Method for detecting oxidized tryptophan metabolites at endogenic intoxication

IPC classes for russian patent Method for detecting oxidized tryptophan metabolites at endogenic intoxication (RU 2249219):

G01N33/68 - involving proteins, peptides or amino acids

G01N30/90 - Plate chromatography, e.g. thin layer or paper chromatography

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Method for detecting oxidized tryptophan metabolites at endogenic intoxication / 2249219
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FIELD: medicine, biochemistry.

SUBSTANCE: at testing one should precipitate high-molecular compounds with acetonitrile and register supernatant's spectral characteristics. Supernatant should be applied onto a paper filter, dried and put into solution containing aromatic aldehyde, acetone and concentrated hydrochloric acid taken at weight ratio of 70:5:1 to be kept for 2-3 min. Then it should be once again dried up to detect qualitative and semiquantitative content of oxidized tryptophan metabolites by intensity and chromatic shades. Moreover, by chromatic shades of yellow dyeing it is possible to detect the content of hydroxylated metabolites and by chromatic shades of violet dyeing - that of unhydroxylated ones.

EFFECT: higher significance of detection.

3 ex

The present invention relates to the field of medicine, namely biochemistry, and can be used to assess the severity of endogenous intoxication.

When endogenous intoxication observed poisoning of the body as the end products of metabolism due to delay their removal and accumulation in the body above the physiological norms and intermediate due to metabolic processes (Endogenous intoxication in acute surgical diseases / edited Unelaborate, You. - Yaroslavl: DIA - press, 2000. - s).

There is a method of detection of oxidized products of tryptophan by thin-layer chromatography on silica gel using luttich systems, for example the acetate - isopropanol - 25% ammonia in the ratio of 9:7:4 or chloroform - acetic acid in the ratio 95:5. Manifestation spend reagent van orca containing 0.125 g of the aldehyde, 0.1 ml of 5%ferric chloride and 100 ml of 65%sulfuric acid.

The disadvantages of this method include duration analysis and the need to use special equipment (plates for thin-layer chromatography, a set of reagents for the elution and manifestations, sprayer).

The closest in technical essence to the present invention is a method of determining the content of toxic the x products of low and medium molecular weight in biosubstrate, including deposition of high molecular weight whey proteins and recording the spectral characteristics of the supernatant in the wavelength range 210-300 nm (Malakhov MY Methods of biochemical registration of endogenous intoxication // Efferent therapy.- 1995, vol. 1, No. 1, pp.61-64). Check the spectrum of serum in the ultraviolet region allows you to conduct a comprehensive assessment of toxic products and more than 200 different substances produced by normal and disturbed metabolism. Calculation of number of substances of low and medium molecular weight is carried out by the formula

where:- the amount in conventional units (cu),

E₂₁₀-E₃₀₀the magnitude of optical density at a given wavelength.

The total analysis time is 50 minutes.

The disadvantages of this method include obtaining General characteristics of substances circulating in the blood that belong to different classes of compounds and determining the profile of the spectrogram. This fact restricts the information about the composition, which in turn does not give a complete picture of the severity of endogenous intoxication and does not allow for full implementation of the required remedial measures.

The objective of the proposed technical solution is the development of pic is BA, allows you to quickly and objectively identify the composition of heterocyclic compounds, forming on the spectrogram peak at 280 nm and evaluate their content in biological objects.

The technical result of the proposed method is to improve the accuracy and detection sensitivity by providing identification of oxidized products of tryptophan - tryptamine, O-aminophenol, indole and skatole in biological environments, as well as simplifying and reducing the time of analysis.

The technical result is achieved in that the method of determination of oxidized metabolites of tryptophan in endogenous intoxication includes sampling, precipitation with acetonitrile macromolecular compounds and obtaining the supernatant. Quantitative and qualitative content of oxidized metabolites of tryptophan conducted by the spectral characteristics of the supernatant.

The distinctive techniques of the proposed method are: the supernatant is applied on filter paper, dried, immersed in a solution containing an aromatic aldehyde, acetone and concentrated hydrochloric acid, taken in a weight ratio of 70:5:1, incubated for 2-3 minutes, then re-dried. Intensity of staining judge qualitative and quantitative composition of metabolites.

Techniques Nan the value of the supernatant on a paper filter, drying and placed in a solution containing an aromatic aldehyde, acetone and concentrated hydrochloric acid, taken in a weight ratio of 70:5:1, achieved stable fixation and expression of the studied substances on paper.

The intensity of staining of oxidized metabolites of tryptophan allows to reliably determine the qualitative and quantitative composition of heterocyclic compounds.

The authors of the proposed method is not known and the available literature is not found the use of such composition reagent for analysis of oxidized products of tryptophan - tryptamine, O-aminophenol, indole, skatole.

All these differences allow to draw a conclusion on the conformity of the proposed technical solution the criteria of the invention of “novelty.”

Comparison of the proposed technical solution is not only the prototype, but also other technical solutions in biochemistry not allowed to reveal in them the features distinguishing the claimed solution not only from the prototype method, but also from other similar technical solutions.

We have not found information that was said about the definition of oxidized metabolites of tryptophan in endogenous intoxication techniques of the proposed method.

The proposed method allows to obtain perceived by the applicant, the technical result improved accuracy of the detection sensitivity of the content of oxidized metabolites of tryptophan in biological environments and reducing the time of analysis.

This allows to conclude that the technical solutions according to the criterion of “inventive step”.

The method constituting the invention, intended for use in medicine, namely in biochemistry. The possibility of its fulfillment is confirmed as described in the application techniques and equipment, therefore, the proposed solution meets the criteria of the invention “industrial applicability”.

The inventive method is as follows.

After deposition of high-molecular compounds of a sample of blood serum is applied on the filter paper in the amount of 2-10 µl and dried in air, and then placed in a mold with projavitelnyi solution. The composition of the developer: aromatic aldehyde, acetone and concentrated hydrochloric acid, taken in a weight ratio of 70:5:1. The exposure is carried out in 2-3 minutes, followed by re-drying in the air.

In the presence of oxidized products of tryptophan spot samples turn from purple to yellow. Intensity and color shades judge qualitative and quantitative composition of metabolites. So about the degree of hydroxylation is judged by the intensity of yellow colour. The intensity of staining increases in the number of mono-, di-, tioxaprofen. The intensity of staining dehydrosilybin met is Bolotov tryptophan serves as a quantitative criterion of their content in the serum, this discoloration is from slightly blue-violet to bright purple.

As for hydroxylated, and do not contain hydroxyl groups of metabolites of tryptophan composed calibration scales, which give the opportunity to determine pilgrammage number of analyzed metabolites.

The total analysis time is less than 30 minutes. The proposed method is illustrated by examples of specific performance.

Example 1.

Patient N., the diagnosis of pancreatic necrosis, widespread purulent peritonitis. The total content of substances of low and medium molecular weight, as determined by the method prototype is as follows: in the lymph 15,177 conventional units (cu), in serum - 9,389 USD Received values to 1.27 times their level in healthy man. The content of substances at 280 nm was particularly high in the lymph and serum, which exceeded the donor's serum 1.6 times. These figures indicate severe clinical course of the disease.

The analysis by the present method allowed to detect the presence of compounds with lemon-yellow coloration, which were identified as derivatives of skatole. Semiquantitative assessment is characterized +++.

The presence of oxidized metabolites by the present method in the lymph and serum of the patient, also the et on the presence of intermediate products of metabolism and characterized by profound disturbances of metabolic processes and heavy endotoxicosis, that demonstrates the need for the application of methods of extracorporeal detoxification.

Example 2.

Patient S Diagnosis of iron deficiency anemia. The content of substances of low and medium molecular weight (UNSM) in serum was 10,296 USD, which is 1.3 times higher than their content from the donor. The identification of these substances at a wavelength of 280 nm was 1.82 times higher than that of the donor.

The analysis by the proposed method allowed to detect the presence of compounds dominant gray-violet color, which was verified as derivatives of indole with an aldehyde group. Semiquantitative their content assessed ++. From the above it follows that the claimed method in the serum was determined a significant level of oxidized products of tryptophan, indicating that the expression of endogenous intoxication.

Example 3.

Patient C. with a diagnosis of CHD, 3 rd functional class angina and rest. When entering the total content VNSM in serum was 10,218 USD, at 280 nm - 0,895 that 1.32 and 2.28 times respectively higher than the level of the donor.

The use of the proposed method revealed that in the serum of the patient there are connections, giving violet staining, indicating the presence of indolyl-3-acetic or indolyl-3-propionic acid. According to the intensity of the color and its content is consistent with +++. Therefore, the proposed method has revealed the presence of the intermediate products of metabolism - oxidized compounds of tryptophan, which indicates the severity of the patient with severe endogenous intoxication.

1.5 months after treatment fetal therapy metabolic status of the patient has clearly improved: the percentage of total VNSM halved and amounted to $ 5,088, the peak in the spectrogram at 280 nm decreased 3 - fold decreased levels of oxidized products of tryptophan. The claimed method detected trace coloring compounds, which indicated a sharp decrease in the content of products perverted metabolism.

3 months after treatment, although a common pool VNSM slightly increased slightly increased content of aromatic substances structure defined at 280 nm. However, these measures were within the values of the donor. I would especially like to note the correlation decreases oxidized products of tryptophan with the clinical condition of the patient.

Results were examined in the dynamics of 156 patients, including 20 patients with pancreatic necrosis, 80 with coronary heart disease, 16 with iron deficiency anemia, 34 - with malignant tumors of the large intestine, 6 with acute leukemia. In all patients, the study of oxidized metabolites of tryptophan conducted simultaneously is built by the present method and the prototype. The total content of toxic products in the blood serum, as determined by the method prototype (if admitted to hospital), amounted to 12.3 and 10.8 USD Received data was not able to determine the qualitative composition of compounds circulating in biological media, in the field of 270-280 nm. Research by the present method has allowed to establish that the 20 patients with pancreatic necrosis, 30 with malignant tumors of the colon and 6 with acute leukemia in serum was present connection, giving lemon-yellow staining intensity +++that indicated the presence of derivatives of skatole and testified to the deep metabolic processes and heavy endotoxicosis. 20 patients of coronary artery disease and 10 with iron deficiency anemia proposed method revealed that the serum contains substances that give gray-purple staining intensity ++verified as derivatives of indole with an aldehyde group.

Analysis of the claimed method from 30 patients with coronary artery disease, 6 with iron deficiency anemia were given purple color and testified to the presence of oxidized products of tryptophan.

The proposed method allows you to quickly and cost-effectively determine the presence of oxidized heterocyclic compounds, which are toxic products that cause deep the e metabolic disorders in the body of the patient, until lesions of various organs and systems.

The method of determination of oxidized metabolites of tryptophan in endogenous intoxication, including sampling, precipitation with acetonitrile macromolecular compounds and recording the spectral characteristics of the supernatant, wherein the supernatant is applied on filter paper, dried, immersed in a solution containing an aromatic aldehyde, acetone and concentrated hydrochloric acid, taken in the ratio of 70:5:1, incubated for 2-3 min, then re-dried and intensity and hue determine the qualitative and semiquantitative content of oxidized metabolites of tryptophan, with color shades of yellow staining determine the content of hydroxylated metabolites and color shades of purple coloring - content dehydrosilybin metabolites.