Erythrocyte shape reversion from echinocytes to discocytes: Kinetics via fast‐measurement NMR diffusion‐diffraction

Abstract

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...

Real-time study of shape and thermal fluctuations in the echinocyte transformation of human erythrocytes using defocusing microscopy

“…This last conclusion corroborates the work done in Ref. 9, where an increase of cholinecointaining phospholipid concentration of a shape-recovered RBC after a Mg 2þ depletion was observed, compared with the control, suggesting a change in the membrane lipidic distribution.…”

“…These deformations are produced at a constant volume and the erythrocyte can recover its original form through the removal of the drug from the medium. 3,4,8,9 Drugs effects in RBCs have been studied principally through Electron Microscopy. These observations have led to a significant understanding of the morphologies with a very high concordance with theoretical models.…”

The z-spectrum from human blood at 7T

“…At increased ionic strength (or osmolarity) red blood cells can form echinocytes (or burr cells) losing their discoid shape and gaining small protrusions on their surface, associated with separation of the lipid bilayer from the underlying membrane skeleton ( Liu et al., 1989 ), and similar effects are observed in vivo in various situations where the cells are under stress. Previous work using 1 H NMR suggests that echinocyte formation is associated with changes in the distribution of phospholipids between the two leaflets of the cell membrane ( Pages et al., 2010 ). It has recently been shown that reconstituted egg phospholipids show an NOE -1.7ppm peak ( Zhang et al., 2017a ).…”

Anisotropic diffusion in stretched hydrogels containing erythrocytes: evidence of cell‐shape distortion recorded by PGSE NMR spectroscopy

“…This approach was claimed to require only one transient per free induction decay (FID) to obtain ‘good’ artefact suppression . Pagès et al demonstrated that this experiment could be used to probe reversion of RBC echinocytes to discocytes by monitoring changes in water diffusion every ~5 min . A slightly simplified version of this pulse sequence was also used to measure diffusion in RBC suspensions in order to monitor morphological evolution of discocytes to echinocytes and spherocytes upon metabolic inhibition with NaF; this allowed estimation of the average cell diameter with a time resolution of a few minutes …”