Determination of Glucose-6-Phosphate Dehydrogenase

Introduction

• Glucose-6-Phosphate Dehydrogenase (G6PD) is an important enzyme of the pentose phosphate pathway, also known as the hexose monophosphate shunt.
• G6PD catalyzes the first and rate-limiting step of this pathway and helps convert glucose-6-phosphate into 6-phosphoglucono-δ-lactone.
• The reaction produces NADPH, which is essential for protecting cells against oxidative damage.
• In red blood cells (RBCs), G6PD is particularly important because the pentose phosphate pathway is their major source of NADPH.
• NADPH helps maintain reduced glutathione (GSH), which protects hemoglobin and the RBC membrane from reactive oxygen species.
• G6PD deficiency is an inherited enzyme disorder that can make RBCs more susceptible to oxidative stress and hemolysis.
• G6PD deficiency may cause hemolytic anemia, jaundice, or hemoglobinuria, particularly after infections, certain medications, or consumption of fava beans.

---

Principle 

1. G6PD catalyzes the oxidation of glucose-6-phosphate in the presence of NADP⁺.
2. The reaction produces NADPH.
3. NADPH absorbs light at 340 nm.
4. The increase in absorbance at 340 nm is measured using a spectrophotometer.
5. The rate of increase in absorbance is directly proportional to the G6PD activity in the sample.

Reaction:

Glucose-6-phosphate + NADP⁺ → 6-Phosphogluconolactone + NADPH + H⁺

---

Sample Required

Specimen

• Whole blood
• Usually collected in an EDTA tube

The test is generally performed on red blood cells because G6PD activity is particularly important for assessing erythrocyte enzyme deficiency.

Sample Preparation

The blood sample is processed according to the manufacturer’s protocol or laboratory procedure to obtain the appropriate erythrocyte preparation or hemolysate.

Important: The exact specimen preparation, reagent volumes, incubation time, temperature, and calculation formula depend on the analytical method and commercial reagent kit being used.

---

Reagents Required

Typical reagents include:

1. Glucose-6-phosphate
2. NADP⁺
3. Phosphate buffer
4. Appropriate stabilizers or activators, depending on the method
5. Hemolysate/sample
6. Distilled or deionized water

Commercial G6PD assay kits may contain these components in ready-to-use or separately supplied reagent forms.

---

Equipment Required

• Spectrophotometer
• Cuvettes
• Micropipettes
• Test tubes
• Test-tube rack
• Centrifuge, if required
• Timer
• Water bath or incubator, if required
• Personal protective equipment (PPE)

---

Procedure

The G6PD activity is commonly measured by a spectrophotometric method based on the formation of NADPH.

A. Sample Collection

1. Collect 2–3 mL of venous blood in an EDTA tube.
2. Mix the blood gently by inverting the tube several times.
3. Use a fresh sample whenever possible and avoid improper storage.

B. Preparation of Hemolysate

1. Separate the red blood cells according to the laboratory protocol.
2. Wash the RBCs with normal saline if required by the method.
3. Prepare the RBC hemolysate using the recommended hemolysis solution.
4. Mix thoroughly to obtain a uniform hemolysate.

C. Preparation of Reaction Mixture

1. Label two cuvettes as Test and Blank.
2. Add the required amount of phosphate buffer to both cuvettes.
3. Add NADP⁺ and glucose-6-phosphate according to the reagent manufacturer’s instructions.
4. Add the required amount of RBC hemolysate to the Test cuvette.
5. Add the appropriate blank solution to the Blank cuvette.
6. Mix both cuvettes gently.

D. Spectrophotometric Measurement

1. Set the spectrophotometer to 340 nm.
2. Adjust the instrument using the appropriate blank.
3. Place the Test cuvette in the spectrophotometer.
4. Record the initial absorbance.
5. Measure the absorbance again at the specified time intervals, such as every 1 minute.
6. Continue measurements for the period specified by the assay method.
7. Calculate the change in absorbance per minute (ΔA₃₄₀/min).

---

Calculation

The G6PD activity is calculated from the rate of increase in absorbance at 340 nm.

G6PD activity ∝ ΔA₃₄₀/min

The final result is commonly expressed as:

G6PD activity (U/g Hb) = Enzyme activity / Hemoglobin concentration

---