SU905787A1 - Method of determination of adenosintriphosphorous acid in blood - Google Patents

Description

one

The invention relates to a technique for determining adenosine triphosphate acid (ATP) in the blood and can be used in biochemical analyzes in research and clinical laboratories, mainly in the study of individual links of the adenyl system.

The closest solution to the invention is a method for determining ATP in blood by preparing a protein-free centrifugate sample with the subsequent addition of hexokinase to the reaction mixture. The accuracy of the known method 10-15% U.

The disadvantage of this method is its complexity, since the method for determining ATP is based on a multistage method and on the repeated measurement of optical densities. In addition, expensive reagents and equipment are required to determine ATP.

The purpose of the invention is to simplify the method.

The goal is achieved by the fact that according to the method of determining ATP in blood by preparing a protein-free centrifugal sample followed by the addition of hexokinase to the reaction mixture, glucose, glucose oxidase: Schm method is added after the hexokinase addition and without its addition, the resulting samples are triple with standard glucose solution, and ATP is determined by the formula

,

ti

F

st

where C is ATP concentration, mg.%, K is ATP conversion factor; The expression of the control sample Eg-extinction of the sample after the addition of hexokinase. E ..- extinction standard solution. The method is carried out in the following manner. The quantitative determination of LTP is carried out by reducing the glucose content in erythrocytes, which occurs as a result of the reaction of glucose + ATP to glucose 6-phosphate + ATP. The process is catalyzed by the enzyme hexokinase. The ATP concentration was calculated according to the formula where C is the amount of ATP, mg%, K is the ATP conversion factor} the extinction of the control sample E is the extinction of the sample after the addition of hexokinase; EL.-extinction of standard solution. The amount of glucose is determined using a gozo-oxidative method. Determination of ATP content in errocytes is carried out as follows. The bled blood is transferred to a cooled to 2-4C tube containing 3-4 drops of heparin solution. C trifugate at 4-2 ° C W min at 2500-3000 rpm. The plasma is aspirated and ejected. The tubes are placed in an ice bath. A double object of a 15% solution of trichloroacetic acid (TCA) is added to the compacted erythrocyte sediment in a test tube. After 0 min, centrifuged again for 10 min at 3000 rpm M solution of triethanolamine STEA buffer (pH 7.5-7.6), 0.35 ml of 0.1 M solution of MgCl4, 0.4 ml of the protein-free centrifugate. The contents of the test tubes are adjusted to pH 7.4 by 1-2 m capsules mi of 5 M solution of CLSO. Then 0.05 ml of I, O-1.5% solution of hexokinase is added. A similar procedure is carried out with the control where, instead of a solution of hexokinase, 0.05 ml of TEA buffer is taken.The control and experimental samples are placed in a thermostat for 20-30 min at 37 C. Then, the glucose content is measured in samples at 90 ° C (control and experiment) take 4.5 ml of distilled water, add 3 ml of dianisidine reagent (3 ml of 0.4 M potassium phosphate buffer at pH 5.9) and 1 ml of a% alcohol solution of 0-dianisidine is added and brought to water up to 100 ml, the reagent is prepared on the day of analysis), 0.3 ml of the solution from the corresponding test tubes is poured into the control and test tubes. In the previous step of the analysis, 0.1 ml of freshly prepared 0.1% glucose oxidase solution, 0.1 ml of freshly prepared 0.1% peroxidase solution or 0.5% hemoglobin solution are added and incubated for 30 minutes at 45 ° WITH. After incubation, 2 ml of 50% sulfuric acid solution is added to the tubes. At the same time, a standard solution of 4.7 ml of distilled water, 3 ml of dianisidine reagent, 0.03 ml of 100 mg% glucose solution, 0.1 ml of 0.1% glucose oxidase solution, 0.1 ml of r, 1% is prepared. a solution of peroxidase incubated for 30 min at 45 ° C. After incubation, 2 ml of a 50% sulfuric acid solution is added. After cooling the contents of the tubes to room temperature, the extinction of the solutions is measured on a photoelectrocolorimeter in a cuvette with a distance between working faces of 10 mm. The measurement of optical density is conducted against a compensation liquid containing 5 ml of water, 3 ml of dianisidine reagent and 2 ml of a 50% aqueous solution of sulfuric acid, with a green light filter. The calculation of the content is carried out according to the above formula, while the minimum ATP content of the proposed method is 15% and the accuracy of determining ATP is 12-14%. According to the proposed method, 65 studies of the ATP content in erythrocytes of rats were conducted. The study was conducted with 13 healthy animals (control group) and with 52 lead poisoned rats. In the control group, the following ATP content in red blood cells was obtained, mg%: 37.5; 44.5; 48.7; 50.5; 55.4; 60.8; 69.0; 70.9; 86.2; 94.0; 101.4; 111.5; 114.1. The average ATP content is 72.7 mg /%.

The average error of the arithmetic mean ± 7,4 mg%.

The standard deviation is ± 26.5 mg%.

The coefficient of variation is 36.5%.

Marked variability should be attributed to the individual characteristics of the animal, in particular

due to the different levels of exchange in the red blood cells at the time of decatisation of rats,

The results of determining the ATP content in spectrocytes in lead-poisoned rats at various times after the windows of the seed are shown in the table.

13 Notes M is the arithmetic mean of the mean, the arithmetic quadratic deviation. Thus, the ATP concentration in the spectrocytes of the rats of the control group is 77 mg%. In the last periods of lead intoxication, the level of ATP in the erythrocytes of white rats increases. Example. The freshly bled blood of a laboratory animal (rat), 4-8 ml, is placed in a test tube cooled to and moistened with heparin. To isolate red blood cells, the sample is centrifuged for 10 minutes at 2500 rpm. The plasma separated by centrifugation is sucked off. To precipitate proteins, spectrocytes are mixed with a pre-cooled 15% solution of TCA in a 1: 1 ratio (1.5 ml of erythrocytes and 1.5 ml of TCA), and the tube is placed for 10 minutes in an ice bath, and then centrifuged again for 10 min at 2500 rpm The obtained protein-free centrifugate is collected in a clean tube. Reagents are poured into two test tubes (control and test) to conduct hexokinase peaKiyiH. Then both tubes are placed in a thermostat at 37 ° C for 20 minutes. As a result, the hexokinin solution is added to the test tube of hexokinase.

13 - the number of observations; i m is the average error, g is the average .. hexokinase to the reaction: glucose + ATP pt-g of leukemia-6-phosphate + ATP, as a result of which glucose is consumed with spectrocytes. In the control vial, the glucose level does not change, since hexokinase is absent in the reactive system. The TEA buffer used (0.05 m, pH 7.5-7.6) is prepared as follows. 4.65 g of triethanol-amine hydrochloride are dissolved in 200 ml of distilled water and 11 ml of 1N is added. NaOH solution, then the resulting solution is brought to 500 ml with distilled water. After thermostating, the glucose content in the control and test tubes is determined by the glucose oxidase method. To do this, from the control and test tubes, take 0.3 ml of the solution contained in them and pour them into test tubes in which 4.5 ml of distilled water and 3.0 ml of dianisidine reagent are added. The pH is then checked and, if necessary, adjusted to 5.9, and 1 ml of a 0.1% glux-zooxidase solution and a 0.1% peroxidase solution are poured into both tubes in O. At the same time

Claims (1)

Claim __ A method for determining adenosine triphosphoric acid (ATP) in the blood by preparing a protein-free · centrifuge sample, followed by adding hexokinase to the reaction mixture, characterized in that, in order to simplify the method, glucose is determined by the glucose oxidase method after the addition of hexokine-α. without adding it, the obtained samples are colorimetric with a standard glucose solution, and ATP ^ is determined by the formula ” 44-¾). C is the concentration of ATP> K is the conversion factor of ATP, mg%; E ^ - extinction of the control sample ·, E _about - extinction of the sample after adding hexokinase; Εςγ- extinction of a standard solution.