SU1355933A1 - Method of detecting sialic acids in biological material - Google Patents

Abstract

This invention relates to biochemistry, is intended for the study of biochemical processes in the body of humans and animals, and for diagnostic and prognostic purposes in clinical practice. The purpose of the invention is to increase the sensitivity of the method. The studied material is hydrolyzed in a 0.05 M solution of sulfuric acid. at 80 С 1 h. At the same time, the release of sialic acids, which are part of the glycoproteins and glycolipids of the studied sample, occurs. After cooling, the hydrolyzate is subjected to periodate oxidation by adding a solution of 0.025 M of iodic acid in 0.125 M HCl to a final concentration of 5 mmol / l. Excess iodic acid, which interferes with the further determination of sialic acid, is destroyed by thiourea or acetylthiourea. The optical density of the colored upper organic phase was measured at 550 nm on a spectrophotometer. The concentration of sialic acid is calculated according to a calibration graph. 1 of FIG. CO ate with co stock

Description

The invention relates to the field of biochemistry, namely to the analysis of biological objects, and can be used in the study of biochemical processes in humans and animals, as well as in clinical practice for diagnostic and prognostic purposes.

The purpose of the invention is to increase the sensitivity of the method.

The drawing shows a calibration graph.

The method is carried out as follows.

The test material is hydrolyzed in a 0.05 M solution of sulfuric acid at 80 ° C for 60 minutes. In this case, the release of sialic acids, which are part of the glycopro teids and glycolipids of the test sample, occurs.

After cooling to room temperature, the hydrolyzate is subjected to periode oxidation. To do this, a freshly prepared solution of 0.5.025-M iodic acid in 0.125 M HC1 is added to a specific volume of hydrolyzate containing 3–25 nmol of sialic acid, to a final concentration of 5 mmol / l, and the sample is stirred for 30 minutes at 37 ° C.

An excess of iodic acid, which interferes with the further determination of sialic acid, is destroyed by thiourea or acetyl thiourea, which are added to the test sample at a concentration of 44-88 and 38-75 mmol / l, respectively. The sample is stirred, after which 39-50 µmol / l of thiobar are added. bituminous acid (pH 9.0), again mixed and placed in a boiling water bath for 7-10 minutes. Then the sample is cooled for 1–2 minutes in a water bath with ice and the same is kept in a thermostat at

J / C, after which the color is extracted

butanol containing 0,69-1,03 mol / o-phosphoric acid. The extraction was performed by vigorously shaking the sample, followed by centrifugation at 2000-3000 rpm for 3-5 minutes.

The colored upper organic phase is separated and its optical density is measured at 550 nm on. spectrometer photometer. The control in this case is a 0.05 M solution of sulfuric acid, treated in a similar way.

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The concentration of sialic acid calculated according to the calibration schedule.

The method is illustrated by the following examples.

Example 1. Quantitative determination of sialic acid in serum using thiourea

0.8 ml of a 0.05 M solution of sulfuric acid and 0.005 ml of the test serum are introduced into a centrifuge tube, covered with a glass stopper on top and placed in a thermostat at 80 ° C for 60 minutes. At the end of the hydrolysis, the sample is cooled to room temperature, 0.2 ml of a 0.025 M solution of iodic acid in 0.125 M HCl is added, stirred and held in a thermostat for 30 minutes at 37 ° C. Then 0.2 ml of 2.5% aqueous is added. thiourea solution, the sample is agitated, 1.0 ml of distilled water and 0.6 ml of a 0.2 M solution of thiobarbituric acid, brought to a pH of 9.0 with 1 M solution of NaOH, are added. The sample is stacked on top of a glass stopper and placed in a boiling water bath for 10 min. After this, the sample is cooled for 2 minutes in an ice-water bath and kept in the thermostat at 37 ° C for the same amount. The resulting colored product (chromogen) is extracted with 2 ml of n-butanol containing 5 by volume of 85% o-phosphoric acid. Extraction is carried out by vigorously shaking the sample, followed by centrifuging it at 3000 rpm for 5 minutes.

The optical density of the colored organic phase is measured at spectrum 7

The SF-26 rrophotometer at 550 nm against an idle sample, which is abrasive, as well as experienced, but does not contain short. Coloring is stable for several hours.

Since the volume of the organic phase is insufficient to fill the standard -. The optical cell (10 mm) of the SF-26 spectrophotometer, when measuring the optical density, requires an optical attachment for photometry of small volumes of the solution.

The concentration of sialic acids in the test serum is calculated by the formula

X a 0,2, (1)

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where a is the sialic acid found by calibration (in this graph, the amount of sialic acid, 93 nmol). Further, according to the form of the acid in the test sample, le (1) calculates the concentration of sialo, 2 is the conversion factor. howl acid in test serum

Dp build calibration gra- (3.59 mmol / l),

The phica is prepared with a series of dilutions of basicPerp 3. A quantitative solution of a standard solution containing the determination of sialic acids in erythro100 mg / l (323.3 mmol / l) sialic cells using thiourea.

acid. Each working dilution of Heparinized blood is centrifuged to form as described above. .Holdering at 3000 rpm for 7 min, plasma

This test is processed by Liu's analogs, and the upper layer of sediment of uniform Helen instead of the working dilution of sialo-agents, containing mainly

0.8 ml of 0.05 Mleukocytes are removed and removed. Then sediment

sulfuric acid solution. After the measurement, the erythryctes are thrice as low as 0.9%.

rhenium optical density of sodium chloride solution by

Build a calibration graph (see even-centrifugation at the described retej).

Optical density (D) exploring-B centrifuge tube taken

of the blood sample of the blood sample 0,300.20 - erythrocyte mass, added According to the calibration schedule, 0.7 ml of 0.9% NaCl solution is found.

the amount of sialic acids (in this 1 ml of 0.1 M solution of hydrochloric acid,

case, 81 nmol). Then, according to the form, they are thoroughly mixed, and the cover (1) calculates the concentration of sialo-yut on top of the glass stopper and of the strong acids in the sample 25 the tag is placed in a thermostat at 80 ° C

(2.16 mmol / l). for 60 min At the end of the hydrolysis

Example 2. A quantitative op-sample is cooled to room temperature with the definition of sialic acids in serum, after which 0.1 ml is added.

blood test using acetylthiomoce 30% K FeCCN GN 0, and

the guilt. 30 O, 1 ml of 40% aqueous solution of acetic acid Preliminary hydrolysis of the studied zinc, mix thoroughly

serum sample and perio-and centrifuged at 3000 rpm in

Data oxidation of the sample is carried out ana-for 7 minutes. In another clean process, in example 1. Then 0.8 ml of transparent liquid is taken into the tube, 0.8 ml of a 1.5% solution of the suction fluid, 0.2 ml is added.

acetyl thioureas, shake up, to -0.025 M sodium periodate solution in

Add 0.4 ml of distilled water with a 0.5 M solution of sulfuric acid and a 0.6 MP 0.2 M solution of thiobarbiturum; hold for 30 minutes at 37 C.

acid, made up with 1 M solution. Then 0.2 ml of 2.5% is added.

rum NaOH to a pH of 9.0, top it with a solution of thiourea, a sample of a glass-cap stirred and placed in a boil, add 1.0 ml of distilled

bath for 10 min. Then the water and 0.6 mp of 0.2 M thiobarprob solution are cooled for 2 minutes in a water bath of a bituminous acid brought to 1 M

with ice and as much. Hold with NaOH to pH 9.0. Sample on a thermostate at 37 ° C, add 2 rds on top of a glass stopper and ..

n-butanol containing 5% by volume is passed into a boiling water bath at

85% o-phosphoric acid, energy-10 min. Then the sample is cooled for 2 minutes

but they are shaken and centrifuged with a 5 min w ice water bath and as many

at 3000 rpm are kept in a thermostat at 37 ° С,

The optical density is abbreviated. 2 ml of n-butanol is added; the organic phase containing is measured on a 5% v / v spectrometer with an SF-26 85% o-phosphoric rrophotometer at 550 nm anti-acid, shaken vigorously and a centrifugal sample is processed. trifuge 5 min at 3000 rpm. c, as well as experienced, but the serum contains non-optical density of the colored. Color stable during the organic phase is measured on a spectra of several hours. With an SF-26 Photometer at 550 nm against

The optical density of the test sample that is used to process the serum sample. , 540. Pos, as experienced, but red blood cells are not

the calibration graph is found to contain - (instead of the erythrocyte mass

take 0.1 MP 059% solution of NaCl), the concentration of sialic acid in rocites is calculated by the formula

X a.25, (2)

where a is the amount of sialic acid found in the sample under calibration;

25 - conversion factor.

Optical density of the erythrocyte sample of the donor, 370. Calibration graphs show the amount of sialic acid (in this case, 32 nmol). Further, according to formula (2), the concentration of sialic acids in the test sample is indicated: erythrocytes. (333.0 μmol / l),

PRI me R 4. Quantitative determination of sialic acid in erythrocytes using acetylthio-h, evins.

Erythrocyte mass production, hydrolysis and periodate oxidation of the sample are carried out as in Example 3.

The excess of the periodate is destroyed by the addition of 0.8 ml of a 1.5% aqueous solution of acetylcholamide, the sample is agitated, 0.4 ml of distilled water and 0.6 ml of a 0.2 M solution of thiobarbituric acid made up with 1 M NaOH solution are added. to pH 9.0, cover the top. glass stopper and placed in a boiling water bath for 10. The sample is then cooled for 2 minutes in an ice-water bath and incubated for the same at 37 ° C, 2 ml of n-butanol containing 5% by volume 85% o-phosphoric acid is added, shaken vigorously and centrifuged 5 min at 3000 rpm

The optical density of the colored organic phase was measured on an SF-26 spectrophotometer at 550 nm against a blank, which is processed, just like the experimental one, but does not contain red blood cells (0.1 ml of 0.9% pacTBopa NaGl instead of erythrocyte mass) , Optical density of the sample under study: donor's erythrocyte sample, 315.

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Calibration graphs show the amount of sialic acid (in this case, 46 nmol). Then, according to formula (2), the concentration of sialic acids in the test sample, erythrocytes (261.5 µmol / l) is calculated.

The proposed method is quite specific, reproducible, gives the correct results, has greater sensitivity compared with the known method. The use of thiourea and acetyl thiourea increases the sensitivity of the proposed method by 20 and 26%, respectively.

This makes it possible to more subtly detect changes in the concentration of sialic acid in the body under various pathological conditions.

In addition, the proposed method is less toxic in comparison with the known one, since it excludes the use of highly toxic sodium arsenite, as well as a highly volatile, aggressive mixture of butanol with hydrochloric acid.

Form ula invention

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The method of determining sialic acids in a biological material by its hydrolysis with sulfuric acid, treating with iodic acid, destroying excess iodic acid, adding thiobarbituric acid, followed by extraction of the colored product with butanol, and spectrophotometrically, In order to increase the sensitivity of the method. a, destruction of excess iodic acid is carried out by adding thiourea at a final concentration of 44- 88 mmol / l or acetyl thiourea at a final concentration of 88-75 mmol / l,

thiobarbituric acid is taken at a concentration of 39-50 µmol / l, and o-phosphoric acid at a final concentration of 0,69-1,03 mol / l is additionally introduced into butanol.

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Claims (1)

Claim Method - determination of sialic acids in biological material by hydrolysis of it with sulfuric acid, treatment with iodic acid, destruction of excess iodic acid, addition of thiobarbituric acid, followed by extraction of the colored product with butanol and spectrophotometry, about characterized in that, in order to increase the sensitivity of the method, the destruction of the excess of iodic acid is carried out by adding thiourea in a final concentration of 4488 mmol / l or acetylthiourea in a final concentration of 88-75 mmol / l, thiobarbituric acid is taken in a concentration of 39-50 mmol / l, and o-phosphoric acid is additionally added to butanol in a final concentration of 0.691.03 mol / l.