Method for determination of concentration of serotonin and histamine in biological fluid

FIELD: medicine, analytical biochemistry.

SUBSTANCE: invention relates to laboratory methods of investigations. Method involves sampling specimen from patient to be inspected, extraction of serotonin and histamine from a specimen, chromatography of extract and determination of concentration of serotonin and histamine by the fluorescence intensity value. Saliva is used as biological fluid. Saliva by volume 1 ml is extracted with 4 ml of 1 N hydrochloric acid solution, 2 g of anhydrous potassium carbonate and 5 ml of mixture of butanol and chloroform in the ratio 3:2 are added, extract is shaken up and centrifuged. Organic phase (4 ml) is sucked off from extract and passed through chromatography column (diameter is 3 mm, height is 16 mm) filled with ion-exchange resin KB-4 or KB-4P-2 or Bio Rex-70 in H+-form, size of granules is 0.1 ± 0.02 mm. Histamine is eluted with 4 ml of 0.1 N hydrochloric acid at the rate of eluting solution 0.4 ml/min. Histamine concentration is determined by reaction with ortho-phthalic aldehyde dissolved in ethanol. Serotonin concentration is determined by reaction with ninhydrin in organic passed through column. Method provides assaying the saliva concentration of serotonin and histamine with high precision.

EFFECT: improved assay method.

 

The invention relates to the field of medicine, to biochemical research and evaluation methods changes the level of serotonin and histamine in the body.

Determination of serotonin and histamine is widely used in the diagnosis of various pathological processes.

It is known that the formation of serotonin in the body increases with carcinoid syndrome, malignant tumors of the prostate, rectum, allergic diseases, tuberculosis, schizophrenia. Reduced serotonin levels observed in thrombocytopenic Purpur, leucosis, collagenosis, rheumatoid polyarthritis. For diagnostic purposes usually examine typically explore the concentration of serotonin in the blood and 5-oxindoles acid in the urine.

Known pathological processes in which the role of the education and liberation of histamine. This is different allergic diseases, emotional stress, response to x-ray irradiation, hypoxia.

The relationship between histamine and serotonin in the development of many pathological processes are not well understood. Therefore, of particular interest is their simultaneous determination in one sample.

The literature describes several methods for simultaneous determination serotina and histamine in a sample of blood.

The closest p the technical essence and the achieved result is the method of determining the content of histamine and serotonin in whole blood (Meshcheryakova S.A., Gerasimov CI Fluorimetric method for the determination of histamine and serotonin in the sample. Laboratory work, 1974, No. 11, s-672).

The well-known principle of the method is based on the extraction of serotonin and histamine from the blood of sulphate of zinc in an alkaline medium, purification from impurities by extraction with butanol and chromatography on a column containing alternately cellulose, transferred to the aqueous phase, which carry out the reaction of ortho-phthalaldehyde reagent. Intensity of fluorescence quantitatively evaluate the concentration of histamine and serotonin.

The disadvantage of this method of determining the content of serotonin and histamine is the fact that, as a biological material that is taken from people in the study use the blood, the fence of which Vienna is traumatic for a person, there is damage to a blood vessel, sometimes with subsequent circulatory disorders, infection in the blood. These circumstances, as well as pain and negative emotions are often the cause of a failure patients from the survey, especially in those cases when it is necessary to conduct a series of tests, with short intervals of time.

In addition, when sampling blood from a vein requires a specially prepared room with trained medical personnel, sterile instrument to the patients and disposable syringes, which leads to the increase in the cost of the survey.

The technical result, which directed the establishment of this invention is to increase the efficiency of the method of diagnosis by simplifying and cheapening of the discovery process, serotonin and histamine.

The technical result is achieved in that in the method for determining the concentrations of serotonin and histamine in the biological fluid, including a sample of the examined person, the extraction from it of serotonin and histamine, chromatography was carried out with the extract and the concentration of serotonin and histamine in fluorescence intensity, as a biological fluid using a saliva extraction of 1 ml of saliva spend 4 ml of 1 n solution of chloric acid, then add 2 g of anhydrous potassium carbonate and 5 ml of a mixture of butanol and chloroform in the ratio 3:2, shaken, centrifuged, sucked off 4 ml of the organic phase and passed through the chromatographic column with with a diameter of 3 mm and a height of 16 mm and placed in her ion exchange resin KB-4 KB-4P-2, or Bio-Rex - 70 N+form, granule size of 0.1±0.02 mm, histamine elute with 4 ml of 0.1 G. of hydrochloric acid at speed eluting solution, 0.4 ml/min, the concentration of histamine is determined by the reaction with ortho-phthalic aldehyde, dissolved in ethanol, concentric the Yu serotonin is determined by the reaction with ninhydrin in the past through the column the organic phase.

The method is as follows.

As the use of biological material the saliva of the person in front of the sample of which the subject washes mouth with boiled water and obsessive cloth.

In centrifuge tubes containing 4 ml of 1 n solution of chloric acid, make 1 ml of saliva, mix thoroughly, leave for 30 min for extraction and, if necessary, can be left in the refrigerator for 1-2 days. Then stirred and centrifuged 15 min at 3000 rpm, If necessary, the material can be stored in a frozen state. In this case the losses of the analyte during storage for one month does not exceed 3-5%.

Further to 4 ml of the extract was added to 2 g of potassium carbonate and 5 ml of a mixture of butanol and chloroform in the ratio 3:2, shaken for 3 minutes After that, the samples centrifuged 3 min at 3000 rpm and sucked off 4 ml of the organic phase and passed through the chromatographic column with a diameter of 3 mm and a height of 16 mm and placed in her ion exchange resin KB-4 KB-4P-2, or Bio-Rex - 70 in H+form, granule size of 0.1±0,02 mm Column was washed with 1 ml of ethanol, 3 ml of water and elute histamine 4 ml of 0.1 G. of hydrochloric acid at speed eluting solution, 0.4 ml/min For recovery column was washed with 10 ml of 1 N. hydrochloric acid, then with 10 ml of water. Store ion the second resin should be in the water.

To the eluate poured 0.15 ml of 5 n sodium hydroxide solution, 0.1 ml of 0.1% solution of ortho-phthalic aldehyde in ethanol, and mix thoroughly. After 4 minutes poured 0.5 ml of 1.5 M solution of phosphoric acid, stirred. The amount of fluorescence is measured at a wavelength of 470 nm using excitation filter with maximum transmission at 365 nm fluorimetry BEAN-130. The intensity of the fluorescence is stable for 30 minutes.

For the determination of serotonin to the organic phase passed through a chromatographic column, add a 0.33 M solution of sodium phosphate for holding the pH to 6.5-7. Then poured 0.1 ml of 0.1 M solution of ninhydrin, mixed and placed in a thermostat at 30 minutes at a temperature of 75°C. is Cooled in water and in an hour, measure the fluorescence at a wavelength of 470 nm. The wavelength of excitation 365 nm.

Specific fluorescence intensity and a calibration curve to determine the concentration of serotonin and histamine, increased concentration which indicates an increase of the level of these substances in the body, increasing their concentration in the blood, and decrease the content of any of these substances in the saliva shows respectively about the decrease in their number in the body.

The method of determining the concentration of serotonin by reaction with ninhydrin in a known way not sufficiently specific.

To increase the specificity often use different ways of extraction with organic solvents. But in this case, the method according to the specificity inferior reaction with ortho-phthalic aldehyde. To increase the specificity of applicants used column ion-exchange chromatography in combination with butanol extraction in an alkaline medium at high concentrations of salts in solution.

Known application of chromatographic methods for the determination of histamine. Thus manufacturing alternately pulp by a known method time-consuming.

The applicants proposed use of commercially available ion-exchange resin. As a result of the applicants research it was found that the optimum characteristics for the selection of histamine from saliva have a weak cation - exchange resin CB-4 and CB-4P-2, Bio-Rex-70.

Applicants further studies were commissioned with the specific composition of the saliva and the presence of substances that can form fluorescent products when we used wavelengths. To provide the necessary specificity of the method we have chosen the optimum conditions of extraction and chromatographic parameters column, the size of the granules of resin.

Applicants selected diameter of the chromatographic column 3 mm, height 16 mm, Corot movement eluting solution of 0.4 ml per minute. As an eluting solution proposed 0.1 N. hydrochloric acid.

When the extraction proposed by the authors in the prototype of a mixture of salts with alkali applicants replaced by anhydrous potassium carbonate. As shown by studies conducted, this salt promotes maximum extraction of histamine and serotonin and the minimum extraction hampering its determination of impurities in our proposed method of extraction and chromatography considering the specifics of the used biological material of saliva.

Of all known methods of deposition of proteins in saliva was found to be the most effective method using perchloric acid. Since, as has been established by the applicants, perchloric acid (or rather the perchlorate anion) interferes with the adsorption of histamine on ion-exchange resin, it was proposed to besiege it by adding potassium carbonate. This salt does two things: creates a high concentration of salts and optimum pH for extraction of histamine in butanol and besieging potassium perchlorate. Used by applicants method of deproteinization has the advantage over that used in the prototype, because serotonin is rapidly destroyed in an alkaline environment, which may be significant for the simultaneous processing of many samples.

During extraction, the applicants have not used expensive heptane, treb is in store time-consuming cleanup. For dissolved ortho-phthalic aldehyde instead of toxic methanol applied ethanol.

Conducted by the applicant's analysis of the prior art, including searching by the patent and scientific and technical information sources, and identify sources that contain information about the equivalents of the claimed invention, has allowed to establish that the applicant had not discovered similar, characterized by signs of an identical all the essential features of the claimed invention.

The definition of a list of the closest analogues of the technical solution (prototype) has identified a set of essential in relation to perceived technical result of the distinctive features in the claimed method for diagnosis of hypergastrinemia set forth in the claims.

Therefore, the claimed invention meets the criterion of “novelty”.

To check the compliance of the claimed invention, the criterion of “inventive step” by the applicant conducted an additional search of the known solutions to identify signs that match the distinctive features of the prototype of the features of the proposed method for diagnosis of hypergastrinemia.

The search results showed that the claimed invention is not apparent to the expert in the obvious way from the prior art, as defined by the applicant.

Consequently the nutrient, the claimed invention meets the criterion of “inventive step”.

The criteria of the invention “industrial applicability” is confirmed by the fact that the proposed method can be successfully, efficiently and used in medical institutions of Russia and CIS.

The method for determining the concentrations of serotonin and histamine in the biological fluid, including a sample of the examined person, the extraction from it of serotonin and histamine, chromatography was carried out with the extract and the concentration of serotonin and histamine in fluorescence intensity, wherein the biological fluid using saliva, extraction of 1 ml of saliva spend 4 ml of 1 n solution of perchloric acid, then add 2 g of anhydrous potassium carbonate and 5 ml of a mixture of butanol and chloroform in the ratio 3:2, shaken, centrifuged, sucked off 4 ml of the organic phase and passed through the chromatographic column with a diameter of 3 mm and height 16 mm, placed in her ion exchange resin KB-4 KB-4P-2, or Bio-Rex - 70 N+form, granule size of 0.1±0.02 mm, histamine elute with 4 ml of 0.1 n hydrochloric acid at speed eluting solution, 0.4 ml/min, the concentration of histamine is determined by the reaction with ortho-phthalic aldehyde, dissolved in ethanol, the concentration of serotonin is determined by react and with ninhydrin in the past through the column the organic phase.



 

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FIELD: medicine, analytical biochemistry.

SUBSTANCE: invention relates to laboratory methods of investigations. Method involves sampling specimen from patient to be inspected, extraction of serotonin and histamine from a specimen, chromatography of extract and determination of concentration of serotonin and histamine by the fluorescence intensity value. Saliva is used as biological fluid. Saliva by volume 1 ml is extracted with 4 ml of 1 N hydrochloric acid solution, 2 g of anhydrous potassium carbonate and 5 ml of mixture of butanol and chloroform in the ratio 3:2 are added, extract is shaken up and centrifuged. Organic phase (4 ml) is sucked off from extract and passed through chromatography column (diameter is 3 mm, height is 16 mm) filled with ion-exchange resin KB-4 or KB-4P-2 or Bio Rex-70 in H+-form, size of granules is 0.1 ± 0.02 mm. Histamine is eluted with 4 ml of 0.1 N hydrochloric acid at the rate of eluting solution 0.4 ml/min. Histamine concentration is determined by reaction with ortho-phthalic aldehyde dissolved in ethanol. Serotonin concentration is determined by reaction with ninhydrin in organic passed through column. Method provides assaying the saliva concentration of serotonin and histamine with high precision.

EFFECT: improved assay method.

FIELD: medicine.

SUBSTANCE: method involves studying blood samples with venous blood mixed with vital stain like methylene blue. Degree of vital stain absorption by erythrocytes is determined by applying photocolorimetry. The value drop being more than 25%, extracorporal detoxication is to be predicted as ineffective.

EFFECT: simplified method.

6 tbl

FIELD: medicine, infectology.

SUBSTANCE: one should detect the level of terminal stable metabolites of nitrogen oxide (NOx) in whole blood. At its value ranged 39.6-86.0 mcM/l one should evaluate hemorrhagic fever accompanied with renal syndrome (HFRS) of average severity, at NOx value ranged 86.7-141.5 mcM/l - severe form of HFRS and at its values ranged 88.2-128.6 mcM/l at the background of pronounced clinical picture - as complicated disease flow. The method enables to shorten the terms for carrying out the assays.

EFFECT: higher accuracy of evaluation.

3 ex, 1 tbl

FIELD: medicine, biochemistry.

SUBSTANCE: in blood serum one should detect the level of lactoferrin and biliary acids. At their ratio being equal to 5-17 it is necessary to detect chronic hepatitis of high activity.

EFFECT: higher accuracy of detection.

3 ex

FIELD: medicine, dermatology, clinical laboratory diagnostics.

SUBSTANCE: the present method deals with detecting the focus of neutrophilic phagocytic activity lesion in capillary blood. At the values of cells' capacity to phagocytosis in percentage and phagocytic number on the 10th d of therapy being below 20% and 3.3, correspondingly one should evaluate therapeutic efficiency to be low, if it is above 40% and 4.0, correspondingly - as high.

EFFECT: higher accuracy of evaluation.

2 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: method involves studying blood serum, processing obtained data and setting disease diagnosis. The study is carried out by preparing dried blood serum sample as suspension in Vaseline oil and doing the infrared spectroscopy analysis in the bandwidth of 120-1000 cm-1 and determining absorption strip peak heights having maximum at 1180; 1165; 1160; 1150; 1130; 1070; 1025 cm-1 and then calculating the following two ratio groups, the first of which is ratio of peak height with maximum at 1165 cm-1 to 1150 cm-1; 1160 cm-1 to 1130 cm-1; 1070 cm-1; 1025 cm-1. The second group has ratio peak having maximum at 1165 cm-1 to 1160 cm-1; 1180 cm-1 to 1130 cm-1; 1065 cm-1; 1070 cm-1. The obtained three-dimensional distribution of the first group is projected to frontal plane for calculating two-dimensional coordinates and comparing to flat reference diagnostic images of hepatic pathologies and to a normal reference diagnostic image represented as flat polygons which boundaries are given by the following values. The norm is represented by X(-2.3;2.0;4.0;4.0) and Y(1.6;0.8;0.8;1.6), respectively. Oncology is represented by X(1.7;1.7;0.0;0.0) and Y(1.9;1.25; 1.25;1.9). Hepatites are represented by X(1.9;2.2;1.8;1.4 and 1.9;1.8;4.0) and Y(1.9;1.9; 0.5;0.5 and 08;0.5;0.8). Cirrhosis is represented by X(1.9;2.6;1.4) and Y(1.6;0.8;0.4). Diseases are differentiated by interpreting point position within particular area. Three-dimensional distribution of the second group is projected to frontal plane and compared to diagnosis images of pathology and norm. Coordinate values of the second group are as follows: norm - X(1.8;2.9;2.5;1.5), Y(2.7;2.0;1.2;1.6); oncological cases - X(0.27;0.67;0.63), Y(0.27;0.67;0.3); hepatitis - X(1.5;2.5;2.4;1.2), Y(1.6;1.2;0.2;0.9); cirrhosis - X(1.1;0.9;0.9). Final diagnosis of pathology is set when particular data values belong to the corresponding pathology zone in both cases.

EFFECT: high accuracy of diagnosis.

FIELD: medicine.

SUBSTANCE: method involves studying biological material by applying infrared spectroscopy techniques. The obtained data are processed and diagnosis is set. Blood serum is used as the biological material. The study is carried out by preparing dried blood serum sample as suspension in Vaseline oil and doing the infrared spectroscopy analysis in the bandwidth of 120-1000 cm-1 and determining absorption strip peak heights having maximum at 1170; 1165; 1160; 1150; 1140; 1060; 1050; 1040; 1025 and then calculating the following ratio values like peak height with maximum at 1160 cm-1 to 1140 cm-1; 1165 cm-1 to 1150 cm-1; 1040 cm-1 to 1025 cm-1. The obtained distribution of this group is projected to frontal plane for calculating two-dimensional coordinates and comparing to flat reference diagnostic images of prostate pathologies and to a normal reference diagnostic image represented as flat polygons which boundaries are given by the following values. The norm is represented by X(-1.15;-0.9;0.45;0.0;-0.65) and Y(0.99;4.2;0.9;0.46), respectively. Pathology by X(-1.15;-1.15;0.35;0.0;0.65) and Y(0.99;-0.03; 0.48;0.09;0.46). The norm and pathology are differentiated. Additional mathematical processing is carried out on infrared spectra of blood serum samples of patients belonging to pathology image according to parameter values. First of all, three-dimensional distribution is calculated as peak having maximum at 1160 cm-1 to one having maximum at 1150 cm-1; 1170; 1160 cm-1; 1160 cm-1 to 1025 cm-1. It is projected then to frontal plane and compared to diagnosis images of prostate adenoma and images of prostate carcinoma. The second group relationships the following values are used: oncological cases - X(0.28;0.77;1.24;0.96), Y(0.75;0.46;-0.13;-0.02); adenoma - X(0.28;1.24;2.21;1.24;0.77), Y(0.75;1.24;-0.12;-0.13;0.46). Differential diagnosis of pathologies is set by interpreting point position within particular pathology image.

EFFECT: high accuracy of differential diagnosis.

FIELD: medicine.

SUBSTANCE: method involves determining mean cytochemical coefficient of lipid accumulation in peripheral blood leukocytes in conditional units before beginning therapy application (MCC1) and in 2-3 or 5-6, or 10-12, or 20-24 months of therapy application (MCC2). Therapy effectiveness coefficient is calculated in conditional units from formula K= MCC2/MCC1. The value being equal to or greater than 1, leprosy therapy is predicted to be effective.

EFFECT: simplified prognosis method.

1 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: method involves determining infrared radiation absorption coefficient in blood plasma in bandwidth of 1543-1396 cm-1. The infrared radiation absorption coefficient is determined in %. The value being equal to 29.7±1.1%, catarrhal cholecystitis is diagnosed. The value being 26.4±1.4%, phlegmonous cholecystitis is diagnosed. The value being 21.2±1.8%, gangrenous cholecystitis is diagnosed. The value being equal to 18.6±0.5%, gangrenous perforated cholecystitis case is diagnosed. The value in norm is equal to 32.4±0.8%.

EFFECT: high accuracy and specificity of diagnosis.

FIELD: medicine.

SUBSTANCE: method involves pouring venous blood treated with heparin into five conic test-tubes in the amount of 0.1 ml. The first three of them contain 0.1 ml of non-colored latex suspension with particle size of 1.5 mcm, the fourth one contains 0.1 ml of medium 199 and 0.1 ml of 0.1% aqueous solution of tetrazole nitro blue, the fifth one contains .1 ml of latex suspension and 0.1 ml of 0.1% aqueous solution of tetrazole nitro blue. The first test-tube is incubated in thermostat for 5 min at37°C, the second one for 30 min, the third one for 1 h, the fourth and the fifth one for 40 min. Smears are prepared from 0.2 ml of incubation mixture on glasses and dried at 37°C, fixed in burner flame, stained with 0.1% aqueous solution of tetrazole nitro blue, repeatedly dried and studied with microscope under immersion with magnification of 90x10. Test results are evaluated from absorption activity in phagocytosis reactions in determining the number of phagocytes, phagocytic number, phagocytic integral index and phagocytosis rate values. Tetrazole nitro blue test response is determined by counting formazan-positive cell number, calculating cytochemical activity index and tetrazole nitro blue test stimulation index.

EFFECT: accelerated test; high accuracy and low cost of examination.

1 dwg, 3 tbl

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