RU2652273C2 - Method for determination of erythrocytes m-cholinoreactivity - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for blood erythrocytes M-cholinoreactivity determination includes taking blood (0.2 ml) from a human or an animal, preparation of an atropine solution at a concentration of 0.4 mg/ml, blood sample incubation in a hypoosmotic medium in the absence (control sample) and presence of atropine in a final concentration of 15×10^-6 mg/ml (test sample), determination of the optical density of sample supernatant, calculation of M-cholinoreactivity value as the percentage by which the hemolysis in the test samples is lower than in the control samples.

EFFECT: method is universal, technically simple, allows to carry out measurements and observe indicator dynamics in the same organism in different functional states and under experimental influences, to obtain reliable results.

4 ex, 3 tbl, 2 dwg

Description

The method for determining the erythrocyte M-cholinergic reactivity relates to experimental biology and medicine, to laboratory research methods in physiology and hematology. It consists in a biochemical determination of the reactivity of red blood cells to cholinergic substances based on the inhibition of osmotic hemolysis in the presence of an atropine M-cholinergic receptor blocker. The method can be used in experimental studies and laboratory diagnostics.

Cholinergic mechanisms, along with adrenergic ones, occupy a significant place in the regulation of body functions. Numerous studies have shown the presence of various subtypes of M-cholinergic receptors on the membranes of cardiomyocytes, smooth muscle cells, and others [12, 13, 14], including on the membranes of blood cells [5, 8, 11, 16, 17].

One of the well-known methods for determining the cholinergic activity of the body is the atropine test method. The essence of the method is to determine the degree of change in heart rate and blood pressure when atropine is introduced into the body. Reactivity is assessed by the deviation of the indicators from the initial values measured at rest [4]. The main disadvantages of this method is the presence of a number of side effects with the introduction of the drug into the body: significant and prolonged tachycardia, dilated pupil and disturbance of accommodation, intestinal atony, etc.

A known method for evaluating M-cholinergic reactivity by changing the amplitude and contraction rate of isolated strips of tissues and organs (myocardium, uterus, aorta) when exposed to acetylcholine. Registration of contractions is performed using a myocytograph with a flow chamber [6, 7]. The disadvantages of working with isolated organs are the need for expensive equipment, the need for surgical intervention in the body, the limited duration of work with the drug, the inability to track the change in the studied property of the same organism in different functional states.

There is a method for determining the erythrocyte M-cholinergic reactivity, which consists in determining the rate of agglutination of human red blood cells in the presence of acetylcholine and atropine [5]. The method is based on the data on the presence of M-cholinergic receptors in erythrocyte membranes [16, 17] and consists in carrying out the erythrocyte agglutination reaction in standard serum of group 0αβ (I) in the absence of acetylcholine (control) and when it is present in incubation medium at concentrations of 5.5 × 10 ^-10 - 5.5 × 10 ^-6 M. The disadvantage of this method is the necessity of using hemagglutinating serums, which is possible when working with human blood, but is not applicable in working with laboratory animals because of the specificity of agglutination factors. The acquisition of species-specific sera for laboratory animals is expensive.

The objective of the invention is the creation of a method for assessing the M-cholinergic reactivity of red blood cells, which is convenient and easy to implement, can be used to work with human blood, as well as laboratory animals under various functional conditions of the body and in a variety of experimental models, will quickly obtain reliable results.

The problem is solved using the proposed method for determining the M-cholinergic reactivity of red blood cells, consisting of a modification of the method for assessing β-adrenergic reactivity of erythrocyte membranes by R. Stryuk and Dlusskaya I.G. (2003) [2].

The prototype method [2] is based on determining the degree of inhibition of hypoosmotic hemolysis in the presence of β-blocker. To do this, determine the optical density of the supernatant of the control (hypotonic solution of 0.4% NaCl) and the experimental sample (hypotonic solution of 0.4% NaCl together with β-blocker). β-adrenoreactivity is evaluated by the percentage of optical density of the experimental samples relative to the control samples. High values of β-adrenergic reactivity of the membranes mean low sensitivity of the adrenergic receptors of membranes, and low values of β-adrenergic reactivity of the membranes mean high sensitivity of adrenergic receptors. The method is recommended for studying the nature of arterial hypertension. The disadvantages of the method [2] are: the inability to assess reactivity to other regulatory factors, in particular cholinergic nature; analysis of the results based on the inverse relationships between decreased hemolysis, adrenoreactivity and sensitivity of cell membrane receptors.

To determine M-cholinergic reactivity, it is proposed to use an atropine blocker of M-cholinergic receptors instead of a β-blocker. The rationale for this approach is the data on the presence of M-cholinergic receptors on the membranes of human and animal blood cells [5, 9, 11, 16, 17], as well as the fact that M-cholinergic receptors in different organs and tissues of humans and animals have similar structure [12]. The ability of acetylcholine through M-cholinergic receptors to activate signaling cascades has been proven, leading to the activation of inosiol-3-phosphate-dependent Ca ²⁺ channels, which increases the entry of Ca ²⁺ into red blood cells and the K ⁺ exit from them [14]. The Ca ²⁺ entering the erythrocyte activates protein kinase C, which phosphorylates the proteins of bands 4.1 and 4.9, which increases the microviscosity of the erythrocyte membrane [10, 15]. The increase in membrane microviscosity also occurs due to the destruction of the lipid bilayer and the decrease in protein-lipid contacts under the influence of the Ca ²⁺ -calmodulin complex and Ca ²⁺ -calmodulin-dependent protein kinase [4]. These changes in the properties of the membrane can reduce the resistance of red blood cells to hemolysis. Thus, atropine, as a blocker of M-cholinergic receptors, is able to affect the resistance of red blood cells to hemolysis in the direction of its increase.

The essence of the modification of the method Stryuk R.I. and Dlusskaya I.G. [2] consists in the fact that instead of an adrenoreactive substance (propranolol), 0.1 ml of a solution of atropine M-anticholinergic antagonist at a concentration of 0.4 mg / ml is introduced into the experimental sample to obtain a final concentration in the erythrocyte incubation medium of 15 × 10 ^-6 mg / ml When choosing a concentration, literature data were taken into account [5, 8]. The erythrocyte M-cholinergic reactivity is calculated as the degree of decrease in osmotic hemolysis in the presence of atropine based on the difference in the optical density of the supernatant of the control (incubation without atropine) and experimental (incubation with atropine) samples.

The proposed method will make it possible to determine the M-cholinergic reactivity of erythrocytes based on the inhibition of osmotic hemolysis by atropine in an incubation medium, which will make it possible to objectively evaluate the property of cholinergic reactivity without side effects from the administration of atropine into the body, without surgical intervention and excision of organs to prepare isolated tissues, does not require expensive, technically complex equipment and specific hemagglutinating serums in the case of studies on laboratory animals. The use of a small volume of blood (200 μl per analysis) makes it possible to determine the indicator in the same organism many times under various functional conditions and in a variety of experimental models.

Distinctive features of the claimed method in comparison with other methods for assessing cholinergic reactivity [4, 5, 6, 7] and the prototype method [2] are: the ability to determine the M-cholinergic reactivity of human and animal blood red blood cells in a biochemical way, which makes the method universal, and its use Along with the determination of β-adrenoreactivity of erythrocyte membranes, it expands the laboratory base for the study of regulatory processes and the properties of red blood cells; determination of M-cholinergic reactivity of red blood cells in an incubation medium, which excludes the introduction of drugs (atropine, acetylcholine) that can cause side effects; the ability to take a small amount of blood (200 μl per study) allows you to monitor the dynamics of the indicator in the same organism in different functional states and experimental conditions, makes it possible to repeat the study and clarify the result if necessary; the lack of need for preliminary preparation, in technically complex and expensive equipment and reagents, which makes the method easy to implement and cost-effective.

The technical result from the implementation of the proposed method is to simplify the assessment of the M-cholinergic reactivity of human and animal blood red blood cells in various functional states while increasing the reliability of the results.

The proposed method is implemented as follows. Reagents are being prepared:

концентрированного фосфатного буфера необходимо взять

натрий фосфорнокислый двузамещенный 12-водный (Na₂HPO₄*12H₂O),

натрий фосфорнокислый однозамещенный 2-водный (NaH₂PO₄*2H₂O) и

натрий хлористый (NaCl) и растворить в

дистиллированной воды. Полученный раствор концентрата буфера можно хранить при температуре не выше 15°С в течение 1 месяца. Для определения взять 10 мл концентрата буфера и довести до

дистиллированной водой;1. Phosphate buffer: for preparation

concentrated phosphate buffer must be taken

disubstituted sodium phosphate 12-aqueous (Na ₂ HPO ₄ * 12H ₂ O),

monosubstituted sodium phosphate 2-aqueous (NaH ₂ PO ₄ * 2H ₂ O) and

sodium chloride (NaCl) and dissolve in

distilled water. The resulting buffer concentrate solution can be stored at a temperature not exceeding 15 ° C for 1 month. For determination, take 10 ml of buffer concentrate and bring to

distilled water;

2. A solution of atropine (0.4 mg / ml): To prepare a solution of atropine, take 2 ml of a solution of atropine sulfate ((C ₁₇ H ₂₃ NO ₃ ) ₂ 3H ₂ SO ₄ ) with a concentration of 1 mg / ml and bring its volume up to 5 ml with distilled water. From this solution for determination, take 0.1 ml;

3. Anticoagulant: heparin sodium (5000 U / ml) - 0.02 ml.

Working process:

For research, you need to get 0.2 ml of whole blood. In laboratory rats, blood is taken from the tail vein according to the generally accepted method [1]. A person receives the necessary amount of blood from a finger in the traditional way [3]. 0.2 ml of whole blood is introduced into a test tube with 0.2 ml of physiological saline and 0.02 ml of heparin, mix gently. A blood sample thus prepared is suitable for further processing for 3 hours at room temperature.

From each analyzed blood sample, 4 samples are prepared: two control and two experimental. To do this, take 4 clean centrifuge tubes and add reagents and test blood to them according to the scheme

After centrifugation, the optical density of the supernatant of all samples at a wavelength of 540 nm is determined.

The value of the indicator of M-cholinergic erythrocytes (M-CRE) is proposed to calculate by the formula

where M-ChRE - the value of the indicator of M-cholinergic erythrocytes, rel. units; Eo ₁ and Eo ₂ - the optical density of the experimental samples, units opt. tight .; Ek ₁ and Ek ₂ are the optical densities of control samples, units opt. tight

According to this formula, M-CRE is defined as the percentage by which hemolysis in the experimental samples is lower than in the control. This allows us to determine precisely the reactivity of hemolysis to the atropine present in the medium and, in contrast to the prototype method [2], go from the analysis of inverse relationships to the analysis of direct relationships between the magnitude of M-CRE and the sensitivity of M-cholinergic receptors. That is, to consider high values of M-ChRE as an indicator of high sensitivity of M-cholinergic receptors, low values of M-ChRE - as an indicator of low sensitivity of M-cholinergic receptors.

Using the proposed method, 117 male non-linear rats were examined, blood was taken to determine the background or initial M-CRE to identify the actual values of the indicator, compare them with the values of β-ARE determined by the prototype method [2], analyze the dynamics of the indicator M-CRE in conditions of a change in the functional state of the body and its regulatory apparatus.

Example 1

Analysis of the background values of M-CRE, the degree of their variation in the sample of nonlinear rats.

Blood sampling was carried out from the caudal vein of animals according to the generally accepted method [1]. 0.2 ml of whole blood was introduced into a test tube with 0.2 ml of physiological saline and 0.02 ml of heparin, carefully mixed. From the prepared blood sample, 2 control and 2 experimental samples were prepared. All samples were prepared on phosphate buffer (2.5 ml). 0.1 ml of distilled water and 0.05 ml of the analyzed blood were added to control samples. In experimental samples - 0.1 ml of atropine solution (0.4 mg / ml) and 0.05 ml of the analyzed blood. Samples were gently mixed and incubated at room temperature for 30 minutes. Then the samples were centrifuged for 10 minutes at 1500 rpm. The supernatant was collected and its optical density was measured on an APEL DD-303UV spectrophotometer. M-CRE is calculated as the degree of decrease in osmotic hemolysis in the presence of atropine based on the difference in the optical density of the supernatant of the control and experimental samples.

As a result of the determination of M-CRE in this way, the actual values of the indicator were obtained, on the basis of which the variational series was constructed (Fig. 1). As can be seen from the diagram, the minimum and maximum values are 1.7 and 17.0 rel. units respectively. The variation series in the range from minimum to maximum corresponds to the normal distribution. The center of the variation series are two distinct peaks, which account for 25% and 31% of all the M-CRE values of the sample. The boundary of these classes corresponds to the average value of M-CRE - 8.2 ± 0.29 rel. units

As a result of the cluster analysis by the k-means method, individuals were grouped by the value of M-CRE. Animals with an indicator value from 6.8 to 10.6 rel. units The group with low M-CRE included individuals with values from 1.7 to 6.6 rel. units, and in the group with high M-HRE - animals with values of M-HRE between 11.1 and 17.0 rel. units The average values for groups with low, medium and high M-CRE are 4.9 ± 0.21, 8.5 ± 0.14 and 13.55 ± 0.43 rel. units respectively (tab. 1). A cluster group with an average M-CRE is the largest (61 pcs.), The smallest (19 pcs.) - A group with high M-CRE. This suggests that among males of non-linear rats in 52% of cases animals with average M-CRE are found, and only in 16% of cases individuals with high M-CRE. About 84% of the values of M-CRE are in the range of medium and low values (from 1.7 to 10.6 rel. Units).

When comparing the background values of M-CRE with the values of β-ARE, determined in the same animals by the method of Stryuk R.I. and Dlusskaya I.G. (2003) [2] found that the rat M-RRE values are on average 5 times lower, and the scatter of their values is narrower than β-RRE values. Also, in most animals, medium and high values of β-ARE are determined. Thus, the parameters of M-CRE and β-ARE, determined in the same animals and the same blood samples, differ significantly in absolute values, the nature of variation in the sample and can be considered as two independent indicators.

Example 2

The study of M-CRE with the introduction of atropine.

The task was to determine whether M-CRE changes when atropine is administered to animals at a dose of 1 mg / kg bw In 12 male rats, the background M-CRE was determined - 11.4 ± 2.25 rel. units After administration of atropine, M-CRE decreased on average in the group to 6.3 7 ± 1.17 rel. units (p <0.0607), which indicates a decrease in the sensitivity of M-cholinergic receptors, since a significant part of those that are present on the membranes are blocked by introduced atropine. That is, the introduction of a blocker of M-cholinergic receptors into the body reduces the M-cholinergic activity of red blood cells.

Example 3

The study of M-CRE and β-ARE in acute stress.

The task was to assess changes in M-CRE, as well as to compare β-ARE, under acute stress. Indicators were determined in a state of calm wakefulness before stress. Then the animals were subjected to acute emotional and pain stress, which was caused by immobilizing the animals in Plexiglas cases for 1 h in combination with electrodermal tail irritation in a stochastic manner by threshold AC values. From the data in Table 2 it follows that acute stress causes a 2-fold decrease in M-Chre (p <0.01), while β-ARE decreases by 31.2% from the level at rest (p <0.05) . Therefore, indicators change unidirectionally, but to a different extent, which confirms their independent significance and variability with a change in the functional state of the body.

Example 4

The study of M-CRE and β-ARE during stimulation of the noradrenergic system.

The task was to find out in which direction and to what extent M-CRE, as well as β-ARE, change with increasing activity of the noradrenergic neurotransmitter system of the body. In 24 male rats, the background (initial) M-CRE was determined - 8.3 ± 0.46 rel. units, and also for comparison β-ARE - 48.4 ± 1.56 rel. units The noradrenergic system was stimulated by four-fold administration of maprotiline (10 mg / kg bw intraperitoneally). From the table. 3 and fig. Figure 2 shows that, against the background of activation of the noradrenergic system, an increase in both indicators was noted, the proportion of individuals with high values of both indicators increased in the sample of animals. So, M-CRE increased by 83% (p <0.001), and β-ARE by 22% (p <0.001) compared to the initial state. As can be seen from the results, the stimulation of the noradrenergic system is accompanied by an almost twofold increase in M-CRE, in contrast to β-ARE. Consequently, M-CRE changes in experimental conditions independently of β-ARE and carries its own information load, and an increase in the value of the indicator indicates an increase in the sensitivity of M-cholinergic receptors under the conditions of the applied experimental exposure.

The proposed method for determining the erythrocyte M-cholinergic reactivity is universal, technically simple, easily reproducible, allows measurements from the same organism in the dynamics of functional states and in a variety of experimental models, and makes it possible to quickly obtain reliable results. The method complements and expands the methodological base of experimental studies of regulatory processes and the properties of red blood cells, with additional refinements, it can be applied to human blood and used for diagnostic purposes as an independent indicator or in combination with the determination of beta-adrenergic reactivity of membranes.

Used Books

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Claims (1)

A method for determining the erythrocyte M-cholinergic reactivity based on the reduction of erythrocyte hypoosmotic hemolysis in the presence of atropine, including blood sampling (0.2 ml) in humans or animals for analysis, preparation of an atropine solution with a concentration of 0.4 mg / ml, incubation of a blood sample in a hypoosmotic medium in the absence (control sample) and the presence of atropine in a final concentration of 15 × 10 ^-6 mg / ml (experimental sample), followed by determination of the optical density of the supernatant of the samples and calculation of the erythrocyte M-cholinergic reactivity of red blood cells as a percentage, for which hemolysis in experimental samples is lower than in control ones, characterized in that the erythrocyte M-cholinoreactivity is determined by the biochemical method when blood samples are incubated in the presence of atropine, and the percentage by which hemolysis in experimental samples is taken is the erythrocyte M-cholinoreactivity lower than in the control.

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