Hydrogen peroxide diffusion across the red blood cell membrane occurs mainly by simple diffusion through the lipid fraction

Hydrogen peroxide (H₂O₂) is an oxidant produced endogenously by several enzymatic pathways. While it can cause molecular damage, H₂O₂ also plays a role in regulating cell proliferation and survival through redox signaling pathways. In the vascular system, red blood cells (RBCs) are notably efficient at metabolizing H₂O₂. In addition to a robust antioxidant defense, we recently determined that human RBCs also have a high membrane permeability to H₂O₂ that is independent of aquaporin 1 or aquaporin 3. In this work, we sought to further investigate the permeation mechanism of H₂O₂ through the membrane of human RBCs. First, we explored the role of other erythrocytic membrane proteins in H₂O₂ transport, including urea transporter B and ammonia transporter Rh proteins. However, no differences were found in H₂O₂ permeability in RBCs lacking these proteins compared to control RBCs. We then focused on the hypothesis that H₂O₂ diffuses through the lipid bilayer. To test this, we studied H₂O₂ permeability in RBCs from patients with Gaucher disease (GD), which accumulate sphingolipids in the membrane, affecting RBC morphology and deformability. We found that RBCs from GD patients exhibited lower H₂O₂ membrane permeability. In another approach, we treated normal RBCs with hexanol, which fluidizes the lipid fraction of the RBC membrane, and observed an increase in the permeability to H₂O₂. In contrast, hexanol had no effect on the rate of water efflux by aquaporin 1. Together, these results support the hypothesis that H₂O₂ diffusion through the RBC membrane occurs primarily through the lipid fraction.