This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Huang, Y. Articles by Assmann, G. Search for Related Content PubMed PubMed Citation Articles by Huang, Y. Articles by Assmann, G. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CHOLESTEROL

(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1412-1418.)
© 1995 American Heart Association, Inc.

Articles

Cholesterol Efflux, Cholesterol Esterification, and Cholesteryl Ester Transfer by LpA-I and LpA-I/A-II in Native Plasma

Yadong Huang; Arnold von Eckardstein; Shili Wu; Gerd Assmann

From the Institut für Arterioskleroseforschung an der Universität Münster (Y.H., S.W., G.A.) and the Institut für Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfälische Wilhelms-Universität Münster (A. von E., G.A.), Germany.

Correspondence to Yadong Huang, Institut für Arterioskleroseforschung an der Universität Münster, Domagkstr 3, D-48149 Münster, Federal Republic of Germany.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract HDLs encompass structurally heterogeneous particles that fulfill specific functions in reverse cholesterol transport. Two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (2D-PAGGE) of normal plasma and subsequent immunoblotting with anti–apolipoprotein (apo) A-I antibodies differentiates an abundant particle with electrophoretic -mobility and less abundant particles with electrophoretic pre-ß-mobility (preß₁–LpA-I, preß₂–LpA-I, preß₃–LpA-I). Immunodetection with anti–apoA-II antibodies identifies a single particle with -mobility. To differentiate -migrating HDL without apo A-II (–LpA-I) from those with apoA-II (–LpA-I/A-II), we combined 2D-PAGGE with immunoadsorption of apoA-II. Incubation of plasma with [³H]cholesterol-labeled fibroblasts in combination with immunosubtracting 2D-PAGGE allowed us to analyze the role of –LpA-I and –LpA-I/A-II in the uptake and esterification of cell-derived cholesterol in native plasma. Depending on the duration of incubations with cells, -LpA-I took up two to four times more [³H]cholesterol than –LpA-I/A-II. Irrespective of the duration of incubation, two to three times more [³H]cholesteryl esters accumulated in –LpA-I than in –LpA-I/A-II. Subsequent incubations in the presence of an inhibitor of lecithin:cholesterol acyltransferase led to preferential accumulation of [³H]cholesteryl esters in –LpA-I/A-II. In conclusion, our data indicate that –LpA-I is more effective than –LpA-I/A-II in both uptake and esterification of cell-derived cholesterol. Moreover, –LpA-I/A-II appears to accumulate cholesteryl esters, at least partially, from –LpA-I.

Key Words: apoA-II • HDL subclasses • reverse cholesterol transport • cholesterol efflux

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Several epidemiological and clinical studies have demonstrated an inverse correlation between the plasma concentration of HDL cholesterol and the risk of coronary heart disease (reviewed in Reference 1¹ ). The protection of the vessel wall from atherosclerosis by HDL is usually attributed to the ability of this lipoprotein fraction to transport excess cholesterol from peripheral cells to the liver (reviewed in References 2 through 4^{2 3 4} ). HDL encompasses structurally and functionally heterogeneous particles that can be classified according to a variety of properties, including hydrated density, apolipoprotein composition, and charge (reviewed in Reference 5⁵ ). Sequential immunoaffinity chromatography differentiates lipoproteins by their apolipoprotein composition. By anti–apoA-I and anti–apoA-II immunoaffinity chromatography, HDL can be separated into LpA-I, which contains apoA-I but not apoA-II, and LpA-I/A-II, which contains both apoA-I and apoA-II.⁶ It is not clear whether LpA-I and LpA-I/A-II differ in their ability to release cholesterol from cells. Barbaras et al⁷ and Barkia et al⁸ reported that LpA-I but not LpA-I/A-II promotes cholesterol efflux from Ob1771 adipocytes and bovine aortic endothelial cells and concluded that the apoA-II in LpA-I/A-II neutralizes the beneficial effect of apoA-I in reverse cholesterol transport. By contrast, neither Johnson et al⁹ nor Oikawa et al¹⁰ found differences in the ability of LpA-I and LpA-I/A-II to remove cholesterol from hepatoma cells, fibroblasts, smooth muscle cells, and bovine endothelial cells. However, when incubating total serum with radiolabeled rat hepatoma cells, de la Llera Moya et al¹¹ found that the ability of serum to promote cholesterol efflux is more strongly correlated with the plasma concentration of LpA-I than with that of LpA-I/A-II. It was proposed that the conflicting results of these in vitro studies are due to differences in the structure and function of HDL subclasses occurring during the isolation of lipoproteins from plasma.¹¹

2D-PAGGE of plasma in the sequence agarose gel electrophoresis polyacrylamide gradient gel electrophoresis, and subsequent immunoblotting, differentiates HDL subclasses according to antigenicity, charge, and size.^{5 12 13} Immunodetection with anti–apoA-I antibodies distinguishes preß₁–LpA-I, preß₂–LpA-I, preß₃–LpA-I, and –LpA-I,^{12 13 14} and immunodetection with anti-apoE antibodies distinguishes -LpE and -LpE.⁵ Immunodetection with anti–apoA-II detects a single particle with -mobility called –LpA-II.⁵ In combination with prior incubation of plasma with cells that have been labeled with large amounts of [³H]cholesterol, 2D-PAGGE has helped to unravel the roles of different HDL subclasses in reverse cholesterol transport. Recently, this method has identified two quantitatively minor HDL subclasses as initial acceptors of cell-derived cholesterol, namely, preß₁–LpA-I, which contains apoA-I as the only apolipoprotein,^{12 13} and -LpE, which contains apoE as the only apolipoprotein.¹⁵ After uptake by preß₁–LpA-I, cell-derived cholesterol is further transferred via preß₂–LpA-I, preß₃–LpA-I, and -migrating HDL to LDL.^{12 13 15} A proportion of cell-derived cholesterol is esterified during its passage through preß₃–LpA-I, and another in -migrating HDL after being recycled from LDL.^{13 16 17}

In contrast to immunoaffinity chromatography, 2D-PAGGE does not depend on the prior isolation of lipoproteins and may hence be less affected by artifacts. However, since LpA-I/A-II and the majority of LpA-I exhibit electrophoretic -mobility and do not differ in size,⁵ we modified 2D-PAGGE to allow us to differentiate the role of LpA-I and LpA-I/A-II in reverse cholesterol transport. Plasma proteins were first separated by agarose gel electrophoresis. The gel strip was then transferred to a PAGGE gel covered with an anti–apoA-II antiserum containing agarose gel. By this immunosubtracting 2D-PAGGE, we could investigate native plasma in regard to the different roles of LpA-I and LpA-I/A-II in reverse cholesterol transport.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Plasma Samples
Three normolipidemic individuals with the phenotype apoE3/3 participated in this study. Characteristics of their lipid metabolism are summarized in the Table. Blood samples were collected after overnight fasting and were cooled immediately on ice. Plasmas and sera were obtained by centrifugation at 4°C (2000g, 15 minutes), divided into aliquots, and frozen at -70°C, since in previous studies it was found that freezing and thawing only once did not affect the ability of normal plasma to take up, transfer, and esterify cell-derived cholesterol.^{11 12 16 18} Serum was used for the quantification of lipids, apolipoproteins, and HDL subfractions. LCAT activity was determined in EDTA plasma. For the experiments in which plasma was incubated with cells, streptokinase was used as the anticoagulant at a final concentration of 150 U/mL blood.

View this table: [in this window] [in a new window]   Table 1. Lipids, Lipoproteins, Apolipoproteins, and LCAT Activities of the Plasmas Analyzed

Quantification of Lipids, Apolipoproteins, and HDL Subfractions
Serum concentrations of triglycerides and cholesterol were quantified enzymatically with an autoanalyzer (Hitachi/Boehringer). HDL cholesterol was measured after precipitation of apoB-containing lipoproteins with phosphotungstic acid/MgCl₂ (Boehringer Mannheim).¹⁹ LDL cholesterol was calculated from the Friedewald formula.²⁰ Concentrations of apoA-I, apoA-II, and apoB were determined with a modified commercially available turbidimetric assay (Boehringer Mannheim).²¹ LpA-I was quantified with a commercially available differential electroimmunoassay (Hydragel LpA-I, Sebia).²² The concentration of LpA-I/A-II was calculated as the difference between total apoA-I and LpA-I.²³ LCAT activity was determined as the esterification of [³H]cholesterol that was incorporated into apoA-I–containing proteoliposomes.²⁴ ApoA-I was isolated from HDL by fast protein liquid chromatography with a MonoQ5-column (Pharmacia) as the immobile phase. ApoA-I was eluted within 32 minutes with a linear gradient of buffer A (15 mmol/L Tris-HCl, 10 mmol/L NaCl₂, and 5 mol/L urea, pH 7.2) and buffer B (15 mmol/L Tris-HCl, 1 mol/L NaCl₂, and 5 mol/L urea, pH 7.2) as the mobile phase.

Cell Culture
Normal human skin fibroblasts were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum in dishes of 3.5-cm diameter as described previously.²⁵ At the state of near confluence, fibroblasts were labeled for 72 hours at 37°C with 0.5 mCi (1,2-³H)-cholesterol (New England Nuclear; 51.7 Ci/mmol) that had been complexed with fetal calf serum. The final specific radioactivity in the labeled cells amounted to 1.3±0.4x10⁷ cpm/mg cell UC (mean±SD).

Nondenaturing Two-Dimensional Electrophoresis
The distribution of apoA-I– and apoA-II–containing lipoproteins in normal plasma was analyzed by 2D-PAGGE in which agarose gel electrophoresis was followed by PAGGE.^{12 13} Briefly, in the first dimension, 20 µL of plasma samples was separated by electrophoresis at 4°C in a 0.75% agarose gel with a 50 mmol/L merbital buffer (pH 8.7, Serva). Bromphenol blue was added to a standard sample to visualize albumin in the native gel. The electrophoresis was stopped when the albumin/bromphenol blue marker had migrated 6 cm. Agarose gel strips containing the preseparated lipoproteins were then transferred to a 2% to 20% polyacrylamide gradient gel. Separation in the second dimension was performed at 40 mA for 4 to 5 hours at 10°C. During this time, the endogenous plasma albumin, which because of bromphenol blue added to the cathodic buffer (300 µL/L buffer) was visible in the native gel as a faint blue band, had migrated 10 cm. The proteins separated in the polyacrylamide gradient gel were electroblotted onto a nitrocellulose membrane. ApoA-I– and apoA-II–containing lipoproteins were detected by use of sheep antibodies against human apoA-I or human apoA-II (Boehringer Mannheim), respectively, which had been biotinylated according to the manufacturer's recommendations (Sigma), and streptavidin–biotinylated horseradish peroxidase complex (Amersham).

Differentiation of –LpA-I and –LpA-I/A-II by Immunosubtracting 2D-PAGGE
To analyze –LpA-I and –LpA-I/A-II separately, we modified the 2D-PAGGE procedure described above. Agarose gel electrophoresis was followed by immunosubtracting electrophoresis in a 2% to 20% polyacrylamide gradient gel covered by a 4-cm-broad 0.6% agarose gel that contained the -globulin fraction of an anti-–apoA-II antiserum (Boehringer Mannheim) at a final concentration of 4% (vol/vol). This gel immunoprecipitates all apoA-II–containing lipoproteins, so that LpA-I but not LpA-I/A-II migrated into the polyacrylamide gel. Electrophoresis and Western blotting were performed under the conditions described for 2D-PAGGE. Complete removal of LpA-I/A-II was ascertained by anti–apoA-II immunoblotting. In control experiments, LpA-I/A-II and LpA-I were separated by 2D-PAGGE using polyacrylamide gradient gels covered with a 4-cm-broad agarose gel without antiserum.

Cholesterol Efflux Experiments
To determine the cholesterol efflux from cells into the different lipoprotein subtractions, 1 mL plasma was incubated for different time intervals with [³H]cholesterol-labeled fibroblasts that had been washed six times with PBS (pH 7.4). To separately determine cholesterol efflux into –LpA-I and –LpA-I/A-II, 10 µL labeled plasma was separated either by anti–apoA-II immunosubtractive 2D-PAGGE (for –LpA-I) or by 2D-PAGGE (for –LpA-I plus –LpA-I/A-II). In parallel, an aliquot of 10 µL unlabeled plasma of the same donor was run in the same gel. After completion of 2D-PAGGE, one half of the gel containing the labeled sample was stored at 4°C. Proteins of the other half of the gel were electroblotted onto a nitrocellulose membrane to immunolocalize apoA-I–containing lipoproteins. This immunoblot was then used as a template to localize preß₁–LpA-I, –LpA-I, and –LpA-I/A-II plus –LpA-I in the native gels. These lipoproteins were cut out, and their lipids were extracted by incubation with chloroform/methanol (2:1) for 72 hours. In some experiments, UC and CEs were separated by thin-layer chromatography using silica gel plates (Merck) as the immobile phase and hexane:ether 6:4 (vol/vol) as the mobile phase. The radioactivity in lipids, UC, and CEs was counted by scintillation chromatography. The radioactivity of -migrating particles in anti–apoA-II–immunosubtracting 2D-PAGGE was assigned to –LpA-I; the radioactivity of -migrating particles in 2D-PAGGE was assigned to –LpA-I plus –LpA-I/A-II. The difference between the radioactivities in these two HDLs was assigned to –LpA-I/A-II.

Every experiment was performed at least three times with different plasma aliquots of each of the three donors (Table). For every experiment, separate dishes with labeled fibroblasts were used. Interassay coefficients of variation in the recovery of radioactivity in the various lipoproteins of a specific plasma were below 20%. As reported previously,¹⁸ 79% to 86% of the radioactivity in apoB-free plasma was recovered in apoA-I–containing HDL subfractions separated by 2D-PAGGE.

General Procedures
Protein concentrations were measured according to the method of Lowry et al²⁶ with BSA as the standard. The data were calculated as mean±SD. In some instances, percent values are presented. They represent the amount of radioactivity in one particle as the percentage of total radioactivity in preß₁–LpA-1+–LpA-I+–LpA-I/A-II.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Characterization of ApoA-I– and ApoA-II–Containing Lipoproteins in Normal Plasma by 2D-PAGGE
Fig 1 presents the distribution of apoA-I– and apoA-II–containing lipoproteins, respectively, after separation of normal plasma by 2D-PAGGE. As described previously,^{12 13 14} apoA-I can be immunochemically detected in a major HDL subfraction with -mobility as well as in three minor subfractions with pre-ß-mobility, which differ by their size (preß₁–LpA-I, preß₂–LpA-I, and preß₃–LpA-I) (Fig 1a). LDL also reacted with anti–apoA-I antibodies. ApoA-II is detectable only in a single particle that has -mobility and is entirely colocalized with the -migrating apoA-I–containing particle (Fig 1b). We therefore call this particle –LpA-I/A-II.

View larger version (100K): [in this window] [in a new window]   Figure 1. 2D-PAGGE and immunoblotting of (a) apo A-I– and (b) apoA-II–containing lipoproteins in normal human plasma. Nondenaturing 2D electrophoresis was performed in the order agarose gel electrophoresis PAGGE. After electroblotting onto nitrocellulose membranes, apoA-I– and apoA-II–containing lipoproteins were detected by use of biotinylated polyclonal sheep antibodies against either human apoA-I or human apoA-II and streptavidin–biotinylated horseradish peroxidase complex.

Differentiation of –LpA-I and –LpA-I/A-II in Normal Plasma by Immunosubtracting 2D-PAGGE
–LpA-I was separated from –LpA-I/A-II by anti–apoA-II–immunosubtracting 2D-PAGGE (Fig 2). Under these conditions, apoA-I remained immunodetectable in particles with either pre-ß- or -mobility (Fig 2a). The complete removal of apoA-II–containing particles, ie, –LpA-I/A-II, was confirmed by anti–apoA-II immunoblotting (Fig 2b; compare Fig 2d). The staining of the -migrating apoA-I–containing particle, ie, –LpA-I, was less intense than that of –LpA-I plus –LpA-I/A-II in the nonimmunosubtracting 2D-PAGGE (Fig 2c). By contrast, the optical density of preß₁–LpA-I and preß₂–LpA-I did not differ between the two experiments (Fig 2a and 2c). These results suggested that anti–apoA-II–immunosubtracting 2D-PAGGE can be used as a rapid method for separating –LpA-I from –LpA-I/A-II.

View larger version (83K): [in this window] [in a new window]   Figure 2. Immunosubtracting 2D-PAGGE and immunoblotting of apoA-I– and apoA-II–containing lipoproteins in normal human plasma. For anti–apoA-II immunosubtracting 2D-PAGGE (a and b), agarose gel electrophoresis was followed by electrophoresis in a 2% to 20% polyacrylamide gradient gel covered by an agarose gel of 4-cm width containing 4% sheep anti–human apoA-II antiserum. In the control experiment (c and d), the polyacrylamide gradient gel was covered by a 4-cm-broad agarose gel without anti–apoA-II antibodies. After electroblotting onto nitrocellulose membranes, (a and c) apoA-I– and (b and d) apoA-II–containing lipoproteins were detected by use of biotinylated polyclonal sheep antibodies against either human apoA-I or human ApoA-II and a streptavidin–biotinylated horseradish peroxidase complex.

Cholesterol Efflux From Cells Into –LpA-I and –LpA-I/A-II Determined by Immunosubtracting 2D-PAGGE
Fig 3 presents the distribution of radioactive cholesterol in various lipoproteins of normal plasma after 1 minute of incubation with [³H]cholesterol-labeled fibroblasts (open bars) and 1 minute of incubation without cells (hatched bars). After both nonimmunosubtractive 2D-PAGGE (Fig 3a) and anti–apoA-II–immunosubtractive 2D-PAGGE (Fig 3b), identical amounts of radioactivity were recovered in preß₁–LpA-I. This suggests that anti–apoA-II–immunosubtracting 2D-PAGGE does not affect the cholesterol efflux into preß₁–LpA-I. Significantly different amounts, however, were recovered in –LpA-I plus –LpA-I/A-II (Fig 3a) and –LpA-I (Fig 3b). After 1 minute of incubation with radiolabeled cells, the percentage of [³H]cholesterol in preß₁–LpA-I, –LpA-I, and –LpA-I/A-II amounted to 30%, 55%, and 15%, respectively. After a further 1-minute incubation of plasma in the absence of cells, the radioactivity in preß₁–LpA-I decreased to 5% and simultaneously increased in –LpA-I and –LpA-I/A-II to 72% and 23%, respectively (Fig 3c). These results demonstrate that preß₁–LpA-I and –LpA-I are more potent acceptors of cell-derived cholesterol than –LpA-I/A-II. The results of the incubations without cells also demonstrate that the transfer of cholesterol from initial acceptors to –LpA-I is more efficient than that to –LpA-I/A-II.

View larger version (15K): [in this window] [in a new window]   Figure 3. Bar graphs showing uptake and transfer of cell-derived [3H]cholesterol through various lipoproteins in normal human plasma. Incubations of normal plasma with radiolabeled fibroblasts were performed for 1 minute (open bars), and incubations without cells for another 1 minute (hatched bars). After incubations, aliquots of 10 µL of samples were separated in parallel by (a) nonimmunosubtracting 2D-PAGGE and (b) anti–apoA-II–immunosubtracting 2D-PAGGE. With anti–apoA-I immunoblots of 2D-electropherograms as templates, apoA-I–containing lipoproteins were localized in the native gels and removed. Their lipids were extracted, and the radioactivity was counted. Data in c were calculated from the data presented in a and b. Each bar shows the mean±SD of three experiments as counts per minute released into the various lipoproteins.

During prolonged incubation with [³H]cholesterol-labeled cells, the radioactivity in preß₁–LpA-I increased little after the first minute (Fig 4a). By contrast, the radioactivity continued to increase in both –LpA-I and –LpA-I/A-II (Fig 4b). During the initial 5 minutes, the radioactivity in –LpA-I was threefold to fourfold higher than in –LpA-I/A-II. After more than 10 minutes of incubation, the amounts of radioactive cholesterol in –LpA-I and –LpA-I/A-II differed by a factor of two. The ratio of 2:1 between the radioactivity in –LpA-I and that in –LpA-I/A-II remained unchanged up to 1 hour of incubation (data not shown). These data underline that –LpA-I is more effective in taking up cell-derived cholesterol than is –LpA-I/A-II.

View larger version (25K): [in this window] [in a new window]   Figure 4. Graphs showing time course of changes in the distribution of cell-derived [3H]UC in various lipoproteins of normal human plasma. Plasma samples were incubated with [3H]cholesterol-labeled fibroblasts for different time intervals. After incubations, the samples were separated by nonimmunosubtracting or immunosubtracting 2D-PAGGE as described in the legend to Fig 3. Subsequent to lipid extraction, UC and CEs were separated by thin-layer chromatography. Their radioactivity was counted separately. a, Changes of radioactivity in preß1–LpA-I found after nonimmunosubtracting () or anti–apoA-II immunosubtracting 2D-PAGGE (). b, Time-dependent changes of radioactivity in –LpA-I () and –LpA-I/A-II (). Each point shows the mean±SD of three experiments as counts per minute released into the various lipoproteins.

Esterification of Cell-Derived Cholesterol in –LpA-I and –LpA-I/A-II
Fig 5 shows the appearance of radioactive CEs in –LpA-I and –LpA-I/A-II after incubation of plasma with [³H]cholesterol-labeled fibroblasts for different time intervals. At any incubation time up to 1 hour, the amounts of radioactive CEs in –LpA-I were twofold to threefold higher than in –LpA-I/A-II.

View larger version (20K): [in this window] [in a new window]   Figure 5. Graph showing esterification of cell-derived [3H]cholesterol in –LpA-I and –LpA-I/A-II. The experiment was performed as described in Fig 4. Circles indicate the appearance of [3H]CEs in –LpA-I; triangles, the appearance of [3H]CEs in –LpA-I/A-II. Each point shows the mean±SD of three experiments as counts per minute released into the various lipoproteins.

Transfer of Radioactive CEs From –LpA-I to –LpA-I/A-II
To investigate the transfer of radioactive CEs from –LpA-I and –LpA-I/A-II, we first incubated plasma with [³H]cholesterol-labeled fibroblasts for 20 minutes. After incubation, the samples were removed, supplemented with 2 mmol/L of the LCAT inhibitor DTNB, and then incubated in the absence of cells for different time intervals. After 20 minutes of incubation with radiolabeled cells, the amounts of radioactive CEs in –LpA-I was about twofold higher than in –LpA-I/A-II. During the subsequent incubation of plasma in the absence of cells, radioactive CEs decreased gradually in –LpA-I and increased slightly in –LpA-I/A-II (Fig 6). After 30 minutes, the amount of [³H]CEs was significantly higher than the initial amount (P<.05, Student's t test).

View larger version (21K): [in this window] [in a new window]   Figure 6. Graph showing transfer of [3H]CEs between –LpA-I and –LpA-I/A-II. After 20 minutes of incubation with [3H]cholesterol-labeled fibroblasts, normal plasmas were supplemented with 2 mmol/L DTNB (final concentration) to inhibit LCAT and then incubated without cells for different time intervals. Plasma samples were then separated by nonimmunosubtracting or anti–apoA-II–immunosubtracting 2D-PAGGE as described in the legend to Fig 3. Subsequent to lipid extraction, CEs and UC were separated by thin-layer chromatography, and their radioactivity was counted by liquid scintillation spectrophotometry. Each point shows the mean±SD of three experiments as counts per minute released into the various lipoproteins.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Various separation methods have been used to investigate the structural and functional heterogeneity of HDL.⁵ Among these, 2D-PAGGE differentiates HDL subfractions according to their charge and size.¹² In combination with prior short incubations of plasma with [³H]cholesterol-labeled cells, this method greatly helped our understanding of the role of quantitatively minor HDL subfractions such as preß₁–LpA-I, preß₃–LpA-I, and LpE, since it allows the analysis of native plasma without prior isolation of lipoproteins.^{12 13 15 27 28 29} However, a major drawback of 2D-PAGGE is that it fails to distinguish between the components of the bulk fraction of HDL that has electrophoretic -mobility and is heterogeneous in its apolipoprotein composition. For example, sequential immunoaffinity chromatography differentiates apoA-I–containing particles with apoA-II, ie, LpA-I/A-II, from those without apoA-II, ie, LpA-I.^{6 30 31 32} Studies with isolated lipoproteins yielded inconclusive data on the role of LpA-I and LpA-I/A-II in reverse cholesterol transport.^{5 7 8 9 10 30 31 32} Some authors have found that LpA-I but not LpA-I/A-II promotes cholesterol efflux,^{7 8} while others have found no differences between these particles.^{9 10} These discrepancies have been attributed to possible differences in the regulation of cholesterol homeostasis in various cells³⁰ and to artifactual changes in the composition of isolated lipoproteins.¹¹ In this study, we combined 2D-PAGGE with an immunoadsorption step that helped us to differentiate –LpA-I and –LpA-I/A-II. In combination with incubations of plasma with radiolabeled cells, anti–apoA-II immunosubtracting 2D-PAGGE has at least two advantages over sequential immunoaffinity chromatography: First, it allows analysis of small volumes of plasma from different individuals. Second, it allows direct comparison of the cholesterol efflux abilities of –LpA-I and –LpA-I/A-II in native plasma. Thereby, artifactual changes in lipoprotein composition caused by the isolation procedure are avoided. Moreover, the potential influence of additional plasma factors on the role of –LpA-I and –LpA-I/A-II in reverse cholesterol transport is also monitored. Using immunosubtracting 2D-PAGGE, we actually provide further evidence for different roles of –LpA-I and –LpA-I/A-II in reverse cholesterol transport.

The rapid uptake of [³H]cholesterol during incubation of plasma with radiolabeled cells and the rapid loss of [³H]cholesterol during incubations without cells has once more confirmed that preß₁–LpA-I is an initial acceptor of cellular cholesterol.^{12 13} During incubations either with or without cells, two to four times more radioactivity accumulated in –LpA-I than in –LpA-I/A-II (Fig 3). One may argue that the enhanced accumulation of radioactivity in –LpA-I simply reflects nonproductive exchange between labeled cholesterol in cell membranes and unlabeled cholesterol in plasma lipoproteins. However, although a single LpA-I particle contains 10% to 60% more UC than a single LpA-I/A-II particle, LpA-I/A-II particles are present in plasma at a higher number and account for 1.4- to 2.6-fold more UC in plasma.^{33 34 35 36} Thus, since our experiments have been performed on total plasma, nonproductive equilibration of labeled and unlabeled cholesterol as the only basis of [³H]cholesterol efflux from cells would have led to the preferential accumulation of radiolabel in LpA-I/A-II. Therefore, our data indicate that –LpA-I is the more effective contributor to cholesterol efflux. This is in agreement with data reported by de la Llera Moya and colleagues,¹¹ who determined the "cholesterol efflux potential" of serum using Fu5AH rat hepatoma cells in a 4-hour incubation. They found that cholesterol efflux is more closely correlated with LpA-I (r=.57) than with LpA-I/A-II (r=.26). Comparing the regression coefficients, they suggested that the cholesterol efflux potential of LpA-I is double that of LpA-I/A-II. This estimation is in agreement with the data we obtained by incubations for longer than 10 minutes (Fig 4).

In this study, we found that incubation of plasma with [³H]cholesterol-labeled fibroblasts leads to the accumulation of twofold to threefold more [³H]CEs in –LpA-I than in –LpA-I/A-II (Fig 5). Previously, Francone et al¹⁶ and Miida et al¹⁷ reported that cell-derived cholesterol is esterified predominantly in preß₃–LpA-I, whereas LDL-derived cholesterol is esterified in –LpA-I. We recently showed that only a minor proportion of cell-derived cholesterol is esterified directly during its passage through preß₃–LpA-I, whereas the majority is transferred to LDL without prior esterification, then to be recycled to –LpA-I for esterification.¹³ In view of these conflicting views, the pronounced accumulation of [³H]CEs in –LpA-I may be the result of either preferential uptake of CEs from preß₃–LpA-I or preferential esterification in –LpA-I. The latter explanation is in agreement with the previous observation that LpA-I contains more LCAT than LpA-I/A-II.^{30 37} Since esterification of cholesterol in the plasma compartment is considered to be the driving force for net cholesterol efflux, probably by maintaining a cholesterol concentration gradient between plasma membranes and lipoprotein surfaces,^{38 39 40} the more effective esterification of cholesterol in –LpA-I is one possible explanation for the better ability of –LpA-I to take up cellular cholesterol. In agreement with this model, Ohta and colleagues³⁷ found that the higher ability of LpA-I to promote cholesterol efflux from macrophages is associated with its higher content of LCAT compared with LpA-I/A-II.

Incubations of plasma in the presence of an LCAT inhibitor revealed that –LpA-I serves as a net donor of CEs to other lipoproteins, whereas –LpA-I/A-II serves as a net acceptor of CEs (Fig 6). Our data do not allow us to conclude whether –LpA-I/A-II receives CEs from –LpA-I or from other lipoproteins that are involved in the esterification of cholesterol, such as preß₃–LpA-I. Miida and colleagues¹⁷ reported that CEs or preß₃–LpA-I are transferred to a subgroup of -HDL, from which they are less available for CETP-mediated transfer to LDL. By contrast, CEs that are generated from LDL-derived cholesterol in another subgroup of -HDL appear to be much more available for transfer by CETP.¹⁷ These observations raise the interesting question of whether –LpA-I/A-II corresponds to this sink for CEs, which are generated in preß₃–LpA-I. Since transfer of CEs to apoB-containing lipoproteins is considered to be the most important pathway for the catabolism of HDL cholesterol in men,^{2 3} since LpA-I/A-II is catabolized more slowly than LpA-I,²¹ and since CEs in LpA-I/A-II are less available for selective uptake by the liver⁴¹ and hydrolysis by hepatic lipase,^{42 43} the accumulation of CEs in –LpA-I/A-II may indicate a delayed catabolism of CEs and, thereby, impaired reverse cholesterol transport.

In summary, our studies on reverse cholesterol transport in native plasma have demonstrated that –LpA-I is more effective than –LpA-I/A-II in both uptake and esterification of cell-derived cholesterol. Moreover, CEs are transferred from –LpA-I to other lipoproteins but accumulate in –LpA-I/A-II. Future studies will have to prove whether this accumulation of CEs in –LpA-I/A-II implies impaired reverse cholesterol transport. Such a negative role of apoA-II is reflected by the previous observations that mice overexpressing either murine apoA-II or both human apoA-I and human apoA-II are more susceptible to diet-induced atherosclerosis than control mice and human apoA-I transgenic mice, respectively.^{44 45}

	Selected Abbreviations and Acronyms

 

apoA-I = apolipoprotein A-I CE = cholesteryl ester CETP = cholesteryl ester transfer protein LCAT = lecithin:cholesterol acyltransferase LpA-I = lipoprotein A-I without A-II LpA-I/A-II = lipoprotein A-I with A-II 2D-PAGGE = two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis UC = unesterified cholesterol

	Acknowledgments

 
This work was supported by grants from the Wissenschaftsministerium Nordrhein-Westfalen (Benningsen-Foerder-Preis) and the Deutsche Forschungsgemeinschaft (Ec 116, 3-1) to Dr von Eckardstein. We gratefully acknowledge Dr Ali Chirazi for determination of LCAT activity and Claudia Heinrichs and Cornelia Elsenheimer for the measurement of lipids and apolipoproteins. We are indebted to Dr Paul Cullen for critically reading our manuscript.

Received February 21, 1995; accepted June 9, 1995.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Gordon D, Rifkind BM. Current concepts: high density lipoproteins: the clinical implications of recent studies. N Engl J Med. 1989;321:1311-1315. [Medline] [Order article via Infotrieve]
2. Eisenberg S. High density lipoprotein metabolism. J Lipid Res. 1984;25:1017-1058. [Medline] [Order article via Infotrieve]
3. Tall AR. Plasma high density lipoproteins: metabolism and relationship to atherogenesis. J Clin Invest. 1990;86:379-384.
4. Johnson WJ, Mahlberg FH, Rothblat GH, Phillips MC. Cholesterol transport between cells and high density lipoproteins. Biochim Biophys Acta. 1987;1085:273-298.
5. von Eckardstein A, Huang Y, Assmann G. Physiological role and clinical relevance of high-density lipoprotein subclasses. Curr Opin Lipidol. 1994;5:404-416. [Medline] [Order article via Infotrieve]
6. Cheung MC, Albers JJ. Characterization of lipoprotein particles isolated by immunoaffinity chromatography: particles containing AI and AII and particles containing AI but no AII. J Biol Chem. 1984;259:12201-12209. [Abstract/Free Full Text]
7. Barbaras R, Puchois P, Fruchart JC, Ailhaud G. Cholesterol efflux from cultured adipose cells is mediated by LpAI particles but not by LpAI:AII particles. Biochem Biophys Res Commun. 1987;142:63-69. [Medline] [Order article via Infotrieve]
8. Barkia A, Puchois P, Ghalim N, Torpier G, Barbaras R, Ailhaud G, Fruchart CJ. Differential role of apolipoprotein AI-containing particles in cholesterol efflux from adipose cells. Atherosclerosis. 1991;87:135-146. [Medline] [Order article via Infotrieve]
9. Johnson WJ, Kilsdonk EPG, van Tol A, Phillips MC, Rothblat GH. Cholesterol efflux from cells to immunopurified subfractions of human high density lipoprotein: LpA-I and LpA-I:A-II. J Lipid Res. 1991;32:1992-2000.
10. Oikawa S, Mendez AJ, Oram JF, Bierman EL, Cheung MC. Effects of high density lipoprotein particles containing apoA-I, with or without apoA-II, on intracellular cholesterol efflux. Biochim Biophys Acta. 1993;1165:327-334. [Medline] [Order article via Infotrieve]
11. de la Llera Moya M, Atger V, Paul JL, Fournier N, Moatti N, Giral P, Friday KE, Rothblat GH. A cell culture system for screening human serum for ability to promote cellular cholesterol efflux. Arterioscler Thromb. 1994;14:1056-1065. [Abstract/Free Full Text]
12. Castro GR, Fielding CJ. Early incorporation of cell-derived cholesterol into pre-ß-migrating high density lipoprotein. Biochemistry. 1988;27:25-29. [Medline] [Order article via Infotrieve]
13. Huang Y, von Eckardstein A, Assmann G. Cell-derived unesterified cholesterol recycles between low density lipoproteins and different high density lipoproteins for its effective esterification. Arterioscler Thromb. 1992;13:445-458. [Abstract/Free Full Text]
14. Asztalos BF, Sloop CH, Wong L, Roheim PS. Two-dimensional electrophoresis of plasma lipoproteins: recognition of new apoA-I-containing subpopulations. Biochim Biophys Acta. 1993;1169:291-300. [Medline] [Order article via Infotrieve]
15. Huang Y, von Eckardstein A, Wu S, Maeda N, Assmann G. A plasma lipoprotein containing only apolipoprotein E and with gamma-mobility on electrophoresis releases cholesterol from cells. Proc Natl Acad Sci U S A. 1994;91:1834-1838. [Abstract/Free Full Text]
16. Francone OL, Gurakar A, Fielding CJ. Distribution and functions of lecithin:cholesterol acyltransferase and cholesterol ester transfer protein in plasma lipoproteins. J Biol Chem. 1989;264:7066-7072. [Abstract/Free Full Text]
17. Miida T, Fielding CJ, Fielding PE. Mechanism of transfer of LDL-derived free cholesterol to HDL subfractions in human plasma. Biochemistry. 1990;29:10469-10474. [Medline] [Order article via Infotrieve]
18. von Eckardstein A, Huang Y, Wu S, Funke H, Noseda G, Assmann G. Reverse cholesterol transport in plasmas of patients with different forms of familial high density lipoprotein deficiency. Arterioscler Thromb Vasc Biol. 1995;15:691-703. [Abstract/Free Full Text]
19. Assmann G, Schriewer H, Schmitz G, Hagele E. Quantification of high-density lipoprotein cholesterol by precipitation with phosphotungstic acid/MgCl₂. Clin Chem. 1983;29:2025-2029.
20. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low density lipoprotein in plasma without use of a preparative ultracentrifuge. Clin Chem. 1972;18:449-456. [Abstract]
21. Sandkamp M, Tambyrajah B, Schriewer H, Assmann G. Simplified turbidimetric determination of apolipoproteins A-I, A-II and B using a microtitre method. J Clin Chem Clin Biochem. 1988;26:685-689. [Medline] [Order article via Infotrieve]
22. Parra HJ, Mezdour H, Ghalim N, Bard N, Fruchart JC. Differential electroimmunoassay of human LpA-I lipoprotein particles on ready-to-use plates. Clin Chem. 1990;36:1431-1435. [Abstract/Free Full Text]
23. Rader DJ, Castro G, Zech LA, Fruchart JC, Brewer B. In vivo metabolism of apolipoprotein AI on high density lipoprotein particles LpA-I and LpA-I:A-II. J Lipid Res. 1991;32:1849-1859. [Abstract]
24. Pritchard PH, Mcleod R, Frohlich J, Park MC, Kudchodkar BJ, Lacko AG. Lecithin:cholesterol acyltransferase in familial HDL-deficiency. Biochem Biophys Acta. 1988;958:227-234. [Medline] [Order article via Infotrieve]
25. Fielding CJ, Frohlich J, Moser K, Fielding PE. Promotion of sterol efflux and net transport by apolipoprotein E in lecithin:cholesterol acyltransferase deficiency. Metabolism. 1982;31:1023-1028. [Medline] [Order article via Infotrieve]
26. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275. [Free Full Text]
27. Francone OL, Fielding CJ. Initial steps in reverse cholesterol transport: the role of short-lived cholesterol acceptors. Eur Heart J. 1990;11(suppl E):218-224.
28. Kawano M, Miida T, Fielding CJ, Fielding PE. Quantitation of preß-HDL-dependent and nonspecific components of the total efflux of cellular cholesterol and phospholipid. Biochemistry. 1993;32:5025-5028. [Medline] [Order article via Infotrieve]
29. Fielding PE, Kawano M, Catapano AL, Zoppo A, Marcovina S, Fielding CJ. Unique epitope of apolipoprotein A-I expressed in pre-ß-1 high-density lipoprotein and its role in the catalyzed efflux of cellular cholesterol. Biochemistry. 1994;33:6981-6985. [Medline] [Order article via Infotrieve]
30. Fruchart JC, Ailhaud G, Bard JM. Heterogeneity of high density lipoprotein particles. Circulation. 1993;87(suppl III):III-22-III-27.
31. Cheung MC. Characterization of apolipoprotein A-containing lipoproteins. Methods Enzymol. 1986;129:130-139. [Medline] [Order article via Infotrieve]
32. Fruchart JC, Ailhaud G. Apolipoprotein A-containing particles: physiological role, quantification and clinical significance. Clin Chem. 1992;38:793-797. [Abstract/Free Full Text]
33. Kilsdonk EPC, Van Gent E, Van Tol A. Characterization of human high-density lipoprotein subclasses LpA-I and LpA-I/A-II and binding to HepG2 cells. Biochim Biophys Acta. 1990;1045:205-212. [Medline] [Order article via Infotrieve]
34. Barkia A, Puchois P, Ghalim N, Torpier G, Barbaras R, Ailhaud G, Fruchart JC. Differential role of apolipoprotein AI-containing particles in cholesterol efflux from adipose cells. Atherosclerosis. 1991;87:135-146.
35. Ohta T, Hattori S, Nishiyama S, Matsuda I. Studies on the lipid and apolipoprotein compositions of two species of apoA-I-containing lipoproteins in normolipidemic males and females. J Lipid Res. 1988;29:721-728. [Abstract]
36. James RW, Pometta D. Postprandial lipemia differentially influences high density lipoprotein subpopulations LpA-I and LpA-I,AII. J Lipid Res. 1994;35:1583-1591. [Abstract]
37. Ohta T, Nakamura R, Ikeda Y, Shinohara M, Miyazaki A, Horiuchi S, Matsuda I. Differential effect of subspecies of lipoprotein containing apolipoprotein A-I on cholesterol efflux from cholesterol-loaded macrophages: functional correlation with lecithin:cholesterol acyltransferase. Biochim Biophys Acta. 1992;1165:119-128. [Medline] [Order article via Infotrieve]
38. Glomset JA. The plasma lecithin:cholesterol acyltransferase reaction. J Lipid Res. 1968;9:155-167. [Abstract]
39. Fielding CJ, Fielding PE. Cholesterol transport between cells and body fluids: role of plasma lipoprotein and the plasma cholesterol esterification system. Med Clin North Am. 1982;66:363-373. [Medline] [Order article via Infotrieve]
40. Fielding CJ. The origin and properties of free cholesterol potential gradients in plasma, and their relation to atherogenesis. J Lipid Res. 1984;25:1624-1628. [Medline] [Order article via Infotrieve]
41. Pieters MN, Castro G, Schouten R, Duchateau D, Fruchart JC, van Berkel TJC. Cholesterol esters selectively delivered in vivo by high density lipoprotein subclass LpA-I to rat liver are processed faster into bile acids than are LpA-I/A-II derived cholesteryl esters. Biochem J. 1993;292:819-823.
42. Thuren T, Wilcox RW, Sisson P, Waite P. Hepatic lipase hydrolysis of lipid: regulation by apolipoproteins. J Biol Chem. 1991;266:4853-4861. [Abstract/Free Full Text]
43. Zhong S, Goldberg IJ, Bruce C, Rubin E, Breslow JL, Tall A. Human apoA-II inhibits the hydrolysis of HDL triglyceride and the decrease of HDL size induced by hypertriglyceridemia and cholesterol ester transfer protein in transgenic mice. J Clin Invest. 1994;94:2457-2467.
44. Schultz JR, Verstuyft JG, Gong EL, Nichols AV, Rubin EM. Protein composition determines the antiatherogenic properties of HDL in transgenic mice. Nature. 1993;364:73-74. [Medline] [Order article via Infotrieve]
45. Warden CH, Hedrick CC, Qiao JH, Castellani JW, Lusis AL. Atherosclerosis in transgenic mice overexpressing apolipoprotein A-II. Science. 1993;261:469-472.[Abstract/Free Full Text]

This article has been cited by other articles:

				E. M. M. Ooi, G. F. Watts, P. J. Nestel, D. Sviridov, A. Hoang, and P. H. R. Barrett Dose-Dependent Regulation of High-Density Lipoprotein Metabolism with Rosuvastatin in the Metabolic Syndrome J. Clin. Endocrinol. Metab., February 1, 2008; 93(2): 430 - 437. [Abstract] [Full Text] [PDF]

				J. Delgado-Lista, F. Perez-Jimenez, T. Tanaka, P. Perez-Martinez, Y. Jimenez-Gomez, C. Marin, J. Ruano, L. Parnell, J. M. Ordovas, and J. Lopez-Miranda An Apolipoprotein A-II Polymorphism (-265T/C, rs5082) Regulates Postprandial Response to a Saturated Fat Overload in Healthy Men J. Nutr., September 1, 2007; 137(9): 2024 - 2028. [Abstract] [Full Text] [PDF]

				J. Ji, G. F. Watts, A. G. Johnson, D. C. Chan, E. M. M. Ooi, K.-A. Rye, A. P. Serone, and P. H. R. Barrett High-Density Lipoprotein (HDL) Transport in the Metabolic Syndrome: Application of a New Model for HDL Particle Kinetics J. Clin. Endocrinol. Metab., March 1, 2006; 91(3): 973 - 979. [Abstract] [Full Text] [PDF]

				N. Fournier, A. Cogny, V. Atger, D. Pastier, D. Goudouneche, A. Nicoletti, N. Moatti, J. Chambaz, J.-L. Paul, and A.-D. Kalopissis Opposite Effects of Plasma From Human Apolipoprotein A-II Transgenic Mice on Cholesterol Efflux From J774 Macrophages and Fu5AH Hepatoma Cells Arterioscler. Thromb. Vasc. Biol., April 1, 2002; 22(4): 638 - 643. [Abstract] [Full Text] [PDF]

				L. Saucan and E. A. Brinton Lp A-I and Niacin: New Views of an Antiatherogenic Duo Arterioscler. Thromb. Vasc. Biol., November 1, 2001; 21(11): 1707 - 1709. [Full Text] [PDF]

				F. M. van 't Hooft, G. Ruotolo, S. Boquist, U. de Faire, G. Eggertsen, and A. Hamsten Human Evidence That the Apolipoprotein A-II Gene Is Implicated in Visceral Fat Accumulation and Metabolism of Triglyceride-Rich Lipoproteins Circulation, September 11, 2001; 104(11): 1223 - 1228. [Abstract] [Full Text] [PDF]

				A. Tailleux, M. Bouly, G. Luc, G. Castro, J.-M. Caillaud, N. Hennuyer, P. Poulain, J.-C. Fruchart, N. Duverger, and C. Fievet Decreased Susceptibility to Diet-Induced Atherosclerosis in Human Apolipoprotein A-II Transgenic Mice Arterioscler. Thromb. Vasc. Biol., November 1, 2000; 20(11): 2453 - 2458. [Abstract] [Full Text] [PDF]

				M. Jauhiainen, J. Huuskonen, M. Baumann, J. Metso, T. Oka, T. Egashira, H. Hattori, V. M. Olkkonen, and C. Ehnholm Phospholipid transfer protein (PLTP) causes proteolytic cleavage of apolipoprotein A-I J. Lipid Res., April 1, 1999; 40(4): 654 - 664. [Abstract] [Full Text]

				M. Lee, A. von Eckardstein, L. Lindstedt, G. Assmann, and P. T. Kovanen Depletion of Preß1LpA1 and LpA4 Particles by Mast Cell Chymase Reduces Cholesterol Efflux From Macrophage Foam Cells Induced by Plasma Arterioscler. Thromb. Vasc. Biol., April 1, 1999; 19(4): 1066 - 1074. [Abstract] [Full Text] [PDF]

				A. Ribeiro, D. Pastier, D. Kardassis, J. Chambaz, and P. Cardot Cooperative Binding of Upstream Stimulatory Factor and Hepatic Nuclear Factor 4 Drives the Transcription of the Human Apolipoprotein A-II Gene J. Biol. Chem., January 15, 1999; 274(3): 1216 - 1225. [Abstract] [Full Text] [PDF]

				P. J. Pussinen, M. Jauhiainen, J. Metso, L. E. Pyle, Y. L. Marcel, N. H. Fidge, and C. Ehnholm Binding of phospholipid transfer protein (PLTP) to apolipoproteins A-I and A-II: location of a PLTP binding domain in the amino terminal region of apoA-I J. Lipid Res., January 1, 1998; 39(1): 152 - 161. [Abstract] [Full Text]