Inhibition of sickling after reduction of intracellular hemoglobin concentration with an osmotic pulse: characterization of the density and hemoglobin concentration distributions.

Hemoglobin S polymerization is markedly dependent on intracellular hemoglobin concentration. In the studies presented here, sickle RBC were subjected to a transient osmotic stress, which induced a short period of increased membrane permeability and allowed partial efflux of Hb S. Morphological sickling of the resulting hypochromic RBC was inhibited. The response of RBC to this osmotic pulse is influenced by the presence of a polyanion, which in these experiments was either inositol hexaphosphate (IHP, 27 mM or 46 mM) or pyrophosphate (69 mM or 95 mM). The decrease in MCHC, measured manually, ranged from 3.1 +/- 1.7 (1 SD) to 6.3 +/- 2.8 g/dl, depending on the conditions used during modification. Parallel electronic analysis of RBC indices demonstrated a comparable decrease in MCHC which was due to both an increased MCV and a decreased MCH. Since the modified cell population is quite heterogeneous, cells were analyzed using discontinuous stractan gradients and/or a laser-based instrument which measures the hemoglobin concentration (HC) of individual cells. For most treatment conditions, the modified cells have a bimodal HC distribution with one peak centered at about 20 g/dL and the other peak corresponding to the unmodified cells. With the higher concentration of IHP, however, many cells had an intermediate HC. For modified RBC with a bimodal HC distribution (27 mM IHP, 69 mM PP, 95 mM PP), inhibition of morphological sickling was proportional to the change in HC and there were no subpopulations with an increased tendency to undergo sickling. However, the intermediate density cells present when RBCs were treated with the higher concentration of IHP underwent sickling at a higher oxygen partial pressure than control cells.