RU2542436C1 - Method of biochemical diagnostics of microelement imbalance in agricultural ungulate animals - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: method comprises preparation of samples of bio-indicators, the determining in them of content of trace elements, and the assessment of the obtained results. The bio-indicators are used as sample of buttermilk, isolated from milk of agricultural ungulate animals. The total content of copper, molybdenum and tungsten is determined in it. The ratio of the total contents of Cu/Mo and Mo/W is determined. When the values of the ratio Cu/Mo are not exceeding 1, and the ratio Mo/W are not exceeding 250, it is considered that the imbalance of trace elements in the body of animals and their habitat is absent. In case of exceeding of the specified values the presence of trace element imbalance is diagnosed.

EFFECT: method enables to diagnose quickly and accurately microelementosis of imbalance of Cu, Mo and W in agricultural ungulate animals.

5 cl, 2 ex

Description

The invention relates to the field of veterinary medicine and the protection of the human environment, since the production of environmentally friendly livestock products directly affects the quality of human food. The method can be used for early diagnosis, including erased clinical manifestations that complicate the diagnosis by conventional diagnostic methods.

In modern geochemical ecology, the interaction between living organisms and their environment is considered through a stream of atoms of chemical elements and their compounds. Entering the body through food chains, chemical elements perform various functions, their compounds are partially transformed during metabolism, accumulated by tissues, or excreted with excreted air. In this case, the biological role of trace elements plays an essential role.

According to the theory put forward by academician V.V. Kovalsky, individual chemical elements enter the body mainly in combination with others, they have specific functions. It is believed that each trace element has clearly defined points of application in the general metabolism of substances. It is with these properties of chemical elements that the development in animals and humans of a special group of diseases (microelementoses) is associated with an excess, deficiency or imbalance in the entry of chemical elements into the body.

[Kovalsky V.V. Geochemical ecology. M .: Nauka, 1974. 300 p.].

In this application, we consider 3 chemical elements: Cu, Mo and W. The first two of them are “true trace elements” - both hyper- and hypomicroelementoses are known for them. The biological role of tungsten is less studied.

Copper is a typical metal prone to complex formation. The copper content in the Earth's crust is 47 mg / kg, and in sea water - 3 μg / l. Copper 4–9 mg / kg dry matter is present in terrestrial plants, and 5–30 mg / kg in terrestrial animals. Copper-concentrating organisms exist, such as Ascidia (Busicon), arthropods, and crustaceans. The blood of the mollusks contains hemocyanin, which carries out the transfer of oxygen to the cells. The discovery of copper in the body of mollusks was made by B. Bizio in 1833.

[Vinogradov A.P. Biogeochemical provinces and endemia // Dokl. USSR Academy of Sciences, 1938. V. 18. No. 4/5. S.820].

Copper is an indispensable trace element necessary for the normal functioning of organisms, including plants, animals and humans. Copper compounds are easily absorbed into the blood from the gastrointestinal tract (5-10% in adult animals and 15-30% in young animals). Copper is characterized by homeostasis, physiologically regulated due to its absorption and excretion. The concentration of copper in the blood serum of animals is almost equal to 1000 μg / L, and in whole blood approaches 1200-1300 μg / L.

The importance of copper in the metabolic processes of the animal organism began to be revealed as a result of the work of the Wisconsin group of researchers in the USA, which showed that a lack of copper is the cause of the so-called milk anemia in rats. This observation led to the establishment of the value of copper for the processes of hematopoiesis and the use of iron in the body [Hart E.V., Waddell J., Elvehjem C.A. Irin in Nutrition. VII. Copper as supplement to iron for hemoglobin in the rat // J. Biol. Chem., 1928. Vol. 77. No. 2. P.797-812.]. Following these works, there were reports of endemic diseases in sheep and cattle in Florida, Holland and Australia, associated with a lack of copper in pasture plants and prevented by the inclusion of small amounts of copper compounds in the diet of animals. Subsequent studies have revealed the widespread prevalence of copper failure among various animal species. It was found that copper, in addition to participating in hematopoiesis, is also necessary for the normal course of many physiological processes - pigmentation and keratinization of the coat, osteogenesis, myelin formation, reproductive function and others.

Molybdenum was discovered in 1778 by C. Scheele as a metal similar to graphite and lead. Its content in the Earth's crust is 1.1-1.5 mg / kg. Terrestrial plants contain Mo 0.2-8 mg / kg dry weight, and animal organisms contain 0.2-2 mg / kg. In the late 19th and early 20th centuries, biological nitrogen fixation involving molybdenum was discovered.

Molybdenum refers to trace elements whose biological importance is considered undeniable. This element is especially important in the nitrogen metabolism of plants, animals and humans. The necessity and specificity of molybdenum has been proven for a number of redox enzyme systems involved in the processes of nitrate reduction and biological nitrogen fixation in plants and microorganisms, as well as in purine metabolism reactions in the animal body.

Water-soluble Mo ⁶⁺ , introduced in the form of salts of molybdenum acid (sodium and ammonium molybdate), Mo from insoluble compounds such as MoO ₃ , CaMoO ₄ , as well as from herbs and diet, is rapidly and almost completely absorbed in the gastrointestinal tract various laboratory and farm animals. Only in small quantities Mo is absorbed from the gastrointestinal tract when administered in the form of MoS ₂ . When rats were injected with a solution of sodium molybdate (Na ₂ MoO ₄ ) (pH 7.0-7.5), its absorption from the gastrointestinal tract of rats was approximately 85%. Mo is found in all organs of mammals, but the liver is richest in them.

The average molybdenum content in the blood is 14.7 ± 1.2 μg / l [Ermakov V.V., Safonov V.A., Tyutikov S.F. Geographic features of the variation of trace elements in the blood of cattle // Biogeochemistry of elements and compounds of toxicants in the substrate and food chains of agro- and aquatic systems. Tyumen: TSAA, 2007. S.100-103]. According to the results of our studies, the content of molybdenum in the blood of cattle depends on the region and varies from 10 to 30 μg / l. The last figure is typical for cows from the molybdenum biogeochemical province of the North Caucasus (Tyrnyauz). Molybdenum compounds, absorbed in the gastrointestinal tract, are excreted from animals with a single-chamber stomach, mainly with urine. Moreover, the degree of excretion depends on the level of sulfates. Elevated sulfate concentrations inhibit or block Mo reabsorption in the renal tubules. From the body of cows and sheep, molybdenum is excreted in the urine. For humans, the total accumulation rate of the trace element is 32, and the half-life is 27.5 days.

Tungsten - a chemical element of the VI group of the periodic table, serial number 74, atomic mass 183.85; refractory heavy metal in light gray. In nature, it consists of a mixture of five stable isotopes with mass numbers 180, 182, 183, 184 and 186.

Tungsten is not common in nature; however, it was found that some archaebacteria and bacteria have enzymes that include tungsten in their active center. There are obligate tungsten-dependent forms of archaebacteria-hyperthermophiles that live around deep-water hydrothermal springs.

Tungsten dust, like most other types of metal dust, irritates the respiratory system. There is evidence of the antagonism of molybdenum and tungsten at the molecular level when they act on the activity of milk xanthine oxidase (CSR - EC 1.17.3.2). A group of thermophilic bacteria is known, where tungsten-containing enzymes play an important role in their life.

The current level of technological development is characterized by the following patents-analogues of our method.

A known method for predicting the development of postoperative complications, including the study of blood by determining the activity of its protein components, namely xanthine oxidase. If the enzyme activity in blood serum is exceeded above 1.25 ± 0.04 cu or a decrease below 1.16 ± 0.03 judge the possibility of complications [US Pat. RF №2222014, G01N 33/48, publ. 01/10/2004].

The method is very controversial from the point of view of biochemistry, since it is known that the Mo- and Cu-containing enzyme xanthine oxidase in the body of higher mammals catalyzes the conversion of hypoxanthine to xanthine and further to uric acid [Zaitsev S.Yu., Konopatov Yu.V. Biochemistry of animals. Fundamental and clinical aspects: Textbook. - St. Petersburg: Publishing House "Lan", 2004. - 384 p.]. This biochemical reaction proceeds actively in the liver, kidneys and intestinal mucosa. In addition to these organs, high amounts of xanthine oxidase are found in blood and milk. However, a number of factors interfere with the reliable determination of the enzyme in serum. In addition, the question arises about the very relationship between the activity of the enzyme (especially in such a narrow interval) and the presence of a pathological condition in the body. The method is interesting, but it only allows you to assign a person to a risk group, and not to diagnose a specific pathology in him.

A known method for the diagnosis of disorders of the hepatobiliary system in newborns and young children from the perinatal risk group. The method also, like the previous one, relates to medicine. It is characterized by the fact that in children in the first 12 months of life, the activity of xanthine oxidase in the blood serum is determined. With enzyme activity values of 10.2 nmol / (s · ml) and lower, morphofunctional disorders of the hepatobiliary system caused by damage to the parenchymal liver tissue are diagnosed. According to the authors, the use of this method allows you to make a diagnosis in the early stages in children after perinatal hypoxia.

The method suffers from the disadvantages of the previous one. In addition, in this case we are not talking about the “norm”, since all children knowingly suffered perinatal hypoxia.

[Pat. RF №2389029, G01N 33/68, publ. 05/10/10].

A known method for assessing the microelement status of a region is carried out by bioindicating the territory, while the muscle tissue of wild ungulates and their agricultural analogs are used as bioindicators, the content of trace elements in it is determined, the results obtained are compared with critical levels of elements in food products, the boundaries of which indicate a deterioration in the microelement status of the region. The method is tested on the example of 4 elements: Zn, Cu, Mn, Se.

The disadvantage of this method is the conduct of a complex biopsy of internal organs, the associated material costs and loss of animal productivity.

[Pat. RF №2280869, G01N 33/50, publ. 07/23/06].

According to US Pat. RF №2477483 (G01N 33/50, publ. 03/10/2013), previously developed by us and taken as a prototype, a method for the diagnosis of chronic microelementoses of agricultural ungulates is characterized by taking wool samples by cutting it from the tail of the tail, quarting them to obtain an average sample in quantity 1.0-1.4 g, sample preparation of the sample, including repeated processing in bidistilled water at 30-40 ° C with constant jelling, subsequent drying at 105-110 ° C to an air-dry state, by ashing the sample in a concentrated mixture nitric and perchloric acids, an analysis of the concentrations of trace elements and a comparison of the results with data on critical concentrations of elements, beyond which are judged from the presence of the disease. The analysis of trace element concentrations is carried out by atomic absorption spectroscopy, and the upper critical concentrations for strontium and copper equal to 10–11 mg / kg and for molybdenum, lead, and mercury — 0.20–0.26 mg / kg, are used to evaluate the results. and for cadmium - 0,033 mg / kg.

The method was tested on the example of determination of Cu, Mo, Sr, Pb, Cd, however, the possibility of assessing not only Cu-Mo and Mo-W imbalances in the body of ungulates was absent. Nor was it possible to consider the antagonistic relationships of these chemical elements in the environment.

The objective of the invention is to expand the number of types of biological indicators in the in vivo diagnosis of microelement imbalance in agricultural ungulates and increase the representativeness of the results of the assessment of their habitat.

The problem is solved in that in the method of biochemical diagnosis of the imbalance of Cu, Mo and W in agricultural ungulates, including the preparation of samples of bioindicators, determining the content of trace elements in them and evaluating the results obtained, a buttermilk sample extracted from milk of agricultural ungulates is used as bioindicators, determine in it the gross content of copper, molybdenum and tungsten, find the ratio of the gross contents of Cu / Mo and Mo / W, and with values of the ratio Cu / Mo not exceeding 1, and the ratio Mo / W, not exceeding 250, consider that there is no imbalance of microelements in the animal organism and their habitat, and if these values are exceeded, the presence of microelement imbalance is diagnosed.

Typically, a bio-indicator sample is prepared by sedimenting a milk sample in a refrigerator for at least 24 hours at a temperature of no more than 3-5 ° C, followed by separation of the fat component of milk, separation, oil separation and buttermilk sampling. It is advisable to take milk samples from at least 1% of the lactating individuals of the dairy herd, and to establish the microelement imbalance in the region, take at least 3 milk samples per 1000 ha of agricultural land.

The determination of trace elements is carried out by mass spectrometry with inductively coupled argon plasma.

Lactating females of agricultural ungulates, usually cattle, take a sample of milk with a volume of at least 500 ml. For a statistically reliable diagnosis of microelement imbalance in animals, it is enough to take milk samples from 1% of lactating individuals of the dairy herd (but not less than 3 individuals in the case of a small farm). To be sure of the regional microelement imbalance, it is necessary to study at least 3 milk samples for every 1000 ha of agricultural land (hayfields and pastures). In this case, the alimentary binding of animals should be in the absence of imported feed.

Buttermilk is separate from other dairy products, because they get it not from milk, like all other dairy products, but by churning (or churning) butter from cream or sour cream. As a result of this process, a turbid, sour-tasting liquid is obtained, which, in many of its properties, is similar to serum, which contains useful, biologically active substances.

Buttermilk contains proteins, fats, milk sugar - lactose, organic acids. There are many different elements in it - potassium, sodium, calcium and magnesium, phosphorus and iron, as well as B vitamins, vitamins E and PP, and even a little vitamin C.

Buttermilk in large quantities contains Cu-Mo-xanthine oxidase, as well as the previously discovered new (for higher mammals) enzyme, W-xanthine oxidase. Our laboratory studies have shown that to increase the reliability of the assessment of the Cu / Mo habitat imbalance, these Mo / W ratios are needed. [Ermakov V.V., Tyutikov S.F., Danilova V.N. and others. The relationship of copper, molybdenum and tungsten in the biogeochemical food chain // Materials VII Intern. initial-practical. conf. “Heavy metals and radionuclides in the environment” (October 5-7, 2012). T.1. S.435-441].

In a plastic or glass container with a screw cap, it is delivered to the laboratory, where it is further sampled.

Sample preparation is carried out in two stages:

The first stage (initial sample preparation):

1. Milk is kept in the refrigerator for at least 24 hours at 3-5 ° C.

2. Carefully skim cream in an amount of at least 50 ml.

3. Using a shuttle apparatus, the butter is separated.

4. A sample of buttermilk (a translucent whitish liquid remaining after separation of the oil) is taken with a volume of at least 20 ml, which enters the second stage of analytical sample preparation.

Analytical sample preparation and instrument determination of copper, molybdenum and tungsten are carried out in accordance with MUK 4.1.1483-03 dated 06/29/2003 “Control methods. Chemical factors. "Determination of the content of chemical elements in diagnosed biosubstrates, preparations and biologically active additives by mass spectrometry with inductively coupled argon plasma." The data obtained are averaged by conventional mathematical methods and determine the ratio of the total contents of Cu / Mo and Mo / W. For Cu / Mo ratios of at least 1, and Mo / W ratios of at least 250, it is believed that there is no imbalance of chemical elements in the animal body and their environment. If these critical values are exceeded, the presence of microelement imbalance is diagnosed.

Example 1

Milk samples (500 ml each) were taken from AOZT Nika, Chernyansky District, Belgorod Region, at 10 cows of the dairy herd. For analysis, 50 ml of the obtained buttermilk was used. The results of the analysis of the concentrations of trace elements in buttermilk gave the following results (μg / L): Cu - from 102.3 to 148.1; Mo is from 73.3 to 109.2 and W is from 0.1 to 0.4. Used mass spectrometric method of analysis.

The ratio Cu / Mo was in the range 1.4-1.7, and Mo / W was in the range 733-273. Based on the calculated average statistical relations of chemical elements, we state:

1) the lack of cows chronic Cu-Mo and Mo-W imbalance,

2) the absence of chronic Cu-Mo and Mo-W imbalance in their environment.

Example 2

At the Tyrnyauz market in the Elbrus region of the Kabardino-Balkarian Republic, milk of local cows was purchased (number of samples 6). The area of grazing and haying does not exceed 1,500 ha. The number of substances in the samples and the analysis method are similar to example 1. The results of the analysis of the concentrations of trace elements in buttermilk gave the following results (μg / l): Cu - from 583.3 to 702.1; Mo is from 697.7 to 893.2 and W is from 3.8 to 4.8. Based on the calculated ratios of concentrations of chemical elements, we ascertain 1) the presence of chronic Cu-Mo and Mo-W imbalance in cows and 2) the presence of chronic Cu-Mo and Mo-W imbalance in their environment.

Thus, this diagnostic method allows in vivo to establish the presence in animals of microelement imbalance of various etiologies, as well as to establish a violation of the natural ratios of elements in their environment. It is relatively simple and allows you to examine a significant number of animals and a large region as a whole almost simultaneously.

The method can be used for environmental mapping, identifying adverse areas of the studied regions and differentiated assessment of regional pollution by tungsten.

Claims (5)

1. The method of biochemical diagnosis of the imbalance of Cu, Mo and W in agricultural ungulates, including the preparation of samples of bioindicators, determining the content of trace elements in them and evaluating the results, characterized in that as a bioindicator use a sample of buttermilk isolated from milk of farm ungulates in it, the gross content of copper, molybdenum and tungsten, find the ratio of the gross contents of Cu / Mo and Mo / W, and with Cu / Mo ratios not exceeding 1, and Mo / W ratios not exceeding 250, they believe that there is no imbalance of microelements in the animal organism and their habitat, and if these values are exceeded, the presence of microelement imbalance is diagnosed. 2. The method according to claim 1, characterized in that the preparation of the bio-indicator sample is carried out by settling a milk sample in the refrigerator for at least 24 hours at a temperature of 3-5 ° C, followed by separation of the fat component of milk, separation, separation of oil and sampling buttermilk. 3. The method according to claim 1, characterized in that milk samples are taken from at least 1% of the lactating individuals of the dairy herd. 4. The method according to claim 1, characterized in that at least 3 milk samples per 1000 ha of agricultural land are taken to establish the trace element imbalance in the region. 5. The method according to claim 1, characterized in that the determination of trace elements is conducted by mass spectrometry with inductively coupled argon plasma.