RU2814934C1 - Method for assessing endogenous intoxication in animals - Google Patents

Abstract

FIELD: medicine; veterinary medicine.

SUBSTANCE: invention refers to medicine, namely to veterinary science, and can be used to assess endogenous intoxication in animals. Test material used is whole blood, wherein erythrocyte concentration and haematocrit are pre-set, then coagulate large-molecular compounds with 15% TCA solution and centrifuge to obtain supernatant. Optical density and concentration of low and medium molecular weight substances (LMMWS) are determined in the supernatant. Derived LMMWS values in whole blood are recalculated taking into account the erythrocyte concentration and the haematocrit value. LMMWS is determined in percentage volume of erythrocytes and in conversion to 10 billion erythrocytes, and if their values increase by more than 10% in relation to values of healthy animals, the presence of pathology and intoxication of the animal's body is determined.

EFFECT: method provides simplified and more effective assessment of endogenous intoxication in animals due to absence of stage of blood separation into plasma and erythrocyte mass and elimination of error related to variability of erythrocyte mass density during its production.

1 cl, 3 tbl, 3 ex

Description

The invention relates to veterinary medicine and can be used to determine the amount of endogenous intoxication in animals.

There is a known method for diagnosing the degree of endotoxemia [Stepanova I.P., Dmitrieva L.M., Patyukov A.G., Vysokogorsky V.E., Atavina O.V. Method for assessing endotoxicosis of the body // Patent for invention RU 2257576 C2, 07.27.2005. Application No. 2003122548/15 dated July 18, 2003.], including a study of blood plasma for the content of VNSMM and total albumin concentration, while additionally determining the content of VNSMM in red blood cells, LII and calculating the intoxication index (II) using the formula:

where S238-298(plasma), S238-298(erythrocytes) are the content of VNSMM of plasma and red blood cells, respectively, arb. units;

TCA - total concentration of blood plasma albumin, g/l;

LII - leukocyte intoxication index, arb. units,

The disadvantages of this method include the need to separate blood plasma and red blood cells, determine the concentration of albumin in plasma, and the leukocyte index of intoxication, which is determined by routine calculation of the leukocyte formula. In addition, the leukocyte profile in different animal species is not the same, which can significantly affect the indicators of endotoxemia, so this approach cannot be universal for all animal species.

There is a known method for determining endogenous intoxication by the content of substances of low and medium molecular weight in the blood [Rubtsov G.K., Bezruchko N.V., Gengin M.T., Vasilkov V.G., Borisova E.Yu., Anopin K.D. ., Vasilyeva A.D., Sadovnikova D.G., Kozlova G.A., Kruchinina A.D., Gamzin S.S. Method for determining the oxidative modification of proteins in a pool of substances of average molecular weight in blood serum, plasma, erythrocytes and urine // Patent for invention RU 2525437 C1, 08/10/2014. Application No. 2012153044/15 dated 12/07/2012], in which blood plasma is freed from high molecular weight proteins by boiling followed by centrifugation, then spectrophotometry of an aqueous solution of the supernatant is performed in the wavelength range from 200 to 330 nm.

A disadvantage of the method can also be considered the need to separate plasma and red blood cells, the need to boil samples, as a result of which some high-molecular compounds are destroyed, which distorts the true concentration of VNSMM.

There is a known method for assessing the degree of endotoxemia [Gavrilov V.B., Bidula M.M., Furmanchuk D.A., Konev S.V., Aleynikova O.V. Assessment of intoxication of the body by the imbalance between the accumulation and binding of toxins in the blood plasma // Clinical laboratory diagnostics - 1999. - No. 2. - P.13-17] using data on the content of VNSMM in the blood plasma and the total concentration of albumin, from which the intoxication criteria (CI) are calculated using the formulas:

KH1=1000* (D254/OKA)

KI2=1000* (D280/OKA),

where D254, D280 is the VNSMM content, determined by the optical density of the plasma at wavelengths of 254 and 280 nm (arbitrary units);

TCA - total concentration of blood plasma albumin (g/l).

The disadvantages of the method include the need to obtain blood plasma, determine albumin in it, and the lack of taking into account the absorption of VNSMM by erythrocytes.

There is a method for determining the level of endogenous intoxication (prototype) [Malakhova M. Ya. Method of recording endogenous intoxication: A manual for doctors. St. Petersburg, 1995. P. 34.], by determining the concentration of VNSMM in plasma and erythrocyte mass after precipitation of high-molecular compounds with a 15% solution of trichloroacetic acid, spectrometry of the resulting supernatant in the wavelength spectrum 310 ... 238 nm and calculating the area under the curve.

The disadvantages of the method include the need to separate blood into plasma and red blood cells. Also, when assessing the content of VNSMM in the erythrocyte sediment, the concentration of blood cells is not taken into account, which can significantly affect the content of VNSMM in the supernatant and leads to unreliable data.

The technical result consists in simplifying the assessment method due to the absence of the stage of separating blood into plasma and erythrocyte mass, as well as increasing the efficiency of assessment by eliminating the error associated with the variability of the density of erythrocyte mass when it is obtained.

The technical result is achieved by the fact that the method for assessing endogenous intoxication in animals, including determining the concentration of substances of low and medium molecular weight, calculating criteria and judging the presence of endotoxemia, according to the invention, whole blood is used as the test material, and the concentration of erythrocytes in it is preliminarily determined and hematocrit index, after which large molecular compounds are coagulated with a 15% TCA solution, centrifuged and a supernatant is obtained, the optical density in the separated supernatant is determined in the ultraviolet wavelength spectrum and the concentration of VNSMM in whole blood is determined, the resulting digital values for the content of VNSMM in whole blood are recalculated taking into account concentration of erythrocytes and hematocrit index, determine the saturation of erythrocytes and the percentage volume of erythrocytes VNSMM, using the following formulas:

,

where VNSMMOE - substances of low and medium molecular weight in the percentage volume of erythrocytes, conventional units, HA - hematocrit index, %.

,

where VNSME - substances of low and medium molecular weight in terms of 10 billion erythrocytes, conventional units, RBC - concentration of erythrocytes per liter of blood 10 ¹² ,

when the values increase by more than 10% in relation to the values of healthy animals, the presence of pathology and intoxication of the animal’s body is judged.

The method is carried out as follows.

The essence of the invention is that the severity of metabolic intoxication is determined by the concentration of substances of low and medium molecular weight (LMW) in whole blood, which are mainly absorbed on erythrocyte proteins. In relation to the prototype, a similar approach is used - determining the concentration of VNSMM in the blood after precipitation of large molecular compounds with a TCA solution. A significant difference is that the blood is not divided into plasma and erythrocyte mass, while the hematological parameters of the blood are first studied, and the digital values of the optical density of the resulting supernatant are recalculated taking into account the hematocrit and erythrocyte concentration.

The technically proposed method includes the following steps:

1) in a sample of stabilized blood, the concentration of erythrocytes and the hematocrit are determined using an automatic hematological analyzer;

2) 0.6 ml of blood, 0.6 ml of physiological solution or distilled water are taken into a 2 ml Eppendorf tube and 0.6 ml of a 15% TCA solution is added (that is, in a ratio of 1: 1: 1), the mixture is homogenized under ultrasonic bath or on a vortex shaker for 5-10 minutes, then centrifuge at 10 thousand vol. within 10 minutes;

3) after precipitation of the precipitate, 0.6 ml of the supernatant is sucked off and diluted in glass test tubes with 5.4 ml of distilled water (1:9 dilution);

4) using quartz cuvettes on a spectrophotometer, the optical density of the resulting solution is determined in the wavelength range 298...238 nm with a step of 4 nm (other step options are possible: 2, 6, 8, 10 nm). As a control, against which the change in optical density is assessed, a 1.5% TCA solution in distilled water is used (15% TCA solution is diluted with distilled water 1:9);

5) optical density indicators at different wavelengths are summed and multiplied by the step size, calculating the area under the curve. The resulting digital result reflects the concentration of VNSMM in whole blood (VNSMMK) conventional units (arb. units);

6) determine the saturation of erythrocytes and the percentage volume of erythrocytes VNSMM, using the following formulas:

,

where VNSMMOE - substances of low and medium molecular weight in the percentage volume of erythrocytes, conventional units, HA - hematocrit index, %.

,

where VNSME - substances of low and medium molecular weight in terms of 10 billion erythrocytes, conventional units, RBC - concentration of erythrocytes per liter of blood 10 ¹² .

Unlike these analogues, the developed method has the following advantages and differences:

1. Whole blood is used as the test material, i.e. there is no stage of blood separation into plasma and red blood cells;

2. Before sedimentation of large molecular compounds in stabilized blood, the concentration of erythrocytes and the hematocrit are determined using a hematological analyzer;

3. The obtained digital values for the content of VNSMM in whole blood are recalculated taking into account the concentration of erythrocytes and the hematocrit index, which eliminates errors associated with the variability of the density of erythrocyte mass when it is obtained.

Example 1.

The level of endogenous intoxication in cows with signs of ketonemia was assessed by determining VNSMM in the erythrocyte mass according to M.Ya. Malakhova and in whole blood using a developed method. For this purpose, blood was obtained from clinically healthy (n=10) animals with signs of ketosis (β-hydroxybutyric acid content more than 1.2 mmol/l). As is known, ketonemia in cows is accompanied by severe metabolic disorders, intoxication and accumulation of low molecular weight compounds in the blood. The research results are shown in Table 1.

Table 1. Concentration of VNSMM in the blood of healthy cows and those with ketosis, arb. units Index Healthy (n=10) Patients (n=10) According to M.Ya. Malakhova VNSMMEM 22.8±0.7 26.2±0.7 ^** Developed method VNSMMK 8.6±0.4 10.5±0.3 ^** VNSMMOE 23.2±0.7 38.4±5.4 ^** VNSMME 128.1±5.6 165.5±5.5 ^*** **P≤0.01; ***P≤0.001 significant in relation to healthy animals

It was found that the concentration of VNSMM in the erythrocyte mass (VNSMMEM) in sick cows was higher by 14.9% (P≤0.01) compared to clinically healthy animals (Table 1). The concentration of VNSMM in whole blood (VNSMMK) in sick animals was higher by 22.1% (P≤0.01), in terms of the percentage volume of erythrocytes (VNSMMW) by 65.5% (P≤0.01), and on 10 billion red blood cells (VNSME) by 29.2% (P≤0.001).

Example 2.

A comparative assessment of the level of endogenous intoxication was carried out in cows with a normal course of the postpartum period (n=10), acute purulent-catarrhal endometritis (n=10) and metritis arising from retained placenta (n=10). The research results are shown in Table 2.

Table 2. Indicators of endotoxicosis in cows with a normal course of the postpartum period and with obstetric pathology, standard units. Index With normal course (n=10) Endometritis (n=10) Metritis after retained placenta (n=10) According to M.Ya. Malakhova VNSMMEM 32.3±1.0 37.8±1.3 ^** 43.3±1.0 ^*** Developed method VNSMMK 11.1±0.4 13.8±0.3 ^*** 17.1±1.4 ^*** VNSMMOE 35.1±0.9 47.3±1.6 ^*** 49.2±1.6 ^*** VNSMME 197.0±5.4 227.0±4.7 ^*** 254.3±8.2 ^*** **P≤0.01; ***P≤0.001 significant in relation to healthy animals

The concentration of VNSMMEM in cows with postpartum purulent catarrhal endometritis after the normal course of the birth process was determined according to the method of M.Ya. Malakhova (Table 3) was higher by 17.0% (P≤0.01), while the proposed method was characterized by higher differences. Thus, the concentration of VNSMMK was higher by 23.4% (P≤0.001), VNSMMOE by 34.8% (P≤0.001), VNSMMME by 15.2% (P≤0.001) compared to animals that did not have obstetric pathology. The level of endotoxicosis in cows with signs of uterine inflammation that arose against the background of retained placenta was characterized by an increase in VNSMM in the erythrocyte mass by 34.1% (P≤0.001), VNSMMK by 54.1% (P≤0.001), VNSMMOE by 40.2% (P≤0.001), VNSMMME by 29.1% (P≤0.001).

Example 3.

The third series of experiments included blood from clinically healthy reindeer (n=10) and with intense edemagenic infestation (n=10). The results of the analysis are shown in Table 3.

Table 3. Concentration of VNSMM (arbitrary units) in the blood, determined by traditional and developed methods in healthy reindeer and with gadfly infestation. Index Healthy (n=10) Patients (n=10) According to M.Ya. Malakhova VNSMMEM 36.4±1.7 40.3±0.8 ^* Developed method VNSMMK 13.7±0.5 15.6±0.4 ^* VNSMMOE 25.1±0.6 29.6±0.9 ^*** VNSMME 132.6±5.6 154.6±4.4 ^** *P≤0.05; **P≤0.01; ***P≤0.001 significant in relation to healthy animals

It was found that during gadfly infestation, the concentration of VNSMMEM was characterized by an increase by 10.7% (P≤0.05), and the concentration of VNSMMEM in whole blood by 14.6% (P≤0.05). When recalculating the indicator taking into account blood hematocrit, the differences between healthy and infected deer were 17.9% (P≤0.001), and when calculated per unit of red blood cells - 16.6% (P≤0.01).

Thus, we can conclude that the proposed method makes it possible to effectively determine the amount of endogenous intoxication in animals. The invention is a more sensitive way to assess the level of endotoxemia, which is confirmed by a more significant difference in indicators between sick and healthy animals. An increase in values by more than 10% relative to the values of healthy animals indicates the presence of pathology and intoxication of the body.

Claims (6)

A method for assessing endogenous intoxication in animals, including determining the concentration of substances of low and medium molecular weight (LMW), calculating criteria and judging the presence of endotoxicosis, characterized in that whole blood is used as the test material, which is not divided into plasma and erythrocyte mass, with in this case, the concentration of erythrocytes and the hematocrit index are first established in it, after which large-molecular compounds are coagulated with a 15% TCA solution, centrifuged and a supernatant is obtained, the optical density in the separated supernatant is determined in the ultraviolet wavelength spectrum and the concentration of VNSMM in whole blood (VNSMMK) is determined, the resulting digital values for the content of VNSMM in whole blood are recalculated taking into account the concentration of erythrocytes and the hematocrit index, the saturation of erythrocytes and the percentage volume of erythrocytes VNSMM are determined using the following formulas: , where VNSMMOE - substances of low and medium molecular weight in the percentage volume of erythrocytes, conventional units, HA - hematocrit index, %, , where VNSME - substances of low and medium molecular weight in terms of 10 billion erythrocytes, conventional units, RBC - concentration of erythrocytes per liter of blood 10 ¹² , when the values increase by more than 10% in relation to the values of healthy animals, the presence of pathology and intoxication of the animal’s body is judged.