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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 medicines. 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 settlements contributes to the preserved custom of related marriages, which leads to the accumulation in the population of homozygous women, giving severe clinical manifestations diseases more often than heterozygous carriers and increasing the likelihood of having homozygous males, as well as the widespread prevalence of tropical malaria in these places in the past.

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.

At 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 the activity of G-6-PD, Clinical signs primaquine anemia.

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, the body temperature rises, there are sharp headache, pain in the extremities, vomiting, sometimes - diarrhea. Shortness of breath occurs, decreases arterial pressure. 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 is not yet clear 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 antiseptics when processing 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 with diabetes 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 takes 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, dilute 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.

Erroneous 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.

E.A. Skornyakova, A.Yu. Shcherbina, A.P. Prodeus, A.G. Rumyantsev

Federal State Institution Federal Research Center for Children's Hematology, Oncology and Immunology of Roszdrav,
RSMU, Moscow

Some primary immunodeficiency states are located at the intersection of several specialties, and often patients with one or another defect are observed not only by an immunologist, but also by a hematologist. For example, a group of defects in phagocytosis includes congenital deficiency of glucose-6-phosphate dehydrogenase (G6PD). This most common enzymatic deficiency is the cause of a spectrum of syndromes, including neonatal hyperbilirubinemia, hemolytic anemia, and recurrent infections characteristic of phagocytic pathology. In some patients, these syndromes can be expressed to varying degrees.

Epidemiology
G6PD deficiency occurs most frequently in Africa, Asia, the Mediterranean, and the Middle East. The prevalence of G6PD deficiency correlates with the geographic distribution of malaria, leading to the theory that carriage of G6PD deficiency provides partial protection against malaria infection.

Pathophysiology
G6PD catalyses the conversion of nicotinamide adenine dinucleotide phosphate (NADP) to its reduced form (NADPH) in the pentose phosphate pathway of glucose oxidation (see figure). NADPH protects cells from free oxygen damage. Since erythrocytes do not synthesize NADPH in any other way, they are the most sensitive to the aggressive effects of oxygen.
Due to the fact that due to G6PD deficiency, the greatest changes occur in erythrocytes, these changes are the most well studied. However, the abnormal response to certain infections (such as rickettsiosis) in these patients raises questions about abnormalities in the cells of the immune system.

Genetics
The gene encoding glucose-6-phosphate dehydrogenase is located in distal long arm of the X chromosome. Over 400 mutations have been identified, most of which occur sporadically.

Diagnostics
Diagnosis of G6PD deficiency is made by quantitative spectrophotometric analysis or, more commonly, a rapid fluorescent spot test that detects the reduced form (NADPH) as compared to NADP.
In patients with acute hemolysis, tests for G6PD deficiency may be false negative because older red blood cells with lower levels of the enzyme have undergone hemolysis. Young erythrocytes and reticulocytes have a normal or subnormal level of enzymatic activity.
G6PD deficiency is one of a group of congenital hemolytic anemias and should be considered in children with a family history of jaundice, anemia, splenomegaly, or cholelithiasis, especially those of Mediterranean or African origin. Testing should be considered in children and adults (especially males of Mediterranean, African, or Asian ancestry) with an acute hemolytic reaction due to infection, use of oxidative drugs, ingestion of legumes, or exposure to naphthalene.
In countries where G6PD deficiency is common, screening of newborns is carried out. WHO recommends newborn screening in all populations with an incidence of 3-5% or more in the male population.

Hyperbilirubinemia of the newborn
Hyperbilirubinemia of newborns is twice as high as the average in the population, in boys with G6PD deficiency and in homozygous girls. Quite rarely, hyperbilirubinemia is observed in heterozygous girls. The mechanism of neonatal hyperbilirubinemia in these patients is not well understood.
In some populations, G6PD deficiency is the second most common cause of kernicterus and neonatal death, while in other populations the disease is almost non-existent, reflecting the varying severity of mutations specific to different ethnic groups.

Acute hemolysis
Acute hemolysis in patients with G6PD deficiency is caused by infection, consumption of legumes, and intake of oxidative drugs. Clinically, acute hemolysis is manifested by severe weakness, pain in abdominal cavity or back, it is possible to increase body temperature to febrile numbers, jaundice that occurs due to an increase in the level of indirect bilirubin, dark urine. In adult patients, cases of acute renal failure have been described.
Drugs that cause an acute hemolytic reaction in G6PD-deficient patients compromise the antioxidant defenses of red blood cells, leading to their breakdown (see table).
Hemolysis usually lasts for 24-72 hours and ends by 4-7 days. Special attention should be given to the appointment of oxidative drugs to lactating women, since, being secreted with milk, they can provoke hemolysis in a child with G6PD deficiency.
Although G6PD deficiency may be suspected in patients with a history of a hemolysis episode after ingestion of legumes, not all of them will develop such a reaction later.
Infection is the most common cause development of acute hemolysis in G6PD-deficient patients, although the exact mechanism is not clear. It is assumed that leukocytes can release oxygen free radicals from phagolysosomes, which is the cause of oxidative stress for erythrocytes. Salmonella, rickettsial infection, beta-hemolytic streptococcus, Escherichia coli, viral hepatitis, type A influenza virus most often cause the development of hemolysis.

Chronic hemolysis
In chronic hemolytic anemia, which is usually due to sporadic mutations, hemolysis occurs during red blood cell metabolism. However, under conditions of oxidative stress, acute hemolysis may develop.

Immunodeficiency
Glucose-6-phosphate dehydrogenase is an enzyme found in all aerobic cells. Enzymatic deficiency is most pronounced in erythrocytes, however, in patients with G6PD deficiency, not only erythrocyte functions suffer. Neutrophils use reactive oxygen species for intra- and extracellular killing of infectious agents. Therefore, for the normal functioning of neutrophils, a sufficient amount of NADPH is required to provide antioxidant protection to the activated cell. With NADPH deficiency, early apoptosis of neutrophils is observed, which in turn leads to an inadequate response to certain infections. For example, rickettsiosis in such patients occurs in a fulminant form, with the development of DIC and a high rate of death. According to the literature, the induction of apoptosis in G6PD-deficient cells in in vitro studies is significantly higher than in the control. There is a correlation between the increase in apoptosis and the number of "breakdowns" during "doubling" of DNA. However, the disorders that occur when there is insufficient antioxidant protection in granulocytes and lymphocytes have been little studied.

Therapy
The treatment of patients with G6PD deficiency should be based on the principle of avoiding possible trigger factors in order to prevent the development of acute hemolysis.
Hyperbilirubinemia of newborns, as a rule, does not require a special approach in therapy. As a rule, the appointment of phototherapy gives a quick positive effect. However, in patients with G6PD deficiency, it is necessary to control the level of bilirubin in the blood serum. With an increase to 300 mmol / l, an exchange transfusion is indicated to prevent the development of kernicterus and the onset of irreversible disorders of the central nervous system.
Therapy for acute hemolysis in patients with G6PD deficiency does not differ from that for hemolysis of another genesis. With a massive breakdown of erythrocytes, hemotransfusion may be indicated to normalize gas exchange in tissues
It is very important to avoid prescribing oxidative drugs that can cause acute hemolysis and worsen the condition. When diagnosing a mutation in a heterozygous woman, it is advisable to conduct prenatal diagnosis in a male fetus.

Recommended reading
1. Ruwende C., Hill A. Glucose-6-phosphate dehydrogenase deficiency and malaria // J Mol Med 1998;76:581-8.
2. Glucose 6 phosphate dehydrogenase deficiency. Accessed July 20, 2005, at: http://www.malariasite.com/malaria/g6pd.htm.
3. Beutler E. G6PD deficiency // Blood 1994;84:3613-36.
4. Iwai K., Matsuoka H., Kawamoto F., Arai M., Yoshida S., Hirai M., et al. A rapid single-step screening method for glucose-6-phosphate dehydrogenase deficiency in field applications // Japanese Journal of Tropical Medicine and Hygiene 2003;31:93-7.
5. Reclos G.J., Hatzidakis C.J., Schulpis K.H. Glucose-6-phosphate dehydrogenase deficiency neonatal screening: preliminary evidence that a high percentage of partially deficient female neonates are missed during routine screening // J Med Screen 2000;7:46-51.
6. Kaplan M., Hammerman C., Vreman H.J., Stevenson D.K., Beutler E. Acute hemolysis and severe neonatal hyperbilirubinemia in glucose-6-phosphate dehydrogenase-deficient heterozygotes // J Pediatr 2001;139:137-40.
7. Corchia C., Balata A., Meloni G.F., Meloni T. Favism in a female newborn infant whose mother ingested fava beans before delivery // J Pediatr 1995;127:807-8.
8. Kaplan M., Abramov A. Neonatal hyperbilirubinemia associated with glucose-6-phosphate dehydrogenase deficiency in Sephardic-Jewish neonates: incidence, severity, and the effect of phototherapy // Pediatrics 1992;90:401-5.
9. Spolarics Z., Siddiqi M., Siegel J.H., Garcia Z.C., Stein D.S., Ong H., et al. Increased incidence of sepsis and altered monocyte functions in severely injured type A- glucose-6-phosphate dehydrogenase-deficient African American trauma patients // Crit Care Med 2001;29:728-36.
10. Vulliamy T.J., Beutler E., Luzzatto L. Variants of glucose 6-phosphate dehydrogenase are due to missense mutations spread throughout the coding region of the gene // Hum Mutat 1993; 2,159-67.

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 for different stages observations of the patient may be errors associated, in particular, with the fact that high reticulocytosis can mask the deficiency of G-6PD, 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.

The most common fermentopathy is glucose-6-phosphate dehydrogenase deficiency- detected in approximately 300 million people; in second place is a deficiency in pyruvate kinase activity, found in several thousand patients in the population; other types of enzymatic defects in erythrocytes are rare.

Prevalence

Glucose-6-phosphate dehydrogenase deficiency unevenly distributed among the population different countries: most often found in residents of European countries located on the Mediterranean coast (Italy, Greece), among Sephardi Jews, as well as in Africa and Latin America. The lack of glucose-6-phosphate dehydrogenase is widely recorded in the former malarial regions of Central Asia and Transcaucasia, especially in Azerbaijan. It is known that patients with tropical malaria, who have a deficiency of glucose-6-phosphate dehydrogenase, died less often, since erythrocytes with enzyme deficiency contained fewer malarial plasmodia than normal red blood cells. Among the Russian population, deficiency of glucose-6-phosphate dehydrogenase activity occurs in approximately 2% of people.

Although deficiencies in this enzyme are ubiquitous, the severity of the deficiency varies among ethnic groups. The following variants of enzyme deficiency in erythrocytes have been established: A + , A ", B + , B" and the Canton variant.

• The variant of glucose-6-phosphate dehydrogenase B + is normal (100% G-b-PD activity), most common among Europeans.


• The variant of glucose-6-phosphate dehydrogenase B "is Mediterranean; the activity of red blood cells containing this enzyme is extremely low, often less than 1% of the norm.


• Variant of glucose-6-phosphate dehydrogenase A + - enzyme activity in erythrocytes is almost normal (90% of the activity of variant B +)


• The variant of glucose-6-phosphate dehydrogenase D A "is African, the activity of the enzyme in erythrocytes is 10-15% of the norm.


• Variant of glucose-6-phosphate dehydrogenase Canton - in the inhabitants of Southeast Asia; enzyme activity in erythrocytes is significantly reduced.

It is interesting to note that the "pathological" enzyme of variant A" is very close in electrophoretic mobility and some kinetic properties to normal variants of glucose-6-phosphate dehydrogenase B + and A +. The differences between them lie in stability. It turned out that in young erythrocytes the activity of the variant enzyme A almost does not differ from that of variant B. However, in mature erythrocytes, the picture changes dramatically.This is due to the fact that the half-life in erythrocytes of the enzyme variant A is approximately 5 times (13 days) less than the enzymes of variant B (62 days). there is insufficient activity of glucose-6-phosphate dehydrogenase variant A" is the result of a much faster than normal denaturation of the enzyme in erythrocytes.

Frequency different types deficiency of glucose-6-phosphate dehydrogenase varies in different countries. Therefore, the frequency of people who “respond” with hemolysis to the action of provoking factors varies from 0 to 15%, and in some areas reaches 30 %.

Deficiency of glucose-6-phosphate dehydrogenase is inherited recessively, linked to the X chromosome. Women can be either homozygous (enzyme activity in erythrocytes is absent) or heterozygous (enzyme activity is 50%) carriers of the defect. In men, the activity of the enzyme is usually below 10/o, which causes pronounced clinical manifestations of the disease.

Pathogenesis of glucose-6-phosphate dehydrogenase

Glucose-6-phosphate dehydrogenase is the first enzyme of pentose phosphate glycolysis. The main function of the enzyme is to reduce NADP to NADPH, which is necessary for the conversion of oxidized glutathione (GSSG) to the reduced form. Reduced glutathione (GSH) is required to bind reactive oxygen species (peroxides). Pentose phosphate glycolysis provides the cell with energy.

Insufficiency of enzyme activity reduces the energy reserves of the cell and leads to the development of hemolysis, the severity of which depends on the amount and variant of glucose-6-phosphate dehydrogenase. Depending on the severity of the deficiency, 3 classes of G-6-PD variants are distinguished. Deficiency of glucose-6-phosphate dehydrogenase is linked to the X chromosome and is inherited recessively. Male patients are always hemizygous, while female patients are homozygous.

The most important function of the pentose cycle is to ensure sufficient production of reduced nicotinamide adenine dinucleotide phosphate (NADP) to convert the oxidized form of glutamine to the reduced form. This process is necessary for the physiological deactivation of oxidant compounds such as hydrogen peroxide that accumulate in the erythrocyte. With a decrease in the level of reduced glutathione or the activity of glucose-6-phosphate dehydrogenase, which is necessary to maintain it in a reduced form, under the influence of hydrogen peroxide, oxidative denaturation of hemoglobin and membrane proteins occurs. Denatured and precipitated hemoglobin is found in the erythrocyte in the form of inclusions - Heinz-Ehrlich bodies. Erythrocytes with inclusions are quickly removed from the circulating blood either by intravascular hemolysis, or Heinz bodies with part of the membrane and hemoglobin are phagocytosed by cells of the reticuloendothelial system and the erythrocyte takes the form of a “bitten” (degmacyte).

Symptoms of glucose-6-phosphate dehydrogenase

The disease can be found in a child of any age. Five clinical manifestations of glucose-6-phosphate dehydrogenase deficiency in erythrocytes are identified.

1. Hemolytic disease of the newborn, not associated with serological conflict (group or Rh incompatibility).

Associated with variants of glucose-6-phosphate dehydrogenase B (Mediterranean) and Canton.

Most common in newborn Italians, Greeks, Jews, Chinese, Tajiks, Uzbeks. Possible provoking factors of the disease are the intake of vitamin K by the mother and child; the use of antiseptics or dyes in the treatment of the umbilical wound; use of diapers treated with naphthalene.

Newborns with glucose-6-phosphate dehydrogenase deficiency in erythrocytes have hyperbilirubinemia with signs of hemolytic anemia, but evidence of serological conflict between mother and child is usually absent. The severity of gierbilirubinemia may be different, the development of bilirubin encephalopathy is possible.

1. Chronic non-spherocytic hemolytic anemia

It is found mainly in northern Europeans.

Seen in older children PI adults; increased hemolysis is noted under the influence of intercurrent infections and after taking medications. Clinically, there is constant moderate pallor of the skin, mild icterus, and slight splenomegaly.

1. Acute intravascular hemolysis.

Occurs in apparently healthy children after taking medications, less often in connection with vaccination, viral infection, diabetic acidosis.

Currently, 59 potential hemolytics have been identified in glucose-6-phosphate dehydrogenase deficiency. The group of drugs that necessarily cause hemolysis include: antimalarial drugs, sulfa drugs, nitrofurans.

Acute intravascular hemolysis develops, as a rule, 48-96 hours after the patient has taken a drug that has oxidizing properties.

Drugs that cause hemolysis in individuals with insufficient activity of glucose-6-phosphate dehydrogenase in erythrocytes

The severity of hemolysis varies depending on the degree of enzyme deficiency and the dose of the drug taken.

Clinically during an acute hemolytic crisis general state the child is severe, severe headache, febrile fever are noted. The skin and sclera are pale icteric. The liver is most often enlarged and painful; the spleen is not enlarged. Repeated vomiting with an admixture of bile, intensely colored stools are observed. A typical symptom of acute intravascular hemolysis is the appearance of urine the color of black beer or a strong solution of potassium permanganate. With very intense hemolysis, acute renal failure and DIC may develop, which can lead to death. After the withdrawal of drugs that cause a crisis, hemolysis gradually stops.

1. Favism.

Associated with eating fava beans (Vicia fava) or inhaling pollen from some legumes. Favism may occur upon first contact with the beans, or may be observed in individuals who have previously consumed these beans, but had no manifestations of the disease. Boys predominate among the patients. Favism most often affects children aged 1 to 5 years, in children early age the process is particularly difficult. Relapses of the disease are possible at any age. The time interval between the consumption of fava beans and the development of a hemolytic crisis ranges from several hours to several days. The development of a crisis may be preceded by prodromal signs: weakness, chills, headache, drowsiness, back pain, abdominal pain, nausea, vomiting. Acute hemolytic crisis is characterized by pallor, jaundice, hemoglobinuria, which persists for up to several days.

1. Asymptomatic form.

Laboratory data

In the hemogram of patients with glucose-6-phosphate dehydrogenase deficiency, normochromic hyperregenerative anemia of varying severity is detected. Reticulocytosis can be significant, in some cases reaching 600-800%, normocytes appear. Anisopoikilocytosis, basophilic puncture of erythrocytes, polychromasia are noted, fragments of erythrocytes (schizocytes) can sometimes be seen. At the very beginning of the hemolytic crisis, as well as in the period of hemolysis compensation after a special blood smear staining, Heinz-Ehrlich bodies can be found in erythrocytes. During the crisis, in addition, there is leukocytosis with a shift of the leukocyte formula to the left.

Biochemically, there is an increase in the concentration of bilirubin due to indirect, a sharp increase in the level of free plasma hemoglobin, hypohaptoglobinemia.

In the bone marrow punctate, a sharp hyperplasia of the erythroid germ is detected, the number of erythroid cells can reach 50-75% of the total number of myelokaryocytes, and erythrophagocytosis is detected.

To verify the insufficiency of glucose-6-phosphate dehydrogenase in erythrocytes, methods of direct determination of enzyme activity in erythrocytes are used. The study is carried out in the period of hemolysis compensation.

To confirm the hereditary nature of the disease, the activity of glucose-6-phosphate dehydrogenase must also be determined in the patient's relatives.

Differential Diagnosis

It is carried out with viral hepatitis, other fermentopathies, autoimmune hemolytic anemia.

Treatment of glucose-6-phosphate dehydrogenase

It is necessary to exclude medicines provoking hemolysis. Folic acid is recommended.

With a decrease in hemoglobin concentration less than 60 g / l, replacement therapy erythrocyte mass (quality requirements and calculation of the volume of erythrocyte mass are presented below).

Splenectomy is used only with the development of secondary hypersplenism, since the operation does not lead to the cessation of hemolysis.

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