After cooling to room temperature, cholesterol was extracted with 1 ml of hexane. The process was repeated four times. The hexane layers were transferred to a round-bottomed flask and dried under vacuum. The extract was redissolved in 1 ml of hexane and was stored at -20°C until analysis. Total cholesterol was determined on a silica fused capillary column (HP-5, 30 m × 0.32 mm I.D. × 0.25 μm thickness) using a Hewlett-Packard 5890A gas chromatography (Palo Alto, CA) equipped with a flame ionization detector.