mRNA expression levels presented were calculated relative to the average of the housekeeping gene cyclophilin and further normalized to the relative expression levels of the respective Carfilzomib clinical controls. Bile collection and assessment of biliary excretion of bile acids, phospholipids, and cholesterol Bile was collected by cannulation of the gallbladder in mice anesthetized by intraperitoneal injection of hypnorm (fentanyl/fluanisone, 1 mg/kg) and diazepam (10 mg/kg). During the bile collection, body temperature was maintained using a humidified incubator. Bile collection was performed for 30 min, and secretion rates were determined gravimetrically. Biliary bile salt, cholesterol, and phospholipid concentrations were determined and the respective biliary excretion rates calculated as described previously (23, 24).

Fecal sterol analysis Mice were individually housed, and feces were collected over a period of 24 h and separated from the bedding. Fecal samples were dried, weighed, and thoroughly ground. Aliquots thereof were used for determination of neutral sterol and bile acid content by gas-liquid chromatography as described (23, 24). HDL kinetics studies HDL kinetics studies were performed essentially as published previously (21). Autologous HDL was prepared from pooled mouse plasma by sequential ultracentrifugation (density 1.063 < d < 1.21). After extensive dialysis against sterile PBS containing 0.01% EDTA, HDL was labeled with 125I-tyramine-cellobiose (TC) and cholesteryl hexadecyl ether (cholesteryl-1,2,-3H; Perkin Elmer Life Sciences) as previously described (25).

For kinetic studies, 0.4 ��Ci of 125I and 0.7 million dpm of the 3H tracer were injected into the tail veins of fasted wild-type mice treated with AdNull or AdhApoE3. Blood samples were drawn by retroorbital bleeding at 5 min and at 1, 3, 6, 11, and 24 h after injection. Plasma decay curves for both tracers were generated by dividing the plasma radioactivity at each time point by the radioactivity at the initial 5-min time point after tracer injection. Fractional catabolic rates (FCRs) were determined from the area under the plasma disappearance curves fitted to a bicompartmental model using the SAAM II program (26). The use of 3H-cholesteryl ether does not affect turnover rates in vivo compared with 3H-cholesteryl ester-labeled HDL (21).

Organ uptake of HDL apolipoproteins (125I) and HDL-CEs (3H-cholesteryl ether) was determined by measuring the counts recovered in each organ expressed as a percentage of the injected dose, which was calculated by multiplying the initial plasma counts (5-min time point) with the estimated plasma volume (3.5% of total body weight). Brefeldin_A Selective uptake into organs was determined by subtracting the percentage of the injected dose of 125I-HDL recovered in each organ from the percentage of the injected dose of 3H-HDL-CE. In vivo RCT studies In vivo RCT studies were performed essentially as published previously (27).