**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*, et al.*, 1955, Lohr & Waller, 1974).

These reactions are illustrated below:

78 Dehydrogenases

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

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

Currently, primaquine, which causes hemolysis in G6PD-deficient patients, is the only radical cure of *Plasmodium vivax* infections (Burgoine*, et al.*, 2010). Therefore, screening to detect G6PD deficiency is very important. Various tests can be used for the detection of G6PD deficiency, which are based on the assessment of the NADPH production capacity of G6PD. The most frequently used tests that measure NADPH production are the fluorescent spot test, cytochemical assay and spectrophotometric assay. However, fluorescent spot test and the spectrophotometric assay are not reliable for the detection of heterozygous females. In addition, DNA analysis can be done to detect G6PD deficiency for the homozygous, hemizygous, and heterozygous-deficient patients. However, we have to design primers for

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-

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 *340* 

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

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

**7. Detection methods of G6PD deficiency** 

all mutations (Peters & Van Noorden, 2009).

luminescence (via a living organism) and others (Bernard, 2002).

*nm* if NADPH is produced (Beutler & Baluda, 1966).

**7.2. Spectrophotometric assay** 

**7.1. Fluorescent spot test** 

drug that can be safely given to G6PD deficiency patients is required.

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:

$$\text{6-phosphate} + \text{NADP} + \xrightarrow{\text{6PGD}} \text{R\"bubose-5-Phosphate} + \text{NADPH} + \text{H}^+ + \text{CO}\_2$$

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.

Glucose-6-Phosphate Dehydrogenase

Deficiency and Malaria: A Method to Detect Primaquine-Induced Hemolysis *in vitro* 81

concentration and amount of oxygen. Despite the fact that there are several methods in order to diagnose G6PD deficiency, these methods do not determine primaquine sensitivity in patients with G6PD deficiency every time. Therefore, lack of primaquinebased treatment by considering only G6PD deficiency can be very dangerous in terms of health of patients with malaria and the epidemiology of the disease. On the other hand, treatment of primaquine-sensitivive individuals with primaquine can cause death. Hence, in the Centers for Disease Control and Prevention (CDC) report (Hill*, et al.*, 2006), it was highlighted that there was an urgent need to develop new *in vitro* methods for determining hemolysis that indicate primaquine sensitivity before treatment of patients with this drug. 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 advantage of this method is that it can determine primaquineinduced hemolysis before treatment with primaquine and its capacity to determine

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.

**Figure 1.** *In vitro* Primaquine-Induced hemolysis

G6PD deficiency.

**8. Conclusion** 
