RU2210775C1 - Method for predicting hyperhistaminemia - Google Patents

Abstract

FIELD: medicine, biochemistry. SUBSTANCE: as biological material one should apply human saliva; centrifuge tubes containing 4 ml 10%-trichloroacetic acid solution are supplemented with 1ml saliva and centrifuged, one should add 0.1 ml 57%-perchloric acid and 5 ml ether to 4 ml extract, ether is thrice removed, ether is added and shaken, then samples are supplemented with 2 g potassium carbonate and 5 ml butanol, after centrifuging process 4 ml organic phase is removed and passed through chromatographic column at 3 mm diameter and 10 mm height with ion-exchange resin inside it, one should elute 2 ml 0.2 H hydrochloric acid, then eluate is supplemented with 0.5 ml diazure-active, 1 ml sodium carbonate and 0.15 ml 5 H sodium hydroxide. Then due to colorimetric technique one should measure optic density and by calibrating curve detect histamine concentration. EFFECT: higher efficiency of diagnostics.

Description

 The invention relates to medicine, to biochemical studies and methods for diagnosing hyperhistaminemia.

 The definition of histamine is widely used in the diagnosis of various pathological processes.

 It is known that under the influence of histamine, a biogenic amine that is involved in neurohumoral regulation of the tone of blood vessels and organs with smooth muscles, the permeability of the vascular wall increases sharply, digestive secretion and excretory glands become more active, spasm of smooth muscle cells occurs, and connective tissue grows in parenchymosis lungs, liver, etc. There is an allergization of the human body.

 Factors such as hypoxia, trauma, emotional stress, x-rays, and other radiation lead to the release of a large amount of histamine in the blood.

 The closest in technical essence and the achieved result is a method for determining the histamine content in whole blood (modification S. Meshcheryakova) (Kamyshnikov BC Handbook of clinical and biochemical laboratory diagnostics. - Minsk: Publishing house "Belarus", 2000, volume 2, p. 442-444).

 The principle of the known method is based on the extraction of histamine from blood with perchloric acid, purification from impurities by extraction with a mixture of butanol and chloroform, and transfer to the aqueous phase with which the orthophthaldehyde reagent is reacted.

 The condensate is stabilized with phosphoric acid and fluorimetric; the concentration of histamine is quantified by the luminescence intensity.

 A disadvantage of the known method for determining the histamine content is that, as the biological material that people take when determining histamine, use blood, the sampling of which from a vein is traumatic for humans, damage to the blood vessel, sometimes with subsequent circulatory disorders, infection in blood. These circumstances, as well as pain and negative emotions, often cause patients to refuse examination, especially in cases where a series of tests with short intervals of time is necessary.

 In addition, when taking human blood from a vein, a specially prepared room with trained medical personnel, sterile instruments and disposable syringes, as well as such expensive equipment as fluorimeters, are required to determine the concentration of histamine.

 The technical result, the achievement of which the creation of this invention is directed, is to increase the efficiency of the diagnostic method by simplifying and cheapening the process of detecting histamine, and as a result, diagnosing hyperhistaminemia.

 The technical result is achieved by the fact that in the method of diagnosing hyperhistaminemia, based on the collection of biological material and determining the concentration of histamine in it, human saliva is used as biological material for collection, in which a change in the concentration of histamine has a positive correlation with a change in the concentration of histamine in the blood , in a centrifuge tube containing 4 ml of a 10% trichloroacetic acid solution, 1 ml of saliva was added, extracted, 0.1 ml was added to 4 ml of the extract 57 % perchloric acid, 5 ml of ether, ether is aspirated three times, ether is added and shaken, then 2 g of potassium carbonate and 5 ml of butanol are added to the samples, after centrifugation, 4 ml of the organic phase are aspirated and passed through a chromatographic column with a diameter of 3 mm and with a height of 10 mm and with an ion-exchange resin placed in it, then 0.5 ml of 4% sodium nitrite is added to the eluate, and after heating and subsequent cooling of the eluate, 0.5 ml of diazoreaktiv, 1 ml of sodium carbonate and 0.15 ml are added 5 n. sodium hydroxide, then the optical density is measured by the colorimetric method and the histamine concentration is determined from the calibration curve, the increased concentration of which indicates hyperhistaminemia.

 The method is as follows.

 As biological material, human saliva is used, before the sampling of which the examinee rinses the oral cavity with boiled water.

 In centrifuge tubes containing 4 ml of a 10% solution of trichloroacetic acid, 1 ml of saliva is added, mixed thoroughly, left for 30 minutes for extraction, and if necessary, can be left in the refrigerator for 1-2 days. Then it is mixed and centrifuged for 15 minutes at 3000 rpm. If necessary, the material can be stored frozen. In this case, the loss of the test substance during storage for one month does not exceed 3-5%.

 Then, 0.1 ml of 57% perchloric acid, 5 ml of ether are added to 4 ml of the extract and shaken for 1 min. The ether is suctioned off, another 5 ml of ether is added, and again shaken. This washing is repeated 3 times. Then 2 g of potassium carbonate and 5 ml of butanol are added to the samples, shaken for 3 minutes. After that, the samples are centrifuged for 3 min at 3000 rpm, 4 ml of the organic phase is sucked off and passed through a chromatographic column with a diameter of 3 mm and a height of 10 mm and with an ion-exchange resin placed in it. The column is washed with 1 ml of ethyl alcohol, 3 ml of water and histamine is eluted with 2 ml of 0.2 N. of hydrochloric acid. To restore efficiency, the column is washed with 10 ml of 1 N. hydrochloric acid, then 10 ml of water. Store ion exchange resin in water.

 0.5 ml of 4% sodium nitrite is poured into the extract, stirred and placed in a boiling water bath for 2 minutes. Then it is cooled in ice water and 0.5 ml of diazoreaktiv obtained by mixing 10 ml of a 0.1% solution of para-nitroaniline in 0.1 N are added. hydrochloric acid with 1 ml of 4% sodium nitrite, 1 ml of sodium carbonate and 0.15 ml of sodium hydroxide, stirring after adding each reagent.

 The optical density is measured on a photocolorimeter at a wavelength of 540 nm in cuvettes 10 mm wide.

 The histamine concentration is determined by a specific optical density and a calibration curve, the increased concentration of which indicates hyperhistaminemia.

 The colorimetric method for determining the concentration of histamine in the proposed method does not require expensive equipment, but it is not specific enough (Klimkina N.V., Plitman S.I. Colorimetric method for determining histamine in the blood and organs of laboratory animals. In: Biochemical studies in hygiene. - M., 1973, p. 68-72).

 To increase the specificity, various extraction options with organic solvents are often used. But even in this case, the colorimetric method is inferior in specificity to the fluorescent one. To increase the specificity of the applicants used column ion-exchange chromatography in combination with extraction with butanol in an alkaline medium at a high concentration of salts in solution.

 The use of chromatographic methods for the determination of histamine is known. In this case, the production of succinic cellulose according to the known method is laborious (Meshcheryakova SA, Gerasimova Ts.I. Fluorimetric method for determining histamine and serotonin in one sample. Laboratory, 1974, 11, p.670-672).

 Applicants have proposed the use of an ion-exchange resin produced by industry. As a result of the studies carried out by the applicants, it was found that the weakest cation exchangers — ion exchange resins KB-4 and KB-4P-2, Bio Rex-70 — possess the most optimal characteristics for histamine release from saliva.

 When extraction proposed by the authors in the prototype, a mixture of salts with alkali, the applicants replaced by anhydrous potassium carbonate. As the studies conducted by us showed, it is this salt that contributes to the maximum extraction of histamine and the minimum extraction of impurities interfering with its determination in the extraction and chromatography method we proposed, taking into account the specificity of the biological material used - saliva.

 The applicants selected a chromatographic column diameter of 3 mm, a height of 10 mm, a speed of movement of the eluting solution of 0.5 ml per minute. As an eluting solution proposed 0.2 N. hydrochloric acid.

 Of all the known methods for the precipitation of proteins in saliva, the most effective method using 10% trichloroacetic acid turned out to be the most effective. Moreover, the applicants found that the salts of trichloroacetic acid are partially extracted into butanol and interfere with the adsorption of histamine on the ion-exchange resin.

 Therefore, the applicants have proposed removing trichloroacetic acid by extraction with diethyl ether from an acidic medium that is created with perchloric acid. Since, as was found by the applicants, perchloric acid (more precisely, perchlorate anion) also interferes with the adsorption of histamine on an ion-exchange resin, it was proposed to precipitate it by adding potassium carbonate. This salt has two functions: it creates a high concentration of salts and the optimal pH for the extraction of histamine in butanol and precipitates potassium perchlorate.

 The analysis of the prior art by the applicant, including a search by patent and scientific and technical sources of information, and the identification of sources containing information about analogues of the claimed invention, allowed to establish that the applicant did not find an analogue characterized by features identical (identical) to all essential features of the claimed invention.

 The determination from the list of analogues of the closest technical solution (prototype) made it possible to identify a set of essential distinguishing features with respect to the technical result seen in the claimed method for diagnosing hyperhistaminemia set forth in the claims.

 Therefore, the claimed invention meets the criterion of "novelty."

 To verify the conformity of the claimed invention to the criterion of "inventive step", the applicant conducted an additional search for known solutions to identify signs that match the distinctive features of the proposed method for the diagnosis of hyperhistaminemia from the prototype.

 The search results showed that the claimed invention does not follow for a specialist explicitly from the prior art determined by the applicant.

 Therefore, the claimed invention meets the criterion of "inventive step".

 The criterion of the invention "industrial applicability" is confirmed by the fact that the proposed method for the diagnosis of hyperhistaminemia can be successfully, with great efficiency used in medical institutions in Russia and the CIS.

Claims (1)

 A method for the diagnosis of hyperhistaminemia, based on the collection of biological material and determination of histamine concentration in it, characterized in that human saliva is used as biological material for collection, in which a change in the concentration of histamine has a positive correlation with a change in the concentration of histamine in the blood, into centrifuge tubes, containing 4 ml of a 10% solution of trichloroacetic acid, 1 ml of saliva is added, extracted, 0.1 ml of 57% perchloric acid, 5 ml of ether are added to 4 ml of extract, producing three times the ether is sucked off, the ether is added and shaken, then 2 g of potassium carbonate and 5 ml of butanol are added to the samples, after centrifugation, 4 ml of the organic phase are aspirated and passed through a chromatographic column with a diameter of 3 mm and a height of 10 mm and with ion exchange placed in it resin, elute 2 ml of 0.2 N. hydrochloric acid, then 0.5 ml of 4% sodium nitrite is added to the eluate, and after heating and subsequent cooling of the eluate, 0.5 ml of diazoreaktiv, 1 ml of sodium carbonate and 0.15 ml of 5 N are added. sodium hydroxide, then the optical density is measured by the colorimetric method and the histamine concentration is determined from the calibration curve, the increased concentration of which indicates hyperhistaminemia.