Erythrocyte Hb-S concentration. An important factor in the low oxygen affinity of blood in sickle cell anemia

The blood in sickle cell anemia has a very low oxygen affinity and, although 2,3-diphosphoglycerate (2,3-DPG) is increased, there is doubt as to whether this is the only factor responsible. In this study of 15 patients with sickle cell anemia (Hb SS) no correlation was found between oxygen affinity (P(50) at pH 7.13) and 2,3-DPG in fresh venous blood. Whole populations of Hb SS erythrocytes were therefore separated, by an ultracentrifuge technique, into fractions of varying density. The packed red cell column was divided into three fractions; a bottom fraction rich in deformed cells or irreversibly sickled cells (ISC), with a very high mean corpuscular hemoglobin concentration (MCHC); a middle fraction containing cells with the highest content of fetal hemoglobin; and a top fraction containing reticulocytes and discoid cells but free of deformed cells. Oxygen affinity was shifted to the right in all layers (mean P(50) (pH 7.13)+/-1SD: top 46.3+/-2.9 mm Hg: middle 49.8+/-4.9 mm Hg; bottom 61.0+/-5.8 mm Hg) compared with normal blood (top 32.1+/-0.7 mm Hg: bottom 30.1+/-0.5 mm Hg). 2.3-DPG was increased in the top fraction, but was low or normal in the bottom fraction (top 21.8+/-3.4 mumol/g Hb: middle 17.7+/-2.2 mumol/g Hb; bottom 13.8+/-3.1 mumol/g Hb; normal whole blood 14.3+/-1.2 mumol/g Hb). The level of 2,3-DPG in top fractions could not account for the degree of right shift of P(50), and in the middle and bottom fractions the even greater right shifts were associated with lower levels of 2,3-DPG. Top fraction cells depleted of 2,3-DPG had a higher, but still abnormally low, oxygen affinity. A strong relationship was found between oxygen affinity and MCHC. The fractions with the greatest right shift in P(50) had the highest MCHC (top 32.4+/-2.0; middle 36.2+/-3.1; bottom 44.6+/-3.2 g/100 ml, respectively) and the plot of P(50) vs. MCHC showed a positive correlation (r = 0.90, P < 0.001). The red cell population in sickle cell anemia is not homogeneous but contains cells of widely varying Hb F content, 2,3-DPG, and hemoglobin concentration. Paradoxically, the cells with the lowest O(2) affinity have the lowest 2,3-DPG, but they also have the highest concentration of Hb S. The dense, deformed cell called the ISC is but the end stage in a process of membrane loss and consequent increase in hemoglobin concentration. The P(50) of Hb SS blood is, to a large extent, determined by the presence of these cells (r = 0.85, P < 0.001). Increased concentration of Hb S in the cell favors deoxygenation and crystallization even at relatively high P(o2). Lowered affinity for oxygen appears to be closely associated with Hb S concentration and not with 2,3-DPG content.