Effect of cholesterol extraction of calcifying vesicles by ethanol on ultrastructural appearance of vesicles. The transmission electron microscopic image of ethanol treated calcifying vesicles was obtained from a cholesterol-fed rabbit. To reduce the number of cholesterol micelles, calcifying vesicle fractions were centrifuged at 250,000 × g for 30 min. To solubilize cholesterol, the resulting pellet was suspended in an aliquot of water and then mixed with alcohol to a final concentration of 60%. The mixture was centrifuged at 10,000 rpm for 15 min in a microfuge and the resulting pellet was processed for electron microscopy. The ultrastructural images did not differ from those obtained from untreated vesicles. The alcohol treatment which presumably solubilized free cholesterol did not alter the LDL-C content in vesicle fraction.