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

Abstract

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 (DCO2) in RBCs and related our results to cell shape. DCO2 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 DCO2. By imaging the spread of H+ ions from a photochemically-activated source (6-nitroveratraldehyde), DCO2 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 DCO2 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.

Single-cell O 2 exchange imaging shows that cytoplasmic diffusion is a dominant barrier to efficient gas transport in red blood cells

“…4D). To arrive at this value, we applied a previously published method (14) that probes the dissipation of cytoplasmic solute gradients at the subcellular level and therefore, is not sensitive to membrane properties. Given that O 2 and CO 2 have similar diffusive properties in plasma, a substantial resistance to gas transport must reside in the cytoplasm.…”

“…4D). A previously described diffusion-reaction model for RBC H + dynamics estimated D c,CO2 to be 48.9 ± 6.1 μm 2 /s (14), indicating substantially restricted diffusion relative to diffusivity in water (2,500 μm 2 /s). This degree of cytoplasmic obstruction is sufficient to explain slow O 2 unloading without having to implicate another barrier, such as the membrane (Eq.…”

“…In contrast, permeation across the membrane bilayer has been postulated to be rate limiting for gas flows (2), and a parallel pathway in the form of gas channels (8) is considered necessary for efficient gas exchange (9)(10)(11)(12)(13). If, contrary to this paradigm, resistance to gas flow was imposed mainly by the cytoplasm, then gas exchange would be exquisitely sensitive to cell shape (diffusion pathlength) and MCHC (tortuosity imposed by Hb "crowding") (14,15), with major implications for our understanding of RBC disorders. A comprehensive appraisal of gas handling must also consider RBC heterogeneity because many hematological disorders manifest a spectrum of phenotypes among circulating cells.…”

“…, and H + (14). The principle underpinning this technique is that H + ions are unable to diffuse freely in the highly buffered environment of RBC cytoplasm; instead, transport occurs solely aboard buffer molecules (i.e., facilitated diffusion).…”

“…S3). Cytoplasmic CO 2 diffusivity (D c,CO2 ) could thus be calculated by comparing D H app measurements in the presence and absence of CO 2 /HCO 3 − buffer (14,28). Cytoplasmic H + diffusion was triggered by photolytically uncaging H + ions in one region of an RBC from the donor 6-nitroveratraldehyde (NVA; 1 mM) ( Fig.…”