Erythrocyte Shape Reversion from Echinocytes to Discocytes: Kinetics Via Fast-measurement NMR Diffusion-diffraction

Pulsed field-gradient spin-echo (PGSE) NMR spectroscopy via q-space plots can characterize erythrocyte shapes and their evolution. The present study employed PGSE NMR to investigate shape reversion from advanced echinocytic to normal discocytic shapes due to depletion and then readdition of Mg(2+). In q-space plots of the data, the diffusion-diffraction minima disappeared for Mg(2+)-depleted erythrocytes and reappeared during the shape recovery process, but with lower definition than for control cells. Shape estimates from PGSE NMR spectra and light microscopy were in excellent agreement after application of a scaling/correction factor. (31)P NMR was used to probe the biochemical processes activated in erythrocytes after depletion or addition of Mg(2+); it showed the activation of the nonoxidative part of the pentose phosphate pathway. Experimental conditions were optimized to bypass this pathway without any influence on the q-space plots. The release of choline from phosphatidylcholine in the outer leaflet of the plasma membrane of the cells, observed using (1)H spin-echo NMR, showed a higher rate for shape-recovered than for control cells. This points to a change in phospholipid asymmetry in the plasma membrane. This variation in asymmetry affected the mean cell shape and hence influenced the average alignment of the erythrocytes with the static magnetic field and so affected the shapes of the q-space plots.