RU2579317C1 - Method for diagnosis of disturbed energy metabolism of lymphocytes with community-acquired pneumonia in children - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention can be used for assessing degree of breakdown of energy metabolism blood lymphocytes in children with community-acquired pneumonia (CAP). For this purpose, flow cytometry is used for accounting of lymphocytes with reduced membrane potential of mitochondria. With values from 0 to 35% normal level is diagnosed, 36 to 75% - subcompensated disorder, not requiring specific therapy, 76% and more - decompensated disorders requiring in complex treatment of community-acquired pneumonia assignment of en-ergotropic preparations.

EFFECT: using the given method enables detecting the disturbed cell supply at earlier stage, if no changes succinate dehydrogenase activity, which enables its use in clinical practice for separation of the risk groups of children with CAP requiring metabolic correction.

1 cl, 1 tbl, 4 ex

Description

The invention relates to medicine, namely to pediatrics, pulmonology, and can be used to assess the degree of impaired energy metabolism of blood lymphocytes in children with community-acquired pneumonia (VP) by increasing the percentage of lymphocytes with a reduced mitochondrial membrane potential (Δψ) (MPM).

Despite the improvement of methods of prevention, diagnosis and treatment, to date, there have been no significant trends towards a decrease in the proportion of community-acquired pneumonia (VP) in the incidence of the child population [V.K. Tatochenko Pneumonia in children: diagnosis and treatment // Attending physician. - 2008. - No. 8. - S. 5-9.].

The main pathogenetic link in diseases of the bronchopulmonary system is a disorder of external and tissue respiration, a decrease in oxidative processes and the development of oxygen deficiency, which leads to impaired hemostasis and all types of metabolism. Numerous studies have established that the development and course of many diseases is accompanied by a decrease in the energy supply of lymphocytes [Shabelnikova E.I. Morpho-functional characteristics of mitochondria of lymphocytes in children with various forms of insufficiency of cellular energy exchange: author. dis. ... cand. honey. sciences. - M., 2005. - 28 p.].

It has been established that in many diseases in children oxidative phosphorylation disorders occur associated with a decrease in the activity of the mitochondrial electron transport system, and it is in these children that acute bronchopulmonary pathology proceeds with a complicated course and is torpid to standard etiopathogenetic therapy [Klembovsky AI, Sukhorukov BC Mitochondrial insufficiency in children // Arch. pathology. - 1997. - No. 5 (59). - S. 3-7]. A reserve for improving the course of the disease in children is the early diagnosis of metabolic disorders in lymphocytes.

A number of authors have suggested that there are various types of individual energy status of the organism and the existence of a latent form of its relative individual insufficiency - “energy-deficient diathesis”, which leaves its mark on the course of various diseases and requires specific energy -otropic correction [Sukhorukov B.C. Violation of cellular energy metabolism in children. - M.: Medicine, 2000. - 80 p.].

A known method for the diagnosis of disorders of energy production in mitochondria by the activity of the enzymes aspartate aminotransferase (ACT), alanine notransferase (ALT), creatine phosphokinase (CPK), lactate dehydrogenase (LDH) in the serum of adolescents living [RU 2465599 C2, 08.28.2010]. The disadvantage of this method is that it is necessary to determine several enzymes, it does not allow to assess the significance of changes in indicators for children with CAP.

There is a method for measuring mitochondrial activity of lymphocytes [RU 2302635 C1 12.28.2005], which consists in measuring the activity of succinate dehydrogenase in individual populations of lymphocytes by flow cytometry. However, the disadvantage of this method is the need for large economic and time costs due to the need to use more reagents for phenotyping of subpopulations of lymphocytes.

In the inner mitochondrial membrane there are elements of the respiratory chain, including succinate dehydrogenase, therefore, a violation of the processes of energy formation can change the transmembrane proton potential. There is evidence of impaired functional state of cells associated with a change in MPM [A. Perelman et al. JC-1: alternative excitation wavelengths facilitate mitochondrial membrane potential cytometry // Cell Death and Disease. - 2012. - 3. - C. 1-7 .; Kondratyeva E.B., Lobanova E.G. The effect of ozone on the membrane potential of platelet mitochondria // Medical almanac. - 2013. - No. 3 (27). - S. 58-59 .; RU 2503003 C1 08/10/2012].

The closest analogue of the claimed invention is a method for determining cellular energy metabolism in children with pneumonia, which consists in a cytochemical determination of the activity of redox enzymes - succinate dehydrogenase (SDH), glutamate dehydrogenase (GDG), alpha-glycerophosphate dehydrogenase (α-GFDGDGDG), blood cells [Kondratova I.Yu., Senatorova A.S. Cellular energy metabolism in children of the first year of life with complicated pneumonia // International Medical Journal. - 2008. - No. 4. - S. 36-39].

The disadvantage of this method is:

- subjectivity of derived measurements;

- the requirement for longer time and financial costs, since it is necessary to determine several enzymes involved in the redox reaction system in the Krebs cycle;

- the lack of differentiation of the degree of violation of the energy metabolism of lymphocytes for the need to correct energy deficiency and maximize the effectiveness of treatment of community-acquired pneumonia in children.

The objective of the invention: improving diagnosis, simplifying the interpretation of data and increasing the accuracy of determining violations of the energy metabolism of lymphocytes in community-acquired pneumonia in children.

The technical result is the ability to more quickly diagnose violations of the energy supply of lymphocytes in children with CAP by taking into account the percentage of lymphocytes with reduced MPM (Δψ) by flow cytometry for the timely correction of detected violations.

Positive effect: the method is more accurate, objective - it allows you to analyze from 5 thousand to 25 thousand lymphocytes, in contrast to light microscopy, where records are kept in 50-200 cells, it is an integral indicator of impaired lymphocyte energy supply, makes it possible to justify the appointment of drugs that improve functional activity of mitochondria.

The technical result is achieved in that in children in heparinized blood the percentage of lymphocytes with reduced MPM was determined using JC-1 dye (5.5 ′, 6.6′-tetrachloro-1.1 ′, 3.3 ′ tetraethylbenzimidazolecarbocanine iodide / chloride) (Bector Dikcenson, USA). JC-1 is a cationic dye whose uptake by mitochondria is directly related to the size of the mitochondrial membrane potential [Michael E. Widlansky et al., Ltered Mitochondrial Membrane Potential, Mass, and Morphology n the Mononuclear Cells of Humans with Type 2 Diabetes // Transl. Res. - 2010 .-- 156 (1). - P. 15-25].

The method is as follows: from the cubital vein in the morning, on an empty stomach, after 12-14 hours after the last meal, take 1 ml of blood in a heparinized tube. 800 μl of blood is layered on 400 μl of ficoll-verographin, centrifuged at 1500 rpm for 15 minutes, a lymphocyte ring is collected, which is centrifuged for 5 minutes at 1000 rpm, the supernatant is drained.

Cells are stained with JC-1 stain, after which they are incubated for 15 minutes at 37 ° C, then washed twice with buffer, and then subjected to analysis on a BD FACS Calibur cytometer (USA) in the Cell Quest Pro program.

Based on the results obtained, an analysis of the gradation of indicators is carried out and ranges of MPM values are calculated.

Depending on the degree of decrease in MPM, children are divided into 3 degrees:

1 degree - the level of MMM from 0 to 35% (normal);

Grade 2 - MPM level from 36 to 75% (subcompensated disorders that do not require specific energy therapy);

Grade 3 - MPM level of 76% or more (decompensated disorders that require the appointment of energy-stimulating agents in the complex treatment of CAP).

To disclose the essence of the invention, the results of a study of the percentage of detection of lymphocytes with reduced MPM in the peripheral blood upon admission to the hospital in 55 children with CAP are presented, indicators in 30 healthy children, comparable by sex and age, served as control.

In parallel, a study was conducted of the activity of the enzymes succinate dehydrogenase (LDH), alpha-glycerophosphate dehydrogenase (α-GPDH), lactate dehydrogenase (LDH) in peripheral blood lymphocytes by a quantitative method [Narcissov R.P. The use of paranitrotetrazolium violet for the quantitative cytochemical determination of human lymphocyte dehydrogenases // Archive of Anatomy, Histology, Embryology. - 1969. - No. 5. - S. 85-91], based on the reaction of the reduction of tetrazolium salts and precipitation of diformazan at the sites of enzyme activity. The activity of dehydrogenases was expressed by the average number of granules in one cell. The following indicators were taken as the norm: LDH - 20-24 granules / cell, α-GPDH - 11-15 granules / cell, LDH - 11-17 granules / cell (Table 1).

On the 1st day of admission, in children with CAP, a reduced degree 1 MPM was detected in 1 child (1.8%), degree 2 - in 35 people. (63.7%), 3 degrees - in 19 people. (34.5%), i.e. in 98.2% of cases, a decrease in M1TM lymphocytes was detected.

A decrease in the activity of LDH on the 1st day of hospitalization was detected in 61% of patients, α-GPDH in 45.5%, LDH in 30.9% of patients.

Using the proposed method for determining the percentage of lymphocytes with a reduced MPM, reflecting the functional and energy state of the lymphocyte, it is possible to identify among patients with CAP children with impaired lymphocyte energy supply for early diagnosis of energy exchange disorders, the need and timely appointment of energy corrective therapy.

As a result of the processing of analytical data, it was found that the method is simpler in comparison with the known analogues and allows to determine violations of the energy metabolism of lymphocytes with increased accuracy.

The advantages of the proposed method:

1. An increase in the percentage of lymphocytes with reduced MPM allows us to judge the violation of the energy state of lymphocytes at an early stage of the disease.

2. The change in MPM is ahead of the change in the indices of redox enzymes according to cytochemical studies.

3. Allows you to analyze simultaneously from 5 thousand to 25 thousand lymphocytes.

4. It is an integral indicator of the energy state of lymphocytes and allows you to identify conditions that require metabolic support.

Clinical examples:

Example 1. The child Ksenia P., 4 years. No complaints. For organs without features. Diagnosis: healthy. In a cytochemical study: LDH 26.7 g / cell, α-GFDH 12.0 g / cell, LDH 11.1 g / cell. The proportion of lymphocytes with reduced MPM is 24.7%. Conclusion: violations of the energy metabolism of lymphocytes were not detected.

Example 2. The child Daniel T., 4 years. He was admitted to the clinic of the Scientific Research Institute of OMiD with the diagnosis: Pneumonia of community-acquired, acute course, focal in the lower lobe on the right, unspecified etiology, moderate. Complaints about unproductive cough, runny nose, fever up to 39 ° C, loss of appetite. He became acutely ill when an unproductive frequent cough, a runny nose, and an increase in body temperature to febrile numbers appeared. Received symptomatic therapy. Upon receipt, the state of moderate severity. The skin is pale, expressed periorbital cyanosis. Auscultation in the lungs is hard breathing, moist, small bubbling rales on both sides, more on the right. BH 26 per minute. Heart rate 110 per minute. In the hemogram Hb - 118 g / l, Er - 4.4 · 10 ¹² / l, Le - 6.0 · 10 ⁹ / l, n - 7%, s - 55%, e - 0%, 6 - 0% , l - 32%, m - 4%, plasma cells - 2%, ESR - 14 mm / h. In a cytochemical study: LDH 37.6 g / cell., Α-GFDH 19.9 g / cell., LDH 20.4 g / cell. - recorded an increase in the activity of redox enzymes. An examination revealed 93.9% of lymphocytes with a reduced transmembrane potential of mitochondria, which corresponds to 3 degrees of impaired energy exchange - “decompensated energy deficiency” of lymphocytes. On the 10th day of treatment, there was no improvement in cytochemical parameters: LDH - 15.2 g / cell, α-GFDH - 8.8 g / cell, LDH - 17.7 g / cell. with reduced MMM, which required the appointment of energy-stimulating therapy.

Example 3. The child Cyril V., 5 years old. He was admitted to the clinic of the Research Institute of OMiD with a diagnosis of community-acquired pneumonia, acute course, left-side lower lobe, unspecified etiology, moderate severity. Complaints of prolonged cough, frequent unproductive in the daytime, nasal congestion. The disease began with a rise in temperature to 38.7 ° C, cough, runny nose. Received symptomatic therapy without effect. At admission: a state of moderate severity. The skin is pale, periorbital cyanosis. Heart sounds are loud, rhythmic. Heart rate of 92 per minute. Auscultation in the lungs breathing hard, wet rales on both sides. BH 24 per minute. In the hemogram: Hb - 143 g / l, Er - 5.11 · 10 ¹² / l, Le - 15.9 · 10 ⁹ , p - 1%, s - 51%, e - 2%, 6-0%, l - 39%, m - 7%, ESR - 5 mm / h. In a cytochemical study: LDH - 24.56 g / cell., Α-GFDH - 13.12 g / cell., LDH - 26.56 g / cell. - indicators of LDH and α-GPDH are within normal limits, an increase in LDH indicates the beginning of the activation of the compensatory anaerobic pathway for energy production. An examination revealed 78.3% of lymphocytes with a reduced transmembrane potential of mitochondria, which corresponds to the 3rd degree of impaired energy transfer “decompensated energy deficiency”. On the 10th day of treatment: LDH - 17.1 g / cell., Α-GFDH - 16.4 g / cell., LDH 23.7 g / cell. - a decrease in the activity of redox enzymes required the appointment of energetic therapy.

Example 4. The child Anastasia K., 7 years old. He was admitted to the clinic of the Research Institute of OMiD with a diagnosis of community-acquired pneumonia, bilateral, in the lower lobe on the left, in S7.8 on the right, focal, acute course, moderate severity, mycoplasma etiology. Complaints: rise in body temperature to 37.4 ° -38.0 ° C, unproductive frequent cough, weakness, loss of appetite. On admission, standing severity. Skin: pale, periorbital shadows. Heart rate 96 in 1 minute. BH 26 in 1 minute. Lung percussion: pulmonary sound. Auscultation of the lungs: hard breathing, weakened in the lower parts, moist, creaking rales on both sides in the lower parts, single dry rales are heard. In the hemogram: Hb - 140 g / l; Er - 5.07 · 10 ¹² / l; Le - 13.3 · 10 ⁹ / l; p - 4%; s - 64%; l - 30%; m - 1%; bases - 1%; ESR - 35 mm / h, toxic granularity of neutrophils ++. In a cytochemical study: LDH - 19.4 g / cell., Α-GFDH - 12.28 g / cell., LDH - 21.68 g / cell. An examination revealed 59.4% of lymphocytes with a reduced transmembrane potential of mitochondria, which corresponds to 2 degrees of energy deficiency “subcompensated disturbances”. On the 10th day of treatment, an improvement in cytochemical parameters: LDH - 21.5 g / cell., Α-GFDH - 16.5 g / cell., LDH 21.8 g / cell. With MPM - 48.39% indicates no need. appointments of energy-stimulating therapy.

Thus, the method allows to reliably determine violations of the energy supply of cells at an earlier stage in the absence of changes in the activity of LDH, which allows its use in clinical practice to highlight the risk groups of children with CAP requiring metabolic correction. This method is short, easy to reproduce, highly sensitive.

Literature

Kurashvili L.V., Bulavkin Yu.V. Kondratova I.Yu., Senatorova A.S. Cellular energy metabolism in children of the first year of life with complicated pneumonia // International Medical Journal. - 2008. - No. 4. - S. 36-39.

Senatorova A.S., Sukhorukov V.S., Kondratova I.Yu. Energy-deficient conditions as a risk factor for the complicated course of pneumonia in children of the first year of life // Odessa Medical Journal. - 2009. - No. 4 (114). - S. 43-47.

Claims (1)

A method for diagnosing the degree of violation of the energy metabolism of blood lymphocytes in children with community-acquired pneumonia by increasing the percentage of lymphocytes with a reduced mitochondrial membrane potential (Δψ), characterized in that the method of flow cytometry counts lymphocytes with a reduced membrane potential of mitochondria and diagnoses it from 0 to 35% normal level, 36 to 75% - subcompensated disorders that do not require specific therapy, 76% or more - decompensated disorders that require complex treatment in ebolnichnyh pneumonia destination energotropic drugs.