Method of blood dichlorobromomethane measurement
SUBSTANCE: method starts with blood sample alkalisation with 10% sodium hydroxide to pH 8-10; dichlorobromomethane is recovered from the sample by hexane extraction; the extract is separated centrifugally at 7000-7500 rpm and analysed with gas chromatography in a partition gas chromatograph with an electron capture detector, while dichlorobromomethane is measured by a calibration diagram.
EFFECT: high sensitivity and accuracy of the method for blood dichlorobromomethane measurement.
1 ex, 5 tbl
The invention relates to medical Toxicological research, in particular to sanitary toxicology, and can be used for the quantitative determination dichlorobromomethane in biological fluids such as blood.
Known microdiffusion method suitable for the determination of chlorinated hydrocarbons in various biosubstrate - blood, urine in the range of 5-35 mg%. Quantitative determination is carried out spectrophotometrically by the intensity of color. The detection error is ±5% .
For the concentration and recovery of chlorinated hydrocarbons from biological fluids used method of gas chromatographic analysis of the equilibrium vapor phase 1,2-dichloroethane . The disadvantage of this method is its low sensitivity: limit of detection of 10 ág. The accuracy is 5-10%.
Also there is a method of gas chromatographic determination of halogenated substances in water, in particular, dichloromethane . The measurement of its concentration carried out by gas chromatography using electron-capture detector. Concentration dichlorobromomethane of water carry gas extraction at temperatures up to 80°C in a confined space. Further assembled gas is injected into the chromatograph evaporator, and the number of dicarbamate is to establish on a calibration curve. However, this method is not applicable to the determination of dichloromethane in the blood, because the number of operations required to heat up to the temperature at which happens clotting of blood.
Another known method of gas chromatographic determination halogen-substituted methane in water is the way in which produce vapor extraction present in water volatile components, their concentration on the sorbent, thermal desorption and separation on the column with sorbent, the entrance of which is placed a layer of sulfonic cation exchanger, with subsequent detection of the separated components of the detector of electron capture .
However, the mentioned known method, it is impossible to determine halogen-substituted methane, in particular dichloromethane, in the blood, because it has such an operation, such as thermal desorption.
The technical result achieved by the invention, is to provide simultaneously high sensitivity and accuracy of the method for determining dichloromethane in the blood.
The technical result is achieved by the proposed method for quantitative determination dichloromethane in the blood, characterized by the fact that initially carry out alkalization of the blood samples of 10%sodium hydroxide solution to a pH of 8-10, produce the removing of dihl brometane from the sample by extraction of its hexane, separate the extract by centrifugation, to produce a study of gas chromatographic analysis, and the number dichlorobromomethane set via a calibration curve.
This technical result is achieved due to the following.
Introduction in a blood sample of 10%aqueous sodium hydroxide solution due to the fact that this reagent does not interfere with the determination and does not cause error.
Bringing the pH in the blood sample to a pH of 8-10 explained by the fact that only with the high pH provides a high degree of extraction dichloromethane, and simultaneously provides the denaturation of low molecular weight proteins, which increases the accuracy of determination.
Use as an extracting solvent hexane provides the high sensitivity of the proposed method.
Thanks to the operation of centrifugation is on the one hand the destruction of proteins that define a complex matrix composition of the sample which may adversely affect the accuracy and sensitivity of the method, and on the other hand - is the separation of the extract. Mode centrifugation recommended from 7000 rpm to 7500 rpm When using modes less than 7000 rpm is incomplete separation of the extract from the assay, when the modes centrifugation over 7500 rpm the centrifugal acceleration occurs, in addition to the besieged who I proteins, for analysis, the deposition of compounds of molecular weight greater than that of the compounds that lead to overly optimistic results. And only when using the specified limits centrifugation guaranteed quality of the analysis (high sensitivity and precision).
The combination and sequence of these operations, their modes and allowed to reach a high degree of accuracy and sensitivity of the proposed method.
The proposed method is implemented as follows.
Example. A sample of blood in quantities of 1 cm3containing dichloromethane, alkalinized with 10% sodium hydroxide solution to a pH of 8-10. Next, make the extraction of 1 cm3hexane by shaking for 1-2 minutes Then the mixture is centrifuged at 7500 rpm for 2 minutes After removal of the protein by centrifugation and separation of the extract, the latter is poured into a test tube and chromatographic on the chromatograph "Crystal 2000" with electron capture detector. Conduct quantitative determination dichloromethane in the prepared samples by gas chromatography on a calibration curve.
The calibration graph is constructed as follows. Blood samples taken from a control group of children, volume 1 cm3containing specified concentrations of dicarbamate is on, alkalinized with 10% sodium hydroxide solution to a pH of 8-10 and extracted with a 1 cm3hexane. The obtained extracts - standard solutions - chromatographic on gaschromatographie "Crystal 2000" with electron capture detector.
Optimization of gas chromatographic parameters to determine dichlorobromomethane in blood was carried out using a hardware-software complex based on gas chromatograph "Crystal 2000" with electron capture detector. Weight selection dichlorobromomethane achieved capillary column Optima-5 - 25m*0,32mm*5,0µm at temperature: column - 50°C; evaporator - 200°C, detector 250°C; flow rate of carrier gas 1 (nitrogen) - 20 cm3/min; flow rate of carrier gas was 30 cm3/min. With the same parameters chromatograph is determined and quantitative content dichloromethane in the blood.
In laboratory tests the dependence of the degree of extraction dichloromethane from pH and nature of organic solvents. The results are shown in table 1.
|The degree of extraction dichlorobromomethane of blood samples from the pH and nature of organic solvents|
|Defined ingredient||Organic solvent||The range of pH start extraction||The degree of extraction, %|
|Dichloromethane in the blood||heptane||2-3||42|
The data in table 1 show that the most effective extractant for the extraction dichloromethane is hexane at pH 8-10.
For denaturation of low molecular weight proteins and increase the degree of extraction of the sample of blood is alkalinized with 10% sodium hydroxide solution to a pH of 8-10.
Another proof of the above results are presented in t the blitz 2, extraction dichlorobromomethane of blood organic solvents when the selected optimal extraction conditions (pH=8-10).
|The average values of the degree of extraction dichloromethane from blood samples of organic solvents for n=5 and p=0,95|
|Solvent||Content dichlorobromomethane, mcg|
|Introduced||Found||The degree of extraction, %|
|Note: n - number of definitions; p - probability|
The result of this experiment, it is confirmed once again that the most complete extraction of dichloromethane from the blood takes place by extraction with hexane in SEL is offered by the environment (pH 8-10).
To determine the sensitivity, then analyzed blood samples from children by the method of supplementation with different content in them dichloromethane. Blood samples containing defined concentrations dichlorobromomethane, alkalinized with 10% sodium hydroxide solution to a pH of 8-10 and extracted with a 1 cm3hexane within 1-2 minutes After separation of the extract and removal of proteins by centrifugation at 7000-7500 rpm for 2 min analyzed extracts chromatographic at least 5 times on the chromatograph "Crystal 2000M with electron capture detector.
Via a calibration curve and determine the content of the component in the samples. The results are shown in table 3.
|No. blood samples||The content defined dichlorobromomethane, mg/cm3|
Thus the detection sensitivity for dichlorobromomethane were 0.002 mg/cm3. Prob is tenderly definition dichlorobromomethane of 0.004 µg in the analyzed sample volume.
During testing it was found that the measurement error for dichlorobromomethane the proposed method is 24,01%. The data are given in table 4.
|The range of values of precision, repeatability, reproducibility|
|The name of the component to be determined and the measurement range, µg/cm3||The measure of repeatability (relative standard deviation repeatability), σr, %||The measure of reproducibility (relative standard deviation of reproducibility) σR, %||Accuracy rate (bounds the relative error with probability P=0,95), ±δ, %|
|Dichlorobromomethane, from 0.002 to 0.01 on.||2,91||9,92|
Also, in laboratory tests determined the limits of repeatability and reproducibility of the determination dichlorobromomethane the proposed method, which characterize the accuracy of the measurement results. Data on these indicators are given in table 5.
|The limits of repeatability and reproducibility of the determination dichlorobromomethane the proposed method with a confidence level of p=0,95|
|The name of the component to be determined and the measurement range, µg/cm3||The limit of repeatability (relative value allowable discrepancies between the two results of parallel measurements), rn, %||Limit intralaboratory reproducibility (relative value allowable discrepancies between the two measurement results obtained in one laboratory, but in different conditions),, %|
|Dichlorobromomethane, from 0.002 to 0.01 on.||of 8.06||2,75|
Laboratory results showed that the proposed method indeed characterized by a high sensitivity (from 0.002 ág/cm3), high accuracy (24,01%) and provides high limits of repeatability and reproducibility. This allows to recommend it for use in medical laboratories.
Sources of information
1. Galuskina I.D., Filov VA Pre the treatment and definition of organic poisons in the body. "Medicine", 1971
2. Guidelines on the detection and determination of 1,2-dichloroethane in a biological material by gas chromatography. M: the Ministry of health of the USSR, 1978
3. Guidelines for gas chromatographic determination of halogenated compounds in water. MUK 4.1.646-96. Approved by the state Committee of sanitary and Russia 31.10.1996,
The method of quantitative determination of dichloromethane in the blood, characterized by the fact that initially carry out alkalization of the blood samples of 10%sodium hydroxide solution to a pH of 8-10, produce removing dichloromethane from the sample by extraction of its hexane, separating the extract by centrifugation at 7000-7500 rpm and produce his study of gas chromatographic analysis on a gas chromatograph with electron capture detector, and the number dichlorobromomethane set via a calibration curve.
SUBSTANCE: excitory cells of the immune system are recovered that is followed with primary incubation of the cells with an investigated substance to produce a primary-incubation supernatant or mixed cells and supernatant; secondary incubation of the target cells with the supernatant or mixed cells and supernatant wherein the secondary-incubation target cells are understood as human tumour cells or cell lines of an oncogenetic origins; the target cells are analysed where the analysis is specified in a group including an expression analysis of specific proteins and an apoptosis and/or necrosis analysis.
EFFECT: improvement of the method.
SUBSTANCE: invention relates to field of medicine, namely to orthopedics. In order to estimate state of bone tissue in case of immobilisation osteoporosis in a laboratory animal examined are homogenates: bone, muscular, bone marrow of any extremity and peripheral blood. Biochemical and integral parametres are determined. Five factor variables F1-F5 are calculated using values of biochemical and integral parametres, constant values of factor coefficients of biochemical parametres and free coefficients. After that calculated is the value of discriminant function, whose value is used to estimate bone state as normal or conclusion about presence of immobilisation osteoporosis is made.
EFFECT: method increases accuracy and efficiency, has high stability of immobilisation osteoporosis recognition.
1 ex, 3 tbl
SUBSTANCE: blood plasma is analysed for cytochrome oxidase activity, erythrocyte 2,3- biphosphoglycerate and lactic acid concentrations. It is followed by calculating an oxygenation coefficient K by formula: K=(C1+C2):A, where C1 is the erythrocyte 2,3- biphosphoglycerate concentration, mol/l; C2 is the erythrocyte lactic acid concentration, mol/l; A is plasma cytochrome oxidase activity, mol/l. The K value withint 1.0 <K≤3.0 enables to diagnose tissue hypoxia, while in case of the K values being 3.0<K≤5.0 cardiovascular hypoxia is diagnosed. If the oxygenation coefficient is 5.0 and more, blood hypoxia is diagnosed.
EFFECT: use of the method enables the differential diagnostics of blood, tissue and cardiovascular hypoxia.
SUBSTANCE: method for prediction of duration of a recurrence-free interval in patients with rectal cancer consisting in the fact that the surgical removal of a peritumoural recurrence is followed with biochemical analysis of the concentration of polypeptide hormone prolactin, and its specified tissue value enables to determine duration of the recurrence-free interval.
EFFECT: method allows predicting duration of the recurrence-free interval following the removal of the recurrence.
SUBSTANCE: for an assay, 5-7 cm3 of blood is taken, and before extraction the sample is pre-treated with 2 cm3 of 60% sulphuric acid; the extraction process is executed with 30 cm3 of n-hexane once, and gas chromatography is preceded with single treatment of n-hexane extract with 10 cm3 of concentrated sulphuric acid.
EFFECT: invention provides higher reliability of α-HCCH, γ-HCCH test results and twofold reduced time of sample preparation.
1 ex, 1 tbl
SUBSTANCE: method includes introduction of sample into sample hole of the device, the device being adjusted for delivering said sample into lateral flow matrix, which has multitude of varieties of IgE antigens, immobilised in respective positions in result window. Method additionally includes possibility of sample movement along lateral flow matrix through immobilised multitude of IgE antigen varieties into the second position lower on flow from the first position, introduction of liquid buffer into lateral flow matrix for mobilisation of labeled reagent, which is adapted for binding aHTH-IgE-antibody and is dried in lateral flow matrix in position higher on flow from introduction of filtered sample into lateral flow matrix, and possibility of movement of labeled reagent, mobilised by liquid buffer, along lateral flow matrix through multitude of immobilised varieties of antigens IgE into position lower on flow from the first position.
EFFECT: simplification of process.
46 cl, 1 ex, 1 tbl, 8 dwg
SUBSTANCE: in patient's saliva concentration of alfa-interferon is determined. If alfa-interferon concentration is from 78.62 to 120.58 pg/ml, compensated form of carious process course is predicted. If alfa-interferon concentration is from 22.659 to 41.821 pg/ml, decompensated form of carious process course is predicted.
EFFECT: application of method allows to predict carious process course in children reliably.
SUBSTANCE: in patient's saliva concentration of alfa-interferon is determined. If concentration of alfa-interferon from 22.659 to 41.821 pg/ml is determined, increase of caries intensity for not less than two teeth after 6 months is predicted.
EFFECT: application of method allows to predict carious process course in children reliably.
SUBSTANCE: invention relates to medicine, in particular, to vascular surgery. Phased surgical treatment of frontal arteries in patients with symptomatic bicarotid stenoses, including at the first stage reconstruction of one internal frontal artery, at the second stage - another internal frontal artery, is carried out. In order to predict character of reperfusion syndrome course, carried out are clinical-instrumental examination and determination in blood serum of level of primary, secondary and final isopropanol-soluble (ISPP, ISSP, ISFP), heptane-soluble (GSPP, GSSP, GSFP) products of lipid peroxidation and ascorbate-induced lipid peroxidation (AOA-1, AOA-2) as well as level of biologically active substances - serotonin, nitrites, nitrates in time periods before the second stage of aversion carotid endarterectomy, as well as on 1-3, 4-7 and 8-14 day after its performance. Using values of ISPP, ISSP, ISFP, GSPP, GSSP, GSFP, AOA-1, AOA-2, serotonin, nitrites and nitrates levels with respect to norm in said periods of time, favourable or unfavourable reperfusion syndrome course is predicted.
EFFECT: method increases reliability of reperfusion syndrome prediction after phased surgical treatment of frontal arteries in patients with symptomatic bicarotid stenoses.
SUBSTANCE: invention concerns a method of antibody-dependent cellular cytotoxicity (ADCC) assay.
EFFECT: simplicity and safety as compared with the analysis, higher sensitivity and real-time realisability of the method.
42 cl, 1 ex, 10 dwg
FIELD: medicine, hepatology.
SUBSTANCE: one should detect the level of hepato-specific enzymes (HSE) in blood plasma, such as: urokinase (UK), histidase (HIS), fructose-1-phosphataldolase (F-1-P), serine dehydratase (L-SD), threonine dehydratase (L-TD) and products of lipid peroxidation (LP), such as: dienic conjugates (DC), malonic dialdehyde (MDA). Moreover, one should detect the state of inspecific immunity parameters, such as: immunoregulatory index (IRI) as the ratio of T-helpers and T-suppressors, circulating immune complexes (CIC). Additionally, one should evaluate the state of regional circulation by applying rheohepatography (RHG), the system of microhemocirculation with the help of conjunctival biomicroscopy (CB) to detect intravascular index (II). In case of increased UK, HIS levels up to 0.5 mcM/ml/h, F-1-P, L-SD, L-Td, LP products, CIC by 1.5 times, higher IRI up to 2 at the norm being 1.0-1.5, altered values of regional circulation, increased II up to 2 points at the norm being 1 point, not more one should diagnose light degree of process flow. At increased level of UK, HIS up to 0.75 mcM/ml/h, F-1-P, L-SD, L-TD, LP products, CIC by 1.5-2 times, increased IRI up to 2.5, altered values of regional circulation, increased II up to 3-4 points one should diagnose average degree of process flow. At increased level of UK, HIS being above 0.75 mcM/ml/h, F-1-P, L-SD, L-TD, LP products, CIC by 2 and more times, increased IRI being above 2.5, altered values of regional circulation, increased II up to 5 points and more one should diagnose severe degree of process flow.
EFFECT: higher accuracy of diagnostics.
FIELD: medicine, infectology, hepatology.
SUBSTANCE: in hepatic bioptate one should detect products of lipid peroxidation (LP), such as: dienic conjugates (DC), activity of antioxidant enzymes, such as: catalase (CAT)and superoxide dismutase (SOD). One should calculate by the following formula: C = DC/(SOD x CAT)x100, where DC - the content of dienic conjugates, SOD - activity of superoxide dismutase, CAT - activity of catalase. At coefficient (C) values being above 65 one should predict high possibility for appearance of cirrhosis, at 46-645 - moderate possibility and at 14-45 -low possibility for appearance of cirrhosis.
EFFECT: higher accuracy of prediction.
FIELD: medicine, clinical toxicology.
SUBSTANCE: at patient's hospitalization one should gather the data of clinical and laboratory values: on the type of chemical substance, patient's age, data of clinical survey and laboratory values: body temperature, the presence or absence of dysphonia, oliguria being below 30 ml/h, hemoglobinuria, erythrocytic hemolysis, exotoxic shock, glucose level in blood, fibrinogen and creatinine concentration in blood serum, general bilirubin, prothrombin index (PTI), Ph-plasma, the state of blood clotting system. The state of every sign should be evaluated in points to be then summed up and at exceeding the sum of points being above "+20" one should predict unfavorable result. At the sum of "-13" prediction should be stated upon as favorable and at "-13" up to "+20" - prediction is considered to be doubtful.
EFFECT: higher accuracy of prediction.
2 ex, 3 tbl
FIELD: medicine, juvenile clinical nephrology.
SUBSTANCE: disease duration in case of obstructive pyelonephritis should be detected by two ways: either by detecting the value of NADPH-diaphorase activity, as the marker of nitroxide synthase activity in different renal department and comparing it to established norm, or by detecting clinico-laboratory values, such as: hemoglobin, leukocytes, eosinophils, urea, beta-lipoproteides, lymphocytes, neutrophils, the level of glomerular filtration, that of canalicular reabsorption, urinary specific weight, daily excretion of oxalates, arterial pressure, and estimating their deviation against average statistical values by taking into account a child's age.
EFFECT: higher efficiency of detection.
7 dwg, 1 ex, 6 tbl
FIELD: clinical medicine, pulmonology.
SUBSTANCE: one should carry out complex estimation of interleukin-1β) concentration in blood, saliva, bronchoalveolar liquid. Moreover, one should detect distribution coefficient (DC) for IL-1β as the ratio of IL-1β blood content to IL-1β salivary content. At increased IL-1β blood content by 10 times and more, by 2 times in saliva, unchanged level of bronchoalveolar IL-1β, at DC for IL-1β being above 1.0 one should predict bronchial obstruction. The method enables to conduct diagnostics of the above-mentioned disease at its earlier stages.
EFFECT: higher efficiency of prediction.
FIELD: medicine, diagnostics.
SUBSTANCE: the present innovation deals with genetic trials, with diagnostic field of oncological diseases due to analyzing DNA by altered status of gene methylation that take part in intracellular regulation of division, differentiating, apoptosis and detoxication processes. One should measure the status of methylation in three genes: p16, E-cadherine and GSTP1 in any human biological samples taken out of blood plasma, urine, lymph nodes, tumor tissue, inter-tissue liquid, ascitic liquid, blood cells and buccal epithelium and other; one should analyze DNA in which modified genes of tumor origin or their components are present that contain defective genes, moreover, analysis should be performed due to extracting and purifying DNA out of biological samples followed by bisulfite treatment of this DNA for modifying unprotected cytosine foundations at keeping 5-methyl cytosine being a protected cytosine foundation followed by PCR assay of bisulfite-treated and bisulfite-untreated genes under investigation and at detecting alterations obtained according to electrophoretic result of PCR amplificates, due to detecting the difference in the number and electrophoretic mobility of corresponding fractions at comparing with control methylated and unmethylated samples containing normal and hypermethylated forms of genes one should diagnose oncological diseases. The method provides higher reliability in detecting tumors, detection of remained tumor cells after operation.
EFFECT: higher efficiency of therapy.
1 cl, 3 dwg, 4 ex
FIELD: medicine, gastroenterology.
SUBSTANCE: one should carry out diagnostic studying, moreover, on the 5th -6th d against the onset of exacerbation in case of gastric and duodenal ulcerous disease one should detect the content serotonin, histamine and acetylcholine in blood, then during 2-3 wk one should conduct medicinal therapy to detect serotonin, histamine and acetylcholine level in blood again and at serotonin content being by 2-3 times above the norm, histamine - by 1.15-1.4 times above the norm and acetylcholine - by 20-45% being below the norm one should predict the flow of gastric and duodenal ulcerous disease as a non-scarring ulcer.
EFFECT: higher accuracy of prediction.
SUBSTANCE: method involves taking blood from ulnar vein (systemic blood circulation) and from large vein of the injured extremity proximal with respect to lesion focus (regional blood circulation). Spontaneous NST-test value is determined and difference is calculated in systemic and regional blood circulation as regional-to-systemic difference. The difference value is used for predicting clinical course of pyo-inflammatory disease in extremities.
EFFECT: high accuracy of diagnosis.
4 cl, 2 tbl
FIELD: medicine, gastroenterology.
SUBSTANCE: one should introduce biologically active substance, moreover, in patient's blood serum one should detect the content of acetyl choline and choline esterase activity followed by 2-h-long intragastric pH-metry at loading with biologically active substance as warm 40-45%-honey water solution at 35-40 C, and at increased content of acetyl choline being above 1.0 mM/l, choline esterase being above 0.5 mM/l/30 min and pH level being 6.0-6.9 it is possible to consider apitherapy to be useful for treating ulcerous duodenal disease.
EFFECT: higher efficiency and accuracy of detection.
FIELD: medicine, gastroenterology.
SUBSTANCE: it has been suggested a new method to detect pharmacological sensitivity to preparations as acidosuppressors. After the intake of the preparation a patient should undergo fibrogastroduodenoscopy 3 h later, then, through endoscopic catheter one should introduce 0.3%-Congo red solution intragastrically and the test is considered to be positive at keeping red color that indicates good sensitivity to the given preparation, and in case of dark-blue or black color the test is considered to be negative that indicates resistance to this preparation. The suggested innovation widens the number of diagnostic techniques of mentioned indication.
EFFECT: higher efficiency of diagnostics.