Sustained low micromolar hydrogen peroxide exposure induces sequential red blood cell dysfunction

Red blood cells (RBCs) are continuously exposed to oxidative stress throughout their lifespan and during ex vivo storage. Most experimental oxidative stress models rely on supraphysiological bolus oxidant addition, yet this approach does not replicate the sustained, low-level oxidant exposure encountered physiologically. Understanding the temporal sequence of RBC oxidative injury under physiologically relevant conditions is essential for identifying early biomarkers of dysfunction and developing targeted interventions.

We employed a glucose oxidase (GX)-based system to generate sustained hydrogen peroxide (H₂O₂) at low micromolar concentrations (0.8-8 µM) and examined the temporal progression of oxidative damage in human RBCs over 24 hours. Healthy donor RBCs exposed to GX (1-10 mU/mL) were evaluated for oxidative burden, antioxidant status, hemoglobin oxidation, relative hemoglobin release, membrane integrity/viability by calcein fluorescence, deformability by ektacytometry, phosphatidylserine (PS) externalization by Annexin V binding, and vesiculation by microscopy.

Sustained H₂O₂ exposure induced rapid glutathione depletion (within 6 hours) followed by progressive methemoglobin formation. Single-cell analyses demonstrated a strong inverse relationship between intracellular oxidative autofluorescence and cellular dysfunction. GX induced concentration-dependent impairment of RBC deformability, with 10 mU/mL causing significant membrane rigidity and hemoglobin release indicative of membrane lysis by 24 hours. Time-dependent vesiculation and release of CD235a- and Band 3-labeled microvesicles occurred with vesiculating RBCs exhibiting higher oxidative burden than non-vesiculating cells. Notably, PS externalization was absent on both vesiculating RBCs and their shed microvesicles.

These findings define a temporal hierarchy of oxidative injury under physiologically relevant conditions and demonstrate that vesiculation and PS externalization are mechanistically uncoupled under sustained oxidative stress. This model provides a framework for identifying early biomarkers of RBC dysfunction that may guide the development of targeted interventions to optimize blood storage and mitigate oxidative injury.