pH and cell volume effects on H2O and phosphoryl resonance splitting in rapid-spinning NMR of red cells

Two resonances are seen in the ¹H-NMR spectrum of water in erythrocyte suspensions spun at the magic angle, a broad signal from water inside the cells and a sharp signal from extracellular water. The splitting is a result of a true chemical shift difference between the two populations, as bulk magnetic susceptibility effects are negated at the magic angle. The pH dependence of this chemical shift difference in erythrocyte suspensions was investigated. Splittings of 16.7 ± 0.1, 18.9 ± 0.9, and 21.0 ± 0.2 Hz were observed at pH 6.0, 7.0, and 8.5, respectively; however, this was accompanied by a change in the mean cell volume. To account for any contribution from the volume change, the osmolality of the pH 6.0 and 8.5 suspensions was adjusted to equalize the cell volume between samples at the three pHs. Under these conditions, the splitting was 18.3 ± 0.1 and 18.6 ± 0.1 Hz at pH 6.0 and 8.5, respectively. Thus the observed chemical shift difference between the two water resonances was independent of pH. Therefore the splitting of the water resonance was concluded to be directly proportional to the protein concentration within the cell. Measurements of the magnetic susceptibility difference between the two compartments were also carried out, yielding a value of 2.0 ± 0.2 × 10⁻⁷ (SI units) for erythrocytes in isotonic saline at pH 7.0.