Anemia, due to deficient activity of glucose-6-phosphate dehydrogenase

Deficit activities glucose-6-fosfatdegidrogenazы (G-6-FD) - Is the most common genetic abnormality of red blood cells, resulting in hemolytic crisis, associated with taking a number of drugs means. Without crises, most patients observed a state of total compensation, although the individual has a permanent place hemolytic anemia.

The first description deficit activity of G-6-PD was made in 1956 g. in persons, taking prophylactic antimalarial drug primaquine. Regardless of these studies 1957 g. It was discovered deficiency of G-6-PD in red blood cells of the patient, which are periodically observed hemolytic crises without taking any drugs.

Currently described more 250 mutantnyh different form G-6-FD. They differ from each other in the electrophoretic mobility of the enzyme, ego related to the media - glucose-6-phosphate and nikotinamidadenindinukleotidfosfatu (NADf) A consequence of the reduced affinity is insufficient enzyme activity in a, when the concentration of substrates is strictly limited by the rate of their formation in the previous reaction. The lack of activity is not, in most cases, loss of the enzyme as such even though there may be also cases. Most often, the absence or reduction of enzyme activity - the result of the presence of his patient in a pathological form of inactive.

Structural gene and gene control, obuslovlivaûŝie syntheses of G-6-FD, located on the X-chromosome, however inheritance deficiency of this enzyme activity in erythrocytes always is linked with the X-chromosome.

There are two main forms of mutant, in which amino acid substitutions do not relate to the active centers, so both widespread mutations are normal. They differ in electrophoretic mobility, however, affinity to the substrate they are equally. In modern nomenclature, one of these forms, common in Europe, BB called form, and the other, found in Africa - A form. Currently described and other mutant forms of, who also did not differ from each other in the kinematic parameters, but have different electrophoretic mobility.

Clutch enzyme with the floor gives a significant predominance of men among persons with symptomatic disease. It is observed in homozygous males, this pathology inherited from the mother with her X-chromosome, homozygous women (inherited the disease from both parents) and in some heterozygous females, inheriting the disease from a parent with the expressed mutant phenotype.

Frequently deficit activity of G-6-PD occurs in Europe, located on the Mediterranean coast, Greece, Italy, as well as in some Latin American countries, Africa, etc..

Maybe, that the extremely high accumulation of an abnormal gene in a number of settlements contributes to the custom of consanguineous marriages, which leads to the accumulation of a population of homozygous women, giving severe clinical manifestations of the disease often, than heterozygous carriers, and increase the probability of homozygous males, and also widely used in the past in these places falciparum malaria.

According to the current hypothesis, person, with a deficit of G-6-PD, easier to tolerate tropical malaria and rarely die from the disease. In favor of this hypothesis is the fact of unequal spread of the parasite among women heterozygous red blood cells. It was found, in normal cells that contain a much larger quantity of parasite, than deficient. There are two explanations for this alleged fact. The first of these: erythrocyte deficit of G-6-PD is not able to withstand the effects of the parasite and immediately splits in the phagocytic macrophages, where he killed Plasmodium. Another view suggests neobhodmost for the development of the parasite reduced glutathione, the amount of which is reduced in erythrocytes deficient G-6-PD.

Etiology and pathogenesis

The first stage of drug exposure - its conversion in the body, transition to the active form, which can cause changes in the structure of the membrane of the erythrocyte. The active form of the drug reacts with oxyhemoglobin. This produces a certain amount of hydrogen peroxide.

Reduced glutathione peroxidase system using neutralizes some portion peroxide, a reducing reaction is oxidized glutathione.

In healthy people, acute hemolytic crisis develops with the introduction of significant amounts of the drug (toksicheskaya dose). The crisis may occur when, when the system of reduced glutathione is unable to cope with the excess of complexes formed and oxidized glutathione. At deficiency of glucose-6-phosphate dehydrogenase and NADP impaired recovery, despite the normal activity of glutathione reductase, violated his recovery, because there is no normal hydrogen source. Reduced glutathione can not resist the oxidative action of the usual therapeutic doses of drugs. This leads to the oxidation of hemoglobin, loss of heme from the hemoglobin molecule, precipitation of the globin chains. Spleen cells from the red blood cells releases Heinz. At the same time it lost part of the surface of red blood cells, which leads to their death.

In the pathogenesis of hemolytic anemia, associated with the consumption of faba bean, many more obscure. Primahinovaya anemia (favizm) It develops only some individuals deficient activity of G-6-PD. Probably, for the manifestation of anemia requires a combination of two enzymatic defects. Maybe, that it is a lack of neutralizing some individuals toxic substances, contained in faba beans, or of the formation of a metabolite, causing disturbances in the sulfhydryl groups of red blood cells. In healthy individuals receiving small amounts of horse bean causes severe hemolytic anemia, since in the presence of reduced glutathione erythrocytes able to counteract the toxic effect of metabolites. Inheritance of this deficit, apparently, autosomal dominant. With the combination of extraordinary transformation in the body of toxic substances, contained in faba beans, deficient activity of G-6-PD show clinical signs favism.

Clinical manifestations

WHO experts are divided variants of G-6-PD in accordance with the clinical manifestations in patients with homozygous and level of activity in the erythrocytes into four classes.

First grade - Options, accompanied by chronic hemolytic anemia.

Second class - Options with the level of G-6-PD erythrocyte 0- 10 % from the norm, carriage which causes hemolytic anemia is the lack of crisis, and crises, associated with taking drugs or drinking horsebean.

Third-class - Variants with activity levels in erythrocytes 10-60 % from the norm, in which the light can be observed clinical manifestations, associated with the intake of drugs.

Fourth grade - Options for a normal or near normal level of activity, not accompanied by clinical pathology.

At birth, the child has hemolytic anemia, Related to the first, and the second class deficiency of G-6-PD.

The level of activity of G-6-PD in red blood cells do not always correlate with the severity of clinical manifestations. In many embodiments, the first class is determined 20-30 % level of enzyme activity. On the other hand, at a zero level of activity in some patients did not observe any clinical symptoms. It's connected, At first, with the properties of enzymes lutantnyh, Secondly, most likely, at a rate of neutralization of medicaments apparatus liver cytochrome patient.

The most common deficiency of the activity of G-6-PD does not provide clinical manifestations without special provocation hemolytic crisis. In most cases, hemolytic crisis begins after receiving sulfonamides (norsulfazola, streptotsid, sulfadimetoksina, sodium sulfatsil, etazola, biseptola), antimalarials (primaquine, xinina, akrihina), nitrofuran drugs (furazolidona, furadonina, furagina, 5-NOK, nevigramona), drugs of isonicotinic acid (tuʙazida, ftivazida), PAS-sodium, and nitroglycerine.

From antimalarials deficiency of activity of G-6-PD can be assigned delagil, of sulfa - ftalazol. A number of drugs, in large doses cause hemolytic crisis, in small doses it can be used at the deficit activity of G-6-PD. These include acetylsalicylic acid, amidopirin, fenacitin, levomicetin, Streptomycin, antidiabetic sulfa drugs.

All drugs, can cause hemolytic crisis, denaturation of hemoglobin catalyzed oxidation with molecular oxygen.

Clinical manifestations of the disease may occur in the second or third day from the start of medication. First, there is a slight yellowness sclera, dark urine. Upon termination of the drug during this period heavy hemolytic crisis does not develop. If the treatment continues, 4-5-th day of hemolytic crisis may occur with the release of urine black or sometimes brown, which it is associated with intravascular erythrocytolysis. Hemoglobin may be reduced by 2-3 %.

In severe disease the body temperature rises, appear sharp headache, pain in the extremities, vomiting, sometimes - diarrhea. There is shortness of breath, reduced blood pressure. Often increased spleen, sometimes - liver.

In rare cases, kidney failure develops, associated with the sharp decline in renal filtration and renal tubular obstruction by blood clots.

Laboratory findings

At a blood analysis revealed anemia with increased reticulocytes. There has been an increase in the number of leukocytes with a shift to myelocytes. In some patients,, especially in children, white blood cell count can sometimes rise to significant digits (100 T in 1 l and more). The number of platelets unchanged. When stained with crystal violet red blood cells during severe hemolytic crisis revealed a large number of Heinz bodies.

Revealed a sharp irritation of red bone marrow germ. Increase the content of free hemoglobin Serum, often elevated levels of bilirubin due to indirect. With benzidine sample revealed the presence of hemoglobin in the urine without red blood cells, sometimes found hemosiderin.

In some forms of deficiency of glucose-6-phosphate dehydrogenase hemolysis observed restraint, t. it is. hemolytic crisis breaks, despite, that the patient continues to take the drug, that caused hemolytic crisis. Ability to self-limitation of hemolysis caused by increased almost to normal values ​​in the level of enzyme activity reticulocytes. In most forms of it is considerably reduced.

In children, severe hemolytic crises occur more frequently, than in adults. In marked deficiency of the activity of G-6-PD sometimes they occur immediately after birth. This hemolytic disease of the newborn, not associated with immunological conflict. It can occur just as hard, as hemolytic anemia, rezusnesovmestimostyu caused maternal and fetal. Perhaps the presence of kernicterus with severe neurological symptoms.

The pathogenesis of these crises has not been studied. It is not found, whether these crises occur spontaneously due to the lack of physiological activity of the enzyme glutathione peroxidase, at birth, or is the reason for their use of certain antiseptics in processing the umbilical cord of the child. Maybe, that sometimes crises associated with taking the mother of some drugs.

In some cases hemolytic crisis deficiency of activity of G-6-PD arise against the background of infectious diseases: Flu, salmonellosis, Viral Hepatitis. Crises can be triggered as acidosis in diabetes mellitus or renal failure.

A small percentage of patients with deficiency of activity of G-6-PD observed constant hemolytic anemia, associated with taking drugs. In these cases, there are a slight increase in spleen, moderate normochromic anemia with high content of reticulocytes, erythrokaryocytes in the bone marrow and bilirubin. Worsening of the disease or possible after receiving the above drugs, or a background infections.

Diagnostics

The basis for the diagnosis of this disease is the determination of red blood cell enzyme activity of G-6-PD in the proband and his family. Qualitative methods, used for this purpose, It should recommend two of the most simple methods.

Method Bernstein It makes it possible not only to diagnose deficiency of activity of G-6-PD at all hemizygous males, Women homozygous, but some estimate the degree of deficiency of this enzyme in heterozygous women. This method can be revealed about 50 % heterozygous females. The advantage of this method is its suitability for use in the mass screening of the population in field conditions.

The method is based on the bleaching of the dye 2,6-dichlorophenolindophenol and when reconstituted. In the presence of G-6-PD oxidizes glucose-6 phosphate and reconstitution to form NADP NADPH. This substance restores phenazine methosulfate, which in turn restores 2,6-dichlorophenolindophenol. Phenazine methosulfate acts in the reaction as a very active carrier of electrons from NADPH to the dye. Without phenazine methosulfate reaction proceeds for several hours, and in the presence of phenazine methosulfate discoloration occurs in 15- 30 m.

Reagents.

1. The solution NADP: 23 mg NADP dissolved in 10 ml of water.
2. A solution of glucose-6-phosphate (G-6-F): 152 mg of the sodium salt of glucose-6-phosphate is dissolved in 10 ml of water. The barium salt of glucose-6-phosphate must be previously transferred to the sodium salt. To this weighed 265 mg barievoy salts glucose-6-phosphate, dissolved in 5 ml of water, add 0,5 ml 0,01 M hydrochloric acid solution and 1 mg of dry sodium sulfate. The precipitate was centrifuged. The supernatant was neutralized 0,01 M sodium hydroxide solution and adjusted with distilled water to 10 ml.
3. Phenazine methosulfate solution: 2 mg of phenazine methosulfate dissolved in 100 ml Tris-buffer 0,74 M; pH 8,0.
4. The dye solution of 2,6-dichlorophenol (sodium salt): 14,5 mg of dye was dissolved in 100 ml buffer solution of tris-hydrochloric acid (0,74 M; pH 8,0). The buffer solution is prepared from 1,48 Simply entails M Tris-gidroksimetilaminometana (42,27 d of 250 ml of water) and 1,43 M solution of hydrochloric acid (2 ampoules fiksanala, contains 0,1 eq, adjusted with water to 135 ml). K 230 ml Tris gidroksimetilaminometala added 110 ml of hydrochloric acid, pH is adjusted to 8,0 and water added to 460 ml.

Before application, a mixture of reagents: 1 no. NADP solution (1), 1 no. solution G-6-P (2), 2 no. phenazine methosulfate solution (3) and 16 no. solution of 2,6-dihlorfenolinodofenola (4).

Method.

The tube, contains 1 ml distilled water, contribute 0,02 ml of blood.

After the onset of hemolysis added 0,5 ml reagents. The results are taken into account through 30 m. The reaction is regarded as normal, with full bleaching of the dye. Where, when there is no discoloration of the dye (It is intensely blue-green I color), response is evaluated as a sharply positive. If the color intensity decreases, but the blue-green color is, the reaction is deemed polo-positive. Where, when there is an obvious discoloration, but when compared with the control group remained greenish, reaction regard as a plus-minus.

Rezkopolozhitelnye positive reaction occur in hemizygous males and homozygous women. Sometimes heterozygous women give a positive reaction, but most plus-minus. Besides, plus or minus the reaction is sometimes observed in perfectly healthy people with some decrease in enzyme activity on the background of the disease or medication. Plus-minus reactions should be taken into account, and to check the activity of the enzyme quantitative method only if you suspect the presence of hemolytic anemia in women, due to deficiency of activity of glucose-6-phosphate dehydrogenase. It should not be taken into account, plus-minus reactions at mass screening.

False positive reactions It can be expressed in patients with anemia due to the fact, what 0,02 ml of blood, introduced into the tube, They contain a small amount of erythrocytes and, Consequently, a small amount of enzyme. In this case, the tube containing distilled water should be added to two or three pipette (by 0,02 ml) blood, to the color of the tube prior to the addition of the dye does not differ from the control.

The method of fluorescent spots Beutler и Mitchell It based on specific fluorescence reduced NADP in the long wavelength ultraviolet light (440- 470 nm), assessed visually in fixed terms.

Reagents.

1. Tris-HCl buffer 0,5 M; pH 8,0: 60,55 dissolved in Tris 800 ml distilled water, add 20 mL of concentrated HCl, pH adjusted to 8,0 through 2 M HCl and topped up with water to 1 ml; solution to store 36 days at 4 ° C.
2. A solution of glucose-6-phosphate 20 M: 6 mg disodium glucose-6-phosphate is dissolved in 1 ml distilled water; to eat 2 days at 4 ° C.
3. The solution NADP 10 M: 8 mg NADP dissolved in 1 ml distilled water; to eat 10 days at a temperature 4 ° C.
4. An aqueous solution of saponin 1 % to eat 20 days at a temperature 4 ° C.
5. The solution of oxidized glutathione (10 ml): 2,4 mg of glutathione was dissolved in 1 ml distilled water; to eat 10 days at 4 ° C.

Method.

Before definitely ready incubation mixture, mixing 1 no. solution of glucose-6-phosphate, 1 no. OVER-F solution, 2 no. saponin solution, 5 no. buffer 1 no. simply entails glutathione. Blood (0,01 ml) introduced in vials or cell haemagglutination board and add 0,2 ml incubation mixture. Through 15 min micropipette selected one drop incubation mixture (0,02 ml) from each sample and applied to them on a chromatographic paper in the form of a spot diameter of 10-12 mm. The spots were dried in air at room temperature, and browsing the ultraviolet light to evaluate fluorescence. Controls are samples with known normal blood. Quality control reagent does not contain blood.

Evaluation of results.

Absence of fluorescence corresponds to the absence of activity, Fluorescence nalïçïe (intense blue glow) - The presence of activity, a faint glow - intermediate reaction. Subject to the conditions of the experimental method does not give false negative results. The source of false-positive diagnosis can be surveyed from severe anemia, but to a much lesser extent, than the method of Berstein. Even with severe anemia observed intermediate reactions, not a lack of fluorescence.

Using a quantitative method for determining the activity of G-6-PD makes it possible to identify the decrease in activity not only in hemizygous and homozygous patients, but among women heterozygous. Because, that the number of reticulocytes and color component influence the level of enzyme activity, it is recommended to amend the results, taking into account these indicators.