Unilamellar vesicle incubations

Figure 2 (A) Effect of incubation temperature on uptake of doxorubicin into 200 nm EPC/cholesterol (55 45 mol/mol) large unilamellar vesicles (LUVs) exhibiting a transmembrane pH gradient (pH 4 inside, 7.8 outside). Doxorubicin was added to LUVs (D/L = 0.3 wt wt) equilibrated at 21°C, 37°C, and 60°C. (B) Effect of cholesterol on the uptake of doxorubicin at 20 into lOOnm LUVs exhibiting a transmembrane pH gradient (pH 4.6 inside, 7.5 outside). Lipid compositions were EPC and EPC/cholesterol (1 1 mol/mol). The initial drug-to-lipid ratio was 100 nmol/pmol. Source Prom Refs. 12 (A), 21 (B).

LCAT, whereas LCAT activity with HDL2 as a substrate is minimal. Others have confirmed that HDL3 is a better substrate for LCAT than HDL2 (Bll, M28). Hamilton et al. (H5) noted that nascent disk-shaped HDL secreted by rat liver were better substrates for LCAT than mature spherical HDL isolated from plasma. Synthetic discoidal complexes of apoA-I, phosphatidylcholine, and cholesterol were better substrates for LCAT than unilamellar vesicles of phosphatidylcholine and cholesterol, incubated in the presence of apoA-I (M32). 

The vesicles, mitochondria, and transfer protein are incubated in buffer containing 1 % fatty acid-poor bovine serum albumin (BSA). The transfer is terminated by sedimenting the mitochondria by centrifugation. An aliquot of the vesicles in the supernatant is then counted. The inclusion of BSA in the incubation increases the vesicle recovery. The extent of transfer is determined from the decrease in the 32P/3H ratio of the unilamellar vesicles. 

Several investigators have studied the physiological relation between these activities under a variety of in vitro conditions. Zilversmit and colleagues [68] demonstrated that transfer of CE from reconstituted lipoproteins to small, unilamellar vesicles proceeded on an equimolar, reciprocal basis with TG transfer. However, Fielding and coworkers [74] clearly showed that CETP can mediate the net transfer of CE in the complete absence of TG. Generating CE by co-incubating LCAT, A-1 and UC-PC liposomes, they found that CE accumulated up to a finite limit that was... 

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