-LpE) in human plasma

Gamma-LpE ( g -LpE), a sphingomyelin-rich lipoprotein that contains apolipoprotein (apo) E as its only protein component, has been proposed to play a role in cellular cholesterol efflux by acting, like preb 1 -LpA-I, as an initial acceptor of cell-derived cholesterol. In order to further characterize the presence of g -LpE in human plasma, we have separated g -LpE by two-dimensional non-denaturing polyacrylamide-gradient gel electrophoresis and detected its presence by immunoblotting with 125 I-labeled polyclonal anti-apoE antibody. Five species of g -LpE were routinely detected in human plasma, ranging in mean particle diameter from 9.5 to 16.5 nm. The largest proportion of g -migrating apoE was associated with g 2 -LpE having a diameter of 13.0 nm. Neither the amount of g -LpE apoE (representing less than 1–2% of total plasma apoE) nor the number of g -LpE subfractions was different in serum vs. plasma, or was affected by the presence of agents able to inhibit protein dimerization. g -LpE subfractions were present in the plasma of patients having different apoE phenotypes (i.e., apoE 2/2, 3/3, or 4/4). Incubation of plasma at 37 8 C (90 min) caused a significant decrease in plasma g -LpE ( . 80%) that was not dependent on LCAT or CETP activity. Storage (at 2 70 8 C) of hypertriglyceridemic but not normolipidemic plasma resulted in an increase in g -LpE. Freezing of postprandial plasma samples, containing increased amounts of triglyceride-rich lipoproteins (TRL) enriched in apoE, also caused an increase in g -LpE. Incubation of VLDL (d , 1.006 g/ml) with lipase resulted in the production of g -migrating apoE. These results demonstrate that: 1 ) different g -LpE subfractions exist in human plasma; 2 ) the amount of apoE associated with g -LpE subfractions is dependent on in vitro conditions of plasma storage; and 3 ) TRL can act as a source of g -LpE apoE in vitro. —Krimbou, L., M. Tremblay, H. Jacques, J. Davignon, and J. S. Cohn. In vitro factors affecting the concentration of gamma-LpE ( g -LpE) in human plasma. J. Lipid Res. 1998. 39: 861–872. Supplementary key words HDL • cholesterol efflux • apoE Apolipoprotein (apo) E is an arginine-rich glycoprotein (34,200 Da) that plays a pivotal role in lipoprotein metabolism (1). It mediates the delivery of lipids to cells by acting as a ligand for a number of cellular lipoprotein receptors, and also participates in the removal of cholesterol from cells by acting as a mediator or acceptor of excess cellular cholesterol (2). The importance of apoE in the pathogenesis of atherosclerosis has been strikingly demonstrated by the presence of spontaneous atherosclerosis in experimental animals made deficient in apoE (3, 4), and conversely by protection or regression of atherosclerosis in apoE-deficient animals supplemented with apoE (5–7). In normolipidemic plasma, the majority of apoE is associated with apoB or apoA-I-containing lipoproteins (8). Recent studies have, however, demonstrated the existence of lipoproteins similar in size to high density lipoproteins (HDL), containing apoE as their only protein component (e.g., g -LpE and preb 1 -LpE) (9–11). Gamma ( g )-LpE are spherical plasma lipoproteins, 12– 16 nm in diameter, that have g -mobility when separated by agarose gel electrophoresis (9, 10). g -LpE are rich in sphingomyelin and apoE and have been proposed to play a role in reverse cholesterol transport by acting, together with preb 1 -LpA-I (small apoA-I-only-containing HDL), as initial acceptors of cell-derived cholesterol (9, 10). g -LpE and preb 1 -LpA-I are thus regarded as antiatherogenic lipoproteins as they have the ability to potentiate the efflux of excess cholesterol from peripheral cells and to mediate the transport of this cholesterol back to the liver for eventual excretion into the bile (12, 13). Abbreviations: apo, apolipoprotein; CAD, coronary artery disease; d, density; CETP, cholesteryl ester transfer protein; DTNB, 5,5-dithiobis2-nitrobenzoic acid; ELISA, enzyme-linked immunosorbent assay; EC, esterified cholesterol; EDTA, ethylene-diamine-tetraacetate; FC, free cholesterol; FPLC, fast protein liquid chromatography; HDL, high density lipoprotein; IDL, intermediate density lipoprotein; LCAT, lecithin:cholesterol acyltransferase; LDL, low density lipoprotein; PBS, phosphate-buffered saline; RCT, reverse cholesterol transport; TRL, triglyceride-rich lipoprotein; VLDL, very low density lipoprotein. 1 To whom correspondence should be addressed. by gest, on N ovem er 6, 2017 w w w .j.org D ow nladed fom