RU2256920C2 - Acetylcholine determination method - Google Patents

Abstract

FIELD: analytical methods in biology.

SUBSTANCE: invention relates to chemical analysis methods for biological materials and, in particular, concerns a method for determining acetylcholine in biological specimens consisting in incubation of a specimen, centrifugation, take-off of supernatant, preparation of alkaline hydroxylamine reagent by adding equal volume of 3.5 n NaOH to hydroxylamine chloride solution, mixing of thus prepared reagent with test specimen, and addition of iron chloride solution followed by measurement of optical density. Invention is characterized by that, before addition of 3.5 n NaOH, hydroxylamine chloride solution is mixed with 10-14% solution of Na₂CO₃, incubation of specimen is performed after the latter is mixed with alkaline hydroxylamine reagent at 1:2 ratio for 90-120 min at 33-37°C, centrifugation is preceded by adding aqueous hydrochloric acid solution, and 0.74 M iron chloride solution is added to taken off supernatant.

EFFECT: increased determination accuracy.

2 tbl, 3 ex

Description

The invention relates to methods for the chemical analysis of biological materials and can be used to determine the content of acetylcholine in biological samples.

A known method for the determination of erythrocyte acetylcholinesterase, adopted as an analogue, is that acidic citrate dextrose is added to whole blood (1).

A known method of certain acetylcholine is adopted as a prototype, which includes diluting a solution of hydroxylamide chloride and adding a 3.5 N NaOH solution, mixing with the analyzed sample and incubating, mixing with hydrochloric acid and staining with a solution of iron chloride, followed by measurement of optical density (2).

However, the prototype method has limited accuracy in the determination of acetylcholine.

The aim of the invention is to improve the accuracy of determination of acetylcholine content in biological samples.

The technical result is achieved in that in the preparation of an alkaline hydroxylamine reagent, an additional 10-14% solution of Na ₂ CO ₃ is additionally used, and incubation is carried out at a temperature of 33-37 ° C for 90-120 minutes, followed by centrifugation of the acidified solution.

The method is as follows.

In a solution of hydroxylamine chloride (14%) add 10-14% solution of Na ₂ CO ₃ and mixed in equal volumes with a 3.5N NaOH solution. An alkaline hydroxylamine reagent is obtained. Mix with the analyzed homogenized sample in a ratio of 2: 1. Incubate in a thermostat at a temperature of 33-37 ° C for 90-120 minutes. It is mixed with hydrochloric acid and centrifuged at 3000-5000 rpm for 8-12 minutes. The supernatant is collected and an 0.74 M solution of iron chloride is added in equal amounts. The optical density is measured spectrophotometrically at a wavelength of 450-480 microns. At the same time, standard acetylcholine solutions are determined to construct a calibration curve.

The method is confirmed by the following examples.

Example 1. Take a sample of the mucous membrane of the rat’s stomach, weighing 50.0 mg, homogenize in 1.0 ml of distilled water, centrifuge for 15 minutes at 5000 rpm. The supernatant was taken in a volume of 0.5 ml of a 14% solution of hydroxylamine chloride, an additional 10% solution of Na ₂ CO _{3 was} added and mixed in equal volumes with a 3.5N NaOH solution to obtain an alkaline hydroxylamine reagent. The reagent is mixed with the analyzed sample in the ratio of 0.5 ml of sample and 1.0 ml of alkaline hydroxylamine reagent. Incubate in a thermostat at a temperature of 35 ° C for 120 minutes. It is mixed with hydrochloric acid 1.0 ml and centrifuged at 5000 rpm for 10 minutes. 0.5 ml of the supernatant was collected and 0.5 ml of a 0.74 M solution of iron chloride was added. The optical density of the sample is measured spectrophotometrically at a wavelength of 480 μm. Each determination is carried out with the definition of standard solutions of acetylcholine 0.1; 0.2; 0.3; 0.4 mg / ml to build a calibration curve for calculating the acetylcholine content in the determined sample.

Example 2. Take 1.0 ml of human gastric juice. To a 14% solution of hydroxylamine chloride, a 14% solution of Na ₂ CO _{3 was} added and mixed in equal volumes with a 3.5N NaOH solution. Mix with the analyzed sample and the ratio of 1.0 ml of sample and 2.0 ml of alkaline hydroxylamine reagent. Incubate in a thermostat at a temperature of 33 ° C for 90 minutes. It is mixed with 1.0 ml of hydrochloric acid and centrifuged at 4000 rpm for 8 minutes. 5 ml of the supernatant were collected and 0.5 ml of a 0.74 M solution of iron chloride was added. The optical density is measured spectrophotometrically at a wavelength of 450 μm. A calibration curve of acetylcholine solutions is built (0.1; 0.2; 0.3; 0.4 mg / ml) to calculate the acetylcholine content in the determined sample.

Example 3. Take 0.5 ml of human serum. In a 14% hydroxylamine chloride solution, a 12% Na ₂ CO ₃ solution was added and mixed in equal volumes with a 3.5 N NaOH solution. An alkaline hydroxylamine reagent is obtained. Mix with the analyzed sample in the ratio of 0.5 ml of sample and 1.0 ml of alkaline hydroxylamine reagent. Incubate in a thermostat at a temperature of 37 ° C for 100 minutes. It is mixed with hydrochloric acid and centrifuged at 3000 rpm for 12 minutes. 0.3 ml of the supernatant was collected and 0.3 ml of a 0.74 M solution of ferric chloride was added. The optical density is measured spectrophotometrically at a wavelength of 470 μm. Each determination was carried out taking into account the calibration curve of standard acetylcholine solutions (0.1; 02; 0.3; 0.4 mg / ml). The acetylcholine content in the determined sample is calculated.

The reproducibility of the method is illustrated in table 1.

Table 1 Sample Number Measurement number Measurements M ± m 1 1 0.480 0.478 ± 0.003 2 0.473 3 0.480 2 1 0.475 0.475 ± 0.003 2 0.480 3 0.470 3 1 0.484 0.482 ± 0.002 2 0.480 3 0.481 4 1 0.482 0.479 ± 0.002 2 0.476 3 0.478 5 1 0.484 0.482 ± 0.001 2 0.480 3 0.482

As can be seen from table 1, the quantitative indicators of the content of acetylcholine in the 5 compared samples do not differ significantly, which proves the high reproducibility of the method.

Comparison of the accuracy of determination of acetylcholine content when using the inventive and known methods are shown in table 2.

table 2 Sample Number The proposed method Known method 1 0.480 0.390 2 0.482 0.278 3 0.484 0.400 4 0.476 0.420 5 0.481 0.370 6 0.478 0.345 7 0.485 0.420 8 0.479 0.340 nine 0.481 0.400 10 0.480 0.365 M ± m 0.481 ± 0.002 0.373 ± 0.024

From table 2 it follows that the accuracy of determination of acetylcholine by the claimed method in comparison with the prototype method is higher by 29%.

Sources of information

1. Vinnitskaya K.B., Kuznetsov V.A. Laboratory science, 1985, No. 7, 396-400.

2. Kamyshnikov V.S. Handbook of clinical and biochemical laboratory diagnostics, pp. 472-474.

Claims (1)

A method for determining acetylcholine, including incubating the analyzed sample, centrifuging, supernatant collection, preparation of the alkaline hydroxylamine reagent by adding to the solution hydroxylamine chloride in an equal volume of 3.5 N NaOH solution, mixing the resulting alkaline hydroxylamine reagent with the analyzed sample, adding hydrochloric acid solution, staining with the chloride solution iron with subsequent measurement of optical density, characterized in that in the preparation of an alkaline hydroxylamine reagent before adding a 3.5 N NaOH solution, the hydroxylamine chloride solution is mixed with a 10-14% Na ₂ CO ₃ solution, the test sample is incubated after mixing it with the obtained alkaline hydroxylamine reagent for 90-120 minutes at a temperature of 33-37 ° C while the alkaline hydroxylamine reagent is mixed with the analyzed sample in a ratio of 2: 1, hydrochloric acid solution is added before centrifugation and 0.74 M solution of iron chloride is added to the selected supernatant.