RU2310196C2 - Method for determination of sympatic-adrenal system activity - Google Patents

Abstract

FIELD: medicine, in particular ecological physiology and endocrinology.

SUBSTANCE: method for determination of sympatic-adrenal system (SAS) activity in human and animals includes blood analysis. Three blood samples diluted by 200 times are prepared, wherein the first one contains isotonic sodium chloride solution; the second (control) one contains fitohemagglutinine solution; and the third (tested) one contains fitohemagglutinine and propanolole solution. Then samples are incubated for 10 min and number of free non-agglutinated erythrocytes is calculated under microscope in Gorjaev's chamber. Activity index (AI) is calculated according to formula: AK = (Er-PP)/(Er-Contr)[100 %, wherein Er is erythrocyte amount in 0.85 % NaCl solution; Contr - is erythrocyte amount in fitohemagglutinine solution; and PP is erythrocyte amount in fitohemagglutinine and propanolole solution. When AK is less than 80 % SAS is recognized as non-active one.

EFFECT: method of increased accuracy.

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Description

The invention relates to physiology and medicine and can be used in environmental physiology, endocrinology, as well as in clinical and experimental studies for biotesting various hormonal and other biologically active substances, including phytoecdysteroids.

Known radioimmunological methods, NMR and fluorescence probes for determining the concentration of free and receptor-related catecholamines (Handbook of a Practitioner / Ed. A. Vorobyov, M .: Medicine, 1992).

The disadvantage of these methods is the need to use sophisticated medical equipment, specially equipped rooms and specially trained personnel, expensive reagents.

A known method for determining the state (sympatho-adrenal system) of SAS by anti-hemolytic effect when using the method of hypoosmotic hemolysis of red blood cells in combination with the PP test (Sominsky V.N., Sokovnin V.M., Okun K.V. Kinetics of the interaction of propranolol with the membrane erythrocytes in individuals with a different antihemolizing effect of this drug // Cosmic Biology and Aerospace Medicine, 1988, No. 3, S.86-88; Sominsky V.N., Berdysheva L.V., Bluma R.K. et al. red blood cells for intravital assessment of functional state adrenoceptor // Fiziol. Zh. of the USSR. Setchenov 1989, №2. s.189-193).

The disadvantage of this method is the need for a relatively large amount of blood and complex mathematical processing.

A known method for assessing the state of the sympathoadrenal system of a person (SU 1422158, A1 09/07/1988), selected for the prototype, the essence of which is as follows. The red blood cells are separated from the patient’s blood, washed three times in a cooled physiological saline solution, 8-10 mg of EDTA are added to 1-2 ml of erythrocyte suspension and catecholamines (A and HA) are extracted in 0.4 N perchloric acid for 30 minutes After separation of the white flocculent precipitate by centrifugation, the resulting supernatant is poured and the pH is adjusted to 8.5. Adsorption column chromatography is carried out using alumina as a sorbent. Catecholamines are eluted by adding 3.5 ml of 0.25 N twice. acetic acid to obtain acetic eluate and further oxidation of catecholamines. The eluate is oxidized with potassium ferrocyanide in phosphate buffer pH 4.2-6.2 to obtain oxidation products A and HA. Fluorescence A and HA are measured, the difference between the experimental and control samples is recorded, and the severity of the disease is judged by their value.

The known method is multi-stage and time-consuming, which affects the accuracy of the research results.

The objective of the invention is to develop a new method for determining the status of the sympatho-adrenal system.

The technical result of the proposed method is to increase the accuracy of the assessment of SAS, this allows you to use the method for monitoring and rapid assessment of SAS animals and humans.

The method is based on the method of phytohemagglutination proposed by N.A. Moiseenko, L.I. Irzhak for a qualitative description of a heterogeneous population of red blood cells (Moiseenko N.A., Irzhak L.I. Agglutination of rabbit red blood cells in case of intense erythropoiesis // Journal of General Biol., 1972. T. 33. No. 6, S.779-786.4).

The erythrocyte membrane contains β-adrenergic receptors and may reflect the state of adrenergic receptors in other organs and tissues. We suggested using the phytohemagglutination method in combination with the PP test. The PP test allows you to compare the initial control blood sample with phytohemagglutinins (PHA) with imitation of its in vitro state under stress.

The technical result is achieved by the fact that in the method for determining the functional activity of the sympatho-adrenal system (CAC) of animals and humans, including a blood test, according to the invention, three blood samples are diluted 200 times, the first with isotonic sodium chloride solution, the second control with a solution of phytohemagglutinins; the third experimental one - with a solution of phytohemagglutinins and propranolol, incubation of samples for 10 minutes, quantitative counting under a microscope in the Goryaev’s chamber of free non-agglutinated red blood cells, the activity index (AK) is calculated by the formula:

where Er is the number of red blood cells in a 0.85% NaCl solution;

Counter. - the number of red blood cells in the PHA solution;

PP - the number of red blood cells in a solution of PHA and propranolol,

And with AK below 80%, CAC is considered inactive.

The method is as follows.

The invention uses propranolol (PP), a non-selective β-adrenergic receptor blocker and a salt extract in a 0.85% NaCl solution of phytohemagglutinin from plant pea (Pisum sativum L.) (Moiseenko N.A., Irzhak L.I. Rabbit erythrocyte agglutination in case of stress erythropoiesis // Journal of General Biol., 1972. T. 33. No. 6, S.779-786). A PHA solution is prepared by extracting them from milled seeds of pea (Pisum sativum L.) with a 0.85% NaCl solution for 2 days in a domestic refrigerator at t = 4C °. The dry weight of the sample to the volume of NaCl solution as 1: 5. The resulting extract is filtered through an anesthetized filter.

Blood with an anticoagulant from a person or an experimental animal, thoroughly mixed 200 times diluted, is collected into erythrocyte melangers and a red blood cell agglutination reaction is quantified.

In the first sample, the blood is diluted with 0.85% NaCl and the total number of red blood cells is determined.

The second control sample is diluted with a FAG solution and the amount of free red blood cells not involved in the agglutination reaction is determined.

The third test sample is diluted with a solution of FAG + PP and determine the number of free red blood cells.

Counting red blood cells that have not undergone agglutination is carried out after 10 minutes of incubation under a microscope in a Goryaev chamber in five large squares. The number of agglutinated red blood cells is calculated from the difference between the total number of red blood cells (sample 1) and the number of free non-agglutinated red blood cells in samples 2 and 3. Experimental sample 3 allows you to compare the initial agglutinability of the control sample in the PHA solution with an in vitro simulation of its state under stress.

The ability of β-adrenoreceptors to bind to PP determines the activity index of the sympathoadrenal system using a simple mathematical formula that compares the degree of red blood cell agglutination in the control (with PHA) and experimental (with a solution of PHA and propranolol) samples.

where AK is the activity of CAC;

Er - the number of red blood cells in a 0.85% NaCl solution;

Counter. - the number of red blood cells in the PHA solution;

PP - the number of red blood cells in a solution of PHA and propranolol.

It was experimentally established that the sympatho-adrenal system is not activated if the activity indicator is below 80%, i.e. There are significant differences in the control and experimental samples.

Example 1

They take blood with an anticoagulant from an intact rat, collect in erythrocyte melangers, while blood is collected up to the 0.5 mark, up to the 101 mark - PHA solution (control sample), the experimental sample - PHA with PP: up to the 0.5 mark - blood, up to 1 - PP with a concentration of 10 ^-6 mol / liter, up to label 101 - PHA solution. The contents of the melangers are mixed during this time. Free non-agglutinated red blood cells are counted in 5 large squares.

The total number of red blood cells in 5 large squares of Er. - 495; in counter. - 312, in the PP test - 371.

CAC is not activated.

Example 2

Evaluate the CAS according to the above method with the introduction of an experimental rat 20-hydroxyecdysone (20 E) at a dose of 20 mg / kg from the plant of the serpent crowned coronata L. coronata L. (Volodin V.V., Volodina S.O. Method for the production of ecdysteroids. Pat. 2153346. Russia. MKI ³ A61K 35/78. Declared March 29, 1999. No. 99106351/14, Publish. July 27, 2000 // B.I. 2000. No. 21).

Er - 571; PP - sample 399; control - 297 er.

CAC is not activated.

Example 3

Assess CAC according to the above method after exercise (swimming for 40 minutes) in an experimental rat.

Er = 480; counter. sample - 247; PP test 273 er.

CAS is moderately activated.

Example 4

Assess CAS according to the above method with the introduction of an experimental rat thyroxine at a dose of 30 mg / kg of body weight.

Er = 580; counter. sample 352; PP test 368 er.

CAC is significantly activated.

It was experimentally established and confirmed that the closer the AK indicator to 100%, the higher the activity of the CAC. The smallest CAS activity in the intact rat and in the rat, which was introduced 20-E; in animals, with the introduction of thyroxine and physical activity (stress factor), a higher CAS activity was observed, maximum with the introduction of thyroxine.

Thus, the proposed method is simple and does not require special training and can be carried out in parallel with the clinical determination of the total number of red blood cells in the blood.

The method is easily implemented in conventional clinical laboratories, does not require sophisticated equipment. The amount of blood necessary for analysis can be taken from blood wastes with an anticoagulant, which usually remain in clinical laboratories after a series of standard tests, or obtained by piercing the skin of people, laboratory and wild animals. It is only important that they be stored without freezing for no more than 24 hours after they are removed from the body. The high sensitivity of the proposed method allows its use to assess the degree of adaptability of people and animals to the conditions of activity and the environment, as well as the functional reserves of the body.

Claims (1)

A method for determining the functional activity of the sympathoadrenal system (CAS) of animals and humans, including a blood test, characterized in that they prepare three blood samples diluted 200 times, the first with an isotonic sodium chloride solution, the second control with a solution of phytohemagglutinins; the third experimental one - with a solution of phytohemagglutinins and propranolol, incubation of samples for 10 min, quantitative counting under a microscope in a Goryaev chamber of free non-agglutinated red blood cells, and the activity index (AK) is calculated by the formula

where Er is the number of red blood cells in a 0.85% NaCl solution; Counter. - the number of red blood cells in the PHA solution; PP - the number of red blood cells in a solution of PHA and propranolol, and with AK below 80%, CAC is considered inactive.