**8. Conclusion**

80 Dehydrogenases

Grinberg, 1981).

*al.*, 2003).

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

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

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 G6PDdeficient 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 *in vitro* primaquine-nduced hemolysis. Primaquine solution was prepared in 0.1 M Tris buffer (pH 7.4). Primaquine concentration was used between 1 and 4 mM in experiments. Different concentrations of primaquine were added into tubes containing 2% erythrocytes that were prepared before. Tubes were then placed and rotated in a rotator tube for 2 hours at 37°C. The rotation speed was less than 2 rpm. This is another important step for detection of *in vitro* primaquineinduced hemolysis. After 2 hours, the supernatant was collected for the heme concentration, which was then determined spectrophotometrically. Hemoglobin released from erythrocyte induction of primaquine-induced hemolysis and compared with complete lysis (100% hemolysis, control group) obtained by adding 5 mM Tris-HCl (Fig. 1) (Allakhverdiev &

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*, et* 

This method demonstrated that the *in vitro* model of primaquine-induced hemolysis can be only maintained by using 2% hematocrit in physiological conditions. Primaquine leads to hemolysis at concentrations between 1 and 4 mM. Other factors that induce primaquine-derived hemolysis are exposure time, incubation temperature, drug

NADP reduced per minute) per miligram of soluble protein at 37°C.

determined by light microscopy (Peters & Van Noorden, 2009).

**7.4.** *In vitro* **primaquine-induced hemolysis methods** 

**7.3. Cytochemical staining assay** 

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 *in vitro* independently from the variants of G6PD deficiency. The principle of the method is based on the quantitative detection of hemolysis by incubation of erythrocytes obtained from G6PD-deficient patients with primaquine in low hematocrit while rotating the culture in a hybridization oven for 2 hours at 37°C. By considering primaquine-induced hemolysis in patients with G6PD deficiency, it can be determined whether these patients may be treated with primaquine or not. The advantages of this method are that it can determine the primaquine-sensitivity in patients with G6PD deficiency before treatment with primaquine. Using this method not only on G6PD deficiency patients but also on patients that suffer from other diseases that may cause primaquine-induced hemolysis constitutes another advantage of the method.

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