RU2229133C1 - Method for detecting velocity in development of lipid peroxidation products in erythocytic membranes in farm animals - Google Patents

Abstract

FIELD: biological chemistry, hematology. SUBSTANCE: due to decreased volume of erythrocytic membranes at their incubation (by 6.7 times against a prototype) and sampling to carry out chromatic reaction with hot thiobarbituric acid, and, also, due to specifying formulas to calculate velocities in development of induced and spontaneous lipid peroxidation products in erythrocytic membranes in farm animals. The possibility is achieved to detect the velocity of lipid peroxidation in erythrocytic membranes easily under conditions of biochemical laboratories. EFFECT: higher efficiency of detection. 4 ex, 4 tbl

Description

The invention relates to the field of veterinary medicine, in particular to hematology, and in particular to laboratory methods for determining the rate of lipid peroxidation in erythrocyte membranes of farm animals.

There is a method for determining the rate of formation of lipid peroxidation products in erythrocyte membranes, which is based on measuring the content of malondialdehyde (MDA) formed during the oxidation of membrane lipids by the color reaction with thiobarbituric acid (EM Stroev, VG Makarova "Biological Workshop Chemistry ": Textbook for pharmaceutical universities and faculty. -M: Higher school, 1986.-S.211-213). The specified method was taken as a prototype.

The method includes obtaining a erythrocyte sediment by centrifuging blood samples; 3-fold washing of the erythrocyte sediment with physiological solution of sodium chloride and reprecipitation in the same centrifugation mode; complete hemolysis of erythrocytes by adding an equal volume of distilled water to 0.5 ml of erythrocyte sediment and holding for 30 minutes; obtaining samples of erythrocyte membranes from hemolized red blood cells by centrifugation and careful pipetting of a supernatant with a gray layer of erythrocyte membranes; preparation of three test samples: 0.3 ml of Mohr's salt solution (4x10 ^-5 M freshly prepared solution), Tris-buffer (Tris-Hcl buffer, 0.04 M solution with a pH of 7.4), ascorbic acid are added to the first test tube 2.6 mM freshly prepared solution) and 0.1 ml of the obtained suspension of erythrocyte membranes, 0.3 ml of Tris buffer, 0.6 ml of distilled water and 0.1 ml of suspension of erythrocyte membranes are added to the second test tube, add to the third (control) the same reagents as in the first test tube, after which 1 ml of trichloroacetic acid is immediately added. Then the samples are incubated for 20 min in a water bath at 37 ° C, the reaction is stopped, adding 1 ml of trichloroacetic acid solution to both test tubes, all samples are centrifuged, the supernatant liquid is drained (2 ml), 1 ml of thiobarbituric acid solution is added, the samples are placed for 10 min in a boiling water bath and cooled in ice water. The optical density of the obtained samples is measured against a control on a spectrophotometer at a wavelength of 532 nm or a photoelectrocolorimeter at a wavelength of 540 nm (green filter) in a cuvette with a layer thickness of 1 cm and the rate of lipid peroxidation in erythrocyte membranes is calculated by the formulas

и

,

and

,

where X1 is the rate of MDA formation in the sample in the presence of prooxidants: Mohr's salt and ascorbic acid (induced lipid peroxidation), nmol per hour; X2 - the rate of formation of MDA in the sample in the absence of prooxidants (spontaneous lipid peroxidation), nmol per hour; 3 - sample volume, ml; 6 - conversion factor for 1 hour; 0.156 is the extinction of 1 nmol of malondialdehyde at 532 nm; E1 and E2 are the optical density of the first and second samples, respectively, against the control.

The disadvantage of this method is that when it is used to determine the rate of formation of lipid peroxidation products in the erythrocyte membranes of farm animals, a precipitate (suspension) after the interaction of substances in the discharge with thiobarbituric acid is observed, which affects the optical density measurement results. However, there is no repeatability of the results when analyzing the same sample.

The objective of the present invention is to select the optimal volume of samples of erythrocyte membranes of farm animals that can be used for incubation, and to clarify the conditions for the subsequent steps to identify the products of lipid peroxidation reaction, which ultimately will ensure repeatability of the results of determination of reaction products and improve accuracy. This is the technical result.

The technical result of the invention is achieved by conducting aqueous hemolysis of red blood cells with ice water, reducing the volume of suspension of red blood cells in experimental samples (6.7 times compared with the prototype), using hot thiabarbituric acid to conduct a color reaction and clarifying the formula for calculating the result.

The method is as follows:

- receive blood from the jugular vein of animals using an anticoagulant;

- blood is centrifuged for 10 min at 3000 rpm, precipitating red blood cells;

- the obtained erythrocyte sediment is washed with a 0.9% sodium chloride solution until the color disappears in the supernatant and reprecipitated in the same centrifugation mode;

- complete erythrocyte hemolysis is performed: 0.5 ml of erythrocyte sediment is taken into a clean centrifuge tube and an equal volume of distilled water cooled to 0 ° C is dispensed from the pipette with a strong jet and left to stand for 30 minutes;

- receive samples of erythrocyte membranes - the samples are centrifuged for 30 min at 3000 rpm, the supernatant with a gray layer of erythrocyte membranes is carefully aspirated and transferred to a clean tube;

- carry out the incubation of samples of erythrocyte membranes - 3 tubes are prepared for each membrane sample: 0.6 ml of Mohr's salt solution (4x10 ^-5 M freshly prepared solution), ascorbic acid (2.6 mm freshly prepared solution) are added to the first test tube, 0.77 ml Tris-buffer (Tris-Hcl buffer, 0.04 M solution with a pH of 7.4), and 0.03 ml of the obtained suspension of erythrocyte membranes; 0.77 ml of Tris buffer, 0.03 ml of suspension of erythrocyte membranes and 1.2 ml of distilled water are poured into the second tube; in the third (control) add the same reagents as in the first test tube, after which 2 ml of a 40% trichloroacetic acid solution are immediately added; the prepared samples are placed in a thermostat and incubated for 20 min at 37 ° C;

- stop the reaction by adding to the test tubes 2 ml of trichloroacetic acid solution;

- the contents of the samples are prepared for a color reaction - all samples are centrifuged for 15 min at 3000 rpm, then the supernatant is carefully taken with a pipette with an elongated end;

- carry out a color reaction: 3.0 ml of the obtained supernatant are measured in warm tubes, 1.5 ml of 0.8% freshly prepared hot solution of thiobarbituric acid are added to each of them, and the samples are placed for 10 min in a boiling water bath. After incubation, the tubes are cooled in ice water;

- measure the optical density of the experimental samples against the control on a photoelectrocolorimeter at a wavelength of 540 nm (green filter) in a cuvette with a layer thickness of 1 cm and calculate the rate of formation of the end product of lipid cross-oxidation of malondialdehyde according to the formulas

и

,

and

,

where X1 is the rate of MDA formation in the sample in the presence of prooxidants - Mohr's salt and ascorbic acid (induced lipid peroxidation), nmol per hour; X2 - the rate of formation of MDA in the sample in the absence of prooxidants (spontaneous lipid peroxidation), nmol per hour; 4.0 - the total volume of medium for incubation of the obtained membrane samples, ml; 3.0 - sample volume for color reaction with thiobarbituric acid, ml; 4,5 - sample volume, ml, taken for photometry; 6 - conversion factor for 1 hour; 0.156 is the extention of 1 nmol MDA at 532 nm; E1 and E2 are the optical density of the first and second samples, respectively, against the control.

The experiments to determine the rate of formation of lipid peroxidation products in the erythrocyte membranes of farm animals were carried out on heifers of two to six months of age at the farm of the Vil'gorod Scientific and Experimental Station of the Komi Scientific Center of the Ural Branch of the Russian Academy of Sciences (Syktyvkar).

Example 1. During the experiments, it was noted that the contents of the samples after the reaction of thiobarbituric acid with products that were formed after incubation of a sample of membranes of red blood cells of heifers at the rate of 0.1 ml per 1.0 ml of incubation medium or 0.2 ml per 2.0 ml of medium at 37 ° C for 20 min, was cloudy and contained particles of sediment. The appearance of sediment in the sample affected the results of the analysis during photometry. Therefore, a study was conducted of the rate of formation of lipid peroxidation products in erythrocyte membrane samples in heifers depending on the change in the volume of freshly obtained membrane samples taken for incubation. It was found that the maximum rate of formation of lipid peroxidation products or malondialdehyde is observed when 2.0 ml of 0.03 ml of the erythrocyte membrane preparation is added to the incubation medium (Table 1).

Example 2. During the experiments, the need for accurate selection of the supernatant after stopping the formation of lipid peroxidation products in animal erythrocyte membrane samples by adding 40% trichloroacetic acid to the incubation medium and their subsequent centrifugation for 15 minutes was proved. It was noted that after passing this reaction, a light grayish coating appears on the walls of the tubes, which, when the contents are drained from the tubes, easily enters the supernatant. In this regard, the rates of formation of lipid peroxidation products are 1.5–3.0 times higher than those obtained by careful sampling (Table 2).

Example 3. When using the method, taken as a prototype to determine the rate of formation of reaction products of lipid peroxidation reactions, there is a scatter in values in a parallel study of one sample of membranes (table. 3, animals No. 1 and 2). Our proposed method provides the optimal probability of coincidence of the results obtained when analyzing the same samples of erythrocyte membranes in animals (Table 3, animals No. 3-6).

Note: * - to determine the rate of formation of products of lipid peroxidation in the membranes of red blood cells of heifers used the method taken as a prototype

Example 4. A method for determining the rate of formation of lipid peroxidation products in erythrocyte membranes of farm animals was used to identify lipid peroxidation products in membranes of erythrocyte membranes of heifers kept from the first day of life under different conditions of motor load: forced movement on a unit with a moving floor - twice per day for 30 minutes at a speed of 20 m per minute or at free content. It was found that the rate of formation of TBA-active products in the erythrocyte membranes of heifers of 125 days old, which are on the first day of life under conditions of forced movement, is 3.5 times or more higher than the rate of formation of products of lipid peroxidation in the membranes of red blood cells in heifers of 107 days age contained from the first day of life in standard cells in free content (table. 4.).

Thus, the proposed method for determining the rate of lipid peroxidation in the erythrocyte membranes of farm animals can be used to identify the features of the functioning of erythrocyte membranes of animals in the process of adaptation of their body to changing conditions (the influence of the intensity of the motor load, temperature, the inclusion of new feed in the diet, etc. .d.).

Claims (1)

A method for determining the rate of formation of lipid peroxidation products (lipid peroxidation) of erythrocyte membranes of farm animals (CFS), which includes obtaining a erythrocyte sediment from venous blood, performing complete aqueous hemolysis of red blood cells, obtaining membranes by centrifugation, preparing prototypes based on aqueous suspension of membranes and studying lipid peroxidation in the presence and without the presence in the experimental sample of prooxidants: a solution of Mohr's salt, ascorbic acid, as well as precipitation with trichloroacetic acid (TCA), with the following color reaction with thiabarbituric acid (TBA) to measure the optical density at 532 or 540 nm and the calculation of the results, characterized in that the hemolysis of red blood cells is carried out with ice water, as a test sample, a suspension of red blood cells is taken 6.7 times smaller in volume than in the standard method, hot TAC is used to conduct the color reaction, and the calculation of the rate of formation of lipid peroxidation products in the membranes of red blood cells of the SCC is carried out according to the formula

and

, where X1 is the rate of formation of malondialdehyde (MDA) in the presence of prooxidants (induced lipid peroxidation); X2 - the rate of formation of MDA in the absence of prooxidants (spontaneous LPO), in nmol / h; 0.156 - extinction level of 1 nmol MDA at 532 nm; E1 and E2 are the optical density, respectively, with and without the addition of prooxidants.