Permeability of Human Red Blood Cell Membranes to Hydrogen Peroxide

Abstract

Hydrogen peroxide (H2O2) and other reactive species are important physiological mediators in the vascular system. Enzymatic production of H2O2 is involved in regulating cell growth, proliferation and vasodilation. Whereas endothelial cells are important sources of H2O2, red blood cells (RBC) are considered the most important sinks of H2O2 in the vasculature. However, little is known about the permeability of their membrane to H2O2. The permeability coefficient of human RBC membranes to H2O2 was determined using the enzyme latency method, based on measuring the rate of H2O2 decomposition in lysed vs whole cells. If the passage through the membrane is the rate limiting step in H2O2 decomposition, then a difference is observed that can be used to calculate the permeability coefficient. Additional experiments were done to differentiate between simple diffusion through the lipid fraction and facilitated diffusion through protein channels. The lack of reported permeability coefficients for lipid membranes prompted us to do experiments with phospholipid-cholesterol liposome membranes that indicated that simple diffusion is a slow process. Determination of partition coefficients in different solvents mimicking different depths of the membrane indicate that the low permeability of lipid membranes to H2O2 is caused mainly by its very low solubility in the acyl region of the bilayer. The activation energy of permeation through RBC membranes suggested that protein channels were involved in facilitating H2O2 diffusion through the membrane. Inhibitors of hAQP3 and hAQP1 had no effect in H2O2 consumption rate, suggesting that other membrane proteins may be involved. Although the RBC membrane presents a significant barrier to H2O2 passage, especially in comparison with other solutes such as oxygen and nitric oxide, the permeability is still high enough to support the role of RBC as sinks of H2O2 in circulation.