SU1529109A1 - Method of determining injure of membranes of erythrocytes - Google Patents

Abstract

The invention relates to medicine and biochemistry and can be used to determine damage to erythrocyte membranes. The goal is to increase the sensitivity of the method. Erythrocyte membranes, obtained by hypoosmotic shock and treated with ultrasound, are incubated with ergocalciferol solution at 37 ° C for 60 min and chemiluminescent analysis of the incubation mixture is carried out. Chemiluminescence is induced by ferrous ions. Record the light sum of the "fast" chemiluminescence flash. At a light sum level above 700 imp / min, damage to erythrocyte membranes is judged.

Description

This invention relates to medicine and biochemistry, in particular, to methods for assessing red cell membrane damage.

The purpose of the invention is to increase the sensitivity of the method by using the preparation of erythrocyte membranes treated with ergocalciferol and Fe

Example 1. 5 ml of venous blood is taken and red blood cells are taken from it, while heparin (2000 eDo / ml) is used as an anticoagulant. For this, the blood is centrifuged at 300 g for 10 min, containing plasma and the top layer of red blood cells containing. leukocytes are removed. Then it is washed three times by adding four times the volume of cooled saline, each time the cells are precipitated by centrifugation

at 300 g for 10 min and remove the supernatant together with the upper layer of erythrocytes.

Erythrocyte membranes were isolated by hypo-osmotic hemolysis, with 5 mM containing 1 mM EDTA being taken as the hemolyzing medium. One milliliter of precipitated erythrocytes are rapidly and vigorously mixed with 20 ml of hemol cooled to the hemolyte and kept at this temperature, stirring occasionally for 40 minutes. The hemolysate is centrifuged at 20,000 g for 20 minutes. The supernatant was removed, and the erythrocyte membrane pellet was washed three times with 20 volumes of 10 mM Tris-HCl buffer (pH 7.4, at 20 ° C), each time the membrane was precipitated by centrifugation at 20,000 g for 20 minutes. Membrane pellets

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roocytes are transferred to volumetric centrifuge tubes and adjusted with 10 mM Tris-HC1 buffer pH 7, D volume up to 1.0 ml. The resulting suspension of erythrocyte membranes is treated with ultrasound at a frequency of 44 kHz with an output electrical generator power in the operating frequency range at an active load of 360 W, 0.2 ml of 0.5% alcoholic solution of ergocapdiferol (final concentration 0.08%) was added to 0.2 MP of an ultrasound-treated erythrocyte membrane suspension (final concentration 0.08%), bringing the incubation mixture to 4 ml 10 mM Tris-HC1-buffer (pH 7.4), and incubated at 37 ° C for 60 minutes. Then, 1 ml of an aqueous solution of ferrous sulfate (PeSO) is added to the incubation mixture at its final concentration in a 5 ml cuvette equal to 5 10 M, and a fast flash is initiated by a chemiluminometer operating in the photon counting mode chemiluminescence of the incubation mixture at 37 ° С The recorded light sum reflects the intensity of free radical processes corresponding to the level of the content of lipid hydroperoxides, an increase in the concentration of which indicates and red cell membrane damage,

It is significant that erythrocyte membranes without ergocalciferol solution do not give a quick flash of chemiluminescence to the introduction of iron ions,

When examining the erythrocyte membranes of practically healthy people (50 people), it was found that the light sum of fast chemiluminescence was 6200t80. containing erythrocyte membrane, up to 12000-17500 imp./min. Consequently, at the level of the light sum of the rapid chemiluminescence flash of the incubation mixture above 7000 imp. / Min, damage to the erythrocyte membranes is judged.

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EXAMPLE 2 Red blood cells are prepared as described in Example 1. Red blood cells are mixed with a solution of digitonin at its final concentration in an incubation medium of 2 μg / ml; 10 µg / ml and 20 µg / ml Incubation mixture contains 1 ml of red blood cell sediment; 8.98 ml of saline and 20 μl of blood plasma containing the appropriate concentration of digitonin. After 20 minutes of incubation, the mixture is centrifuged and the resulting precipitate of digitonin-treated erythrocytes, as well as the standard sample, is used for hypoosmotic hemolysis (as described in Example 1) and the subsequent determination of the light sum of the fast Fe-induced chemiluminescence

A similar study was conducted using the prototype method.

With greater damage to erythrocyte membranes, a more significant increase in the light sum of their chemiluminescence is observed, measured by the proposed method 7883 imp./min 10982 imp./min, 15117 imp, / min, which confirms the adequacy and sufficient sensitivity of the proposed method.

Claims (1)

Invention Formula The method for determining damage to erythrocyte membranes by treating erythrocytes with chemical reagents that induce chemiluminescence, followed by recording the level of chemiluminescence, which means that, in order to detect the sensitivity of the method, the erythrocyte membranes are treated with ultrasound and incubated in an alcohol solution by a heart-lung system, and a non-cardiac maker will be incubated in an alcohol solution by a heart-lung system and a non-cardiac maker. hemilu-. The minescence is induced by ferrous ions, then the light sum of a fast flash of chemiluminescence is recorded, and the presence of damage to the membranes is determined at a light sum of flash of 7000 counts / min.