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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1755-1763

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1755-1763.)
© 1995 American Heart Association, Inc.

Articles

Lipoproteins Containing Apolipoprotein A-IV but Not Apolipoprotein A-I Take Up and Esterify Cell-Derived Cholesterol in Plasma

Arnold von Eckardstein; Yadong Huang; Shili Wu; Ahmad Saadat Sarmadi; Sigrid Schwarz; Armin Steinmetz; Gerd Assmann

From the Institut für Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfälische Wilhelms-Universität Münster (A.von E., A.S.S., G.A.), FRG; the Institut für Arterioskleroseforschung an der Universität Münster (Y.H., S.W., G.A.); and the Abteilung für Endokrinologie und Stoffwechsel, Zentrum Innere Medizin, Philipps-Universität Marburg (S.S., A.S.).

Correspondence to Arnold von Eckardstein, Institut für Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfälische Wilhelms-Universität Münster, Albert-Schweitzer-Strasse 33, D-48129 Münster, FRG.


*    Abstract
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 *Abstract
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Abstract Two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (2D-PAGGE) identifies distinct apoA-I– or apoE-containing subclasses of high-density lipoproteins (HDLs), each of which plays a different role in reverse cholesterol transport. In this study we used 2D-PAGGE to investigate the role of apoA-IV–containing lipoproteins in reverse cholesterol transport in native plasma. Incubation of 2D electrophoretograms with anti–apoA-IV antibodies identified up to three subclasses of particles. The smaller particle subclasses, LpA-IV-1 and LpA-IV-2, were found in every plasma sample. The largest particle subclass, LpA-IV-3, was observed in fewer than 10% of the plasmas analyzed. 2D-PAGGE of apoA-I–deficient plasma and apoA-I–depleted plasma and anti–apoA-I immunosubtracting 2D-PAGGE of normal plasma revealed that LpA-IV-1 and LpA-IV-2 do not contain apoA-I. The importance of LpA-IV-1 and LpA-IV-2 for uptake and esterification of cell-derived cholesterol was investigated using pulse-chase incubations of plasma with [3H]cholesterol-labeled fibroblasts followed by anti–apoA-I immunosubtracting 2D-PAGGE. During 1-minute pulse incubation with cells, [3H]cholesterol was taken up by {gamma}-LpE >LpA-IV-1 >pre-ß1-LpA-I >LpA-IV-2 (">" denotes "more than"). During subsequent chase incubation without cells, proportionately less radioactivity disappeared from LpA-IV-1 and LpA-IV-2 than from pre-ß1-LpA-I and {gamma}-LpE. During 5-minute pulse incubations, radioactive cholesteryl esters were formed in pre-ß3-LpA-I >{alpha}-LpA-I >LpA-IV-1 >LpA-IV-2. The fractional esterification rate was highest in pre-ß3-LpA-I and lowest in {alpha}-LpA-I. Subsequent chase led to the disappearance of [3H]cholesteryl esters from pre-ß3-LpA-I and, to a lesser extent, from LpA-IV-1 and LpA-IV-2 but to an increase of [3H]cholesteryl esters in {alpha}-LpA-I and LDL. Similar pulse-chase experiments with apoA-I–deficient plasma revealed that LpA-IV-1 and LpA-IV-2 take up and esterify cell-derived cholesterol even more effectively than in normal plasma. We conclude that LpA-IV-1 and LpA-IV-2 are apoA-I–free lipoproteins that are important contributors to reverse cholesterol transport.


Key Words: lecithin:cholesterol acyltransferase • HDL subfractions • reverse cholesterol transport • cholesterol efflux • nondenaturing two-dimensional electrophoresis


*    Introduction
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 *Introduction
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The reverse cholesterol transport model is widely used to explain the protective role of HDLs in atherogenesis. In this model, HDLs mediate the efflux of excess cholesterol from peripheral cells into plasma as well as the esterification of cholesterol in plasma by LCAT. From HDL, CEs are subsequently delivered to the liver by several mechanisms. In humans, the most important pathway apparently is the exchange of CEs with triglycerides from LDL and VLDL that is mediated by CETP (reviewed in References 1 through 31 2 3 ). HDLs, however, encompass 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 44 ). 2D-PAGGE of plasma in the sequence agarose gel electrophoresis followed by PAGGE and subsequent immunoblotting characterizes HDL-subclasses by charge, size, and antigenicity.4 Immunodetection with anti–apoA-I antibodies differentiates pre-ß1-LpA-I, pre-ß2-LpA-I, pre-ß3-LpA-I, and {alpha}-LpA-I.2 5 6 7 Previously, pulse-chase experiments identified the quantitatively minor HDL subclass pre-ß1-LpA-I as being the initial acceptor of cell-derived cholesterol.2 5 6 From pre-ß1-LpA-I, which contains apoA-I as the only apolipoprotein, cell-derived cholesterol is rapidly transferred to LDL via pre-ß2-LpA-I->pre-ß3-LpA-I->{alpha}-LpA-I.2 5 6 LCAT esterifies cholesterol either directly during its passage through pre-ß3-LpA-I or in {alpha}-LpA-I after recycling from LDL.2 6 8 9

The absence of premature atherosclerosis in some patients with apoA-I deficiency10 11 stimulated us to search for HDL subclasses that are free of apoA-I but that fulfill important functions in reverse cholesterol transport. We previously identified {gamma}-LpE, a lipoprotein that contains apoE as the only protein and that also serves as an initial acceptor of cell-derived cholesterol.12 Other apoA-I–free lipoproteins that may play some role in reverse cholesterol transport are those containing apoA-IV (reviewed in Reference 1313 ). ApoA-IV is secreted by enterocytes into the lymph fluid as a component of chylomicrons.14 15 16 17 18 In fasting plasma, apoA-IV is found in HDL and in the lipoprotein-free fraction.16 17 18 19 20 21 22 23 Sequential anti–apoA-IV and anti–apoA-I immunoaffinity chromatography has identified apoA-IV–containing particles that also contain apoA-I (LpA-I/A-IV) and particles containing ApoA-IV alone (LpA-IV).23 The latter particle contains more than 90% of the apoA-IV in fasting plasma. Both LpA-I/A-IV and LpA-IV are poor in lipids, which are almost entirely phospholipids and triglycerides.23 Both particles contain activities for LCAT and CETP and stimulate cholesterol efflux from cells in vitro.23 24 To better understand the in vivo relevance of apoA-IV–containing lipoproteins in reverse cholesterol transport, we analyzed the contribution of LpA-IV to the uptake, esterification, and transfer of cell-derived cholesterol in native plasma. Similar to previously reported experiments that helped to investigate the role of apoA-I- and apoE-containing HDL subclasses, we used pulse-chase incubations of native plasma with [3H]cholesterol-labeled fibroblasts and subsequent 2D-PAGGE.2 4 5 6 12 Since LpA-IV contains little UC,23 nonspecific equilibration of labeled and unlabeled molecules does not contribute much to the appearance of radioactivity in these lipoproteins. However, since apoA-IV–containing lipoproteins have electrophoretic properties similar to those of {alpha}-LpA-I and pre-ß1-LpA-I,4 we modified the 2D-PAGGE system by introducing an electrophoretic immunoadsorption step. This anti–apoA-I immunosubtracting 2D-PAGGE allowed us to investigate the role of LpA-IV in reverse cholesterol transport in native plasma. In addition, to test this procedure against a valid control and to demonstrate the importance of apoA-I–free lipoproteins in reverse cholesterol transport, we also analyzed the plasma of an apoA-I–deficient patient.11


*    Methods
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Plasma Samples
Electrophoretic analyses and pulse-chase experiments were performed on plasma samples from three normolipidemic individuals and a woman with apoA-I deficiency.11 Table 1Down summarizes characteristics of the lipid metabolism of the normolipidemic proband and the apoA-I–deficient patient whose data have been selected for presentation in Tables 2 through 5DownDownDownDown. All four subjects had the phenotypes apoA-IV-1/1 and apoE3/3.25 Moreover, we analyzed 20 different plasma samples by nondenaturing 2D electrophoresis and subsequent anti–apoA-IV immunoblotting to investigate possible effects of the apoA-IV polymorphism on the electrophoretic appearance of apoA-IV–containing lipoproteins. Blood samples were collected after overnight fasting and immediately cooled on ice. Plasma and sera were obtained by centrifugation at 4°C (3000g, 15 minutes), divided into aliquots, and frozen at -70°C. 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.


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  		Table 1. Characteristics of Lipoprotein Metabolism of the Probands Analyzed


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  		Table 2. Uptake and Transfer of Cell-Derived [3H]Cholesterol Through Various Lipoproteins in a Normoalphalipoproteinemic Plasma


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  		Table 3. Uptake and Transfer of Cell-Derived [3H]Cholesterol Through Various Lipoproteins in ApoA-I–Deficient Plasma


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  		Table 4. Esterification of Cell-Derived [3H]Cholesterol Through Various Lipoproteins in Normoalphalipoproteinemic Plasma


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  		Table 5. Esterification of Cell-Derived [3H]Cholesterol Through Various Lipoproteins in ApoA-I–Deficient Plasma

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). LDL cholesterol was calculated by using the Friedewald formula.26 Concentrations of apoA-I, apoA-II, and apoB were determined with a modified commercially available turbidimetric assay (Boehringer Mannheim).27 ApoA-IV was quantified by electroimmunodiffusion using a rabbit anti-human apoA-IV antiserum.28 Plasma activities of LCAT and CETP were determined as described previously.25

Isolation of Lipoproteins
LpA-IV and LpA-I/A-IV were isolated from plasma by sequential anti–apoA-IV and anti–apoA-I immunoaffinity chromatography.23

Nondenaturing 2D Electrophoresis
The distribution of apoA-I–, apoE-, and apoA-IV–containing lipoproteins in normal plasma was analyzed by 2D-PAGGE, whereby agarose gel electrophoresis was followed by PAGGE.5 6 Briefly, in the first dimension, 20 µL of each plasma sample was separated by electrophoresis at 4°C in a 0.75% agarose gel using 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 bromphenol blue had been 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 PAGGE were electroblotted onto a nitrocellulose membrane. ApoA-I– and apoE-containing lipoproteins were detected by the use of sheep antibodies against human apoA-I or human apoE (Boehringer Mannheim), respectively, which had been biotinylated following the manufacturer's recommendations (Sigma), and streptavidin-biotinylated horseradish peroxidase complex (Amersham). ApoA-IV–containing lipoproteins were identified by the use of a rabbit anti-human apoA-IV antiserum,28 a biotinylated donkey anti-rabbit antiserum (Amersham), and streptavidin–horseradish peroxidase.

Demonstration of LpA-IV by Anti–ApoA-I Immunosubtracting 2D-PAGGE
To circumvent contamination of LpA-IV with {alpha}-LpA-I and pre-ß1-LpA-I, we modified the 2D-PAGGE procedure described above. Agarose gel electrophoresis was followed by immunosubtracting electrophoresis in a 2% to 20% polyacrylamide gradient gel that was covered by a 4-cm broad 0.6% agarose gel that contained the {gamma}-globulin fraction of a goat anti–apoA-I antiserum (Boehringer Mannheim) at a final concentration of 4% (vol/vol). This immunoprecipitating gel prevented all apoA-I–containing lipoproteins from migrating into the polyacrylamide gel. Electrophoresis and Western blotting were performed under the conditions described for 2D-PAGGE. Complete removal of apoA-I–containing lipoproteins was ascertained by anti–apoA-I immunoblotting.

Cell Culture
Normal human skin fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum using dishes of 3-cm diameter as described previously.6 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), which had been complexed with fetal calf serum. The final specific radioactivity in the labeled cells amounted to 1.3±0.4x107 counts per minute (cpm)/mg cell UC (mean±SD).

Cholesterol Efflux Experiments
To determine the cholesterol efflux from cells into the different lipoprotein subfractions, 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 measure radioactivity in LpA-IV, 20 µL plasma was separated by anti–apoA-I immunosubtractive 2D-PAGGE. To determine radioactivity in {gamma}-LpE, pre-ß1-LpA-I, and {alpha}-LpA-I, 20 µL plasma was separated by 2D-PAGGE. In parallel, an aliquot of 20 µL unlabeled plasma from the same donor was run in the same gel. After completion of 2D electrophoresis, one half of the gel containing the labeled sample was stored at 4°C. Proteins in the other half of the gel were electroblotted onto a nitrocellulose membrane to immunolocalize apoA-I–, apoE-, or apoA-IV–containing lipoproteins. These immunoblots were then used as templates to localize pre-ß1-LpA-I, pre-ß3-LpA-I, {alpha}-LpA-I, {gamma}-LpE, LpA-IV-1, LpA-IV-2, and LDL in the native gels. Since pre-ß3-LpA-I was not detectable in every anti–apoA-I immunoblot, positive immunoblots of previous experiments were used for localization. The lipoproteins were cut out and their lipids extracted by incubation with chloroform/methanol (2:1, vol/vol) for 72 hours.29 In some experiments UC and CEs were separated by thin-layer chromatography on silica gel plates (Merck) as the immobile phase and hexane/ether (6:4, vol/vol) as the mobile phase. The radioactivity in the lipids was counted by scintillation spectrometry.

Every experiment was performed three times on each plasma sample from three normolipidemic donors and the apoA-I–deficient patient. 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,30 about 90% of the radioactivity in apoB-free plasma was recovered in apoA-I– and apoE-containing HDL subfractions separated by nondenaturing 2D-PAGGE.

Other Procedures
Protein concentrations were measured according to Lowry's method31 using bovine serum albumin as the standard. SDS-PAGE was performed according to the protocol published by Laemmli.32 For statistical comparisons we used the Student's t test.


*    Results
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 *Results
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Characterization of ApoA-I–, ApoE-, and ApoA-IV–Containing Lipoproteins in Normal Plasma by 2D-PAGGE
Fig 1Down presents the distribution of apoA-I–, apoE-, and apoA-IV–containing lipoproteins, respectively, after separation of normal plasma by 2D-PAGGE. As described previously,2 5 6 7 apoA-I was detected in a major HDL subfraction with {alpha}-mobility as well as in three minor subfractions with pre-ß mobility that differ by their size (pre-ß1-LpA-I, pre-ß2-LpA-I, and pre-ß3-LpA-I) (Fig 1aDown). Pre-ß3-LpA-I was immunodetectable in only a few Western blots. LDL also reacted with anti–apoA-I antibodies. ApoE was detectable in a particle that is mainly colocalized with {alpha}-LpA-I and in a quantitatively minor subfraction with electrophoretic {gamma}-mobility that we previously termed {gamma}-LpE.10 In some Western blots, LDL immunoreacted with anti-apoE (Fig 1bDown). ApoA-IV was visualized in three particles (Fig 1cDown and 1dDown). Two of them, termed LpA-IV-1 and LpA-IV-2, form sharp bands that exhibit electrophoretic "slow {alpha}-mobility"; ie, on agarose gel electrophoresis they migrate faster than pre-ß-LpA-I but slower than {alpha}-LpA-I (Fig 1cDown). We observed these two particles in all of 23 analyzed normolipidemic samples. The third particle, termed LpA-IV-3, forms a diffuse spot, has electrophoretic {alpha}-mobility, and is larger than LpA-IV-1 and LpA-IV-2. In contrast to LpA-IV-1 and LpA-IV-2, LpA-IV-3 was observed in only 2 of 23 normolipidemic plasmas analyzed (Fig 1dDown).



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  		Figure 1. 2D electrophoresis and immunoblotting of apolipoprotein (apo) A-I– (a), apoE- (b), and apoA-IV–containing lipoproteins (c and d) in normoalphalipoproteinemic human plasma. Nondenaturing 2D electrophoresis of 40 µL normal plasma was performed in the sequence agarose gel electrophoresis->nondenaturing polyacrylamide gel electrophoresis. After electroblotting to nitrocellulose membranes, apoA– and apoE-containing lipoproteins were detected using biotinylated polyclonal sheep antisera against either human apoA-I (a) or human apoE (b) and streptavidin-biotinylated horseradish peroxidase complex. ApoA-IV–containing lipoproteins were visualized by the use of a rabbit anti-human apoA-IV antiserum, a biotinylated donkey anti-rabbit IgG antiserum, and streptavidin–horseradish peroxidase (c and d). LpA-IV-1 and LpA-IV-2 (c and d) were observed in every plasma sample and LpA-IV-3 (d) only in a few samples. The asterisk denotes the position of pre-ß3-LpA-I, which was only detectable in a minority of anti–apoA-I immunoblots.

Electrophoretic Differentiation of LpA-IV and LpA-I/A-IV
To analyze which of the electrophoretic LpA-IV particles correspond to LpA-I/A-IV and LpA-IV, both LpA-I/A-IV and LpA-IV were isolated from plasma by sequential immunoaffinity chromatography and separated by either 2D-PAGGE or SDS-PAGE. 2D-PAGGE and subsequent anti–apoA-IV immunoblotting of LpA-I/A-IV did not lead to the visualization of any particle (Fig 2aDown). After SDS-PAGE of 100 µg lyophilized LpA-I/A-IV, apoA-I was identified on protein staining (Fig 3aDown, lane 1), whereas apoA-IV became detectable only by anti–apo-IV immunoblotting (Figure 3bDown, lane 1). Thus, LpA-I/A-IV appears to contain only trace amounts of apoA-IV that escaped detection by 2D-PAGGE and anti–apoA-IV immunoblotting. In contrast, 2D-PAGGE and anti–apoA-IV immunoblotting of LpA-IV revealed the presence of LpA-IV-1 and LpA-IV-2 (Fig 2bDown). On SDS-PAGE of LpA-IV, apoA-IV was already detectable in Coomassie Blue–stained gels. No apoA-I was detectable in this particle either on protein staining (Fig 3aDown, lane 2) or immunoblotting (Fig 3bDown, lane 2). In another attempt to differentiate LpA-IV and LpA-I/A-IV, plasma of an apoA-I–deficient patient was separated by 2D-PAGGE. As expected, we did not find any anti–apoA-I–immunoreactive particles (Fig 4aDown), whereas apoE-containing particles including {gamma}-LpE were present (Fig 4bDown). We observed apoA-IV–containing particles with electrophoretic properties similar to those of LpA-IV-1, LpA-IV-2, and LpA-IV-3 (Fig 4cDown). Together these data indicate (1) that LpA-IV-1 and LpA-IV-2 do not contain apoA-I and (2) that apoA-I does not appear to be an obligate constituent of LpA-IV-3. Moreover, LpA-I/A-IV could not be localized in our nondenaturing 2D electrophoretograms.



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  		Figure 2. Nondenaturing 2D-PAGGE and anti–apoA-IV immunoblotting of LpA-I/A-IV (a) and LpA-IV (b). LpA-IV and LpA-I/A-IV were isolated from plasma by sequential anti–apoA-IV and anti–apoA-I immunoaffinity chomatography.23 2D-PAGGE was performed as described in Fig 1Up. Note that LpA-I/A-IV does not contain amounts of apoA-IV high enough for immunnodetection, whereas LpA-IV contains LpA-IV-1 and LpA-IV-2.



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  		Figure 3. SDS-PAGE of LpA-IV and LpA-I/A-IV. LpA-IV and LpA-I/A-IV were isolated from plasma by sequential immunoaffinity chomatography. For SDS-PAGE, 100 µg lipoproteins per lane were lyophilized and separated in a 12% acrylamide gel according to the recommendations of Laemmli.32 a, Coomassie Blue-staining; b, anti–apoA-I and anti–apoA-IV immunoblot. a and b, lanes No. 1 contain LpA-I/A-IV; lanes No. 2 LpA-IV. Note that LpA-I/A-IV contained amounts of apoA-I high enough for detection by protein staining (a, lane 1). In this particle, apoA-IV became visible only after anti–apoA-IV immunoblotting (b, lane 1). By contrast, LpA-IV contained apoA-IV at amounts that allowed its detection by protein staining (a, lane 2). In this sample, apoA-I did not become visible after anti–apoA-I immunoblotting (b, lane 2).



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  		Figure 4. Demonstration of apoA-I– (a), apoE- (b), and apoA-IV– (c) containing lipopoteins in the plasma of a patient with apoA-I deficiency. ApoA-I–deficient plasma11 30 was separated by 2D-PAGGE as described in Fig 1Up. Anti–apoA-I immunoblotting does not detect lipoproteins (a). Anti-apoE immunoblotting identified two particles, one with {gamma}-mobility ({gamma}-LpE) and another with {alpha}-mobility ({alpha}-LpE) (b). Anti–apoA-IV immunoblotting detected LpA-IV-1 and LpA-IV-2 and a lipoprotein with the electrophoretic properties of LpA-IV-3 (c).

Cholesterol Efflux From Cells Into LpA-IV-1 and LpA-IV-2
LpA-IV-1 and LpA-IV-2 exhibit electrophoretic mobilities that are similar to those of pre-ß1-LpA-I and {alpha}-LpA-I (cf Fig 1Up). To isolate LpA-IV-1 and LpA-IV-2 from 2D-PAGGE-gels without contamination of apoA-I–containing lipoproteins, we used anti–apoA-I immunosubtracting 2D-PAGGE. This method led to the detection of LpA-IV-1 and LpA-IV-2 but not of LpA-IV-3 (Fig 5aDown). The complete removal of apoA-I by the immunoadsorbing agarose gel was confirmed by anti–apoA-I immunoblotting (Fig 5bDown).



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  		Figure 5. Demonstration of apoA-IV–containing lipoproteins by anti–apoA-I immunosubtracting 2D-PAGGE. Anti–apoA-I immunosubtracting 2D-PAGGE is a modification of 2D-PAGGE, whereby agarose gel electrophoresis is followed by nondenaturing electrophoresis in a 2% to 20% polyacrylamide gradient gel, which is covered by a 4-cm-wide agarose gel containing 4% anti–apoA-I antiserum. Anti–apoA-IV immunoblotting detected LpA-IV-1 and LpA-IV-2 (a). Subsequent incubation of the blot with anti–apoA-I antibodies verified the absence of apoA-I–containing lipoproteins (b).

Table 2Up presents the distribution of radioactive cholesterol in various lipoproteins of a normal plasma after a 1-minute pulse incubation with [3H]cholesterol-labeled fibroblasts and a 1-minute chase incubation without cells. {gamma}-LpE, pre-ß1-LpA-I, {alpha}-LpA-I, and LDL were isolated from the gel after 2D-PAGGE, LpA-IV-1 and LpA-IV-2 after anti–apoA-I immunosubtracting 2D-PAGGE. LpA-IV-1 took up 30% more radioactivity than did pre-ß1-LpA-I (P<.05) and 30% less than {gamma}-LpE (P<.01). Only small amounts of [3H]cholesterol appeared in LpA-IV-2. During the subsequent chase incubation only 25% and 33% of the initial radioactivity disappeared from LpA-IV-1 and LpA-IV-2 (NS), respectively, whereas the radioactivity decreased by 65% in {gamma}-LpE and pre-ß1-LpA-I (both P<.01). In parallel radioactivity increased by about 80% in LDL and about 30% in {alpha}-LpA-I.

Table 3Up presents the distribution of radioactive cholesterol in various lipoproteins of apoA-I–deficient plasma. After a 1-minute pulse, similar amounts of radioactivity were detectable in LpA-IV-1 and {gamma}-LpE. Significantly higher amounts of [3H]cholesterol accumulated in LpA-IV-1 of apoA-I–deficient plasma compared with LpA-IV-1 of normal plasma (P<.05; cf Table 2Up). Considerably less radioactivity was detectable in LpA-IV-2; only background radioactivity was found in pre-ß1-LpA-I. As described previously,32 considerable amounts of radioactivity were also found in a particle with the electrophoretic properties of {alpha}-LpA-I; however, that particle did not contain apoA-I. Because LpA-IV-1 and LpA-IV-2 comigrate with this fraction, the radioactivity is at least partially attributable to LpA-IV-1 and LpA-IV-2. During a 1-minute chase, only 25% of the initial radioactivity disappeared from LpA-IV-1 (NS) but 75% disappeared from {gamma}-LpE (P<.01). Almost all of the radiolabel appeared in LDL.

Esterification of Cell-Derived Cholesterol in LpA-IV
Table 4Up summarizes the outcomes of our studies on the relative contribution in normal plasma of pre-ß3-LpA-I, {alpha}-LpA-I, LpA-IV-1, LpA-IV-2, and LDL to the esterification of cell-derived [3H]cholesterol and transfer of [3H]CEs. After a 5-minute pulse-incubation with [3H]cholesterol-labeled fibroblasts, radioactive CEs were recovered in pre-ß3-LpA-I, {alpha}-LpA-I, LpA-IV-1, LpA-IV-2, and LDL. Pre-ß3-LpA-I contained the highest amount of [3H]CE and had the highest FER (ie, ratio of [3H]CE to [3H]UC+[3H]CE). {alpha}-LpA-I contained twice as much [3H]CE as did LpA-IV-1 and LpA-IV-2 (P<.01). However, the FER was higher in apoA-IV–containing particles. During the subsequent 5-minute chase the incubation without cells but in the presence of the LCAT-inhibitor DTNB, 65% of [3H]CE initially present in pre-ß3-LpA-I disappeared (P<.01) compared with 37% and 21% of the [3H]CE initially present in LpA-IV-1 and LpA-IV-2, respectively (both P<.01). In contrast, {alpha}-LpA-I and LDL accumulated significant amounts of [3H]CE during this chase incubation.

Table 5Up summarizes the results of pulse-chase experiments that were performed to investigate cholesterol esterification and CE transfer in apoA–deficient plasma. Both LpA-IV-1 and LpA-IV-2 contributed to the esterification of cholesterol in apoA-I–deficient plasma. Neither the amounts of radioactive CE nor the FERs in LpA-IV-1 and LpA-IV-2 differed significantly between normal and apoA-I–deficient plasmas (cf Table 4Up). LpA-IV-3 also accumulated cell-derived [3H]cholesterol, but significantly lower proportions were esterified compared with LpA-IV-1 and LpA-IV-2 (P<.01). The transfer of [3H]CE from LpA-IV-1 and LpA-IV-2 to other lipoproteins was faster in apoA-I–deficient plasma than in normal plasma. In contrast, LpA-IV-3 accumulated [3H]CE during the chase incubation.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Previous studies with apoA-IV–containing lipoproteins either isolated from plasma22 23 or reconstituted in vitro24 33 34 35 36 37 38 39 have provided considerable evidence that apoA-IV contributes to reverse cholesterol transport. In fasting plasma, apoA-IV is associated with lipoproteins of high or very high density.22 23 Incubation of apoA-IV with phospholipids or with cell membranes leads to the formation of small HDL-like particles.33 34 In vitro, apoA-IV activates LCAT.35 36 As an activator of a putative HDL-conversion factor, apoA-IV is involved in remodeling of HDL.37 ApoA-IV in both native and reconstituted lipoproteins has been shown to promote cholesterol efflux from various cells.22 23 24 38 39 Since apoA-IV easily dissociates from lipoproteins both in vivo and in vitro,22 23 24 40 41 42 little is known of the role of apoA-IV–containing lipoproteins in reverse cholesterol transport in vivo. Thus, it is unclear how much apoA-IV is to be found in apoA-I–containing lipoproteins21 22 23 43 and whether apoA-IV interacts with cellular HDL-binding sites.22 24 44 45 46 In this study we investigated for the first time the role of apoA-IV–containing lipoproteins in the uptake, esterification, and transfer of cell-derived cholesterol in native plasma.

2D-PAGGE and subsequent anti–apoA-IV immunoblotting discriminated three particles: LpA-IV-1, LpA-IV-2, and LpA-IV-3. Like pre-ß1-LpA-I and {gamma}-LpE but unlike {alpha}-LpA-I, both LpA-IV-1 and LpA-IV-2 form sharp bands, probably because they are homogeneous lipid-poor particles.23 Since LpA-IV-1 and LpA-IV-2 were detectable in LpA-IV, in apoA-I–free, and in apoA-I–deficient plasmas but not in LpA-IV/A-I, we conclude that these particles do not contain apoA-I. Our data do not allow any definite conclusion on the electrophoretic appearance of LpA-I/A-IV. The absence of LpA-IV-3 in apoA-I–depleted plasma might indicate that LpA-I/A-IV occurs principally as LpA-IV-3. However, we found an LpA-IV-3–like particle in the plasma of an apoA-I–deficient patient and were unable to detect LpA-IV-3 in LpA-I/A-IV. ApoA-I is probably not an obligate constituent of LpA-IV-3. The finding of LpA-IV-1 and LpA-IV-2 in every plasma sample, including those from apoA-I–deficient individuals, supports the data of Duverger et al, who concluded that most of the apoA-IV in fasting plasma resides on particles free of apoA-I.23 This is in contrast to other authors who reported that apoA-IV predominantly occurs in apoA-I–containing particles.21 22 43

In combination with prior incubation of plasma with [3H]cholesterol-labeled fibroblasts, 2D-PAGGE has previously helped to improve our understanding of how important reverse cholesterol transport is to the quantitatively minor HDL-subfractions such as pre-ß1-LpA-I, preß3-LpA-I, and {gamma}-LpE.2 4 5 6 12 30 Since these particles are very poor in UC, any appearance of radioactivity within them indicates the specific movement of [3H]cholesterol from cell membranes to the particles rather than nonspecific exchange of radiolabeled and unlabeled cholesterol molecules. LpA-IV is also a lipid-poor particle.23 Therefore, it is unlikely that uptake of [3H]cholesterol by LpA-IV-1 and LpA-IV-2 reflects nonproductive equilibration but rather indicates that these particles serve as initial acceptors of cell-derived cholesterol into the plasma compartment. Interestingly, LpA-IV-1 of normal plasma took up more radioactivity than pre-ß1-LpA-I and less radioactivity than {gamma}-LpE (Table 2Up). In this context it is also noteworthy that we previously found that the plasmas of patients with Tangier disease or apoA-I deficiency accumulate considerable amounts of cell-derived cholesterol in a fraction with the electrophoretic properties of small {alpha}-LpA-I (ie, {alpha}-LpA-I3), although apoA-I was immunologically undetectable in this region of the gel.30 Because the electrophoretic properties of LpA-IV-1 and LpA-IV-2 are similar to those of {alpha}-LpA-I3, it is likely that these apoA-IV–containing particles contributed to this phenomenon. This also indicates that apoA-IV–containing lipoproteins are important contributors to the residual activity of HDL-deficient plasmas to release cholesterol from cells.30

During chase-incubations of either normal or apoA-I deficient plasma, significantly smaller proportions of [3H]cholesterol were removed from LpA-IV-1 and LpA-IV-2 than from pre-ß1-LpA-I and {gamma}-LpE. Unlike pre-ß1-LpA-I and {gamma}-LpE, which transfer cell-derived cholesterol to other particles for further esterification and catabolism,4 5 6 8 9 12 47 both LpA-IV-1 and LpA-IV-2 are involved in the esterification of cholesterol (Table 4Up). With respect to the FER, LpA-IV-1 and LpA-IV-2 are less effective than pre-ß3-LpA-I but more effective than {alpha}-LpA-I, two other lipoproteins containing LCAT activity.6 8 9 Like pre-ß3-LpA-I, LpA-IV-1 and LpA-IV-2 serve as net donors of CE to other lipoproteins.6 8 However, the fractional transfer rate is significantly lower in LpA-IV-1 and LpA-IV-2 than in pre-ß3-LpA-I. This raises the question of whether LpA-IV-1 and LpA-IV-2 catabolize cholesterol by mechanisms other than CETP-mediated transfer of CE to apoB-containing lipoproteins, which has been thought to be the most important pathway for removal of CEs from HDL.48 In this regard it is noteworthy that turnover studies have shown that apoA-IV is catabolized much more rapidly than apoA-I.49 Since apoA-IV mediates binding of lipoproteins to liver cells,44 45 46 it is possible that the short-lived LpA-IV catabolizes cholesterol of peripheral cells by hepatic uptake and degradation, whereas the long-lived LpA-I catabolizes cholesterol by transfer to LDL and VLDL, which are more rapidly removed by the liver than HDL.

In conclusion, this study demonstrates for the first time the importance of apoA-IV–containing lipoproteins in reverse cholesterol transport in native plasma. Other than {gamma}-LpE, LpA-IV-1 and LpA-IV-2 provide further examples of apoA-I–free HDL subclasses help to release cholesterol from cells. Unlike pre-ß1-LpA-I, and {gamma}-LpE, LpA-IV-1 and LpA-IV-2 are initial acceptors of cell-derived cholesterol and are also involved in the production and removal of CEs. LpA-IV-1 and LpA-IV-2 may catabolize cell-derived cholesterol directly instead of by transferring CEs to apoB-containing lipoproteins. Like {gamma}-LpE, LpA-IV-1 and LpA-IV-2 may serve as a backup system for reverse cholesterol transport and may contribute to the antiatherogenic role of HDL when apoA-I–containing particles are absent or severely reduced in concentration.10 11 12 23


*    Selected Abbreviations and Acronyms
 
apo = apolipoprotein
CE(s) = cholesteryl ester(s)
CETP = cholesterol ester transfer protein
2D-PAGGE = two-dimensional polyacrylamide gradient gel electrophoresis
FER = fractional esterification rate
LCAT = lecithin:cholesterol acyltransferase
Lp = lipoprotein
PAGE = polyacrylamide gel electrophoresis
UC = unesterified cholesterol


*    Acknowledgments
 
This work was supported by the grants from Wissenschaftsministerium Nordrhein-Westfalen (Benningsen-Foerder-Preis) and Deutsche Forschungsgemeinschaft to Dr von Eckardstein (Ec 116,3-1) and to Dr Steinmetz (Ste 381,5-1). We gratefully acknowledge the excellent technical assistance of Anja Merschjann, Sabine Motzny, and Martina Plüster. We are grateful to Dr G. Noseda for providing us with plasma of the apoa-I–deficient patient and to Dr Paul Cullen for skillful review of the manuscript.

Received June 8, 1995; accepted August 2, 1995.


*    References
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 *References
 

  1. Eisenberg S. High density lipoprotein metabolism. J Lipid Res. 1984;25:1017-1058. [Medline] [Order article via Infotrieve]
  2. Fielding CJ, Fielding PE. Molecular physiology of reverse cholesterol transport. J Lipid Res. 1995;36:211-228. [Abstract]
  3. Johnson WJ, Mahlberg FH, Rothblat GH, Phillips MC. Cholesterol transport between cells and high density lipoproteins. Biochim Biophys Acta. 1992;1085:273-298.
  4. 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]
  5. 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]
  6. Huang Y, von Eckardstein A, Assmann G. Cell-derived cholesterol cycles between different HDLs and LDL for its effective esterification in plasma. Arterioscler Thromb. 1993;13:445-458. [Abstract/Free Full Text]
  7. 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]
  8. 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]
  9. 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]
  10. Assmann G, von Eckardstein A, Funke H. High density lipoproteins, reverse transport of cholesterol, and coronary heart disease: insights from mutants. Circulation. 1993;87(suppl III):III-28-III-34.
  11. Römling R, von Eckardstein A, Funke H, Motti C, Fragiacomo G, Noseda G, Assmann G. A nonsense mutation in the apolipoprotein A-I gene is associated with high density lipoprotein deficiency but not with coronary heart disease. Arterioscler Thromb. 1994;14:1915-1923. [Abstract/Free Full Text]
  12. 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]
  13. Tenkanen H, Ehnholm C. Molecular biology of apolipoprotein A-IV. Curr Opin Lipidol. 1993;4:95-99.
  14. Weisgraber KH, Bersot TP, Mahley RW. Isolation and characterization of an apoprotein from the d <1.006 lipoproteins of human and canine lymph homologous with the rat A-IV apoprotein. Biochem Biophys Res Commun. 1978;85:287-292. [Medline] [Order article via Infotrieve]
  15. Utermann G, Beisiegel U. Apolipoprotein A-IV, a protein occurring in human mesenteric lymph chylomicrons and free in plasma: isolation and quantitation. Eur J Biochem. 1979;99:333-343. [Medline] [Order article via Infotrieve]
  16. Beisiegel U, Utermann G. An apolipoprotein homolog of rat apolipoprotein A-IV in human plasma: isolation and partial characterization. Eur J Biochem. 1979;93:601-608. [Medline] [Order article via Infotrieve]
  17. Green PHR, Glickman RM, Riley JW, Quinet E. Human apolipoprotein A-IV: intestinal origin and distribution in plasma. J Clin Invest. 1980;65:911-919.
  18. Bisgaier CL, Sachdev OP, Megna L, Glickmann M. Distribution of apolipoprotein A-IV in human plasma. J Lipid Res. 1985;26:11-25. [Abstract]
  19. Weinberg RB, Spector MS. The self association of apolipoprotein A-IV: Evidence for an in vivo circulating dimeric form. J Biol Chem. 1985;260:14279-14286. [Abstract/Free Full Text]
  20. Vezina CA, Milne RW, Weech PK, Marcel YL. Apolipoprotein distribution in human lipoproteins separated by polyacrylamide gradient gel electrophoresis. J Lipid Res. 1988;29:573-585. [Abstract]
  21. Lagrost L, Gambert P, Boquillon M, Lallemant C. Evidence for high density lipoproteins as the major apolipoprotein A-IV-containing fraction in normal human serum. J Lipid Res. 1989;30:1525-1534. [Abstract]
  22. Nowicka G, Brüning T, Böttcher A, Kahl G, Schmitz G. Macrophage interaction of HDL subclass separated by free flow isotachophoresis. J Lipid Res. 1990;31:1947-1961. [Abstract]
  23. Duverger N, Ghalim N, Ailhaud G, Steinmetz A, Fruchart J-C, Castro G. Characterization of apoA-IV-containing lipoprotein particles isolated from human plasma and interstitial fluid. Arterioscler Thromb. 1993;13:126-132. [Abstract/Free Full Text]
  24. Bisgaier CL, Siebenkas MV, Hesler CB, Swenson TL, Blum CB, Marcel YL, Milne RW, Glickman RM, Tall AR. Effect of neutralizing cholesteryl ester transfer protein on the redistribution of apolipoproteins A-IV and E among human lipoproteins. Lipid Res. 1989;30:1025-1031. [Abstract]
  25. von Eckardstein A, Funke H, Chirazi A, Chen-Haudenschild C, Schulte H, Schönfeld R, Köhler E, Schwarz S, Steinmetz A, Assmann G. Sex-specific effects of the glutamine/histidine polymorphism in apolipoprotein A-IV on high density lipoprotein metabolism. Arterioscler Thromb. 1994;14:1114-1120. [Abstract/Free Full Text]
  26. 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]
  27. 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]
  28. Steinmetz A, Czekelius P, Thiemann E, Motzny S, Kaffarnik H. Changes of apolipoprotein A-IV in the human neonate: evidence for different induction of apolipoproteins A-I and A-IV in the postpartum period. Atherosclerosis. 1988;69:21-27. [Medline] [Order article via Infotrieve]
  29. Folch JM, Lees M, Sloane GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957;226:497-509. [Free Full Text]
  30. 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]
  31. 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]
  32. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond). 1970;227:680-685. [Medline] [Order article via Infotrieve]
  33. Jonas A, Steinmetz A, Churgay L. The number of {alpha}–helical segments of apolipoproteins A-I, E, and A-IV determines the size and functional properties of their reconstituted lipoprotein particles. J Biol Chem. 1993;268:1596-1602. [Abstract/Free Full Text]
  34. Hara H, Yokoyama S. Interaction of free apolipoproteins with macrophages: formation of high density lipoprotein-like lipoproteins and reduction of cellular cholesterol. J Biol Chem. 1991;266:3080-3086. [Abstract/Free Full Text]
  35. Chen CH, Albers JJ. Activation of lecithin cholesterol acyltransferase by human apolipoproteins E-2, E-3, and A-IV. Biochim Biophys Acta. 1985;836:279-285. [Medline] [Order article via Infotrieve]
  36. Steinmetz A, Utermann G. Activation of lecithin cholesterol acyltransferase by human apolipoprotein A-IV. J Biol Chem. 1985;260:2258-2264. [Abstract/Free Full Text]
  37. Barter PJ, Rajaram OV, Chang LBF, Rye KA, Gambert P, Lagrost L, Ehnholm C, Fidge NH. Isolation of a high density lipoprotein conversion factor from human plasma: a possible role of apolipoprotein A-IV as its activator. Biochem J. 1988;254:179-184. [Medline] [Order article via Infotrieve]
  38. Stein O, Stein Y, Lefevre M, Roheim PS. The role of apolipoprotein A-IV in reverse cholesterol transport studied with cultured cells and liposomes derived from an ether analog of phosphatidylcholine. Biochim Biophys Acta. 1986;878:7-14. [Medline] [Order article via Infotrieve]
  39. Savion N, Gamliel A. Binding of apolipoprotein A-I and apolipoprotein A-IV to cultured bovine aortic endothelial cells. Arteriosclerosis. 1988;8:178-184. [Abstract/Free Full Text]
  40. Bisgaier CL, Sachdev OP, Lee ES, Williams KJ, Blum CB, Glickman RM. Effect of lecithin:cholesterol acyltransferase on distribution of apolipoprotein A-IV among lipoproteins of human plasma. J Lipid Res. 1987;28:693-703. [Abstract]
  41. Steinmetz A, Barbaras R, Ghalim N, Clavey V, Fruchart J-C, Ailhaud G. Human apolipoprotein A-IV binds to apolipoprotein A-I/A-II receptor sites and promotes cholesterol efflux from adipose cells. J Biol Chem. 1990;265:7859-7863. [Abstract/Free Full Text]
  42. Weinberg RB, Spector MS. The self association of human apolipoprotein A-IV: evidence for an in vivo circulating dimeric form. J Biol Chem. 1985;260:4914-4921. [Abstract/Free Full Text]
  43. Malmendier CL, Gambert P, Meunier S, Morgado P, Desgres J, d'Athis P, Lallemant C. In vivo metabolism of apolipoproteins A-IV and A-I associated with high density lipoprotein in normolipidemic subjects. J Lipid Res. 1991;32:801-808. [Abstract]
  44. Ghiselli G, Crump WL, Gotto AM. Binding of apo A-IV phospholipid complexes to plasma membranes of rat liver. Biochem Biophys Res Commun. 1985;139:122-129.
  45. Dvorin E, Gorder NL, Benson DM, Gotto AM. Apolipoprotein A-IV: a determinant for binding and uptake of high density lipoproteins by rat hepatocytes. J Biol Chem. 1986;261:15714-15720. [Abstract/Free Full Text]
  46. Weinberg RB, Patton CS. Binding of human apolipoprotein A-IV to human hepatocellular plasma membranes. Biochim Biophys Acta. 1990;1044:255-261. [Medline] [Order article via Infotrieve]
  47. Miida T, Kawano M, Fielding CJ, Fielding PE. Regulation of the concentration of pre-ß high density lipoprotein in normal plasma by cell membranes and lecithin:cholesterol acyltransferase activity. Biochemistry. 1992;31:11112-11117. [Medline] [Order article via Infotrieve]
  48. Tall AR. Plasma cholesteryl ester transfer protein. J Lipid Res. 1993;34:1255-1274. [Medline] [Order article via Infotrieve]
  49. Rader DJ, Schäfer JR, Lohse P, Verges B, Kindt M, Zech LA, Steinmetz A, Brewer HB Jr. Rapid in vivo transport and catabolism of human apolipoprotein A-IV-1 and slower catabolism of the apoA-IV-2 isoform. J Clin Invest. 1992;92:1009-1017.



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