RU2371723C1 - Method of assessment of erythrocyte resistance to functional stress - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention can be applied in assessment of human disadaptive processes at various pathological states. Method involves determination of met-hemoglobin, membrane-linked hemoglobin and oxidised nucleotide content and total optic erythrocyte density before and after functional sampling in the form of short-term local ischemia. Coefficient of erythrocyte resistance to ischemia K is defined on the basis of obtained data. If K is below 0 then erythrocyte resistance to ischemia is high, K values within 0 to 0.1 indicate standard resistance, and K over 0.1 indicates low resistance. Invention allows for assessment of erythrocyte resistance to functional stress, such as tissue hypoxia.

EFFECT: fast, reliable and objective detection of different types of cell adaptation to ischemia.

3 ex, 1 tbl

Description

The present invention relates to medicine, namely to biochemistry and physiology, and can be used to evaluate maladaptive processes in humans in various pathological conditions.

-микроглобулина, трофобластического β1-1-гликопротеида, плацентарного

-микроглобулина. Устанавливаемую фазу стрессового состояния (тревоги, резистентности, истощения) и норму диагностируют по величине рассчитываемого коэффициента (Патент №2288475 РФ, МПК G01N 33/74, G01N 33/68. Способ диагностики фаз стрессовых состояний /В.Н.Морозов, А.А.Хадарцев, Ю.В.Карасева, В.И.Морозова, А.В.Хапкина. - Опубл. 27.11.2006, Бюл. №33).A known method for the diagnosis of phases of stressful states by determining blood parameters that reflect the functional state of the body: the concentration of glucocorticoids (cortisol) and fertile factors:

-microglobulin, trophoblastic β1-1-glycoprotein, placental

-microglobulin. The established phase of the stress state (anxiety, resistance, exhaustion) and the norm are diagnosed by the value of the calculated coefficient (Patent No. 2288475 of the Russian Federation, IPC G01N 33/74, G01N 33/68. Method for the diagnosis of phases of stressful conditions / V.N. Morozov, A.A. .Khadartsev, Yu.V. Karaseva, V.I. Morozova, A.V. Khapkina. - Published on November 27, 2006, Bull. No. 33).

The disadvantages of this method include the duration of the determination, and the use of expensive equipment and reagents, which prevents its use in wide medical practice.

Closest to the proposed is a method for determining the types of cell reactivity in children by assessing the resistance of red blood cells to functional load (Patent No. 2231792 of the Russian Federation, MKI ⁷ G01N 33/49, G01N 33/50, G01N 33/74. Method for determining cell reactivity in children / O .I. Zaitseva, V.P. Tereshchenko, E.I. Prakhin. - Published on June 27, 2004).

The essence of the known method lies in the fact that the type of cellular reactivity is determined by the level of chlortetracycline probe fluorescence spectroscopy of erythrocyte membranes using pharmacological preparations: acetylcholine, adrenaline, dexamethasone. To implement the known method, a sample of frozen red blood cells obtained from whole venous blood was washed three times with physiological saline, after which erythrocyte membranes were isolated. By adding physiological solution, a suspension of erythrocyte membranes was obtained, in samples of which one of the indicated pharmacological preparations was administered in physiological doses in vitro. After measuring the level of fluorescence according to the formula, the response rate of erythrocyte membranes was calculated, by which the type of cell reactivity was established:

- cholinergic - cell structures are in a state of stability;

- cholinadrenergic synergism - resistance to extreme impacts;

- adrenergic - lability of regulatory mechanisms;

- chinadrenergic type hyposynergism - reduced reactivity.

The disadvantages of this method include the fact that it is intended to determine the type of cell reactivity only in children and cannot be used for other age groups. In addition, the known method is time-consuming, involves the use of expensive equipment, and it also requires the involvement of a neurologist to assess the subject's autonomic homeostasis.

The objective of the proposed technical solution is to develop a method for assessing the resistance of red blood cells to functional load - tissue hypoxia.

The technical result of the proposed method is its simplification and reduction of determination time.

The technical result is achieved by the fact that the method of assessing the resistance of red blood cells to functional load includes taking a blood sample, isolating red blood cells, obtaining a supernatant, conducting a functional test and determining the physicochemical parameters of red blood cells.

Distinctive techniques of the proposed method are to determine the content of methemoglobin, membrane-bound hemoglobin, oxidized nucleotides and the total optical density of red blood cells.

The difference between the method also lies in the fact that these indicators are determined twice - before and after the functional test.

A distinctive technique is also the fact that a “cuff” test is used as a functional test, providing short-term local ischemia.

The authors of the proposed method found that the resistance of red blood cells to ischemia is determined by the coefficient calculated by the formula:

K = 1/4 · (ΔМНb + ΔmcHb + ΔNuc + ΔOp) · 0.1,

where: K is the coefficient of resistance of red blood cells to ischemia;

Δ is the difference in the values of the indicators after and before the functional test;

ΔМНb is the difference in the values of methemoglobin content,%;

ΔmcHb is the difference in the values of the membrane-bound hemoglobin content,%;

ΔNuc is the difference in the values of the content of oxidized nucleotides, cu;

ΔOr. - the difference in the values of the total optical density, cu;

0.1 is a constant coefficient.

The authors of the proposed method found that the value of the coefficient K allows us to judge the nature of the resistance of red blood cells to ischemia. So, when the value of the coefficient K is less than "0", the resistance of red blood cells to ischemia is estimated to be high, from "0" to 0.1 - normal, above 0.1 - reduced.

A comparative analysis with the prototype showed that the proposed method differs from the known above methods and, therefore, meets the criteria of the invention of "novelty."

Comparison of the claimed technical solution not only with the prototype, but also with other solutions did not allow us to identify signs that distinguish the claimed solution not only from the prototype method, but also from other similar solutions in this and related fields of medicine.

In the available literature, there is no way to assess the compensatory ability of a cell - an erythrocyte to tissue hypoxia (ischemia), not by a single parameter, but by the functional state of red blood cells, which is characterized by the physicochemical parameters proposed for determination - methemoglobin, membrane-bound hemoglobin, oxidized nucleotides, total optical density of red blood cells.

Clinical observations and analysis of the results allowed the authors of the proposed method to evaluate the resistance of red blood cells to ischemic effects, which can be used to judge the cellular compensatory-adaptive reactions of the body in ischemic and hypoxic conditions.

This allows us to conclude that the technical solution meets the criterion of "inventive step".

The method comprising the claimed invention is intended for use in medicine, namely in biochemistry and physiology. The possibility of its implementation is confirmed by the examples and means described in the application, therefore, the proposed solution meets the criteria of the invention "industrial applicability".

The inventive method is as follows.

In the morning on an empty stomach, from the cubital vein, blood is drawn into a test tube containing 3.8% sodium citrate solution. Blood sampling is carried out twice: before and after a 5-minute clamping of the brachial artery with the cuff of the Korotkov apparatus, so that its indicators are 10 mm Hg. the column exceeded the systolic pressure of the patient (cuff test). Repeated blood sampling is carried out from the ulnar vein of the arm, which was not subjected to ischemia.

To determine the content of methemoglobin and membrane-bound hemoglobin, the blood is hemolyzed with distilled water and the values of these parameters are determined by spectral characteristics.

So, the content of methemoglobin is carried out at a wavelength of 630 nm (Reference on laboratory research methods under the editorship of L.A. Danilova, “Peter.” - 2003. - P.74), and the content of membrane-bound hemoglobin - at a wavelength of 536 nm (Toktamysova Z.S., Birzhanova N.Kh. Determination of membrane-bound hemoglobin. - J. "Biophysics". - 1990. - T.35, issue 6. - p. 1119-1120).

The content of methemoglobin and membrane-bound hemoglobin is expressed as a percentage.

The total optical density and the content of oxidized nucleotides are determined in the erythrocyte supernatant after sedimentation of the erythrocyte suspension by trichloroacetic acid and the UV spectra were taken in the wavelength range of 220-300 nm and are expressed in units of optical density - cu (Kuznetsova E.E., Gorokhova V.G. et al. Method for determining the state of erythrocyte membranes. IPC G01N 33/48, 33/52. RF patent No. 2296992, 04/10/2007, Bull. No. 10).

To determine the difference in magnitude (Δ) of each test indicator, it is necessary to subtract its value from the value after the functional test before the functional test.

The obtained differences of the determined indicators (Δ) are summed, averaged and multiplied by a constant coefficient of 0.1. The obtained indicator, the authors of the proposed method identified the coefficient of resistance of red blood cells to ischemia - K.

K = 1/4 · (ΔМНb + ΔmcHb + ΔNuc + ΔOp) · 0.1,

Where:

ΔМНb is the difference in the values of methemoglobin content,%;

ΔmcHb is the difference in the values of the membrane-bound hemoglobin content,%;

ΔNuc is the difference in the values of the content of oxidized nucleotides, cu;

ΔOr - the difference in the values of the total optical density, cu;

0.1 is a constant coefficient.

When K is less than "0", the resistance of red blood cells to ischemia is estimated to be high, from "0" to 0.1 - normal, above 0.1 - reduced.

The proposed method is illustrated by examples of specific performance.

Example 1. Donor M., almost healthy. A cuff test was performed. In the obtained blood samples, the studied parameters were determined before and after the sample.

MHb: before - 1.0%, after 1.4% ΔMHb = 0.4% mcHb: before - 3.6%, after 0.9% ΔmcHb = -2.7% Nuc: before - 0.36 cu, after 0.34 cu ΔNuc = -0.002 c.u. Or: before - 4.53 cu, after - 4.79 cu ΔOr = 0.26 c.u.

The resulting differences (Δ) are summed, averaged and multiplied by 0.1.

K = ((0.4-2.7-0.002 + 0.26) / 4) · 0.1 = - 0.0515

The calculated coefficient K reflects the high resistance of red blood cells to ischemia.

Example 2. Patient V., medical history 1223, diagnosis: coronary heart disease, II functional class, angina pectoris.

A cuff test was performed on the patient. In the obtained blood samples, the studied parameters were determined before and after the sample.

MHb: before - 2.7%, after 3.4% ΔМНb = 0.7% mcHb: before - 8.2%, after 10.9% ΔmcHb = 2.7% Nuc: before - 0.6 cu, after 0.057 cu ΔNuc = -0.003 cu Or: before - 1.768 cu, after - 1.549 cu ΔOr = -0.219 c.u.

The resulting differences (Δ) are summed, averaged and multiplied by 0.1.

K = ((0.7 + 2.7-0.003-0.219) / 4) 0.1 = 0.079

The calculated value of the coefficient K in this patient made it possible to assess the resistance of red blood cells to ischemia as normal.

Example 3. Patient M., medical history 1264, diagnosis: coronary heart disease, III functional class of exertional angina.

A cuff test was performed on the patient. In the obtained blood samples, the studied parameters were determined before and after the sample.

MHb: before - 0.1%, after 0.4% ΔМНb = 0.3% mcHb: before - 10%, after 14.3% ΔmcHb = 4.3% Nuc: before - 0.05 cu, after 0.188 cu ΔNuc = 0.138 c.u. Or: before - 2.902 cu, after - 4.33 cu ΔOp = 1,428 c.u.

K = ((0.3 + 4.3 + 0.138 + 1.428) / 4) 0.1 = 0.1542

The obtained indicator "K" indicates a reduced resistance of red blood cells to ischemia in this patient.

After a complex of therapeutic measures, patients V. and M. received the following results:

Patient B.

MHb: up to - 0.3%, after 0.1% ΔMHb = -0.2% mcHb: before - 8.4%, after 9.3% ΔmcHb = 0.9% Nuc: before - 0.105 cu, after 0.121 cu ΔNuc = 0.016 c.u. Or: before - 2.342 cu, after - 2.579 cu ΔOr = 0.237 c.u.

K = ((0.9 + 0.016 + 0.237-0.2) / 4) 0.1 = 0.0238

Patient M.

MHb: before - 3.2%, after 4.0% ΔМНb = 0.8% msb: up to 8.1%, after 9.8% ΔmcHb = 1.7% Nuc: before - 0.04 cu, after 0.049 cu ΔNuc = 0.009 c.u. Or: before - 2.389 cu, after - 2.844 cu ΔOr = 0.455 cu

K = ((0.8 + 1.7 + 0.009 + 0.455) / 4) 0.1 = 0.0741

Therefore, the measures of therapeutic effect led to a significant improvement in the resistance of red blood cells to ischemic test in these patients with coronary artery disease.

40 patients with a diagnosis of coronary heart disease aged 46 to 71 years (mean age 55 ± 1.3 years) were examined. Of these, 20 patients with angina of exertion of the II and III functional classes according to NYHA. The control group consisted of donors - 15 healthy people.

A comparative analysis of the averaged data showed that the coefficient of erythrocyte resistance to ischemia in donors is significantly different from the coefficients found in patients with coronary artery disease with different severity (functional class) of angina pectoris (Table).

The erythrocyte resistance coefficient - K Donors (15) II functional class (20) III functional class (20) average, including: 0.0002377 ± 0.01 0.043105 ± 0.01 * 0.078855 ± 0.018 * K <0 -0.0299 ± 0.01 -0.069975 ± 0.03 -0.039933 ± 0.018 0 <K <= 0.1 0.036678 ± 0.01 0.046491 ± 0.01 0.047488 ± 0.007 K> 0.1 0 0.12612 ± 0.009 * 0.160156 ± 0.02 * * p <0.05 compared with donors

Thus, the developed method allows you to quickly, efficiently and objectively identify the different nature of cellular adaptation to ischemia, which makes it possible to widely use it in practical medicine. The value of the coefficient K is an evaluation criterion for the resistance of red blood cells to ischemia not only in healthy people, but also in patients. The study of individual types of resistance and response to environmental factors at the cell level will allow pathogenetically sound approach to the early diagnosis and treatment of many pathological processes.

Claims (1)

A method for assessing the resistance of red blood cells to functional load, including taking a blood sample, isolating red blood cells, obtaining a supernatant and conducting a functional test with subsequent determination of the physicochemical parameters of red blood cells, characterized in that the content of methemoglobin, membrane-bound hemoglobin, oxidized nucleotides and the total optical density of red blood cells to and after a functional test, which is used as a short-term local ischemia, then determine Difference values for each indicator after and before functional test, after which the resistance of erythrocytes to ischemia is set according to the formula
K = 1/4 (ΔMHb + ΔmcHb + ΔNuc + ΔOp) 0.1
where K is the coefficient of resistance of red blood cells to ischemia;
Δ is the difference in the values of the indicators after and before the functional test;
ΔMHb is the difference in the values of methemoglobin content,%;
ΔmcHb is the difference in the values of the indicators of the content of membrane-bound hemoglobin,%;
ΔNuc - the difference between the values of indicators of the content of oxidized nucleotides, cu;
ΔOp - the difference in the values of the total optical density, cu;
0,1 - constant coefficient,
and when the coefficient K is less than 0, the resistance of red blood cells to ischemia is estimated to be high, from 0 to 0.1 - normal, above 0.1 - reduced.