Method for determining oxidative protein modification in substance pool of average molecular weight in blood serum, plasma, erythrocyte, and in urine

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to clinical biochemistry, and aims at determining oxidative protein modification in a substance pool of an average molecular weight in a biological medium accompanying any pathological conditions by biochemical examination. The biological medium specified in the blood plasma, erythrocyte or urine is sampled; proteins are deposited by adding 10% trichloroacetic acid; if observing sedimentation, a centrifugation cycle follows at 1000 rpm for 15 minutes; thereafter, 2,4-dinitrophenylhydrazine 0.05 M in hydrochloric acid 2 M is added; the sample is centrifuged at 1000 rpm for 20 minutes; and if observing sedimentation, the sediment is washed twice in ethanol-ethylacetate (1:1), dried on a water bath for 10 minutes and dissolved in urea 8 M; the sample is kept in a boiling water bath for 10 minutes until dissolved completely; the prepared solution is analysed by spectrophotometry. The method is applicable both for single use, and for monitoring of the postoperative oxidation protein modification and average molecule levels.

EFFECT: method provides higher information value of biochemical tests, reducing consumption of the biological material.

9 tbl, 2 ex

 

The invention relates to medicine, in particular for clinical biochemistry and is intended for the determination of oxidative modification of proteins in the pool substances average molecular weight (average molecules) serum or blood plasma, erythrocytes, urine under any pathological conditions by biochemical tests.

Currently widely studied mechanisms of lipid peroxidation and its role in the normal functioning of cells and the pathogenesis of various diseases. However, reactive oxygen species can cause free-radical oxidation not only of lipids and proteins that makes a significant contribution to the formation of oxidative stress of any Genesis. The study of oxidative modification of proteins in the pool substances average molecular weight, which can manifest, and oxidant and Pro-oxidant properties that will allow us to further explore the mechanisms of development of kriticheskih States and deepen clinical and laboratory diagnosis of the severity of endotoxemia [1].

A known method for determining endogenous intoxication [2], which consists in determining the level of molecules of average weight in the serum. However, this method does not allow to assess the degree of oxidative modification of serum proteins, the essential contribution which contribute substance average molecular weight.

The most effective is m to the claimed invention is a biochemical method for determining protein oxidative modification of human serum [3], consisting of a reaction between the oxidized amino acid residues of proteins with 2,4-dinitrophenylhydrazine (2,4-FGD) with the formation of derivatives of 2,4-dinitrophenylhydrazine.

This method involves drawing blood on an empty stomach, then to 0.1 ml of serum prilisaetsa 20% solution of trichloroacetic acid. To the denatured protein is added an equal volume (1 ml) 0.1 M 2,4-FGD dissolved in 2 M HCl. In the control sample is added instead of the 2,4-FGD equal volume of 2 M HCl. Incubation is carried out at room temperature for 1 hour. Then the samples centrifuged at 3000 rpm for 20 minutes. The precipitate is washed 3 times with a solution of ethanol-ethyl acetate (1:1) for the extraction of lipids and 2,4-FGD, which did not react with carbonyl groups in oxidized proteins. The precipitate is dried to eliminate the remaining solvent ethanol-ethyl acetate and then dissolved in 8 M urea solution. Urea is poured to the precipitate in a volume of 2.5 ml and incubated in a boiling water bath for 5 minutes until dissolved. The optical density of the formed dinitrophenylhydrazones recorded on the spectrophotometer at the following wavelengths: 356, 370, 430, 530 nm.

However, this method is only one biological environment - serum, only one parameter of oxidative modification of proteins.

There is also the technical difficulty is one character: this reagent, as 0.1 M 2,4-FGD dissolved in 2 M HCl, to make difficult because of the practical insolubility of 2,4-FGD - he always precipitates. You can conduct heat, but, over time, the sediment again falls.

The task of the invention is to increase the informativeness of biochemical tests of molecules of average weight (MSM) and oxidative modification of proteins (BIP) in serum, erythrocytes by examining them together in the same sample and reducing the consumption of biological material. The proposed method can work in other biological media, plasma, urine. Thus, the application of this invention will enable a greater understanding of the mechanisms of oxidative modification of proteins in patients in critical conditions due to a comprehensive study of basic biological marker environments used in clinical diagnostic laboratories, blood serum, erythrocytes, and urine. The serum can be replaced by the plasma.

The technical result of the claimed invention is the determination of oxidative modification of proteins in the pool substances average molecular weight serum, erythrocytes, characterized in that both samples produce the definition of middle-sized molecules, and oxidative modification of proteins, being the m dissolution denaturirovannykh proteins produced in 0.05 M solution of 2,4-FGD (dinitrophenylhydrazine) in 2 M HCl (hydrochloric acid), then the sample is centrifuged at 1000 rpm for 20 minutes, then the precipitate is washed 2 times with a mixture of ethanol-ethyl acetate (1:1), after drying and dissolution which stand the sample in boiling water for 10 minutes. In the method for determining the oxidative modification of proteins in the pool substances average molecular weight instead of blood serum as a biological material can be used in the blood plasma.

Also the problem is solved by a method for determining the oxidative modification of proteins in urine pool substances average molecular weight, including the sampling of biological media, the deposition of proteins, centrifugation, addition of urea solution, characterized in that a urine sample for determination of oxidative modification of proteins take 0.5 ml of liquid from the upper part of the tube, then treated with 0.05 M solution of 2,4-FGD 2 M HCl, the sample was centrifuged at 1000 rpm for 20 minutes.

The inventive method is carried out as follows. Produce blood collection by venipuncture on an empty stomach. The blood is centrifuged to separate the serum from the formed elements.

Spend the dilution of serum in 10 times (in glass tubes, including all stages hereinafter in glass tubes) by adding 0.1 ml of serum, 0.9 ml of saline. Then to precipitate white what s in the sample prilisaetsa 0.5 ml of 10% solution of trichloroacetic acid and stirring. Spend centrifugation for 15 minutes at 1000 rpm.

The supernatant liquid is taken in the amount of 0.5 ml was added to 4.5 ml of distilled water and determine the optical density of the resulting solution at a wavelength of 254 nm. The value of the extinction of the sample corresponds to the level of molecules of average weight, the content of which is expressed in units of optical density (adept).

To the precipitate denaturirovannykh protein add 1 ml of 0.05 M 2,4-FGD dissolved in 2 M HCl. Incubation is carried out at room temperature for 1 hour. Then the samples are centrifuged at 1000 rpm for 20 minutes.

The precipitate is washed successively with 2 times with a mixture of ethanol-ethyl acetate (1:1) for the extraction of lipids and 2,4-FGD, which did not react with carbonyl groups in oxidized proteins. After each washing with a solution of ethanol-ethyl acetate (1:1) residue dried on a water bath for 10 minutes to eliminate the remaining solvent ethanol-ethyl acetate and then dissolved in 2.5 ml of 8 M urea solution, soaking the samples in a boiling water bath for 10 minutes to dissolve.

Similarly spend stroke method with urine and red blood cells. However, the urine has a specific character.

The peculiarity of the method in the urine. For the analysis we take no precipitate denaturirovannykh proteins, because he, as a rule, poorly formed, and 0.5 ml of p is obliki. Stage wash solution ethanol-ethyl acetate (1:1) absent. Then do everything in accordance with the method: add 2.5 ml of 8 M urea solution, soaking the samples in a boiling water bath for 10 minutes.

To determine the oxidative modification of proteins in the pool substances average molecular weight in erythrocytes, first the red blood cells obtained from the blood as follows: blood is centrifuged at 3000 rpm for 15 minutes, the plasma is sucked off, and the red cells washed 3 times with saline solution volume : 9 ml RBC mass is used in accordance with the progress of the method. Blood should be taken with anticoagulant, such as heparin or Trilon B.

Implementation of the method can be implemented in the form of a clinical assay kit. In this case, the description of the method use the following notation: reagent A - 10% solution of trichloroacetic acid; reagent B - 0.05 M 2,4-FGD dissolved in 2 M HCl; the reagent In a solution of ethanol-ethyl acetate (1:1); the reagent G - 8 M urea solution.

The optical density of the formed dinitrophenylhydrazones recorded on the spectrophotometer at the following wavelengths: 356, 370, 430, 530 nm (against water). Healthy individuals without clinical signs of any disease (control group - 13 people aged 32,69±2,99 years) indicators of this parameter, the levels of MSM are presented in table 1. Values more than these levels (upper limit of normal range) allow to speak about the increased levels of these parameters, indicating that the increase of endogenous intoxication, manifested, including increased oxidative modification of proteins in the analyzed biological environments.

Analysis of OMB definitions at different wavelengths shows that the greatest differences in their levels in different biological environments are at 530 nm. This allows us to recommend this wavelength as needed base. However, depending on the available in this clinical diagnostic laboratory equipment, you can choose any of these wavelengths, or use them all (356, 370, 430, 530 nm) in the complex.

Table 1.
The magnitude of molecules of average weight (MSM) and the levels of oxidative modification of proteins (BIP) in the control group norm.
Biochemical testsBiological environment
SerumErythrocytesUrine
MSM, units of optical density0,13±0004 0,15±0,0040,31±0,03
OMB, units of optical density (356 nm)0,325±0,030,38±0,030,57±0,09
OMB, units of optical density (370 nm)0,435±0,020,41±0,020,66±0,07
OMB, units of optical density (430 nm)0,64±0,050,49±0,020,65±0,035
OMB, units of optical density (530 nm)0,175±0,020,45±0,030,26±0,01

Below are clinical examples of the use of the proposed method.

Example 1. Patient W., 62, entered the City clinical hospital of emergency medical aid to them. Gasparina Penza (CCH SMP them. Gasparina) diagnosed with chronic cholecystitis.

The patient was performed venipuncture and collection of morning urine, on an empty stomach. The condition of the patient and preparation for laparoscopic cholecystectomy (LCE) required a comprehensive laboratory examination. To assess the expression of endogenous intoxication determined the levels of MSM and oxidative modi the paths protein (BIP) in the serum, erythrocytes in pre-LCE, immediately after LCE, on the 1st and 5th day of observations in the urine until LCE, 1 and 5 days after surgery. To obtain serum and erythrocytes taken the blood was divided into two portions: in the test tube, which was scheduled receipt of erythrocytes was added EDTA.

Oxidative modification of proteins in the human serum pool substances average molecular weight was determined as follows.

1. In the serum.

Taken, the blood was centrifuged to separate the serum from the formed elements. Spent the dilution of serum in 10 times (in glass tubes, including all stages below) by adding 0.1 ml of serum, 0.9 ml of saline. Then for the deposition of protein in the sample was added 0.5 ml of 10% solution of trichloroacetic acid and stirring. Conducted centrifugation for 20 minutes at 1000 rpm.

The supernatant liquid was collected in the amount of 0.5 ml was added to 4.5 ml of distilled water and determined the optical density of the resulting solution at a wavelength of 254 nm. The value of the extinction of the sample corresponded to the level of molecules of average weight, the content of which is expressed in units of optical density (adept).

To the precipitate denaturirovannykh protein was added to 1 ml of 0.05 M 2,4-FGD dissolved in 2 M HCl. Incubation was carried out at room is the temperature for 1 hour. Then the sample was centrifuged at 1000 rpm for 20 minutes.

The precipitate was washed successively with 2 times with a mixture of ethanol-ethyl acetate (1:1) for the extraction of lipids and 2,4-FGD, which did not react with carbonyl groups in oxidized proteins. After each washing with a solution of ethanol-ethyl acetate (1:1) precipitate was dried on a water bath for 10 minutes to eliminate the remaining solvent ethanol-ethyl acetate and then dissolved in 2.5 ml of 8 M urea solution, soaking the samples in a boiling water bath for 10 minutes to dissolve.

2. In erythrocytes.

For analysis in erythrocytes blood (taken with EDTA) was centrifuged at 3000 rpm for 15 minutes, the plasma was aspirated, and the erythrocytes were washed 3 times with saline solution (9 ml). Next, similarly conducted the course of the method, as in the serum.

3. In the urine.

The move method is generally similar to its implementation in the serum. However, to determine the OMB did not take precipitate denaturirovannykh proteins, because it was poorly formed, and 0.5 ml of liquid from the tube. Stage wash solution ethanol-ethyl acetate (1:1) was absent. Then did everything in accordance with the method of adding 2.5 ml of 8 M urea solution, soaking the samples in a boiling water bath for 10 minutes.

The optical density of the formed dinitrophenylhydrazone (serum shelter is, erythrocytes, urine) were recorded on a spectrophotometer at the following wavelengths: 356, 370, 430, 530 nm (against water). The results obtained are presented in tables 2-4.

Dynamics of molecules of average weight (MSM) and oxidative modification of proteins (BIP) was analyzed on the background of the generally accepted criteria of endogenous intoxication - leukocyte index of intoxication (LII) and nuclear index shift (table 5).

Table 2.
The magnitude of molecules of average weight (MSM) and the levels of oxidative modification of proteins (BIP) in the blood serum of the examined patient with chronic cholecystitis.
Biochemical testsNormaThe level parameter values
DolhaImmediately after LCE1 day after LCE5 days after LCE
MSM, units of optical density0,13±0,0040,171780,136190,157610,17495
OMB, units of optical density (356 nm) 0,325±0,030,1580,2180,2260,121
OMB, units of optical density (370 nm)0,435±0,020,3040,2470,2260,299
OMB, units of optical density (430 nm)0,64±0,050,2510,1560,1810,241
OMB, units of optical density (530 nm)0,175±0,020,2280,0920,0710,143

Table 3.
The magnitude of molecules of average weight (MSM) and the levels of oxidative modification of proteins (BIP) in erythrocytes have examined a patient with chronic cholecystitis
Biochemical testsNormaThe level parameter values
Until LCEImmediately after LCE1 day after LCE5 days after LCE
MSM, units of optical density0,15±0,0040,2380,2450,210,233
OMB, units of optical density (356 nm)0,38±0,030,2910,2610,3040,27
OMB, units of optical density (370 nm)0,41±0,020,2610,2830,3150,258
OMB, units of optical density (430 nm)0,49±0,020,6840,5880,610,494
OMB, units of optical density (530 nm)0,45±0,030,930,881,050,66

Table 4.
The magnitude of molecules of average weight (MSM) and the levels of oxidative modification of proteins (BIP) in urine have examined a patient with chronic cholecystitis.
Biochemical testsNormaThe level parameter values
Until LCE1 day after LCE5 days after LCE
MSM, units of optical density0,31±0,030,238530,557870,18253
OMB, units of optical density (356 nm)0,57±0,090,3080,5340,313
OMB, units of optical density (370 nm)0,66±0,070,7180,4580,3
OMB, units of optical density (430 nm)0,65±0,0350,4231,0840,509
OMB, units of optical density (530nm) 0,26±0,010,280,3120,414

td align="left"> The index of the nuclear shift
Table 5.
Leukocyte index of intoxication and the index of the nuclear shift in the examined patient with chronic cholecystitis.
Biochemical testsNormaThe level parameter values
Until LCEImmediately after LCE1 day after LCE5 days after LCE
LEAH on Calf-Caliph1,59±0,094,133,385sm 1,8571,703
LEAH on Ostrovsky1,79±0,063,3482,846sm 1,8571,703
LEAH on the chemical1,09±0,062,8792,3341,3740,749
0,02±0,0040,13240,104500

The obtained survey data testified to the presence of endogenous intoxication in patients that revealed elevated levels of LEAH to 1 day of observations, on the 5th day after the operation, their values were close to normal (except for LII on the chemical, which was below the norm by 1.5 times).

While the levels of MSM and BIP serum, erythrocytes and urine showed themselves as more sensitive tests of endotoxemia: to 5 days of observations the number of their figures differed from the norm. MSM were increased in the serum - 1.4 times, in erythrocytes 1.6 times in urine - 1.7 times. The level of BIP at 530 nm, offered by us, as a basic wavelength for studies in serum was less than the standard 1.2 times, erythrocytes and urine is above 1.5 times and 1.6 times, respectively. This may indicate some imbalance BIP in the studied biological environments of the organism in chronic cholecystitis.

Example 2. Patient K., 51, entered the City clinical hospital of emergency medical aid to them. Gasparina Penza (CCH SMP them. Gasparina) with a diagnosis of acute cholecystitis.

The patient was performed venipuncture and collection outran the th urine, on an empty stomach. Heavy condition of the patient and preparation for laparoscopic cholecystectomy (LCE) required a comprehensive laboratory examination. To assess the expression of endogenous intoxication determined the levels of MSM and oxidative modification of proteins (BIP) in serum, erythrocytes at stages immediately after LCE, 3 and 5 days of observations in the urine for 5 days after surgery. To obtain serum and erythrocytes taken the blood was divided into two portions: in the test tube, which was scheduled receipt of erythrocytes was added EDTA.

Oxidative modification of proteins in the human serum pool substances average molecular weight was determined as follows.

1. In the serum.

Taken, the blood was centrifuged to separate the serum from the formed elements. Spent the dilution of serum in 10 times (in glass tubes, including all stages below) by adding 0.1 ml of serum, 0.9 ml of saline. Then for the deposition of protein in the sample was added 0.5 ml of 10% solution of trichloroacetic acid and stirring. Conducted centrifugation for 15 minutes at 1000 rpm.

The supernatant liquid was collected in the amount of 0.5 ml was added to 4.5 ml of distilled water and determined the optical density of the resulting solution at a wavelength of 254 nm. Values of the extinction of the sample corresponded to the level of molecules of average weight, the contents of which are expressed in units of optical density (adept).

To the precipitate denaturirovannykh protein was added to 1 ml of 0.05 M 2,4-FGD dissolved in 2 M HCl. Incubation was carried out at room temperature for 1 hour. Then the sample was centrifuged at 1000 rpm for 20 minutes.

The precipitate was washed successively with 2 times with a mixture of ethanol-ethyl acetate (1:1) for the extraction of lipids and 2,4-FGD, which did not react with carbonyl groups in oxidized proteins. After each washing solution ethanol-ethyl acetate (1:1) precipitate was dried on a water bath for 10 minutes to eliminate the remaining solvent ethanol-ethyl acetate and then dissolved in 2.5 ml of 8 M urea solution, soaking the samples in a boiling water bath for 10 minutes to dissolve.

2. In erythrocytes.

For analysis in erythrocytes blood (taken with EDTA) was centrifuged at 3000 rpm for 15 minutes, the plasma was aspirated, and the erythrocytes were washed 3 times with saline solution (9 ml). Next, similarly conducted the course of the method, as in the serum.

3. In the urine.

The move method is generally similar to its implementation in the serum. However, to determine the OMB did not take precipitate denaturirovannykh proteins, because it was poorly formed, and 0.5 ml of liquid from the tube. Stage wash solution ethanol-ethyl acetate (1:1) was absent in the. Then did everything in accordance with the method of adding 2.5 ml of 8 M urea solution, soaking the samples in a boiling water bath for 10 minutes.

The optical density of the formed dinitrophenylhydrazone (serum, blood, urine) were recorded on a spectrophotometer at the following wavelengths: 356, 370, 430, 530 nm (against water). The results obtained are presented in tables 6-8.

The dynamics of MSM and BIP were analyzed on the background of the generally accepted criteria of endogenous intoxication - leukocyte index of intoxication (LII) and nuclear index shift (table 9).

The obtained survey data testified to the presence of endogenous intoxication in patients that revealed elevated levels of LEAH to 3 days of observation, on the 5th day after the operation, their values were close to normal (with the exception of LEAH on Calf-Caliph, a decrease of 2.2 times, and the index of nuclear displacement, increased 4.2 times).

While the levels of MSM and BIP serum, erythrocytes and urine showed themselves as more sensitive tests of endotoxemia: to 5 days of observations the number of their figures differed from the norm. MSM were increased in erythrocytes - 1.3 times, in the urine - 1.9 times. The level of BIP at 530 nm, offered by us, as a basic wavelength for the study was above normal in erythrocytes and urine - 1.4 times and 1.9 times, respectively. It may Weiterstadt about the imbalance in the BIP in the studied biological environments of the organism in acute cholecystitis.

Table 6.
The magnitude of molecules of average weight (MSM) and the levels of oxidative modification of proteins (BIP) in the blood serum of the examined patient acute cholecystitis.
Biochemical testsNormaThe level parameter values
Immediately after LCE3 days after LCE5 days after LCE
MSM, units of optical density0,13±0,0040,2670,2480,1297
OMB, units of optical density (356 nm)0,325±0,030,4420,5540,521
OMB, units of optical density (370 nm)0,435±0,02of 0.3370,319 0,655
OMB, units of optical density (430 nm)0,64±0,050,5830,7080,63
OMB, units of optical density (530 nm)0,175±0,020,2050,280,16
Table 7.
The magnitude of molecules of average weight (MSM) and the levels of oxidative modification of proteins (BIP) in the red blood cells in the examined patient acute cholecystitis.
Biochemical testsNormaThe level parameter values
Immediately after LCE3 days after LCE5 days after LCE
MSM, units of optical density0,15±0,0040,2050,21580,1905
OM is, units of optical density (356 nm)0,38±0,030,3420,3310,299
OMB, units of optical density (370 nm)0,41±0,020,4190,3560,302
OMB, units of optical density (430 nm)0,49±0,020,4350,50,485
OMB, units of optical density (530 nm)0,45±0,030,4220,456of 0.615

Table 8.
The magnitude of molecules of average weight (MSM) and the levels of oxidative modification of proteins (BIP) in the urine in the examined patient acute cholecystitis.
Biochemical testsNorma The level parameter values
5 days after LCE
MSM, units of optical density0,31±0,030,5808
OMB, units of optical density (356 nm)0,57±0,091,231
OMB, units of optical density (370 nm)0,66±0,071,226
OMB, units of optical density (430 nm)0,65±0,0351,2
OMB, units of optical density (530 nm)0,26±0,010,486
Table 9.
Leukocyte index of intoxication and the index of the nuclear shift in the examined patient acute cholecystitis.
Biochemical testsNormaLevel meant the th parameters
Immediately after LCE3 days after LCE5 days after LCE
LEAH on Calf-Caliph1,59±0,092,7862,6250,725
LEAH on Ostrovsky1,79±0,062,5712,1251,778
LEAH on the chemical1,09±0,061,1571,3180,924
The index of the nuclear shift0,02±0,0040,04350.1330,0847

Using the proposed method is examined 12 patients with cholecystitis, including 5 patients with acute and 7 patients with chronic cholecystitis. These patients, who were held laparoscopic cholecystectomy (LCE). Observations in the studied groups were conducted before and immediately after LCE, 1, 3, 5 day post-operative period.

Research conducted by the and clinical basis of the Department of anesthesiology and intensive care and emergency medical services (Arise) State budgetary educational institution of additional professional education "Penza Institute the physicians of the Ministry of health and social development of the Russian Federation (state budgetary educational institution of additional professional education of PIUV the health Ministry of Russia) in City clinical hospital of emergency medical aid to them. Gasparina Penza (CCH SMP them. Gasparina) in the surgical Department. Preparation of biological material for analysis was carried out in the Express lab, Department of resuscitation and intensive therapy CCH SMP them. Gasparina, also one of the clinical bases of the Department Arise state budgetary educational institution of additional professional education of PIUV the health Ministry of Russia.

Values MSM was determined on a spectrophotometer SF-2000, the level of BIP was estimated using KLF-3 in the laboratory of the Department of biochemistry PSPU them. Vchelyshkov.

The proposed method allows to increase the information content of the biochemical test - determination of the level of molecules of average weight in serum, plasma, erythrocytes and urine, which is one of the most commonly used markers of endogenous intoxication. This proposal will expand the understanding of the mechanisms of oxidative modification of proteins in patients in critical States, in particular, to use it to Express the laboratories of the departments of intensive care, for a small amount of the obtained biological medium (blood, urine) and time of analysis.

Sources of information:

1 Ryabov GA, Azizov, Y.M., Dorokhov SR, Kulabukhov V.V., Titov I.A., Pasechnik, I.N., Soto T.B., Citizens VY Oxidative modification of proteins in blood plasma of patients in critical States // Anesthesiology and resuscitation. - 2000. No. 2. - p.72-75.

2. The invention "METHOD of determination of ENDOGENOUS INTOXICATION" patent No. 2193780, priority from 26.04.2001, published 27.11.2002.

3. Dubinin E.E., Burmistrov S.O., Moves D.A., Porotov I.G. Oxidative modification of proteins in human serum, the method of its determination // Questions of medical chemistry. - 1995. No. 1. - p.24-26.

The method for determining the oxidative modification of proteins in the pool substances average molecular weight in the biological environment, characterized by the fact that produce fence biological environment, selected from blood plasma, blood or urine precipitate proteins by adding a 10% solution of trichloroacetic acid, and in the case of sediment spend centrifugation at 1000 rpm for 15 minutes, then add 0.05 M solution of 2,4-dinitrophenylhydrazine in 2 M hydrochloric acid, after which the sample is centrifuged at 1000 rpm for 20 minutes, and in the case of precipitation the precipitate is washed 2 times with a solution of ethanol-ethyl acetate (1:1), then dried on a water bath for 10 minutes and then dissolved in 8 M urea solution, soaking the samples in a boiling water bath for 10 minutes to fully dissolve the texts, subsequent analysis of the solution by spectrophotometric method.



 

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1 tbl, 4 dwg

FIELD: medicine.

SUBSTANCE: method is based on a method of observing turbidimetric fibrin clot formation with optical transmission of an incubation medium recorded by ultraviolet radiation band 230 to 320 nm by means of UV-spectrophotometre as a fibrin-polymer detector.

EFFECT: invention enables higher accuracy and sensitivity of the method.

4 ex, 4 dwg

FIELD: medicine.

SUBSTANCE: for thrombin production measurement, a layer of said sample contacts with a fluorogenic substratum of thrombin where the thickness of said layer is 0.05 to 5 mm, while the surface area is 10 to 500 mm2. Further, the thrombin production environment in said sample is provided. It is followed by measuring the fluorescence emitted from the layer surface by a fluorescent group released by the fluorescent substratum as a result of an enzymatic action of produced thrombin on said fluorogenic substratum. Besides, the invention ensures a kit for measuring the thrombin activity in the sample.

EFFECT: higher measuring accuracy.

29 cl, 12 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: blood is examined. A hematocrit level (H), erythrocyte count (E), thrombocyte count (T) are determined. Said parametres are evaluated. In the event if they keep within the determined limits for the patients with acute coronary syndrome (ACS), then adenosine phosphate induced (ADP-induced) clotting time test samples are prepared. Citrated blood sample 0.4 ml is prepared of whole blood and divided on two samples 0.2 ml. Each of these samples is introduced in a measuring cell, recalcified at temperature 37°C for 2 minutes. Then a magnetic ball mixer is placed in each cell. The measurement is activated, and in three seconds the ADP solution 0.1 ml is introduced. After a clotting reaction, a process time duration is recorded separately for each sample. An arithmetical mean of the derived values is calculated (A). The derived values of each of said parameters are scored. Total score Σ=A+H+E+P shows the risk of recurrent thrombotic events. If Σ=4 points, the low risk is observed; the value Σ=5-6 points shows the medium risk, while Σ=7-10 points - the high risk.

EFFECT: method provides more objective risk evaluation of recurrent thrombotic events in the patients with ACS with its simplicity and low cost.

1 ex, 1 tbl

FIELD: mining.

SUBSTANCE: proposed method comprises tripping the video camera in hole with turbid medium on logging cable over tubing. Logging zone is isolated by packer and web in tubing suspension wherein suspended rock is settled by gravity. Visualised logging is executed in column of laminated optically clear fluid by displacement of video camera inside tubing suspension. Logging results are used to estimate operating conditions of the hole. If required, extra web is used to confine the logging zone. In compliance with first version, logging device comprises tubing suspension with packer separating the logging zone from hole behind-the-packer chamber, logging cable, video camera suspended from logging cable inside the suspension. Web is hooked to video camera to fit in suspension seat and to be detached at camera tripping in hole along suspension to logging zone. Web and seat in suspension are furnished with locking elements. Web has a central hole with gland for logging cable to slide there over and can have filter cells. Said web features OD smaller than tubing ID. Video camera is equipped with centring skid. Logging cable is coated with polyamide shell over logging length. In compliance with second version, logging device comprises tubing suspension with packer separating the logging zone from hole behind-the-packer chamber, logging cable, video camera suspended from logging cable inside the suspension. Web is secured to video camera to slide by its edges over suspension wall. Video camera is equipped with centring skid. Gland seals are arranged over web edges. Said web can be made of filter material. Said web features OD smaller than tubing ID.

EFFECT: lower costs, accelerated analysis.

15 cl, 2 dwg

FIELD: metallurgy.

SUBSTANCE: proposed method comprises revealing the dependence between first parameter, an aluminium oxide porous layer depth index, and colour parameter, an index of light reflected from said porous layer. Getting of first info, tolerance for said first parameter of aluminium oxide porous layer that features structure irregular within tolerances. It includes the step of making the mould to be controlled. Note here that said mould has porous layer of aluminium oxide on it surface. It includes the step of getting the colour parameters, an index of light reflected from said porous layer of controlled mould. Besides it comprises the step of revealing the fitness of the first controlled mould parameter proceeding from obtained colour parameter and first colour info.

EFFECT: non-destructive easy control over fine irregular structure.

7 cl, 9 dwg, 2 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to analytical chemistry, namely to photometric methods of determining rare earth elements in natural objects and technical materials. method includes decomposition of sample by its melting with mixture of anhydrous soda and borax, processing decomposed sample with hydrochloric acid, precipitation of metal hydroxides, washing residue of metal hydroxides with ammonium hydroxide, elimination interfering impact of titanium compounds by addition of hydrogen peroxide before precipitation of hydroxides, elimination of interfering impact of iron and aluminium by masking iron by addition of ascorbic acid and masking aluminium by addition of sulfosalicylic acid, conversion of non-soluble compounds of rare earth elements into soluble compounds, transfer of rare earth elements into dyed compounds with arsenazo III and further photometry.

EFFECT: invention makes it possible to reduce time for analysis performance, as well as reduce labour consumption of analysis and increase its accuracy.

5 cl, 1 tbl

FIELD: chemistry.

SUBSTANCE: described is a method for spectrophotometric determination of metal cations in aqueous solutions using photochromic organic compounds selected from spiropyrans and chromenes, the method being based on placing ashless filter paper into a metal salt solution, placing said paper into acetonitrile after drying, holding said paper therein and removing it, and adding a solution of a photochromic compound in acetonitrile to the obtained solution of metal ions.

EFFECT: simple method which does not require synthesis of water-soluble photochromic functionalised compounds or polymers and enables to use a wide range of already synthesised photochromic spiro compounds and chromenes to determine content of metal ions in aqueous solutions.

1 tbl, 9 ex, 4 dwg

FIELD: instrument making.

SUBSTANCE: spectrometer comprises the following components serially arranged on one optical axis: a source of light emission with a continuous spectrum, a collimator, a polariser, a cylindrical lens or a cylindrical mirror, a device of disturbed full internal reflection with a reflecting element, a dispersing device, a focusing lens and a light-sensitive photomatrix installed in the lens focus.

EFFECT: possibility to produce a spectrum of surface plasmon resonance in a continuous optical range of wavelengths in real time mode and in increased sensitivity of a device.

6 cl, 6 dwg

FIELD: chemistry.

SUBSTANCE: method involves treating a water sample with potassium iodide solution; successive measurement of optical density of a chlorine dioxide sample at pH=7 and chlorite ions and chlorine dioxide at pH=2.5; determining from calibrated curves concentration of chlorine dioxide at pH=7 and total concentration of chlorite ions and chlorine dioxide at pH=2.5; calculating concentration of the chlorite ions using the formula: (C216,86C167,46)×16,86, where C1 is concentration of chlorine dioxide at pH=7, mg/dm3; C2 is total concentration of chlorine dioxide and chlorite ions at pH=2.5, mg/dm3; 67.46 is the oxidative equivalent of chlorine dioxide corresponding to pH=7; 16.86 is the oxidative equivalent of chlorite ions corresponding to pH=2.5.

EFFECT: invention enables to cut analysis time and increases reliability of results thereof.

FIELD: food industry.

SUBSTANCE: caviar (W) is loaded onto the charging tray (6), light from the light radiator (11) is directed at the caviar (W), the image of at least part of the caviar (W) (while the caviar (W) is being irradiated with the light radiator (11) light) is taken with the help of an image taking device (12). The image (Pa) taken by the image taking device (12) is processed, one measuring the transmission parameter of light passing through the caviar (W) and determining the caviar (W) maturity relying on the measured light transmission parameter.

EFFECT: one can accurately determine caviar maturity using the described caviar maturity device and method.

17 cl, 3 dwg, 10 ex

FIELD: measurement equipment.

SUBSTANCE: invention is based on usage of at least one sensor reacting at availability of a detected substance and which is radiated with at least one source of light and at least one photodetector.

EFFECT: invention makes it possible to detect availability and/or concentration of a detected substance without usage of complex optoelectronic equipment.

15 cl, 13 dwg

FIELD: measurement equipment.

SUBSTANCE: in calibration device with at least one sensor and with at least one exciting source, a control device to control the measurement system, a calibration medium and a drive that is intended to move the calibration medium under control of the control device in measurement system calibration mode to the position, in which the calibration medium is on the trajectory of beams in the measurement system, and when being changed over to the operating mode to check the analysed objects, and mainly bank-notes, to the position out of the trajectory of beams in the measurement system, calibration medium has more than one reference, and drive has the possibility of moving the calibration medium under control of the control device to the position, in which the calibration medium is on the trajectory of beams in the measurement system and one of the references of the calibration medium covers the active zone of the measurement system and in which the measurement system measures properties of that reference located in its active zone, as well as the possibility of at least single movement of calibration medium under control of the control device to the specified distance or to the distance that is divisible by that specified distance, to the position in which the active zone of the measurement system covers the other reference and in which the measurement system measures the properties of that reference located in its active zone; at that, the above specified distance corresponds to length of individual references of calibration medium in the direction, in which the drive moves the calibration medium.

EFFECT: invention allows providing accurate calibration at lower costs.

5 cl, 4 dwg

FIELD: measurement equipment.

SUBSTANCE: use of two calibration media is provided at calibration of the measurement system. The first high-quality calibration medium (10) with a standard reference, which does not depend on the calibrated measurement system, is used once, for example at manufacture or after repair of the measurement system at the first calibration stage, at which the measurement system is calibrated and adjusted. At the next calibration stage, the second calibration medium with a non-standard reference is used, which is a component of the measurement system, for the purpose of shaping and storing measurement signals from the second calibration medium (6) at least with one sensor (3) of the measurement system. In the calibration mode, when performing subsequent calibration processes, only the second calibration medium is used for the purpose of repeated shaping of measurement signals from the second calibration medium with sensor (3). In case of discrepancies between measurement signals formed at subsequent calibrations and stored measurement signals formed at the second calibration stage, sensor (3) is adjusted; for that purpose, correction coefficients compensating the detected differences are determined.

EFFECT: invention ensures the possibility of accurate and long-term stable calibration.

2 dwg, 7 cl

FIELD: veterinary science.

SUBSTANCE: the method deals with irradiating urine, milk or cow's udder with visible light waves and/or long-wave infrared beams ranged 400-2500 nm, measuring the intensity of passed, passed/reflected light beams followed by multi-elemental, regression analysis. Either presence or absence of mastitis should be detected based upon the spectra of mentioned light beams. Device includes a light source, a spectroscope, an optic modulator, integrating sphere and a holder for samples The present innovation enables to rapidly and accurately predict mastitis in cows.

EFFECT: higher efficiency.

11 cl, 10 dwg

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