Method of biochemical identification of highly-toxic anticholinesterase poisons

FIELD: medicine.

SUBSTANCE: method includes the determination of an inhibiting impact of a solution of highly-toxic anticholinesterase poison on cholinesterase activity, represented by acetylcholinesterase of human blood erythrocytes, in the presence and in the absence of a substrate, represented by acetylthiocholiniodide. The inhibiting impact of the solution of highly-toxic anticholinesterase poison on the activity of acetylcholinesterase of the human blood erythrocytes is determined first in the presence of the substrate, and then in its absence. An analysed sample is preliminarily diluted by 10 or 100 times to obtain the inhibiting effect on acetylcholinesterase of the human blood erythrocytes in the presence and in the absence of the substrate within 25-75%. After that, the value of the protective effect of acetylthiocholiniodide and the obtained calculated value is compared with table values, determined experimentally in advance for each particular highly-toxic anticholinesterase poison. If the calculated values coincide with the table values, a conclusion about the presence of a respective highly-toxic anticholinesterase poison in the analysed sample is made.

EFFECT: increase of safety.

2 ex

 

The invention relates to medicine, namely to toxicology, relates to a method of biochemical identification highly toxic anticholinesterase poisons - O-isopropyl-methyl-fluoro-phosphonate, pinacolyl-methyl-fluoro-phosphonate, O-ethyl-S-diisopropyl-aminoethyl-methyl-thio-phosphonate, O-isobutyl-S-diethyl-aminoethyl, which can be used in emergency situations (accidents at facilities for destruction of chemical weapons) to identify listed hazardous to human poisons.

Currently anticholinesterase poisons are widely used in medicine, agriculture and other areas of the economy. Some of these chemical compounds belong to the first class of danger [1], pose an unusual and extraordinary threat to the life or health when released into the environment and shall be destroyed within implementation of the "Convention on the prohibition of the development, production, stockpiling and use of chemical weapons and on their destruction, of 13.01.1993 G. among such compounds include O-isopropyl-methyl-fluoro-phosphonate, pinacolyl-methyl-fluoro-phosphonate, O-ethyl-S-diisopropyl-aminoethyl-methyl-thio-phosphonate and O-isobutyl-S-diethyl-aminoethyl-methyl-thio-phosphonate the Ability to identify these dangerous highly toxic poisons is of practical importance.

One of the most sensitive methods for the detection�Oia anticholinesterase poisons is a biochemical technique based on the use of cholinesterase. The classical scheme of determination of anticholinesterase activity used in most of the technical means proposed by Aldridge [2] and is in the preliminary contact of the enzyme with anticholinesterase poison, and then measure the catalytic activity of cholinesterase. Its main disadvantage is the relatively low specificity. In a sense it's ironic, because it is considered that the higher the sensitivity, the greater its specificity [3, 4].

The fact that high sensitivity in the case of a biochemical method using cholinesterase, reaches a value of 1·10-7mg/ml (i.e. 1·10-8%), indicates a very high analytical selectivity. But even in a very pure solvent with a basic substance content not less than 99.9% at the concentrations of 1·10-8% as impurities there are a large number of different chemical compounds [5]. Therefore, the specificity of the interaction, for example, the enzyme acetylcholinesterase inhibitors of the specified enzyme is very high, if this set of various compounds, the enzyme reacts with the analyte substance. Therefore, the low specificity of the biochemical method is explained not so much analytical capability,�awns of enzyme used, how about the fact that these capabilities were not used in full.

The analytical signal obtained by the implementation of the classical method of detection of anticholinesterase poisons, expressed as the degree of oppression or the amount of residual catalytic activity (in % of the initial activity or absolute values of the activity, is a function of the free, not associated with the poison of the enzyme and does not assume almost any information about the chemical nature of the analyzed poison. This is because the same degree of inhibition of catalytic activity of cholinesterase can be obtained as when the contact of the enzyme with any anticholinesterase pesticide at a concentration of 1·10-5M or even inorganic compounds at a concentration of 1·10-1M, and with highly toxic anticholinesterase poisons at a concentration of 1·10-9M.

To increase the specificity of the biochemical method of analysis anticholinesterase poisons using cholinesterase proposed different versions, each distinguished by use of cholinesterase different biological origin and the application of some chemical compounds (effectors) that alter the affinity to cholinesterase anticholinesterase poison [6].

As the effector used, for example, chloride of barium. So, was the offer�n a method of identifying highly toxic anticholinesterase poisons when they act on butirilholinèsterazu amid the presence of barium chloride and in its absence [7]. Then identify the relationship of the constants of inhibition, i.e. the identification of the effect of the presence of barium chloride, allows to judge about the presence in the sample of individual highly toxic anticholinesterase poisons.

The known method of identification of highly toxic anticholinesterase poisons that include the use as a test enzyme acetylcholinesterase of erythrocytes of human blood and propionylcarnitine the optic ganglia of the squid [8]. The method consists in determination of the inhibitory effect of the test solution, and suspicious content anticholinesterase poison, on two different cholinesterase biological origin with subsequent calculation of the ratio of inhibition constants and comparing the obtained result with the table values defined in advance for all highly toxic topical anticholinesterase poisons.

A significant drawback of these analog techniques is that none of them allows to identify all the dangerous anticholinesterase poisons.

The known method of identification of anticholinesterase poisons involving the use of four cholinesterase various biological origin - acetylcholinesterase of erythrocytes in human blood, serum butyrylcholinesterase horse blood, the ACO�ilpolistirene aphids (cereals and sugar beet) and cholinesterase flies, identifying relevant highly toxic anticholinesterase poisons [9]. This method biochemical identification anticholinesterase poisons the closest achieved technical result of the claimed method and adopted as a prototype method.

The disadvantages of the prototype method is:

- relatively low sensitivity due to reduced affinity acetylcholinesterase aphids and cholinesterase flies highly toxic anticholinesterase poisons;

- the lack of commercial availability acetylcholinesterase aphids and cholinesterase flies;

- the complexity of working with enzymes in insects.

The aim of the invention is to provide a method of biochemical identification highly toxic anticholinesterase poisons to be destroyed in accordance with the Convention, namely-isopropyl-methyl-fluoro-phosphonate, pinacolyl-methyl-fluoro-phosphonate, O-ethyl-S-diisopropyl-aminoethyl-methyl-thio-phosphonate, O-isobutyl-S-diethyl-aminoethyl-methyl-thio-phosphonate, easy to apply and effective in the identification of these dangerous to humans highly toxic anticholinesterase poisons.

The identifying characteristics of the specific interaction of highly toxic anticholinesterase poison with cholinesterase is a bimolecular rate constant of interaction of the inhibitor with the farms�Tom, to calculate which it is necessary to know the concentration of the poison. However, the analysis of samples suspected of contamination with highly toxic anticholinesterase poisons, the concentration of substances contained in them, is not known.

It is known that the bimolecular rate constant of reaction of the enzyme with the inhibitor in the absence (k2) and the presence of the substrate (k2S) is calculated by the formula [9]:

where: Aost - residual enzyme activity after exposure to the inhibitor, %;

t - time contact with the inhibitor, min;

C - concentration of the inhibitor, M/L.

If necessary, calculate the ratio of k2/k2S(that is, determine the protective effect of the substrate) when working with the same initial solution are first dilution of the sample, wherein the inhibiting effect on the enzyme in the presence of the substrate is 25-75%.

Then after dilution in 10 or 100 times to determine the inhibitory effect in the absence of substrate. In this regard, to calculate the magnitude relationship of the constants k2/k2S, that is, to calculate the magnitude of the protective effect (P) of the substrate, it is necessary to consider only the breeding, but to know the concentration. The value of the ratio of the constants k2/k2Sdepends on the chemical nature of the inhibitor and can be used for, combined with led�tion of highly toxic anticholinesterase poisons.

Therefore, the purpose of the invention is achieved by calculating the protective effect of substrate acetylthiocholine on the inhibition of acetylcholinesterase of erythrocytes human blood solutions highly toxic anticholinesterase poison, distinguished by the contents of the poison in 10 or 100 times. The calculated value of the magnitude of the protective effect of the substrate is compared with tabular values obtained in advance for each specific anticholinesterase poison, and the coincidence of the calculated values from table and make a conclusion of what exactly the poison is contained in the sample.

Example 1. The procedure for implementing the inventive method biochemical identification of highly toxic anticholinesterase poisons to be destroyed under the Convention.

When implementing the proposed method to 1.0 ml of an aqueous solution of the enzyme acetylcholinesterase of erythrocytes human blood is added 1.0 ml of 0.1 M phosphate buffer (pH of 7.5), 0.5 ml of an aqueous solution of substrate acetylthiocholine at a concentration of 5.78% was established mg/ml, 0.5 ml water and 1.0 ml of the analyzed solution containing highly toxic anticholinesterase compound, namely-isopropyl-methyl-fluoro-phosphonate, pinacolyl-methyl-fluoro-phosphonate, O-ethyl-S-diisopropyl-aminoethyl-methyl-thio-phosphonate or On-isobutyl-S-diethyl-aminoethyl-methyl-thio-phosphonate. Incubated for 3 min at temp�the temperature of 37°C, and then add 0.5 ml water and 0.5 ml of an aqueous solution of Ellman's reagent at a concentration of 2·10-2M and immediately measure the optical density (D1) against that of the reference solution, characterized in that instead of 1.0 ml of enzyme solution using 1.0 ml of water. In the control sample is analyzed solution use water. Then the analyzed sample was diluted 10 or 100 times depending on the properties of the poison) and determine the magnitude of the inhibitory effects in the absence of substrate. To 1.0 ml of an aqueous solution of the enzyme is added 1.0 ml of distilled water, 1.0 ml of 0.1 M phosphate buffer (pH 7.5) and 1.0 ml of the investigated solution, diluted 10 or 100 times. Incubated for 3 min at 37°C, and then added 0.5 ml of Ellman, 0.5 ml of the substrate solution and after 3 min measure the optical density.

The magnitude of the protective effect (P) calculated by the formula:

where: A1OST and And2OST - the residual activity of the enzyme, determined by incubation of the enzyme with the analyzed sample in the absence and in the presence of the substrate, respectively, % of baseline value;

t1and t2- contact time of the enzyme with the inhibitor when defining A1OST and And2OST, respectively, min;

K1and K2- the factors taking into account the dilution of the sample when determining�attachment A 1OST and And2OST, respectively.

The degree of inhibition of the enzyme in determining the residual activity in the absence and in the presence of the substrate should be in the range of 25-75%.

Size Aost calculated according to the formula:

where: D is the optical density of the control sample;

D1- optical density of test sample.

Measure the optical density of the control and experimental samples is carried out using a spectrophotometer at a wavelength of 412 nm.

The calculated value of the magnitude of the protective effect of the substrate (P) is compared with tabular values defined for O-isopropyl-methyl-fluoro-phosphonate, pinacolyl-methyl-fluoro-phosphonate, O-ethyl-S-diisopropyl-aminoethyl-methyl-thio-phosphonate, About-isobutyl-S-diethyl-aminoethyl-methyl-thio-phosphonate, and the coincidence of the evidence and make a conclusion about the presence of the corresponding inhibitor.

Example 2. Identification of highly toxic anticholinesterase poisons to be destroyed in accordance with the Convention, using the proposed method.

In the experiment using the proposed method were performed to identify hazardous highly toxic anticholinesterase poisons, namely-isopropyl-methyl-fluoro-phosphonate, pinacolyl-methyl-fluoro-phosphonate, O-ethyl-S-diisopropyl-aminoethyl-methyl-thio-phosphonate, Isuzu�Il-S-diethyl-aminoethyl-methyl-thio-phosphonate, why advance experimentally established values of the protective effect (P) of the substrate, shown in the table.

In the identification of O-ethyl-S-diisopropyl-aminoethyl-methyl-thio-phosphonate previously found dilution of the sample, the suspect on the contents of the specified highly toxic anticholinesterase poison, exerting inhibitory effect on acetylcholinesterase of erythrocytes of human blood in the presence and in the absence of the substrate in the range of 25-75%. In the experiment, the value obtained 70% inhibition, i.e., A1OST is 30%. Then, the sample had to be diluted 100 times and obtained the value of the effect of oppression in the absence of the substrate, equal to 45%, And2OST is 55%. The time of oppression was 3 min.

The calculation of the size of the protective effect (P) produced by using the formula (2):

The result, according to the table, indicates the presence in the sample of highly toxic anticholinesterase poison - O-ethyl-S-diisopropyl-aminoethyl-methyl-thio-phosphonate.

In the identification of O-isopropyl-methyl-fluoro-phosphonate previously found dilution of the sample, the suspect on the contents of this highly toxic anticholinesterase poison, exerting inhibitory effect on acetylcholinesterase of erythrocytes of human blood in the presence�Wii and in the absence of the substrate in the range of 25-75%. In the experiment, the value obtained 70% inhibition, i.e., A1OST is 30%. Then, the sample had to be diluted 10 times and the obtained value of the effect of oppression in the absence of the substrate, equal to 40%, And2OST is 60%. The time of oppression was 3 min.

The calculation of the size of the protective effect (P) produced by using the formula (2):

The result, according to the table, indicates the presence in the sample of highly toxic anticholinesterase poison - O-isopropyl-methyl-fluoro-phosphonate.

Thus, experimentally proved the possibility of identification using the proposed method dangerous highly toxic organophosphorus poisons to be destroyed according to the Convention:

- O-isopropyl-methyl-fluoro-phosphonate;

- - Pinacolyl-methyl-fluoro-phosphonate;

- O-ethyl-S-diisopropyl-aminoethyl-methyl-thio-phosphonate;

- O-isobutyl-S-diethyl-aminoethyl-methyl-thio-phosphonate.

The claimed invention meets the criterion of "novelty", as the first to identify dangerous highly toxic anticholinesterase poisons is proposed to use a method comprising the determination of the protective effect of acetylthiocholine on the enzyme acetylcholinesterase of erythrocytes of human blood in the interaction of the inhibitor with the farms�Tom.

The claimed invention meets the criterion of "inventive step", as known and available sources of information containing descriptions of biochemical methods of identification anticholinesterase compound, there is no information pointing to the possibility and desirability of a definition for this purpose, the protective effect of substrate (acetylthiocholine) on the enzyme (acetylcholinesterase of erythrocytes of human blood).

The claimed invention, the criterion of "suitability for use" is confirmed by the results of the above examples showed that the proposed method with the simplicity of the event and the availability of reagents used, and the equipment to identify widely known anticholinesterase poisons representing when released into the environment an unusual and extraordinary threat to the life and health of people. The proposed method can find application in emergency situations (accidents at facilities for destruction of chemical weapons) to identify hazardous to human poisons.

Method biochemical identification of highly toxic anticholinesterase poisons, namely-isopropyl-methyl-fluoro-phosphonate, pinacolyl-methyl-fluoro-phosphonate, O-ethyl-S-diisopropyl-aminoethyl-methyl-thio-phosphonate, About-isobutyl-S-diethyl-aminoethyl-methyl-thio-phosphonate, by determining inhib�tion of the influence of a solution of highly toxic anticholinesterase poison on cholinesterase activity, characterized in that the use of cholinesterase acetylcholinesterase of erythrocytes of human blood and determine the inhibiting effect of solutions of highly toxic anticholinesterase poison, distinguished by the contents of the poison in 10 or 100 times, the activity of acetylcholinesterase of erythrocytes of human blood, first in the presence of the substrate, which is used as acetylthiocholine, and then in his absence, and the magnitude of the protective effect of acetylthiocholine R is calculated by the formula:

where: A1OST and And2OST - the residual activity of the enzyme, determined by incubation of the enzyme with the analyzed sample in the absence and in the presence of the substrate, respectively, % of baseline value;
t1and t2- contact time of the enzyme with the inhibitor when defining A1OST and And2OST, respectively, min;
K1and K2- the factors taking into account the dilution of the sample when defining A1OST and And2OST, respectively,
size Aost calculated according to the formula:

where: D is the optical density of the control sample;
D1- optical density of test sample,
measure the optical density of the control and experimental samples is carried out using a spectrophotometer at a wavelength of 412 nm, then the resulting�th calculated value of the magnitude of the protective effect of acetylthiocholine R is compared with tabular values defined experimentally in advance for the O-isopropyl-methyl-fluoro-phosphonate, pinacolyl-methyl-fluoro-phosphonate, O-ethyl-S-diisopropyl-aminoethyl-methyl-thio-phosphonate, O-isobutyl-S-diethyl-aminoethyl-methyl-thio-phosphonate, and the coincidence of the evidence and make a conclusion about the presence of this dangerous highly toxic anticholinesterase poison.



 

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