Recent interest in therapies for sickle cell anemia based on elevating fetal Hb has made accurate estimates of the sparing effect offetal Hb (Hb F) and other non-sickle Hbs on sickle Hb (Hb S) polymerization essential. We have developed a technique, using HbCO as surrogate for HbO2, that enables us to assess the solubility of Hb S as a function of ligand saturation under conditions that mimic those of the sickling disorders. Equimolar mixtures of unliganded Hb S with Hb F or normal Hb A2 were isosoluble. Solubilities for equimolar mixtures with normal (Hb A) or abnormal (Hb C) Hbs were also identical but were lower than in the prior case. Thus, the sparing effect of both Hb F and Hb A2 should be considered in therapeutic strategies designed to modify Hb S polymerization. Hemolysates, stripped of 2,3-bisphosphoglycerate, from sickle cell disease patients with Hb (F + A2) levels varying from 6 to 25%, as well as from a sickle trait individual, were used to evaluate equilibrium solubiity as a function of ligand saturation over the range of pathophysiologic interest (25-70%). Our results show that the sparing effect of Hb (F + A2) increases relative to that of Hb A as ligand saturation increases, and that in the absence of ligand, '30% Hb (F + A2) is essentially isosoluble with the 60% Hb A of sickle trait. Although detailed knowledge of expected therapeutic benefits is confounded by the heterogeneity of Hb F distribution and other variables, these data should provide a framework for estimating likely clinical benefit from pharmacologic efforts to modulate globin gene expression.Sickle cell anemia exists in individuals who are homozygous for a point mutation (GAG --GTG) at codon 6 of the 3-globin gene, which results in a Glu-6 -* Val substitution in P-globin. are, thus, said to have a "sparing" effect on intracellular Hb S polymerization (3, 4). For Hbs F and A2, this solubilization can be described well with a model that assumes complete exclusion of either the homotetramer (a2-y2 or a2&2) or the hybrid heterotetramer (a238y or a2138) from the nascent polymer (5-7). For Hbs A and C, the data are compatible with exclusion of only homotetramers (a2I3A and a2K) from the polymer. Hybrid heterotetrameric forms (a2f3Sf3A and a2138S,c) appear to be incorporated into the polymer, but with a copolymerization probability roughly one-half that of Hb S (8-10). These copolymerization tendencies explain why the solubility enhancement induced by Hb F or A2 in admixture with Hb S is greater than that for Hb A or C.The solubilizing properties of various non-S Hbs have been established by use of a chemical reducing agent (dithionite) to completely deoxygenate mixtures of oxy-Hb S and the oxy form of other hemoglobins (Hb X), followed by centrifugation to fractionate the resulting semisolid gel into a tightly packed polymer pellet and a supernatant consisting of soluble Hb S and Hb X. The concentration of all Hb species in the supernatant is by definition the saturation concentration (csat), or equilibrium solubility, under g...

We report the hematologic and clinical features of four adult patients (Pts.) with sickle cell anemia and iron-limited erythropoiesis. Two of the Pts. had spontaneous iron deficiency (chronic GI bleeding, low-grade hemoglobinuria). In the other two Pts. iron restriction was induced by periodic RBC aphereses as part of a pilot protocol designed to decrease intracellular HbS polymerization by MCHC reduction. Iron-limited erythropoiesis was defined by reduction in red cell indices (MCV range 60.4-67 fl) in the presence of low serum ferritin (range < 10-20 ng/ml). In these Pts. iron restriction did not cause clinically significant worsening of the anemia (Hb 7.8-9.0 g/dl). In two Pts. the anemia actually improved. Other hematologic effects of iron restriction were: decreased MCHC, reticulocyte count, RDW, and dense cells. A reduced hemolytic rate was suggested by a lowering of serum bilirubin and LDH. In one of the Pts. the 51Cr RBC T1/2 survival increased from 12 to 16 days. The intracellular HbS polymer fractions (fp) were determined at 25% O2 by Csat and with the use of the conservation of mass equation. The baseline fp values ranged from 0.48-0.53. After iron restriction they ranged from 0.33-0.48. The fp decreased even though iron-limited erythropoiesis also lowered the Hb F concentration in three of our Pts. In one of the two Pts. with induced iron depletion, hospitalization days for pain crises decreased from an average of 4.5 days/month (2 year baseline period) to an average of 0.5 days/month in the 3 year follow-up after iron depletion. The second patient with induced iron restriction experienced the rapid healing of a leg ulcer. Controlled iron restriction should be explored as a therapeutic strategy in selected SS patients.

Solubilities of deoxygenated sickle cell hemoglobin (deoxy-Hb S), at varying pH and temperature over a range of concentrations encompassing those found in erythrocytes, were measured. The technique involved ultracentrifugation, which gave values of the supernatant concentration and the mass of the sedimented material. The data establish that the solubility of deoxy-Hb S is the saturation concentration and is independent of initial concentration. The mass of the pellet phase increases linearly with initial concentration. Moreover, the saturation concentration represents the critical concentration above which monomers are in equilibrium with polymers. These polymers are the putative cause of erythrocyte deformation associated with sickle cell anemia. The solubility-pH profiles of deoxy-Hb S at various temperatures, unlike those of other proteins, show no minima at the isoelectric pH, but instead snow a marked decrease in solubility below pH 7.0, indicating the predominance of polymerization over the expected increase in solubility. Deoxy-Hb S, within specified ranges of temperature and pH, possesses a negative temperature coefficient of solubility, a property characteristic of hydrophobic interactions. The saturation concentration is, however, temperature independent at conditions close to physiological. The enthalpy of polymerization (3.5 kcal/mol) is temperature independent from 60 to 220 for all pH values between 6.45 and 7.40. In the range of 220 to 38°, this parameter becomes less endothermic, having a value of 2.5 kcal/mol at pH 6.45 and a value of zero at pH 7.20. Such behavior of the system suggests a phase transition near 220. Within the range of.conditions examined the polymerization is entropically driven.Sickle cell hemoglobin (Hb S) is a variant of normal adult hemoglobin (Hb A) in which a substitution of valine for glutamic acid occurs at position 6 in both ,3 chains. Deoxygenated solutions of Hb S, at concentrations comparable to those within erythrocytes, form liquid crystals or tactoids. These nematic crystals of deoxy-Hb S, the putative cause of erythrocyte sickling, have been characterized optically by their birefringence and polarization dichroism (1, 2). Structural information derived from studies of both x-ray diffraction and optical diffraction of electron micrographs has provided evidence that these paracrystalline arrays are composed of helical microtubules packed into square lattices (3, 4).The term lowest gelling point has been used (5) to denote the concentration at which a solution of deoxy-Hb S loses its fluidity. Binary mixtures of Hb S with Hb A, and with mutant hemoglobins, have been characterized by their "lowest gelling point" as well. More recent experiments, using somewhat different techniques, have evaluated the effect of a diverse population of hemoglobins, both liganded and unliganded, on what has been termed minimum gelling concentration (6). Gelation of deoxy-Hb S has also been studied by sedimentation equilibrium experiments using either interference (7) or schlie...

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