*4.3.1. Some Important G6PD Variants*

70 Dehydrogenases

**4.1. Erythrocytes** 

*et al.*, 1999). As an inhibitory effect, one of the products of G6PD reaction NADPH is an effective inhibitor (Luzzatto, 1967). Increase in NADP and decrease in NADPH as a result of whichever oxidative event in cells effect prepotently to increase G6PD activity (Luzzatto & Testa, 1978). Consequently, G6PD is the most important enzyme in biosynthesis reactions owing to enzyme property as NADPH reducer in its critical role in the cytoplasm (Koehler & Van Noorden, 2003).

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

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

membrane damage occur (Ruwende & Hill, 1998, Peters & Van Noorden, 2009).

**4.2. The importance of pentose phosphate pathway for erythrocytes** 

**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*, et al.*, 1991, Greene, 1993). G6PD B+ is the most commonly found enzyme type and it is used as a standard for normal enzyme activity and electrophoretic mobility. For identification of other variants, G6PD B+ is used. The rate at

generating reducing capacity (Greene, 1993).

**4. The effect of G6PD on erythrocyte metabolism** 

**4.3.1.1 G6PD 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.

**4.3.1.2 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).

**4.3.1.3 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 halflife 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*, et al.*, 1988). Second is a silent mutation result of a cytosine to thymine mutation at nucleotide number 1311 (Beutler, 1990). There are many similar Class II variants in the Mediterranean region (Cagliari, Sassari, El Fayoum), South Asia (Hong Kong, Canton, Mahidol), and elsewhere. Most of these emerge as a consequence of point mutations resulting in single amino acid mutations that have variable effects on activity and electrophoretic mobility (Luzzatto & Battistuzzi, 1985, Luzzatto, 1989, Beutler, 1990, Beutler, 1991, Beutler, 1992).

Glucose-6-Phosphate Dehydrogenase

Deficiency and Malaria: A Method to Detect Primaquine-Induced Hemolysis *in vitro* 73

**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

**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).

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

**5.5. 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*, et al.*, 2011). In addition, β-glucosides in bean seeds, maturity stage of fava beans attain very high amounts causing a severe course of hemolytic crisis (Katz & Schall, 1979, Greene, 1993, Beutler, 1994). Often, in the G6PD Mediterranean variant, acute and a very severe hemolytic crisis are seen due to fava bean ingestion, even capable of causing death (Fairbanks, 1999, Luzzatto, 2001). In favism, damage in erythrocytes is similar to oxidative damage of drugs. Fava beans include visin, konvisin, ascorbic acid and L-Dopa, which have oxidative properties. The most commonly cited konvisin and visin glycosides during digestion fava beans by β-glycosidase or acid hydrolysis demolished to the active agents, which are converted to "divisine" and "izouramil." Divisine and izouramilin reduce the level of the GSH and NADPH in vitro conditions and damage the cell membrane by the formation of cross-connection with Heinz bodies; it also has been shown to inhibit Ca2+- ATPase and catalase (Arese & De Flora, 1990, Beutler, 1994, Gaetani*, et al.*, 1996, Luzzatto, 2001). 24-48 hours after ingesting foods like fava beans, characteristic symptoms occur in the form of pallor, jaundice and hemoglobinuria (Ninfali*, et al.*, 2000). In addition, jaundice,

precipitate hemolysis in patients with G6PD deficiency (Beutler, 1994).

**5.4. Chronic nonspherocytic anemia** 

susceptibility to infection (Beutler, 1994, Luzzatto, 2001).
