Fluorescence measurements of the labile iron pool of sickle erythrocytes

Abstract

Sickle erythrocytes have increased ferritin and increased molecular iron on the inner membrane leaflet, and we postulated that cytosolic labile iron is also elevated. We used the fluorescent metallosensor, calcein, and a permeant Fe2+ chelator to estimate labile cytoslic Fe2+, and calcein plus an Fe3+ chelator to estimate total cytosolic labile iron (Fe2+ + Fe3+). We measured membrane nonheme iron by its reactivity with ferrozine. As estimated by calcein and Fe2+ chelator, the mean +/- SD labile Fe2+ concentration was significantly lower in hemoglobin (Hb) SS (n = 29) than hemoglobin AA (n = 17) erythrocytes (0.56 +/- 0.35 microM versus 1.25 +/- 0.65 microM; P <.001). In contrast, as estimated by calcein and Fe3+ chelator, total erythrocyte labile iron was similar in hemoglobin SS (n = 12) and hemoglobin AA (n = 10) participants (1.75 +/- 0.41 microM versus 2.14 +/- 0.93 microM; P =.2). Mean membrane nonheme iron levels were higher in hemoglobin SS cells than hemoglobin AA cells (0.0016 x 10-4 versus 0.0004 x 10-4 fmol/cell; P =.01), but much lower than the mean amounts of total labile iron (1.6-1.8 x 10-4 fmol/cell) or hemoglobin iron (18 000-19 000 x 10-4 fmol/cell). Both membrane iron and total labile iron were much less than the mean amount of iron potentially present in erythrocyte ferritin as calculated from results of other investigators (15 x 10-4 versus 34 x 10-4 fmol/cell in HbAA versus HbSS erythrocytes). We conclude that cytosolic labile iron is not elevated in hemoglobin SS erythrocytes and that elemental membrane iron is present in only trace amounts.