RU2134420C1 - Method of determination of erythrocyte peroxide resistance - Google Patents

Abstract

FIELD: medicine, biochemical methods of analysis. SUBSTANCE: peroxide resistance of erythrocytes is measured on the basis of three parameters: (1) optical density of a sample subjected for peroxide hemolysis; (2) optical density corresponding 100% hemolysis and (3) optical density indicating the spontaneous (without H₂O₂) hemolysis of erythrocytes. Method involves the use of a single batch of erythrocyte suspension. Optical density is determined at wavelength 536 nm. Value of peroxide resistance is measured based on erythrocyte suspension dilution. EFFECT: increased precision and sensitivity of method. 1 tbl, 1 dwg, 1 ex

Description

 The invention relates to biochemical research methods, and in particular to methods for determining the peroxide resistance of red blood cells.

 Peroxide oxidation (PO) is a normal component of the oxidative processes that constantly occur in the body. Excessive activation of the software has a distinctly damaging effect at the level of subcellular formations.

 The invention is of interest for sports biochemistry, since almost any sports activity is accompanied by activation of software, which, due to the aforementioned damaging effect, negatively affects the physical performance of the body. Therefore, controlling the intensity of software in the body of an athlete is an important task in the biochemistry of sports. This control is conveniently carried out on the basis of assessing the hemolytic resistance of red blood cell membranes. Phospholipid structures of cell membranes are among the biological objects most sensitive to changes in the level of PO. At the same time, red blood cells are readily available for research.

 Of the methods described in the literature for determining the hemolytic resistance of red blood cells, they turned out to be closest to the proposed one [1, 2].

 The method [2] was taken as the closest analogue. It is as follows. 2 ml of a 0.068% hydrogen peroxide solution was added to 2 ml of a 5% suspension of red blood cells and 0.16 ml of a 0.2% sodium azide solution. A erythrocyte suspension and solutions of hydrogen peroxide and sodium azide were prepared in buffered saline with a pH of 7.4.

 Thus, two experimental samples were prepared. The third - control sample, designed to determine spontaneous hemolysis of red blood cells, consisted of the same volumes of a suspension of red blood cells and a solution of sodium azide, but instead of a solution of hydrogen peroxide, an equal volume of buffered saline was taken with a pH of 7.4.

The experimental and control samples were incubated for 1 hour at 37 ^o C. To obtain 100% erythrocyte hemolysis, 0.2 ml of a 4% saponin solution was added to one of the experimental samples. After incubation, all samples (experimental and control) were centrifuged 10 'at 2000 rpm. The supernatant was used to determine the degree of hemolysis. To do this, 2.7 ml of a transforming solution was added to 0.3 ml of the supernatant (see Instruction of the USSR Ministry of Health dated 07.06.82 “Determination of blood hemoglobin by the hemoglobin cyanide method”) and then the optical density E _{541 was} determined, which is proportional to the amount of hemoglobin in the sample.

The degree of hemolysis A (in%), determined under standard conditions, under the influence of hydrogen peroxide was calculated by the formula
A = 100 (a - c) / (b - c), (1)
where a is the optical density of the experimental sample E ₅₄₁ ;
b - optical density at full (100%) hemolysis under the influence of saponin;
in - the value of the optical density of the control sample is a characteristic of spontaneous hemolysis of red blood cells in the experience.

 The average peroxide resistance of erythrocytes calculated in this way for a group of healthy people is A = 8.9 + 1.13 [2].

 The disadvantages of the method taken as the closest analogue. As follows from the description, the initial substrate for determining peroxide resistance is a suspension of red blood cells.

 Obviously, even with very careful suspension and careful selection of the erythrocyte suspension for sample preparation, the number of red blood cells inevitably turns out to be different in all samples, especially during mass flow measurements. Therefore, the assessment of the degree of hemolysis of erythrocytes in the experimental sample with respect to the sample with 100% hemolysis (see formula (1)) is accompanied by a significant error, especially if slight hemolysis is observed in the experimental sample or there is a sufficiently strong spontaneous hemolysis of red blood cells in the control sample .

 Thus, the heterogeneity of the erythrocyte suspension and the need to prepare a separate sample for 100% hemolysis negatively affect the accuracy of the method. After all, the number of red blood cells in the sample, in which 100% hemolysis is carried out by adding saponin, and in other samples can vary significantly, which removes the accuracy and reproducibility of the method.

 In the course of determination, it is necessary to repeatedly manipulate small volumes of substrate and reagents used. The measurement error of these volumes also negatively affects the accuracy of the final result.

 The method taken as the closest analogue is mainly used in clinical biochemistry, where the result is compared with normal values of the degree of hemolysis for a healthy person.

 The table below shows (for comparison with the data obtained by the proposed method) the results of determining peroxide resistance by the method [2].

 The proposed method for determining the peroxide resistance of red blood cells. The developed method takes into account the disadvantages of the closest analogue. Before proceeding to its detailed consideration on the basis of a specific example, we should dwell on several practical characteristics of the proposed method.

 1. A 4.4% suspension was prepared from twice erythrocytes washed with physiological saline. For example, 2 ml of suspension was prepared from red blood cells contained in 0.2 ml of whole blood.

 2. In a series of special experiments, the initial working concentration of hydrogen peroxide, 2.2%, was selected empirically, which provides a convenient value for measuring the optical density of the hemolysate (from 0.1 to 0.8). The concentration of hydrogen peroxide was controlled by extinction at λ = 230 nm using a molar extinction coefficient.

3. In method [2], sodium azide is used to inhibit erythrocyte catalase in order to directly evaluate the resistance of erythrocyte membranes to peroxidation initiated by H ₂ O ₂ . The proposed method has a slightly different goal: to evaluate the overall resistance of erythrocyte membranes, including the action of erythrocyte catalase, which decomposes hydrogen peroxide. Therefore, sodium azide in the proposed method was not used.

4. As already noted, in the method [2] apply a transforming solution that gives a colored complex with hemoglobin Fe ³⁺ . The optical density E _{541 of the} resulting solution allows us to judge the concentration of hemoglobin and, therefore, the degree of hemolysis of red blood cells. In the proposed method, a transforming solution was not used. The degree of hemolysis was judged by the optical density E ₅₃₆ , which directly reflects the concentration of hemoglobin. The measurements were carried out on an SF-46 spectrophotometer, which ensured the accuracy of reading the readings to the third decimal place.

An example of a specific application of the proposed method
10 samples of the same donated blood were taken. In a spectrophotometric cuvette with a volume of 1 ml and with a thickness of an absorbing liquid layer of 1 cm, 0.8 ml of buffered saline solution (pH 7.4) is added, 0.1 ml of a suspension of red blood cells is added. The contents are mixed.

 All three parameters of each sample ("a", "b" and "c"), appearing in formula (1), are determined directly in the same spectrophotometric cell.

First, the value "b" is determined, the characteristic of spontaneous hemolysis. In a series of preliminary experiments, it was found that under the conditions of the described determination for 60 ', spontaneous hemolysis is practically proportional to the incubation time. Immediately after the above mixing, the sample in a spectrophotometric cuvette is centrifuged (5 ', 3000 rpm) and the absorbance of the supernatant is determined at λ = 536 nm. Get E ₀ - the zero point in the graph below. Next, the precipitate is agitated and after 30 'incubation at 37 ^o C again centrifuged and determine the optical density E ₃₀ . Then, according to the graph, taking into account the linear dependence, E _{60 is} obtained by extrapolation. As an example, the drawing shows data related to the definition of "c" for the first sample (first row of the table, II). E ₀ = 0.010; E ₃₀ = 0.035.

As follows from the drawing, the value of E ₆₀ = 0,060. Obviously, the operation of linear extrapolation is not necessarily related to the construction of the graph. In this case, E ₆₀ = 2 E ₃₀ - E ₀ . Hence, taking into account dilution with the subsequent addition of H ₂ O ₂ (the procedure is described below), we have
c = 0.9 E ₆₀ = 1.8 E ₃₀ - 0.9 E ₀ . (2)
The procedure is as follows: 0.1 ml of a 2.2% hydrogen peroxide solution is added and, with constant shaking of the cuvette and stirring of the contents of the sample, incubate for 30 'at the same temperature. Then, after centrifugation (5 ', 3000 rpm), the optical density corresponding to the value of "a" is determined.

 Next, a glass rod soaked in a 4% saponin solution is introduced into the cuvettes, after which the sediment is stirred up. Preliminary experiments showed that this operation and the subsequent 10 'exposure provide 100% hemolysis. Upon reaching 100% hemolysis, the samples are again centrifuged (5 ', 3000 rpm) and the supernatant is photographed, determining the value of "b". The data obtained are shown in the table at the end of the description.

 The left side of the table presents the results of the determination of peroxide resistance of 10 samples of the same donor blood by the method [2], in the right - by the proposed method. Comparison of the obtained results shows that our method provides higher accuracy and reproducibility of the results. The average error of determination is 3.8%. While the error of the method [2] is 13%. At the same time, an increase in accuracy is accompanied by a simplification and cheaper method (the transforming solution used in [2] is not used in it).

Thus, the elimination of the shortcomings of the closest analogue is achieved through the individualization of each sample. The erythrocyte suspension placed in the spectrophotometric cell no longer leaves it and is used first to determine spontaneous hemolysis, then to determine hemolysis caused by the addition of H ₂ O ₂ , and finally, to determine the complete hemolysis due to the addition of saponin.

 The scope of the proposed method is beyond the scope of sports biochemistry. It is of interest for all cases, including clinical ones, in which it is important to have accurate data on the dynamics of changes in red blood cell peroxide resistance.

Literature
1. Pokrovsky AA, Abrarov A.A. To the question of peroxide resistance of red blood cells. - Q. Food, 1964. - N 6. - S. 44-46.

 2. Benisovich V.I., Idelson L.I. The formation of peroxides of unsaturated fatty acids in the membrane of red blood cells in the disease of Markiath-Miqueli. - Q. honey. Chemistry, 1973. - N 6. - S. 596-599.

Claims (1)

A method for determining red blood cell peroxide resistance, including determination of spontaneous hemolysis of a suspension of red blood cells, hemolysis caused by the addition of H ₂ O ₂ , and 100% hemolysis caused by a detergent, and peroxide resistance is calculated based on these values, characterized in that it is centrifuged after mixing a portion of the suspension of red blood cells 5 min at 3000 rpm, determine the resistance of erythrocyte membranes, including the action of erythrocyte catalase by assessing the magnitude of spontaneous hemolysis, hemolysis caused by ext by applying H ₂ O ₂ , and then 100% hemolysis in the same portion of a erythrocyte suspension placed in a spectrophotometric cuvette, the optical density of the supernatant is determined at a wavelength of λ = 536 nm, and the value of peroxide resistance is determined taking into account the dilution of the suspension red blood cells.

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