Sterol composition of plasma and tissues
Following a four-hour fast of 12-week-old mice fed on regular rodent chow diet; blood and tissues were collected under isofluorane anesthesia from each of the Abcg8+/+, Abcg8+/- and Abcg8-/- mice (n = 3 for each group). Blood was collected from the retro-orbital venous plexus by heparinized glass tube. The animals were sacrificed by cervical dislocation for tissue collection. Plasma (100 μl) was saponified in 1 N NaOH for one hour, 1.5 mL water added and sterols extracted with three sequential portions of 1.5 mL ethyl acetate, containing 70 μg 5α-cholestane as an internal standard, pooled and dried. Mouse tissues (liver, spleen and brain) were weighed, homogenized in 1 mL phosphate buffer saline (PBS) with a dounce homogenizer (15 strokes at 500 rpm) and a small aliquot of the whole homogenate assayed for protein concentration. Aliquots for sterol analysis from whole tissue homogenates were extracted as described above. Dried sterol samples were derivatized as trimethylsilylethers, redissolved in 10 μL hexane and 2 μL samples were injected into a capillary column (26 m length, 0.32 mm ID, 0.45 mm OD) coated with liquid CpWAX 57 CB (Chrompak, Bridgewater, NJ, USA) in a Hewlett-Packard Gas Chromatograph equipped with a flame ionization detector [28]. Concentrations of plasma and tissue sterols were reported as mg/dL and μg/g wet weight tissue, respectively.