Applied Chemical Engineering

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

Karima Jalil Hamza

Department of Chemistry, College of Science, University of Baghdad, Baghdad, Iraq

Maysoon Khalid Hussein

Department of Chemistry, College of Science, University of Baghdad, Baghdad, Iraq

DOI: https://doi.org/10.59429/ace.v9i1.5885

Keywords: Beta-thalassemia major; Glycolytic enzymes; Hexokinase; Pyruvate kinase; Phosphofructokinase; PFKP gene expression; Erythrocyte metabolism; Pyruvate kinase activators; Biomarkers

---

Abstract

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.

---

References

[1]. Van Wijk R, van Solinge WW. The energy-less red blood cell is lost: erythrocyte enzyme abnormalities of glycolysis. Blood 2005;106(13):4034-4042.

[2]. Beutler E. Red cell metabolism: a manual of biochemical methods. 3rd ed. Grune & Stratton; 1984.

[3]. Grace RF, Zanella A, Neufeld EJ, Morton DH, Eber S, Yaish H, Glader B. Erythrocyte pyruvate kinase deficiency: 2015 status report. Am J Hematol. 2015;90(9):825-830.

[4]. Mor I, Cheung EC, Vousden KH. Control of glycolysis through regulation of PFK1. Cold Spring Harb Symp Quant Biol 2011;76:211-216.

[5]. Rivella S. Iron metabolism under conditions of ineffective erythropoiesis in β-thalassemia. Blood 2019;133(1):51-58.

[6]. Vinchi F, Muckenthaler MU, Da Silva MC, Balla G, Balla J, Jeney V. Atherogenesis and iron: from epidemiology to cellular level. Front Pharmacol. 2014;5:94.

[7]. De Franceschi L, Bertoldi M, De Falco L, Santos Franco S, Ronzoni L, Turrini F, Colancecco A, Camaschella C, Cappellini MD, Iolascon A. Oxidative stress modulates heme synthesis and induces peroxiredoxin-2 as a novel cytoprotective response in β-thalassemic erythropoiesis. Antioxid Redox Signal. 2011;14:1097-1108.

[8]. Matte A, Federti E, Kung C, Kosinski PA, Narayanaswamy R, Russo R, Federico G, Carlomagno F, Desbats MA, Salviati L, Leboeuf C, Valenti MT, Turrini F, Janin A, Yu S, Beneduce E, Ronseaux S, Iatcenko I, Dang L, Ganz T, Jung CL, Iolascon A, Brugnara C, De Franceschi L. The pyruvate kinase activator mitapivat reduces hemolysis and improves anemia in a β-thalassemia mouse model. J Clin Invest. 2021;131:e144206.

[9]. Kuo KHM, Layton DM, Lal A, Al-Samkari H, Bhatia J, Kosinski PA, Tong B, Lynch M, Uhlig K, Vichinsky EP. Safety and efficacy of mitapivat, an oral pyruvate kinase activator, in adults with non-transfusion dependent alpha-thalassaemia or beta-thalassaemia: an open-label, multicentre, phase 2 study. Lancet. 2022;400(10351):493-50.

[10]. Mutlag SS, Alyasiri QK, Hussein AJ. Causes and clinical management of abdominal pain in pediatric patients. Adolescencia e Saude 2025;20(2):15-23.

[11]. Sahib HS, Mohsin RK, Mutlag SS. Analyzing the issue of overweight and children health. Revista Latinoamericana de Hipertension 2021;15(3):260-266.

[12]. Mutlag SS. The association between iron over load and tanner stage retardation in the females with b-thalassemia major. International Journal of Research in Pharmaceutical Sciences 2020;11(1):546-552.

[13]. Hashim ZA, Mutlag SS. Ophthalmologic changes in thalassemia patients. Journal of Pharmaceutical Sciences and Research 2017;9(11):2237-2239.

[14]. Beutler E. Red cell enzyme defects as nondiseases and as diseases. Blood 1979;54:1-7.

[15]. Valentine WN, Tanaka KR, Paglia DE. Hemolytic anemias and erythrocyte enzymopathies. Ann Intern Med 1985;103:245-257.

[16]. Taher AT, Weatherall DJ, Cappellini MD. Thalassaemia. Lancet 2018;391:155-167.

[17]. Fibach E, Rachmilewitz EA. Pathophysiology and treatment of patients with beta-thalassemia. Exp Hematol 2017;52:1-6.

[18]. Kattamis A, Forni GL, Aydinok Y, Viprakasit V. Changing patterns in epidemiology of β-thalassemia. Eur J Haematol 2020;105:692-703.

[19]. Semenza GL. HIF-1: upstream and downstream of cancer metabolism. Curr Opin Genet Dev 2010;20:51-56.

[20]. Tannahill GM, Curtis AM, Adamik J, Palsson-McDermott EM, McGettrick AF, Goel G, Frezza C, Bernard NJ, Kelly B, Foley NH, Zheng L, Gardet A, Tong Z, Jany SS, Corr SC, Haneklaus M, Caffrey BE, Pierce K, Walmsley S, Beasley FC, Cummins E, Nizet V, Whyte M, Taylor CT, Lin H, Masters SL, Gottlieb E, Kelly VP, Clish C, Auron PE, Xavier RJ, O'Neill LAJ. Succinate is an inflammatory signal that induces IL-1β through HIF-1α. Nature. 2013;496(7444):238-242.

[21]. O'Neill LAJ, Kishton RJ, Rathmell J. A guide to immunometabolism for immunologists. Nat Rev Immunol 2016;16:553-565.

[22]. Kelly B, O'Neill LAJ. Metabolic reprogramming in macrophages and dendritic cells. Cell Res 2015;25:771-784.

[23]. Vogel C, Marcotte EM. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat Rev Genet 2012;13:227-232.

[24]. Liu Y, Beyer A, Aebersold R. On the dependency of cellular protein levels on mRNA abundance. Cell 2016;165:535-550.

[25]. Schwanhäusser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, Chen W, Selbach M. Global quantification of mammalian gene expression control. Nature. 2011;473(7347):337-342.

[26]. Maier T, Güell M, Serrano L. Correlation of mRNA and protein in complex biological samples. FEBS Lett 2009;583:3966-3973.

[27]. Kung C, Hixon J, Kosinski PA, Cianchetta G, Histen G, Chen Y, Hill C, Gross S, Si Y, Johnson K, DeLaBarre B, Luo Z, Gu Z, Yao G, Tang H, Fang C, Xu Y, Lv X, Biller S, Su SM, Yang H, Popovici-Muller J, Salituro F, Silverman L, Dang L. AG-348 enhances pyruvate kinase activity in red blood cells from patients with pyruvate kinase deficiency. Blood. 2017;130(11):1347-1356.

[28]. Grace RF, Rose C, Layton DM, Galactéros F, Barcellini W, Morton DH, van Beers EJ, Yaish H, Ravindranath Y, Kuo KHM, Sheth S, Kwiatkowski JL, Barbier AJ, Bodie S, Silver B, Hua L, Kung C, Hawkins P, Jouvin MH, Bowden C, Glader B. Safety and efficacy of mitapivat in pyruvate kinase deficiency. N Engl J Med. 2019;381(10):933-944.

[29]. Al-Samkari H, Galactéros F, Glenthøj A, Rothman JA, Andres O, Grace RF, Morado-Arias M, Layton DM, Onodera K, Verhovsek M, Barcellini W, Chonat S, Judge MP, Zagadailov E, Xu R, Hawkins P, Beynon V, Gheuens S, van Beers EJ. Mitapivat versus placebo for pyruvate kinase deficiency. N Engl J Med. 2022;386(15):1432-1442.

[30]. van Zwieten R, Verhoeven AJ, Roos D. Inborn defects in the antioxidant systems of human red blood cells. Free Radic Biol Med 2014;67:377-386.

[31]. Koralkova P, van Solinge WW, van Wijk R. Rare hereditary red blood cell enzymopathies associated with hemolytic anemia. Int J Lab Hematol 2014;36:338-352.

[32]. Sahib HS, Mohsin RK, Mutlag SS. The burden of obesity on children health, its correlation to their age. Revista Latinoamericana de Hipertension 2021;15(2):151-157.

[33]. Sahib HS, Mohsin RK, Mutlag SS. Assessment the concentration of malondialdehyde MDA and polyubiquitin c protein in patients with breast and gastric cancer. Revista Latinoamericana de Hipertension 2021;15(3):232-238.

[34]. Mutlag SS. Metabolic acidosis impairs clearance of upec-uti. Research Journal of Pharmacy and Technology 2020;13(1):330-334.

[35]. AL-SARRAY A, El-YASSIN H, JAWAD A. The Prime Mutation of β-Thalassemia and Disease Severity in (Wasit and Maysan) South of Iraq. J Nat Sc Biol Med 2023;14:221-224.

[36]. Hamed OM, Al-Taii RA, Jankeer MH. Biochemical and Genetic Study in Blood of β-Thalassaemia Children in Mosul City, Iraq. Iraqi Journal of Science 2021;62:2501-2508.

[37]. Al-Husseiny IA, Al-Jumaili EF. Comparative Analysis of the Age, Gender, and Ferritin Levels with Hepcidin in Iraqi Patients Suffering Beta Thalassemia (Major and Intermedia). HAYATI J Biosci 2025;32:1518-1528.

[38]. Majeed MS. Evaluation of some Biochemical and Endocrine Profiles in transfusion-dependent Iraqi major β-thalassemia patients. Iraqi Journal of Science 2017;58:639-645.