RU2646161C1 - Method of obtaining biological reference material for producing standard samples of composition of liquid biological medium and material obtained using said method - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology. Multi-element reference material and a method for its preparation are proposed. Method for obtaining a multi-element reference material comprises formation of groups of animals according to the number of toxic materials that are part of the reference preparation obtained, introduction into the animal body of a toxic material – metal in the form of its water-soluble salt and sampling of the liquid biological medium of the animal after analysis for the content of the toxicant therein. Toxic metal selected from a group consisting of mercury, cadmium, and lead is introduced into the body of the animal, and blood or urine is sampled from at least two groups of animals as the liquid biological medium. Urine or blood taken from each group of animals is mixed with known concentrations of toxic material, to obtain a composite liquid biological medium containing at least two toxic metals, followed by freezing and lyophilisation thereof.

EFFECT: claimed inventions shorten the duration of the method of obtaining multi-element reference materials with the required content of toxic material.

2 cl, 2 ex

Description

The group of inventions relates to biotechnology, and in particular to a method for producing a biological multi-element reference material for the production of standard samples (CO) of the composition of blood or urine containing at least two toxic metals selected from the range of mercury, cadmium, lead, and also to the material obtained by the proposed method, and can be used in toxicology, medicine, veterinary medicine in determining the content of these toxic metals in the blood or urine.

The following terminology is used in the invention.

Biomaterial - a part of organs, tissues, cellular or subcellular structures of the body.

Reference material - “the reference material may be used as the material (substance) of the standard sample, i.e. a material (substance), sufficiently homogeneous and stable with respect to one or several specific properties, used in accordance with the purpose in the measuring process. " [P.50.2.056-2007.GSI. Samples of materials and substances are standard. Terms and Definitions; ISO VIM: 1993].

Standard sample (composition) - (the term is specified taking into account its definitions given in GOST 8.315-97, as well as in international regulatory documents) a sample of a substance or material whose chemical composition or physical properties are:

- typical for a specific group of substances or materials;

- identified with the necessary accuracy;

- differ in high constancy;

- certified by a certificate.

Toxic - poisonous, causing negative changes in the structures and / or functions of molecular, supramolecular, cellular formations, tissues, organs and the body as a whole.

Toxic metals (hereinafter referred to as HM) are a conditional group of metals that do not have a biological function in the body, but are prone to bioaccumulation, toxic even in small quantities [Pollution of the Arctic 2002. Program for monitoring and evaluating the environment of the Arctic. AMAP. Oslo. - 2002-110 c.], Which include lead, mercury, cadmium, arsenic, thallium, vanadium, nickel, chromium, uranium and sometimes other metals.

Seeding - the introduction of a toxic metal in one way or another into the body of an animal.

Multi-element reference material - a reference material containing several TM.

Matrix of the chemical composition of a substance [material] (object of analytical control): A component or set of components that form a given substance or material of an analytical control and are its basis [GOST P 52361-2005].

To determine the content of HM in various types of materials, analysis methods are used, in particular, neutron activation, atomic emission with a direct current arc, atomic emission with inductively coupled plasma, mass spectrometric with inductively coupled plasma, atomic absorption with electrothermal atomization, atomic absorption with flame atomization, inversion voltammetric, which require the use of appropriate comparison materials - standard samples containing analysis my TM in the specified amounts.

Known standard samples made on the basis of aqueous solutions of TM salts [see, for example, State Standard Sample (hereinafter GSO) Mercury 7879-2001, GSO Cadmium 7874-2000; GSO Lead 7877-2000].

These standard samples are designed to test the performance of analytical equipment, calibrate it and can ensure the reliability of the analysis results in cases where the test material is also an aqueous solution of TM or close to that in its properties.

However, these standard samples are of little use for the analysis of biomaterials, which are far in properties from aqueous solutions and have a complex matrix structure. It is extremely important to obtain accurate analysis results that the maximum possible similarity of the matrix of the standard sample and the material under study is achieved, due to which the similarity of conditions (chemical bonds, valencies, molecular structures, etc.) in which the TM is in the standard sample and in the material under study is achieved.

So, biological reference materials are known (CO of the blood composition containing lead, state registration number GSO 9104-2008, certificate number 3677; CO of the blood containing mercury, state registration number GSO 9653-2010, certificate number 1579) obtained from animal blood or a person to whom, after selection, water-soluble salts of TM are added.

However, the TM introduced into the reference material under consideration does not undergo natural assimilation by a living organism, and therefore the completeness of the TM forms is not achieved, as well as the adequacy of the matrix of standard and studied samples.

As the closest analogues of the claimed method and material, a method for producing a biological reference material and a reference material described in RU 2567046 are selected.

The method under consideration includes dosed parenteral administration of three TMs into the animal’s body - cadmium, mercury and lead by injection of a solution containing water-soluble salts of these TMs, selection of the liquid biological medium, in particular, the blood of the animal after analysis of the content of each of these TMs, as well as freezing selected blood and its lyophilization. The reference material under consideration is a brown crystalline powder of an organo-mineral matrix (blood lyophilisate) with mercury, cadmium and lead embedded in the indicated matrix.

The obtained reference materials of the blood composition have a matrix close to the matrix of the studied blood samples, since HMs are introduced into the reference material as a result of natural assimilation by the animal’s body. At the same time, a natural diversity of forms of TM introduced into the matrix of the reference material is achieved, in particular mercury, in which the TM can be in a living organism.

The advantage of this method is also the possibility of obtaining a multi-element reference material containing the three indicated TM, which is suitable for the preparation used for the preparation of standard samples for the content of mercury, cadmium and lead used in the analysis of the liquid biological environment most demanded in modern toxicology.

However, according to the method under consideration, based on a single seed, a multi-element reference material of only one composition can be obtained, and for the preparation of a number of multi-element reference materials with different contents of HM, a new seed of animals must be made each time.

This significantly increases the labor costs and the duration of the process of obtaining multi-element reference materials with different content of HM.

The problem to be solved during the implementation of the claimed invention is the reduction of labor costs and the duration of the process of obtaining multi-element reference materials with different required content of HM. as well as obtaining multi-element reference materials of the claimed method.

The essence of the invention in relation to the proposed method lies in the fact that the method of obtaining a biological reference material for the production of standard samples of the composition of the liquid biological medium includes the introduction into the body of an animal of a toxic metal in the form of its water-soluble salt and the selection of the animal’s liquid biological medium after analysis of the toxic metal content . Moreover, according to the invention, a toxic metal selected from the series of mercury, cadmium, lead is introduced into the animal’s body, different toxic metals are introduced to different groups of animals with one or more animals in the group, blood or urine is taken as a liquid biological medium, while liquid biological medium from at least two groups of animals, the selected liquid biological medium is mixed to obtain a composite liquid biological medium containing at least two toxic metals, and then carry out t freezing the resulting composite liquid biological medium, followed by lyophilization.

The invention with respect to the biological multi-element reference material is that it is obtained by the claimed method.

In the inventive method, the TMs used to prepare the multi-element reference material are embedded in its matrix as a result of natural metabolic processes occurring in the animal organism, which provides a high degree of adequacy of the matrix of the reference material and the studied blood or urine samples. At the same time, a natural variety of HM forms is introduced, embedded in the matrix of the reference material, in particular, mercury, which can be in the form of organic and inorganic compounds, as well as in metallic dispersed form.

The advantage of the proposed method is that it allows you to get a multi-element reference material containing at least two TM from a number of mercury, cadmium, lead, in the required concentrations, which is suitable for the preparation of standard samples used in the analysis most demanded in modern toxicology liquid biological medium for the content of two or three of these TM.

Essentially important in the method is that to obtain a multi-element reference material using composite blood or urine obtained by mixing blood or urine taken from two or three groups of animals, inoculated in each group with one of the TM included in the multi-element reference material.

Moreover, according to the claimed method, by mixing blood or urine taken from the indicated two or three groups of animals, a reference material is obtained containing, respectively, two or three TM from the above series of TM.

The content of a specific TM in the blood or urine of an animal depends on the dose and the duration of its seeding with said TM, which is carried out by parenteral or alimentary administration of a water-soluble salt of said TM into the animal’s body and is determined by analysis of a sample of blood or urine.

Moreover, by mixing the blood or urine of animals of different groups with a specific content of one of TM in them in a different volume ratio, a number of multi-element reference materials with different required contents of two or three TM in them can be obtained on the basis of one seed of animals of each group.

That is, obtaining multi-element reference materials of various compositions using the proposed method is achieved by a simple technological method and does not require the use of several seed cycles. This ensures high accuracy in achieving the required concentrations of HM in the reference material.

Thus, the technical result of the invention in relation to the method is to reduce labor costs and the duration of the process of obtaining multi-element reference materials with the required different content of HM.

Moreover, the inventive method allows to provide the required content of TM in the reference material with high accuracy.

The inventive biological multi-element reference material for the preparation of standard samples of blood or urine is in the first case a crystalline powder of brown color, and in the second case, a loose gray mass, with two or three HMs embedded in the matrix of these materials and characterized in that it is obtained in in accordance with the claimed method.

The inventive reference material may have a different content of HM, which is regulated by mixing the blood or urine of animals of different groups with a specific content of one of HM in them in a selected volume ratio. Thus, it is possible to produce standard samples of the composition of blood or urine with the required concentrations of these three HMs on the basis of the claimed reference material.

Thus, the technical result achieved by the implementation of the claimed invention in relation to the material is to obtain a multi-element reference material containing at least two HM from a series of mercury, cadmium, lead, intended for the preparation of standard samples of the composition of blood or urine containing these HM .

The inventive method is as follows.

Two or three groups of animals are formed according to the number of TM, which are part of the resulting reference material. The number of animals in the group is one individual or more. As animal donors for obtaining reference material in accordance with the claimed method can be used such animals as rats, rabbits, goats, etc.

The animals are prepared for the process of obtaining the biological reference material, keeping the animals in quarantine for the time necessary for its adaptation to the conditions of detention.

Dosed seeding of animals of each group is carried out with one of TM selected from a number of mercury, cadmium, and lead. The seed is carried out by alimentary or parenteral administration to the animal's body of a water-soluble salt of one of these TM.

As water-soluble salts of mercury, cadmium, lead can be used, in particular, acetates or nitrates of the indicated TM, characterized by a sufficiently high resistance and solubility in water.

Blood or urine of animals in each group is analyzed for the content of one of TM in it.

Blood or urine of animals in each group is selected upon reaching a concentration of HM in blood or urine exceeding the concentration of this HM in the reference material, taking into account the subsequent decrease in the concentration of each HM in the total volume of composite blood or urine.

According to the method, it is also possible to select both blood and urine from animals in each group.

Mix blood or urine taken from each group of animals with known concentrations of TM in them, choosing the volume ratio of these portions, based on the content of each of the TM in composite blood or urine, corresponding to the required content in the resulting reference material.

The volume ratio of blood or urine can, in particular, be determined using the following ratios, which are based on three metals.

или

or

,

,

where V _i is the volume of composite blood or urine containing three metals - "a", "b" and "d" in concentrations a _i , b _i , d _i .

A, B, D - the concentration of the same elements in the original blood or urine,

V _a = V _i * a _i , / A is the volume of the initial blood or urine with metal "a",

V _b = V _i * b _i / B is the volume of the initial blood or urine with the metal "b".

V _d = V _i * d _i / D is the volume of the original blood or urine with the metal "d",

wherein a≤A, b≤B, d≤D.

The resulting composite blood or urine is packaged, frozen and lyophilized.

As a result of the method, a biological reference material is obtained, which is a lyophilizate of composite blood or urine of animals in the form of a dry product with the required content of HM selected from the above series.

The material is stored and transported in a sealed glass container at a temperature of + 5 ± 2 ° C.

The following are examples of embodiments of the inventions.

Example 1

Received a biological multi-element reference material for the preparation of standard samples of the composition of blood containing mercury, cadmium, lead.

Three groups of rats weighing 180-210 g were formed, 5 animals each.

In each group, enteral inoculation of rats was carried out by introducing into the animal organism the acetate salt of one of TM for 30 days with drinking water.

In this case, the animals of the first group were injected with mercury acetate in a daily dose of 0.5 mg / kg, the animals of the second group were administered cadmium acetate in a daily dose of 1.5 mg / kg, the animals of the third group were injected with lead acetate in a daily dose of 15 mg / kg.

At the end of the seed period, the blood of animals of each of the three groups was analyzed in order to determine the content of the corresponding TM in it and blood was sampled from the indicated groups of animals.

Blood lead and cadmium were determined by atomic absorption spectroscopy on an MGA-915 spectrometer, as well as inductively coupled plasma mass spectrometry (ICP-MS) on an ELEMENT-2 analyzer, and the mercury concentration was studied on RA-915 analyzers, “Julia -2 ". In all cases, at least four measurements of the determined indicator were made (n = 4).

As a result, three types of blood were obtained, containing, respectively, mercury at a concentration of 60.0 ± 9.3 μg / dm ³ (hereinafter referred to as blood of the first type), cadmium at a concentration of 16.3 ± 2.1 μg / dm ³ (hereinafter referred to as blood second type), lead at a concentration of 456 ± 18.8 μg / dm ³ (hereinafter referred to as blood of the third type).

To obtain composite blood, the blood of the three indicated species was mixed in a 5 cm ³ vial. In this case, two options were obtained (option 1 and option 2) of composite blood with different contents of TM by mixing different volumes of these three types of blood. For calculations, we used relations (1) and (2).

Option 1 of composite blood was obtained by mixing 2.50 cm ^{3 of} blood of the first type, 1.25 cm ^{3 of} blood of the second type and 1.25 cm ^{3 of} blood of the third type (the ratio of blood volumes of the first, second and third types in volume fractions was 0 respectively , 50: 0.25: 0.25).

The estimated concentration of mercury, cadmium, lead in composite blood according to option 1 was 30 μg / dm ³ , 4.1 μg / dm ³ and 114 μg / dm ^3, respectively.

The obtained composite blood according to option 1 was analyzed for the content of mercury, cadmium and lead. The concentration of these TM in the specified composite blood was 28.5 ± 3.50 μg / dm ³ , 3.9 ± 0.48 μg / dm ³ , 110 ± 9.1 μg / dm ^3, respectively.

Composite blood according to option 1 was packaged in 5 cm ³ , frozen and subjected to freeze drying.

Option 1 of the reference material was obtained — a brown crystalline powder.

To control the content of HM in this reference material, the obtained powder was dissolved in 5 cm ^{3 of} distilled water to obtain restored blood. The indicated recovered blood was analyzed for the content of mercury, cadmium, and lead in it, which amounted to 27.9 ± 3.80 μg / dm ³ , 4.1 ± 0.44 μg / dm ³ , 123 ± 11.4 μg / dm, respectively ³ .

The control data showed that the concentration of HM in option 1 of the reference material is close to their calculated values.

Variant 2 of composite blood was obtained by mixing 1.00 cm ^{3 of} blood of the first type, 1.5 cm ^{3 of} blood of the second type and 2.50 cm ^{3 of} blood of the third type, (the ratio of blood volumes of the first, second and third types in volume fractions was respectively 0.2: 0.30: 0.5).

The estimated concentration of mercury, cadmium, lead in composite blood according to option 2 was 12 μg / dm ³ , 4.9 μg / dm ³ and 228 μg / dm ^3, respectively.

The resulting composite blood according to option 2 was analyzed for the content of mercury, cadmium and lead. The concentration of these TM in the specified composite blood was 13.4 ± 1.90 μg / dm ³ , 5.2 ± 0.67 μg / dm ³ , 239 ± 20.0 μg / dm ^3, respectively.

Composite blood according to option 2 was packaged in 5 cm ³ , frozen and subjected to freeze drying.

Option 2 of the reference material was obtained — a brown crystalline powder.

To control the content of HM in this reference material, the obtained powder was dissolved in 5 cm ^{3 of} distilled water to obtain restored blood. The indicated recovered blood was analyzed for the content of mercury, cadmium, and lead in it, which amounted to 13.0 ± 0.5 μg / dm ³ , 5.2 ± 0.41 μg / dm ³ , 232 ± 21.0 μg / dm, respectively ³ .

The control data showed that the concentration of HM in option 2 of the reference material is close to their calculated values.

Example 2

Received a biological multi-element reference material for the preparation of standard samples of the composition of urine containing lead, cadmium and mercury.

Three groups of rats weighing 180-210 g were formed, 5 animals each.

In each group, parenteral (intramuscular) seeding of rats was carried out by introducing into the animal organism the acetate salt of one of TM during the first three days of a ten-day experiment.

In this case, the animals of the first group were injected with mercury acetate in a daily dose of 3.0 mg / kg, the animals of the second group were injected with cadmium acetate in a daily dose of 0.5 mg / kg, the animals of the third group were injected with lead acetate in a daily dose of 45 mg / kg.

At the end of the inoculation period on the 10th day, the urine of animals of each of the three groups was analyzed in order to determine the content of the corresponding TM in it and urine was collected from the indicated groups of animals.

Urine lead was determined by inversion voltammetry on the ABA-2 and ABA-3 analyzers, cadmium content was determined by atomic absorption spectroscopy on the MGA-915 spectrometer, as well as by ICP-MS on the ELEMENT-2 analyzer, the mercury concentration was studied on RA analyzers -915, "Julia-2." In all cases, at least four measurements of the determined indicator were made (n = 4).

As a result, three types of urine were obtained, containing, respectively, mercury in a concentration of 124.0 ± 3.2 μg / dm ³ (hereinafter referred to as urine of the first type), cadmium in a concentration of 124.0 ± 3.2 μg / dm ³ (hereinafter urine second type), lead at a concentration of 319 ± 3.8 μg / dm ³ (hereinafter urine of the third type).

To obtain composite urine, the urine of the three indicated species was mixed in a 5 cm ³ vial. In this case, two options were obtained (option 1 and option 2) of composite urine with different TM contents in it by mixing different volumes of these three types of urine. For calculations, we used relations (1) and (2).

Option 1 of composite urine was obtained by mixing 1.25 cm ^{3 of} urine of the first type, 2.50 cm ^{3 of} urine of the second type and 1.25 cm ^{3 of} urine of the third type (the ratio of the volumes of urine of the first, second and third types in volume fractions was 0 respectively , 25: 0.50: 0.25).

The estimated concentration of mercury, cadmium, lead in composite urine according to option 1 was 31.0 μg / dm ³ , 5.06 μg / dm ³ and 79.8 μg / dm ^3, respectively.

The resulting composite urine according to option 1 was analyzed for the content of mercury, cadmium and lead. The concentration of these TM in the specified composite urine was 32.9 ± 2.60 μg / dm ³ , 5.46 ± 0.44 μg / dm ³ , 75.6 ± 5.1 μg / dm ^3, respectively.

The composite urine of option 1 was packaged in 5 cm ³ , frozen and subjected to freeze drying.

Variant 1 of the urine reference material was obtained, which was a loose grayish mass.

To control the content of HM in this reference material, the obtained dry product was dissolved in 5 cm ^{3 of} distilled water to obtain recovered urine. The indicated recovered urine was analyzed for the content of mercury, cadmium, and lead in it, which amounted to 31.2 ± 2.50 μg / dm ³ , 4.78 ± 0.98 μg / dm ³ , 76.8 ± 1.3 μg, respectively / dm ³ .

The control data showed that the concentration of HM in option 1 of the reference material is close to their calculated values.

Option 2 of composite urine was obtained by mixing 0.25 cm ^{3 of} urine of the first type, 4.00 cm ^{3 of} urine of the second type and 0.75 cm ^{3 of} urine of the third type (the ratio of the volumes of urine of the first, second and third types in volume fractions was 0 respectively 05: 0.80: 0.15).

The estimated concentration of mercury, cadmium, lead in composite urine according to option 2 was 6.2 μg / dm ³ , 8.1 μg / dm ³ and 47.9 μg / dm ^3, respectively.

The resulting composite urine according to option 2 was analyzed for the content of mercury, cadmium and lead. The concentration of these TM in the specified composite urine was 5.9 ± 0.47 μg / dm ³ , 8.21 ± 1.95 μg / dm ³ , 47.86 ± 5.4 μg / dm ³ , respectively.

The composite urine of option 2 was packaged in 5 cm ³ , frozen and subjected to freeze drying.

Variant 2 of the urine reference material was obtained, which was a loose grayish mass.

To control the content of HM in this reference material, the obtained dry product was dissolved in 5 cm ^{3 of} distilled water to obtain recovered urine. The indicated recovered urine was analyzed for the content of mercury, cadmium, and lead in it, which amounted to 4.6 ± 0.35 μg / dm ³ , 7.86 ± 2.04 μg / dm ³ , 51.2 ± 1.7 μg, respectively / dm ³ .

The control data showed that the concentration of HM in option 2 of the reference material is close to their calculated values.

Examples 1 and 2 show that, based on a single seed of animals, biological multi-element reference materials with different TM contents can be obtained.

Claims (2)

1. A method of obtaining a biological reference material for the production of standard samples of the composition of a liquid biological medium, comprising introducing a toxic metal into the animal’s body in the form of its water-soluble salt and selecting the animal’s liquid biological medium after analysis of the toxic metal content in it, characterized in that the animal’s body a toxic metal selected from the range of mercury, cadmium, lead is introduced; different toxic metals are introduced to different groups of animals with the number of animals in the group of one or more, in Blood or urine is taken from the liquid biological medium, while the liquid biological medium is taken from at least two groups of animals, the selected liquid biological medium is mixed to obtain a composite liquid biological medium containing at least two toxic metals, and then the resulting composite liquid is frozen biological environment, followed by lyophilization. 2. Biological reference material, characterized in that it is obtained by the method according to p. 1.