Glycolytic Enzyme Profile in Beta-Thalassemia Major: Coordinated Alterations in Hexokinase, Pyruvate Kinase, and Phosphofructokinase with Implications for Therapeutic Targeting

Background: Mature erythrocytes depend exclusively on glycolysis for ATP production, making glycolytic enzymes critical for red blood cell survival. Beta-thalassemia major is characterized by oxidative stress and metabolic perturbations that may affect glycolytic enzyme function. Objective: To characterize the glycolytic enzyme profile (hexokinase [HK], pyruvate kinase [PK], phosphofructokinase [PFK]) and PFKP gene expression in beta-thalassemia major patients and to evaluate inter-enzyme correlations indicative of coordinated metabolic regulation. Methods: This case-control study included 42 patients with beta-thalassemia major and 30 healthy controls. Serum enzyme levels were measured by ELISA, and PFKP gene expression was assessed using RT-qPCR with the 2⁻ΔΔCt method. Results: Patients demonstrated significantly elevated HK (327.71 ± 282.40 vs. 131.47 ± 158.67 pg/mL; p = 0.0012) and PFK (1.83 ± 1.52 vs. 0.51 ± 0.53 ng/mL; p < 0.0001). A very strong positive correlation existed between HK and PFK (r = 0.874, p < 0.0001), with moderate correlations among all enzymes. PFKP gene expression did not correlate with serum protein levels (r = 0.006, p = 0.968). Conclusion: Beta-thalassemia major exhibits coordinated elevation of glycolytic enzymes, with PFK showing the highest diagnostic potential. The discordance between gene expression and protein levels suggests post-transcriptional regulation. These findings support therapeutic strategies targeting glycolytic metabolism, including pyruvate kinase activators. HIGHLIGHTS Hexokinase and phosphofructokinase are significantly elevated in patients with beta-thalassemia major. A very strong correlation between HK and PFK (r = 0.874) suggests coordinated regulation of glycolysis. All three glycolytic enzymes show significant intercorrelations (r = 0.509–0.874). PFKPgene expression does not correlate with serum PFK protein levels, indicating post-transcriptional control. Findings support the use of pyruvate kinase activators as potential therapeutic agents in thalassemia.