Isolation and analysis of lipoproteins secreted by rat liver hepatocytes.
A procedure has been developed for the small-scale isolation and characterization of lipoproteins secreted by cultured rat liver hepatocytes. The lipoproteins in the culture medium were separated into VLDL, LDL, HDL and a fraction with d greater than 1.21 on single-spin density-gradients. The lipoproteins were removed from the gradients by adsorption onto Cab-O-Sil, a hydrated colloidal silica. The lipid components were extracted from the silica with CHCl3/CH3OH and the apoproteins solubilized in a buffer that contained 2% sodium dodecyl sulfate and 6 M urea. The proteins were analyzed on 3-20% acrylamide electrophoresis gels that contained 1% sodium dodecyl sulfate. The two major rat-plasma lipoproteins, VLDL and HDL, were well separated by the gradients. The Cab-O-Sil was shown to bind 90-95% of the HDL and VLDL in the fractions from the gradient. The recovery of the lipid components was essentially quantitative. The recovery of the apolipoproteins was only about 60% but with very good precision. Over a 20 h period, the lipid phosphorus associated with secreted lipoproteins increased linearly. The secretion of apolipoprotein A1 and apolipoprotein E associated with HDL and apolipoprotein B associated with VLDL also increased as a nearly linear function with time. The secretion of apolipoprotein E associated with VLDL was linear only up to approx. 6 h. The availability of this procedure should greatly facilitate further studies on the characterization of lipoproteins secreted by hepatocytes and mechanisms that regulate lipoprotein synthesis and secretion.
D000818:Animals; D016632:Apolipoprotein A-I; D001053:Apolipoproteins / Q000032:analysis; D001055:Apolipoproteins B; D001057:Apolipoproteins E; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D008074:Lipoproteins / Q000302:isolation & purification* / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000032:analysis; D051381:Rats; D013997:Time Factors
D016632:Apolipoprotein A-I; D001053:Apolipoproteins; D001055:Apolipoproteins B; D001057:Apolipoproteins E; D008074:Lipoproteins; D010713:Phosphatidylcholines