A 28-year-old male presents with recurrent infections, jaundice, and dark urine after consuming fava beans. Clinical examination reveals splenomegaly. Hemoglobin is 9.2 g/dL with reticulocytosis. Which investigation is most appropriate to confirm the underlying enzymatic defect in the pentose phosphate pathway?

## Diagnosis: G6PD Deficiency **Key Point:** G6PD deficiency is the most common enzyme defect of the pentose phosphate pathway (PPP), affecting ~400 million people worldwide. The clinical presentation of hemolytic anemia triggered by oxidative stress (fava beans, infections, sulfonamides) is pathognomonic. ### Why G6PD Activity Assay is Diagnostic G6PD catalyzes the first committed step of the PPP: $$\text{Glucose-6-phosphate} \xrightarrow{\text{G6PD}} \text{6-phosphogluconolactone}$$ This reaction generates NADPH, which is essential for: - Glutathione reduction (antioxidant defense) - Protection against oxidative stress in RBCs **High-Yield:** Direct measurement of G6PD enzyme activity in RBCs is the gold standard confirmatory test. It quantifies the enzymatic deficiency and correlates with clinical severity. ### Pentose Phosphate Pathway Context | Enzyme | Function | Deficiency Consequence | |--------|----------|----------------------| | G6PD | Generates NADPH | ↓ Glutathione reduction → oxidative hemolysis | | 6-PGD | Continues NADPH generation | Rare; milder phenotype | | TK (Transketolase) | Non-oxidative phase | Wernicke-Korsakoff (thiamine deficiency) | **Clinical Pearl:** G6PD activity is often falsely normal during acute hemolytic episodes (young RBCs with higher enzyme levels predominate). Retest 2–4 weeks after recovery for accurate diagnosis. **Mnemonic:** **NADPH** — the critical product of PPP. G6PD deficiency → ↓ NADPH → ↓ reduced glutathione → RBC oxidative damage.