Interconversion between apolipoprotein A - I - contain i ng lipoproteins of pre - beta and alp ha electrophoretic mobilities Steven

Apolipoprotein (apo) A-I-containing lipoproteins can be separated into two subfractions, pre-beta HDL and alpha HDL (high density lipoproteins), based on differences in their electrophoretic mobility. In this report we present results indicating that these two subfractions are metabolically linked. When plasma was incubated for 2 h at 37"C, apoA-I mass with pre-beta electrophoretic mobility disappeared. This shift in apoA-I mass to alpha electrophoretic mobility was blocked by the addition of either 1.4 mM DTNB or 10 mM menthol to the plasma prior to incubation, suggesting that 1ecithin:cholesterol acyltransferase (LCAT) activity was involved. There was no change in the electrophoretic mobility of either pre-beta HDL or alpha HDL when they were incubated with cholesterolloaded fibroblasts. However, after exposure to the fibroblasts, the cholesterol content of the pre-beta HDL did increase approximately sixfold, suggesting that pre-beta HDL can associate with appreciable amounts of cellular cholesterol. Pre-beta HDLlike particles appear to be generated by the incubation of alpha HDL with cholesteryl ester transfer protein (CETP) and either very low density lipoproteins (VLDL) or low density lipoproteins (LDL). This generation of pre-beta HDL-like particles was documented both by immunoelectrophoresis and by molecular sieve chromatography. I Based on these findings, we propose a cyclical model in which 1) apoA-I mass moves from pre-beta HDL to alpha H D L in connection with the action of LCAT and the generation of cholesteryl esters within the HDL, and 2) apoA-I moves from alpha HDL to pre-beta HDL in connection with the action of CETP and the movement of cholesteryl esters out of the HDL. Additionally, we propose that the relative plasma concentrations of pre-beta HDL and alpha HDL reflect the movement of cholesteryl esters through the HDL. Conditions that result in the accumulation of HDL cholesteryl esters will be associated with low concentrations of pre-beta HDL, whereas conditions that result in the depletion of HDL cholesteryl esters will be associated with elevated concentrations of pre-beta HDL. This postulate is consistent with published findings in patients with hypertriglyceridemia and LCAT deficiency.-Kunitake, S. T., C. M. Mendel, and L, K. Hennessy. Interconversion between apolipoprotein A-Icontaining lipoproteins of pre-beta and alpha electrophoretic mobilities. J. Lipid Res. 1992. 33: 1807-1816. Supplementary key words cholesterol acyltransferase cholesteryl ester transfer protein pre-beta HDL alpha HDL lecithin: High density lipoproteins (HDL) are a collection of discrete species that vary in composition and structure. For the most part, the functions of these species are illdefined; however, it is possible that each may perform a unique task in the body. One of the chief processes in which HDL appear to participate is reverse cholesterol transport (1, 2), the multi-step process in which cholesterol is transported from the periphery to the liver. This process may involve several HDL species. One way that HDL species can be differentiated is on the basis of their electrophoretic mobility. We have previously shown that immunoisolated apoA-I-containing lipoproteins (Lp(A-I)) can be divided into two major subfractions, those with alpha electrophoretic mobility, "alpha HDE, and those with pre-beta mobility, "pre-beta HDC' (3).* We first isolated and characterized pre-beta HDL by preparative electrophoresis after isolation of Lp(A-I) from plasma ( 3 ) . Although the majority of apoA-I-containing lipoproteins in plasma are alpha HDL, pre-beta HDL are also normal components of human plasma. Observations made earlier on serum (4) and centrifugally isolated HDL Abbreviations: VLDL, very low density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein; LCAT, lecithin:cholesterol acyltransferase; C ETP, cholesteryl ester transfer protein; Lp(A-I), apoA-I-containing lipoprotein; TBS, Tris-buffered saline; PBS, phosphate-buffered saline. 'To whom reprint requests should be addressed at: Cardiovascular Research Institute, University of California, San Francisco, 3rd and Parn a s u s Avenues, San Francisco, CA 94143. *The precise definition of HDL is those lipoproteins ultracentrifugally isolated between the density interval 1.063 to 1.21 g/ml. Lipoproteins isolated by selected affinity immunosorption all contain apoA-I. As apoA-I is the dominant protein in HDL, we used HDL as shorthand for the immunoisolated particles. We then named the alpha HDL and pre-beta HDL subfractions based on their general electrophoretic mobilities. These terms are not intended to imply any structural feature or lipid content to the two subfractions. Journal of Lipid Research Volume 33, 1992 1807 by gest, on O cber 8, 2017 w w w .j.org D ow nladed fom (5, 6) are consistent with the presence of pre-beta HDL as we have defined them. They are also present in the plasma of mice (7), monkeys (8), and dogs (9). Particles similar to pre-beta HDL have been identified in the plasma of patients with 1ecithin:cholesterol acyl transferase (LCAT) deficiency (10, ll), in the plasma of patients undergoing chronic hemodialysis (12), and in the medium of cultured HepG2 cells (13). The concentration of prebeta HDL in plasma can range from virtually nonexistent in patients who have undergone small bowel resection (K. LaSala, S. T. Kunitake, and J. P. Kane, unpublished results) to more than half of the apoA-I mass in the plasma of hypertriglyceridemic subjects (14-16), although the average concentration of pre-beta HDL is about 5% of the apoA-I in the plasma of normolipidemic individuals (14, 16). Furthermore, pre-beta HDL have been postulated to serve a key role in the process of reverse cholesterol transport, possibly acting as the initial acceptors of cellular unesterified cholesterol (17-21). We have shown that pre-beta HDL are small, proteinrich lipoproteins that are composed predominantly of apoA-I, without detectable apoA-11, and structurally distinct from alpha HDL (3, 22). Subsequently, three subfractions of apoA-I-containing lipoproteins whose electrophoretic mobilities differ from the electrophoretic mobility of alpha HDL, when resolved by twodimensional gel electrophoresis, have been identified (18, 19). During preparative isolation of pre-beta HDL by starch block electrophoresis, only the peak fractions are recovered; particles with mobilities differing from the peak pre-beta HDL are excluded, possibly accounting for differences in the description of pre-beta HDL species. Using this method, the vast majority of pre-betamigrating apoA-I, isolated from the plasma of fasting normolipidemic subjects, appears to reside in particles of approximately 60 kDa (S. T. Kunitake, C. M. Mendel, and L. K. Hennessy, unpublished results). Pre-beta HDL and alpha HDL appear to be metabolically linked. We have reported on the movement of prebeta HDL to alpha HDL when plasma is incubated (23). In the present study we investigated the interconversion of apoA-I between pre-beta HDL and alpha HDL. Specifically, we monitored the movement of apoA-I mass between pre-beta HDL and alpha HDL apparently linked to the movement of cholesteryl esters into and out of the total pool of plasma HDL (HDL compartment).