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Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:149-159

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:149-159.)
© 1996 American Heart Association, Inc.

Articles

Plasma Concentration of Apolipoprotein E in Intermediate-Sized Remnant-Like Lipoproteins in Normolipidemic and Hyperlipidemic Subjects

Jeffrey S. Cohn; Michel Tremblay; Mireille Amiot; Daniel Bouthillier; Madeleine Roy; Jacques Genest, Jr; Jean Davignon

From the Hyperlipidemia and Atherosclerosis Research Group and the Cardiovascular Genetics Laboratory (J.G.), Clinical Research Institute of Montreal, Quebec, Canada.

Correspondence to Dr J.S. Cohn, Hyperlipidemia and Atherosclerosis Research Group, Clinical Research Institute of Montreal, 110 Pine Ave W, Quebec, Canada, H2W 1R7.


*    Abstract
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 *Abstract
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Abstract Triglyceride-rich lipoprotein (TRL) remnants have been strongly implicated in the pathogenesis of atherosclerosis. To further investigate plasma remnant lipoprotein metabolism, we have determined the plasma concentration of apolipoprotein (apo) E (by polyclonal enzyme-linked immunoassay) in remnant-like lipoproteins, isolated by automated gel filtration chromatography as a fraction intermediate in size between VLDL and HDL. In normolipidemic subjects (n=12), 1.2±0.1 mg/dL (33±2%, mean±SE) of total plasma apoE was associated with this fraction (termed ISL apoE). In hypercholesterolemic (type IIa, n=12), hypertriglyceridemic (type IV, n=12), and mixed hyperlipidemic (type IIb, n=12) subjects, mean ISL apoE concentrations were 2.1±0.2, 2.5±0.2, and 3.8±0.4 mg/dL, respectively (P<.001 versus normal values) (45±2%, 32±2%, and 44±2% of total). ISL apoE was 8.7±1.4 mg/dL (42±3%) in type III dyslipidemic subjects (apoE2/2, n=8). ISL apoE was positively correlated with plasma triglyceride (r=.41, P<.01), and at any given level of plasma triglyceride, subjects with an apoE2/2 or apoE3/2 phenotype tended to have a higher concentration of ISL apoE (P<.01) than apoE3/3 or E4/3 individuals. ISL apoE was also correlated (P<.001) with total plasma cholesterol (r=.66), TRL cholesterol (r=.49), TRL apoE (r=.47), LDL apoB (r=.42), and LDL+HDL triglyceride (r=.74). These results suggest that (1) a significant proportion of plasma apoE resides within an intermediate-sized remnant-like lipoprotein fraction in both normolipidemic and hyperlipidemic subjects; (2) plasma remnant lipoprotein accumulation is associated with an elevation in ISL apoE concentration; and (3) ISL apoE concentration is significantly correlated with various proatherogenic lipid parameters and may itself be a potentially important atherogenic index.


Key Words: triglyceride-rich lipoproteins • apoE phenotype • FPLC • atherosclerosis • coronary artery disease


*    Introduction
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 up arrowAbstract
 *Introduction
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Increasing evidence suggests that TRL remnants play a significant role in the pathogenesis of atherosclerosis. In experimental animals, diet-induced hyperlipidemia and atherosclerosis are characterized by the presence in plasma of cholesterol-enriched remnant lipoproteins (ß-VLDL).1 Patients with familial dysbetalipoproteinemia (type III hyperlipoproteinemia), caused by impaired apoE-mediated clearance of TRL remnants, also have a marked accumulation of plasma remnants (ß-VLDL) and an increased incidence of peripheral vascular disease and CAD.2 The atherogenicity of mutant apoE-induced dyslipoproteinemia is supported experimentally by the observation that transgenic mice expressing dysfunctional apoE have a markedly increased susceptibility to atherosclerosis.3 4 In addition, results of angiographic intervention studies have provided clinical evidence that elevated levels of remnant lipoproteins are associated with progression of CAD.5 6 7

TRL remnants are produced in the circulation from triglyceride-rich apoB-48 containing chylomicrons of intestinal origin or from apoB-100-containing VLDL of hepatic origin. Triglycerides in the core of TRL are hydrolyzed by lipoprotein lipase on the surface of vascular endothelial cells, resulting in the formation of smaller and more dense TRL remnants. These lipoproteins are rapidly cleared from the circulation as a result of recognition of apoE by specific hepatic lipoprotein receptors or, in the case of apoB-100 TRL, are efficiently converted to smaller LDL. The importance of apoE in mediating the plasma clearance of apoB-containing lipoproteins is reflected by the marked increase in VLDL and IDL in apoE-deficient mice produced by gene targeting.8 9 These animals develop spontaneous aortic atherosclerosis as a result of pronounced plasma accumulation of atherogenic cholesterol-rich lipoproteins.

Investigation of TRL remnant lipoprotein metabolism and its relationship to CAD has been hampered by the lack of precise methods to detect the presence in plasma of remnant lipoproteins. This is because they are normally removed very rapidly from the circulation and hence are found at very low plasma concentrations. Second, although TRL remnants are less triglyceride-rich and smaller in size than chylomicrons or VLDL, they are difficult to differentiate from their newly secreted precursors. One distinguishing feature is their increased content of apoE, and an intermediate-sized (smaller than VLDL and larger than HDL) apoE-containing lipoprotein fraction has been identified using 6% agarose gel filtration chromatography in normolipidemic,10 familial hypercholesterolemic,11 lipase-deficient12 and apoA-I-deficient subjects.13

To further investigate plasma remnant lipoprotein metabolism, we have adapted the methodology of Gibson et al12 and in the present study have measured the plasma apoE concentration of intermediate-sized remnant-like lipoproteins (termed ISL apoE) in relative and absolute terms in both normolipidemic and hyperlipidemic individuals.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
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Subjects
Hyperlipidemic subjects (26 men, 20 women) were selected from the Lipid Clinic of the Clinical Research Institute of Montreal. They were grouped according to their type of hyperlipoproteinemia14 : type IIa patients (n=12), LDL cholesterol concentration >3.8 mmol/L, plasma triglyceride concentration <2.3 mmol/L; type IIb (n=12), LDL cholesterol >3.8 mmol/L, triglyceride >2.3 mmol/L; type III (n=8), triglyceride >2.3 mmol/L, total plasma cholesterol >6.2 mmol/L, apoE2/2 phenotype, and presence of ß-VLDL on agarose gel electrophoresis; type IV (n=12), triglyceride >2.3 mmol/L, LDL cholesterol <3.4 mmol/L (none of these patients had an LDL apoB >120 mg/dL); and type V (n=2), triglyceride >10 mmol/L with chylomicrons in fasting plasma as evidenced by agarose gel electrophoresis. Cutoff points of 2.3 mmol/L for triglycerides and 3.8 mmol/L for LDL cholesterol were chosen because they approximately represented the 75% percentile, based on the Lipid Research Clinics population study.15 Normolipidemic subjects (5 men, 7 women) were selected from laboratory volunteers: LDL cholesterol <3.4 mmol/L, triglyceride <2.3 mmol/L. Six patients were taking lipid-lowering medication (1 type IIb [statin], 3 type III [fibrate], 2 type IV [fibrate]). The apoE profile of these individuals did not differ significantly from those of untreated patients of the same phenotype, and they were analyzed together. Twelve patients were glucose intolerant or diabetic, 19 were hypertensive, and 16 were obese. The mean fasting lipid levels and the distribution of men and women in each group are shown in Table 1Down. Ten of the normolipidemic subjects had an apoE3/E3 phenotype and 2 had an apoE3/E2 phenotype. Seven of the type IIa subjects were apoE3/E3, 2 were apoE3/E2, 2 were apoE4/E3, and 1 was apoE4/E4. Eight of the type IIb subjects were apoE3/E3, 2 were apoE3/E2, and 2 were apoE4/E3. Six of the type IV subjects were apoE3/E3, 1 was apoE3/E2, 4 were apoE4/E3, and 1 was apo4/E4. Both type V patients were apoE3/E3.


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  		Table 1. Plasma Lipid and Lipoprotein ApoE Concentrations in Normolipidemic and Hyperlipidemic Subjects

Chromatographic Separation of Plasma Lipoproteins
Venous blood was drawn into tubes containing EDTA (final EDTA concentration, 1.5 mg/mL). All subjects were fasted overnight (12 to 14 hours) before blood was drawn. Plasma was obtained by centrifugation at 3000 rpm (15 minutes, 4°C). Lipoproteins were separated by automated gel filtration chromatography on a Pharmacia LKB Biotechnology Inc FPLC system. Plasma samples (1 mL) were manually transferred to a 2-mL sample loop with two washes of 0.5 mL saline solution. They were programmed (Liquid Chromatography Controller LCC-500 Plus) to be loaded and separated on a 50-cm column (16 mm ID) packed with cross-linked agarose gel (Superose 6 prep grade, Pharmacia). The column was eluted with 0.15 mol/L NaCl (0.01% EDTA, 0.02% sodium azide, pH 7.2) at a rate of 1.0 mL/min, and 25 minutes after addition of the sample, ninety 1-mL fractions were collected sequentially. The total run time for each sample, including washes before and after, was 150 minutes. Sample elution was monitored spectrophotometrically at an OD of 280 nm. Three peaks of triglyceride or cholesterol were identifiable for every plasma sample, corresponding to TRL, LDL, and HDL lipoprotein fractions, as has been described for FPLC separations of human plasma.16 17 A characteristic FPLC profile for the plasma of a normolipidemic individual is shown in Fig 1Down. TRL triglyceride and cholesterol routinely eluted between fractions 8 and 18, LDL between 24 and 35, and HDL between 38 and 48. Some settling of the sepharose gel occurred during the 8-month duration of this study, which resulted in the appearance of lipoprotein fractions in gradually earlier elution fractions. This effect was not pronounced, however, and did not affect the quantitation of lipoprotein fractions.



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  		Figure 1. Line graph shows separation of plasma lipoproteins by automated gel filtration chromatography. Triglyceride and cholesterol peaks correspond to lipoprotein fractions identified at the top of the figure. Also shown is the OD profile at 280 nm. The amount of apoE in each fraction is represented by the filled circles. The plasma sample was from a normolipidemic male (aged 35 years) with a plasma cholesterol of 4.3 mmol/L, a plasma triglyceride of 0.8 mmol/L, and an apoE concentration of 3.9 mg/dL.

Plasma apoE characteristically eluted as three overlapping chromatographic peaks (Fig 1Up). The first peak coeluted with TRL triglyceride and cholesterol and was defined as TRL apoE. The last peak eluted 1 to 3 fractions earlier than HDL lipid and was defined as HDL apoE. ApoE that eluted between these fractions (thus intermediate in size between TRL and HDL) was defined as ISL apoE. LDL-sized lipoproteins also eluted in these fractions; however, they made only a small contribution to total ISL apoE (see "Discussion"). The overlapping nature of apoE-containing lipoprotein fractions reflected the fact that apoE-containing lipoproteins in plasma represented a "size continuum."

To define the point of separation between apoE-containing fractions, the FPLC fraction with the lowest amount of apoE between peaks (the nadir) was taken as the cutoff point, and apoE in this fraction was included in the larger-sized fraction (the TRL fraction for the nadir between TRL and ISL and the ISL fraction for the nadir between ISL and HDL). For some separations, an apoE nadir did not exist between lipoprotein fractions, in which case the OD profile was used to define cutoff points. Thus, the last FPLC fraction containing TRL apoE was defined as the nadir in OD between VLDL and LDL, and the last FPLC fraction containing ISL apoE was defined for normotriglyceridemic individuals as the fourth fraction before the OD nadir between the LDL and plasma protein peaks. In hypertriglyceridemic individuals (triglyceride >2.3 mmol/L), the ISL and HDL apoE fractions eluted a few fractions later because of the presence of somewhat smaller apoE-containing lipoproteins. The last FPLC fraction containing ISL apoE was thus defined as the second fraction before the OD nadir between the LDL and plasma protein peaks. This number of fractions was found to be the average difference between apoE and OD profiles for six normotriglyceridemic and six hypertriglyceridemic subjects, who were selected because they had clearly defined apoE and absorbance nadirs between ISL and HDL.

Agarose gel electrophoresis was used to confirm the identity of eluted FPLC fractions (Fig 2Down). TRL fractions (8 to 18) contained lipoproteins with pre-ß electrophoretic mobility, characteristic of plasma VLDL. In certain situations, ie, for type I and III patients after an overnight fast (or for subjects in the fed state), they contained (particularly fractions 8 to 10) triglyceride-rich chylomicrons of intestinal origin, which were observed at the origin after separation by agarose gel electrophoresis. ISL fractions contained lipoproteins with slow pre-ß or ß mobility. HDL-containing fractions had {alpha} migrating lipid-stained bands, and apoE was detected in a slow {alpha} migrating region (barely visible for subject B in Fig 2Down). Fractions were appropriately pooled or stored individually at 4°C for 1 week or less before measurement of lipids or apolipoproteins. Recovery of plasma after FPLC separation was assessed by expressing cholesterol found in recovered fractions as a percentage of cholesterol in whole plasma. Cholesterol recovery was 94.0±5.7% for normolipidemic subjects (n=11), 96.0±4.8% for hypercholesterolemic subjects (n=12), and 92.1±6.4% for hypertriglyceridemic subjects (n=11). Plasma concentration of apoE in each lipoprotein fraction was determined by expressing apoE in each pooled lipoprotein fraction as a percentage of total recovered apoE and then multiplying this percentage by total plasma apoE concentration.



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  		Figure 2. Agarose gel (0.5%) electrophoretic separation of lipoproteins isolated by automated gel filtration chromatography (FPLC) from the plasma of two subjects (A and B). Subject A was a type IIb hyperlipoproteinemic patient (plasma cholesterol concentration, 7.03 mmol/L; plasma triglyceride, 3.98 mmol/L; plasma apoE, 5.86 mg/dL). Subject B was a normolipidemic subject (plasma cholesterol concentration, 4.77 mmol/L; plasma triglyceride, 1.47 mmol/L; plasma apoE, 3.1 mg/dL). Top panels show the electrophoretic separation of lipoprotein bands stained for lipid with Sudan black. Middle panels show the presence of apoE (detected by horseradish peroxidase–conjugated antibody) after transfer of samples onto a nitrocellulose membrane. Identified on the left of these panels are the origin (0) and the {alpha} and ß migrating lipoproteins. Bottom panels show the apoE and OD (at 280 nm) FPLC elution profiles for the two subjects. The three OD peaks represent (from left to right) TRL, LDL, and plasma proteins. The numbered arrows indicate the fractions that were separated in the sample lanes numbered 1 through 8 in the top panels. For subject A, samples 1 through 8 represent fractions 11, 13, and 15 (containing TRL apoE); 18, 21, and 27 (ISL apoE); and 36 and 40 (HDL apoE). For subject B, samples 1 through 8 represent fractions 13 (TRL apoE), 20, 23, 25, 27, 29, 32 (ISL apoE), and 43 (HDL apoE). The outside lanes (without numbers) contained whole plasma diluted 1 in 10 or the same plasma undiluted.

Quantitation of ApoE
ApoE was measured by a noncompetitive polyclonal enzyme-linked immunoassay developed in our laboratory according to the method of Bury et al.18 Immunopurified goat polyclonal anti-human apoE antibody (supplied by Dr J. Ordovas, Boston, Mass) was used as both the capture and detection antibody. Ninety-six well polystyrene plates (Nunc-Immuno Plate Maxisorp) were coated with immunopurified antibody (1.1 µg per well) dissolved in PBS (10 mmol/L sodium phosphate, 0.15 mol/L NaCl, 1 mg/mL NaN3, pH 7.4). The outer rows of each plate were not used to avoid outside-well variability. Plates were sealed with SealPlate adhesive film and incubated for 3 hours at 37°C, followed by overnight incubation at 4°C. Coated plates were used within 2 months of preparation. Before each assay, plates were inverted and washed three times (PBS containing 0.5 mL/L Tween 20, Bio-Rad Laboratories) with an automated microplate washer (EL402, Bio-Tek Instruments), and residual binding sites were blocked for 1 hour at room temperature with 200 µL PBS containing 0.1% casein (BDH Laboratory Supplies) and 0.01% merthiolate (Eastman).

A standard curve (ranging from 0.5 to 10.0 ng) was prepared for each assay by making appropriate dilutions of a plasma standard (stored at -70°C), which had been calibrated with the use of recombinant apoE (Crystal Chem). The mass of the recombinant apoE primary standard was determined by amino acid analysis. Standards (100 µL) were applied to microtiter plates in duplicate, together with two control plasmas (low and high, stored at -70°C) diluted 1:2000 with sample buffer (PBS, 0.1% casein, 0.01% merthiolate, 0.5 mL/L Tween 20). Normolipidemic plasma samples were routinely diluted 1 in 2000 with sample buffer and were also applied (100 µL) in duplicate. Hypertriglyceridemic samples were diluted 1:4000 or 1:6000, depending on their triglyceride concentration.

ApoE was detected by incubating plates for 2 hours at 37°C on an orbital shaker (Bellco Biotechnology). After vacuum aspiration of samples and six automated washes with PBS-Tween, horseradish peroxidase-conjugated antibody (100 µL) diluted appropriately (according to previous titration assay) was added to each well. Plates were incubated for 2 hours at 37°C. After six washes with PBS-Tween, color was developed by addition of 100 µL of freshly prepared substrate solution (sodium phosphate/citrate buffer, pH 5.6, containing 2.4 g/L o-phenylenediamine hydrochloride, 0.021% H2O2). The reaction was stopped after 30 minutes with 100 µL of 2.5 mol/L H2SO4. Color development was measured at 490 nm with an automated microplate reader (EL310, Bio-Tek Instruments). Standard curves were prepared by plotting absorbance at 490 nm as a function of apoE concentration. A second-order polynomial curve was fitted to the data with Sigmaplot software (Jandel Scientific), and absorbance values were converted to concentration measurements by regression analysis.

The accuracy of our apoE ELISA was assessed by comparison with the assay of Bury et al.18 ApoE was determined on 40 fresh normolipidemic plasmas in the laboratory of Dr Maryvonne Rosseneu in Belgium (mean apoE concentration, 3.7±0.2 mg/dL), which were sent frozen for analysis in Montreal. The mean apoE concentration of these samples was found to be 4.8±0.3 mg/dL. The two assays compared favorably with a correlation coefficient (r) of .87 (P<.001; y=0.63x+0.69). The precision (reproducibility) of our apoE ELISA was reflected by a within-assay variation (n=20) of 4.0% and a between-assay variation of 7.0% for a control plasma (apoE, 4.56±0.32 mg/dL) measured on every plate during the 8-month analysis period (n=59).

To ensure that apoE could be detected equally well in different lipoprotein fractions, serial dilutions of a control plasma, FPLC-isolated TRL, ISL, and HDL fractions of two hypertriglyceridemic plasmas (triglyceride concentration, 5.67 and 6.45 mmol/L), and an ultracentrifugally isolated hypertriglyceridemic VLDL (d<1.006 g/mL) fraction were assayed for apoE with the standard assay procedure. The reactivity of apoE in different fractions was similar, as reflected by the parallel nature of the dilution curves (Fig 3Down).



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  		Figure 3. Line graph shows comparison of the reactivity of apoE in normolipidemic plasma with that of apoE in different lipoprotein fractions from two hypertriglyceridemic plasmas. Serial dilutions were made of a normolipidemic (triglyceride concentration, 0.54 mmol/L; total cholesterol, 3.88 mmol/L) control plasma ({bullet}) and the FPLC-isolated TRL fractions ({circ}), FPLC-isolated ISL fractions ({triangledown}), and FPLC-isolated HDL fractions ({square}) of two hypertriglyceridemic plasmas (triglyceride concentration, 5.67 and 6.45 mmol/L). The apoE reactivity of a VLDL (d<1.006 g/mL) fraction ({diamond}) isolated by ultracentrifugation from one of the hypertriglyceridemic plasmas is also shown. Serial dilutions were applied to a single microtiter plate, and color development was monitored at 490 nm as described in "Methods." Parallelism of the dilution curves shows the equal reactivity of apoE of different lipoprotein fractions in this assay system.

Lipid and Lipoprotein Analyses
Lipoproteins were separated by ultracentrifugation at d=1.006 g/mL to obtain VLDL and by precipitation of apoB in the d>1.006 g/mL fraction to isolate HDL.19 Plasma and lipoprotein cholesterol and triglyceride concentrations were determined enzymatically on an autoanalyzer (Cobas Mira, Roche). LDL+HDL triglyceride was obtained by measuring triglyceride in the d>1.006 g/mL fraction. Plasma and LDL (d>1.006 g/mL) apoB and plasma apoA-I were measured by nephelometry (Behring Nephelometer 100 Analyzer). Lp(a) was measured with a commercial ELISA (Macra EIA Kit, Strategic Diagnostics Industries, Inc). ApoE phenotypes were determined by tube gel isoelectric focusing electrophoresis of delipidated VLDL20 or by immunoblotting of plasma separated by minigel electrophoresis.21 Plasma lipoproteins were separated by agarose gel electrophoresis on a Beckman Paragon Electrophoresis System (Beckman Instrs, Inc) and were visualized with Sudan black staining. The presence of apoE in electrophoretically separated samples was detected with a horseradish peroxidase-labeled polyclonal apoE antibody with the use of enhanced chemiluminescence (ECL Western blotting detection reagents, Amersham).

Statistical Analysis
Mean apoE concentrations were compared by Student's unpaired t test. Pearson correlation coefficients (r) were used to describe the correlation between individual plasma parameters. The slopes of the relationship between plasma triglyceride concentration and apoE concentration for patients with different phenotypes were compared by partial F test of identity of two regressions.22


*    Results
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 up arrowIntroduction
 up arrowMethods
*Results
down arrowDiscussion
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Plasma apoE elution profiles obtained with our FPLC system (Figs 1Up and 2Up) were similar to those reported previously with 6% agarose gel filtration chromatography.10 Three overlapping peaks of apoE were identifiable for all subjects studied and were defined as the TRL, ISL, and HDL apoE lipoprotein fractions. The cutoff points for each fraction were defined (where possible) by the nadirs of the apoE elution profile or alternatively by inspection of the OD profile (see "Methods"). These cutoff points are shown for the apoE profiles in Figs 4Down, 6Down, and 7Down, and they were used to calculate the relative and absolute amounts of total plasma apoE in each lipoprotein fraction.



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  		Figure 4. Line graphs show plasma lipoprotein apoE distribution ({bullet}) of four representative normolipidemic subjects. The OD profile ({circ}) for each plasma is shown to identify the position of TRL, LDL, and plasma protein fractions. The intermediate-sized apoE lipoprotein fraction is indicated by the shaded area. The proportion of total plasma apoE found in each fraction and the calculated apoE concentration for these fractions is shown for each subject.



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  		Figure 6. Line graphs show plasma lipoprotein apoE distribution ({bullet}) of six representative hyperlipidemic patients. Top panels show data from two type IIa patients (triglyceride concentration, 0.66 and 1.70 mmol/L; total cholesterol, 6.84 and 6.81 mmol/L [left to right, respectively]). Middle panels contain data from two type IIb patients (triglyceride, 2.61 and 3.14 mmol/L; cholesterol, 6.78 and 6.17 mmol/L [left to right, respectively]), and bottom panels contain data from two type IV patients (triglyceride, 6.29 and 6.51 mmol/L; cholesterol, 6.72 and 5.14 mmol/L [left to right, respectively]). The OD profile ({circ}) for each plasma is shown to identify the position of TRL, LDL, and plasma protein fractions. The intermediate-sized apoE lipoprotein fraction is indicated by the shaded area. The proportion of total plasma apoE found in each fraction and the calculated apoE concentration for these fractions are shown for each subject.



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  		Figure 7. Line graphs show plasma lipoprotein apoE distribution ({bullet}) of two representative type III patients. The patient on the left had a triglyceride concentration of 2.77 mmol/L and a total cholesterol concentration of 6.15 mmol/L; the patient on the right had a triglyceride concentration of 4.84 and a total cholesterol concentration of 9.31 mmol/L. Both patients had an apoE2/2 phenotype and a prominent ß-VLDL band when their plasma was separated by agarose electrophoresis. The OD profile ({circ}) for each plasma is shown to identify the position of TRL, LDL, and plasma protein fractions. The intermediate-sized apoE lipoprotein fraction is indicated by the shaded area. The proportion of total plasma apoE found in each fraction and the calculated apoE concentration for these fractions are shown for each subject.

Agarose gel electrophoresis was used to characterize eluted FPLC fractions, as shown for two subjects (A and B) in Fig 2Up. Subject A was a type IIb hyperlipoproteinemic patient, and subject B was normolipidemic. ApoE elution profiles for each subject are shown in the bottom panels, together with OD profiles (absorbance at 280 nm) that clearly identify the position of TRL (peaking at fraction 11 or 12) and LDL (peaking at fraction 27 or 28) in Fig 2Up. For subject A, agarose gel-separated fractions were chosen with the aim of characterizing the TRL and early ISL apoE–containing fractions. For subject B, fractions were chosen with the aim of characterizing the later ISL apoE–containing fractions, which also contained the bulk of LDL. The results for both subjects were very consistent. TRL-FPLC fractions contained lipoproteins with pre-ß electrophoretic mobility, as detected by lipid staining or by immunoblotting with anti-apoE antibody. TRL in eluted fractions migrated further than pre-ß migrating VLDL in whole plasma (outside lanes in Fig 2AUp and 2BUp), possibly due to the absence of plasma proteins in these fractions, which normally retard lipoprotein electrophoretic migration. Early ISL-FPLC fractions (lane 4 in Fig 2AUp, lane 2 in Fig 2BUp) contained lipid-stained pre-ß migrating lipoproteins, whereas later ISL-FPLC fractions (lane 6 in Fig 2AUp, lanes 3 through 7 in Fig 2BUp) contained ß migrating lipid-stained lipoproteins. In contrast, apoE-containing ISL lipoproteins migrated with slow pre-ß or ß migration. In agreement with the apoE elution profiles, the majority of ISL apoE detected by immunoblotting was associated with early ISL-FPLC fractions rather than late ISL fractions, suggesting that a significant proportion of ISL apoE was associated with lipoproteins larger than LDL. HDL-FPLC fractions contained lipoproteins with {alpha}-mobility, as detected by lipid staining (lane 8 in Fig 2AUp and 2BUp); however, apoE-containing HDL had slow {alpha} or pre-ß mobility and was found in earlier (larger-sized), lipid-poor HDL-FPLC fractions (lane 7, Fig 2AUp).

The plasma apoE elution profiles of four representative normolipidemic subjects are shown in Fig 4Up. The intermediate-sized ISL apoE lipoprotein fraction is indicated by the shaded area. The proportion of total plasma apoE found in each fraction and the calculated apoE concentration for these fractions are shown for each subject. Four individual profiles have been presented in Fig 4Up to show that (1) the intermediate-sized apoE-containing fraction was a consistent feature of normolipidemic plasma; (2) TRL apoE was a relatively minor component; and (3) the majority of plasma apoE in normolipidemic subjects was found in HDL, although considerable interindividual differences existed in the relative contribution of HDL (range, 49% to 76%). The mean percentage of total plasma apoE found in each lipoprotein fraction for the 12 normolipidemic subjects is shown diagrammatically in Fig 5Down, and mean lipoprotein apoE concentrations are given in Table 1Up.



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  		Figure 5. Bar graph shows distribution of total plasma apoE between TRL, ISL, and HDL lipoprotein fractions in normolipidemic and hyperlipidemic subjects. ApoE in each lipoprotein fraction was expressed as a percentage of total apoE recovered after gel filtration chromatography. Mean results are presented for each patient group. White bars indicate TRL apoE; lightly shaded bars, ISL apoE; and heavily shaded bars, HDL apoE.

Plasma apoE profiles for 2 type IIa, 2 type IIb, and 2 type IV patients are shown in Fig 6Up, and profiles for 2 type III patients are shown in Fig 7Up. ApoE in intermediate-sized lipoproteins is indicated by the shaded area. The range of the x axis is greater in the graphs for type III and type IV patients. Mean data for each patient group are presented in relative terms in Fig 5Up and in absolute terms in Table 1Up. Comparing the mean data for different patient groups in Fig 5Up, it is apparent that the proportion of total plasma apoE in TRL increased as the proportion in HDL decreased. Whereas HDL was the predominant carrier of apoE in normolipidemic subjects, TRL was the most predominant carrier in type IV and type V subjects (54±3% and 76%, respectively). As for the normolipidemic subjects, a significant proportion of plasma apoE in the hyperlipidemic patients was associated with remnant-sized lipoproteins (45±2%, 44±2%, 42±3%, and 32±2% in types IIa, IIb, III, and IV, respectively).

Mean ISL apoE concentrations for different patient groups are shown and statistically compared in Table 1Up. The highest concentrations of ISL apoE were found in type III patients (8.7±1.4 mg/dL). ISL apoE was significantly higher in all patient groups compared with normolipidemics, and mean ISL apoE was significantly (P<.05) higher in type IIb compared with type IIa and type IV patients. In comparison to normolipidemic subjects, hyperlipidemic patients had significantly higher levels of total, TRL, and ISL apoE. HDL apoE was not significantly different between normolipidemic, type IIa, and type IIb patients but was higher in type III and significantly lower in type IV patients.

Individuals with higher plasma triglyceride concentration (and hence TRL triglyceride) tended to have higher plasma apoE, TRL apoE, and ISL apoE concentrations (Table 1Up). Statistically significant positive correlations were observed between plasma triglyceride and total, TRL, and ISL apoE concentrations (Table 2Down). Comparing type IIa and type IIb patients, the type IIb patient group had by definition a significantly higher mean plasma triglyceride concentration (P<.001) and significantly higher mean concentrations of total, TRL, and ISL apoE (but not HDL) concentrations. Differences in ISL apoE concentration were not, however, totally dependent on plasma triglyceride concentration, as reflected by the comparison of type IIb and type IV patient groups. Despite a significantly higher mean plasma triglyceride concentration, type IV patients did not have a significantly greater mean total or ISL apoE concentration (Table 1Up).


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  		Table 2. Correlation Between Plasma and Lipoprotein ApoE Concentrations and Other Plasma Lipoprotein Parameters

ISL apoE concentration was found to be significantly correlated with various plasma lipid parameters. Statistically significant relations between plasma ISL apoE concentration and plasma cholesterol (r=.66, P<.001), triglyceride (r=.41, P<.01), HDL cholesterol (r=-.47, P<.001), and LDL+HDL triglyceride concentrations (r=.55, P<.001) are shown in Fig 8Down. Type III patients were not included in this analysis to avoid the strong effect of apoE2 homozygosity on plasma lipid levels. As shown in Table 2Up, ISL apoE was also significantly correlated with TRL cholesterol, TRL triglyceride, TRL apoE, total plasma apoB, and LDL apoB but not with Lp(a), apoA-I, or the ratio of VLDL cholesterol to total plasma triglyceride. TRL apoE concentration was most strongly correlated with TRL cholesterol and TRL triglyceride concentrations. HDL apoE correlated with HDL cholesterol and total apoA-I.



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  		Figure 8. Scatterplots show relations between plasma ISL apoE concentration (CONC.) and plasma cholesterol (r=.66, P<.001), triglyceride (r=.41, P<.01), HDL cholesterol (r=-.47, P<.001), and LDL+HDL triglyceride concentrations (r=.55, P<.001) for all individuals (n=50) excluding the type III hyperlipoproteinemics.

In view of the documented effect of apoE phenotype23 24 25 on total plasma apoE concentration, the present data set was used to analyze the effect of apoE phenotype on lipoprotein apoE concentrations. Of the 58 patients studied, 8 had an apoE2/2 phenotype (the type III patients), 7 were apoE3/2, 33 were apoE3/3, 8 were apoE4/3, and 2 were apoE4/4. In this analysis, the 2 apoE4/4 patients were included in the apoE4/3 group. To determine the effect of apoE phenotype independent of triglyceride concentration, the relationship between triglyceride concentration and apoE concentration was compared for subjects grouped according to apoE phenotype. Correlation coefficients and the slopes of the linear regressions are shown in Table 3Down. A statistically significant positive correlation was observed between both total and TRL apoE concentrations and plasma triglyceride for all phenotypes. A strong positive correlation between plasma triglyceride and ISL apoE concentrations was only observed for apoE3/2 and apoE2/2 individuals. In the case of HDL apoE, a positive correlation was found for apoE2/2 individuals and a weak negative correlation was found for the apoE4/3 group. These data suggest that at any given level of triglyceride, apoE2/2 and apoE3/2 individuals tended to have a higher level of TRL apoE and ISL apoE than apoE3/3 or apo4/3 individuals. In the case of HDL apoE, at any given level of plasma triglyceride, apoE2/2 individuals tended to have a higher and apoE4/3 individuals tended to have a lower HDL apoE concentration.


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  		Table 3. Correlation Between Plasma Triglyceride Concentration and ApoE Concentration in Total Plasma and in Lipoprotein Fractions for Subjects Grouped According to ApoE Phenotype


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
*Discussion
down arrowReferences
 
The results of the present study have shown that a significant proportion of plasma apoE resides within an intermediate-sized remnant-like lipoprotein fraction in both normolipidemic and hyperlipidemic subjects. In normolipidemic individuals, one third of plasma apoE (33±2%) was found in this fraction, while in hypercholesterolemic (type IIa) patients, ISL apoE represented 45±2% of total apoE. These results are in general agreement with those of Gibson et al,11 who showed that apoE in intermediate-sized lipoproteins accounted for an average 24% of total apoE in normolipidemic subjects and 49% in patients homozygous for familial hypercholesterolemia (with a mean LDL cholesterol level of 620 mg/dL [16.1 mmol/L]). In addition, we have found that patients with hypertriglyceridemia (type IV), mixed hyperlipidemia (type IIb), and type III hyperlipoproteinemia had 32±2%, 44±2%, and 42±3% of total apoE in intermediate-sized lipoproteins, respectively. In absolute terms, mean apoE concentration in intermediate-sized lipoproteins was 1.2±0.1 mg/dL in normolipidemic subjects and was significantly (P<.001) greater in all hyperlipidemic patient groups (Table 1Up). The highest levels of ISL apoE were observed in patients with type IIb (3.8±0.4 mg/dL) and in those with type III hyperlipoproteinemia (8.7±1.4 mg/dL), reflecting a quantitatively significant involvement of this lipoprotein fraction in plasma apoE metabolism in these patients.

We have referred to apoE in intermediate-sized lipoproteins (separated by FPLC) as being associated with "remnant-like" lipoproteins, and we have labeled it ISL apoE. This interpretation is supported by three observations: (1) ISL apoE eluted with lipoproteins smaller than TRL and larger than HDL, with a size distribution that favored lipoproteins larger than LDL, consistent with it being associated with partly catabolized TRL remnants; (2) this lipoprotein fraction had slow pre-ß or ß migration on agarose gel electrophoresis (Fig 2Up), which is characteristic of remnant lipoproteins26 27 ; and (3) the highest levels of ISL apoE were observed in type III hyperlipoproteinemic patients, who are known to have markedly elevated levels of circulating remnant lipoproteins.2 It must be noted, however, that this intermediate-sized lipoprotein fraction is heterogeneous in nature and contains a number of different lipoprotein species such as IDL, Lp(a) (which characteristically elutes between fractions 18 and 25), and also large and small LDL. The association of apoE with Lp(a) has recently been demonstrated with the use of immunoaffinity chromatography,28 and two-dimensional nondenaturing gradient gel electrophoresis has revealed the association of apoE with LDL.29 The proportion of ISL apoE associated with these lipoproteins is probably relatively small, however, and preliminary two-dimensional nondenaturing gradient gel electrophoresis analyses in our own laboratory have suggested that apoE associated with LDL rarely represents more than 15% of ISL apoE (data not shown). Irrespective of the relative contribution of LDL-bound apoE to ISL apoE, it can be argued that LDL apoE is still remnant-like in nature since it is associated with a triglyceride-depleted lipoprotein that is a product of VLDL catabolism.

The presence of elevated levels of ISL apoE in patients with type III hyperlipoproteinemia, in association with increased levels of ß-VLDL, suggests that plasma remnant lipoprotein accumulation is associated with an elevated ISL apoE concentration. This is supported by the observation that patients with hepatic lipase deficiency,30 whose dyslipidemia is characterized by the plasma accumulation of remnant lipoproteins, also have an accumulation of apoE in intermediate-sized lipoproteins.12 Furthermore, we have observed an increase in ISL apoE (data not shown) in subjects with double pre-ß lipoproteinemia, who have an increased concentration of remnant lipoproteins in their VLDL fraction, as evidenced by the presence of a second slow migrating pre-ß band in their d<1.006 g/mL density fraction.26 27 Plasma remnant lipoprotein accumulation in type III hyperlipoproteinemics,2 hepatic lipase-deficient patients,30 or apoE-deficient patients31 has invariably been associated with an increase in the level of VLDL triglyceride and cholesterol, and in the present study, ISL apoE was significantly correlated with VLDL cholesterol (r=.49, P<.001) and VLDL triglyceride (r=.39, P<.01) (type III patients excluded, n=50). It is also pertinent that type III hyperlipoproteinemia and hepatic lipase deficiency are characterized by a marked increase in LDL and HDL triglyceride concentration,2 30 and in the present study, ISL apoE was strongly correlated with LDL+HDL (d>1.006 g/mL) triglyceride (r=.74, P<.001). The ratio of VLDL cholesterol to total triglyceride (a parameter that predicts the presence of ß-VLDL in type III hyperlipoproteinemic patients)32 did not, however, correlate with ISL apoE (with apoE2/2 patients excluded from the analysis).

In addition to ISL apoE data, the present study has provided information concerning the plasma concentration of apoE in the TRL and HDL fractions of different patient groups. For all subjects combined, plasma TRL apoE concentration was strongly dependent on total and VLDL triglyceride concentration (as documented by others33 34 ), while HDL apoE was positively related to HDL cholesterol levels (Table 2Up). Plasma concentration of HDL apoE has previously been shown to be inversely associated with the presence of coronary heart disease and has been suggested to be of value in the assessment of coronary risk.35 No statistically significant difference was observed between mean HDL apoE levels in normolipidemics, type IIa, or type IIb patients, although HDL apoE concentration was significantly higher in type III patients and significantly lower in type IV patients compared with the other groups (Table 1Up). VLDL and HDL apoE concentrations have been reported in previous studies, but the majority of these used sequential or density gradient ultracentrifugation to isolate plasma lipoprotein fractions,33 34 36 37 38 39 40 a technique that causes 20% to 40% of total apoE to be dissociated from lipoprotein particles41 and that results in the appearance of apoE in the d>1.21 g/mL density fraction.10 33 34 In contrast, gel filtration chromatography results in minimal dissociation of apoE from lipoproteins. Gel filtration chromatography is therefore a preferred method for the determination of plasma lipoprotein apoE distribution, and automation with the use of an FPLC system, as in the present study, provides the added advantage of timely and reproducible plasma separations.

It is well known that the polymorphism of apoE affects plasma lipid and lipoprotein concentrations,42 particularly the plasma concentration of apoE.23 24 25 From data obtained in 2018 randomly selected 35-year-old Dutch men, Smit et al24 calculated that in comparison to the {epsilon}3 allele, the average effect of the {epsilon}2 allele was to raise apoE concentration by 2.1 mg/dL, whereas the {epsilon}4 allele reduced apoE by 0.6 mg/dL. Even in a highly selected group of subjects, we observed a similar effect of apoE polymorphism on total apoE concentration. By normalizing our data for plasma triglyceride concentration (Table 3Up), we found that type III subjects with an apoE2/2 phenotype had a higher concentration of apoE than subjects with an apoE3/2 phenotype (P<.05), who in turn tended to have a higher concentration than apoE3/3 or apoE4/3 subjects. This was also true for lipoprotein apoE concentrations. In the case of ISL apoE, apoE2/2 and apoE3/2 subjects tended to have significantly (P<.05) higher levels of ISL apoE than apoE3/3 or apoE4/3 subjects after adjustment for plasma triglyceride. It is interesting to note that apoE2/2 individuals (type III) had a significant proportion of plasma apoE in their HDL fraction (Figs 5Up and 7Up), which was positively correlated with plasma triglyceride concentration. In contrast, no correlation was observed for apoE3/2 or apoE3/3 subjects, whereas a weak although statistically significant negative relationship was found for apoE4/3 individuals. These data are consistent with the finding that apoE4 has a higher affinity for VLDL than HDL.43 44 Furthermore, the accumulation of apoE in the HDL fraction of type III individuals, in comparison with the other groups (Table 1Up), may reflect the disulfide linkage of apoE2 (which contains two cysteine residues) with apoA-II to form an apoE-A-II complex within HDL.45 The apoE phenotype is, however, only one of many interacting factors affecting the plasma distribution of apoE. Ultimately, the amount of apoE in each lipoprotein fraction is dependent on (1) the total plasma apoE concentration, (2) the apoE phenotype, and (3) the relative concentration and composition of different lipoprotein species, which can affect the exchange of apoE within the circulation.

Patients with premature CAD often have an abnormal lipoprotein profile characterized by one or more of the following features: an increase in the concentration of VLDL cholesterol and triglyceride, an increase in IDL cholesterol (reflecting the plasma accumulation of remnant lipoproteins), an increase in the concentration of small LDL (rich in apoB and relatively poor in cholesterol), and a reduction in HDL concentration.5 46 47 This lipoprotein pattern is characteristic of patients with familial combined hyperlipidemia48 and is often associated with impaired glucose tolerance, central obesity, and hypertension.49 Increasing experimental evidence suggests that remnant lipoprotein accumulation is not just an associated characteristic of the atherogenic lipoprotein profile, but that remnants themselves can be directly and independently atherogenic. Recently, Phillips et al6 presented data from the Montreal Heart Institute Nicardipine Study, a controlled clinical trial designed to evaluate the effect of a calcium antagonist (antianginal and antihypertensive agent) on angiographically assessed progression of coronary artery atherosclerosis. Derived measures of remnant lipoprotein cholesterol concentration were found by multivariate analysis to be independently associated with lesion progression and CAD-related clinical events. These data support earlier findings from the National Heart, Lung, and Blood Institute Type II Coronary Intervention Study,5 which evaluated the effect of cholestyramine (a bile acid–binding resin) on coronary artery atherosclerosis and in which IDL cholesterol concentration was associated with progression of CAD. The accumulation of remnant lipoproteins in the postprandial state has also been independently associated with CAD progression.7 50 The results of these studies together provide strong support for the concept that remnant lipoproteins are potentially atherogenic. They also point out the need for alternative and more direct methods for assessing the presence in plasma of remnant lipoproteins. One approach has been to measure cholesterol in a remnant-like population of VLDL particles isolated with an apoB-100 monoclonal antibody specific for a conformationally sensitive epitope.51 The measurement of apoE concentration in intermediate-sized lipoproteins is an alternative approach that may have inherent pathophysiological relevance, since apoE has been implicated in the formation of lipid-laden foam cells52 53 and in the accumulation of TRL by human atherosclerotic plaques.54 In the present study, ISL apoE concentration correlated positively with various proatherogenic parameters (eg, total plasma cholesterol, triglyceride, and apoB) and inversely with antiatherogenic parameters (eg, HDL cholesterol), providing evidence that ISL apoE may itself be a potentially important predictor of coronary disease.

In summary, the results of this study suggest that (1) a significant proportion of plasma apoE resides within an intermediate-sized remnant-like lipoprotein fraction in both normolipidemic and hyperlipidemic subjects; (2) plasma remnant lipoprotein accumulation is associated with an elevation in ISL apoE concentration; and (3) ISL apoE concentration is significantly correlated with various proatherogenic lipid parameters and may itself be a potentially important atherogenic index. Additional studies are presently under way to establish the ability of ISL apoE concentration to independently predict the presence of CAD and to assess the extent to which ISL apoE concentration can be affected by diet and drug therapy.


*    Selected Abbreviations and Acronyms
 
apo = apolipoprotein
CAD = coronary artery disease
ELISA = enzyme-linked immunosorbent assay
FPLC = fast protein liquid chromatography
ISL apoE = apolipoprotein E in an intermediate-sized remnant-like lipoprotein fraction
Lp(a) = lipoprotein(a)
OD = optical density
PBS = phosphate-buffered saline
TRL = triglyceride-rich lipoprotein
ß-VLDL = ß-migrating VLDL


*    Acknowledgments
 
This study was supported by a joint university-industry grant from the Medical Research Council of Canada and Ciba-Geigy Canada Ltd (UI-11407) and by La Succession J.A. De Sève. Dr Genest was supported by a scholarship from the Medical Research Council of Canada. The excellent technical assistance of Hélène Jacques, Claudia Rodriguez, and Louis-Jacques Fortin is gratefully acknowledged. We would like to thank Drs Jose Ordovas and Ernst Schaefer for their kind gift of immunopurified goat polyclonal antibody against human apoE. The help of Dr Maryvonne Rosseneu in verifying our apoE assay was also appreciated. We would especially like to thank the head nurse of the Lipid Clinic of the Clinical Research Institute of Montreal, Denise Dubreuil, for her assistance in obtaining patients' blood samples. Dr Anne Minnich's help with statistical analysis and in critical evaluation of our manuscript was also appreciated.

Received June 6, 1995; accepted October 26, 1995.


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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
up arrowResults
up arrowDiscussion
*References
 

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