AbstractNon-enzymatic net transport of cholesteryl esters (CE) from HDL to VLDL in exchange for triglycerides has been reported in vitro. Likewise, in vivo observations have been adduced favoring the same process in human subjects and implicating LCAT product lipoproteins as carriers. Exchange of CE among lipoproteins is thought not to occur. In experiments in which LCAT generated radioactive CE are formed from cholesterol dispersion in plasma, exchange to near equilibrium is observed. To better define this process, a labeling procedure employing lecithin: 3H-unesterified cholesterol single bilayer vesicles was devised. The order of lipoprotein labeling with isotopic CE is HDL > VLDL > LDL. After 19 hours of incubation, esters reached equilibrium. Control experiments with vesicles treated by purified LCAT showed that this result could not be explained by distribution of discrete ester-rich vesicle products or their binding to lipoproteins. Subfractionation of labeled lipoproteins on agarose columns confirmed completeness of equilibration, except for the HDL subfraction of smallest size, which is incompletely equilibrated. These results indicate that LCAT-generated CE are capable of equilibration among human lipoproteins.Key Words: high-density lipoproteinslecithinlow-density lipoproteinsunesterified cholesterol vesicles;very-low-density lipoproteins
Plasma 1ecithin:cholesterol acyltransferase (LCAT) activity is increased during the clearance phase of alimentary lipemia induced by a high-fat test meal in normal subjects.Ultracentrifugal fractionation of high density lipoproteins (HDL) into HDL2, HDL,, and very high density (VHD) subfractions followed by analyses of lipid and protein components has been accomplished at intervals during alimentary lipemia to seek associations with enzyme changes.HDLz lipids and protein increased substantially, characterized primarily by enrichment with lecithin.HDL,, which contain the main LCAT substrates, revealed increased triglycerides and generally reduced cholesteryl esters which were reciprocally correlated, demonstrating a phenomenon previously observed in vitro by others.Both changes correlated with LCAT activation, but partial correlation analysis indicated that ester content is primarily related to triglycerides rather than LCAT activity.The VHD cholesteryl esters and lysolecithin were also reduced.Plasma incubation experiments with inactivated LCAT showed that alimentary lipemic very low density lipoproteins (VLDL) could reduce levels of cholesteryl esters in HDL by a nonenzymatic mechanism.In vitro substitution of lipemic VLDL for postabsorptive VLDL resulted in enhanced reduction of cholesteryl esters in HDL, and VDH, but not in HDL2, during incubation.Nevertheless, augmentation of LCAT activity did not result, indicating that cholesteryl ester removal from substrate lipoproteinsis an unlikely explanation for activation.Since VHD and HDL3, which contain the most active LCAT substrates, were also most clearly involved in transfers of esters to VLDL and low density lipoproteins, the suggestion that LCAT product lipoproteins are preferentially involved in nonenzymatic transfer and exchange is made.The main determinant of ester transfer, however, appears to be the level of VLDL, both in vitro and in vivo.
A procedure for the extraction of human erythrocyte lipids using chloroform–isopropanol 7:11 (v/v) is described. It is simple and reproducible, affords almost quantitative extraction of cholesterol and phospholipid, uses a single extraction tube, and yields an extract only slightly contaminated by heme.
