The formation of echinocytes by the insertion of oxygenated sterol compounds into red cell membranes

Human red cells incubated with oxygenated sterol compounds (OSC) at a concentration of 2.5 × 10−5M in medium containing lipoprotein-depleted serum become echinocytic. The formation of echinocytes by OSC is impeded by low temperature, replacement of lipoprotein-depleted serum by bovine serum albumin, or lipoprotein-containing serum, but not by the presence of free cholesterol. Echinocyte formation is apparent within 2 min of exposure of red cells to OSC, reaches a peak at 5–150 min, and declines to a nadir at 2–8 hr. This reversal to normal shape is followed by a second phase of echinocyte formation, with the percentage of abnormally shaped red blood cells increasing progressively over the next 40 hr. Following a 24-hr incubation with OSC, the oxysterols can be detected in approximately equal amounts in extracts of whole red cells or red-cell stroma. The proportion of OSC in the incubation mixture inserted into erythrocyte membranes ranges from 2.4% in the case of 25-hydroxycholesterol to 36% for 7β-hydroxycholesterol. A non-echinocyte-forming OSC such as 20α-hydroxycholesterol enters red cell membranes with an efficiency equal to or greater than that of potent echinocyte-forming OSC. Virtually all OSC membrane insertion occurs within 1 hr of red cell exposure, and the amount of membrane-inserted OSC remains constant over the next 23 hr. OSC insertion into red cell membranes is impeded by the presence of lipoproteins, but not by the presence of free cholesterol. We postulate that the early formation of echinocytes by membrane-inserted OSC is a consequence of asymmetric expansion of the outer lamella of the red-cell membrane lipid bilayer due to the presence of bulky hydrophilic groups on sterol molecules inserted within the hydrophobic environment of the membrane hydrocarbon chains. Early disappearance of echinocytes may be a consequence of the “flip-flop” and equilibrium redistribution of membrane-inserted OSC between the outer and inner membrane halves. OSC may be involved in red cell echinocyte formation in certain human diseases.