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 but free of deformed cells. Oxygen affinity was shifted to the right in all layers (mean P 50 (pH 7.13)ñ1SD: top 46.3ñ2.9 mm Hg; middle 49.8ñ4.9 mm Hg; bottom 61.0ñ5.8mm Hg) compared with normal blood (top 32.1ñ0.7 mm Hg; bottom 30.1ñ0.5 mm Hg). 2,3-DPG was increased in the top fraction, but was low or normal in the bottom fraction (top 21.8ñ3.4 æmol/g Hb; middle 17.7ñ2.2 æmol/g Hb; bottom 13.8ñ3.1 æmol/g Hb; normal whole blood 14.3ñ1.2 æmol/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 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 32.4ñ2.0; middle 36.2ñ3.1; bottom 44.6ñ3.2 g/100 ml, respectively) and the plot of P 50 vs. MCHC showed a positive corelation (r=0.90,P<0.001). The red cell popualtion in sickle cell anemia is not homogeneous but contains cells of widely varying HB F content, 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 Poý. Lowered affinity for oxygen appears to be closely associated with Hb S concentration and not with 2,3-DPG content (AU)

Irreversibly sickled erythrocytes: a consequence of the heterogeneous distribution of hemoglobin types in sickle-cell anemia

The amount of fetal hemoglobin (Hb F) in erythrocytes of patients with sickle cell anemia (Hb SS disease) was measured by two methods: (a) photometry of individual cells strained for Hb F by the Kleihauer-Betke technique; and (b) chemical assay of alkali-resistant hemoglobin in cells distributed according to specific gravity by ultracentrifugation. Irreversibly sickled cells (ISC), which could be identified directly during photometry and which were found to gather in high concentration at the bottom of ultracentrifuged cell columns, contained significantly less Hb F than non-ISC. Cell content of total Hb was constant regardless of cell size, shape, or ultracentrifugal behavior: thus absolute amounts of Hb F and S varied reciprocally from cell to cell. In experiments designed to estimate age, at formation, and rate of destruction of ISC, Hb SS blood was incubated with selenomethionine-75Se (which labels reticulocytes) of 51Cr (which labels erythrocytes at random) and reinfused. Sequential blood samples were separated by ultracentrifugation into fractions rich in reticulocytes, non-ISC, ans ISC; and chronological changes in the specific activity of each fraction were determined. Analogous information was obtained from radioautography of sequential blood samples after reinfusion of whole blood labeled with amino acids-3H: this technique permitted direct visual characterization of labeled erythrocytes as ISC of non-ISC, all of which had been reticulocytes at the time of reinfusion. The transformation of non-ISC into ISC, presumably a manifestation of membrane damage, proved to begin soon after cell release from the marrow; and ISC subsequently underwent rapid removal from the circulating blood. It is therefore apparent from these studies that, in Hb SS disease, relatively small reciprocal changes in the amounts of the two major hemoglobins carry predictive importance: (a) net synthesis of Hb F is least in erythroid cells destined to become ISC; and (b) these irreversibly deformed erythrocytes suffer preferential destruction. (AU)