RU2247986C1 - Method for detecting detergent in sample - Google Patents

Abstract

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: the suggested method is based upon registration the rate of hemolysis in isotonic water solution that contains erythrocytic suspension either in the presence of a sample or without it. For this purpose the above-mentioned isotonic water solution containing standard erythrocytic suspension should be supplemented with glycine buffer at pH being 2-2.5 that induces acidic hemolysis. Detergent should be determined by the increase of acidic hemolysis rate in the presence of a sample. The method enables to improve detection in case of low concentrations of detergent in a sample.

EFFECT: higher sensitivity of detection.

2 ex, 2 tbl

Description

The invention relates to medicine and can be used in traumatology and dentistry to assess the toxicity of bone cement and plastics by the yield of monomers, in biochemistry to determine detergents in biological fluids and tissues, in the field of sanitation and hygiene for the detection of detergents in aqueous media, in pharmacology for testing the hemolytic activity of drugs.

The toxicity of detergents as surfactants lies in their ability to destroy biological membranes by disorganization of the lipid bilayer. This ability of detergents is used to identify and quantify them in aqueous solutions.

There is a well-known generally accepted method for determining detergent in a sample by recording the rate of hemolysis in an isotonic aqueous solution (Kaler G.V., Rachkovsky L.I., Okun I.M. Hemolysis of red blood cells with detergents - a mathematical model and analysis of concentration and kinetic curves. // Cytology. - 1986. - T.28. - No. 9. - S.954).

The disadvantage of this method is its low sensitivity. The low sensitivity of the method is due to the fact that registering the hemolysis rate induced by the detergent is possible only when a certain threshold concentration of detergent in the sample is reached.

The objective of the invention is to increase the sensitivity of the method for determining detergent in the sample at low concentrations of detergent.

The problem is solved in that in the known method for determining the detergent in the sample, including recording the hemolysis rate in an isotonic aqueous solution containing a standard suspension of red blood cells, in the presence of a sample and without it, according to the invention, glycine buffer with a pH of 2-2.5 is first added to the solution inducing acid hemolysis, and detergent is determined by increasing the rate of acid hemolysis in the presence of a sample.

The inventive method is based on the first discovered effect of the acceleration of acid hemolysis with a detergent. To induce acid hemolysis in an isotonic aqueous solution containing a standard suspension of red blood cells, glycine buffer with a pH value of 2-2.5 is added. The buffer is used to prevent the possible effect of the sample containing the detergent on the acidity of the medium. The method has greater sensitivity compared to the prototype, since an increase in the rate of acid hemolysis in the presence of detergent can be detected at lower concentrations of detergent in the sample compared to the prototype, where detergent hemolysis rate is recorded in the presence of detergent (see table 1).

A quantitative assessment of the detergent content in the sample is possible according to the following formula:

X = K (V ₀ −V _k ), where

K is the conversion factor, μM / dЕ 800 / min.

V ₀ is the rate of hemolysis in the presence of a sample, dЕ 800 / min.

V _to - the rate of acid hemolysis without sample, dЕ 800 / min.

The method can be carried out, for example, as follows.

Approximately 1 ml of laboratory rabbit citrate blood is obtained. Blood is washed three times with saline. To do this, it is centrifuged for 5 minutes at a speed of 1500 rpm./min, the plasma is drained and 10-15 ml of physiological saline are added to red blood cells; after mixing, the suspension is centrifuged for 5 minutes at a speed of 1500 rpm, the supernatant is drained and the procedure is repeated two more times. A physiological solution is added to the suspension of washed red blood cells to obtain a standard suspension of red blood cells: the optical density of a standard suspension at a wavelength of 700-800 nm should be 0.560 ± 0.010 after it is diluted 8 times with physiological saline. 0.6 ml of physiological saline is added to a spectrophotometer thermostatted at 37 ° C, the cell is heated for 3 minutes, after which 0.1 ml of a standard suspension of red blood cells and 0.1 ml of 0.1 M glycine buffer with a pH value are sequentially added to it 2-2.5 (the total volume of the incubation mixture is 0.8 ml). The acid hemolysis rate is recorded every 5 seconds of incubation according to the decrease in optical density at a wavelength of 700-800 nm. The maximum difference in optical density values calculated per unit time corresponds to the control hemolysis rate (hemolysis rate without sample) - Vк (dE 800 / min). An isotonic detergent aqueous solution (sample) is added to another 0.6 ml temperature-controlled cuvette of the spectrophotometer in another. The cuvette is heated for 3 minutes, after which 0.1 ml of a standard suspension of red blood cells and 0.1 ml of 0.1 M glycine buffer with a pH value of 2-2.5 are added successively (the total volume of the incubation mixture is 0.8 ml) . The acid hemolysis rate is recorded every 5 seconds of incubation according to the decrease in optical density at a wavelength of 700-800 nm.

The maximum difference in optical density values calculated per unit time corresponds to the experimental hemolysis rate (hemolysis rate in the presence of a sample) - Vo (dE 800 / min).

The method can be illustrated by the following examples.

Example 1. Determination of methyl methacrylate (a toxic component of bone cement and some plastics). To determine the methyl methacrylate according to the proposed method, various concentrations of a 1% solution of methyl methacrylate in physiological saline (sample) were used. The results are presented in table 1.

Table 1
The results of the determination of methyl methacrylate in the sample The concentration of methyl methacrylate (μm) The hemolysis rate according to the claimed method (dЕ800 / min) The prototype hemolysis rate (dЕ800 / min) V _to V _o 1,2 0,020 0,031 0 2,4 0,020 0,040 0 3.6 0,020 0,053 0 4.8 0,020 0,065 0

From table 1 it is seen that according to the claimed method in all samples at low concentrations of methyl methacrylate, an increase in the rate of acid hemolysis in the presence of a detergent (V _o above V _k ) is recorded. By the method of the prototype, the hemolysis rate in the presence of a sample is not recorded. Therefore, the sensitivity of the proposed method is higher in comparison with the prototype.

Quantification of methyl methacrylate in the sample is possible. A crushed plastic sample - 0.5 g of polymethylmethacrylate was incubated for three hours in 2 ml of physiological saline with constant stirring to extract unbound monomer in an isotonic aqueous solution. The quantification of monomethyl methacrylate in the sample was carried out by recording Vo in the presence of a supernatant (sample) and Vc without a sample. The following values were obtained: Vo = 0.031 dЕ 800 / min; Vk = 0.020 dE 800 / min (see table 1). According to the experimental data presented in Table 1, the conversion coefficient K = 111 μM / dЕ 800 / min. In accordance with the calculation formula, the content of methyl methacrylate (X) in the sample is:

X = 111 (0.031-0.020) = 1.2 (μM)

Example 2. The definition of saponin in the sample.

The threshold concentration of the hemolytic action of Merk saponin was determined experimentally and amounts to 7 μg / ml. For the study, a subthreshold concentration of saponin of 3 μg / ml was chosen. The results of the determination of saponin in the sample (0.125% isotonic saponin solution ("Merk")) by the present method and the prototype are presented in table.2.

table 2
The results of the determination of saponin in the sample The concentration of saponin (μg / ml) The hemolysis rate of the present method (dE 800 / min) The rate of hemolysis of the prototype (dE 800 / min) V _to V _o 3 0,020 0,053 0

From table 2 it is seen that when the concentration of detergent (saponin) is less than the threshold, in the presence of the sample, only an increase in the rate of acid hemolysis by the claimed method is recorded. The rate of detergent hemolysis by the method prototype is not recorded, that is, the detergent is not detected. This once again confirms the higher sensitivity of the proposed method.

The inventive method allows not only to increase the sensitivity of detergent determination in the sample, but also to reduce the labor costs for analysis. So in the prototype requires a long incubation (1-6 hours) of detergent with red blood cells and during the incubation, the selection of control and experimental samples, their centrifugation and determination of optical density. In the proposed method, the registration of hemolysis dynamics can be computerized and lasts from 10 to 30 minutes.

Claims (1)

A method for determining a detergent in a sample, comprising registering the hemolysis rate in an isotonic aqueous solution containing a standard suspension of red blood cells, in the presence and absence of a sample, characterized in that glycine buffer with a pH of 2-2.5, which induces acid hemolysis, is added to the solution, and determine the detergent by increasing the rate of acid hemolysis in the presence of a sample.