Glucose 6 phosphate dehydrogenase in erythrocytes. Clinical pharmacology and pharmacotherapy. Etiology and pathogenesis

Hereditary deficiency of erythrocyte enzymes manifests itself most often when exposed to certain toxins and drugs in the form of acute hemolysis, less often chronic hemolysis. Among them, G-6PD deficiency is the most common.

G-6PD is the first enzyme of anaerobic glycolysis or pentose shunt. It plays a large role in the elimination of toxic peroxides in red blood cells. G-6PD is a polymer consisting of 2-6 units; dimer of two chains - the active form of the enzyme; its concentration in the cell depends on the concentration of NADP, which increases under the influence of oxidants, leading to an increase in the activity of G-6PD.

There are over 100 variants of the G-6FD. In persons of different races, different G-6PD isoenzymes are found in erythrocytes, which differ somewhat in their activity and stability. In most cases, enzyme deficiency remains asymptomatic under normal conditions and is manifested by hemolytic crises when taking oxidant medications. Sometimes, with a more pronounced deficiency of G-6PD, hemolysis occurs chronically. It is always carried out with the accumulation of peroxides in erythrocytes, which contribute to the oxidation of hemoglobin (the appearance of Heinz bodies) and lipids of the erythrocyte membrane.

The genetic transmission of G-6PD deficiency is sex-linked. The corresponding gene is located on the X chromosome at a locus close to the locus of color blindness and distant from the locus of hemophilia. Men - carriers of the altered gene always show clinical manifestations of this pathology. In heterozygous women, the manifestations are mild or absent, and vice versa, in rare homozygous women, there is a pronounced enzymopenia.

According to some reports, there are more than 100 million carriers of the pathological gene. G-6PD deficiency is especially prevalent among dark-skinned individuals, including 10% of black Americans and 10-30% of black Africans. This pathology is also common in the Mediterranean basin, in the Middle East, in Saudi Arabia. It is also found in the Far East - in China, Southeast Asia. In some cases, there is a distinct, as it were, protective effect of this pathology against malaria.

Clinic. The severity of the disease is related to the intensity of the deficiency. A small deficiency (within 20% of the norm) can manifest itself as acute drug-induced hemolysis, more pronounced - jaundice of the newborn, chronic hemolysis.

Episodes of acute hemolysis occur almost always under the influence of an oxidant drug, which was first described in the treatment with primaquine. Later, the effect of other antimalarial drugs, sulfonamides, nitrofuran derivatives (furadonin), some analgesics (amidopyrine, aspirin) and other drugs (quinidine, amilgan, benemide, etc.) became known. Insufficiency of the liver and kidneys (with a violation of the release of drugs from the body) favors acute hemolysis due to G-6PD deficiency.

After taking medication, after 2-3 days, hemolysis develops with anemia, fever, jaundice, and in the case of massive hemolysis - hemoglobinuria. Anemia is usually moderate, normochromic, with an increase in the number of reticulocytes; Heinz bodies are found in erythrocytes. Anemia increases by the 10th day. Then, from the 10th to the 40th day (even if the medication is not stopped), repair occurs, anemia decreases, the number of erythrocytes increases with high reticulocytosis (up to 25-30%), reflecting the intensity of bone marrow hematopoiesis. Finally, the so-called equilibrium phase occurs, during which there is no anemia, although hemolysis and active hematopoiesis are still ongoing. The subsequent recovery is due to the fact that the "old" erythrocytes sensitive to the drug are gradually destroyed, and the newly formed ones contain a larger amount of G-6PD and are resistant to hemolysis. However, this resistance is relative (taking large doses of the drug can cause hemolysis) or temporary. These manifestations with a rather favorable course are more characteristic of persons with dark skin. In individuals with white and yellow skin, manifestations of G-6PD deficiency may be more severe. Intensive hemolysis is accompanied by fever, shock, hemoglobinuria, anuria. The severity of manifestations does not decrease if the drug is not canceled. The disease is provoked by many different medicines, and above all those mentioned above, which are sometimes administered in small doses and for a short time. Some infections (flu, viral hepatitis) can also provoke acute hemolysis.

Chronic hemolytic anemia due to G-6PD deficiency occurs only in whites. Anemia is found in newborns and young children. It remains moderately pronounced, sometimes complicated by acute hemolysis or erythroblastopenia. Growth disorders and serious complications characteristic of sickle cell disease and thalassemia are not observed.

As a diagnostic, a simple, indicative test is the detection of Heinz bodies. Spontaneously or after incubation in the presence of phenylhydrazine, a significant proportion of G-6PD-deficient erythrocytes show inclusions, which are precipitates of hemoglobin derivatives. Heinz bodies are nonspecific and occur in patients with other erythrocyte enzymopathies, toxic anemia, and hemoglobin instability. A number of methods for the semi-qualitative determination of G-6PD deficiency make it possible to detect it before the development of hemolysis. Most of them are based on the use of the sensitivity of the colored indicator to the phenomenon of the conversion of NADP to NADH, which occurs under the action of G-6PD. Thus, the Motulski test is based on measuring the discoloration time of cresyl diamond. The Brewer test evaluates the rate of reduction of methemoglobin by methylene blue.

Enzyme activity is quantified using spectrophotometry and colorimetry. When evaluating the results of these tests at different stages of patient observation, there may be errors associated, in particular, with the fact that high reticulocytosis can mask G-6PD deficiency, since these cells contain a larger amount of the enzyme.

Treatment this pathology is symptomatic. In acute hemolysis with a large drop in hemoglobin, blood transfusions are performed. Insufficiently substantiated use of drugs that cause acute hemolysis in G-6PD deficiency should be avoided.

G-6-PD deficiency is a recessively inherited sex-linked disease characterized by the development of hemolysis after drug intake or consumption of horse beans. Mostly men are ill.

Etiology. Patients have a deficiency of G-6-FDG in erythrocytes, which leads to a disruption in the processes of glutathione recovery when exposed to substances with a high oxidizing ability.

Pathogenesis. G-6-FDG deficiency is inherited in a recessive manner. With low activity of the enzyme in erythrocytes, the processes of reduction of nicotinamide dinucleotide phosphate (NADP) and the conversion of oxidized glutathione into reduced one are disrupted. The latter protects the erythrocyte from the action of hemolytic oxidizing agents. Hemolysis under their influence develops inside the vessels according to the type of crisis.

clinical picture. Substances provoking a hemolytic crisis can be antimalarial drugs, sulfonamides, analgesics, nitrofurans, plant products (beans, legumes). Hemolysis occurs 2-3 days after taking the drug. The patient's temperature rises, there is a sharp weakness, abdominal pain, repeated vomiting. Quite often the collapse develops. Dark or even black urine is excreted as a manifestation of intravascular hemolysis and determination of hemosiderin in the urine. Sometimes acute renal failure develops due to blockage of the renal tubules by hemolysis products. Jaundice appears, hepatosplenomegaly is determined.

Diagnostics based on the determination of the activity of G-6-FDG. Immediately after a hemolytic crisis, the result may be overestimated, since erythrocytes with the lowest content of the enzyme are destroyed first.

In the study of blood - normochromic severe anemia, reticulocytosis, a smear contains many normocytes and Heinz bodies (denatured hemoglobin). The content of free bilirubin in the blood increases. The osmotic stability of erythrocytes is normal or increased. The decisive diagnostic method is the detection of a decrease in G-6-PDG in erythrocytes.

Treatment consists in eliminating the factors provoking hemolysis. With the development of hemolytic crises - transfusion of freshly citrated blood, intravenous administration of fluids. In some cases it is necessary to resort to splenectomy.

Immune hemolytic anemias

IHA - diseases associated with a shortening of the life of erythrocytes due to exposure to antibodies, while maintaining the ability of the bone marrow to respond to anemic stimuli. The main symptom of these diseases is a rapid decrease in the number of red blood cells and hemoglobin in the blood.

IGA groups:

Alloimmune (or isoimmune) - anemia associated with exposure to exogenous antibodies to the antigens of the patient's erythrocytes;

Transimmune - IHA associated with exposure to antibodies that cross the placenta and are directed against the antigens of the child's erythrocytes;

Heteroimmune (haptenic) - IHA, developing as a result of fixation on the surface of the erythrocyte of a new exogenous antigen - hapten;

Autoimmune - IHA resulting from changes in the function of the body's immune system.

The incidence of IHA is about 100 cases per 1 million population. The most significant is autoimmune hemolytic anemia.

Deficiency in glucose-6-phosphate dehydrogenase (G-6-PD) activity- this is the most common hereditary anomaly of red blood cells, leading to hemolytic crises associated with taking a number of drugs. Outside of crises, most patients experience a state of complete compensation, although some individuals have persistent hemolytic anemia.

The first description of a deficiency in G-6-PD activity was made in 1956 in individuals taking the antimalarial drug primaquine for prophylactic purposes. Independent of these studies, in 1957, a deficiency of G-6-PD was found in the erythrocytes of a patient who periodically experienced hemolytic crises without taking any drugs.

Currently, more than 250 different mutant forms of G-6-PD have been described. They differ from each other in the electrophoretic mobility of the enzyme, its affinity for substrates - glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (NADP). A consequence of reduced affinity is insufficient activity of the enzyme under conditions when the concentration of substrates is strictly limited by the rate of their formation in previous reactions. The absence of activity does not mean in most cases the loss of the enzyme as such, although such cases can be observed. Most often, the absence or decrease in the activity of the enzyme is the result of its presence in the patient in a pathologically inactive form.

The structural gene and the gene-regulator, which determine the synthesis of G-6-PD, are located on the X chromosome, therefore, the inheritance of a deficiency in the activity of this enzyme in erythrocytes is always linked to the X chromosome.

There are two main mutant forms, in which amino acid substitutions do not involve active sites, and therefore both of these widespread mutations are normal. They differ from each other in electrophoretic mobility, but their affinity for the substrate is the same. According to modern nomenclature, one of these forms, common in Europe, is called the BB form, and the other, observed in Africa, is called the A form. Currently, other mutant forms are also described, which also do not differ from each other in kinematic parameters, but have different electrophoretic mobility.

The linkage of the enzyme with sex gives a significant predominance of men among those with clinical manifestations of pathology. It is observed in homozygous men who inherited this pathology from their mother with her X chromosome, in homozygous women (who inherited the disease from both parents) and in some heterozygous women who inherited the disease from one of the parents with a pronounced mutant phenotype.

Most often, a deficiency of G-6-PD activity occurs in European countries located on the Mediterranean coast, Greece, Italy, as well as in some countries of Latin America, Africa, etc.

It is possible that the extremely high accumulation of the abnormal gene in a number of localities is facilitated by the preserved custom of related marriages, which leads to the accumulation in the population of homozygous women, who give severe clinical manifestations of the disease more often than heterozygous carriers, and increase the likelihood of the birth of homozygous men, as well as wide distribution in past in these places tropical malaria.

Etiology and pathogenesis

The first stage of the effect of the drug is its transformation in the body, the transition to the active form, which can cause changes in the structure of the erythrocyte membrane. The active form of drugs interacts with oxyhemoglobin. This produces some amount of hydrogen peroxide.

The reduced glutathione neutralizes some of the peroxide with the help of the peroxidase system, and the reduced glutathione is oxidized during the reaction.

In healthy people, an acute hemolytic crisis develops with the introduction of a significant amount of the drug (toxic dose). A crisis can occur when glutathione recovery systems are unable to cope with the excess of complexes formed and oxidized glutathione. With a deficiency in the activity of glucose-6-phosphate dehydrogenase and impaired recovery of NADP, despite the normal activity of glutathione reductase, its recovery is impaired, since there is no normal source of hydrogen. Reduced glutathione cannot withstand the oxidative effects of conventional therapeutic doses of drugs. This leads to oxidation of hemoglobin, loss of heme from the hemoglobin molecule, precipitation of globin chains. The spleen releases red blood cells from the Heinz bodies. In this case, part of the surface of erythrocytes is lost, which leads to their death.

There is still much unclear in the pathogenesis of hemolytic anemia associated with the consumption of horse beans. Primaquine anemia (favism) develops only in some individuals with a deficiency of G-6-PD activity. This anemia probably requires a combination of two enzymatic defects. It is possible that we are talking about insufficient neutralization of the toxic substance contained in horse beans in some individuals, or about the formation of some kind of metabolite that causes disturbances in the sulfhydryl groups of erythrocytes. In healthy individuals, taking a small amount of fava beans does not cause severe hemolytic anemia, since in the presence of reduced glutathione, red blood cells are able to counteract the toxic effect of the metabolite. The inheritance of this deficiency appears to be autosomal dominant. When combined with an unusual transformation in the body of a toxic substance contained in horse beans, with a deficiency in G-6-PD activity, clinical signs of primaquine anemia appear.

Clinical manifestations

WHO experts subdivide G-6-PD variants into four classes according to clinical manifestations in homozygous patients and the level of activity in erythrocytes.

First grade- options that are accompanied by chronic hemolytic anemia.

Second class- variants with a level of G-6-PD activity in erythrocytes of 0-10% of the norm, the carriage of which determines the absence of hemolytic anemia outside the crisis, and crises associated with taking medications or eating horse beans.

Third class- variants with an activity level in erythrocytes of 10-60% of the norm, in which mild clinical manifestations associated with taking medications can be observed.

fourth grade- variants with a normal or near-normal level of activity that are not accompanied by clinical pathology.

At the birth of a child, hemolytic anemia is observed, belonging to both the first and second classes of G-6-PD deficiency.

The level of G-6-PD activity in erythrocytes does not always correlate with the severity of clinical manifestations. In many first class variants, a 20-30% level of enzyme activity is determined. On the other hand, at a zero level of activity, some patients do not experience any clinical symptoms. This is connected, firstly, with the properties of lutant enzymes, and secondly, in all likelihood, with the rate of neutralization of drugs by the cytochrome apparatus of the patient's liver.

Most often, the deficiency of G-6-PD activity does not give clinical manifestations without a special provocation of a hemolytic crisis. In most cases, the hemolytic crisis begins after taking sulfanilamide drugs (norsulfazol, streptocide, sulfadimethoxine, sulfacyl sodium, etazol, biseptol), antimalarial drugs (primaquine, quinine, quinine), nitrofuran drugs (furazolidone, furadonin, furagin, 5-NOC, nevigramone ), preparations of isonicotinic acid (tubazid, ftivazid), PASK-sodium, as well as nitroglycerin.

From antimalarial drugs with a deficiency of G-6-PD activity, delagil can be prescribed, from sulfanilamide drugs - fthalazol. A number of drugs that cause hemolytic crises in high doses can be used in small doses in case of deficiency of G-6-PD activity. These include acetylsalicylic acid, amidopyrine, phenacytin, chloramphenicol, streptomycin, antidiabetic sulfanilamide drugs.

All drugs capable of causing hemolytic crises catalyze the oxidative denaturation of hemoglobin by molecular oxygen.

Clinical manifestations of the disease can occur on the second or third day from the start of taking medications. Initially, there is a slight yellowness of the sclera, dark urine. When you stop taking the drug during this period, a severe hemolytic crisis does not develop. If treatment is continued, on the 4-5th day, a hemolytic crisis may occur with the release of black or sometimes brown urine, which is associated with the intravascular breakdown of red blood cells. The content of hemoglobin may decrease by 2-3%.

In a severe course of the disease, body temperature rises, a sharp headache, pain in the limbs, vomiting, and sometimes diarrhea appear. Shortness of breath occurs, blood pressure decreases. The spleen is often enlarged, sometimes the liver.

In rare cases, kidney failure develops, associated with a sharp decrease in renal filtration and blockage of the renal tubules by blood clots.

Laboratory indicators

A blood test reveals anemia with an increase in the number of reticulocytes. There is an increase in the number of leukocytes with a shift to myelocytes. In some patients, especially in children, the number of leukocytes can sometimes rise to significant numbers (100 G in 1 liter or more). The number of platelets does not change. When staining erythrocytes with crystal violet during severe hemolytic crises, a large number of Heinz bodies are found.

A sharp irritation of the red germ of the bone marrow is revealed. The content of free hemoglobin in serum increases, the level of bilirubin is often increased due to indirect. With the help of a benzidine test, the presence of hemoglobin in the urine without red blood cells is detected, sometimes hemosiderin is detected.

In some forms of glucose-6-phosphate dehydrogenase deficiency, self-limiting hemolysis is observed, i.e., the hemolytic crisis ends, despite the fact that the patient continues to take the drug that caused the hemolytic crisis. The ability to self-limit hemolysis is due to an increase in the level of enzyme activity in reticulocytes to almost normal levels. In most forms, it is significantly reduced.

Severe hemolytic crises are more common in children than in adults.. With a pronounced deficiency of G-6-PD activity, they sometimes occur immediately after birth. This is a hemolytic disease of the newborn, not associated with immunological conflict. It can be as severe as hemolytic anemia due to Rh incompatibility between mother and fetus. Perhaps the presence of nuclear jaundice with severe neurological symptoms.

The pathogenesis of these crises is not well understood. It has not yet been clarified whether these crises occur spontaneously due to a physiological deficiency in the activity of the glutathione peroxidase enzyme at birth or whether they are caused by the use of certain antiseptic agents when treating the umbilical cord of a child. It is possible that sometimes crises are associated with the mother taking certain medications.

In some cases hemolytic crises with a deficiency of G-6-PD activity occur against the background of infectious diseases: influenza, salmonellosis, viral hepatitis. Crises can also be triggered by acidosis in diabetes mellitus or kidney failure.

In a small proportion of patients with a deficiency of G-6-PD activity, persistent hemolytic anemia associated with the use of drugs is observed. In these cases, there is a slight enlargement of the spleen, moderate normochromic anemia with an increase in the content of reticulocytes, erythrokaryocytes in the bone marrow and the level of bilirubin. An exacerbation of the disease is possible either after taking the above medicines, or against the background of infections.

Diagnostics

The basis for the diagnosis of this erythrocyte enzyme deficiency is the determination of G-6-PD activity in the proband and its relatives. Of the qualitative methods used for this purpose, two of the simplest methods should be recommended.

MethodBernstein makes it possible not only to diagnose deficiency of G-6-PD activity in all hemizygous men, homozygous women, but also to approximately estimate the degree of deficiency of this enzyme in heterozygous women. This method can identify about 50% of heterozygous women. The advantage of this method is its suitability for use in mass surveys of the population in expeditionary conditions.

The method is based on the bleaching of the dye 2,6-dichlorophenolindophenol a during its recovery. In the presence of G-6-PD, glucose-6 phosphate is oxidized and NADP is reduced to form NADP-H. This substance restores phenazine methasulfate, which in turn restores 2,6-dichlorophenolindophenol. Phenazine methasulfate acts as a very active electron carrier from NADP-H to the dye in this reaction. Without phenazine methasulfate, the reaction proceeds for several hours, and in the presence of phenazine methasulfate, discoloration occurs in 15-30 minutes.

Reagents.

1. NADP solution: 23 mg of NADP is dissolved in 10 ml of water.
2. Glucose-6-phosphate solution (G-6-P): 152 mg of glucose-6-phosphate sodium salt is dissolved in 10 ml of water. The barium salt of glucose-6-phosphate must first be converted to the sodium salt. To do this, weigh 265 mg of barium salt of glucose-6-phosphate, dissolve in 5 ml of water, add 0.5 ml of 0.01 M hydrochloric acid solution and 1 mg of dry sodium sulfate. The precipitate is centrifuged. The supernatant layer is neutralized with 0.01 M sodium hydroxide solution and adjusted with distilled water to 10 ml.
3. Phenazine methasulfate solution: 2 mg of phenazine methasulfate are dissolved in 100 ml of Tris buffer 0.74 M; pH 8.0.
4. 2,6-dichlorophenolindophenol (sodium salt) dye solution: 14.5 mg of dye are dissolved in 100 ml of tris-hydrochloric acid buffer solution (0.74 M; pH 8.0). The buffer solution is prepared from a 1.48 M solution of tris-hydroxymethylaminomethane (42.27 g per 250 ml of water) and a 1.43 M solution of hydrochloric acid (2 ampoules of fixanal containing 0.1 g-eq, adjusted with water to 135 ml). 110 ml of hydrochloric acid are added to 230 ml of tris-hydroxymethylaminometal solution, the pH is adjusted to 8.0 and water is added to 460 ml.

Before use, a mixture of reagents is prepared: 1 part of a solution of NADP (1), 1 part of a solution of G-6-F (2), 2 parts of a solution of phenazine metasulfate (3) and 16 parts of a solution of 2,6-dichlorophenolinodophenol (4).

Methodology.

0.02 ml of blood is added to a test tube containing 1 ml of distilled water.

After the onset of hemolysis, 0.5 ml of the reagent mixture is added. The results are taken into account after 30 minutes. The reaction is regarded as normal if the dye is completely decolorized. In those cases where dye discoloration does not occur (an intense blue-green I color remains), the reaction is assessed as sharply positive. If the intensity of the color decreases, but the blue-green color remains, the reaction is considered positive. In cases where a clear discoloration occurs, but a greenish tint remains when compared with the control, the reaction is regarded as plus or minus.

Strong positive and positive reactions observed in hemizygous men and homozygous women. Sometimes heterozygous women give a positive reaction, but more often plus or minus. In addition, a plus or minus reaction is sometimes observed in perfectly healthy people with a slight decrease in enzyme activity against the background of a disease or medication. Plus-minus reactions should be taken into account and the activity of the enzyme should be checked by a quantitative method only if a woman is suspected of having hemolytic anemia due to a deficiency in glucose-6-phosphate dehydrogenase activity. Plus or minus reactions should not be taken into account in a mass examination.

False positive reaction may be in persons with severe anemia due to the fact that 0.02 ml of blood added to the test tube contains a small amount of erythrocytes and, consequently, a small amount of the enzyme. In this case, two or three pipettes (0.02 ml each) of blood should be added to a test tube with distilled water so that these tubes do not differ in color from the control ones before the addition of the dye.

Fluorescent spot methodBeutlerand Mitchell based on the specific fluorescence of reduced NADP in long-wave ultraviolet light (440-470 nm), assessed visually at fixed times.

Reagents.

1. Tris-HCl buffer 0.5 M; pH 8.0: Dissolve 60.55 Tris in 800 ml distilled water, add 20 ml concentrated HCl, adjust pH to 8.0 with 2 M HCl solution and top up with water to 1 ml; the solution is stored up to 36 days at a temperature of 4°C.
2. Glucose-6-phosphate solution 20 M: 6 mg of glucose-6-phosphate disodium salt is dissolved in 1 ml of distilled water; store up to 2 days at 4°C.
3. 10 M NADP solution: 8 mg of NADP is dissolved in 1 ml of distilled water; store up to 10 days at a temperature of 4 °C.
4. An aqueous solution of saponin 1% is stored for up to 20 days at a temperature of 4 °C.
5. Solution of oxidized glutathione (10 ml): 2.4 mg of glutathione is dissolved in 1 ml of distilled water; store up to 10 days at 4°C.

Methodology.

Before determination, an incubation mixture is prepared by mixing 1 part of glucose-6-phosphate solution, 1 part of NAD-P solution, 2 parts of saponin solution, 5 parts of buffer and 1 part of glutathione solution. Blood (0.01 ml) is added to test tubes or cells of the hemagglutination board and 0.2 ml of the incubation mixture is added. After 15 minutes, one drop of the incubation mixture (0.02 ml) is taken from each sample with a micropipette and applied to chromatographic paper in the form of a spot with a diameter of 10-12 mm. The spots are air dried at room temperature and viewed under ultraviolet light to assess fluorescence. Controls are samples with known normal blood. The reagent quality control does not contain blood.

Evaluation of results.

The absence of fluorescence corresponds to the absence of activity, the presence of fluorescence (intense blue glow) corresponds to the presence of activity, and the weak glow corresponds to an intermediate reaction. Subject to the experimental conditions, the method does not give false negative results. The source of a false positive diagnosis may be severe anemia in the examined, but to a much lesser extent than for the Berstein method. Even with severe anemia, an intermediate reaction is observed, and not the absence of fluorescence.

The use of a quantitative method for determining the activity of G-6-PD makes it possible to detect a decrease in activity not only in hemizygous and homozygous patients, but also in heterozygous women. Due to the fact that the number of reticulocytes and the color index affect the level of enzyme activity, it is recommended to correct the results taking into account these indicators.

ETIOLOGY. A disease that develops as a result of a deficiency of G-6-PD in red blood cells. It is assumed that oxidizing agents, including medicinal ones, in such an erythrocyte reduce the reduced glutathione, which, in turn, creates conditions for oxidative denaturation of enzymes, hemoglobin, constituent components, and the erythrocyte membrane and leads to intravascular hemolysis or phagocytosis. Currently, 59 potential hemolytics have been identified in this type of enzymopathy. The group of drugs that necessarily cause hemolysis in G-6-PD deficiency includes: antimalarial, sulfonamides, nitrofuran derivatives (furadonin, furatsilin, furazolidone), aniline derivatives, naphthalene and its derivatives, methylene blue, phenylhydrazine. Hemolysis in patients with G-6-PD deficiency can be caused by vaccines. The course of the disease usually worsens under the influence of intercurrent infections, especially viral ones. Hemolysis of G-6-PD-deficient erythrocytes can also be caused by endogenous intoxications and a number of plant products.

The structural gene and the gene-regulator, which determine the synthesis of G-6-PD, are located on the X chromosome, therefore, the inheritance of a deficiency in the activity of this enzyme in erythrocytes is linked to the X chromosome. The location of the locus responsible for the synthesis of G-6-PD on the X chromosome is known quite accurately. G-6PD deficiency is inherited as an incompletely dominant, sex-linked trait.

PATHOGENESIS. It is known that in the erythrocyte G-6-PD catalyzes the reaction: glucose-6-phosphate + NADP = 6-phosphogluconate + NADPHBN. Therefore, in erythrocytes with a reduced activity of the G-6-PD enzyme, the formation of reduced nicotinamide adenine dinucleotide phosphate (NADP) and oxygen binding decrease, as well as the rate of methemoglobin reduction and resistance to various potential oxidizing agents - ascorbic acid, methylene blue, etc.

In the mechanism of destruction of erythrocytes, great importance is attached to the reduced content in these cells of the level of reduced glutathione and NADP - substances that are essential for the vital activity of erythrocytes. According to a number of authors, hemolyzing agents lead to the formation of hydrogen peroxides. The occurrence of the latter occurs either as a result of a direct oxidation reaction due to the oxygen of oxyhemoglobin (HbO3), or as a result of the formation of catabolites, i.e. intermediate decay products that directly oxidize hemoglobin to methemoglobin and reduced glutathione to an oxidized form. According to the latter mechanism, catabolites of acetylsalicylic acid, aniline, phenacetin, and sulfonamides influence. By both mechanisms, hemolysis is carried out by acetylphenylhydrazine, primaquine, hydroquine.

In normal cells, medicinal substances activate the reactions of the pentose phosphate cycle, which contributes to an increase in the content of reduced forms of glutathione and NADP in these cells, which are involved in the neutralization of oxidants. In erythrocytes with insufficient G-6-PD activity, this mechanism is absent, therefore, when exposed to oxidizing agents and certain drugs, the activity of thiol enzymes is suppressed, destructive changes in hemoglobin occur, which leads to the hemolytic process.

The direct mechanism of hemolysis, apparently, is to increase the permeability of the erythrocyte membrane in relation to sodium and potassium ions. An increase in the permeability of the erythrocyte membrane with respect to these ions may be due to a decrease in activity, as well as a direct consequence of a violation of the erythrocyte glutathione cycle. First of all, the oldest erythrocytes, in which there is a low content of G-6-PD, undergo decay.

CLINICAL MANIFESTATIONS. The disease can be found in a child of any age. Deficiency of G-6-PD is noted mainly in males, who, as is known, have a single X-chromosome. In women, clinical manifestations are observed mainly in cases of homozygosity, i.e. in the presence of two G-6-PD-deficient chromosomes.

There are five clinical forms of G-6-PD deficiency in erythrocytes: 1) acute intravascular hemolysis - a classic form of G-6-PD deficiency. It occurs everywhere, but more often among representatives of the Caucasoid and Mongoloid races. It develops as a result of medication, vaccination, diabetic acidosis, due to a viral infection. Manifestations of hemolysis usually begin on the 3-6th day after taking a therapeutic dose of a particular drug; 2) favism associated with eating or inhaling the pollen of certain legumes (Vicia fava); 3) hemolytic disease of the newborn, not associated with hemoglobinopathy, with group or Rh incompatibility, sometimes complicated by kernicterus; 4) hereditary chronic hemolytic anemia (non-spherocytic), caused by G-6-PD deficiency in erythrocytes; 5) asymptomatic form.

Hyperbilirubinemia with signs of hemolytic anemia is often observed among newborns with deficiency of G-6-PD erythrocytes, but in these cases, evidence of a serological conflict between mother and child is usually absent (negative Coombs test, no isoimmune antibodies are detected). The disease can proceed benignly when hyperbilirubinemia does not reach a critical level and decreases along with the fading of the intensity of the hemolytic process. In more severe cases, bilirubin encephalopathy may develop.

In older children, G-6-PD deficiency can manifest as chronic (non-spherocytic) hemolytic anemia, which usually worsens with intercurrent infections and after medication. A more common form of manifestation of this hereditary defect is hemolytic crises after taking medications in seemingly healthy children. Acute hemolysis that occurs after taking medications leads to severe anemia, and hemoglobinuria is less common. Despite the relatively favorable course in most cases, some patients experience severe complications in the form of anuria and hypovolemic shock. In typical cases, the general condition of the child is severe, the skin is yellow in color. There is a high fever, severe headache, general weakness. There may be repeated vomiting with an admixture of bile, liquid, intensely colored stools. There may be an increase in the liver, less often - the spleen. In the peripheral blood, anemia with reticulocytosis, leukocytosis with a shift to myelocytes are expressed. Aniso-, poikilocytosis is noted, fragments of erythrocytes (schizocytes), polychromasia, basophilic puncture of erythrocytes are visible.

A characteristic sign of intravascular hemolysis is hyperhemoglobinemia, the blood serum becomes brown when standing due to the formation of methemoglobin. At the same time, hyperbilirubinemia is noted. The content of bile pigments in the duodenal contents, in feces increases, urine can be the color of black beer or a strong solution of potassium permanganate, which is due to the released hemoglobin, methemoglobin, as well as hemosiderin and urobilin. In very severe cases, anuria develops as a result of blockage of the renal tubules by blood and protein clots ("hemolytic kidney"), sometimes there is a micro-obstruction of the nephron with uremia, the development of DIC and death. An unfavorable outcome can also come from coma, when, due to the rapid breakdown of red blood cells, vomiting of bile and a collaptoid state develop. Hemolytic crisis immediately after birth may be accompanied by kernicterus with severe neurological symptoms.

Of the characteristic laboratory signs inherent in enzymopenic hemolytic anemia, it should be noted a decrease in hematocrit, hemoglobin and erythrocytes, an increase in the concentration of bilirubin in the blood due to unconjugated, hyperhemoglobinemia, hypohaptoglobinemia.

In the bone marrow, as in other hemolytic anemias, reactive hyperplasia of the erythrocyte germ is found, the cells of which in severe cases make up 50-70% of the total number of myelokaryocytes.

A special form of manifestation of enzymatic deficiency of erythrocytes is favism, in which hemolytic crises occur in patients when eating Vicia fava beans or even when inhaling the pollen of these plants. It has been established that some cases of favism are also due to hereditary deficiency of G-6-PD. As a result of clinical and experimental observations, it has been found that the time interval between coming into contact with fava beans and the appearance of symptoms of the disease ranges from several hours to several days. In contrast, the interval between drug administration and hemolysis of G-6PD-deficient streets is 2-3 days.

Favism can occur upon first contact with the beans or occurs in individuals who have previously consumed these beans, but they did not have manifestations of the disease. Relapses of favism are not uncommon, and familial cases of this type of hemolytic anemia have been reported.

The nature of the substances contained in beans that cause hemolytic crisis in individuals with G-6-PD deficiency has not yet been fully elucidated. It has been suggested that hemolysis is caused by plant pyrimidines - vicin, convicin, devicin, which, when ingested, contribute to a catastrophic drop in the concentration of reduced glutathione and sulfhydryl groups in the red blood cell. Favism predominantly affects children aged 1 to 14 years, the process is especially difficult in young children, who make up about half of all patients. The ratio of boys and girls with favism is 7:1, which is explained by the peculiarities of the hereditary transmission of G-6-PD deficiency of erythrocytes with the sex (X) chromosome.

The clinic of favism is very variable - from symptoms of mild hemolysis to a super-acute severe hemoglobinuric crisis. The development of a crisis may be preceded by prodromal phenomena in the form of weakness, chills, fever, headache, drowsiness, pain in the lower back, abdomen, nausea, and vomiting.

Acute hemolytic crisis is characterized by pallor, jaundice and hemoglobinuria. An objective examination reveals an increase in the liver, spleen, displacement of the boundaries of the heart and the appearance of anemic noises.

In hospitalized patients, there is a sharp decrease in the number of erythrocytes in the peripheral blood, in most cases this figure is 1-2 10/l. In patients with favism, pathological changes in the urine are often found. Hemoglobinuria is detected within 1-3 days, there is usually no longer hemoglobinuria. Sometimes large amounts of oxyhemoglobin and methemoglobin are found, due to which the urine acquires a dark brown, red or even black color. Severely ill patients may experience oliguria or even anuria with concomitant azotemia. Kidney failure can be fatal.

The diagnosis of G-6-PD deficiency in erythrocytes should be based on direct determination of enzyme/a activity, which is currently available to many laboratories. As a preliminary study, especially in mass analyzes, a semi-quantitative study of the enzyme by various methods based on a change in the color of the medium as a result of the enzyme reaction (the test of Motulsky and Campbell, Bernstein, Fairbanks and Beutler, etc.) is acceptable. In special cases, it is advisable to use other methods - tests for the reduction of methemoglobin, for the stability of reduced glutathione in erythrocytes, for the formation of Heinz bodies, enzyme electrophoresis, etc. To confirm the hereditary nature of the disease, the study of G-6-PD activity should also be carried out in the patient's relatives .

The differential diagnosis of enzymopenic hemolytic anemia is carried out primarily with viral hepatitis, then with hereditary microspherocytosis and immune forms of hemolytic anemia. At the second stage, the type of enzyme absent or reduced in its activity is specified.

TREATMENT. Therapy for hemolytic anemia in children begins immediately, as soon as increased hemolysis is detected. Treatment of acute hemolytic crisis with G-6-PD deficiency consists in the abolition of the drug that caused hemolysis.

With a mild hemolytic crisis with a slight decrease in hemoglobin, mild icterus and hyperbilirubinemia, antioxidants are prescribed (revit, vitamin E preparations). Apply agents that increase the reduced glutathione in erythrocytes, the amount of which decreases during hemolytic crises, xylitol 0.25-0.5 g 3 times a day with riboflavin - 0.6-1.5 mg per day with a 3-time intake . At the same time, phenobarbital (or zixorin) is given in a daily dose, depending on age, to children at 0.005-0.01 g for 10 days. Phenobarbital, having a bilirubin-conjugating effect, induces the glucuronyl transferase system of the liver.

In severe hemolytic crises with severe signs of intravascular hemolysis, prevention of acute renal failure is necessary. Depending on age, a 1-4% sodium bicarbonate solution is injected intravenously, depending on age, at the rate of 5 ml per 1 kg of body weight per day, which prevents the development of metabolic acidosis and acts as a weak diuretic that promotes the excretion of hemolysis products. As a weak diuretic and platelet antiplatelet agent that improves renal beds, a 2.4% solution of eufillin is used intravenously at a rate of 4-6 mg per 1 kg per day in 250-500 ml of isotonic sodium chloride solution. Forced diuresis is supported by a 10% solution of mannitol (1 g per 1 kg of body weight). In the event of a threat of DIC, heparinized cryoplasma is prescribed from 5 to 10 ml per 1 kg of body weight per day. Heparinization is carried out by introducing heparin into a container with thawed plasma at the rate of 1 unit for each milliliter of injected plasma.

Red blood cell transfusion is used only for severe anemia. In cases of prolonged anuria, extracorporeal dialysis is indicated. In the neonatal period, with hyperbilirubinemia, it is necessary to perform an exchange transfusion in order to prevent kernicterus.

Clinical examination of patients with hemolytic anemia as a result of G-6-PD deficiency should be carried out in hematological centers. Prevention of manifestations of the hereditary defect G-6-PD includes its timely recognition, which makes it possible to prevent the prescription of potentially dangerous drugs. Eating fava beans is prohibited. It is necessary to protect the child from intercurrent infections.

Deficiency of glucose-6-phosphate dehydrogenase (G-6-PD) activity is the most common hereditary erythrocyte anomaly leading to hemolytic crises (exacerbation as a result of intensive destruction of erythrocytes) associated with the intake of a number of drugs. Outside of a crisis (exacerbation), the well-being and condition of a person with this disease is fully compensated. It is known that a number of drugs, especially antimalarials, can cause acute hemolytic anemia in some individuals. Drug intolerance is often observed in members of the same family. It has been established that after a hemolytic crisis in people, large inclusions appear in erythrocytes, which are called Heinz bodies. After placing the erythrocytes of persons who have undergone an acute hemolytic crisis due to the intake of any drug in a test tube with the substance acetylphenylhydrazine, many Heinz bodies appear in the erythrocytes (much more than in healthy people). The first description of a deficiency in the activity of the G-6-PD enzyme dates back to 1956. Low activity of the enzyme was found in individuals who took the antimalarial drug primaquine for prophylactic purposes. At the same time, an acute hemolytic crisis developed. Independent of these studies, another scientist in 1957 discovered a deficiency of the same enzyme in the erythrocytes of a young man from Iran who had intermittent hemolytic crises without taking any medication.

The deficiency of the activity of this enzyme is always transmitted linked to the X chromosome. The linkage of the mutant gene with sex gives a significant predominance of men among persons with this disease. It manifests itself in men who inherited this pathology from the mother with her X chromosome, in women who inherited the disease from both parents, and in some women who inherited the disease from one of the parents.

Most often, G-6-PD deficiency occurs in European countries located on the Mediterranean coast - in Greece, in Italy. Enzyme activity deficiency is widespread in some Latin American and African countries.

The first step in the metabolism of the drug in the body is its transition to the active form, which can cause changes in the structure of the erythrocyte membrane. The active form of the drug interacts with hemoglobin. This produces some amount of hydrogen peroxide. In healthy people, an acute hemolytic crisis develops when a significant amount of the drug is administered (toxic dose). A crisis can occur when the recovery systems are unable to cope with the excess hydrogen peroxide produced in red blood cells. At the same time, Heinz bodies appear in red blood cells. The spleen releases red blood cells from these bodies, and part of the surface of red blood cells is lost, which leads to their premature death.

Experts from the World Health Organization subdivide variants of the G-6-PD enzyme deficiency into 4 classes in accordance with the emerging manifestations and the level of enzyme activity in erythrocytes.

1st class- options that are accompanied by chronic hemolytic anemia.

2nd grade- variants with an enzyme activity level in erythrocytes of 0-10% of the norm, the carriage of which determines the absence of hemolytic anemia without exacerbation, and exacerbations are associated with taking medications or eating fava beans.

3rd grade- variants with an enzyme activity level in erythrocytes of 10-60% of the norm, in which there may be mild signs of hemolytic anemia associated with taking medications.

4th grade- Variants with a normal or close to normal level of enzyme activity without any manifestations.

Hemolytic anemia at the birth of a child occurs with a deficiency of the G-6-PD enzyme of both the 1st and 2nd class. The activity of G-6-PD in erythrocytes does not always correspond to the severity of the emerging manifestations of the disease. In many variants of the 1st class, 20-30% enzyme activity is determined, and with zero activity, some carriers do not show any manifestations of the disease. This is due, firstly, to the properties of the mutant enzymes themselves, and secondly, to the rate of drug neutralization in the liver.

Most often, a deficiency in the activity of the enzyme G-6-PD does not give any manifestations without provocation. In most cases, hemolytic crises begin after taking certain drugs, primarily sulfanilamide drugs (norsulfazol, streptocide, sulfadimethoxin, sodium albucide, etazol, biseptol), antimalarial drugs (primaquine, quinine, quinine), nitrofuran derivatives (furazalidon, furadonin, furagin, 5-NOC, blacks, nevigramon), drugs for the treatment of tuberculosis (tubazid, ftivazid), antihelminthic drug niridazole (ambilhar). With a deficiency in the activity of the G-6-PD enzyme, the antimalarial drug delagil can be used, and only fthalazole can be used from sulfanilamide drugs. Some drugs in large doses cause hemolytic crises, and in small doses they can be used with a deficiency in the activity of the G-6-PD enzyme. These drugs include acetylsalicylic acid (aspirin), amidopyrine, phenacetin, chloramphenicol, streptomycin, artan, antidiabetic sulfanilamide drugs.

Manifestations of the disease can occur on the 2-3rd day from the start of the medication. Initially, a slight yellow coloration of the eyes appears, the urine becomes dark. If you stop taking the medicine during this period, then a severe hemolytic crisis does not develop, otherwise, on the 4th or 5th day, a hemolytic crisis may occur with the release of black, sometimes brown urine, which is associated with the breakdown of red blood cells inside the blood vessels. The content of hemoglobin during this period may decrease by 20-30 g/l or more. In a severe course of the disease, the temperature rises, a sharp headache, pain in the limbs, vomiting, and sometimes diarrhea appear. Shortness of breath occurs, blood pressure decreases. The spleen is often enlarged, sometimes the liver.

In rare cases, the massive breakdown of red blood cells provokes intravascular coagulation with the formation of blood clots that close the lumen of the vessels. This, in turn, can lead to impaired blood circulation in the kidneys and the development of acute renal failure.

A blood test reveals anemia with an increase in the number of immature forms of red blood cells (reticulocytes). The number of leukocytes increases. Sometimes, especially in children, the number of leukocytes can become very large (100 x 10 9 / l and above). Platelet levels usually do not change. A special study of erythrocytes during a period of severe exacerbation of the disease reveals a large number of Heinz bodies. As a result of the pronounced destruction of erythrocytes in the blood serum, the content of free hemoglobin increases, the content of bilirubin is often increased. Hemoglobin also appears in the urine.

Children are more likely to experience severe hemolytic crises than adults. With a pronounced deficiency in the activity of the G-6-PD enzyme, hemolytic crises sometimes occur immediately after birth. This is a hemolytic disease of the newborn, not associated with an immunological conflict between him and the mother. It can proceed as severely as hemolytic anemia associated with Rh incompatibility between mother and child, can provoke severe jaundice with severe damage to the central nervous system.

Hemolytic crises with a deficiency in the activity of the G-6-PD enzyme sometimes occur with infectious diseases (influenza, salmonellosis, viral hepatitis), regardless of medication, can be triggered by an exacerbation of diabetes mellitus or the development of renal failure.

A small proportion of individuals with a deficiency in the activity of the enzyme G-6-PD have persistent hemolytic anemia associated with medication. In these cases, there is a slight increase in the spleen, hemoglobin does not decrease so much, the level of bilirubin in the blood slightly increases. In such people, the disease may worsen either after taking the above drugs, or with infectious diseases.

Some individuals with G-6-PD deficiency develop hemolytic anemia associated with eating fava beans - favism. Manifestations of favism consist of signs of rapid destruction of red blood cells, which occurs faster than after taking drugs, and digestive disorders associated with the direct effect of fava beans on the intestines. Hemolytic crises occur within a few hours after eating beans, less often after 1-2 days, their severity depends on the number of beans eaten. Favism is more often complicated by renal insufficiency. Mortality in favism is higher than in drug-induced forms. When pollen is inhaled, hemolytic crises are more likely to be mild, but occur a few minutes after contact with pollen.

Single hemolytic crises are described, caused by the intake of male fern, eating blueberries, blueberries.

The basis for detecting deficiency of the G-6-PD enzyme is the determination of enzyme activity using special research methods.

Treatment insufficiency of the enzyme G-6-PD is necessary only with pronounced signs of acute destruction of erythrocytes. With persistent hemolytic anemia with a deficiency in the activity of G-6-PD of the 1st class, sometimes the spleen is removed. In case of mild hemolytic crises with a slight darkening of urine, slight yellowness of the sclera and a mild decrease in hemoglobin, the drug that caused the crisis should be canceled, riboflavin 0.015 g 2-3 times a day, xylitol 5-10 g 3 times a day, vitamin E preparations.

With pronounced signs of intravascular breakdown of erythrocytes, especially with favism, prevention of acute renal failure is necessary. Prevention of renal failure is carried out only in a hospital or intensive care unit and depends on the severity of the condition.

Erythrocytes are transfused only with severe anemia.

Prevention of hemolytic crises is reduced to the refusal to take drugs that can cause an exacerbation of the disease. In this case, such drugs must be replaced with analogues, which should be done by the attending physician.

Forecast. Individuals with G-6-PD deficiency are practically healthy, and if preventive measures are observed, they can be healthy throughout their lives. The performance of such people does not suffer. Chronic hemolytic anemia associated with G-6PD deficiency is usually mild. As a rule, the performance is fully preserved. The prognosis for acute hemolytic crises depends on the speed of discontinuation of the drug that caused the hemolytic crisis, age, and the state of the cardiovascular system. With favism, the prognosis is worse, but preventive measures make mortality small even in cases complicated by acute renal failure.