Abstract
We have developed and validated a method for in vitro incorporation of radiolabeled cholesteryl esters into low density (LDL) and high density lipoproteins (HDL). Radiolabeled cholesteryl esters dissolved in absolute ethanol were mixed with LDL or HDL in the presence of lipoprotein-deficient serum (LPDS) as a source of core lipid transfer activity. The efficiency of incorporation was dependent on: a) the core lipid transfer activity and quantity of LPDS, b) the mass of added radiolabeled cholesteryl esters, c) the length of incubation, and d) the amount of acceptor lipoprotein cholesterol. The tracer incorporation was documented by repeat density gradient ultracentrifugation, agarose gel electrophoresis, and precipitation with heparin-MnCl2. The radiolabeling conditions did not affect the following properties of the lipoproteins: 1) chemical composition, 2) electrophoretic mobility on agarose gels, 3) hydrated density, 4) distribution of apoproteins on SDS gels, 5) plasma clearance rates, and 6) immunoprecipitability of HDL apoproteins A-I and A-II. Rat HDL containing radiolabeled cholesteryl esters incorporated in vitro had plasma disappearance rates identical to HDL radiolabeled in vivo.
Highlights
We have developed and validated a method for in vitro incorporation of radiolabeled cholesteryl esters into low density (LDL) and high density lipoproteins (HDL)
Pilot studies using human lipoprotein-deficient serum (LPDS) showed little incorporation of cholesteryl [l-I4C]oleateinto lipoproteins. We postulated that this might be related to core lipid transfer activity; this was examined in a number of species (Table 1)
Little activity was found in rat LPDS and highest activities were found in two New World monkey species, the tamarin and spider monkeys, and in the Old World cynomolgus monkey
Summary
We have developed and validated a method for in vitro incorporation of radiolabeled cholesteryl esters into low density (LDL) and high density lipoproteins (HDL). The efficiency of incorporation was dependent on: u) the core lipid transfer activity and quantity of LPDS, 6) the mass of added radiolabeled cholesteryl esters, c) the length of incubation, and d ) the amount of acceptorlipoprotein cholesterol. In vivo and in vitro methods employing LCAT introduce radiolabeled free cholesterol as well as cholesteryl esters into the lipoproteins. This problem can be avoided by using radiolabeled cholesteryl esters but the efficiency of cholesteryl ester incorporation has generally been low [3,4,5,6,7,8,9]. Possible effects of the labeling procedure on the properties of the lipoproteins were studied and plasma clearance rates of lipoproteins labeled in vitro were compared with those radiolabeled in vivo
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