In vitro metabolism of LDL labeled with a nondegradable cholesteryl ester analogue

Low density lipoprotein (LDL) metabolism by human skin fibroblasts was studied using LDL labeled in the neutral lipid fraction with a nonhydrolysable cholesteryl ester analogue 3H-cholesteryl linoleyl ether (3H-CLE-LDL). LDL uptake could be quantitated accurately using 3H-CLE-LDL, since the label accumulated intracellularly due to its resistance to hydrolysis. 3H-CLE-LDL was taken up via apo B,E receptor-mediated endocytosis in a manner similar to 125I-labeled LDL. This was demonstrated by similar rates of uptake of the two differently labeled LDL preparations, saturation kinetics of uptake with respect to 3H-CLE-LDL concentration, regulation of 3H-CLE-LDL uptake by procedures that up-regulate or down-regulate the number of apo B,E receptors, and negligible uptake of 3H-CLE-LDL by receptor-negative cell strains. The major difference between the handling of 125I-LDL and 3H-CLE-LDL was the finding that, while the amount of trypsin-releasable (surface) 3H-CLE increased progressively over a 24-hour experimental period, trypsin-releasable 125I-LDL reached a maximum within 30 minutes. After 24 hours of incubation, the 3H radioactivity released by brief trypsinization was three to four times higher than could be accounted for by 125I radioactivity released by a similar treatment. Possible reasons for this behavior are discussed.