**1. Introduction**

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*, et al.*, 2000). It is estimated that it affects approximately 400 million people worldwide (Noori-Daloii*, et al.*, 2004). This disease is seen most frequently in approximately all of Africa, Asia, and the countries near the Mediterranean Sea (Frank, 2005). G6PD enzyme was demonstrated to play an active role in survival of erythrocytes. It is known that in the pentose phosphate pathway of erythrocytes, glucose-6 phosphate dehydrogenase (G6PD) enzyme provides the production of NADPH and GSH. GSH, produced by pentose phosphate pathway can react with H2O2 and reduce it to H2O. This prevents the generation of oxidative stress within red blood cells; 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, leading to oxidative stress and hemolysis. This is the only mechanism available for the erythrocyte in order to generate reducing equivalence, therefore making it essential for the survival of erythrocytes. In individuals whose G6PD enzyme is deficient, different kinds of hemolysis from mild to severe are seen bound to differences in variants of the disease (Beutler, 1983, Luzzatto, 1989).

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 *Plasmodium* species—*P.* 

© 2012 Allahverdiyev et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*falciparum, P. vivax, P. ovalae, P. malariae and P.knowlesi*—lead to this disease by infecting erythrocytes. Malaria can become a life-threatening condition when it is not treated. 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. As it can be easily seen, malaria and G6PD deficiency share the same geographic distribution. It was shown that G6PD enzyme has various genetic variants and polymorphic frequencies. Highly polymorphic frequencies, which are indicators of G6PD deficiency, are seen in endemic regions for malaria such as Asia, Africa, Central and South America, while in non-endemic regions, these rates decrease, suggesting the relationship between G6PD deficiency and malaria (Haworth*, et al.*, 1988, Organization, 2009, Sutherland*, et al.*, 2010). This relationship reveals two important results. One of them is that G6PD deficiency provides great protection from malaria infection, especially for falciparum infections (Motulsky, 1961, Siniscalco & Bernini, 1961, Ganczakowski*, et al.*, 1995). On the other hand, G6PD deficiency has been recently demonstrated to cause serious problems in fighting against malaria. Primaquine, which is the only drug currently, used in the treatment of Plasmodium infections leads to severe hemolysis in G6PD-deficient patients. This drug may even cause death in G6PD-deficient patients. When primaquine is administered to individuals with G6PD deficiency, its metabolites lead to more severe hemolysis by inducing oxyhemoglobin generation, GSH depletion and stimulation of the hexose monophosphate pathway (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* 67

G6PD deficiency was identified in 1956 by Carson *et al.* (Alving*, et al.*, 1956), and its Xchromosomal inheritance was discerned in the 1950s by Childs *et al.* (Childs*, et al.*, 1958). G6PD was cloned and sequenced by Persico *et al.*(Persico*, et al.*, 1986, Persico*, et al.*, 1986) in 1986 and independently by Takizawa and Yoshida (Takizawa*, et al.*, 1986) G6PD (Misumi*, et al.*, 1982) is in the hexose monophosphate pathway, the only NADPH-generation process in mature erythrocytes, which lack the citric acid cycle. Deficiency of this enzyme in erythrocytes causes various forms of illnesses such as favism, anemia, chronic nonspherocytic hemolytic anemia, drug-sensitive hemolytic anemia, primaquine sensitivity

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*, et al.*, 1996). The gene encoding G6PD is located near the telomeric region of the distal arm of the X chromosome (Pai*, et al.*, 1980, Szabo*, et al.*, 1984, Patterson*, et al.*, 1987) (band Xq28) and a valuable X-linked genetic marker for determination of X chromosome inactivations (Migeon, 1983). G6PD has various polymorphism sites at the G6PD locus like the colorblindness, Xg blood group and the hemophilia A locus and has close linkage at the X chromosome (Boyer & Graham, 1965, Adam*, et al.*, 1967). G6PD is one of a group of genes including fragile X, (Oberle*, et al.*, 1987) color vision (Motulsky, 1988, Filosa*, et al.*, 1993) hemophilia A (Boyer & Graham, 1965) clasped-thumb mental retardation syndrome (MASA), (Macias*, et al.*, 1992) and dyskeratosis congenita (Arngrimsson*, et al.*, 1993) existing on the distal long arm of the X chromosome.

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

**2. Genetic basis of G6PD** 

and jaundice in newborns (Beutler*, et al.*, 1968).

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 *in vitro*. This method provides the determination of G6PD deficiency patients that are susceptible to primaquine independently from the variants of G6PD deficiency. In our studies, it was determined that this method demonstrated high sensitivity for detection of primaquine-induced hemolysis before treatment of malaria patients with primaquine. This chapter aims to represent the relationship between G6PD deficiency and malaria, and to demonstrate the method that has high sensitivity for detection of primaquine-induced hemolysis in patients with malaria whose G6PD enzyme is deficient before their treatment with primaquine.

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 hemolysis before treatment of patients with G6PD deficiency.
