Fig. 3.

Transfer of cholesterol between red cells and cyclodextrin-cholesterol. Erythrocytes (100 μl of packed cells containing ≈80 μg of cholesterol) were allowed to achieve cholesterol mass equilibrium with 9.9 ml of PBS containing methyl-β-cyclodextrin (5 mg/ml) bearing 40–90 μg/ml cholesterol (18). Cells and cyclodextrin were separated by centrifugation. Either the cell or the cyclodextrin compartment was labeled with [³H]cholesterol and then recombined with the other compartment for analysis of transfer kinetics. The transfer reaction at 10°C used 60 μl of packed red cells and 5.94 ml of medium with which it was in mass equilibrium. (a) Transfer of [³H]cholesterol to cyclodextrin complexes from labeled red cells bearing 1.7 (○), 1.1 (▿), or 0.73 (□) times the control cholesterol level. (b) Dependence on red cell cholesterol of the rate of transfer of [³H]cholesterol to cyclodextrin complexes. First-order rate constants were obtained in 15 experiments such as those shown in a.(c) Transfer of [³H]cholesterol from labeled cyclodextrin to red cells. Cyclodextrin-[³H]cholesterol complexes were prepared by equilibrating cyclodextrin-cholesterol with labeled red cells and separating it by centrifugation. The labeled donor preparations were then mixed at 10°C with unlabeled red cells that had been mass equilibrated in parallel with unlabeled cyclodextrin-cholesterol complexes, and the time course of transfer was then determined. (○) Unenriched red cells; (▿) red cells enriched 1.7-fold in cholesterol. (d) Effect of membrane intercalators on red cell [³H]cholesterol efflux. Labeled red cells were added to mass-equilibrated cyclodextrin-cholesterol mixtures to which had been added LPC (10.8 μM; i.e., 0.5 μmol/μmol cell cholesterol) or n-octanol (Oct, 2.1 mM). A high level of LPC was used to compensate for its binding to the cyclodextrin. As widely observed, mild red cell spiculation occurred; however, the low level of free LPC did not extract cholesterol or lyse the cells. n-Octanol did not cause cholesterol extraction, cell lysis, or shape change. The amount of octanol in the membranes could not be calculated because its potential binding to cyclodextrin is unknown. Transfer kinetics in three or four experiments are plotted relative to controls (Con).