RU2362572C2 - Way of depression of concentration of ammonia in blood by means of ammocytes and incapsulated glutamin synthetase - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: for depression of concentration of ammonia in blood by means of the enzyme introduced in erythrocytes of glutamin synthetase, it is incapsulated in erythrocytes using the dialysis method. The obtained ammocytes are entered intravenously to the animals with experimental acute hyperammoniemia.

EFFECT: invention dilates an arsenal of the agents reducing concentration of ammonia at the expense of creation of an agent, possessing high efficiency and not causing immunologic rejection throughout several days.

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Description

Usage: in the field of medical biochemistry, in particular in acute ammonia intoxication.

The essence of the invention: obtaining red blood cells with the included dialysis method glumamine synthetase (ammocytes). Ammocytes are administered intravenously to animals and after 0.5-2 hours the concentration of ammonia in the blood is determined. The method allows to reduce the concentration of ammonia in the blood by 1.5-4 times in the first 2 hours of acute ammonia poisoning.

The invention relates to medical biochemistry and can be used to reduce the concentration of toxic ammonia in the blood using an enzyme incorporated into red blood cells. Carrier erythrocytes filled with the enzyme and designed to reduce the concentration of ammonia in the blood (called by us ammocytes) are not equivalent to the whole erythrocytes in metabolic properties. Ammocytes contain a special enzyme that can neutralize toxic ammonia and which is absent in normal red blood cells. This ability of ammocytes depends on the enzyme used, the method of incorporation of the enzyme into red blood cells and the subsequent processing of newly obtained cells.

There is a method of introducing L-asparaginase into red blood cells as a medicine (Agranenko V.A., Ataullakhanov F.I., Vitvitsky V.M., Kiyatkin A.B., Zhabotinsky AM, Markova N.A., Sinauridze E.I. A method of preserving pharmacocytes with L-asparaginase incorporated. Patent of the Russian Federation No. 1777887, published on November 30, 1992). The aim of the development of this method was to store the drug in canned donated blood for possible future use. The disadvantages of the method are that it is not intended to reduce the concentration of ammonia in the blood.

A known method of reducing the concentration of ammonia in the blood of a mouse using red blood cells and glutamate dehydrogenase encapsulated in them (Sanz S., Lizano C., Luque J., Pinilla M. In vitro and in vivo study of glutamate dehydrogenase encapsulated into mouse erythrocytes by a hypotonic dialysis procedure Life Sci. 1999, 65 (26), 2781-2789). The method was tested in chronic ammonia poisoning in mice. This method is closest to the proposed in this invention and is used as a prototype.

The aim of the invention is to develop a method for reducing the concentration of pathologically high concentrations of ammonia in the blood using ammocytes - erythrocytes with the enzyme glutamine synthetase encapsulated in them. The goal is achieved by strict standardization of the dialysis process of obtaining erythrocyte carriers. The difference of the proposed method from the prototype is that the erythrocyte carriers are loaded with glutamine synthetase.

Example. Receiving and storage of ammocytes with encapsulated glutamine synthetase. For the preparation of carrier erythrocytes with glutamine synthetase inside, erythrocytes obtained by removing plasma from the blood of laboratory rats and then washing them three times were used.

Blood was collected from a rat in a test tube with 2 ml of heparin (40 units / ml), centrifuged for 10 min at 1000 g and 4 ° C. Plasma was removed. The cell pellet was washed three times at 2500 g, 10 min, 4 ° C with a solution containing 10 mM KH ₂ PO ₄ , 3.5 mM KCl, 1.5 mM MgCl ₂ , 145 mM NaCl, pH 7.4.

The washed red blood cells (1 ml) were mixed with 0.3 ml of a buffer solution containing 75 mM KCl, 75 mM Na ₂ HPO ₄ , 10 mM mercaptoethanol, pH 8.0, and 2 units. glutamine synthetase (hematocrit suspension 15%).

The mixture was transferred to a dialysis bag (CelluSep H1, pore size 15,000 Da). Hypotonic dialysis was performed for 3 hours in a dialysis unit in 100 ml of a lysing solution containing 5 mM KH ₂ PO ₄ , 5 mM glucose, 2 mM MgCl ₂ , 1.5 mM ATP, 0.1 mM NAD, 3 mM glutathione, 2.3 mM mercaptoethanol, pH 7.45, at 4 ° C and with continuous stirring.

At the end of dialysis, the mixture was transferred to a plastic bottle and subjected to “hardening”: 10 minutes stirring on a shaker at 100 swings / min and 37 ° C. Then, the cell membranes were closed by adding 0.7 ml of a buffer solution containing 30 mM NaH ₂ PO ₄ , pH 7.4, 30 mM inosine, 30 mM glucose, 30 mM sodium pyruvate, 1.5 mM adenine and 3% NaCl, and incubation for 5 min at 37 ° C and 100 swings / min

Ammonites were washed for 10 min at 4 ° С and 200 g with a solution containing 10 mM KH ₂ PO ₄ , pH 7.4, 3.5 mM KCl, 1.5 mM MgCl ₂ , 145 mM NaCl, 6 mM glucose, resuspended in this solution and for 0– 24 hours was used for administration to rats with experimental acute hyperammonemia.

Each rat of the experimental group was injected intraperitoneally with 0.3 ml of 0.25 M ammonium acetate (dose 2.5 mmol / kg body weight). Immediately before this, control animals were injected intravenously with 0.4 ml of a mixture of 0.9% NaCl and 5 mM glucose, while experimental animals were injected intravenously with 0.4 ml of ammonites with inoculated glutamine synthetase. Blood was taken from these rats after 30 and 60 minutes from the retroorbital venous plexus, after 120 minutes - mixed during decapitation.

The specific activity of glutamine synthetase in ammocytes was 1.35 μmol / min · ml, and the degree of inclusion of the enzyme in the cells was 15.7%.

Changes in the concentration of ammonia in the blood in rats with hyperammonemia over time and after administration of ammocytes with glutamine synthetase are shown in the table.

Table The time after the introduction of ammonia, minutes The concentration of ammonia in the blood, mm Control (introduced 0.9% NaCl and glucose) Experience (introduced ammocytes) thirty 0.94 ± 0.16 (n = 4) 0.52 ± 0.09 (n = 4) 60 0.84 ± 0.02 (n = 2) 0.57 ± 0.00 (n = 2) 120 0.95 ± 0.26 (n = 4) 0.22 ± 0.05 (n = 3)

Claims (1)

A method of reducing blood ammonia concentration using red blood cells with an encapsulated enzyme, characterized in that glutamine synthetase is used as such an enzyme, and that the red blood cells are encapsulated with a dialysis method of glutamine synthetase, the obtained ammocytes are administered intravenously to animals with experimental acute and measure the concentration of ammonia in the blood.