The limited depletion of cholesterol from erythrocyte membranes on treatment with incubated plasma

Abstract

About 35% of the cholesterol of human erythrocyte membranes can be removed by the “preincubated plasma” technique (Murphy, J.R. (1962) J. Lab. Clin. Med. 60, 86–109), in which erythrocytes are extracted with plasma that has been preincubated to esterify a portion of its lipoprotein cholesterol. The limitation on the cholesterol depletion is shown not to be a result of insufficient plasma capacity to take up additional cholesterol or of changes in the plasma during the extraction. The maximum cholesterol depletion from “ghosts” was the same as that from whole cells. “Inside-out” membrane vesicles (Steck, T.L. (1974) in Methods in Membrane Biology (Korn, E., ed.), Vol. 2, pp. 245–281) were utilized to determine if the limitation to cholesterol depletion is a result of the remaining cholesterol being 1located at the membrane inner surface and therefore not accessible to the plasma. No further cholesterol depletion occurred when “inside-out” vesicles, prepared from erythrocytes which were depleted of cholesterol by the usual method, were extracted. Also, “inside-out” vesicles prepared from untreated erythrocytes gave the same cholesterol depletion as is usually attained. The maximal cholesterol depletion was unaffected by a number of modifications of the extracting preincubated plasma: addition of lysolecithin or albumin, dialysis against isotonic buffer, and variation in pH of the preincubated plasma from 6.0 to 9.0. It is concluded that the limitation on the cholesterol depletion is a result of a firm binding of the remaining cholesterol.