The method of increasing the resistance to hemolysis of red blood cells and restore their damaged forms

 

(57) Abstract:

The invention relates to medicine and can be used for stabilization of donor blood, the protection of red blood cells in lung machine, or any pathologies involving damage to erythrocytes. The method is carried out by introducing into the blood natural hydrophilic antioxidant carnosine or its N-acetyl derivative. Technical result: increased resistance to hemolysis of red blood cells and restore their damaged forms without disruption of the native structure of biomembranes. 2 Il., table 2.

The invention relates to medicine and can be used to stabilize blood, the protection of red blood cells in lung machine, or any pathologies, accompanied by damage of erythrocytes.

There is a method of protection of erythrocyte membranes from peroxidation using biosynthetic insulin [1].

This method is used in diabetes, but even within this pathology in patients with angiopathies it is not effective.

There is also known a method of increasing hemolytic stability (protection) of erythrocytes, it is ovadiah used method of ultrasonic hemolysis. With this method the most effective drugs that increase hemolytic resistance cells are alkyl substituted pyrimidines and benzimidazole.

The disadvantage of this method is use to increase hemolytic stability of erythrocytes are not natural compounds and synthetic drugs that violate the functional properties of erythrocytes possess inhibitory activity against membrane-bound enzymes.

The prototype of the present invention, the closest to him on the set of essential features, is a way to increase hemolytic stability of erythrocytes by introducing natural antioxidant, such as tocopherol [3] . As a damaging factor used thermal injury in laboratory animals. The method is realized by the introduction of laboratory animals administered intraperitoneally-tocopherol single dose of 20 mg/kg of body weight to thermal injury, which reduces spontaneous hemolysis of erythrocytes and reduces maintenance decreased hemoglobin in the plasma during the subsequent thermal injury.

The disadvantages of the prototype is to increase the resistance to hemolysis of erythrocytes without wosu of the present invention is to increase the resistance to hemolysis of red blood cells and the recovery of their corrupted forms without disruption of the native structure of membranes of erythrocytes.

This goal is carried out by introducing into the blood hydrophilic natural antioxidant carnosine or its N-acetyl derivative in a certain concentration range.

The method is as follows.

In the blood, which has damaged erythrocytes (poikilocytes, acanthocyte), enter solution carnosine or its N-acetyl derivative (N-acetyl-carnosine) in physiological solution at a certain concentration range, the average of 2-10 mm, which increases the resistance of erythrocytes to acid hemolysis and restoring the shape of red blood cells without disruption of the native structure of their membranes.

The resistance of erythrocytes to acid hemolysis assess physiological solution (pH 5.5) at (23+0,5)oC within 1 h after blood sampling. The dynamics of hemolysis under the action of 2 mm hydrochloric acid see by the change in optical density at 670 nm. The total change in optical density is taken for 100% hemolysed erythrocytes. Then based on these changes in the optical density in each 30-second intervals of hemolysis count % of hemolysis in each 30-second period of time.

Next, build the curve Rosie parameters: Tn- the start time of hemolysis, min; Tto- the end time of hemolysis, min; Tmaxthe time of maximum erythrogramma, min; % max number of hemolyzed erythrocytes in the interest for the time Tmax.

Acid erythrogramma for normal erythrocytes well reproducible, its main parameters: Tn=(1,70,2) min; Tto, (7,40,4) min; Tmax(3,50,1) min; % max (1,32,3) % (data are the averages of 11 observations). Increase or Tnand/or Ttoand/or Tmaxand/or reduction % max indicates an increase in resistance of erythrocytes to hemosiderosis the action of acid. Accordingly decrease or Tnand/or Ttoand/or Tmaxand/or increase % max indicates decreased resistance of erythrocytes to hemosiderosis the action of acid.

Evaluation forms erythrocytes conduct microscopic method after staining blood smears method Romanovsky-Institute [4].

Example 1. The blood of the patient B., 43 years. Diagnosis: alcoholism stage 2, the withdrawal syndrome. The number of erythrocytes with normal form diakopoulos disk does not exceed 20%. The remaining 80% of the erythrocytes are shaped acanthocytes, poikilocytes, moderate anisoles, as seen Nazina or its N-acetyl derivative. In Fig. 2 (curve A) shows erythrogramma the patient without adding carnosine or its N-Actilingua derived. It has the following basic parameters: Tn=1,0 min, Tto=7,0 min, Tmax=3,5 min, % max=23,5%, i.e. the resistance of erythrocytes to hemolysis compared to the norm reduced.

In the presence of carnosine improves the morphology of erythrocytes (recovery of their corrupted forms: significantly reduced the number of poikilocytes, disappear acanthocyte, no anisocytosis), as seen in Fig. 1 (B), which shows the micrograph of red blood cells (increase in 1400 times) patient B. after the addition of carnosine. When conducting hemolysis in the presence of carnosine basic parameters of erythrogramma: Tn=1.5 min, Tto=9,5 min, Tmax=4.5 min, % max=13,6% (see curve B in Fig. 2), indicating that the increased resistance to hemolysis of erythrocytes in the presence of this connection.

The conduct of hemolysis of erythrocytes in the presence of N-acetyl derivative of carnosine also increases the resistance of erythrocytes to hemosiderosis the action of acid. The main parameters erythrogramma in the presence of N-acetyl-carnosine to the red blood cells of the patient B: n=1,5 Mino in the presence of N-acetyl-carnosine is also a normalization of the shape of red blood cells: acanthocyte disappear blood cells have the same size and shape close to normal.

Example 2. The blood of the patient Was, 57 years old. Diagnosis: alcoholism stage 2, the withdrawal syndrome. The number of red blood cells that have a normal form, does not exceed 20%. 80% of the red blood cells have an abnormal shape. Without the addition of carnosine in the blood, or N-acetyl-carnosine settings erythrogramma the following: Tn= 1,0 min, Tto=7,0 min, Tmax=3.0V min, % max=21,3%.

In the presence of carnosine parameters are changed: Tn=1,0 min, Tto=8.5 min, Tmax= 4,0 min, % max=14,1%, i.e. the resistance of red blood cells increases. In the presence of N-acetyl derivative of carnosine basic parameters of erythrogramma were as follows:n=1.5 min, Tto=8,0 min, Tmax=4,0 min, %max=15,2%. That is, the resistance of red blood cells with data similar to carnosine also increases. According to microscopic studies of the shape of the erythrocytes in the presence of carnosine or N-acetyl-carnosine improves the number of cells with normal form increases to 70-80%, acanthocyte disappear, the number of poikilocytes does not exceed 30%.

Table 1 summarizes the aggregated parameters erythrogram 12 patients with alcoholism stage 2 in a state of abstinence, in the guises of the main parameters of erythrogram, obtained without drugs (control), in the presence of carnosine and N-acetyl-carnosine.

Ttoand Tmaxin the presence of carnosine and N-acetyl-carnosine significantly increased, and % max significantly reduced compared with control in the presence of N-acetyl-carnosine, in the presence of carnosine reduction % max was not reliable. These data indicate increased resistance to hemolysis of erythrocytes in the presence of carnosine and N-acetyl-carnosine.

Microscopic study of the effect of carnosine and its N-acetyl derivative in the form of erythrocytes was performed in 30 patients with alcoholism. The improved shape of the erythrocytes was observed in 12 cases with carnosine and 17 cases with N-acetyl-carnosine out of 30 cases, respectively, suggesting that carnosine and N-acetyl derivative is able not only to increase the resistance of erythrocytes to hemosiderosis factors, but also to recover a corrupted form of erythrocytes. While carnosine and N-acetyl - carnosine interchangeable.

Table 2 presents a comparison of the main essential features of the prototype and of the present invention, from which the advantages of the use of carnosine or N-acetyl-carnitine is N-acetyl-carnosine can protect erythrocytes, but to restore their damaged form.

- Carnosine and N-acetyl-carnosine is a hydrophilic compounds that are not embedded in native membranes and, therefore, do not violate their structure and functions.

- Hydrophilic nature of carnosine and N-acetyl-carnosine can facilitate their use in practice: in the artificial circulation, perfusion solutions and solutions for injection.

The list of references.

1. Balashova Ie, Galega E. N., Rudko I. A., Balabolkin M. I., Kubatiev A. A. lipid Peroxidation and antioxidant protection of erythrocytes in patients with diabetes mellitus. Therapeutic archive. 1993, T. 65, No. 10, S. 23 - 27.

2. Braginskaya F. I., Kruglyakov K. E., Smirnov, L. D., Zorin O. M. Zhumabayev So So Increased hemolytic stability of erythrocytes derivatives of nitrogen heterocycles. News of the USSR Academy of Sciences, biological series, 1987, No. 1, S. 154-160.

3. Bekyarova, I., Markov, M. P. Hagan Century, the Protection - tocopherol erythrocytes from hemolysis induced thermal injury. Bulletin of experimental biology and medicine. 1989, T. 107, No. 4, S. 413-415. (Prototype).

4. Nemenova Y. M. Methods of laboratory clinical trials. M:breitenau forms without disruption of the native structure of biomembranes, includes introduction to blood hydrophilic natural antioxidant carnosine or its N-acetyl derivative.

 

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