**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*, et al.*, 1995). This situation reveals two important facts. One of them is that G6PD deficiency provides great protection from malaria, especially for falciparum infections. On the other hand, using antimalarial drugs can cause lifethreatening hemolytic anemia in patients with G6PD deficiency. Hence, malaria patients should be screened for their tendency to G6PD deficiency before their treatment with antimalarial drugs. In this part, we will first summarize the importance of malaria for the world. Then, we will explore the relationship between these two diseases in detail.

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—*P. falciparum, P. vivax, P. ovalae, P. malariae* and *P.knowlesi*—lead to this disease (Wernsdorfer & McGregor, 1988, Sutherland*, et al.*, 2010).

74 Dehydrogenases

**5.6. Neonatal jaundice** 

headache, backache, nausea, fever, and chills are all signs of acute hemolysis (Tyulina*, et al.*, 2000). Favism is most common seen in children between the ages of 2-5, and is also 2-3 times more common in boys than in girls (Luzzatto, 2001). Clinical signs of favism begin earlier and are more severe than drug-induced hemolytic crises. Rarely, as a result of pollen of fava inhalation, hemolysis may occur within hours (Beutler, 1994). While each favism patient must have G6PD deficiency, hemolytic reactions may not occur after ingestion of fava beans in each person with G6PD deficiency. Each individual with G6PD deficiency of the same family could not be affected in the same way when they eat fava bean. On the other hand, changes are observed in the same person at different times. Genetic variations between individuals, differences of fava bean active metabolites may be responsible for these

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*, et al.*, 2004). G6PD Aˉ, G6PD mediterrian, G6PD Canton variants are known as types that cause kernicterus and hyperbilirubinemia (Luzzatto, 2001). Clinically, the jaundice, the level of G6PD in the normal physiological jaundice in newborns occur on the same days, or a little earlier, but it takes as long as 2-3 weeks (Tan, 1981, Luzzatto, 2001). There are two major differences between jaundice due to incompatibility of blood groups and jaundice due to G6PD deficiency. First, the presence of jaundice in G6PD deficiency is very rare during childbirth and usually it begins in the second or third day. Second, according to anemia, jaundice is more pronounced and it is encountered with severe anemia very rarely in the absence of the enzyme (Luzzatto, 1993, Luzzatto, 2001).

variable characteristics (Meloni*, et al.*, 1983, Group, 1989, Luzzatto, 2001).

**6. Malaria and glucose-6 phosphate dehydrogenase deficiency** 

world. Then, we will explore the relationship between these two diseases in detail.

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

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*, et al.*, 1995). This situation reveals two important facts. One of them is that G6PD deficiency provides great protection from malaria, especially for falciparum infections. On the other hand, using antimalarial drugs can cause lifethreatening hemolytic anemia in patients with G6PD deficiency. Hence, malaria patients should be screened for their tendency to G6PD deficiency before their treatment with antimalarial drugs. In this part, we will first summarize the importance of malaria for the *Plasmodium falciparum (P. falciparum)* is the most serious and life-threatening form of the disease. 80% of death cases are reported from patients that have been infected with *P. falciparum*. It was also demonstrated that resistance has been developed in this type of parasites against current antimalarial drugs. It is generally seen in Africa, specifically in sub-Saharan regions. Interestingly, falciparum-derived malaria cases have been recently reported in various parts of the world where this parasite species was believed to be completely eradicated.

*Plasmodium vivax (P. vivax)* constitutes a milder form of the disease. Vivax infections generally do not cause death. However, individuals that suffer from vivax infection also need to be treated. Among all *Plasmodium* species, *P. vivax* is the one that shows the broadest geographic distribution worldwide. Causative agents for 60% of malaria infections are reported as *P. vivax* infections in India. This parasite has a liver stage and can remain in the body for years without causing sickness. If the patient is not treated, the liver stage may re-activate and cause relapses—malaria attacks—after months, or even years without symptoms.

*Plasmodium ovale (P. ovale*) is known as one of the other milder form of the disease. Like *P. vivax*, it generally does not commonly lead to death. Nevertheless, infected individuals require medical therapy. This parasite, similar to *P.vivax*, can live in the liver for long periods without causing symptoms. Therefore, if it is not treated, reactivation of parasites can be observed in the liver and this leads to relapse of the disease

*Plasmodium malariae (P. malariae)* is also another milder form of the disease. It does not commonly lead to death. However, it still requires treatment. This type of *Plasmodium* parasites are reported to stay in the blood of some individuals for several decades.

*Plasmodium knowlesi (P. knowlesi)* causes malaria in macaques, but can also infect humans (Mendis*, et al.*, 2001, Singh*, et al.*, 2004, Mueller*, et al.*, 2007).

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, *Plasmodium* parasites enter the human body when bitten by an infective female mosquito, which is called Anopheles. These mosquitoes become infected with malaria when they take *Plasmodium*-containing blood from an infected person. Approximately one week later, these parasites mix with the mosquito's saliva when the mosquito takes its next blood meal from another person and this individual is injected with *Plasmodium* parasites when they are being bitten (Bozdech*, et al.*, 2003).

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 *Plasmodium* species by destroying these parasites in the liver before they reach the bloodstream and cause disease (Yazdani*, et al.*, 2006).

Glucose-6-Phosphate Dehydrogenase

Deficiency and Malaria: A Method to Detect Primaquine-Induced Hemolysis *in vitro* 77

mechanism for erythrocytes to survive. GSH that is produced by NADP+ reduction reacts with H2O2 and reduce it to H2O. This prevents the generation of oxidative stress within red blood cells. Since oxidative stress is the most important factor for the disruption of red blood cells, these cells are protected from this effect. However, in G6PD deficient erythrocytes, G6PD activity is significantly reduced. In G6PD A (-) variant, enzyme activity level reduces to 10 or 20% of normal levels, while enzyme activity completely disappears in G6PD variant. Therefore, oxidative stress can be induced in erythrocytes whose G6PD enzymes are deficient. In this situation, GSH is not produced and H2O2 is not reduced to H2O and leads to oxidative stress. Hence, it is thought that since malaria parasites are susceptible to oxidative stress, they do not live within the erythrocytes where their maturation occurs (Toncheva & Tzoneva, 1985, Greene, 1993). Additionally, during oxidative stress, the loss of potassium from the cell and from the parasite can cause the death of the parasite (Friedman

According to the second suggestion, *Plasmodium* parasites oxidize NADPH and reduce the level of reduced glutathione (GSH) in erythrocytes. In the situation of G6PD deficiency, this effect becomes more severe and induces oxidative-induced damage within erythrocytes. Moreover, *Plasmodium* parasites break down hemoglobin and release toxic components like iron and these substances lead to hemolysis. Hence, the development rates of *Plasmodium* parasites are diminished. Additionally, red blood cells that are affected by oxidative stress and are damaged are eliminated by the immune system via phagocytosis. This elimination decreases the growth of parasites much more since it occurs during an early ring-stage of parasites' maturation. Therefore, all of these data indicate that G6PD deficiency can provide protection against malaria infections. Considering the relationship between G6PD deficiency and plasmodium infections, research has aimed to develop antimalarial drugs that decrease the level of GSH within erythrocytes and then produce hydrogen peroxide and the other free radical species in order to enhance the inhibition of Plasmodium species (Mehta*, et al.*,

Primaquine is the only effective antimalarial drug that provides inhibition of persistent liver stages of *P. falciparum, P. vivax,* and *P. ovalae* parasites that lead to relapses of malaria

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

2000, Fortin*, et al.*, 2002, Kwiatkowski, 2005, Prchal & Gregg, 2005).

& Trager, 1981).

(Phompradit*, et al.*, 2011).

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 *P. falciparum* and *P. vivax* parasites preferred to invade younger erythrocytes, which possessed high levels of G6PD enzyme. Since enzyme levels are diminished in older erythrocytes, parasites do not prefer to invade these erythrocytes. These studies suggested the protective effect of G6PD deficiency from parasitemia (Allison & Clyde, 1961, Kruatrachue*, et al.*, 1962). In the recent past, Ruwando et al. also carried out a case-control study on more than 2,000 African children and exhibited that risk of contracting malaria in patients that have the African form of G6PD deficiency decreased at a rate of 46 to 58%. In this study, it was suggested that the selective advantage of resistance to malaria was counterbalanced with selective disadvantageous results of G6PD deficiency, and this stopped the rise of malaria frequencies in endemic regions (Ruwende*, et al.*, 1995). In another study, Ninokata et al. (2006) investigated 345 healthy adults for G6PD deficiency on Phuket Island, which had been determined to be a malaria-endemic region and found out that 10% of these individuals had G6PD deficiency. Interestingly, it was observed that none of the individuals had molecular evidence of malaria infection. According to this study, researchers postulated that G6PD deficiency provided an advantageous genetic trait against malaria (Ninokata*, et al.*, 2006).

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*, et al.*, 1989). This demonstrates that these parasites are very susceptible to oxidative stress. It is known that in the pentose phosphate pathway of erythrocytes, glucose-6 phosphate dehydrogenase (G6PD) enzyme has an important role in production of NADPH and GSH. This is the only mechanism for erythrocytes to survive. GSH that is produced by NADP+ reduction reacts with H2O2 and reduce it to H2O. This prevents the generation of oxidative stress within red blood cells. Since oxidative stress is the most important factor for the disruption of red blood cells, these cells are protected from this effect. However, in G6PD deficient erythrocytes, G6PD activity is significantly reduced. In G6PD A (-) variant, enzyme activity level reduces to 10 or 20% of normal levels, while enzyme activity completely disappears in G6PD variant. Therefore, oxidative stress can be induced in erythrocytes whose G6PD enzymes are deficient. In this situation, GSH is not produced and H2O2 is not reduced to H2O and leads to oxidative stress. Hence, it is thought that since malaria parasites are susceptible to oxidative stress, they do not live within the erythrocytes where their maturation occurs (Toncheva & Tzoneva, 1985, Greene, 1993). Additionally, during oxidative stress, the loss of potassium from the cell and from the parasite can cause the death of the parasite (Friedman & Trager, 1981).

76 Dehydrogenases

*al.*, 2006).

*al.*, 2006).

indicator of G6PD deficiency.

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 *Plasmodium* species by destroying these parasites in the liver before they reach the bloodstream and cause disease (Yazdani*, et* 

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

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 *P. falciparum* and *P. vivax* parasites preferred to invade younger erythrocytes, which possessed high levels of G6PD enzyme. Since enzyme levels are diminished in older erythrocytes, parasites do not prefer to invade these erythrocytes. These studies suggested the protective effect of G6PD deficiency from parasitemia (Allison & Clyde, 1961, Kruatrachue*, et al.*, 1962). In the recent past, Ruwando et al. also carried out a case-control study on more than 2,000 African children and exhibited that risk of contracting malaria in patients that have the African form of G6PD deficiency decreased at a rate of 46 to 58%. In this study, it was suggested that the selective advantage of resistance to malaria was counterbalanced with selective disadvantageous results of G6PD deficiency, and this stopped the rise of malaria frequencies in endemic regions (Ruwende*, et al.*, 1995). In another study, Ninokata et al. (2006) investigated 345 healthy adults for G6PD deficiency on Phuket Island, which had been determined to be a malaria-endemic region and found out that 10% of these individuals had G6PD deficiency. Interestingly, it was observed that none of the individuals had molecular evidence of malaria infection. According to this study, researchers postulated that G6PD deficiency provided an advantageous genetic trait against malaria (Ninokata*, et* 

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*, et al.*, 1989). This demonstrates that these parasites are very susceptible to oxidative stress. It is known that in the pentose phosphate pathway of erythrocytes, glucose-6 phosphate dehydrogenase (G6PD) enzyme has an important role in production of NADPH and GSH. This is the only According to the second suggestion, *Plasmodium* parasites oxidize NADPH and reduce the level of reduced glutathione (GSH) in erythrocytes. In the situation of G6PD deficiency, this effect becomes more severe and induces oxidative-induced damage within erythrocytes. Moreover, *Plasmodium* parasites break down hemoglobin and release toxic components like iron and these substances lead to hemolysis. Hence, the development rates of *Plasmodium* parasites are diminished. Additionally, red blood cells that are affected by oxidative stress and are damaged are eliminated by the immune system via phagocytosis. This elimination decreases the growth of parasites much more since it occurs during an early ring-stage of parasites' maturation. Therefore, all of these data indicate that G6PD deficiency can provide protection against malaria infections. Considering the relationship between G6PD deficiency and plasmodium infections, research has aimed to develop antimalarial drugs that decrease the level of GSH within erythrocytes and then produce hydrogen peroxide and the other free radical species in order to enhance the inhibition of Plasmodium species (Mehta*, et al.*, 2000, Fortin*, et al.*, 2002, Kwiatkowski, 2005, Prchal & Gregg, 2005).

Primaquine is the only effective antimalarial drug that provides inhibition of persistent liver stages of *P. falciparum, P. vivax,* and *P. ovalae* parasites that lead to relapses of malaria (Phompradit*, et al.*, 2011).

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*, et al.*, 1955, Bolchoz*, et al.*, 2002, Beutler & Duparc, 2007).

Glucose-6-Phosphate Dehydrogenase

Deficiency and Malaria: A Method to Detect Primaquine-Induced Hemolysis *in vitro* 79

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

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

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

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*, et al.*,

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-

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

of other compounds (Lehninger, 2000).

1955, Lohr & Waller, 1974).

These reactions are illustrated below:

PGDH) occurs according to the reaction:

This is prevented by use of maleimide, an inhibitor of 6-PGDH.

sample solution except the substance to be assayed.

be naturally colored and can be read directly spectrophotometrically.

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.
