Abstract
We have used two-dimensional gel electrophoresis to separate and characterize human plasma apolipoprotein (apo) E-containing lipoproteins in the high density lipoprotein (HDL) size range. Lipoproteins were separated from whole plasma by electrophoresis (according to charge) in a 0.75% agarose gel, and then in the second dimension (according to size) in a 2-15% non-denaturing polyacrylamide gradient gel. ApoE-containing lipoproteins were detected by radiography after electrotransfer of lipoproteins to nitrocellulose membranes and incubation with 125I-labeled affinity-purified polyclonal apoE antibody. ApoE-containing lipoproteins in the HDL size range had a particle size ranging from 9 to 18.5 nm in diameter and could be characterized as having either gamma, pre-beta1-, pre-beta2- or alpha-electrophoretic mobility (designated gamma-LpE, pre-beta1-LpE, pre-beta2LpE, and alpha-LpE respectively). gamma-LpE and a substantial proportion of pre-beta1- and pre-beta2-LpE did not co-migrate with apoA-I, apoA-II, apoC-III, or apoB-100. Subsequent experiments focused on the pre-beta1-LpE, pre-beta2LpE, and alpha-LpE subfractions, which represented > 95% of apoE in HDL-sized lipoproteins. Storage of plasma at 4 degrees C or in vitro incubation of plasma at 37 degrees C caused a relative decrease in pre-beta1-LpE and increase in alpha-LpE. Normolipidemic patients with an apoE 2/2 phenotype tended to have increased levels of alpha-LpE, whereas apoE 4/4 subjects tended to have a greater proportion of HDL-apoE as pre-beta1-LpE. Decrease in plasma HDL apoE concentration after an oral fat load was associated with a reduction in the plasma concentration of all HDL-apoE subfractions. These results demonstrate that: 1) apoE-containing HDL are heterogeneous in size and charge; 2) pre-beta1-LpE is a relatively labile HDL subfraction; 3) HDL-apoE subfraction distribution is dependent on apoE phenotype; and 4) all apoE-containing HDL subfractions participate in the plasma transfer of apoE during the postprandial period.
Highlights
We have used two-dimensional gel electrophoresis to separate and characterize human plasma apolipoprotein E-containing lipoproteins in the high density lipoprotein (HDL) size range
It is generally believed that HDL inhibits atherogenesis by promoting the efflux of excess cholesterol from lipid-laden macrophages [2] and by mediating the plasma transport of excess cholesterol to the liver for eventual excretion in the bile [3,4].HDL has, been shown to mediate other potentially anti-atherogenic functions, such as modulating the plasma clearance of triglyceriderich lipoproteins (TRL) ( 5 ),inhibiting low density lipoprotein (LDL) oxidation [6],and inhibiting the expression of endothelial cell adhesion molecules [7]
Human HDL has been separated by ultracentrifugation into two major density subfractions, designated HDL2 (density (d): 1.063-1.125 g/ml) and HDLS (d: 1.125-1.21 g/ml) [10].Nondenaturing polyacrylamidegradient gel electrophoresis has been used to separate HDL on the basis of particle size into five subfractions, namely HDL?, Abbreviations: apo, apolipoprotein; CAD, coronary artery disease; d, density; ELISA, enzyme-linked immunosorbent assay;EDTA, ethylenediamine-tetraacetate; FPLC, fast protein liquid chromatography; HDL, high density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; LRP, LDL receptor-related protein; phosphate-buffered saliric (PBS), phosphate-buffered saline; RCT, reverse cholesterol transport; TRL, triglyceride-rich lipoprotein; VLDI, very low density lipoprtr tein
Summary
We have used two-dimensional gel electrophoresis to separate and characterize human plasma apolipoprotein (apo) E-containing lipoproteins in the high density lipoprotein (HDL) size range. Human HDL has been separated by ultracentrifugation into two major density subfractions, designated HDL2 (density (d): 1.063-1.125 g/ml) and HDLS (d: 1.125-1.21 g/ml) [10].Nondenaturing polyacrylamidegradient gel electrophoresis has been used to separate HDL on the basis of particle size into five subfractions, namely HDL?, Abbreviations: apo, apolipoprotein; CAD, coronary artery disease; d, density; ELISA, enzyme-linked immunosorbent assay;EDTA, ethylenediamine-tetraacetate; FPLC, fast protein liquid chromatography; HDL, high density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; LRP, LDL receptor-related protein; PBS, phosphate-buffered saline; RCT, reverse cholesterol transport; TRL, triglyceride-rich lipoprotein; VLDI,, very low density lipoprtr tein. It has been suggested that HDL-apoE is involved in a nuniber of aspects of plasma lipoprotein metabolism, including: I ) receptor-mediated delivery of HDL cholesterol to the liver [22]; 2) hepatic lipase-catalyzed hydrolysis of HDL phospholipid [23]; 3) plasma cholesterol esterification [24, 25]; 4 ) plasma cholesteryl ester transfer [26]; 5 ) postprandial triglyceride nietabolism [27]; and 6) efflux of cell-derived cholesterol [28, 29]
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