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 CO(2) and O(2) 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 CO(2) 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 CO(2)/HCO(3)(−) 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.