Rheological changes in human red blood cells under oxidative stress

The present study was designed to evaluate the effects of free iron on normal red blood cells (RBCs) and possible mechanisms responsible for the alteration of the RBC rheological properties. Human RBCs (Ht 5%) were incubated for 1 h at 37°C with 0–2 mM FeSO 4 in the presence of ascorbate. Thiobarbituric acid reactive substances assay was employed to estimate lipid peroxidation and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate to analyze membrane proteins. A cone-plate viscometer, and low-shear and high-shear rheoscopes were used to compare the rheological parameters in Fe 2+ /ascorbate-treated RBCs with those of diamide-treated ones. The incubation of RBCs with free iron caused a dose-dependent increase in lipid peroxidation, enhanced binding of hemoglobin to the cell membrane, diminishing of band 3 content and the concomitant increase in lower molecular weight peptides (approximately 60 and 35 kDa), while the incubation with diamide resulted in spectrin crosslinking. Both diamide and Fe 2+ /ascorbate treatment impaired RBC deformability and aggregation. The suspensions of Fe 2+ -treated RBCs manifested increased viscosity, presumably due to enhanced formation of three-dimensional aggregates. In conclusion, although in both these cases of oxidative stress the ultimate result was the deterioration of membrane structure and functions, the mechanisms of oxidative changes of the RBCs rheological properties were apparently dissimilar.