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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:691-703.)
© 1995 American Heart Association, Inc.

Articles

Reverse Cholesterol Transport in Plasma of Patients With Different Forms of Familial HDL Deficiency

Arnold von Eckardstein; Yadong Huang; Shili Wu; Harald Funke; Giorgio Noseda; Gerd Assmann

From the Institut für Klinische Chemie und Laboratoriumsmedizin, Zentrallaboratorium, Westfälische Wilhelms-Universität (A. von E., H.F., G.A.), and the Institut für Arterioskleroseforschung an der Universität Münster (Y.H., S.W., G.A.), Münster, FRG, and the Ospedale Regionale della Beata Vergine, CH-Mendrisio, Switzerland (G.N.).

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

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract HDLs encompass structurally heterogenous lipoproteins that fulfill specific functions in reverse cholesterol transport. Two-dimensional nondenaturing gradient gel electrophoresis (2D-PAGGE) of normoalphalipoproteinemic plasma and subsequent immunoblotting with anti–apoA-I-antibodies differentiates pre-ß₁-LpA-I, pre-ß₂-LpA-I, pre-ß₃-LpA-I, -LpA-I₂, and -LpA-I₃. Immunodetection with anti-apoE antibodies differentiates -LpE and -LpE. Pulse-chase incubations of plasma with [³H]unesterified cholesterol ([³H]UC)–labeled fibroblasts and subsequent 2D-PAGGE revealed that cell-derived [³H]UC is taken up by pre-ß₁-LpA-I and -LpE. From these initial acceptors, [³H]UC is transferred to LDL via pre-ß₂-LpA-Ipre-ß₃-LpA-I-LpA-I. Some UC is esterified in pre-ß₃-LpA-I, and some is esterified in -LpA-I after its retransfer from LDL. In this study we investigated the effect of various forms of familial HDL deficiency on reverse cholesterol transport. Plasma samples of patients with various forms of HDL deficiency are characterized by the lack of specific HDL subclasses. ApoE-containing HDLs, including -LpE, are present in all kinds of HDL deficiency. However, all forms of LpA-I are absent in apoA-I–deficient plasma, pre-ß₃-LpA-I and -LpA-I from the plasma of patients with Tangier disease (TD), and pre-ß₃-LpA-I and large -LpA-I from the plasma of patients with lecithin:cholesterol acyltransferase (LCAT) deficiency and fish-eye disease (FED). After a 1-minute pulse with labeled fibroblasts, efflux of [³H]UC into HDL-deficient plasmas decreased, compared with normal plasma, by 49% (apoA-I deficiency), 36% (TD), 21% (LCAT deficiency), and 28% (FED). In apoA-I deficiency, only -LpE takes up cell-derived [³H]UC. In the three other HDL-deficiency states, cell-derived [³H]UC is initially taken up by both pre-ß₁-LpA-I and -LpE. The four HDL deficiencies are also characterized by differences in the esterification of cell-derived [³H]UC. No esterification occurs in LCAT-deficient plasma. In FED plasma, [³H]UC is esterified in LDL. In apoA-I deficiency and TD, however, [³H]UC is esterified in lipoproteins free of apoA-I and apoB. In the two latter cases, the transfer of [³H]cholesteryl ester to LDL is enhanced compared with normal plasma. The lack of specific HDL subclasses and the consequent changes in reverse cholesterol transport pathways differently affect net mass efflux of cholesterol from fibroblasts into HDL-deficient plasma. Compared with normoalphalipoproteinemic plasma, net cholesterol efflux from fibroblasts into plasma is reduced by 48%, 12%, 60%, and 34% in apoA-I deficiency, TD, LCAT deficiency, and FED, respectively. Removal of apoB-containing lipoproteins from plasma of patients with apoA-I deficiency, TD, LCAT deficiency, and FED further decreased net cholesterol efflux rates by 77%, 84%, 72%, and 64%, respectively, compared with a reduction of 39% in normoalphalipoproteinemic control plasma. In conclusion, various quantitatively minor HDL subfractions and LDL also present in HDL-deficient plasma effectively contribute to reverse cholesterol transport.

Key Words: HDL subclasses • apoA-I deficiency • familial LCAT deficiency • fish-eye disease • Tangier disease

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Several epidemiological and clinical studies have revealed an inverse correlation between the plasma concentration of HDL cholesterol and the risk of myocardial infarction (reviewed in Reference 1¹ ). The ability of HDL to protect the vessel wall from atherosclerosis has usually been explained by the reverse cholesterol transport model (reviewed in References 2 through 4^{2 3 4} ), in which HDL mediates the flux of excess cholesterol from peripheral cells to the liver. HDL, however, includes structurally and functionally heterogenous lipoproteins that can be differentiated on the basis of density, size, charge, and apolipoprotein composition.^{5 6 7 8} Pulse-chase incubations of plasma with [³H]cholesterol-labeled fibroblasts and subsequent nondenaturing two-dimensional gradient gel electrophoresis (2D-PAGGE) have helped to assign distinct roles to the various HDL subclasses in reverse cholesterol transport. From cell membranes, cholesterol is initially taken up by a subgroup of HDL that contains apoA-I as its only apolipoprotein and is termed pre-ß₁-LpA-I because of its electrophoretic pre-beta mobility⁹ and by another subclass of HDL that contains only apoE and is termed -LpE because of its electrophoretic gamma mobility.¹⁰ From pre-ß₁-LpA-I, cell-derived cholesterol is rapidly transferred to other lipoproteins in the order pre-ß₂-LpA-Ipre-ß₃-LpA-I-LpA-ILDL.¹¹ Details of cholesterol transfer subsequent to its uptake by -LpE are not fully understood, except that cholesterol ultimately accumulates in LDL. In normolipoproteinemic human plasma, lecithin:cholesterol acyltransferase (LCAT) directly esterifies a minor portion of cell-derived cholesterol during its passage through pre-ß₃-LpA-I; most cholesterol, however, is esterified in -LpA-I after recycling from LDL.^{11 12 13}

Several inborn errors of metabolism interfere with the formation of normal HDL.¹⁴ Some mutations in the apoA-I gene prevent synthesis and secretion of apoA-I. Clinically, the patients may present with xanthomatosis, atherosclerosis, and/or corneal opacifications.^{15 16 17 18 19 20} Mutations in the LCAT gene lead to the expression of two different clinical and biochemical phenotypes. Familial LCAT deficiency results from the complete failure to esterify cholesterol in the plasma compartment and is characterized by increases in the ratio of unesterified cholesterol (UC) to cholesteryl ester (CE).^{21 22 23} Affected patients suffer from corneal opacifications and nephropathy with proteinuria and renal insufficiency. By contrast, in fish-eye disease (FED), LCAT fails to esterify cholesterol in HDL but not in the apoB-containing lipoproteins (LpB). Clinically, this selective loss of -LCAT activity is characterized by the presence of massive corneal opacifications, which provide the name of the disease.^{24 25 26} In another form of partial LCAT deficiency characterized by the presence of corneal opacities and a normal UC-CE ratio, the ability of the patient's plasma to esterify radiolabeled cholesterol in VLDL, LDL, and HDL was reduced because of a decrease in LCAT mass.²⁷ The pathogenesis of Tangier disease (TD) is as yet unknown but is thought to mainly involve a disturbance of intracellular lipid transfer processes in macrophages and Schwann cells. Patients with TD present with abnormal tonsils, neuropathy, and hepatosplenomegaly.²⁸ Despite the absence or a severe reduction of HDL, most patients with these familial HDL deficiency syndromes appear not to be at increased risk for coronary disease. Therefore, we hypothesized that the maintenance of reverse cholesterol transport in both HDL-deficient and normal plasma does not depend on the major part of HDL but on the presence of subfractions that might compensate for one another. To prove this hypothesis, we performed pulse-chase incubations of plasma samples from HDL-deficient patients by using [³H]cholesterol-labeled fibroblasts. After separation of these plasma samples by nondenaturing 2D-PAGGE, we monitored the occurrence of radioactive cholesterol and CEs in the various HDL subclasses. This helped us to identify those lipoproteins that are involved in the initial uptake of cell-derived cholesterol as well as its subsequent transfer and esterification.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Subjects
Three normolipidemic probands and four patients with different forms of primary HDL deficiency were included in this study. Characteristics of their lipid metabolism are summarized in Table 1. The 32-year-old Italian woman with apoA-I deficiency is homozygous for a nonsense mutation in codon 32 of the apoA-I gene.²⁹ The truncated protein could not be detected in her plasma. She was not affected by premature atherosclerosis. The 60-year-old German patient with TD has been reported previously.³⁰ The patient with familial LCAT deficiency is homozygous for a mutation in codon 321 of the LCAT gene, which leads to replacement of a Thr by an Ile.²¹ FED was diagnosed in the 30-year-old German woman, who presented with typical corneal opacifications and selective loss of -LCAT activity. Like most of the German patients with FED who have been described,^{24 25} this woman was homozygous for a missense mutation in the LCAT gene, which leads to a ThrIle substitution at residue 123.

View this table: [in this window] [in a new window]   Table 1. Characteristics of Lipoprotein Metabolism of the Probands

Blood Samples
Blood samples were taken after the subjects had fasted overnight and immediately placed on ice. Plasma samples and sera were obtained by centrifugation at 4°C (2000g, 15 minutes), divided into aliquots, and frozen at -70°C. In former studies we found that freezing and thawing did not affect the ability of normal plasma to take up, esterify, and transfer cell-derived cholesterol. Serum was used for the quantification of lipids. LCAT and CE transfer protein activities were determined in EDTA-plasma. For experiments in which plasma was incubated with cells, streptokinase (Sigma Chemical Co) was used as the anticoagulant at a final concentration of 150 U/mL.

Quantification of Lipids, Apolipoproteins, and Lipid Transfer Enzyme Activities
Serum concentrations of triglycerides and cholesterol were quantified with an autoanalyzer (Hitachi/Boehringer). HDL cholesterol concentrations were measured after precipitation of LpB with phosphotungstic acid/MgCl₂ (Boehringer). LDL cholesterol was calculated with the Friedewald formula.³¹ Concentrations of apoA-I and apoB were determined with a modified commercially available turbidimetric assay (Boehringer Mannheim).³² LCAT activity was determined as the amount of esterified [³H]cholesterol that was incorporated into apoA-I–containing proteoliposomes.³³ The plasma activity of CE transfer protein was determined as the amount of [¹⁴C]cholesteryl oleate transferred from artificial apoA-I–containing proteoliposomes to LDL, as reported previously.^{34 35}

Preparation of Lipoproteins
LDL (d=1.019 to 1.063 g/mL) and HDL (d=1.063 to 1.21 g/mL) were isolated from fresh normal human plasma by standard preparative ultracentrifugation techniques and dialyzed against 10 mmol/L sodium phosphate buffer (PBS, pH 7.4) containing 0.15 mol/L NaCl.³⁶ In some experiments as indicated, apoB-free plasma was obtained by precipitation of LpB by phosphotungstic acid/MgCl₂ as recommended by the manufacturer (Boehringer Mannheim). The apoB-free supernatant was subsequently dialyzed against PBS (pH 7.4) and used in experiments for determining net cholesterol efflux. Complete removal of LpB was ascertained by immunoturbidimetry of apoB.³²

Nondenaturing 2D-PAGGE
The distribution of apoA-I– and apoE-containing lipoproteins in the plasma from normoalphalipoproteinemic and HDL-deficient probands was determined by nondenaturing 2D electrophoresis, in which agarose gel electrophoresis was followed by polyacrylamide gradient gel electrophoresis (2D-PAGGE).^{9 10} Briefly, in the first dimension, 20 µL of normal or HDL-deficient plasma was separated by electrophoresis at 4°C on a 0.75% agarose gel with a 50 mmol/L merbital buffer (pH 8.7, Serva). 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 4°C in a buffer system that has been described by Altland and coworkers.³⁷ After separation, the proteins in 2D-PAGGE gels were electroblotted onto a nitrocellulose membrane. ApoA-I– and apoE-containing lipoproteins were detected by the use of sheep antibodies against human apoA-I and human apoE, respectively (Boehringer Mannheim), which were biotinylated according to the manufacturer's recommendations (Sigma). The antigen-antibody complexes were visualized with a streptavidin-biotinylated horseradish peroxidase complex (Amersham) at a dilution of 1:1000. 4-Chloro-1-naphthol was used as the chromogen.

Cell Culture
Normal human skin fibroblasts were cultured in Dulbecco's modification of Eagle's minimum essential medium containing 10% fetal calf serum as described previously.³⁸ After 5 to 10 passages, cells were plated on 3.5-cm-diameter dishes for the pulse-chase experiments. When they were nearly confluent, the cells of some dishes were incubated for 72 hours at 37°C with 0.5 mCi [1,2-³H]cholesterol (51.7 Ci/mmol, New England Nuclear), which was complexed with fetal calf serum. Before the incubations with plasma, fibroblasts were washed six times with PBS (pH 7.4). The final specific radioactivity in the labeled cells then amounted to 5.2±1.4x10⁸ cpm/mg cell protein, or 1.7±0.8x10⁷ cpm/µg cell cholesterol (mean±SD).

Pulse-Chase Incubations With Fibroblasts
In pulse-chase experiments, 1 mL of complete plasma or apoB-free plasma was first incubated with labeled fibroblasts (pulse). After either 1 or 5 minutes of incubation, the plasma was removed and used for the chase incubations, which were performed in the absence of cells. Conditions and time intervals were varied as indicated in "Results." In some instances, LDL (50 µg protein) or 5,5'-dithio-bis(2-nitrobenzoic acid) at a final concentration of 1.5 mmol/L (DTNB, Sigma) was added to complete or apoB-free plasma before starting the chase incubations.

After the chase incubation, the plasma samples were either delipidated by the addition of chloroform/methanol (2:1, vol/vol)³⁹ or used for nondenaturing 2D electrophoresis. Typically, an unlabeled sample from a normoalphalipoproteinemic control subject and a labeled sample from a patient were run in parallel on one gel. One half of the gel containing the 2D electrophoretic pattern of the patient was stored at 4°C. The other half of the gel containing the pattern of the normoalphalipoproteinemic control subject was electroblotted onto a nitrocellulose membrane to immunolocalize the lipoproteins containing apoA-I and apoE. The immunoblot was then used as a template to localize the corresponding lipoproteins in the other half of the gel (Fig 1). These lipoproteins were cut out, and their lipids were extracted with chloroform/methanol (2:1, vol/vol) for 72 hours. In some experiments, the total radioactive cholesterol in various lipoprotein fractions was determined. To separately count their radioactivities, in other experiments UC and CE were first 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.

View larger version (50K): [in this window] [in a new window]   Figure 1. Two-dimensional (2D) electrophoresis and immunoblotting of apoA-I– (a) and apoE- (b) containing lipoproteins in normoalphalipoproteinemic human plasma. Nondenaturing 2D electrophoresis was performed in the sequence agarose gel electrophoresisnondenaturing polyacrylamide gel electrophoresis. After electroblotting to nitrocellulose membranes, apoA-I– and apoE-containing lipoproteins were detected with biotinylated polyclonal sheep antisera against either human apoA-I or human apoE and streptavidin-biotinylated horseradish peroxidase complex. c, Schematic summary of the localization of apoA-I– and apoE-containing lipoproteins in the gel. Rectangular fields represent areas removed from the gel for extraction of lipids from the indicated lipoproteins. Area 1, pre-ß1-LpA-I; 2, pre-ß2-LpA-I; 3, pre-ß3-LpA-I; 4, -LpA-I3; 5, -LpA-I2; 6, -LpE; 7, -LpE; and 8, LDL.

Determination of Cholesterol Net Mass Transfer
Net cholesterol mass transfer from fibroblasts describes the difference in the plasma concentrations of UC after a 2-hour incubation of plasma with and without fibroblasts and was determined as described previously.^{11 40} This test is based on the assumption that esterification of cholesterol will decrease the concentration of UC in both the presence and absence of fibroblasts. However, efflux of cellular cholesterol into the medium causes less of a decrease in UC in the sample that has been incubated with fibroblasts. In brief, after they were washed four times with PBS, dishes with confluently growing fibroblasts that had been preloaded with cholesterol for 48 hours⁴¹ and dishes without cells were incubated with 2 mL of 5% plasma or apoB-free plasma in PBS. The media were removed after a 2-hour incubation at 37°C. Lipids in 1 mL of total medium were extracted with chloroform/methanol (2:1, vol/vol) three times for 6 hours each. Cholesterol mass in the extracts was quantified by the use of a modified fluoroenzymatic assay as reported previously.⁴⁰

General Procedures
Total protein concentrations were measured according to the method of Lowry et al⁴² using bovine serum albumin as the standard. Every experiment was performed three times on plasma samples from each proband. In some instances percent values are presented. They represent the amount of [³H]UC in one particle as a percentage of total [³H]UC in all lipoproteins (ie, -LpE+pre-ß-LpA-I+-LpA-I+LDL). VLDL was not included in this calculation, because this large lipoprotein does not migrate into the polyacrylamide gradient gel.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Characterization of ApoA-I– and ApoE-Containing Lipoproteins in Normoalphalipoproteinemic and HDL-Deficient Plasma Samples
Fig 1 presents the distribution of apoA-I– and apoE-containing lipoproteins after nondenaturing 2D-PAGGE of plasma from a normoalphalipoproteinemic proband. As described previously,^{9 11} apoA-I can be immunochemically detected by 2D-PAGGE in a major HDL subfraction with -mobility (-LpA-I) as well as in three minor subfractions with pre-ß mobility that differ by size (pre-ß₁-LpA-I, pre-ß₂-LpA-I, and pre-ß₃-LpA-I). According to Fielding and colleagues,⁴³ we differentiated -LpA-I into smaller -LpA-I₃ with a Stokes' diameter of 7.5 to 9.5 nm and larger -LpA-I₂ with a Stokes' diameter of 9.6 to 12 nm (Fig 1c). LDL also reacted with anti–apoA-I antibodies. ApoE is immunolocalized in two particles; the bulk of apoE is present in a particle that mainly colocalizes with -LpA-I. A minor subfraction of apoE, however, is present in a distinct lipoprotein with -mobility and a Stokes' diameter of 12 to 14 nm, which we previously described as -LpE.¹⁰ In some Western blots, LDL immunoreacted with anti-apoE.

Fig 2 shows the distribution of apoA-I–containing lipoproteins in the plasma of patients with various forms of familial HDL deficiency. ApoA-I–containing lipoproteins were undetectable in apoA-I deficiency (Fig 2a) but present in TD, FED, and familial LCAT deficiency (Fig 2b through 2d). Plasma of the TD patient contained pre-ß₁-LpA-I and pre-ß₂-LpA-I at apparently normal concentrations but no pre-ß₃-LpA-I or -LpA-I (Fig 2b). Pre-ß₁-LpA-I and pre-ß₂-LpA-I were also present in the plasma of patients with FED and familial LCAT deficiency; in the latter group, these lipoprotein fractions were present in higher concentration (Fig 2c and 2d). Pre-ß₃-LpA-I was undetectable in FED and LCAT deficiency. In contrast to TD and apoA-I deficiency, in LCAT deficiency and FED some small -LpA-I particles with a Stokes' diameter of 7.5 to 9.5 nm, ie, -LpA-I₃, were visible. By contrast with normal plasma, LDL in HDL-deficient plasma did not react with anti–apoA-I antibodies.

View larger version (52K): [in this window] [in a new window]   Figure 2. Nondenaturing two-dimensional electrophoresis and immunoblotting of apoA-I–containing lipoproteins in plasma of patients with apoA-I deficiency (a), Tangier disease (TD) (b), fish-eye disease (FED) (c), and familial lecithin:cholesterol acyltransferase (LCAT) deficiency (d). For further details, see the legend to Fig 1.

Fig 3 presents the distribution of apoE-containing lipoproteins in the plasma of the HDL-deficient patients. -LpE was present in all plasma samples. -LpE was very heterogenous in the various HDL deficiency conditions. In apoA-I deficiency, -LpE covered a considerably larger area of the gel than did -LpE in normal plasma, indicating a greater heterogeneity in size (Fig 3a). TD plasma contained an anti-apoE immunoreactive particle with ß-mobility rather than -mobility on agarose gel electrophoresis (Fig 3b). By contrast with normoalphalipoproteinemic plasma and despite its heterogeneity in size and charge, -LpE never colocalized with anti–apoA-I immunoreactive particles in HDL-deficient plasma. Moreover, LDL was not detected by the use of anti-apoE antibodies.

View larger version (45K): [in this window] [in a new window]   Figure 3. Nondenaturing two-dimensional electrophoresis and immunoblotting of apoE-containing lipoproteins in plasma of patients with apoA-I deficiency (a), Tangier disease (TD) (b), fish-eye disease (FED) (c), and familial lecithin:cholesterol acyltransferase (LCAT) deficiency (d). For further details, see the legend to Fig 1.

Uptake and Transfer of Cell-Derived Cholesterol in Normal and HDL-Deficient Plasma
For practical reasons we performed all subsequently described studies on plasma samples that had been frozen at -70°C, because in a pilot study we had found that freezing and thawing of plasma did not affect the distribution of apoE- and apo A-I–containing subclasses.

To determine their ability to release cholesterol from cells, plasma samples from normoalphalipoproteinemic probands as well as HDL-deficient patients were incubated with [³H]UC-labeled cells for 1 minute (Table 2). Compared with that from normoalphalipoproteinemic plasma, the efflux of cell-derived [³H]cholesterol from HDL-deficient patients was reduced by 49%, 36%, 21%, and 28% in apoA-I deficiency, TD, FED, and LCAT deficiency, respectively (Table 2).

View this table: [in this window] [in a new window]   Table 2. Release of [3H]Cholesterol From Fibroblasts Into Normal or HDL-Deficient Plasma

Anti–apoA-I and anti-apoE immunoblots of 2D electrophoretograms of normal plasma were used as templates to localize apoA-I– and apoE-containing lipoproteins in native 2D electrophoretograms of normal and HDL-deficient plasma samples that had been pulsed with radiolabeled cellular cholesterol. Because the plasma samples of patients with FED and familial LCAT deficiency contained only small -LpA-I (Fig 2c and 2d), -LpA-I was divided into -LpA-I₃ (Stokes' diameter, 7.5 to 9.5 nm) and -LpA-I₂ (Stokes' diameter, 9.6 to 12.0 nm). -LpE was not considered separately for two reasons. First, in normoalphalipoproteinemic plasma, most -LpE colocalizes with -LpA-I₂. Second, -LpE is heterogenous in HDL deficiency. Table 3 summarizes the recoveries of radioactivity extracted from the gels compared with the radioactivity in total plasma, supernatants, and infranatants after precipitation of LpB with phosphotungstic acid/MgCl₂. These recoveries ranged from 80% to 90% and did not differ significantly between plasma samples from HDL-deficient patients and normoalphalipoproteinemic control subjects. Experiments on every sample were performed in triplicate and in independent series. The interassay coefficients of variation of the radioactivity recovered from plasma or the various HDL subfractions were below 20%.

View this table: [in this window] [in a new window]   Table 3. Recovery of [3H]Cholesterol Extracted From Lipoproteins After Nondenaturing Two-Dimensional Electrophoresis

Figs 4 and 5 show the presence of [³H]cholesterol in the various lipoproteins of normal and HDL-deficient plasma after a 1-minute pulse with radiolabeled fibroblasts (open bars) and an additional 1-minute chase without cells (hatched bars). Fig 5 represents the amount of [³H]UC in one particle as a percentage of total [³H]UC in all lipoproteins (ie, -LpE+pre-ß-LpA-I+-LpA-I+LDL). VLDL was not included in this calculation because this large lipoprotein does not migrate into the polyacrylamide gradient gel. Fig 4 gives absolute numbers as counts per minute in the various lipoproteins. Table 4 presents the initial efflux of cholesterol into the various lipoproteins as a percentage of [³H]cholesterol in the cells. After a 1-minute pulse of normal plasma (Fig 5a), the percentages of [³H]cholesterol in -LpE, pre-ß₁-LpA-I, pre-ß₂-LpA-I, pre-ß₃-LpA-I, -LpA-I₃, -LpA-I₂, and LDL amounted to 20±3%, 9±2%, 4±1%, 5±1%, 32±4%, 15±2%, and 15±2%, respectively. After an additional 1-minute chase, the radioactivity in -LpE and pre-ß₁-LpA-I decreased to 7±1% and 5±1% and simultaneously increased in -LpA-I₃, -LpA-I₂, and LDL to 40±4%, 18±2%, and 26±3%, respectively. As reported previously,^{9 10 11} the occurrence of [³H]cholesterol in the cholesterol-poor pre-ß₁-LpA-I and -LpE during the pulse and its disappearance from these particles and increase in -LpA-I during the chase indicate that in normal plasma, considerable proportions of cell-derived cholesterol are taken up first by both -LpE and pre-ß₁-LpA-I and then transferred to -LpA-I (mainly -LpA-I₃) and LDL.

View larger version (39K): [in this window] [in a new window]   Figure 4. Bar graphs showing uptake and transfer of cell-derived [3H]cholesterol through various lipoproteins of plasma from normoalphalipoproteinemic probands and HDL-deficient patients. Pulse incubations with radiolabeled fibroblasts were performed for 1 minute (open bars); chase incubations were performed without cells for another 1 minute (hatched bars). Plasma samples were then separated by nondenaturing two-dimensional (2D) polyacrylamide gradient gel electrophoresis. Anti–apoA-I and anti-apoE immunoblots of 2D electrophoretograms of normal plasma were used to localize lipoproteins (cf Fig 1). These were removed from the native gels, their lipids were extracted, and radioactivity was counted. For further details see the "Methods" section. Panels a, b, c, d, and e represent results obtained with plasma from normoalphalipoproteinemic probands (normal) and patients with apoA-I deficiency (def.), Tangier disease (TD), fish-eye disease (FED), and lecithin:cholesterol acyltransferase (LCAT) deficiency, respectively. Each bar shows the mean and SD of three experiments as counts per minute released into the various lipoproteins.

View larger version (36K): [in this window] [in a new window]   Figure 5. Bar graphs showing uptake and transfer of cell-derived [3H]cholesterol through various lipoproteins of plasma from normoalphalipoproteinemic probands and HDL-deficient patients. The figure shows the percent distribution of radioactivity among the various lipoproteins, as calculated from the data presented in Fig 4. See the legend to Fig 4 for details and explanation of abbreviations.

View this table: [in this window] [in a new window]   Table 4. Fractional Release of Cellular [3H]Cholesterol Into Different Lipoprotein Fractions in Normal and Various HDL-Deficient Plasma

In all forms of HDL deficiency, -LpE took up cell-derived [³H]cholesterol in normal amounts (Figs 4b through 4e and 5b through 5e and Table 4). Because of the reduced efflux of [³H]cholesterol into HDL-deficient plasma, the percentages of radioactivity in -LpE were higher in HDL-deficient plasma than in normoalphalipoproteinemic plasma samples, namely, 38±5%, 32±3%, 36±5%, and 34±5% in apoA-I deficiency, TD, FED, and LCAT deficiency, respectively. After a 1-minute chase, the radioactivity in -LpE decreased to approximately 10% in all forms of HDL deficiency. In contrast to the similar degree of uptake of cell-derived [³H]cholesterol by -LpE in all HDL-deficient plasma, efflux into LpA-I-subfractions varied widely in the different forms of HDL deficiency.

After a 1-minute pulse with apoA-I–deficient plasma (Figs 4b and 5b and Table 4), radioactivity was detectable only in -LpE, LDL, and a fraction with the mobility of -LpA-I₃. The latter particle, however, did not react with anti–apoA-I antiserum (cf Fig 2a). By contrast, no radioactivity was detected in the fraction with -LpA-I₂–like mobility, although this fraction included an apoE-containing lipoprotein (cf Fig 3a). After a 1-minute chase, [³H]cholesterol disappeared from -LpE and the -mobile lipoprotein and accumulated in LDL.

A 1-minute pulse incubation with TD plasma (Figs 4c and 5c and Table 4) led to the regular uptake of [³H]cholesterol by pre-ß₁-LpA-I and -LpE. No radioactivity was found in pre-ß₂-LpA-I, pre-ß₃-LpA-I, or -LpA-I₂, although -LpA-I contained apoE. Like apoA-I–deficient plasma but unlike normal plasma, pulse incubation with TD plasma led to the occurrence of small amounts of [³H]cholesterol in an apoA-I–free fraction with -LpA-I₃–like mobility (Figs 4c and 5c; cf Figs 2b and 3b). During chase incubations, [³H]cholesterol disappeared from all of these initial acceptors and accumulated in LDL (Figs 4c and 5c and Table 4).

Pulse incubations of plasma samples from patients with FED and LCAT deficiency led to efflux of cell-derived [³H]cholesterol into pre-ß₁-LpA-I and -LpE (Figs 4d, 4e, 5d, and 5e and Table 4) followed by a decrease in radioactivity in this fraction during chase incubation. In contrast to plasma from control subjects, apoA-I–deficient patients, and TD patients, high amounts of radioactivity accumulated in the pre-ß₂-LpA-I of both FED and LCAT-deficient plasma samples. As with the other two forms of HDL deficiency, only trace amounts of radioactivity were detected in fractions with the mobility of pre-ß₃-LpA-I and -LpA-I₂, although the latter fraction was anti-apoE immunoreactive.

The percentage of radioactivity in LDL after a 1-minute chase was increased in HDL-deficient compared with normal plasma (26±3%), LCAT-deficient and FED plasma (45±5%), and apoA-I–deficient and TD plasma (60±8%). However, because exogenous [³H]cholesterol and endogenous unlabeled cholesterol within plasma lipoproteins equilibrate by diffusion and 75% of UC in normal plasma and more than 90% of UC in HDL-deficient plasma are present in LDL, higher amounts of [³H]cholesterol in LDL of patients with HDL deficiency may simply reflect the disproportionate distribution of UC among the various lipoproteins in normal versus HDL-deficient plasma. To investigate this possibility, we prolonged the chase incubation periods to various time intervals (0 to 60 minutes) and subsequently precipitated LpB with phosphotungstic acid/MgCl₂. This procedure allowed us to separately determine the specific radioactivity (counts per minute of [³H]UC per microgram of UC) in the apoB-free supernatants and the apoB-containing infranatants (Fig 6). Without chase (time 0), the specific radioactivity of cell-derived [³H]UC in the supernatants of all plasma exceeded that in the infranatants (ie, LDL+VLDL) by a factor 2 to 3. During subsequent chases the specific radioactivity decreased in the supernatants but increased in the infranatants. In control plasma, specific radioactivity in the infranatant exceeded that in the supernatant after 22 minutes (Fig 6a). By contrast, in HDL-deficient plasma a higher specific radioactivity in the infranatant was already observed after 6 to 8 minutes (Fig 6b through 6e). This indicates that the transfer of cell-derived [³H]UC from the initial acceptors to LDL is enhanced in HDL-deficient compared with normal plasma.

View larger version (33K): [in this window] [in a new window]   Figure 6. Time course of changes in the distribution of cell-derived [3H]unesterified cholesterol (UC) in apoB-free () and apoB-containing () lipoproteins of plasma from normoalphalipoproteinemic probands (normal) and various HDL-deficient (def.) patients. Pulse incubations with radiolabeled fibroblasts for 1 minute were followed by chase incubations without cells for the indicated times. ApoB-free and apoB-containing lipoproteins were separated by precipitation with phosphotungstic acid/MgCl2. Subsequent to lipid extraction, radioactive UC and cholesteryl ester were separated by thin-layer chromatography. Specific radioactivity is expressed as counts per minute of [3H]UC per microgram of UC in apoB-free and apoB-containing lipoproteins. Each point shows the mean value and standard deviation of four experiments. TD indicates Tangier disease; FED, fish-eye disease; and LCAT, lecithin:cholesterol acyltransferase.

Esterification of Cell-Derived Cholesterol in Normal and HDL-Deficient Plasma
In subsequent studies, we compared the esterification of cell-derived UC and the transfer of CE to various lipoproteins of normal and HDL-deficient plasma. To obtain amounts of labeled cholesterol sufficient to differentiate between [³H]UC and [³H]CE, pulse incubations of plasma with radiolabeled fibroblasts were prolonged to 5 minutes and chase incubations to 15 minutes (Table 5). To inhibit LCAT, all chase incubations were done in the presence of 1.5 mmol/L DTNB. After a 5-minute pulse incubation, normal plasma esterified 3.6% of the [³H]cholesterol released from cells into plasma. This fractional esterification was increased to 4.3% in apoA-I deficiency and to 5.5% in TD. In FED we observed a slight decrease to 3.1%. As expected, no detectable amounts of [³H]CE were formed in LCAT-deficient plasma.

View this table: [in this window] [in a new window]   Table 5. Esterification of Cell-Derived [3H]Cholesterol and Distribution of [3H]Cholesteryl Ester in Various Lipoproteins of Normal and HDL-Deficient Plasma

In control experiments, approximately 32% of [³H]CE was recovered in pre-ß₃-LpA-I, 57% in -LpA-I, and 11% in LDL (5-minute pulse). A 15-minute chase in the presence of the LCAT inhibitor DTNB resulted in a 32% to 12% decrease of [³H]CE in pre-ß₃-LpA-I, a 57% to 69% increase in -LpA-I, and an 11% to 19% increase in LDL. In the plasma of patients with FED, apoA-I deficiency, and TD, [³H]CEs were detectable only in LDL but not in pre-ß-LpA-I or -LpA-I (Table 5). After pulse incubation, the amount of [³H]CE in LDL was twofold to threefold higher in the plasma of patients with FED, TD, and apoA-I deficiency compared with normal plasma. In contrast to normal plasma, chase incubation with these HDL-deficient plasmas did not significantly increase the amount of [³H]CE in LDL (Table 5).

The more rapid appearance of [³H]CE in LDL raises the possibility that in these HDL-deficient plasmas, cell-derived cholesterol is esterified in LDL or transferred to LDL after generation in lipoproteins that contain neither apoA-I nor apoB. Therefore, pulse-chase incubations were repeated with apoB-free plasma from normoalphalipoproteinemic subjects and HDL-deficient probands (Fig 7). No [³H]CE was generated in apoB-free FED and LCAT-deficient plasma, whereas the amount of [³H]CE gradually increased with prolonged chase incubation in apoB-free supernatants of control plasma, TD plasma, and apoA-I–deficient plasma. Esterification of cell-derived [³H]cholesterol in apoB-free plasma from patients with apoA-I deficiency and TD was increased twofold and threefold, respectively, compared with apoB-free plasma from normal control subjects. Thus, with the exception of FED and LCAT-deficient plasma, esterification of cell-derived [³H]cholesterol occurred in the absence of LpB (Fig 7).

View larger version (25K): [in this window] [in a new window]   Figure 7. Effect of LpB on production and transfer of cholesteryl ester (CE) in normoalphalipoproteinemic (normal) and HDL-deficient (def.) plasma. a, Time-dependent appearance of cell-derived [3H]CE in apoB-free plasma; b and c, bar graphs showing effect of exogenous LDL on the transfer of [3H]CE from apoB-free lipoproteins to LDL. Each point or bar shows the mean and SD of three experiments. Experimental details: a, 1 mL apoB-free plasma from normoalphalipoproteinemic probands or HDL-deficient patients was incubated with radiolabeled fibroblasts for the indicated time intervals. At each indicated time point, 100 µL of sample was removed, and after lipid extraction, the radioactive unesterified cholesterol (UC) and CE were separated by thin-layer chromatography. Shown are data from the Tangier disease (TD) patient (), the apoA-I–deficient patient (), the normoalphalipoproteinemic probands (), and the patients with fish-eye disease and lecithin:cholesterol acyltransferase deficiency (). b and c, After 60 minutes' incubation with radiolabeled fibroblasts, 200 µL apoB-free plasma was removed and incubated for a further 15 minutes with 200 µg LDL. Subsequently, the apoB-free fraction (b) and LDL (c) were separated by precipitation with phosphotungstic acid/MgCl2. After lipid extraction, radioactive UC and CE in the apo B-free supernatants (b) and LDL-containing infranatants (c) were separated by thin-layer chromatography. Open and closed bars represent [3H]CE in lipoproteins before and after chase incubations, respectively.

To further investigate the more rapid transfer of CE to LDL in apoA-I–deficient and TD plasma, apoB-free plasma samples were first pulsed with cell-derived [³H]cholesterol, then supplemented with exogenous LDL, and finally used for 15-minute chase incubations. Exogenous LDL was then precipitated by addition of phosphotungstic acid/MgCl₂ to separately determine [³H]UC and [³H]CE in the apoB-free supernatants (Fig 7b) and in the LDL-containing infranatants (Fig 7c). More than one fourth (29±4%) of [³H]CE was found in the LDL-containing infranatant of normal plasma, whereas 76±3% and 73±3% were detected in the LDL-containing infranatants of TD plasma and apoA-I–deficient plasma, respectively.

Net Cholesterol Efflux From Fibroblasts Into Normal and HDL-Deficient Plasma
We also determined the net mass transfer of UC from fibroblasts to both native and apoB-free plasma during a 2-hour incubation (Table 6). During this period, a net amount of 7.46±1.14 nmol UC per milligram of cell protein was released into 100 µL plasma of normoalphalipoproteinemic probands. Compared with normal plasma, the net transfer into plasma from apoA-I–deficient, TD, FED, and LCAT-deficient patients was reduced to 53±7%, 88±11%, 66±11%, and 41±7%, respectively. Removal of LpB from normal plasma resulted in a 39% decrease of net released cellular cholesterol, from 7.46±1.14 to 4.55±0.73 nmol UC per milligram of cell protein. Removal of LpB from the plasma of patients with apoA-I deficiency, TD, FED, and LCAT deficiency, however, decreased the net cholesterol transport rates by 77%, 84%, 72%, and 64%, respectively. These findings indicate that LpB plays an important role in net cholesterol efflux in HDL-deficient plasmas of various origins.

View this table: [in this window] [in a new window]   Table 6. Cholesterol Net Transport Rates in Normal and HDL-Deficient Plasma Incubated With Human Fibroblasts

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The antiatherogenic effect of HDL has generally been attributed to its ability to mediate the flux of excess cholesterol from peripheral cells to the liver.^{2 3 4} In normoalphalipoproteinemic plasma, this reverse cholesterol transport involves (1) uptake of cell-derived UC by pre-ß₁-LpA-I and -LpE; (2) subsequent transfer of UC to LDL via pre-ß₂-LpA-I, pre-ß₃-LpA-I, and -LpA-I; (3) esterification of UC, mostly in -LpA-I but to a lesser extent in pre-ß₃-LpA-I; and (4) transfer of CE to LDL.^{9 10 11 12 13 43} Patients with various forms of familial HDL deficiency do not lack HDL completely but rather are deficient in distinct HDL subclasses.^{19 44 45 46 47 48} In our study, nondenaturing 2D electrophoresis revealed the absence of pre-ß₃-LpA-I and -LpA-I₂ and the presence of -LpE in all four kinds of familial HDL deficiency. Furthermore, pre-ß₁-LpA-I and pre-ß₂-LpA-I were present in all forms of HDL deficiency except apoA-I deficiency; -LpA-I₃ was detectable in LCAT deficiency and FED but not in TD and apoA-I deficiency. Uptake, transfer, and esterification of cell-derived cholesterol in the plasma of patients with familial HDL deficiency syndromes were significantly different from those of normal plasma (Table 7).

View this table: [in this window] [in a new window]   Table 7. Reverse Cholesterol Transport in Normoalphalipoproteinemic and HDL-Deficient Plasma

Efflux of Cell-Derived Cholesterol
Cholesterol efflux from cells is the result of complex mechanisms that involve the synthesis of cholesterol in the endoplasmic reticulum, hydrolysis of CE in lysosomes and cytosolic lipid droplets, translocation of cholesterol to the cell membrane, and finally desorption of cholesterol from the cell membrane into the plasma compartment.^{4 49} These processes are regulated differently in different cell types and depend on different extracellular stimuli and acceptors of cholesterol, such as the various apolipoproteins and HDL subclasses.⁵⁰ In apoA-I deficiency, cellular cholesterol was taken up by -LpE but not by pre-ß₁-LpA-I. The initial efflux of [³H]cholesterol and the net efflux of cholesterol into plasma were decreased by 50% to 60% compared with normoalphalipoproteinemic plasma. Similarly, Fielding and coworkers (Fielding and Moser⁵¹ and Kawano et al⁵² ) observed a 55% reduction in cholesterol efflux stimulation by plasma that had been depleted of LpA-I by immunoaffinity chromatography. These authors concluded that pre-ß₁-LpA-I contributed to more than half of a plasma's ability to release cholesterol from cells and attributed the residual activity to nonspecific effects of albumin.^{9 51 52} Our data, however, show that -LpE takes up to twofold more [³H]cholesterol than does pre-ß₁-LpA-I, suggesting that this lipoprotein is also a major contributor to the cholesterol efflux–stimulating activity of plasma. The importance of -LpE for the initial efflux of cell-derived cholesterol into plasma is also underlined by our observation that the initial efflux of cell-derived [³H]UC into TD plasma is only slightly higher than that by apoA-I–deficient plasma, although TD plasma contains pre-ß₁-LpA-I.

Transfer of UC to LDL
In normoalphalipoproteinemic plasma, cell-derived cholesterol is taken up by a number of particles and then transferred to LDL. This transfer involves various lipoproteins, including pre-ß₂-LpA-I, pre-ß₃-LpA-I, and -LpA-I. From LDL, UC is either retransferred to -LpA-I for esterification or taken up by cells.^{11 13} These transfer mechanisms of cell-derived cholesterol to LDL were found to be operative in the various HDL deficiency syndromes, suggesting first that -LpE may directly participate in the transfer of cell-derived cholesterol to LDL and second, that the specific absence of pre-ß₂-LpA-I, pre-ß₃-LpA-I, and -LpA-I does not interfere with the transfer of UC from pre-ß₁-LpA-I to LDL.

Esterification of Cholesterol and Transfer of CE to LDL
In normoalphalipoproteinemic plasma, most of the cell-derived cholesterol is esterified in -LpA-I after retransfer from LDL. A smaller amount is esterified during passage through pre-ß₃-LpA-I.^{11 13} In contrast to LCAT deficiency, in which plasma cholesterol esterification activity is completely lost, FED is characterized by the selective failure of plasma to esterify cholesterol in exogenous HDL or apoA-I–containing proteoliposomes while retaining the ability to esterify cholesterol in exogenous LpB.^{53 54 55} In our experiments with FED plasma, CEs accumulated in LDL and were not found in other lipoproteins. These findings further suggest that LDL cholesterol can be directly esterified by the mutant LCAT in FED. By contrast, in apoA-I–deficient and TD plasma, CEs accumulated in LDL after undergoing esterification in lipoproteins containing neither apoA-I nor apoB. The nature of these lipoproteins is unclear at present. Other authors have also detected LCAT activity in the apoA-I– and apoB-free fractions of TD plasma^{30 46 56} and in apoA-IV–containing particles.⁴⁵ Obviously, the newly formed CEs can be effectively transferred from these abnormal particles to LDL.

Familial HDL deficiency is very rare, so we were able to perform only exemplary studies on single patients and could not analyze the effect of possible heterogeneity within a given syndrome. For example, heterogeneity within apoA-I deficiency, LCAT deficiency, FED, or TD arises from allelic variation of the underlying defects in the genes of apoA-I, LCAT, or the as yet unidentified TD gene.^{14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30} Further heterogeneity may originate from variations in the genetic background of an individual and other factors, such as gender, age, concomitant diseases, or genetic polymorphisms. Nevertheless, we believe that the phenomena described in this article can be extrapolated to other patients with the same HDL deficiency syndromes, because they generally cause qualitative rather than quantitative changes in the functioning of a given HDL subfraction.

In this study, we searched only for those lipoproteins that contained apoA-I or apoE. We may, therefore, have overlooked two classes of HDL particles that may play a role in reverse cholesterol transport. First, there is some evidence for the involvement of lipoproteins containing neither apoE nor apoA-I, which are present even in normoalphalipoproteinemic plasma. Thus, lipoproteins that contain apoA-IV but no apoA-I or apoE can release cholesterol from cells.^{57 58} LpA-IV also contains LCAT activity.⁵⁷ Second, because apoA-I is the most abundant apolipoprotein in all subclasses, a lack or severe decrease in apoA-I gives rise to abnormal lipoproteins. Thus, -LpE particles in apoA-I deficiency and TD are heterogenous in size and charge and do not entirely colocalize with apoA-I– or apoE-containing particles in normal plasma. These apoE-containing particles, however, do not appear to contribute substantially to reverse cholesterol transport, as neither pulse nor chase incubations led to the occurrence of radiolabel in those proportions of abnormal -LpE that do colocalize with -LpA-I of normal plasma (Figs 2 through 6). Abnormal HDL that contains apoA-II but no apoA-I has also been identified in some patients with apoA-I deficiency or TD.^{28 29 47 59} In one case, this apoA-II–containing particle was shown to promote cholesterol efflux from cells.⁴⁷ Despite the uncertainty surrounding the role of these minor HDL subfractions in reverse cholesterol transport, they appear either to contribute little to the efflux of [³H]cholesterol from cells or to be entirely colocalized in those electrophoretic fractions that, in normal plasma, contain apoA-I or apoE, as 80% to 90% of the total plasma radioactivity was recovered in LpE, LpA-I, and LDL of both normal and HDL-deficient plasmas.

In summary, our studies demonstrate that the two crucial steps in reverse cholesterol transport—efflux of cellular cholesterol into the plasma compartment and its transfer to LDL for final targeting to the liver—are maintained in all forms of HDL deficiency, although quantitatively and qualitatively modified (Table 7). This may explain why many forms of familial HDL deficiency do not put homozygous carriers at increased risk of coronary disease. Our data suggest that quantitatively minor plasma subfractions, eg, pre-ß₁-LpA-I and -LpE, together with LDL, are important contributors to reverse cholesterol transport. In particular, apoA-I does not play an exclusive role in reverse cholesterol transport, as shown by our in vitro findings in A-I deficiency and the absence of premature atherosclerosis in affected individuals. This is also highlighted by recent observations in transgenic animals that do not express apoA-I but fail to develop atherosclerosis.⁶⁰

	Acknowledgments

 
This project was the topic of the Bennigsen-Foerder-Award from Ministerium für Forschung und Wissenschaft Nordrhein Westfalen to Dr von Eckardstein. Further support was provided by a fellowship from Boehringer Ingelheim Fonds to Dr Huang. We gratefully acknowledge the assistance of Dr Ali Chirazi in the determination of lipid transfer enzyme activities and the help of Dr Paul Cullen in editing the manuscript.

Received July 13, 1994; accepted February 2, 1995.

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