Red blood cell thickness is evolutionarily constrained by slow, hemoglobin-restricted diffusion in cytoplasm

During capillary transit, red blood cells (RBCs) must exchange large quantities of CO₂ and O₂ in typically less than one second, but the degree to which this is rate-limited by diffusion through cytoplasm is not known. Gas diffusivity is intuitively assumed to be fast and this would imply that the intracellular path-length, defined by RBC shape, is not a factor that could meaningfully compromise physiology. Here, we evaluated CO₂ diffusivity (D_CO2) in RBCs and related our results to cell shape. D_CO2 inside RBCs was determined by fluorescence imaging of [H⁺] dynamics in cells under superfusion. This method is based on the principle that H⁺ diffusion is facilitated by CO₂/HCO₃^- buffer and thus provides a read-out of D_CO2. By imaging the spread of H⁺ ions from a photochemically-activated source (6-nitroveratraldehyde), D_CO2 in human RBCs was calculated to be only 5% of the rate in water. Measurements on RBCs containing different hemoglobin concentrations demonstrated a halving of D_CO2 with every 75 g/L increase in mean corpuscular hemoglobin concentration (MCHC). Thus, to compensate for highly-restricted cytoplasmic diffusion, RBC thickness must be reduced as appropriate for its MCHC. This can explain the inverse relationship between MCHC and RBC thickness determined from >250 animal species.