Tuesday, October 21, 2008

Determination of Glucose-6-Phosphate Dehydrogenase in Erythrocytes

The hexose monophosphate shunt is essential in erythrocyte metabolism, liver, adipose tissue and kidney metabolism. The regulation of pentose phosphate is a dehydrogenation step of glucose-6-posphate by G6PD. A decrease in G6PD enzyme in erythrocyte will cause a reduction in NADPH production and haemolysis occurs. G6PD deficiency is an inherited x-linked recessive disease. Its determination is very important since the deficiency of it can cause haemolytic anaemia and neonatal jaundice.

G6PDH is an enzyme of the pentose phosphate pathway that catalyzes the reaction below:

Glucose-6-Phosphate + NADP+ <=> 6-Phosphogluconolactone + NADPH

Individuals whose G6PDH is partly inactive are prone to oxidative stress. This arises because NADPH produced by the pentose phosphate pathway is required for reduction of glutathione, which, in turn, helps reduce peroxides. In the absence of sufficient NADPH, glutathione remains oxidized and peroxides cannot be neutralized.

In this of experiment we have carried out determination of glucose-6-phosphate dehydrogenase in erythrocyte which is a significant enzyme in pentose phosphate pathway. When G6PD is present in erythrocytes at normal level, it can act on glucose-6-phosphate and catalyses NADP+ to NADPH which in turn will emit UV light, absence of UV light indicates a decrease in the enzyme level. A decreased level of G6PD enzyme in erythrocytes will cause reduction of NADPH production and consequently hemolysis will occur (hemolytic anemia).

A mutation present in some populations causes a deficiency in glucose-6-phosphate dehydrogenase with consequent impairment in the generation of NADPH. This impairment is manifested as red cell hemolysis when the susceptible individual is subject to oxidants such as the antimalarial primaquine, aspirin, or sulfonamides or when the susceptible individual eats fava beans (Vicia fava).

The Pentose phosphate pathway supplies RBC with NADPH to maintain the reduced state of glutathione. The inability to maintain reduced glutathione in RBC’s leads to increased accumulation of peroxides, predominantly H202 that in turn results in a weakening of the cell wall and concomitant hemolysis.Therefore, any defect in the production of NADHP could, therefore, have profound effects on erythrocyte survival.

Hemolytic anemia is the red blood cell (RBC) plasma membranes rupture prematurely. The released haemoglobin pours into the plasma and may damage the filtering unit (glomeruli) in the kidneys. The condition may result from inherited defects such as abnormal red blood cell enzymes, or from outside agents such as parasites, toxins, or antibodies from incompatible transfused blood.

The symptom of hemolytic anemia is the reduced number of RBC’s or a decreased amount of haemoglobin in the blood. The person feels fatigued and is tolerant of cold, both of which are related to lack of oxygen needed for ATP and heat production. Also, the skin appears pale due to the low content of red-colored haemoglobin circulating in skin blood vessel. That is why G6PD determination or verification is very significant especially for new born babies as a screening test.

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