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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:763-772.)
© 1996 American Heart Association, Inc.

Articles

Fenofibrate Reduces Plasma Cholesteryl Ester Transfer From HDL to VLDL and Normalizes the Atherogenic, Dense LDL Profile in Combined Hyperlipidemia

Maryse Guérin; Éric Bruckert; Peter J. Dolphin; Gérard Turpin; M. John Chapman

From the Institut National de la Santé et de la Recherche Médicale (INSERM) (M.G., M.J.C.), Unité 321, Pavillon Benjamin Delessert, and the Service d'Endocrinologie-Métabolisme (E.B., G.T.), Hôpital de la Pitié, Paris, France, and The Lipoprotein Group (P.J.D.), Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia, Canada.

Correspondence to Maryse Guérin, Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 321, Pavillon Benjamin Delessert, Hôpital de la Pitié, 83 Boulevard de l' Hôpital, 75651 Paris cedex 13, France.

	Abstract

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Abstract The effect of fenofibrate on plasma cholesteryl ester transfer protein (CETP) activity in relation to the quantitative and qualitative features of apoB- and apoA-I–containing lipoprotein subspecies was investigated in nine patients presenting with combined hyperlipidemia. Fenofibrate (200 mg/d for 8 weeks) induced significant reductions in plasma cholesterol (-16%; P<.01), triglyceride (-44%; P<.007), VLDL cholesterol (-52%; P=.01), LDL cholesterol (-14%; P<.001), and apoB (-15%; P<.009) levels and increased HDL cholesterol (19%; P=.0001) and apoA-I (12%; P=.003) levels. An exogenous cholesteryl ester transfer (CET) assay revealed a marked decrease (-26%; P<.002) in total plasma CETP-dependent CET activity after fenofibrate treatment. Concomitant with the pronounced reduction in VLDL levels (37%; P<.005), the rate of CET from HDL to VLDL was significantly reduced by 38% (P=.0001), whereas no modification in the rate of cholesteryl ester exchange between HDL and LDL occurred after fenofibrate therapy. Combined hyperlipidemia is characterized by an asymmetrical LDL profile in which small, dense LDL subspecies (LDL-4 and LDL-5, d=1.039 to 1.063 g/mL) predominate. Fenofibrate quantitatively normalized the atherogenic LDL profile by reducing levels of dense LDL subspecies (-21%) and by inducing an elevation (26%; P<.05) in LDL subspecies of intermediate density (LDL-3, d=1.029 to 1.039 g/mL), which possess optimal binding affinity for the cellular LDL receptor. However, no marked qualitative modifications in the chemical composition or size of LDL particles were observed after drug treatment. Interestingly, the HDL cholesterol concentration was increased by fenofibrate therapy, whereas no significant change was detected in total plasma HDL mass. In contrast, the HDL subspecies pattern was modified as the result of an increase in the total mass (11.7%) of HDL_2a, HDL_3a, and HDL_3b (d=1.091 to 1.156 g/mL) at the expense of reductions in the total mass (-23%) of HDL_2b (d=1.063 to 1.091 g/mL) and HDL_3c (d=1.156 to 1.179 g/mL). Such changes are consistent with a drug-induced reduction in CETP activity. In conclusion, the overall mechanism involved in the fenofibrate-induced modulation of the atherogenic dense LDL profile in combined hyperlipidemia primarily involves reduction in CET from HDL to VLDL together with normalization of the intravascular transformation of VLDL precursors to receptor-active LDLs of intermediate density.

Key Words: cholesteryl ester • lipoprotein subspecies • HDL • VLDL • dyslipoproteinemia

	Introduction

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The atherogenic dyslipidemia CHL is characterized by elevated levels of plasma VLDL-C, TGs, and LDL-C.^{1 2} The metabolic abnormalities that underlie this disorder involve increased production rates of VLDL apoB and/or LDL apoB^{3 4} ; in contrast, the fractional catabolic rates of LDL are normal.^{3 4 5} In addition, plasma concentrations of HDL-C are reduced,⁶ while plasma CETP mass and activity are elevated.^{7 8} Furthermore, the LDL subspecies profile in CHL is dominated by small, dense LDL particles that are present in plasma at concentrations as much as threefold greater than those typical of normolipidemic subjects.^{6 9} Dense LDL particles possess a low binding affinity for the cellular LDL receptor¹⁰ and display a prolonged residence time in plasma in vivo.^{11 12} Such dense LDL are therefore exposed to a greater degree to biological modification,^{6 13 14} which results in their catabolism by atherogenic pathways, such as those represented by the scavenger receptors of monocyte-derived macrophages.¹³ These observations are consistent with the hypothesis that dense LDL particles are of elevated atherogenicity and contribute significantly to the premature vascular disease typical of CHL.

Fibric acid and its derivatives are widely used in the therapeutic treatment of the atherogenic dyslipidemias, particularly CHL.^{15 16} These compounds are known to inhibit hepatic VLDL synthesis and secretion,¹⁷ to promote the lipolysis of TG-rich lipoproteins through stimulation of lipoprotein lipase activity,^{18 19} and to promote catabolism of LDL particles via the LDL receptor–mediated pathway.^{12 20 21 22} Evidence suggests that fibrates increase excretion of hepatic cholesterol in bile and that endogenous hepatic cholesterol synthesis may be decreased.²³

A second-generation fibric acid derivative, fenofibrate, has hypolipidemic effects on plasma lipid parameters in CHL.^{24 25 26 27} Plasma cholesterol and TG levels are typically reduced by 10% to 30% and 30% to 67%, respectively; equally, VLDL-C and LDL-C are decreased by 30% to 70% and 2% to 29%, respectively, whereas HDL-C is increased by up to 26%. In addition, an augmentation of 3% to 38% in plasma apoA-I levels and a reduction of 10% to 37% in apoB concentrations have been reported.^{24 25 26 27} A major feature of such hypolipidemic action involves modulation of the de novo synthesis, intravascular remodeling, and cellular degradation of plasma lipoproteins.^{9 12 16 17 20} More specifically, fenofibrate not only reduces absolute circulating concentrations of VLDL but also induces a reduction in the size of VLDL particles of hepatic origin; as a consequence of the intravascular remodeling of such VLDLs, receptor-active LDLs are formed, thereby resulting in an increase in the proportion of plasma LDLs that are catabolized by the nonatherogenic LDL receptor pathway.^{12 20} These latter effects have been demonstrated in vivo both in hypertriglyceridemic and hypercholesterolemic patients.^{12 20} It must be emphasized, however, that the precise mechanisms that underlie the fenofibrate-mediated enhanced formation of LDL receptor–active LDL subspecies are as yet undetermined, although some evidence for drug-induced changes in LDL chemical composition and particle size has been provided.²⁰

There is a paucity of information on the principal features of CET between donor and acceptor lipoproteins in CHL and on the potential contribution of such transfer to the atherogenic lipoprotein profile characteristic of CHL patients. As it is now established that CETP-facilitated transfer and exchange of neutral lipids between HDL and apoB-containing lipoproteins (VLDL, IDL, and LDL) play a major role in the determination of the apoA-I– and apoB-containing lipoprotein subspecies profile,^{28 29 30} we hypothesized that the CET to LDL was deficient in CHL patients as a result of the elevated concentrations and high CE acceptor activity of VLDL. We therefore investigated the effect of fenofibrate on CET and exchange between VLDL, LDL, and HDL to assess the potential role of neutral lipid transfer in the fenofibrate-induced formation of receptor-active LDLs in CHL. In parallel, we evaluated the effect of fenofibrate therapy on the quantitative and qualitative features of apoB- and apoA-I–containing lipoprotein subspecies in CHL patients. We observed that fenofibrate significantly reduced CET from HDL to VLDL as a result of reductions in both plasma CETP-dependent CET activity and VLDL concentrations. Moreover, fenofibrate normalized the LDL subspecies profile by inducing a shift from denser LDL toward receptor-active LDL particles of intermediate density. Our data indicate that the modulation of CETP activity is a major factor in the correction of the intravascular metabolism of apoB-containing lipoproteins during drug therapy.

	Methods

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CHL Patients
Two major groups of subjects have increased circulating concentrations of LDL, ie, those with familial hypercholesterolemia and those with a combination of hypercholesterolemia and hypertriglyceridemia, the latter disorder being termed CHL. Individuals in the latter group are distinguished by elevated levels of both LDL and VLDL. As there is to date no single genetic or clinical marker allowing identification of CHL patients, our classification is similar to that proposed by Arad et al,¹ ie, a lipid profile including an LDL-C level >90th percentile for age and sex with a concomitant plasma TG level 150 mg/dL.

Approval by the human subjects review committee of the hospital and informed consent from all participants were first obtained. CHL patients (n=9) with fasting plasma levels of total cholesterol >200 mg/dL and TGs >150 mg/dL were selected and taken off all lipid-lowering agents 6 weeks before the initial blood sampling. All subjects were nondiabetic. Two of the 9 patients had previously presented with premature coronary heart disease; 5 of the subjects were smokers and 4 were nonsmokers. The clinical characteristics of individual CHL patients are presented in Table 1. Secondary hyperlipidemia was excluded by clinical examination and by the determination of hepatic enzyme activities, the plasma levels of creatinine, glucose, thyroid hormones, and blood proteins, and by evaluation of proteinuria. Four patients had the apoE3/E2 heterozygous phenotype.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics and Plasma Lipid Parameters in CHL Patients: Effect of Fenofibrate Treatment

Blood Samples
Blood samples were obtained by venipuncture after an overnight fast and were placed in sterile EDTA-containing tubes (final concentration, 1 g/L) at the time of inclusion into the study and after 8 weeks of fenofibrate treatment (200 mg/d micronized fenofibrate). Plasma was separated by low-speed centrifugation at 4°C. EDTA (0.1 g/L), sodium azide (0.01%), and gentamicin (0.005%) were added to plasma to chelate metal cations and to inhibit microbial growth.

ApoE Phenotyping by Isoelectric Focusing
Plasma samples (10 µL) were delipidated with 2 mL acetone-ethanol (1:1, vol/vol) at -20°C overnight. Precipitated protein was sedimented by centrifugation (15 minutes, 2500 rpm), resuspended in the same volume of diethyl ether, stored at -20°C for 1 hour, and pelleted by centrifugation under the same conditions. After drying, protein pellets were solubilized in 1 mL of 10 mmol/L Tris-HCL buffer, pH 8.6, containing 8 mol/L urea and 10 mmol/L dithiothreitol. Samples were electrophoresed into a gel consisting of 7.5% polyacrylamide, 8 mol/L urea, and 2.8% pharmalyte (pH 4 to 6.5; Pharmacia). Isoelectric focusing was performed at 200 V for 3 hours, after which plasma proteins were electrophoretically blotted onto nitrocellulose sheets for 1 hour at 90 V in a transfer buffer containing 25 mmol/L Tris, 192 mmol/L glycine, and 20% methanol. The nitrocellulose paper was then blocked with 5% nonfat milk in PBS buffer for 30 minutes at room temperature, after which it was probed for 1 hour with a sheep anti-human apoE antibody diluted 1:1000 in PBS buffer containing 0.1% bovine serum albumin. After washing three times in PBS, the blots were subsequently incubated for 15 minutes with a rabbit anti-sheep peroxidase-conjugated IgG diluted 1:15 000 in PBS. Detection was performed by chemiluminescence (ECL, Amersham). Identification of apoE isoforms was determined by reference to an apoE3/E3 standard, the kind gift of Dr K.H. Weisgraber.

Isolation of Plasma Lipoproteins
Lipoproteins were isolated from plasma by density gradient ultracentrifugation with a Beckman SW41 Ti rotor at 40 000 rpm for 44 hours at 15°C by a slight modification of the method of Chapman et al.³¹ Briefly, plasma density was increased to 1.21 g/mL by adding dry solid KBr. A discontinuous density gradient was then constructed as follows: 2 mL of a NaCl/KBr solution (d=1.24 g/mL), 3 mL plasma (d=1.21 g/mL), 2 mL of a solution (d=1.063 g/mL), 2.5 mL of a solution (d=1.019 g/mL), and 2.5 mL of a NaCl solution (d=1.006 g/mL). All density solutions contained sodium azide (0.01%), EDTA (0.01%), and gentamicin (0.005%), pH 7.4. After centrifugation, gradients were collected from the top of the tube in 12 fractions corresponding to VLDL (d<1.017 g/mL), IDL (d=1.018 to 1.019 g/mL), LDL-1 (d=1.019 to 1.023 g/mL), LDL-2 (d=1.023 to 1.0239 g/mL), LDL-3 (d=1.029 to 1.039 g/mL), LDL-4 (d=1.039 to 1.050 g/mL), LDL-5 (d=1.050 to 1.063 g/mL), HDL_2b (d=1.063 to 1.091 g/mL), HDL_2a (d=1.091 to 1.110 g/mL), HDL_3a (d=1.110 to 1.133 g/mL), HDL_3b (d=1.133 to 1.156 g/mL), and HDL_3c (d=1.156 to 1.179 g/mL). Density limits were taken from a standard curve of density versus volume derived from control gradients containing only salt solutions.³¹ All lipoprotein fractions were analyzed for their lipid and protein content. Recovery of cholesterol was 98% and between 90% and 95% for all other lipids. Comparison of lipoprotein mass profiles as a function of density, computed from chemical analyses of lipoprotein subfractions isolated from aliquots of the same plasma fractionated in separate tubes during the same ultracentrifugal run, revealed a high degree of reproducibility; indeed, mass profiles were indistinguishable, with coefficients of variation of <5% for the masses of individual subfractions.³¹

Lipid and Protein Analyses
Lipids in plasma or in isolated lipoprotein fractions were quantified enzymatically with Bio-Merieux kits (69280) for TGs and phospholipids and Boehringer Mannheim kits (38240) for total and free cholesterol. CE mass was calculated as (Total Cholesterol-Free Cholesterol)x1.67 and thus represents the sum of the esterified cholesterol and fatty acid moieties. Protein was measured by the bicinchoninic acid assay reagent (Pierce). Lipoprotein mass corresponded to the sum of the mass of the individual components for each lipoprotein fraction. The coefficients of variation (interassay and intra-assay) for our chemical analyses of cholesterol, TGs, phospholipids, and protein were 1.9%, 3.6%, 3.8%, and 4.6%, respectively.

Estimation of LDL Subspecies Size
LDL particle size was evaluated by electrophoresis in nondenaturing polyacrylamide gradient gels (2% to 16%; Pharmacia Fine Chemicals) in the GE 2/4 LS gel electrophoresis apparatus.^{6 32 33} A set of standard proteins with known hydrated diameters (latex beads, 380 Å; thyroglobulin, 170 Å; apoferritin, 122 Å; and catalase, 104Å) was run on each slab as a reference marker. From the migration distances of the different LDL subfractions (LDL-1 through LDL-5) and those of the calibration proteins, it was possible to calculate the Stokes' diameters of the LDL subspecies with the Stokes-Einstein equation.³⁴ The correlation coefficient for the regression line of the relationship between the logarithm of the diameter of the calibration proteins and their migration distance was typically >.98. The intra-assay coefficient of variation for LDL sizing analyses was <2%.

Preparation of [³H]CE-Labeled HDL
Radiolabeled HDLs were obtained from the d>1.063 g/mL fraction of plasma by ultracentrifugation at 100 000 rpm for 3 hours and 30 minutes at 15°C with a Beckman TL100 centrifuge.^{28 35} The d>1.063 g/mL fraction was incubated for a total of 18 hours at 37°C in the presence of 4 µCi [1,2,6,7-³H]cholesterol in an ethanol solution (specific activity, 71 Ci/mmol) to allow endogenous lecithin:cholesterol acyltransferase to esterify the radioactive cholesterol.³⁵ HDL-containing esterified radiolabeled CE was then isolated by adjusting the density to 1.21 g/mL by adding dry solid KBr followed by centrifugation at 100 000 rpm for 5 hours and 30 minutes at 15°C. Radiolabeled-HDL preparations displayed a specific radioactivity that ranged from 10 000 to 15 000 cpm/µg CE; >95% of the total radioactive free cholesterol added was transformed into labeled CE.³⁵

CETP-Dependent CET Assay
To estimate the maximal transfer activity in plasma, the CETP-dependent esterified cholesterol assay described by Ahnadi et al³⁶ was employed. Briefly, the density of a 1-mL plasma sample was adjusted to 1.21 g/mL by adding dry solid KBr. After ultracentrifugation for 6 hours at 100 000 rpm at 15°C, plasma lipoproteins were removed, and the bottom fraction was extensively dialyzed in Spectrapor membrane tubing at 4°C against a buffer containing 150 mmol/L NaCl, 10 mmol/L Tris base, 1 mmol/L EDTA, and 1 mmol/L sodium azide, pH 7.4. After dialysis, the volume of the bottom fraction was adjusted to 1 mL by adding buffer and was used as a source of CETP. Labeled HDL CE (200 nmol) was mixed with 800 nmol VLDL/LDL CE in the presence or absence of 400 µL of the CETP-containing bottom fraction (final volume, 1 mL). After 0, 0.5, 1, 2, and 3 hours of incubation at 37°C, a 150-µL sample was withdrawn and immediately chilled on ice. Lipoproteins were then precipitated by the phosphotungstate–magnesium chloride procedure. The VLDL/LDL pellets were dissolved in 100 µL of 0.5 mol/L Na₂CO₃, and their radioactivity content was determined. The facilitated transfer of CE from HDL to VLDL/LDL was calculated from the difference between the radioactivity transferred in the presence or absence of CETP. The interassay and intra-assay coefficients of variation for the CETP-dependent CET assay were <2.6%.

Measurement of CET and Exchange Between HDL and ApoB-Containing Lipoproteins
Radiolabeled HDL (equivalent to 1% of the total HDL CE mass present in 1 mL of a subject's plasma) and iodoacetamide (to inhibit endogenous lecithin:cholesterol acyltransferase activity; final concentration, 1.5 mmol/L) were added to each subject's plasma. Aliquots of 1 mL were incubated for 4 hours at 0°C or 37°C. After incubation, lipoproteins were isolated by density gradient ultracentrifugation as described above, and the radioactive content and lipid composition of individual density fractions were determined. Radioactivity was quantified by a Pharmacia Rack Beta 509 for liquid scintillation spectrometry. The recovery of radioactivity was >98% in all experiments. Measurements of CET and exchange between HDL and the apoB-containing lipoproteins were performed in triplicate. Net mass transfer of CE represents the net augmentation or decrease in the CE mass content of individual lipoprotein fractions. Measurement of radioactivity alone is indicative of CET plus exchange. Thus, exchange of CE between lipoprotein particles was calculated as the difference between the mass of labeled CE transferred, which was calculated from the known specific radioactivity of HDL CE and the net mass transfer of CE as determined enzymatically.³⁵ The interassay and intra-assay coefficients of variation for the determination of CET and the exchange between HDL and apoB-containing lipoproteins were <5%.

Determination of CE Exchange and Transfer Rates Between HDL and ApoB-Containing Lipoproteins
Radiolabeled HDL (equivalent to 1% of the total HDL CE mass present in 1 mL of a subject's plasma) and iodoacetamide (to inhibit endogenous lecithin-cholesterol acyltransferase activity; final concentration, 1.5 mmol/L) were added to 3 mL of each subject's plasma. After 0, 0.5, 1, 2, 3, and 4 hours of incubation at 37°C, aliquots of 0.5 mL were withdrawn and immediately chilled on ice. Plasma lipoproteins were then isolated by sequential ultracentrifugation at 100 000 rpm at 15°C. By this method, we successively isolated the VLDL+IDL (d<1.019 g/mL), LDL (d=1.019 to 1.063 g/mL), and HDL (d=1.063 to 1.21 g/mL) fractions by centrifuging for 1.5, 3.5, and 5.5 hours, respectively. The radioactive content and lipid composition of each individual fraction were determined as described above. The kinetics of CE mass transferred, determined on the basis of radioactivity or by measuring CE mass enzymatically, were linear (r=.99) during the initial 4-hour period of incubation.^{28 35} The rate of CET represents the slope of the individual curves and is expressed in micrograms of CE mass transferred per hour per milliliter of plasma for the rate of net CE mass transfer and in micrograms of labeled CE transferred per hour per milliliter of plasma for the rate of CET+exchange. The difference between these two determinations of CET rates represents the rate of CE exchange between HDL and the apoB-containing lipoproteins. The interassay coefficient of variation for the rates of CET and exchange was <5%.

Statistical Analysis
The effects of fenofibrate on plasma lipid levels, on the plasma concentrations and chemical compositions of lipoprotein particles, and on CET between HDL and apoB-containing lipoproteins were determined by comparing these parameters at the time of inclusion into the study with those after 8 weeks of drug therapy with Student's paired t test.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effects of Fenofibrate on Plasma Lipid and Apolipoprotein Parameters
Plasma lipid and apolipoprotein levels in nine CHL patients before and after 8 weeks of fenofibrate treatment (200 mg/d) are shown in Table 1. At inclusion, all CHL patients displayed plasma lipid levels characteristic of CHL as defined by Arad et al,¹ ie, a lipid profile including an LDL-C level greater than the 90th percentile for age and sex with a concomitant plasma TG level 150 mg/dL. Drug therapy significantly lowered plasma cholesterol and TG levels, by 16% (P<.02) and 44% (P<.007), respectively; indeed, all nine CHL patients displayed a reduction in their plasma cholesterol and TG levels. However, marked differences in the response to fenofibrate treatment were observed among the subjects (the percent change in cholesterol levels ranged from -3% to -37% and in TG levels, from -17% to -64%). Such variations in individual response to drug therapy almost certainly reflect the underlying genetic heterogeneity in the patient population. After fenofibrate treatment we observed significant reductions in plasma VLDL-C (-52%; P=.01), LDL-C (-14%; P<.001), and apoB (-15%; P<.009), whereas fenofibrate induced increases in plasma HDL-C (19%; P=.0001) and apoA-I (12%; P=.003) concentrations. Fenofibrate therapy for 8 weeks induced no significant changes in lipoprotein(a) levels in this study.

Effects of Fenofibrate on Plasma Lipoprotein Distribution and Composition
Plasma lipoprotein particles were separated by density gradient ultracentrifugation to yield multiple LDL and HDL subspecies. The correspondence of density subfractions to lipoprotein subspecies was assessed on the basis of chemical, physical, and immunological analyses.³² The distribution of lipoprotein mass among density gradient subfractions and the relative proportions of LDL and HDL subspecies from the plasma of CHL patients before and after fenofibrate treatment are presented in Table 2. After fenofibrate treatment the mean plasma VLDL and IDL (d=1.018 to 1.019 g/mL) mass concentrations decreased by 37% (P<.005) and 55% (P<.05), respectively, and total LDL mass concentration was reduced by 8% (P=.0276). Before treatment, CHL patients displayed a net asymmetry in the LDL profile in which the denser LDL subfractions, LDL-4 (d=1.039 to 1.050 g/mL) and LDL-5 (d=1.050 to 1.063 g/mL), predominated. After fenofibrate treatment the LDL profile was normalized, and the LDL density peak shifted toward the LDL-3 subfraction (d=1.029 to 1.039 g/mL). Indeed, we observed significant increases in both the mean plasma concentration (26%; P<.05) and the relative proportion (37%; P<.007) of LDL-3 concomitant with a decrease in plasma levels of both LDL-4 (-17%) and LDL-5 (-30%), although these latter changes were not significant (P=.068). In addition, it is noteworthy that absolute concentrations of the light, large LDL subspecies (LDL-1 and LDL-2) were decreased by 16.8% after drug treatment (NS). In contrast, such light subspecies accounted for similar proportions of total LDL before and after fenofibrate (18.8% and 17.0%, respectively). Within the hydrated density range of HDL (d=1.063 to 1.179 g/mL), CHL patients treated by fenofibrate displayed increases in the mass HDL_2a (d=1.091 to 1.110 g/mL; 4%), HD_L3a (d=1.110 to 1.133 g/mL; 19%), and HDL_3b (d=1.133 to 1.156 g/mL; 13%). Fenofibrate therapy also induced significant reductions in plasma HDL_2b (d=1.063 to 1.110 g/mL) and HDL_3c (d=1.156 to 1.179 g/mL) levels (-22%; P=.0004 and -25%; P<.05, respectively).

View this table: [in this window] [in a new window]   Table 2. Effect of Fenofibrate Treatment on the Concentrations and Relative Proportions of Plasma Lipoprotein Subspecies in CHL Patients

The mean weight chemical compositions of native lipoprotein subspecies (expressed as percent of their free cholesterol, CE, TG, phospholipid, and protein contents) are shown in Table 3. Analysis of the composition of VLDL, IDL, and each LDL subfraction revealed no significant effect of fenofibrate therapy. However, we observed a significant reduction in the relative proportion of TGs in HDL_2a (P=.006) and HDL_3a (P=.0048) subspecies after fenofibrate treatment, which was equally reflected in an increase in the CE/TG ratio in these subspecies from approximately 3 to 4.5 (Table 3).

View this table: [in this window] [in a new window]   Table 3. Effect of Fenofibrate Treatment on the Chemical Composition of Plasma Lipoprotein Subspecies in CHL Patients

Hydrated particle size was evaluated for each LDL subfraction by electrophoresis in 2% to 16% polyacrylamide gradient gels under nondenaturing conditions. Particle size showed a marked and progressive diminution as density increased (Table 4). Analysis of particle diameters in each LDL subfraction after fenofibrate therapy showed a tendency toward nonsignificant increases in mean particle size in LDL-4 and LDL-5. It is of note that the LDL-1 (d=1.019 to 1.023 g/mL) and LDL-2 (d=1.023 to 1.029 g/mL) subfractions from CHL patients displayed a superior hydrated particle size compared with those of the corresponding subfractions from normolipidemic subjects.⁶

View this table: [in this window] [in a new window]   Table 4. Evaluation of LDL Subfraction Particle Size in CHL Patients Before and After Fenofibrate Treatment

Effect of Fenofibrate on Maximal Activity of Plasma CETP
To determine whether fenofibrate exerted any effect on the maximal plasma activity of CETP, we employed an exogenous assay of CETP activity that accurately reflects plasma CETP mass.^{7 36} CETP activity in the plasma of seven CHL patients was assessed before and after fenofibrate treatment with an exogenous system containing donor HDL and VLDL/LDL acceptor particles isolated from normolipidemic plasma. After 3 hours of incubation, the mean transfer of CE radioactivity was significantly reduced in plasma from fenofibrate-treated patients by 26% (26.0±2.5% and 19.2±2.6% before and after treatment, respectively; P=.0014) (Fig 1A). Thus, fenofibrate induced a reduction of CETP-dependent CET activity in CHL patients. Furthermore, fenofibrate therapy resulted in a decrease in the amount of radioactive CE transferred (range, 12% to 43%) in the seven subjects tested (Fig 1B).

View larger version (15K): [in this window] [in a new window]   Figure 1. Line graphs show the indirect evaluation of plasma CETP mass in CHL patients (n=7) before () and after () 8-week treatment with fenofibrate 200 mg/d. The exogenous assay used HDL donor and VLDL/LDL acceptor particles isolated from control plasmas with aliquots of the CHL patient's plasma serving as the source of CETP (see "Methods"). A, Fenofibrate therapy induced a significant reduction in the mean resulting CET, thereby indicating a reduction of plasma CETP mass concentration after treatment. B, All seven subjects' plasma showed a reduction of radioactive CET after 3 hours of incubation. Values are mean±SD. *P=.0014, **P=.0001 vs CHL patients before treatment.

CET and Exchange Between HDL and ApoB-Containing Lipoproteins
The transfer of radioactive CEs between lipoprotein species is indicative of exchange and/or mass transfer reactions, whereas variations in net mass of CE specifically reflect CET reactions (see "Methods"). Thus, the difference between these two determinations (radioactivity or mass) represents CE exchange reactions. To evaluate the effect of fenofibrate on the reactions of CET and/or the exchange mediated by CETP that occurs between lipoprotein particles, plasma from each CHL patient before and after fenofibrate treatment was incubated with radiolabeled HDL.

First, the rates of transfer and exchange of CE between HDL and the apoB-containing lipoproteins were determined in plasma from CHL patients before and after fenofibrate treatment by measuring the increase or decrease in both radioactive CE and the CE mass content of the VLDL+IDL, LDL, and HDL fractions isolated by sequential ultracentrifugation at each time point of incubation. The effects of fenofibrate treatment on the rates of transfer and exchange of CEs between HDL and apoB-containing lipoproteins are shown in Table 5. After fenofibrate administration, the rate of transfer of HDL CE to VLDL was significantly reduced (-38%; P=.0001). This finding is consistent with that of Mann et al³⁷ in hypertriglyceridemic patients treated with bezafibrate. However, the rate of CE exchange between HDL and LDL remained unchanged after fenofibrate treatment. It is also important to note that the rate of loss of CE from HDL closely approximates the corresponding rate of gain of CE by the apoB-containing lipoproteins.

View this table: [in this window] [in a new window]   Table 5. Effect of Fenofibrate Treatment on the Rates of CE Exchange and Transfer Between HDL and the ApoB-Containing Lipoproteins

Second, the total transfer of radioactive CE and CE mass transferred to the apoB-containing lipoproteins was subsequently quantified after 4 hours of incubation at 37°C after density gradient ultracentrifugation. Fig 2 represents the distribution of radiolabeled CE, expressed in micrograms of labeled CE transferred per milliliter of plasma (calculated from the known specific radioactivity of HDL CE) and CE mass, expressed in micrograms of CE transferred per milliliter of plasma (determined enzymatically) in CHL patients before and after fenofibrate treatment. Evaluation of CET from HDL to VLDL on the basis of radioactivity or by enzymatic determinations gave similar results, indicating that no exchange of CE occurred between HDL and VLDL during the first 4 hours of incubation at 37°C. In contrast, the transfer of radioactive CET from HDL to LDL occurred to a significant degree, during which the LDL fraction received 20% of total labeled CE transferred from HDL to the apoB-containing lipoproteins. However, no net mass transfer of CE from HDL to LDL was detected. Thus, the acquisition of radioactive CEs by the LDL fraction of CHL patients represents only CE exchange between HDL and LDL in the absence of net mass transfer. Fenofibrate treatment induced a significant reduction (-34%) of CET from HDL to VLDL (93.6±16.7 versus 61.9±11.6 µg CE transferred/mL plasma before and after treatment, respectively; P=.0023). However, drug therapy induced no effect on CE exchange between HDL and LDL.

View larger version (21K): [in this window] [in a new window]   Figure 2. Bar graph shows the distribution of radiolabeled CE transferred (solid bars) calculated from the known specific radioactivity of HDL CE and the CE mass transferred (dotted bars) as determined by enzymatic methods after 4 hours of incubation at 37°C in CHL plasmas before and after 8 weeks of fenofibrate (200 mg/d). Labeled CET from HDL to LDL in the absence of CE mass transfer indicates that only reactions of CE exchange occurred between HDL and LDL in CHL patients. Fenofibrate treatment induced a significant reduction of net CE mass transfer from HDL to VLDL. However, drug therapy had no effect on CE exchange between HDL and LDL. Values are mean±SD. *P<.02, **P<.008 vs CHL patients before treatment.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study has demonstrated for the first time that a second-generation fibrate, fenofibrate (in micronized form), can induce significant reductions in both the elevated total plasma CETP-dependent CET activity and in CET from HDL to VLDL in CHL; such total transfer activity is strongly correlated to plasma CETP mass.³⁶ Moreover, fenofibrate quantitatively normalized the atherogenic LDL subspecies profile characteristic of CHL by specifically reducing the plasma levels of both dense LDL subfractions (LDL-4 and LDL-5, -21%) and proportionately increasing those of the intermediate LDL subspecies (LDL-3, 26%) of high receptor-binding affinity.¹⁰ Such changes occurred concomitantly with significant reductions not only in plasma cholesterol (-16%), LDL-C (-14%), and apoB (-15%) but also in plasma TG (-44%) and VLDL-C (-52%) levels. Finally, despite marked increases in HDL-C and apoA-I levels (19% and 12%, respectively), total HDL mass was not modified by fenofibrate; this observation was, however, accounted for by subtle modifications in the HDL subspecies profile, in which the relative proportions of HDL_2a, HDL_3a, and HDL_3b were each significantly increased at the expense of HDL_2b and HDL_3c.

Our new methodological approach to the determination of physiological rates of CE exchange and transfer has allowed us to demonstrate that the rate of CE mass transfer from HDL to VLDL is accelerated twofold in CHL patients (24.1±3.9 µg CE transferred·h^-1·mL^-1 plasma) compared with normolipidemic subjects (12.3±2.8 µg CE transferred·h^-1·mL ^-1 plasma³⁵ ). In contrast, no CE mass transfer from HDL to LDL was detectable in the plasma from CHL patients; radiolabeled CET was observed, however, implying that only reactions of CE exchange occurred between HDL and LDL. The absence of CE mass transfer and the occurrence of CE exchange alone between HDL and LDL thus constitute a characteristic of CHL.³⁸ Indeed, in this respect, CHL patients differ markedly from normolipidemic subjects and from familial heterozygous hypercholesterolemic patients, in whom CE mass transfer from HDL to LDL predominates over that to VLDL.^{28 35} Several mechanisms may underlie the deficiency of CE mass transfer between HDL and LDL in CHL patients. Thus, on the one hand, plasma VLDLs, which qualitatively represent the most active CE acceptors among the apoB-containing particles,³⁵ are present at elevated concentrations (3- to 10-fold) and therefore are at the origin of a diminished CE flux to LDL.³⁸ On the other hand, LDL particles from CHL patients may interact poorly with CETP as a result of their qualitative abnormalities,⁶ which involve both chemical composition and particle size.³⁸ After fenofibrate therapy the overall CE mass transfer to apoB-containing lipoproteins was significantly reduced (-34%; P=.0023), whereas no effect on CE exchange between HDL and LDL was observed. Concomitant with the decrease in CETP activity, plasma VLDL concentrations were lowered by 37%. Thus, the occurrence of CE exchange between HDL and LDL was not directly related to the predominance of VLDL particles in the plasma of CHL patients. As fenofibrate therapy did not qualitatively modify LDL subspecies and thus did not modulate the potential affinity of LDL particles for CETP, we hypothesize that the abnormal chemical composition and particle structure of LDLs in CHL underlie their low affinity for CETP and that in the presence of elevated levels of VLDL acceptors, reactions of CE exchange occur exclusively between HDL and LDL.

To determine whether the reduction of plasma CET after fenofibrate therapy might result at least in part from a reduction in plasma CETP mass, we employed an exogenous assay of maximal plasma CETP activity that accurately reflects plasma CETP mass.^{7 36} This indirect assay indicated that fenofibrate induced a significant reduction (-26%) in plasma CETP concentration. Information on the expression of the CETP gene gained from CETP-transgenic mice suggests that transcription of the CETP gene is induced by a high-cholesterol diet³⁹ and that this response to cholesterol requires DNA sequences contained in the natural flanking regions of the human CETP gene. Furthermore, the proximal promoter of the gene mediates induction of CETP mRNA by dietary cholesterol, thereby indicating that this region contains a sterol upregulatory element distinct from the known sterol response element.³⁹ In this context, it is relevant that evidence has been presented to indicate that fenofibrate inhibits endogenous cholesterol synthesis before mevalonate, indirectly causing a significant reduction of hydroxymethylglutaryl–coenzyme A reductase activity.⁴⁰ This drug may also inhibit acyl coenzyme A–cholesterol acyltransferase activity, thereby reducing conversion of free cholesterol to its ester form.⁴⁰ These observations lead us to suggest that the reduction of cholesterol synthesis by fenofibrate may induce downregulation of human CETP gene expression, resulting in the diminution of plasma CETP concentration.

Analysis of the LDL subspecies isolated by density gradient ultracentrifugation not only revealed that dense, small particles (LDL-4, d=1.039 to 1.050 g/mL and LDL-5, d=1.050 to 1.063 g/mL) predominated in CHL subjects but also that their plasma levels were elevated up to threefold compared with those of normolipidemic subjects.^{6 9} This atherogenic, dense LDL profile ("phenotype B"⁴¹ ) was dramatically normalized by fenofibrate therapy as a result of an absolute increase (26%) in levels of intermediate LDL subspecies (LDL-3, d=1.030 to 1.039 g/mL), a modification that occurred simultaneously with diminution in the plasma levels of LDL-4 and LDL-5 (-17% and -30%, respectively). Such normalization was specifically related to the quantitative modifications of LDL subspecies that apparently occurred in the absence of alterations in the composition or size of LDL particles. Similar findings are reported by Caslake et al,¹² who failed to observe an effect of fenofibrate on the composition of LDL particles in patients with hypercholesterolemia. It is of interest to note that another fibric acid derivative, ciprofibrate, also normalizes the atherogenic dense LDL profile in CHL patients.⁹ This effect involves the promotion of LDL removal via the receptor pathway.²² However, in contrast to fenofibrate, ciprofibrate induced normalization of both the composition and size of LDL subspecies.⁹

Investigation of neutral lipid transfer in the present studies has afforded new insight into the mechanisms underlying the fenofibrate-induced normalization of the LDL subspecies profile. Thus, transfer rates of CE from HDL to VLDL in CHL patients were elevated twofold over normal; in addition, VLDLs were the major acceptors of CE. In contrast, no net mass transfer of CE to LDL was detected before or after fenofibrate therapy. After treatment, the rate of CET from HDL to VLDL was significantly reduced as a result of both reductions in plasma CETP mass and VLDL concentrations. These data are consistent with the hypothesis that the abnormal hepatic formation and intravascular metabolism of TG-rich VLDLs in CHL are major determinants in the formation of dense LDL subspecies in this atherogenic dyslipidemia. We suggest that several mechanisms underlie the shift in LDL subspecies profile induced by fenofibrate: the normalization of the hepatic formation and secretion of VLDL, of the hepatic catabolism of VLDL remnants, of the intravascular remodeling of VLDL as a result of stimulation of lipoprotein lipase and reduction in CETP mass and CET activity and the reduced production of LDL.^{12 19 20 40} Acting in concert, these complex metabolic actions result in the preferential intravascular formation of intermediate-density LDL of high receptor-binding affinity from hepatic VLDL precursors. Overall, our above postulates are entirely consistent with the significant inverse relationship typically observed between TG levels and mean plasma LDL particle size^{42 43} as well as with studies of the Framingham cohort that show that mean plasma LDL particle size can be modulated by a change in plasma TG concentration.⁴³ Finally, our findings allow us to exclude one potential mechanism whereby dense LDL subspecies might be transformed into intermediate LDL particles in CHL plasma and that would involve progressive CETP-facilitated transfer of CE molecules to the hydrophobic core of dense LDL; such a mechanism as this has been proposed to account for the transformation of dense LDL into lighter subspecies in the guinea pig.⁴⁴

Plasma HDL₂ levels are some 20% lower in CHL patients than normolipidemic subjects (146.8±21.0 versus 182.6±39.8 mg/dL, respectively [n=9]; P<.03³⁸ ). All nine CHL subjects in the present study displayed significant increases in plasma HDL-C level after fenofibrate treatment. Earlier studies have typically focused on the effects of fenofibrate on plasma HDL-C without regard to total plasma HDL mass. When total plasma HDL concentrations are considered, however, we observed that no significant modification occurred with drug therapy. The effects of fenofibrate on plasma HDL concentration were, however, heterogeneous among the nine patients studied. Thus, three patients displayed a 5% to 34% increase in total HDL concentration after treatment, three showed a reduction of 7% to 18%, and the last three presented similar HDL levels before and after fenofibrate therapy. Fenofibrate induced an increase in HDL_2a (4%; d=1.091 to 1.110 g/mL), HDL_3a (18%; d=1.110 to 1.133 g/mL), and HDL_3b (13%; d=1.133 to 1.156 g/mL) levels concomitant with reductions in HDL_2b (-22%; d=1.063 to 1.091 g/mL) and HDL_3c (-25%; d=1.156 to 1.176 g/mL). The latter two HDL subfractions quantitatively represented the minor HDL subfractions in CHL patients compared with the HDL_2a, HDL_3a, and HDL_3b subspecies. Thus, HDL_2b and HDL_3c accounted for 33% and 25% of total plasma HDL mass before and after fenofibrate treatment, respectively. Consequently, the net effect of drug treatment was an increase in plasma HDL-C concentrations in the absence of any modifications in total HDL mass. Similar conclusions were reached by Eisenberg et al⁴⁵ in hypertriglyceridemic patients, in whom a reduction in plasma HDL_2a and an increase in HDL_2b levels were found; in addition, treatment of hypertriglyceridemic patients with bezafibrate effected a shift in this HDL pattern as a result of an increase in HDL_2a concentration at the expense of HDL_2b.⁴⁵ Thus, the net effect was an elevation in HDL-C.

During circulation of HDL₃ in plasma, CE accumulates in the hydrophobic core of these particles through the action of lecithin:cholesterol acyltransferase; in consequence, HDL₃ are transformed into the larger lipoprotein subspecies HDL_2a.⁴⁶ CETP is proposed to mediate the heteroexchange of TGs and CE primarily between HDL_2a and the apoB-containing lipoproteins.⁴⁶ Such neutral lipid exchange results in the formation of HDL_2b, which subsequently becomes TG-enriched and deficient in CE. HDL_2b is then transformed back to HDL₃ by hydrolysis of TGs and phospholipids by the action of hepatic lipase (see Fig 3 in Reference 46⁴⁶ ). After fenofibrate therapy, the relative proportions of HDL_2a, HDL_3a, and HDL_3b in plasma increased by 20%, 40%, and 29%, respectively, whereas those of HDL_2b and HDL₃c decreased (-10% and -15%, respectively). In addition, we observed a significant reduction in the relative proportion of TGs in HDL_2a and HDL_3a. These quantitative and qualitative modifications of HDL subspecies are consistent with the drug-induced reduction of CETP activity observed in CHL plasma and do not appear to reflect an increase in hepatic lipase activity, which is essentially unaffected by fenofibrate.⁴⁰

It is of substantial interest to consider the present findings in light of the recent advances in our knowledge of the molecular mechanisms that underlie the lipid-lowering action of the fibrates and of fenofibrate in particular. The fibrates activate a group of transcription factors belonging to the superfamily of nuclear hormone receptors, the so-called peroxisome proliferator–activated receptors.^{47 48} Upon activation, these receptors bind to the response elements of target genes and thus regulate their expression. Several such genes have been identified, including those of apoC-III, apoA-I, apoA-II, apoA-IV, acyl coenzyme A oxidase, and possibly that of lipoprotein lipase.^{49 50 51 52} The transcriptional downregulation of apoC-III and the upregulation of lipoprotein lipase by fenofibrate^{50 51 52} are of major relevance to our present observations, in which circulating levels of VLDLs and their remnants were markedly reduced in CHL patients. Such actions enhance both the intravascular lipolysis of TG-rich lipoproteins as well as their tissue catabolism via apoE-mediated binding to specific cellular receptors.⁴⁹ These insights thus shed new light on one of the key mechanisms implicated in the fenofibrate-induced normalization of the atherogenic dense LDL profile in CHL, ie, the correction of the abnormal hepatic and intravascular metabolism of TG-rich VLDL particles and their remnants.

	Selected Abbreviations and Acronyms

 

CE = cholesteryl ester CET = cholesteryl ester transfer CETP = cholesteryl ester transfer protein CHL = combined hyperlipidemia HDL-C = HDL cholesterol LDL-C = LDL cholesterol PBS = phosphate-buffered saline TG = triglyceride VLDL-C = VLDL cholesterol

	Acknowledgments

 
We are indebted to INSERM, to Laboratoires Fournier, and to the Medical Research Council of Canada (MT-5999 to Dr P.J. Dolphin) for financial support of these studies. Dr M. Guérin was supported by a research fellowship from the Association Claude Bernard.

Received August 10, 1995; accepted January 19, 1996.

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