No abstract

W e have established that 2.3-diphosphoglycerate (2.3-DPG) content and intracellular pH exert separate, but interdependent, effects on the equilibrium solubility (cSJ of deoxyhemoglobin S (deoxy-Hb S) that act in concert to modulate intraerythrocytic polymer formation. In a nonphysiologic c,,~ assay system, a steep dependence of csat on pH in the physiologic range 7.0 to 7.6 was shown for both stripped (Hb) and DPG-saturated deoxy-Hb S (Hb-DPG). The solubility-pH profile for Hb under near-physiologic buffer conditions also showed that C,,~ increased steeply in the same pH range (6.8 to 7.6). The effect of 2.3-DPG on c, , under near-physiologic conditions was evaluated separately. At pH 7.20, the pH of the human red blood cell, c, , values for Hb and Hb-DPG were 19.56 * 0.14 and 17.95 * 0.45 g /dL, respectively, indicating that the solubility of HE ROLE OF red blood cell (RBC) 2,3-diphosphogly-T cerate (2,3-DPG) in the pathophysiology of sickle cell disease has been a point of long-standing dispute. Some workers claim that 2,3-DPG promotes the intracellular polymerization of deoxyhemoglobin S (deoxy-Hb S) that underlies the sickling phenomenon,'-6 whereas others assert that it has no such effect.'-" We recently reexamined this issue with the intention of establishing definitively whether or not 2,3-DPG affects the equilibrium solubility (c,,,) of deoxy-Hb S. Although our initial findingsI2 showed that the binding of 2,3-DPG resulted in a significant decrease in c,,~, the decrement in solubility observed in the physiologic pH range (7.0 to 7.6) was somewhat lower (--1.8 g/dL) than that observed at acid pH (-3.0 g/dL), a reflection of the diminished strength of binding of the allosteric effector a t the elevated pH.I3A more conclusive demonstration of the role of 2,3-DPG in modulating the solubility of deoxy-Hb S was achieved by using a cross-linked derivative of H b S in which the 2,3-DPG binding site was effectively blocked by acylation of the two Lys 82p r e s i d~e s . '~. '~ Under assay conditions in which unmodified deoxy-Hb S showed a substantial decrease in cSaI in the presence of an equimolar amount of 2,3-DPG, the solubility of the cross-linked derivative was unaffected. This finding establishes unequivocally that binding of 2,3-DPG The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with IS U.S.C. section I734 solely to indicate this fact. 0 I990 by The American Society of Hematology. 0006-4971/90/7605-00I 9$3.00/0 Hb-DPG is lower than that of Hb by 8.2% k 2.3%. Thus, binding of 2.3-DPG in the @-cleft promotes the polymerization of deoxy-Hb S, the ultimate determinant of cell sickling. Furthermore, because of the abnormal Bohr effect of sickle blood (approximately double that of normal blood), the intracellular pH of deoxygenated sickle erythrocytes should be -0.28 pH unit higher than that of oxygenated cells (ie, 7.41 v 7.13). At the higher pH. the corresponding c, , for Hb-DPG is 20.22 g/dL, which ...

Recent work has enabled us to quantitate the four variables (2,3-DPG concentration, pHi, non-S hemoglobin composition, and O2 saturation) that modulate the equilibrium solubility (csat) of Hb S inside sickle erythrocytes (SS RBCs). Using measured values of mean corpuscular hemoglobin concentration (MCHC), 2,3-DPG concentration, and %Hb (F+A2), along with estimates of pHiand the Δcsat due to partial oxygenation of SS RBCs in the microcirculation, we calculated the mean polymer fraction (fp) in erythrocytes from 46 SS homozygotes. Values of fp derived from the conservation of mass equation ranged from 0.30 to 0.59. MCHC and %Hb F were major determinants of the magnitude of fp; 2,3-DPG concentration and pHialso contributed, but to a lesser extent. A clinical severity score (CSS) was assigned to each patient based on mean hospitalization rate. There was a weak, but statistically significant, negative correlation between fp and steady state hematocrit (P = .017), but none between fp and whole blood hemoglobin concentration (P = .218). Although there was no correlation between fp and mean number of hospitalization days per year, patients with the greatest number of admissions and hospitalization days were found only among those who had an fp > 0.45. All five patients who died during the follow-up period (median, 7 years; range, 3 to 10 years) had fp values ≥0.48. However, patients with few admissions, low hospitalization days, and long survivals occurred at all fp levels. These results suggest that the clinical course of homozygous SS disease cannot be predicted by mean fpcalculations, which assume a homogeneous distribution of the five variables that modulate intraerythrocytic polymerization. A heterogeneous distribution is more likely; so the amount of polymerized Hb S could vary considerably among cell populations. Factors such as membrane abnormalities and endothelial cell interactions may also contribute to clinical severity.

Astronauts experience severe/invasive disorders caused by space environments. These include hematological and cardiac abnormalities, bone and muscle losses, immunodeficiency, neurological disorders and cancer. Exploiting the extraordinary plasticity of hematopoietic stem cells (HSCs), which differentiate not only to all types of blood cells, but also to various tissues, including muscle, bone, skin, liver, and neuronal cells, we advanced a hypothesis that some of the space-caused disorders might be amenable to hematopoietic stem cell therapy (HSCT) so as to maintain astronauts' homeostasis. If this were achievable, the HSCT could promote human exploration of deep space. Using mouse models of human anemia (β-thalassemia) and spaceflight (hindlimb suspension unloading system), we have obtained feasibility results of HSCT for space anemia, muscle loss, and immunodeficiency. For example, the β-thalassemic mice were successfully transplanted with isologous HSCs, resulting in chimerism of hemoglobin species and alleviation of the hemoglobinopathy. In the case of HSCT for muscle loss, β-galactosidase-marked HSCs, which were prepared from βgalactosidase-transgenic mice, were detected by the X-gal wholemount staining procedure in the hindlimbs of unloaded mice following transplantation. Histochemical and physical analyses indicated structural contribution of HSCs to the muscle. To investigate HSCT for immunodeficiency, β-galactosidase-transformed Escherichia coli was used as the reporter bacteria, and infected to control and the hindlimb suspended mice. Results of the X-gal stained tissues indicated that the HSCT could help eliminate the E. coli infection. In an effort to facilitate the HSCT in space, growth of HSCs has been optimized in the NASA Rotating Wall Vessel (RWV) culture systems, including Hydrodynamic Focusing Bioreactor (HFB).

The effects of various tetrasubstituted ammonium compounds on the solubility of deoxygenated sickle hemoglobin were evaluated. These conclusions were drawn from the slopes of the solubility profiles obtained: (1) for the homologous series of tetraalkylammonium chloride salts (R4NCl), antigelling potency increased with increasing chain length of the R group: Ch3 less than C2H5 less than C3H7 less than C4H9; (2) for halide salts of the tetrabutylammonium cation there was no difference in effectiveness among the anions examined (Br-, Cl-, F-), presumably because of the extremely potent salting-in effect of this cation; (3) substitution of a benzyl for an alkyl group in three tetraalkylammonium chloride salts, C6H5CH2(R)3NCl, where R = CH3, C2H5, or C4H9, potentiated the antigelling capacity by as much as eight-fold. Because they are substantially more water soluble than any other class of noncovalent inhibitors examined to date, further manipulation of the aryl substituent on these compounds may contribute to the design of an effective antisickling agent.