Pentose phosphate pathway and G6PD deficiency

Figure 1Pentose phosphate pathway

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Illustration for biochemistry/pentose phosphate pathway and g6pd deficiency from Wikimedia Commons, titled 'Pentose phosphate pathway': Pentose phosphate pathway. The author and licence are given in the credit line.

The PPP is divided into two interlinked branches: an oxidative phase that generates NADPH and a non‑oxidative phase that interconverts sugar phosphates for nucleotide synthesis. ^[2] G6PD (glucose‑6‑phosphate dehydrogenase) is the first and rate‑limiting enzyme of the oxidative phase and acts as a gatekeeper of the pathway. ^[2]

The oxidative phase begins with G6PD converting glucose‑6‑phosphate (G6P) to 6‑phosphoglucono‑δ‑lactone, which is hydrolysed to 6‑phosphogluconate. 6‑Phosphogluconate dehydrogenase (6PGD) then oxidatively decarboxylates it to ribulose‑5‑phosphate (Ru5P), producing two molecules of NADPH per molecule of glucose oxidised. During this phase, NADP⁺ is the electron acceptor in both reactions, so the oxidative phase is the sole source of NADPH in the PPP.
The non‑oxidative phase is catalysed by transketolase (TKT) and transaldolase (TAL), which rearrange sugar phosphates to generate intermediates that can be fed into glycolysis or used for ribose‑5‑phosphate synthesis.
In the parasite Entamoeba histolytica, which lacks G6PDH, 6PGDH and TAL, an alternative hexose‑pentose interconversion pathway involving TKL, FBA and a PPi‑dependent phosphofructokinase supplies pentose phosphates. When exposed to oxidative stress, metabolites of the non‑oxidative branch such as glycerol and chitin biosynthesis are increased, reflecting a shift of carbon flux away from glycolysis and towards the PPP. ^[1]

NADPH produced by the PPP is a critical reducing equivalent for several cellular processes:

• Glutathione regeneration – NADPH donates electrons to glutathione reductase, maintaining reduced glutathione (GSH) that detoxifies reactive oxygen species (ROS). G6PD‑deficient cells are sensitive to oxidative stress because they cannot generate sufficient NADPH for GSH recycling. ^[2]
• Respiratory burst in phagocytes – NADPH oxidase uses NADPH to generate superoxide during the oxidative burst, a key antimicrobial mechanism.
• Drug metabolism – NADPH is required by cytochrome P450 enzymes for the oxidation of xenobiotics. Inhibition of the PPP reduces NADPH availability, impairing drug detoxification and increasing susceptibility to haemolytic drugs in G6PD‑deficient individuals.

The balance of NADP⁺/NADPH is tightly regulated. Elevated ROS or inhibition of NADPH‑generating pathways raises the NADP⁺/NADPH ratio, which in turn activates G6PD and stimulates the PPP to restore redox balance. ^[2]

G6PD deficiency is an X‑linked inherited disorder characterised by markedly reduced G6PD activity in red cells, leading to haemolytic anaemia when exposed to oxidative stressors such as certain drugs or infections. The deficiency must be considered in the differential diagnosis of haemolytic anaemia and when prescribing potentially haemolytic drugs. In areas with high prevalence, G6PD testing is essential before administering drugs like primaquine or dapsone. ^[3]

The clinical spectrum ranges from asymptomatic mild anaemia to severe, life‑threatening acute haemolytic anaemia (AHA) when red cells are exposed to oxidative stress. The severity of haemolysis is influenced by the specific G6PD variant and the proportion of deficient red cells. For example, the G6PD Coimbra and G6PD Mediterranean variants tend to produce more frequent and severe episodes of AHA than variants with higher mean activity such as G6PD Seattle and G6PD Kalyan‑Kerala. In a heterozygous female, the G6PD Seattle variant can cause ^[1]

The X‑linked inheritance pattern means that males have a single allele; a pathogenic mutation results in all red cells being deficient. Females may be homozygous, heterozygous or mosaic due to X‑chromosome inactivation. Heterozygous females therefore harbour a mixed population of normal and deficient red cells, with a variable ratio that can range widely around an average of 50:50. The number of known mutations exceeds 187, and at least 35 are common polymorphisms worldwide. None of these mutations causes complete loss of G6PD, which would be lethal; instead, they reduce enzyme stability or catalytic efficiency, accelerating the normal decline of activity as red cells age.

The residual activity of the enzyme is a key determinant of clinical risk. For instance, G6PD Mediterranean retains only about 5 % of normal activity, whereas G6PD A‑ retains about 13 %. However, the relationship between residual activity and haemolytic severity is not linear, as qualitative differences in the enzyme also play a role. ^[3]

Oxidative triggers include certain drugs, infections, and foods. Primaquine and other antimalarials are classic examples; they can precipitate AHA in G6PD‑deficient individuals. The risk of drug‑induced haemolysis persists because many clinicians still prescribe these agents without prior G6PD screening, especially in high‑prevalence populations. Other drugs such as rasburicase and dapsone have also been implicated. In a study of dapsone, 10 % of patients developed AHA, but all affected individuals were G6PD deficient, indicating that the risk in G6PD‑normal patients is essentially zero while it is near 100 % in deficient patients. ^[3]

Infections can generate reactive oxygen species that overwhelm the compromised PPP, leading to oxidative damage of haemoglobin. The resulting Heinz bodies are phagocytosed by splenic macrophages, contributing to extravascular haemolysis. The presence of Heinz bodies on a smear is therefore a diagnostic clue to G6PD‑related oxidative haemolysis.

The pathophysiology of drug‑induced haemolysis involves inhibition of G6PD activity or generation of oxidants that exceed the capacity of the PPP to produce NADPH, the reducing equivalent required for glutathione regeneration. Without sufficient NADPH, red cells cannot detoxify peroxides, leading to oxidative damage, Heinz body formation, and ultimately haemolysis.

Chronic granulomatous disease is caused by mutations in genes encoding subunits of the NADPH oxidase complex. The enzyme complex normally transfers electrons from NADPH to molecular oxygen, generating superoxide anion, a key microbicidal oxidant. In CGD, this electron transfer is defective, so phagocytes cannot produce sufficient reactive oxygen species to kill ingested pathogens. Consequently, patients develop granulomatous inflammation and are prone to infections with catalase‑positive organisms such as Staphylococcus aureus, Streptococcus spp., Nocardia, Listeria and Candida.

The clinical presentation is highly variable. Early childhood is the most common period for diagnosis, with recurrent bacterial and fungal infections, especially in the lungs, skin, lymph nodes and liver. Granulomas may form in almost any organ, leading to complications such as obstructive airway disease, intestinal strictures or hepatic fibrosis. Diagnosis is confirmed by functional assays of the respiratory burst (e.g., nitroblue tetrazolium test or dihydrorhodamine flow cytometry) and genetic testing for mutations in the CYBB, CYBA, NCF1, NCF2 or NCF4 genes.

Management of CGD involves prophylactic antibiotics (trimethoprim‑sulfamethoxazole and fluconazole) and antifungals (voriconazole or posaconazole), interferon‑γ to boost innate immunity, and aggressive treatment of infections. Hematopoietic stem cell transplantation offers a potential cure, particularly in patients with severe disease or refractory infections. Gene therapy is an emerging approach, with early trials demonstrating durable correction of the NADPH oxidase defect.

The PPP is vital for cells that require large amounts of NADPH, such as erythrocytes, hepatocytes and rapidly proliferating tumour cells. In erythrocytes, NADPH maintains glutathione in its reduced form, protecting the cell from oxidative damage. In tumour biology, increased PPP flux supports anabolic growth and is often driven by oncogenic mutations; G6PD is overexpressed in many cancers, including diffuse large B‑cell lymphoma.

Clinical relevance of G6PD deficiency

1. Drug safety
   Certain drugs (e.g., rasburicase, primaquine, dapsone) can precipitate haemolysis in G6PD‑deficient individuals. A G6PD test is recommended before prescribing these agents, especially in regions where the deficiency is common. In the context of tumour lysis syndrome, rasburicase is contraindicated in patients with a history of G6PD deficiency, but the recommendation should be to test before administration rather than rely on history alone.

2. Malaria treatment
   Primaquine is essential for radical cure of Plasmodium vivax and for gametocytocidal activity against P. falciparum. WHO recommends G6PD testing before primaquine use, yet this is rarely performed in endemic areas. The lack of testing often leads to withholding primaquine, which hampers malaria elimination efforts. ^[3]

3. Diagnostic strategy
   In acute haemolytic anaemia, a normal G6PD result may be misleading; a repeat test after the acute episode has resolved is advised to avoid misclassification. DNA‑based testing is suitable when a known mutation is identified; otherwise, phenotypic assays are mandatory. ^[3]