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 21 . In q-space plots of the data, the diffusion-diffraction minima disappeared for Mg 21 -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
21; 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 shaperecovered 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 qspace plots. Magn Reson Med 64:645-652, 2010. V C 2010 Wiley-Liss, Inc. Key words: 1 H spin-echo NMR; magnesium depletion; phospholipase D; pulsed-field gradient stimulated-echo NMR; q-space imaging; red blood cell shapeThe discocyte-to-echinocyte-to-spherocyte shape transition of the human erythrocyte (red blood cell; RBC) is emblematic of end-stage differentiation in cells, so it is notable that under special conditions of incubation the process can be reversed (1). Studying this reversion, and time course kinetics provides a different angle on the understanding of the biochemical and cytoskeletal-structural mechanisms underlying the discocyte-to-echinocyte-to-spherocyte shape transition. Another type of reversion has recently been reported for human RBCs, whereby membrane flickering that is inhibited with a sulfhydryl reagent can be reinstated with the membrane intercalating compound, diethyl phthalate (2). The physical techniques used in both studies hold promise for a better (quantitative) description of cell shape dynamics.The asymmetric distribution of lipids across the RBC membrane bilayer is an area of high-research activity (3). In normal human RBCs, the membrane bilayer is composed of amino-phospholipids in the inner monolayer (leaflet) while choline-containing phospholipids reside mostly in the outer leaflet (4). Normal RBCs have a characteristic biconcave disk shape. This is a meta-stable shape that involves the continuous adjustment of the asymmetric transbilayer lipid distribution. A change in the lipid location, and so a variation of the membrane asymmetry, appears to be involved in a change in the cell shap...