SU1725120A1 - Method of estimating toxicity of biological fluids - Google Patents

Abstract

The invention relates to medicine, biology, and can be used to determine the toxicity of biological fluids of a human body. The purpose of the invention is to simplify the method by reducing the number of labor-intensive operations while increasing the accuracy of the method. Toxicity assessment is made by taking a sample of a biological fluid, preparing a filtrate, exposing the filtrate to a suspension of mobile biological micro-objects, followed by recording the dependence of the mobility of the suspension of mobile micro-objects on time. In this case, the sample is subjected to filtration in the foam phase through a filter with a pore diameter of 45 ± 5 A & and a throughput of 0.5 ± 0.1 in the mol. 50000, a suspension of bull spermatozoa in a glucose-citrate solution is used as a suspension of mobile biological microobjects, the viability of bull spermatozoa is determined, and toxicity is determined by the change in viability of the suspension of bull spermatozoa in the filtrate relative to the viability of the bull sperm suspension via glycosocytes in a filtrate relative to the viability of a bull spermatozoa suspension via glycosocyte suspension. The proposed method makes it possible to increase the accuracy of assessing the toxicity of biological fluids of a human body in various diseases accompanied by intoxication. ; ..,. cl

Description

The invention relates to medicine, in particular, it is applicable to determine the toxicity of biological fluids of a person in a variety of diseases accompanied by intoxication.

The purpose of the invention is to simplify the method by reducing the number of laborious oner radios while improving the accuracy of the method.

The method is carried out as follows.

A sample of the studied biological fluid is filtered in the foam phase through a filter with a pore diameter of 45 ± 5 A ° and a throughput of 0.5 ± 0.1 in the mol. 50000. As a suspension of mobile biological micro-objects, a suspension of bull spermatozoa in a glucose-citrate solution is used. The viability of a bull spermatozoa suspension is determined as the product of the average mobility time and the logarithm of the mobility at the time of the beginning of exposure to the filtrate, and toxicity is determined by the change in the viability of the bull sperm suspension in the filtrate relative to the viability of the bull sperm suspension in a glucose citrate solution.

The choice of filter characteristics (namely, a pore diameter of 45 ± 5A ° and a transmission capacity of 0.5 ± 01) is due to the need to remove from the sample of biological fluid high-molecular compounds that cause sperm cell agglutination. This makes it possible to correctly assess the viability of the sperm suspension. In addition, the characteristics of the filter are such that all components responsible for toxicity fall into the filtrate.

Filtration in the foam phase allows the use of small sample volumes, provides better mixing and removal of the filter cake.

Use as a suspension of mobile biological microobjects a suspension of bovine spermatozoa in a glucose-citrate solution is due to the fact that the sperm cell as a mammalian cell, in comparison with parameres by a known method, more adequately responds to changes in the composition of biological fluids of the human body.

Example 1. A patient is taken from a catheter standing in one of the central veins, 10 ml of blood is taken, then it is centrifuged for 15 minutes at a speed of 3000 bpm. The obtained serum is subjected to filtration using a peristaltic pump through a fiber filter with a pore diameter of 45 ± 5 A ° and a flow rate of 0.5 ± 0.1 in the molar area. 50,000.

To determine the degree of toxicity, the filtrate is compared with the control solution. The control sample is the cytocylate medium (sodium citrate, distilled water).

The prototype in the experiment is the filtrate, brought to isotony. 0.2 g of glucose and 0.050 g of sodium citrate are added to 5 ml of the filtrate.

As a suspension of mobile biological micro-objects, granulated bull sperm frozen in liquid nitrogen vapor is used.

Thawing of each granule is carried out in 0.5 ml of glucose-citrate medium at 40 ° C. After thawing, the semen is poured into a flask and mixed thoroughly. Then, in 1 ml of the test sample, 0.2 ml of thawed sperm is added. Prepared experimental and control samples are placed in the measuring cell. The measurement of the dependence of mobility is carried out - values,

equal concentration of motile cells on the average modulus of their speed, from time to time in arbitrary units for the test and control samples. Calculate the viability of the bull sperm suspension in the experimental C ° and control LCD samples as the product of the average mobility time tcp and the logarithm of mobility at the initial time point logmt O

Ж ° т. V logm ° t 0; LCD tcPK logm tK 0. Average mobility time is measured in minutes, and mobility in arbitrary units.

Toxicity (toxicity index) is defined as the ratio of the viability of the test sample to the viability of the control sample.

Ж °

t;

Lcd

100%

0

five

0

five

where 1T is the toxicity index.

For the patient's serum B, diagnosed with pancreatic necrosis, stuffing pouch abscess with a breakthrough into the free abdominal cavity, spilled purulent peritonitis state after operations: tcp ° 21.7 min, tm) 0 438 cu, J0 57.8 min Dsr 51,5 min, tm) .e. ,, 5min, T 44%.

Example 2. Patient A. serum with a diagnosis of pancreatic necrosis, enzymatic peritonitis state after cholecystostomy, drainage and packing of the omental bursa and abdominal cavity: tcp ° 25.1 min, gti-o ° 605, F ° 68, 6 min, tcpK 51.5 min. mt-oK 178 cu, LCD 131.5 min, G 53%.

Example 3. For the patient's serum of a patient I. diagnosed with sepsis, spilled purulent peritonitis, phlegmon of the retroperitoneal space and pararental tissue, the state after operations: tcp ° 12.8 min, mt-o ° 258 u., Ж ° 30, 9 min, tCpK 34.1 min, rm-ca 190u, e „LCD 77.7 min. t 40%.

Example 4. For serum of a healthy person T: tcp ° 36.4 min, mt-o ° 217 cu, ° ° 217.6 min, tcpK 49.1 min, int-oK 167 cu, LCD 167.9 min, 1T 130%.

Example 5. For serum of a healthy person K: tcp ° 51.6 min, mon-o ° 335 cu, Zh ° 339 min, tCpK 62.8 min, 284 cu, LCD 284 min, 1t 119%.

From examples 1-4 it can be seen that patients have a toxicity index value significantly lower (higher intoxication) than in healthy people. Patient I. compared with patients V. and A. the lowest toxicity index, indicating a greater severity of intoxication, which led the patient to death. In all presented patients with syndrome of epdotoxicosis, the value of the toxicity index coincided with clinical and laboratory data.

The toxicity index determined by this method makes it possible to objectively assess the severity of endotoxic acid, the choice of detoxification therapy method and its evaluation.

Example 6. Experimental animal (dog), diagnosis: peritonitis.

Lymph is obtained, sample preparation is carried out analogously to example 1: tcp ° 17.5 min, cu, 6 min, .4 min, bale - 260 cu, lc 77.8 min. T 58.6%.

Example 7. Experimental animal (dog), diagnosis: peritonitis.

The cerebrospinal fluid is obtained, sample preparation is carried out as in Examples 1-4: tcp ° 18.2 min, rt-° ° 512 cu, ° C 49.3 min, tcp 34.2 min, mt-o 258 cu. Lc 81.9 min, 1T 60.2%.

The invention makes it possible to increase the accuracy of the method by estimating the mobility of 1000 or more cells, as well as by determining the viability of the bull sperm suspension as a product of the average mobility time by the logarithm of the mobility at the moment of onset of exposure. In the aggregate, the accuracy of the proposed method is increased, at least by an order of magnitude. The proposed method also makes it possible to simplify the determination of the toxicity of biological fluids.

five

0

five

0

due to the use of a standard sperm suspension, maintained for an unlimited length of time before use in liquid nitrogen, and also due to the possibility of using an instrumental assessment of sperm suspension mobility. The ability to use a sperm suspension is provided by pre-filtration of the sample under study.

Fo rmu l and z o b r e u A method for assessing the toxicity of biological fluids, including a study of the effect of biological fluid on a suspension of mobile biological objects, followed by an assessment of toxicity of changes in the mobility of objects in time immediately after the addition of the test fluid characterized in that, in order to simplify the method by reducing the number of labor-intensive operations while simultaneously improving the accuracy of the method, a sample of the biological fluid is previously subjected to filter s in the foam phase, removing the ingredients with mol.m. over 50,000, as a micro-object, a suspension of bovine spermatozoa in a glucose-citrate solution is used, and viability is defined as the product of the mean mobility time and the logarithm of the mobility value.

Claims (1)

Claim A method for assessing the toxicity of biological fluids, including the study of the effect of a biological fluid on a mobile / biological suspension. microobjects with subsequent toxicity assessment by changing the mobility of objects in time immediately after adding the test fluid, characterized in that, in order to simplify the method by reducing the number of time-consuming operations while improving the accuracy of method 25, a sample of biological fluid is pre-filtered in the foam phase removing ingredients from mol.m. more than 50,000, a suspension of sperm of bovine 30 in a glucose-citrate solution is used as microobjects, and viability is defined as the product of the average mobility time and the logarithm of the mobility value.