The method of determining the rate of formation of products of lipid peroxidation in erythrocyte membranes of farm animals

 

(57) Abstract:

The invention relates to the field of biological chemistry, in particular to Hematology, namely, laboratory methods of determining the rate of formation of products of lipid peroxidation in erythrocyte membranes of farm animals. The essence of the invention is that by reducing the volume of the erythrocyte membrane during incubation (6.7 times compared to the prototype) and selection of content samples for color reaction with hot thiobarbiturate acid, and also due to more accurate formula for calculating the speed of formation of products of induced and spontaneous lipid peroxidation in erythrocyte membranes of farm animals. The technical result consists in the possibilities available in terms of biochemical laboratories to determine the rate of lipid peroxidation in erythrocyte membranes. table 4.

The invention relates to the field of veterinary medicine, in particular to Hematology, namely laboratorni methods of determining the rate of lipid peroxidation in erythrocyte membranes of farm animals.

There is a way to determine the speed spans the deposits formed during the oxidation of membrane lipids malondialdehyde (MDA) by color reaction with thiobarbituric acid (Stroev, E. M., Makarova Century, "Workshop on biological chemistry: a Training manual for formats. universities and FAK. -M: The High. HQ., 1986.-S. 211-213). This method was taken as a prototype.

The method includes obtaining sediment erythrocytes by centrifugation of blood samples; 3 times the flushing of sediment erythrocytes physiological solution of sodium chloride and pereosazhdeniya in the same mode centrifugation; complete hemolysis of erythrocytes by adding 0.5 ml of sediment erythrocytes equal volume of distilled water and incubation for 30 min; obtaining samples of erythrocyte membranes from hemolyzed erythrocytes by centrifugation and careful selection pipette the supernatant liquid with a gray layer of the membranes of red blood cells; preparation of three prototypes: in the first test tube add 0.3 ml of the salt solution Mora (4x10-5M freshly prepared solution), Tris buffer (Tris-Hcl buffer, 0.04 M solution with pH 7.4), ascorbic acid (2.6 mm freshly prepared solution) and 0.1 ml of the obtained suspension of erythrocyte membranes, the second vial is poured 0.3 ml of Tris-buffer, 0.6 ml of distilled water and 0.1 ml of the suspension of erythrocytes, in the third (control) add the same reagents as in the first wading S, stop the reaction by adding both experienced test tubes and 1 ml of a solution of trichloroacetic acid, all samples centrifuged, decant the supernatant (2 ml), was added 1 ml of a solution thiobarbiturate acid, place the samples for 10 min in a boiling water bath and cooled in ice water. Measure the optical density of the obtained samples vs. control on the spectrophotometer at a wavelength of 532 nm or fotoelektrokalorimetry at a wavelength of 540 nm (green filter) in a cell with a layer thickness of 1 cm and calculate the rate of lipid peroxidation in erythrocyte membranes by formulas

and ,

where X1 - rate education in a sample of MDA in the presence of prooxidants: salt Mora and ascorbic acid induced lipid peroxidation), nmol per hour; x2 - rate education in a sample of MDA in the absence of prooxidants (spontaneous lipid peroxidation), nmol per hour; 3 - volume of sample, ml; 6 - the conversion factor for 1 hour; 0,156 - extenze 1 nmole malondialdehyde at 532 nm; E1 and E2 is the optical density, respectively, the first and second samples vs. control.

The disadvantage of this method is that when it is used to determine the speed of Obrosova the emergence of sediment (suspended solids) after interaction in the discharge of substances with thiobarbituric acid, what affects the results of the measurement of optical density. There is no repeatable results when analyzing the same samples.

The present invention is the selection of the optimal amount of sample erythrocyte membranes of farm animals, which can be used for incubation, and clarification of the conditions of the subsequent stages to identify the reaction products of lipid peroxidation, which ultimately will ensure the repeatability of the determination of the reaction products and to improve the accuracy. This is a technical result.

The technical result of the invention is achieved by carrying out the water hemolysis of erythrocytes ice water, reduce the volume of the suspension of erythrocytes in experimental models (6.7 times in comparison with the prototype), use the hot thiobarbiturate acid for carrying out a color reaction and refinement of the calculation result.

The method is as follows:

- receive blood from the jugular vein of the animals with the use of anticoagulant;

the blood is centrifuged 10 min at 3000 rpm, precipitating erythrocytes;

- obtained sediment the erythrocytes promyrtil centrifugation;

- carry a complete hemolysis of erythrocytes: in a clean centrifuge tube taken at 0.5 ml sediment erythrocytes and poured a pipette, a strong jet of equal volume cooled to 0C distilled water and leave to stand for 30 min;

get samples of erythrocyte membranes - samples centrifuged 30 min at 3000 rpm and gently sucked off the supernatant with a gray layer of erythrocyte membranes and transferred to a clean tube;

- carry out the incubation of samples of erythrocyte membranes on each sample membranes prepared 3 tubes: in the first test tube add 0.6 ml of salt solution Mora (4x10-5M freshly prepared solution), ascorbic acid (2.6 mm freshly prepared solution), of 0.77 ml of Tris-buffer (Tris-Hcl buffer, 0.04 M solution with a pH of 7.4), and 0.03 ml of the obtained suspension of erythrocytes; the second vial is poured 0,77 ml of Tris-buffer, of 0.03 ml of the suspension of erythrocytes and 1.2 ml of distilled water; in the third (control) add the same reagents as in the first test tube, and then immediately poured 2 ml of 40% solution of trichloroacetic acid; the prepared sample is placed in thermostated and incubated for 20 min at S;

- stop the reaction by adding to the op who I color reaction - all samples centrifuged 15 min at 3000 rpm, then gently pipette with an elongated end select supernatant;

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

- measure the optical density of the experimental sample against a control fotoelektrokalorimetry at a wavelength of 540 nm (green filter) in a cell with a layer thickness of 1 cm and calculate the rate of formation of the final product changeover lipid oxidation - malondialdehyde by formulas

and ,

where X1 - rate education in a sample of MDA in the presence of prooxidants - salt Mora and ascorbic acid induced lipid peroxidation), nmol per hour; x2 - rate education in a sample of MDA in the absence of prooxidants (spontaneous lipid peroxidation), nmol per hour; 4,0 - total environment for incubation of the samples of the membranes, ml; 3,0 - volume of sample for carrying out a color reaction with thiobarbituric of cyclotide at 532 nm; E1 and E2 is the optical density, respectively, the first and second samples vs. control.

Experiments to determine the rate of formation of products of lipid peroxidation in erythrocyte membranes of farm animals was carried out on the girls two to six months of age farm Belgorodskoi scientific experimental station, Komi scientific center, Ural branch of RAS (, Syktyvkar).

Example 1. During the experiments it was noted that the contents of the samples after the reaction thiobarbiturate acid products, which were formed after incubation, the samples of erythrocyte membranes heifers at a rate of 0.1 ml to 1.0 ml of incubation medium or 0.2 ml to 2.0 ml of the medium when C for 20 min, was turbid and contained particles of sediment. Precipitate in the sample affected the results of the analysis by photometry. Therefore, a study was conducted on the rate of formation of products of lipid peroxidation in samples of erythrocyte membranes in heifers depending on volume changes their designs membranes taken to conduct incubation. Received that the maximum rate of formation of products of lipid peroxidation or malondialdehyde observed in ptx2">

Example 2. In the experiments proved the need for careful selection of the supernatant liquid after stopping the reaction with formation of products of lipid peroxidation in samples of membranes of erythrocytes of animals added to the incubation medium 40% trichloroacetic acid and then centrifuged for 15 minutes Noted that after completion of this reaction on the walls of the tubes a slight grayish tinge that the discharge of the contents of the tubes is easy to fall into the supernatant. In this regard, the magnitude of the rate of formation of products of lipid peroxidation 1.5-3.0 times higher than those obtained with careful sampling (table. 2).

Example 3. When using the method, taken as a prototype to determine the rate of formation of the reaction products of lipid peroxidation, is marked variation in the variables in a parallel study, a test membrane (PL. 3, animals # 1 and # 2). Our proposed method provides the optimal probability of coincidence of the results obtained from the analysis of the same samples of erythrocyte membranes in animals (PL. 3, animals No. 3-6).

Example 4. The method for determining the rate of formation of products of lipid peroxidation in erythrocyte membranes of farm animals used to identify products of lipid peroxidation in samples of erythrocyte membranes heifers contained with the first day of life in different conditions locomotion: forced motion on the unit with the driving floor - twice a day for 30 min with a speed of 20 m / minute or free content. Got that the rate of formation of TBA-active products in erythrocyte membranes heifers 125 days of age, on the first day of life in conditions of forced movement, 3.5 times higher than the rate of formation of products of lipid peroxidation in erythrocyte membranes from heifers 107 days of age, contained on the first day of life in the standard cells on the free content (table. 4.).

Thus, the proposed method of determining the rate of lipid peroxidation in erythrocyte membranes of farm animals can be used to identify specific features of functioning of red blood cell membranes of animals in p is temperature, the inclusion of new forage in the diet, and so on).

The method of determining the rate of formation of products of lipid peroxidation (LPO) of erythrocyte membranes of farm animals (Shi), including the production of sediment red blood cells from venous blood, carrying a full water hemolysis of erythrocytes, obtaining membranes by centrifugation, preparation of test samples on the basis of the water-suspended membranes and GENDER studies in the presence and without the presence in the experimental sample of prooxidants: salt solution Mora, ascorbic acid, and trichloroacetic acid precipitation (THU), followed by a color reaction with thiobarbituric acid (TBA) to measure the optical density at 532 or 540 nm and calculate results characterized in that the hemolysis of erythrocytes hold ice water, as samples taken erythrocyte suspension 6.7 times smaller than in the standard method for carrying out a color reaction using hot TBQ, and the calculation of the rate of formation peroxidation in erythrocyte membranes SHG produced by the formula

and ,

where X1 is the rate of formation of malondialdehyde (MDA) in the presence of prooxidants (induced BY the,156 - the level of extinction 1 nmol MDA at 532 nm;

E1 and E2 is the optical density, respectively, with addition and without addition of prooxidants.

 

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