Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathological disease in humans. This disease is described as a widespread, heritable, X-chromosome linked abnormality (Reclos
In epidemiological studies, it was shown that the prevalence of G6PD deficiency significantly related to malaria. Malaria is known as a parasitic disease that affects 300-500 million people all over the world. It is widespread in tropical and subtropical regions of Asia, Africa and American continents. Five different types of
Therefore, investigations on detection of G6PD deficiency have a vital importance for malaria patients before their treatment with primaquine. On the other hand, the methods that are used for diagnosing G6PD deficiency are unreliable. Even worse is that it is very difficult to distinguish heterozygously-deficient patients from healthy individuals (Peters & Noorden, 2009). All these data indicate that there is an urgent need to develop new methods for reliable detection of G6PD deficiency in order to prevent hemolysis in patients treated with primaquine. Current methods cannot determine primaquine sensitivity in patients with G6PD deficiency every time. However, in our previously researches, we developed a new method for the determination primaquine induced hemolysis
This chapter aims to represent the problems in treatment of malaria patients with G6PD deficiency by using primaquine, different methods for determination of G6PD deficiency and a new method to determine primaquine induced hemolysisbefore treatment of patients with G6PD deficiency.
2. Genetic basis of G6PD
G6PD deficiency was identified in 1956 by Carson
By virtue of fact that G6PD is found in all cells, functional and structural studies have revealed properties of this housekeeping gene (Luzzatto, 2006). G6PD expression level is regulated by hormonal and nutritional factors in only a few tissues. G6PD expression is regulated in liver and adipose tissue, and its activity depends on the rate of fatty acid biosynthesis (Greene, 1993). The G6PD gene region is one of the first regions of the human genome to be completely sequenced (Chen
The X-linkage of the G6PD gene has important implications. This linkage is very stable and linkage with other group locuses is similar in all mammals (Luzzatto & Battistuzzi, 1985, Group, 1989, Luzzatto, 1989, Beutler, 1990). In mice, X-linkage of G6PD was shown by Epstein (Epstein, 1969). Epstein concluded that the G6PD gene is X-linked in the mouse; its synthesis occurs in the oocyte and is dosage-dependent. G6PD is a sex-linked and very polymorphic gene in populations in which males have only one allele (hemizygous) and females have two G6PD alleles. Thus, females can be either normal or deficient (homozygous), or intermediate (heterozygous) phenotypes, whereas males can be either normal or G6PD-deficient phenotype (Luzzatto, 2006). The frequency of the deficient phenotype is higher in males than females owing to males being hemizygous, in which one allele of the gene expresses the deficient phenotype; to arise in females, G6PD-deficiency needs two deficient alleles. However, hemizygous deficient males and homozygous express the same degree of enzyme deficiency level. Since deactivation of one X-chromosome in embryological development in heterozygous females have two populations of red cells (G6PD-normal and G6PD-deficient), with a wide range of total G6PD enzyme activity depending on the relative proportions. If one of the alleles contains deficiency, as a result of random deactivation of X-chromosomes, about half of the cells will be normal and the other half will be deficient, although there is a wide range of variation around that average (Nance, 1964, Rinaldi
The total length of the gene is about 18.5 kb on the X chromosome (xq28) and contains 13 exons.Exon 13 is about 800 nucleotides long and contains the translation stop codon (Nagel & Roth, 1989, Greene, 1993). The protein-coding region is divided into 12 segments, ranging in size from 12 to 236 bp (Martini
The sequence of the whole G6PD gene is known (Chen
The promoter of the G6PD gene contains a TATA-like, TTAAAT sequence, and a great number of stimulatory protein 1 (Sp1) elements (Philippe
The transcribed region from the initiation site to the poly(A) addition site covers 15,860 bp. (Chen
Up to 450 G6PD variants have been identified depending on the enzyme kinetics, physicochemical characteristics, and other parameters (Luzzatto & Battistuzzi, 1985, Chen
3. Structure of G6PD and enzymatic properties
G6PD is a typical cytoplasmic, housekeeping enzyme and has been found in all cells from liver to kidney and organisms, from prokaryotes to yeasts, to protozoa, to plants and animals (Luzzatto & Battistuzzi, 1985, Antonenkov, 1989, Glader, 1999, Notaro
Activation of the enzyme requires NADP+ tightly binding to dimer or tetramer formation of enzyme. G6PD catalyses the first step of the oxidative pentose phosphate pathway and controls reaction velocity (Wrigley
4. The effect of G6PD on erythrocyte metabolism
Erythrocytes, which contain hemoglobin, are blood cells that perform the transfer of oxygen and carbon dioxide between tissues. G6PD is an important enzyme that performs vital functions within all cells of the body (Greene, 1993). The quantity of active G6PD decreases during the life of an erythrocyte and also the older erythrocytes become vulnerable to oxidative stress. G6PD, an enzyme in the oxidative pentose phosphate pathway, converts the nicotinamide adenine dinucleotide phosphate (NADP+) into its reduced form NADPH. It is necessary for the protection against oxidative stress in erythrocytes. The cells cannot eliminate this stress, which causes hemolysis of erythrocytes. Because H2O2 and other reactive oxygen species cannot be reduced, oxidation of hemoglobin to methemoglobin and membrane damage occur (Ruwende & Hill, 1998, Peters & Van Noorden, 2009).
4.2. The importance of pentose phosphate pathway for erythrocytes
G6PD is the key enzyme in the oxidative pentose phosphate pathway. The first step of the pentose phosphate pathway is catalyzed by G6PD. In this step, NADP+ is reduced to NADPH, and ribulose-5-phosphate, a precursor of DNA, RNA, and ATP, emerge from G6P (Turner, 2000). The most important reducing agent in the cytoplasm of cells is NADPH (Koehler & Van Noorden, 2003). The second enzymatic step in this pathway is NADPH production as a consequence of reactions that reduce oxidized glutathione (GSSG) to reduced glutathione (GSH). The only defense against oxidant stress in the red blood cell (RBC) is GSH production (Friedman, 1979, Group, 1989, Peters & Van Noorden, 2009). In unstressed, normal erythrocytes, the G6PD activity is only about 2% of total capacity (Group, 1989, Peters & Van Noorden, 2009). The pentose phosphate pathway’s main function is the generation of reducing capacity through the production of NADPH and ultimately, GSH. This is essential for cell survival and is available in the erythrocyte for generating reducing capacity (Greene, 1993).
4.3. Classification of Glucose-6-Phosphate Dehydrogenase variants
More than 400 variants of G6PD have been distinguished based on their biochemical characteristics, enzyme kinetics, physicochemical characteristics, and other parameters (Luzzatto & Battistuzzi, 1985, Chen
4.3.1. Some Important G6PD VariantsG6PD A+ is the most widely seen variant worldwide and also the first variant in which the nucleotide mutation and amino acid substitution were determined (Beutler, 1990). This Class IV variant has 90% of the enzyme activity of G6PD B+ (Luzzatto, 1989). This variant also called for the African variant cause widely seen in Africa; 20-40% of African men and 20% of African American men have this variant. It is faster than G6PD B+ electrophoretically and it does not cause hemolysis (Beutler, 1989). G6PD A+ derives from a single amino acid substitution of aspartic acid for asparagine at amino acid number 126, and this was the result of an adenine to guanine mutation at nucleotide number 376.G6PD Aˉ is a Class II variant that has 10 to 20% of the activity of G6PD B+ and the same electrophoretically mobility as G6PD A+(Luzzatto, 1989); 11% of African American men have this variant. Its half-life is 13 days. Three types of mutations have arisen with molecular studies. The most common mutation being at nucleotide number 202 is a result of a guanine to adenine mutation at amino acid number 68 substitution of valine to methionine (Beutler, 1989, Luzzatto, 1989, Beutler, 1990). The second one occurs at nucleotide number 680 as a result of a guanine to thymine mutation at amino acid number 227 substitution of arginine to leucine. And the third mutation occurs at the nucleotide number 968, as a result of a thymine to cytosine mutation at amino acid number 323 substitution of leucine to proline (Beutler, 1989). G6PD A + and G6PD Aˉ variants are defined as unique to Africa, but they can also be seen in Caucasian populations from Italy, Spain, Southeast Asia, Middle East and South America (Beutler, 1990).G6PD Mediterranean is a widely seen variant in the Mediterranean region and Middle East. In addition, it is seen in the Indian subcontinent and other regions of the Americas (Beutler, 1991). This Class II variant has less than 10% of the enzyme activity of G6PD B+ and the electrophoretical mobility is similar with G6PD B+ (Luzzatto, 1989). Its half-life is only 8 days and DNA analysis identified two different point mutations. The first mutation is a result of a cytosine to thymine mutation at nucleotide number 563, at amino acid number 188 substitution of serine to phenylalanine (Vulliamy
5. Clinical tables on G6PD deficiency
Depending on G6PD enzyme deficiency are: Hemolytic Anemia (Drug-induced hemolysis), Diabetes mellitus-induced hemolysis and Infection-induced hemolysis; chronic nonspherocytic anemia, Favism and Neonatal jaundice.
5.1. Hemolytic anemia
5.1.1. Mechanism of hemolysis
In some people, for example, the Mediterranean-type, G6PD deficiency from drug intake occurs, although not a permanent hemolytic condition. In erythrocytes, NADPH cannot form with G6PD deficiency and unformed NADPH creates a deficiency in conversion of the oxidized form of glutathione (GSSG), to its reduced form (GSH) (Lachant
The red blood cell (RBC) membrane was adhered to by Heinz bodies, which are particles of denatured protein. These appear in the early stages of drug administration and disappear as hemolysis progresses. Hemolysis usually occurs in blood vessels and hemoglobinuria follows. The increase of reticulocytes emerges in response to this situation and the hemoglobin level begins to increase again within 8-10 days (Beutler, 1994). In severe hemolysis, the patient may complain of back and stomach pain and the urine turns dark. The hemolytic anemia is self-limited when G6PD deficiency is relatively mild because only the older RBCs are destroyed and the young RBCs have normal or nearly-normal enzyme activity (Beutler, 1994).
Table 1 lists the drugs and chemicals that cause clinically significant hemolytic anemia.
5.2. Diabetes mellitus-induced hemolysis
Hemolysis in G6PD deficiency individuals might initiate diabetic ketoacidosis.This situation is not exactly accepted. However, hemolysis formation has been reported when blood glucose levels are normal in diabetic individuals (Beutler, 1994). It has also has been reported that hypoglycemia might precipitate hemolysis in patients with G6PD deficiency (Beutler, 1994).
5.3. Infection-induced hemolysis
Infections are probably the most common cause of hemolysis in people with G6PD deficiency.There are numerous reports about the importance of infection in causing hemolytic anemia. A large number of bacterial, viral and rickettsial infections have been reported as predisposing factors. Infectious hepatitis (hepatitis A), pneumonia and typhoid fever are known to trigger hemolysis. Involving the upper respiratory tract and gastrointestinal system, viral infections have been reported to cause a more severe hemolysis (Luzzatto, 2001). The mechanism of infection-induced hemolysis is not clear, but it is thought to be that during the infection, superoxide anion and H2O2 production by macrophages causes the hemolysis (Glader, 1999, Luzzatto, 2001).
5.4. Chronic nonspherocytic anemia
Class I G6PD variants, such as the absence of precipitating factors in the occurrence of excessive hemolytic anemia, lower still further the remaining enzyme activity. This is observed in people with chronic hemolytic anemia and oxidative stress, even if unstable conditions occur as a result of insufficient enzyme activity in erythrocytes. Granulocyte dysfunction is seen in some cases. In these cases, more severe hemolysis is due to increased susceptibility to infection (Beutler, 1994, Luzzatto, 2001).
Favism is an illness that occurs in G6PD deficiency individuals with acute hemolysis by eating raw beans (Vicia fabu). Wet, dry or frozen fava bean ingestion of grains, even if the mother eats fava beans can cause hemolysis in newborn infants through breast milk may occur (Luzzatto, 2001). Individuals with G6PD deficiency hemolytic effect caused by the beans contained many glycosides that are toxic due to the visin and konvisin (Beutler, 1994, Akhter
5.6. Neonatal jaundice
One of the most threatening consequences of G6PD deficiency is neonatal jaundice (Beutler, 1994). Jaundice in babies with G6PD enzyme deficiency could be mild or severe enough to cause kernicterus, a spastic type of cerebral palsy, and may even cause death (Luzzatto, 1993). In addition, infants with G6PD deficiency, hyperbilirubinemia is more remarkable than anemia. It facilitates this because of the inadequate physiological conjugation in liver in the neonatal period (Moskaug
6. Malaria and glucose-6 phosphate dehydrogenase deficiency
As we mentioned above, there is a strong relationship between malaria and G6PD deficiency diseases. In several epidemiological studies, it was shown that distribution of malaria was nearly the same with distribution of G6PD deficiency (Motulsky, 1961, Siniscalco & Bernini, 1961, Ganczakowski
As it is known, malaria is a parasitic disease that threatens 300-500 million people all over the world. Malaria can be defined as the most deadly vector-borne disease in the world (Myrvang & Godal, 2000). It is widespread in tropical and subtropical regions of Asia, Africa and the American continents. Each year, malaria leads to deaths of millions of people all around the world and a large percentage of deaths are seen in Sub-Saharan regions of Africa. The causative agents of malaria are the Plasmodium parasites, which are transmitted to humans by the bites of infected mosquitoes. If patients are not treated with antimalarial drugs, malaria can easily lead to death. Five different types of Plasmodium species—
When life cycles of Plasmodium parasites are investigated, it is seen that the parasites multiply in the liver of the human body, and then infect erythrocytes. As we mentioned before,
Multiplication of the parasites within erythrocytes enhances the severity of the disease and cause symptoms such as anemia, fever, chills, nausea, flu-like illness, and, in severe cases, coma, and death. Treatment of this disease can be achieved by using antimalarial drugs. Primaquine, which is the most common antimalarial drug, can be used as a primary prophylactic because it prevents primary parasitemia of
As we pointed out before, according to epidemiological studies, the prevalence of malaria deeply relates to glucose-6 phosphate dehydrogenase (G6PD) enzyme deficiency. In these studies, it was demonstrated that 66 of 77 genetic variants that have reached polymorphic frequencies were seen in populations living in tropical and subtropical areas where malaria was endemic. On the other hand, this genetic diversity does not occur in populations living in non-endemic regions of the world for malaria, indicating that high polymorphism is the indicator of G6PD deficiency.
When investigated in terms of cellular biology, we can see that Plasmodium parasite that causes malaria use erythrocytes as host cells. Erythrocytes are also the most affected cells from G6PD deficiency. This situation also suggests the relationship between the two diseases. In several studies, it was demonstrated that G6PD deficiency provides a protection against malaria infections. In one of the early studies, it was indicated that
The exact mechanism of this protection is still unknown. However there are two postulated explanations. According to the first suggestion, it was found that parasites that cause malaria can only survive in conditions with low oxygen levels (Clark
According to the second suggestion,
Primaquine is the only effective antimalarial drug that provides inhibition of persistent liver stages of
However, as we initially mentioned, using primaquine in order to prevent the relapse of malaria can be very dangerous for G6PD deficiency patients since its usage results in very severe hemolysis. In all G6PD variants, activity levels of the enzyme have been diminished and this partially prevents the defense of erythrocytes against oxidative attack. However, when primaquine is administered, its metabolites lead to more severe hemolysis than oxidative damage by inducing oxyhemoglobin generation, GSH depletion and stimulation of the hexose monophosphate pathway. Moreover, primaquine can also induce the generation of Heinz bodies, which are insoluble aggregates that attach to the surfaces of erythrocytes. The most probable mechanism of primaquine-induced hemolysis is the generation of oxyhemoglobin, which forms hydrogen peroxide. Since G6PD enzyme level is low in G6PD-deficient erythrocytes, these peroxides accumulate and lead to denaturation of hemoglobin. Peroxides also generate Heinz bodies that attach to cell membranes of red blood cells. Hemolysis occurs when damaged erythrocytes pass through the spleen. In each pass, red blood cells lose a portion of the cell membrane. After additional passes, membranes of cells completely lose their competency (Beutler
These conditions reach life-threatening scenarios for all G6PD deficiency patients with different genetic variants. Hence, individuals that are required to use antimalarial drugs should be screened very carefully for their tendency to have G6PD deficiency.For effective control and treatment, either a reliable test for detecting G6PD deficiency or an anti-malarial drug that can be safely given to G6PD deficiency patients is required.
7. Detection methods of G6PD deficiency
Currently, primaquine, which causes hemolysis in G6PD-deficient patients, is the only radical cure of
7.1. Fluorescent spot test
Fluorescence is a form of luminescence that uses the physical change of emission of light upon excitation of molecules. There are various different types of luminescence, classified depending on the style of excitation: chemo-luminescence (ending in a chemical reaction) photo-luminescence (fluorescence, phosphorescence and delayed fluorescence), bio-luminescence (via a living organism) and others (Bernard, 2002).
Nicotinamide Adenine Dinucleotide Phosphate (NADPH) is the reduced form of NADP, with absorption maximum at 340 nm and a maximum emission at 460 nm. NADPH concentrations have been studied in great detail using optical methods. A parameter for direct measurements of the G6PD activity is the fluorescence of NADPH. When G6PD shows enough functional activity in erythrocytes, two molecules of NADP+ are reduced to NADPH. After the addition of glucose 6-phosphate and NADP+, blood spot fluoresces at
7.2. Spectrophotometric assay
Spectrophotometric methods are greatly used in biological sciences for quantitative and qualitative measurements due to the fact that these methods do not break down the molecules analyzed and enable us to assay small quantities of matter fundamentally (Lehninger, 2000). Spectrophotometric techniques allow detection of the concentration of a solution by evaluating its absorbance of a specific wavelength by way of a spectrophotometer, which produces light at a chosen wavelength and passes it directly through the sample. Because every molecule have a specific absorption spectrum, we can recognize and characterize its properties or detect its current concentration in the presence of other compounds (Lehninger, 2000).
In the case of enzyme activity measurements, the assay solution contains some other compounds that are required for the reaction to occur. Other compounds in the reaction mix may absorb light at the same wavelength with the enzyme being analyzed. To eliminate the interference of other compounds, the absorbance of a sample solution is compared with blank solution, which is taken as the reference. The blank contains everything found in the sample solution except the substance to be assayed.
In the matter of protein (enzymatic activity or protein concentration) measurements, colorimetric methods are used. Colorimetric measurements are performed by way of quantitative assessment of a colored complex, which is mostly formed by the reaction of a colorless compound and a dye reagent. However, the compound that will be analyzed can be naturally colored and can be read directly spectrophotometrically.
Glucose-6-phosphate dehydrogenase catalyzes the first step in the pentose phosphate shunt, oxidizing glucose-6-phosphate (G-6-P) to 6-phosphogluconate (6-PG). The enzyme activity can be determined quantitatively by spectrophotometer assay method, which is based on the rate of NADPH production from NADP+ in G6PD-deficient patients (Kornberg
These reactions are illustrated below:Nictotinamide adenine dinucleotide phosphate (NADP) is reduced by G6PD in the presence of G-6-P. The rate of formation of NADPH is proportional to the G6PD activity and is measured spectrophotometrically as in increase in absorbance at 340 nm. Production of a second molar equivalent of NADPH by erythrocyte 6-phosphogluconate dehydrogenase (6-PGDH) occurs according to the reaction:This is prevented by use of maleimide, an inhibitor of 6-PGDH.
The Enzyme Commission of the International Union of Biochemistry recommends expressing this in international units (IU) and defines 1 IU as the amount of an enzyme that catalyzes the transformation of 1 micromole of substrate per minute under standard conditions of temperature, optimal pH, and optimal substrate concentration. Specific activity relates activity to total mass of protein to avoid bias through individual differences in weight (Bairoch, 1993). Therefore, G6PD activity was expressed as units (micromoles of NADP reduced per minute) per miligram of soluble protein at 37°C.
7.3. Cytochemical staining assay
The Cytochemical staining assay is based on the intracellular reduction of the tetranitro blue tetrazolium (TNBT) by the G6PD via exogenous electron carrier 1-methoxyphenazine methosulfate and TNBT is reduced to dark-colored water-insoluble formazan, which can be determined by light microscopy (Peters & Van Noorden, 2009).
In vitroprimaquine-induced hemolysis methods
3 cc of venous blood anti-coagulated by 2% heparin solution (126 mM NaCl, 14 mM Na2HPO4, 1 mM KH2PO4, 13,2 mM glucose, pH 7.4) was collected from healthy and G6PD-deficient persons. The blood was washed three times with sterile heparin solution at 3000 rpm for 10 min. Erythrocytes were resuspended in PBS after that hematocrit was adjusted to 2%. This is the one of the most important steps for detection of
The principle of this method is based on conversion of Hemoglobin (Hb) to cyanmethemoglobin by the addition of KCN and ferricyanide, whose absorbance is measured spectrophotometrically as cyanmethemoglobin at 540 nm versus a standard solution. Supernatant of hemolyzed red blood was diluted four-fold (v/v) with distilled water. On the other hand, the control group was diluted twenty-fold (v/v) with distilled water. After that, 50 μL KCN (10% w/v) and 50 μL potassium ferricyanide (2% w/v) were directly mixed and the color was measured at 540 nm. The standard curve was constructed using the standard cyanmethemoglobin solutions in different concentrations (Bhaskaram
This method demonstrated that the
This chapter has aimed to represent the relationship between G6PD deficiency and malaria and to suggest a sensitive method for detection of primaquine-induced hemolysis in patients with G6PD deficiency. As mentioned above, G6PD deficiency is the most common enzymopathologic disorder in humans and it affects 400 million people worldwide. In patients with G6PD deficiency, oxidative stress cannot be prevented since G6PD enzyme is the initial catalyst of the pentose phosphate pathway in erythrocytes that reduces the peroxides to H2O. This situation leads to mild to severe hemolysis, changing depending on genetic variants of the disease. As we mentioned before, according to epidemiological studies, the prevalence of G6PD deficiency deeply relates to malaria. In these studies, it was demonstrated that 66 of 77 genetic variants, which have reached to polymorphic frequencies, were seen in populations living in tropical and subtropical places where malaria was endemic. On the other hand, this genetic diversity does not occur in populations living in non-endemic regions of the world for malaria, indicating that high polymorphism is the indicator of G6PD deficiency and distribution of malaria is nearly the same with distribution of G6PD deficiency. This situation reveals two important results.
One of them is that G6PD deficiency provides partial protection from malaria infections, especially for falciparum infections. In several studies, it was demonstrated that risk of contracting malaria in patients that have G6PD deficiency decreased at a rate of 46 to 58%. On the other hand, using antimalarial drugs can cause life-threatening hemolytic anemia in patients with G6PD deficiency. Since G6PD deficiency does not provide exact protection, these patients still have a risk of contracting malaria. However, using primaquine, which is the only radical cure of Plasmodium infections, can induce more severe hemolysis by generating oxyhemoglobin, GSH depletion and Heinz bodies and enhancing oxidative attack. This threatens the lives of patients with G6PD deficiency. Hence, patients with malaria should be screened for their tendency to G6PD deficiency before their treatment with antimalarial drugs. Common methods that are used for diagnosing G6PD deficiency are unreliable. Even worse is that it is very difficult to distinguish heterozygously-deficient patients from healthy individuals. Additionally, current methods cannot accurately indicate hemolysis, even though they give information about activity of the enzyme. Also, these methods do not determine primaquine sensitivity in patients with G6PD deficiency every time. However, the method that we developed provides the determination of primaquine sensitivity in patients with G6PD deficiency
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