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.
Highlights
The flattened RBC form may facilitate gas exchange in two ways
Restricted cytoplasmic diffusivity can explain the inverse RBC thickness/mean corpuscular hemoglobin concentration (MCHC) relationship observed amongst different animal species, and highlights the potential vulnerability of gas exchange efficiency in diseases that involve a change in RBC shape, such as in spherocytosis
Chemical reactions and membrane transport are recognized to be critically important in determining the rate of gas exchange, and their experimental characterization has informed our current model of RBC physiology
Summary
The flattened RBC form may facilitate gas exchange in two ways. Firstly, it increases the cell’s surface area/volume ratio (ρ), which allows faster membrane transport. The biconcave shape of human RBCs reduces the mean cytoplasmic path-length to 0.9 μm(equal to the cell’s average half-thickness, h), which shortens intracellular diffusion time delays by a factor of seven, compared to a spherical RBC variant (r2/6 ÷h2/2) This seven-fold acceleration will not translate into a meaningful improvement to gas exchange efficiency if gas diffusion coefficients are high, as they are in water. The required experimental approach must be capable of resolving diffusion on the scale of a single intact cell, and exclude contributions from permeation across extracellular unstirred layers and the surface membrane To meet these criteria, we evaluated CO2 diffusivity (DCO2) in intact RBCs by measuring the ability of CO2/HCO3− to facilitate cytoplasmic H+ diffusion[17,18,19]. Restricted cytoplasmic diffusivity can explain the inverse RBC thickness/MCHC relationship observed amongst different animal species, and highlights the potential vulnerability of gas exchange efficiency in diseases that involve a change in RBC shape, such as in spherocytosis
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