Chronic obstructive pulmonary disease (COPD) is a lung disease characterized by persistent impairment of airflow from the lungs. COPD is an insufficiently diagnosed, life-threatening lung disease that interferes with normal breathing and is completely incurable. Currently, COPD affects more than 11 million able-bodied people in Russia. According to WHO, COPD is third in the list of causes of death on our planet [1]. 2.8 million people die of COPD every year in the world [2].

In all industrialized countries, COPD is a major medical and social problem, since the development of this disease is determined by the adverse effects of natural and man-made factors and the work environment [3].

The life expectancy of people with COPD does not exceed an average of 6 years, which is 3.5 times less than with bronchial asthma. Disability occurs on average 10 years after diagnosis, which necessitates early diagnosis of disorders of bronchial conduction and the prognosis of further aggravation of the disease for timely prevention or adequate treatment [4].

Thus, the prevalence, significant economic damage caused by temporary disability, early disability and premature mortality of patients with COPD determine the urgency of the problem and the need to develop a timely forecast of worsening bronchial obstruction in order to prevent its progression and full rehabilitation of patients.

Currently, the Global Initiative for COPD to assess respiratory function and determine bronchial obstructive or restrictive pathology of the lungs, it is recommended to use spirography, which evaluates the existing violations of bronchial patency at the moment and does not allow to predict the further course and progression of COPD [5]. To assess the deterioration of lung function is possible only with repeated spirography after 6 months or more, when time is lost to prevent progression and timely treatment is not prescribed.

In recent years, scientists believe that the main mechanism for the appearance of dyspnea in patients with exercise is hyperinflation (cell hypoxia), which develops already in the early stages of COPD [6]. In general, gas exchange worsens as the disease progresses. Obstruction of the peripheral bronchi leads to a violation of the ventilation-perfusion ratio (VA / Q); in severe illness, obstruction is combined with impaired respiratory muscle function, which helps to reduce ventilation, followed by a delay in the blood of carbon dioxide. Violation of alveolar ventilation and a decrease in pulmonary blood flow lead to further progression of VA / Q imbalance. Such an obstruction of the peripheral airways increasingly impedes the release of air from the lungs into the expiratory phase (the so-called air traps), and as a result hyperinflation develops. Hyperinflation leads to a decrease in inspiratory capacity, especially during physical exertion (in this case, they speak of dynamic hyperinflation), as a result, shortness of breath and limitation of exercise tolerance appear. A longer stay in conditions of reduced oxygen content is accompanied by a transition to a new level of regulation of oxygen homeostasis, which is characterized by the economy of energy metabolism and the development of hypoxia. As hypoxia develops, a gradual damage to the elements of the intracellular respiratory chain, a decrease in the oxygen supply to the cells, is observed.

It is known that adequate provision of cells with oxygen is the main condition for maintaining its viability.

Impaired respiration at the cell level is associated with a change in the membrane potential of mitochondria (MPM), which is expressed in the regulation of ion transport in the direction of individual flows of substrates and metabolites. Considering that the membrane potential of mitochondria is a multifunctional regulator of the activity of enzyme proteins localized in the membrane, an indicator of the hormonal effect at the cell level [7], the assessment of the potential of a living cell is one of the most appropriate methods for studying its functional and energy state.

Mitochondria can be isolated from different biological material by standard methods. Platelets were used as a living cell, which generally characterize a wide range of disturbances in biological processes [8, 9]. In this regard, not only platelets are studied, but also others, including peripheral tissues and blood cells. Thus, changes in the functions of mitochondria were detected in blood platelets [10], in addition, a violation of the function of glutamate transport with a decrease in the rate of 3H-glutamate uptake was detected in platelets compared with healthy people and patients suffering from chronic neurological diseases [8]. All this suggests that in the blood of patients there are factors that cause platelet activation.

Mitochondrial membrane potential - the potential difference between the mitochondrial matrix and the intracellular medium is many times higher than the potential of the plasma membrane. Mitochondria are unique cellular organelles capable of creating a transmembrane electric potential reaching 200 mV, while the charge inside the mitochondria is negative and the pH value of the internal space is about 8. As a result, mitochondria are able to accumulate penetrating components of a cationic nature, as well as weak acids in their anionic form [eleven].

Mitochondrial membrane potential can change under the influence of many intracellular biochemical processes, as well as external stimuli [12, 13]. Therefore, the determination of the membrane potential of mitochondria makes it possible to assess the potential of a living cell, which is one of the most adequate methods for studying its functional state.

A known method for predicting frequent respiratory diseases in children, based on the study of the immunogenetic status of the body. Moreover, the presence of major histocompatibility complexes (HLA - antigens), namely A3, B7, B12, Cw6, is detected in the blood. In the presence of one of the listed antigens or their combination, the possibility of frequent respiratory diseases in a preschool child is predicted [14]. The disadvantage of this method is that it can be used only in children, does not predict the course of COPD in patients already suffering from this disease, and does not allow one to evaluate how quickly irreversible bronchial obstruction will progress in each individual patient.

A known method for the diagnosis of chronic obstructive pulmonary disease by examining the fluid of bronchoalveolar lavage fluid (BALF) allows, based on the determination of the relative number of neutrophils and alveolar macrophages, the assessment of their functional activity, to establish the presence and degree of activity of inflammation in the lungs, to identify a tendency to progression of the process [15]. The method allows to differentiate the disease according to the degree of activity of the inflammatory process in the lungs. However, the known method does not allow to diagnose the oxalate form of chronic obstructive pulmonary disease.

A known method for the diagnosis of inflammatory processes of the bronchopulmonary system, including the analysis of body parameters using instrumental methods of research, the determination of significant indicators, their analysis and diagnosis of the patient's condition, according to which, chemiluminescence, spectrophotometry and rocket electrophoresis are used as instrumental methods, as significant indicators determine spontaneous and induced chemiluminescence, the level of antioxidant activity of blood plasma, contains malon dialdehyde, levels of α ₁ -inhibitor of proteinases and elastase, in the course of analysis of indicators determine the average similar indicators in healthy people and determine the integral indicator K by the formula:

K = Ax / Ak + Bx / Bk + Cx / Ck, where

A is the ratio of the magnitude of the induced and spontaneous chemiluminescence;

B is the ratio of the level of antioxidant activity of blood plasma to the content of malondialdehyde;

C is the ratio of the level of α ₁ proteinase inhibitor and elastase;

x - values of the coefficients of the examined patient;

to - the average value of the coefficients is normal.

In the presence of an integral indicator of K values of less than 125, the inflammatory process in the bronchopulmonary system is diagnosed. When the integral indicator value K = 110-120 and the induced chemiluminescence value of 15-25 mV, chronic obstructive pulmonary disease is diagnosed as an inflammatory process [16]. The disadvantage of this method is the complexity and the need to use expensive equipment.

Despite the patent studies, a direct prototype of the prior art has not been identified.

As mentioned above, respiratory failure at the cell level is associated with a change in the membrane potential of mitochondria, which can be used to judge its functional and energy state.

According to the authors of this application, the determination of the membrane potential of mitochondria makes it possible to assess the potential of a living cell in the study of its functional state in patients with COPD disease at different stages; the magnitude of the values expressing the change in the membrane potential of mitochondria can serve as an estimated indicator for predicting the course of the disease.

The objective of the invention is to create a new reliable method for predicting the progression of disorders of bronchial obstruction in patients with chronic obstructive pulmonary disease by identifying differences in the levels of the membrane potential of platelet mitochondria at different stages of the disease.

The problem is solved as follows: a method for predicting the progression of bronchial obstruction in patients with chronic obstructive pulmonary disease includes taking a patient’s blood samples “before” and “after” a stress test, extracting platelets from samples, followed by isolation of mitochondria from them, processing mitochondrial samples with Safranin dye O and spectrophotometric measurement in samples of optical density at wavelengths of 523 nm and 555 nm, with subsequent calculation, according to the Nernst equation, of the meme mitochondrial potential, then the mitochondrial membrane potential index is determined by which the risk of COPD progression is predicted. So, if the index value of the membrane potential of mitochondria is more than or equal to 1.40 conventional units, a favorable course of COPD is predicted, and when the value of the index of the membrane potential of mitochondria is less than or equal to 1.20 conventional units, an unfavorable course of COPD is predicted.

The calculated unit of the membrane potential index of mitochondria is designated as a conventional unit (cu).

A lack of oxygen (hypoxia) causes a decrease in the energy activity of mitochondria, which is due to the regulation of ion transport in the direction of individual flows of substrates and metabolites, expressed in terms of the value of the mitochondrial potential. The dynamics of these changes is characterized by the mitochondrial membrane potential index (IMPM) before and after the load test (IMPM = MPM before the load / MPM after the load).

The technical result of the invention is that a new reliable and inexpensive way to predict the development of obstructive pulmonary disease; the invention helps to predict the adverse course of the disease in time and prescribe treatment in a timely manner.

The inventive method is developed and tested experimentally in the clinic of the Vladivostok branch of FSBI "NTs FPD" SB RAMS-NII MKVL.

Study participants were divided into groups: a control group and a group with COPD. Patients with allergic, autoimmune, and chronic infectious diseases were excluded from the observation groups. The control group consisted of 20 people who did not have chronic bronchopulmonary pathology and allergic diseases, non-smokers, with normal respiratory function, confirmed by appropriate clinical and laboratory examination methods.

A group with diagnosed bronchopulmonary pathology - COPD was 48 people. Of these, 19 people had a mild degree of impairment of the function of external respiration, 17 people had an average degree of impairment of the internal respiratory function, 12 people had severe impairment of the function of external respiration.

For each patient, bronchial patency was determined and the corresponding membrane potential of platelet mitochondria was measured, and then the index of the membrane potential of platelet mitochondria was calculated.

The proposed method is based on the functional activity of the development of hypoxia caused by submaximal exercise breakdown - physical activity (FN) [17]. To determine exercise tolerance, a six-minute step test (6-MCT) was used, which in pulmonological practice is one of the methods for assessing the functional state of the respiratory system of the body. It is carried out indoors, in a straight corridor, convenient for walking, 30 m long. Before the test, the patient rests for 10 minutes, the heart rate (HR) is calculated. The criterion for the stress test is the submaximal heart rate, which is calculated by the formula of G.M. Yakovleva [18]: Submaximal heart rate = heart rate at rest + K × (215 - age - heart rate at rest), where K is the correction coefficient, it is 0.9 for athletes, 0.8 for healthy, 0.7 for patients, 0.6 for myocardial infarction.

The test is performed at maximum walking speed for 6-12 minutes.

The mitochondrial membrane potential of platelets is determined using the dye Safranin O, which can penetrate the mitochondria, due to the dye having a positive charge.

Biomaterial: blood with an anticoagulant (EDTA - 0.1 M 0.02 ml / ml blood) 8-12 ml. The time from blood sampling to the moment of cell isolation should be no more than 1 hour.

Bronchial patency was assessed by the indicators of forced expiratory volume for 1 second (FEV ₁ ) and forced vital lung capacity (FVC), FEV index ₁ / FVC. FEV ₁ <80% and FEV ₁ / FVC <70% were taken for bronchial obstruction.

Among patients with COPD, 19 people had a mild degree of impaired external respiration (VFD) - FEV, 79.6 ± 2.1% and FEV ₁ / FVC 77.6 ± 1.5%, the average duration of the disease was 9.7 ± 2.3 years, average age 56 ± 1.2 years. FEV, 58.2 ± 4.1% and FEV ₁ / FVC 66.2 ± 2.1%, corresponding to an average degree of HFV disorders, had 17 patients, middle-aged 59.2 ± 2.9 years with a disease duration of 12 , 8 ± 3.1 years. The remaining 12 patients, middle-aged 62.8 ± 4.1 years, had an average disease duration of 18.2 ± 2.5 years and severe impaired respiratory function: FEV1 43.9 ± 2.1%, FEV ₁ / FVC 57.3 ± 2.1% of due.

The method is as follows.

Blood is taken from the ulnar vein into sterile silicone tubes with an anticoagulant (EDTA - 0.1 M 0.02 ml / ml blood). Before analysis, the blood tube is placed in rotamix and gently mixed for 5 minutes.

Then the blood is centrifuged for 30 minutes at 1000 rpm. The result is a separation of blood into 3 layers: red blood cells, white blood cell film and plasma. 60% of platelets are in the plasma layer, and 40% are in the leukemic film and the upper layer of red blood cells. Plasma and leuko film are collected in a dry tube and centrifuged for 30 minutes at 1600 rpm. In this case, two layers are formed: a platelet-rich plasma and a precipitate containing red blood cells, white blood cells and up to 40% of platelets (residual platelets). Platelet-rich plasma is collected in a test tube and centrifuged for 30 minutes at 3200 rpm. The supernatant is removed. Sediment is platelets [19, 20]. 5 ml of isolation medium [21] is added to the platelet pellet and centrifuged for 10 minutes at 4 ° C at 1500 rpm. The supernatant is collected in another tube, the pellet is discarded. The supernatant is centrifuged for 10 min at 1500 rpm to precipitate the destroyed residues. The supernatant was collected in eppendorfs and centrifuged for 15 minutes at 14,000 rpm to precipitate mitochondria.

The supernatant is discarded. The precipitate was resuspended with 1 ml of isolation medium [21] and centrifuged again at 14,000 rpm for 15 minutes. The supernatant is removed. Sediment is a ready-made suspension of mitochondria.

The level of membrane potential of mitochondria is determined using the dye Safranin O [22]. For this, 1 ml of dye medium is added to the precipitate in eppendorf and incubated for 20-30 minutes at a temperature of 0-4 ° C. Then the cells are washed twice with phosphate buffer.

The measurements are carried out in flat-bottomed 96-well plates with a volume of 400 μl on a spectrophotometer at wavelengths of 523 nm and 555 nm [23]. The indicator of the mitochondrial membrane potential of cells is calculated according to the Nernst equation [24].

As a result of the analysis of laboratory parameters in the examined patients with COPD, IMPM was determined depending on the disturbances of bronchial patency in comparison with the control group. As follows from the table, the mitochondrial membrane potential index was different in patients with COPD with varying degrees of bronchial patency.

Table The dependence of bronchial patency on the index of the membrane potential of mitochondria in platelets Indices of bronchial patency Indicators of the membrane potential of mitochondria in platelets (MPM), mV The mitochondrial membrane potential index (IMPM), cu FEV ₁ FEV ₁ / FVC to load after exercise MMM before exercise / MMM after exercise one 2 3 four 5

Table continuation one 2 3 four 5 ≥100% (healthy) ≥80% (healthy) 188 ± 0.075 129 ± 1.18 1.46 ± 0.01 ≥80% of due <70% of due 183 ± 1.28 ** 128 ± 1.10 1.43 ± 0.01 182 ± 1.51 ** *** 157 ± 2.51 *** *** 1.16 ± 0.02 *** <80%, but ≥50% of due <70% of due 172 ± 1.67 *** 119 ± 1.79 *** 1.42 ± 0.02 * 169 ± 1.35 *** *** 150 ± 2,30 *** *** 1.13 ± 0.01 *** <50% <70% of due 164 ± 0.57 *** *** 152 ± 2.08 *** *** 1.08 ± 0.01 *** Note Asterisks indicate the reliability of indicators in patients with impaired respiratory function to the right of the control group and to the left within the group (* - p <0.05; ** - p <0.01; *** - p <0.001).

In the control group, the MMM was 188 ± 0.075 mV, which corresponds to the values previously described in the literature [11]. As a result of the studies, it was found that in the control group the value of the membrane potential after physical activity decreases on average by 28-35% and the values of IMPM were above 1.4 cu

As a result of the analysis of laboratory parameters in patients with COPD, various variants of combining the values of the membrane potential (MPM) and the index of the ratio of the values of mitochondrial potential (IMPM) before and after exercise were revealed. In patients with FEV ₁ ≥80% (mild severity), the MPM level was lower than the control group, averaging 183 ± 1.28 mV, which characterizes a decrease in cell energy activity. Lack of oxygen (hypoxia) causes a decrease in the membrane potential of mitochondria (after exercise), which is expressed in the index of the ratio of mitochondrial potential (IMPM) before and after the load test. The study showed that in 79% of the examined (14 people) COPD (mild severity) IMPM was higher than 1.42 ± 0.01 cu, and in 21% (5 people) IMPM was significantly lower than 1.16 ± 0 , 02 cu and did not exceed 1.17 cu

In patients with FEV ₁ <80%, but ≥50% of the proper, the mitochondrial membrane potential continued to decrease compared to the control group and had a value of 172 ± 1.67 mV, which characterizes the average degree of bronchial obstruction. Against the background of the same MPM values after a stress test, 70% (12 people) had an IMPM lower than the control group and did not exceed 1.14 cu In patients with FEV ₁ <50% of the due, i.e. having severe violations of bronchial patency, a decrease in UTI (164 ± 0.57 mV) was noteworthy, and in 100% (5 people) a decrease in UTI (1.08 ± 0.01 cu). Patients with severe bronchial obstruction have no oxygen consumption adequate to their severity (MPM), and pathological UTI is present. This is an unfavorable combination of factors, on the one hand contributing to the recurrent course of chronic obstructive pulmonary disease, and on the other hand, indicating the formation of mitochondrial dysfunction as a factor in the development of pathological conditions (hypoxia, ischemia, oxidative and nitrosating stress).

During the year, all subjects with COPD were monitored.

All patients underwent a functional examination of external respiration and laboratory research methods of IMPM three times during the year. As a result of dynamic observation in patients with mild COPD with UTI, ≥1.40 c.u. violation of the function of external respiration (ATS) was detected: FEV ₁ was 79.8 ± 2.4% and FEV ₁ / FVC - 78.3 ± 1.4%, which corresponds to stable remission. In patients with mild COPD with IMPI ≤1.21 c.u. disease progression was observed, which was reflected in the indices of FEV ₁ (56.4 ± 2.8%) and FEV ₁ / FVC (64.8 ± 3.0%), which corresponded to the average degree of impairment of FVD. In patients with moderate severity of COPD with UTI, ≥1.42 c.u. during the year, there was no change in FVD, and FEV ₁ was 79.8 ± 2.4%, FEV ₁ / FVC 68.3 ± 1.4%, which corresponds to stable remission. In patients with an initially low rate of IMPM (≥1.14 c.u.), moderate severity of COPD during the year, violations of bronchial obstruction of FEV ₁ <50% of the due, i.e. severe violations of bronchial obstruction, a decrease in IMPI to 1.05 ± 0.01 cu was also noteworthy, which is an unfavorable combination of factors contributing to the sharp progression of the disease.

In conditions when patients with severely impaired bronchial obstruction lack adequate oxygen consumption, UTI reflects the severity of the disease and characterizes, on the one hand, the recurrent course of chronic inflammation, and on the other hand, phased shutdown of individual respiratory chain complexes under hypoxia with a sequential decrease in the mating functions of mitochondria. The value of IMPM characterizes mitochondrial swelling, which is accompanied by an increase in the pore size of the cell cytoplasm and the movement of various substrates and low molecular weight proteins, including cytochrome C, from the cell matrix into the intercellular space. As the membrane potential decreases, cytochrome C molecules begin to leave the membrane surface, as a result, the cell loses its substrate. Respiratory activity in this case is completely suppressed and the cell dies. This process is accompanied by the replacement of normal lung tissue with fibrous tissue, i.e. the constant progression of irreversible bronchial obstruction.

Thus, the dynamics of changes in IMPI can serve as a criterion for assessing the development of the disease, and allows you to predict the risk of progression of COPD, so with IMPI greater than or equal to 1.40.e. - predict a favorable course of COPD, i.e. the decrease in FEV _{1 is} not more than 50 ml per year, and with UTI less than or equal to 1.20 cu - predict an unfavorable course of COPD with a decrease in FEV _{1 of} more than 50 ml per year. A comparison of the results fully confirmed the reliability of the proposed method for predicting the progression of bronchial obstruction. A decrease in the level of the mitochondrial membrane potential index in patients with COPD can be considered as factors in the progression of the disease.

The findings were verified through dynamic follow-up of patients with COPD over the course of a year. In patients with UTI less than or equal to 1.20 c.u. at values unfavorable for this disease, there was a constant deterioration in the function of external respiration.

The invention allows at an earlier date from the onset of the disease to establish the possibility of disease progression (to predict the dynamics of the development of the disease) and proceed to treatment.

Example. Patient V., 52 years old, was admitted to the IMKVL SB RAMS clinic with complaints of a productive cough with a small amount of mucous sputum, shortness of breath of a mixed nature with little physical exertion, remote wheezing at night. From the anamnesis: the above complaints worry for 10 years, does not receive the necessary treatment, periodically takes sucking pills that soften the cough. Over the past 5 years, the above complaints have been bothering 3-4 times a year with an exacerbation duration of more than a month, notes the susceptibility to acute respiratory viral diseases. An objective examination: the condition is satisfactory, the skin is pale pink. Percussion above the lungs is a boxy sound, auscultatory weakening of breathing. BH 18 in 1 min, heart rate 72 in 1 min, blood pressure 130/70 mm Hg, FEV ₁ 64% and FEV ₁ / FVC 81%. For other organs and systems without pathological features. The patient was examined according to the proposed method. According to the results of the study, patient V. determined the membrane potential index, which amounted to 1.08 cu, which indicates severe impairment of the function of external respiration. Evaluation of bronchial patency by the indicators of forced expiratory volume for 1 second (FEV ₁ ) and forced vital capacity of the lungs (FVC), FEV ₁ / FVC index showed: FEV ₁ - 42% and FEV ₁ / FVC - 55%, which indicates severe violation of the function of external respiration, therefore, a conclusion is drawn about the adverse nature of the course of the disease with constant progression and deterioration of indicators characterizing the function of external respiration. After 11 months: auscultation in the lungs, in addition to weakened vesicular breathing, revealed dry low-tone rales with forced expiration, the patient noted a decrease in exercise tolerance and the appearance of shortness of breath when climbing to the 4th floor. When analyzing indicators allowing to assess bronchial obstruction, FEV ₁ was 40% and FEV ₁ / FVC 47%, IMPI amounted to 1.06 cu, which indicates the progression of disorders of bronchial obstruction.

Thus, the assessment of the reactivity of cells by the level of IMPM is an important, non-contraindicated, reliable, informative and accelerated diagnostic method shown to all patients with COPD to predict the progression of bronchial obstruction. The invention allows at an earlier time from the onset of the disease to establish the possibility of disease progression, that is, to predict the dynamics of the development of the disease, and proceed to treatment.

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Claims (1)

A method for predicting the progression of bronchial obstruction in patients with chronic obstructive pulmonary disease, including taking a patient’s blood test “before” and “after” a stress test, isolation of platelets from the samples, followed by isolation of mitochondria, processing of mitochondrial samples with Safranin O dye and spectrophotometric measurement in optical density samples at wavelengths of 523 nm and 555 nm, followed by calculation, according to the Nernst equation, of the membrane potential of mitochondria, then determination the mitochondrial membrane potential index, which predicts the risk of COPD progression, so if the mitochondrial membrane potential index is more than or equal to 1.40 conventional units, a favorable COPD course is predicted, and when the mitochondrial membrane potential index is less than or equal to 1.20 conventional units predict an unfavorable course of COPD.