Decoding the metabolic landscape of pathophysiological stress-induced cell death in anucleate red blood cells

In response to stress, anucleate red blood cells (RBCs) can undergo a process of atypical cell death characterised by intracellular Ca²⁺ accumulation and phosphatidylserine (PS) externalisation. Here we studied alterations in RBC metabolism, a critical contributor to their capacity to survive environmental challenges, during this process.

Metabolomics analyses of RBCs and supernatants, using ultra-high-pressure liquid chromatography coupled to mass spectrometry, were performed after in vitro exposure of RBCs to different pathophysiological cell stressors, including starvation, extracellular hypertonicity, hyperthermia, and supraphysiological ionic stress. Cell death was examined by flow cytometry.

Our data show that artificially enhancing RBC cytosolic Ca²⁺ influx significantly enhanced purine oxidation and strongly affected cellular bioenergetics by reducing glycolysis. Depleting extracellular Ca²⁺ curtailed starvation-induced cell death, an effect paralleled by the activation of compensatory pathways such as the pentose phosphate pathway, carboxylic acid metabolism, increased pyruvate to lactate ratios (methemoglobin reductase activation), one-carbon metabolism (protein-damage repair) and glutathione synthesis; RBCs exposed to hypertonic shock displayed a similar metabolic profile. Furthermore, cell stress promoted lipid remodelling as reflected by the levels of free fatty acids, acyl-carnitines and CoA precursors. Notably, RBC PS exposure, independently of the stressor, showed significant correlation with the levels of free fatty acids, glutamate, cystine, spermidine, tryptophan, 5-oxoproline, lactate, and hypoxanthine.

In conclusion, different cell death-inducing pathophysiological stressors, encountered in various clinical conditions, result in differential RBC metabolic phenotypes, only partly explained by intracellular Ca²⁺ levels and ATP availability.