Application

This kit can be used for detection of Glucose-6-phosphate Dehydrogenase (G6PD) activity in packed red cells.

Detection significance

Glucose-6-phosphate dehydrogenase (G6PD or G6PDH) (EC 1.1.1.49) is a cytosolic enzyme that catalyzes the chemical reaction.

D-glucose 6-phosphate + NADP+ ⇌ 6-phospho-D-glucono-1,5-lactone + NADPH + H+

This enzyme participates in the pentose phosphate pathway, a metabolic pathway that supplies reducing energy to cells (such as erythrocytes) by maintaining the level of the co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH). The NADPH in turn maintains the level of glutathione in these cells that helps protect the red blood cells against oxidative damage from compounds like hydrogen peroxide. Of greater quantitative importance is the production of NADPH for tissues involved in biosynthesis of fatty acids or isoprenoids, such as the liver, mammary glands, adipose tissue, and the adrenal glands. G6PD reduces NADP+ to NADPH while oxidizing glucose-6-phosphate. Clinically, an X-linked genetic deficiency of G6PD predisposes a person to non-immune hemolytic anemia.

Detection principle

G6PD in the sample catalyzes glucose-6-phosphate (G6P) transform into 6-phosphogluconic acid (6PG), at the same time oxidized coenzyme II (NADP) transform into reduced coenzyme II (NADPH), and G6PD activity in the samples can be calculated by measuring the increased rate of OD value.

Experimental instrument

Test tube, Micropipettor, Water bath, Spectrophotometry or Biochemistry analyzer

Pretreatment of sample

1.         The test sample should be packed red cells.

2.         Collect the fasting blood. Heparin, EDTA and sodium citrate can be used as anticoagulants. Don’t use dry tube and procoagulant tube. Centrifuge at 1000 rpm for 5 minutes. Avoid the plasma layer and accurately absorb 20 uL of the packed red blood cells and add 1 mL lysis solution to full dissolution (about 1-2 min, less than 25 min). Avoid hemolysis, or the results will be affect.

3.         Interfering substance: Vitamin C ≤ 0.5 g/L, conjugative bilirubin ≤ 300 μmol/L, hemoglobin ≤ 5 g/L the results won’t be effected.

Operation steps

1) Main performance parameters

2) Operate with Automatic biochemical analyzer

3) Operate with Spectrophotometry

Technical parameter

1. Absorbance of blank reagent: A(optical path=1.0cm)≤1.

2. Sensitivity: The difference of absorbance value △A is less than 0.0030 when testing 1300 U/L samples.

3. Linear range: 50-3000 U/L, r2＞0.990.

4. Precision: the relative deviation is less than 10%.

5. Stability: This kit can be store at 2-8℃ for 12 months with shading light. It can be stable for a month at 2-8℃ with shading light after opening. Dissolved standard can be stable for a week at -20℃ with shading light.

6. Reference range: adult, 1300-3600 U/L; children: 1700-4000 U/L.

Notes

1. The kit is for research use only.

2. Choose a filter with the closest wavelength to the required one if there is no suitable filter.

3. The ratio of sample and reagent can be scaled as required.

4. Do not use components from different batches of kit.