Water chemical shift in ¹H NMR of red cells: Effects of pH when transmembrane magnetic susceptibility differences are low

Abstract

Two resonances are observed for 1 H 2 O in magic angle spinning (MAS) NMR spectra of erythrocyte (red blood cell; RBC) and other cellular suspensions (1-3). The signals are separated by Ϸ18 Hz (Fig. 1), with the broad resonance assigned to intracellular water and the sharp resonance to extracellular water. As the mean residence time for a water molecule inside an RBC is Ϸ10 ms (4), the exchange of water between the two pools is fast on the NMR time scale and a single exchange-averaged peak for water is expected. The explanation for the observed splitting of the water resonance is the separation of the suspension into a system composed of two concentric cylinders inside the MAS rotor as a result of spinning the sample (5). The outer cylinder comprises tightly packed cells plus some interstitial water, and the inner cylinder is cell-free water, or by analogy to centrifugation, the supernatant. The interstitial water (normally Ϸ3%) does not contribute significantly to the observed spectrum and is not considered in this article. As a result of the physical separation of the two water pools, the exchange of water between the supernatant and cells is essentially eliminated, giving rise to separate water signals from each compartment. Bruno et al. (6) have shown that by immobilizing the RBCs in a gel matrix the centrifugation of the cells can be prevented and a single exchange averaged peak is observed for water in 1 H NMR spectra.For cylindrical samples oriented at the magic angle there is no contribution to the splitting of the water peak from a magnetic susceptibility difference between the cells and the suspension medium (3,7). Separate intracellular and supernatant resonances are also seen in 31 P spectra of phosphoryl compounds in RBC suspensions (8), where it has been shown that the separation is brought about through differences in the extent of hydrogen bonding to the phosphoryl oxygen due to the high protein concentration in RBCs, the so-called "hydrogen-bond split-peak effect." In the case of water in MAS experiments, the "splitting" also represents a true chemical shift difference between water in the two compartments, brought about by differences in the extent of hydrogen bonding of water, with the magnitude of the separation being dependent on the intracellular protein concentration, and not the pH of the suspension medium (2,9).Our aim here is to present a model for the chemical shift of water for static samples in a high-resolution NMR tube, namely, one that is aligned with the magnetic field, under conditions of low transmembrane magnetic susceptibility difference. Such samples are those that have not had paramagnetic shift and/or relaxation reagents (e.g., containing Mn 2ϩ , or Dy 3ϩ complexes) added, or made paramagnetic by conversion of hemoglobin to methemoglobin, or where the deoxyhemoglobin concentration is elevated, increasing the magnetic susceptibility (8). Our experiments were carried out so that the pH or osmolality of the suspension buffer was changed to give a range of intracellular protei...

In-cell NMR: Why and how?

“…enucleated) generate chemical shift differences of 0.1-0.2 ppm (Fig. 26) [1566][1567][1568] that vary in a linear fashion with the intracellular hemoglobin concentration.…”

“…Turning to consideration of the water, protein solvation has consequences on water chemical shift and relaxation. The contribution of water-protein exchange has been measured to be about 0.2 parts-per-billion (ppb) per g/L of protein [1567,1570,1620,1621]. To calculate the effective water frequency in protein solution, this exchange contribution has to be combined with the effects of protein magnetic susceptibility, which is of the opposite sign and about twice as large.…”

Spatial reorientation experiments for NMR of solids and partially oriented liquids

“…The technique was also extended to the study of metabolites in liver and muscle tissue, where the linewidth is also dominated by magnetic susceptibility broadening [108]. A similar approach, based on VAS, was employed to separate the contributions of chemical shift and magnetic susceptibility differences to the separate signals observed for intracellular and extracellular water protons in suspensions of erythrocytes [109], with the conclusion that the chemical shift variations often observed in medical MRI are primarily due to magnetic susceptibility effects [110].…”