Generation of the High-Na+, Low-K+ Red Blood Cell (RBC) Fraction of Normal and Sickle Cells - A Possible Terminal State.
Authors: Bookchin Robert M. , Etzion Zipora , Daw Nuala , Tiffert Teresa , Lew Virgilio L. 
Published: April 2020

Abstract
We described earlier a small fraction of sickle (SS) and normal RBC that resisted dehydration when K+-permeabilized in low-K+ media with valinomycin (val) or Ca2+ + A23187 (“valres” and “calres”, together “CVres”) (Bookchin et al, PNAS, 97:8045, 2000). Their resistance to dehydration was shown to result from their high-Na+ and low-K+ contents, but the mechanism of generation of these CVres RBCs and their (patho)- physiological relevance were unknown. We noted then that SS-valres RBCs included a large number of ISC shapes, suggesting that their dehydration and/or membrane damage might play a role. We report now that these RBCs also show a relatively high %Hb-F, as measured by HPLC, comparable to Hb F levels in the corresponding denser SS discocyte fractions; this suggested that the oldest SS RBCs, the SS F-cells, were also more likely to become CVres. Franco et al (Blood, 96:3610, 2000) reinfused biotin-labeled high-density-enriched SS RBCs and detected an older population of light, labeled RBCs, supporting our hypothesis that these cells are derived from the dense cell population and represent a terminal state. To test the possible age-related origin of normal calres and valres RBCs, we isolated these cells, now defined as those retaining a density lower than 1.117 after K+-permeabilization, by spinning the val- or Ca2+ + A23187-treated RBCs through diethylphthalate oil (D=1.117 g/ml) to separate the dehydrated cells (pellets) from the CVres cells (on top of the oil). The fraction of RBCs in pellets and on top of the oil was estimated in each sample from Hb measurements, and comprised ~0.2–0.4% (valres) or 1.0–5.0% (calres) of normal RBCs. The higher yield of calres RBCs was attributed to additional net Na+ gain by RBCs during the isolation procedures by activation of the non-selective cation permeability pathway Pcat induced by elevated [Ca2+]i (Bookchin et al, Blood, 104:439a, 2004). Their Hb A1c content was measured by HPLC to serve as a reliable age-marker (in non-diabetic normal RBCs). The fraction of Hb A1c was 7.08 ± 0.25%[SD] for valres and 8.19 ± 0.35% for calres RBCs (total RBC = 5.77 ± 0.25%), consistent with an advanced age of these CVres RBCs. We recently reported that normal RBCs show an age-dependent decline in Ca2+-pump Vmax (Lew et al, Blood, 102:4206, 2003). Together, these finding are consistent with the following mechanisms involved in CVres RBC generation: The age-decline in PMCA activity results in an increasing density of aging RBCs, by allowing intermittent episodes of [Ca2+]i elevations in the circulation, with transient Gardos channel activation and gradual dehydration by net KCl and water loss. The variable increase in [Ca2+]i together with the resulting RBC dehydration then activates Pcat, resulting in Na+ gain and further K+ loss, with a net gain in NaCl and water and cell swelling overtaking the previous dehydrated state. The resulting state of the cells mirrors the features of CVres RBCs. Our recent findings of generation of valres RBCs from normal RBCs following exposure to various oxidants in vitro (Amer et al, BBA, 1760:793, 2006) raises the possibility that oxidant damage to the RBC membranes may play a contributory role to the above age-related functional alterations, and we are currently testing for such effects.