Metabolic Landscapes in Hereditary Hemolytic Anemias: Dynamics and Therapeutic Opportunities

Red blood cells (RBCs) transport oxygen to tissues and remove carbon dioxide produced during oxygen-dependent energy metabolism, supporting systemic energy homeostasis. RBCs have no organelles and depend on anaerobic glycolysis for energy. Because RBCs lack de novo protein synthesis, they rely on coordinated metabolic pathways, including glycolysis, the Rapoport-Luebering shunt, and the pentose phosphate pathway (PPP), to balance energy production, oxygen release, and antioxidant defense. This is critical for RBC function and survival, as RBCs are continuously exposed to oxidative stress during their ~120-day lifespan resulting in progressive protein and membrane loss that reduce deformability. Loss of deformability ultimately leads to splenic clearance. Hereditary hemolytic anemias comprise a heterogeneous group of disorders caused by defects in hemoglobin (hemoglobinopathies), metabolic enzymes (enzymopathies), membrane structure (membranopathies), or ion channels (channelopathies). Emerging evidence implicates metabolic dysregulation as a shared disease mechanism, highlighting metabolic interventions as promising therapeutic strategy. In this thesis, we investigated the metabolic landscapes of hereditary hemolytic anemias using metabolomics and glucose tracing to characterize pathway dynamics and to evaluate potential targeted metabolic.
In Part I, we monitored glucose metabolism through glycolysis and the PPP using glucose tracing. Chapter 2 studied RBCs from patients with hexokinase (HK) or pyruvate kinase (PK) deficiency. HK-deficient RBCs showed reduced carbon-labeling from glucose downstream of HK, whereas flux through the PPP remained unchanged. PK-deficient RBCs accumulated carbon-labeled glycolytic metabolites upstream of PK and increased flux toward the PPP. Our findings reveal disease-specific metabolic signatures and demonstrate the value of ex vivo glucose tracing for studying RBC glucose metabolism. We then studied RBCs from patients with glucose phosphate isomerase (GPI) deficiency in Chapter 3. Reduced GPI activity caused accumulation of glucose-6-phosphate and depletion of downstream glycolytic intermediates, while later steps in glycolysis were largely preserved. Glucose-6-phosphate was diverted into the PPP, partially compensating for the block at GPI, although incomplete carbon-recycling was suggested by erythrose-4-phosphate accumulation. These findings offer relevant insights for future diagnosis and treatment strategies. In Chapter 4, we studied RBCs from patients with hereditary xerocytosis (HX) due to gain-of-function variants in the mechano-sensitive ion channel PIEZO1. We demonstrated increased glucose consumption, accelerated glycolytic flux with enhanced production of carbon-labeled pyruvate and lactate, and increased turnover of PPP metabolites. These findings indicate an overall upregulation of glucose metabolism, suggesting limited benefit from further stimulation through PK activation.
In Part II, we used metabolomics to characterize metabolic alterations in hereditary hemolytic anemias and to evaluate metabolic interventions. In Chapter 5, we analyzed blood from patients with sickle cell disease treated with the PK activator mitapivat in the ESTIMATE study. Mitapivat affected (end-products of) glycolysis, as well as acylcarnitines and nucleotide metabolism, demonstrating the value of metabolomics in assessing metabolic therapies. Chapter 6 examined hereditary spherocytosis, revealing a relative decreased PK activity. Ex vivo PK activation enhanced glycolysis and altered acylcarnitine and nucleotide metabolism. RBC hydration improved, although deformability did not, supporting further investigation. Chapter 7 compared HX caused by defects in either PIEZO1 or KCNN4. PIEZO1-HX RBCs showed greater impairment in PK activity, stability, and protein levels. PK activation increased enzyme activity and ATP in both subtypes but improved hydration only in KCNN4-HX, indicating subtype-specific responses to therapy. Finally, we identified nicotinamide mononucleotide adenylyltransferase 3 (NMNAT3) deficiency as a novel RBC enzymopathy in two siblings in Chapter 8, as indicated by reduced enzymatic activity and altered nicotinamide adenine dinucleotide (NAD) metabolism. Supplementation with NAD precursors improved hemolytic markers, supporting diagnostic screening for NMNAT3 variants and targeted metabolic treatment strategies.
Overall, this work highlights the central role of metabolic dysregulation in hereditary hemolytic anemias and supports the development of targeted metabolic therapies to improve patient outcomes.