SU1737337A1 - Method for determination of adenosine triphosphoric and 2,3-diphosphoglyceric acids in human blood - Google Patents

Abstract

The invention relates to the field of medicine and biology, namely to the field of photometric laboratory and clinical studies of human blood, and can be used to diagnose various diseases in which the concentrations of ATP and 2,3-diphosphoglyceric acid in erythrocytes change. The aim of the invention is to simplify the method of determination. To do this, blood samples are processed at room temperature, and the blood sample is incubated in physiological saline for 24 hours at 37 ° C before the addition of trichloroacetic acid, followed by the determination of 2,3-diphosphoglyceric acid. 1 tab.

Description

The invention relates to medicine and biology, in particular to photometric laboratory and clinical studies of human blood, and can be used to diagnose various diseases in which ATP and 2,3-diphosphoglycerate (2,3-DPG) concentrations change in red blood cells.

The aim of the invention is to simplify the method.

The method is carried out as follows.

From the patient's finger take 0.1 ml of blood into a test tube with 0.5 ml of 0.9% sodium chloride solution (test tube No. 1) and 0.1 ml of blood into a test tube with 0.9 ml of 5% trichloroaceous acid (tube number 2). Tube N ° 1 was placed in a thermostat for 24 hours at 37 ° C. After incubation, add 0.4 ml of 10% trichloroacetic acid to it, mix thoroughly and centrifuge with tube No. 2 for 15 min at 3 thousand revolutions per minute. Take 0.2 ml of the supernatant from tube No. 2 into the tube with 0, 2 ml of 2N HCI (tube No. 3) and boil it in a water bath for 7 minutes in order to hydrolyze ATP to AMP. Then neutralize the contents of the tube Ms 3 by adding to it 0.2 ml of 2 n solution of NAOH. The inorganic phosphorus in the supernatant from each tube is determined. For this, 1 ml of 5% ammonium molybdate solution in 5% sulfuric acid and 1 ml of 1% solution are added to 0.5 ml of the supernatant from tubes No. 2 and No. 3 (to 0.25 ml of the supernatant from tube No. 1). a solution of ascorbic acid on 0.1 n hydrochloric acid. After 15 min, the optical densities of all samples were measured on a photoelectric colorimeter at a wavelength of 670 nm versus reagent control. Phosphorus concentrations in all samples are calculated from the calibration curve and are expressed in mmol x per liter.

Calculation of ATP and 2,3-DFG concentrations (mmol / l of blood):

ATP (30 Rz - Yu P2) / 2;

2,3-DFG (20P1-ZORz) / 2,

cl

with

vi

SA) VI CJ CO VI

where Pi, P2, P3 are the concentrations of phosphorus in the supernatant from tubes No. 1, No. 2, No. 3, respectively.

Having determined the hematocrit, it is possible to calculate the concentration of ATPi2,3-DFGv in erythrocytes by dividing the obtained values of their concentrations in the blood by the hematocrit value in the SI system.

PRI me R. In practically healthy people (20 people), ATP and 2,3-DFG concentrations in erythrocytes were determined according to the proposed method and prototype. The data obtained are presented in the table.

As can be seen from the obtained data, the differences between the average values of the indices determined by the proposed method and the prototype are not significant (p 0.1). The correlation coefficients between the values of the parameters determined by different methods were 0.87 (p 0.01) for ATP and 0.81 (p 0.01) for 2,3-DFG, which indicates a good agreement between the results obtained by the proposed method and prototype.

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The proposed method allows the determination of ATP and 2,3-DPG concentrations in erythrocytes from a small volume of blood taken from a finger. The method differs from the similar ones by simplifying the method, reducing its labor intensity without loss of accuracy and can be implemented in any clinical laboratory for diagnosing various diseases.

Claims (1)

The invention of the method for determining adenosine triphosphate and 2,3-diphosphoglyceric acid in human blood by adding trichloroacetic acid to a blood sample, mixing, adding 2 N HCI to the supernatant, incubating for 7 minutes in a boiling water bath, neutralizing it, followed by determining inorganic phosphorus to calculate concentrations of adenosine triphosphate and 2,3-diphosphoglyceric acids, characterized in that, in order to simplify the method, the second blood sample is incubated in a thermostat for 24 hours at 37 ° C for efosforilirova- audio organic phosphate by the action of enzymes own erythrocytes.