Pravastatin Modulates Cholesteryl Ester Transfer From HDL to ApoB-Containing Lipoproteins and Lipoprotein Subspecies Profile in Familial Hypercholesterolemia
Abstract Familial hypercholesterolemia (FH) results from genetic defects in the LDL receptor, and is characterized by a marked elevation in plasma LDL and by qualitative abnormalities in LDL particles. Because LDL particles are major acceptors of cholesteryl esters (CEs) from HDL, significant changes occur in the flux of CE through the reverse cholesterol pathway. To evaluate the effects of an HMG-CoA reductase inhibitor, pravastatin, on CE transfer from HDL to apo B–containing lipoproteins and on plasma lipoprotein subspecies profile in subjects with heterozygous FH, we investigated the transfer of HDL-CE to LDL subfractions and changes in both concentration and chemical composition of the apo B– and the apo AI–containing lipoproteins. After pravastatin treatment (40 mg/d) for a 12-week period, plasma LDL concentrations (mean±SD, 745.4±51.9 mg/dL) were reduced by 36% in patients with FH (n=6). By contrast, the qualitative features of the density profile of LDL subspecies in patients with FH, in whom the intermediate (d=1.029 to 1.039 g/mL) and dense (d=1.039 to 1.063 g/mL) subspecies were significantly increased relative to a control group, were not modified by pravastatin. In addition, no significant effect on the chemical composition of individual LDL subfractions was observed. Furthermore, plasma HDL concentrations were not modified, although the density distribution of HDL was normalized. Indeed, the HDL density peak was shifted towards the HDL2 subfraction (ratios of HDL2 to HDL3 were 0.7 and 1.1 before and after treatment, respectively). Evaluation of plasma CE transfer protein (CETP) mass was performed with an exogenous CE transfer assay. Under these conditions, no modification of plasma CETP protein mass was induced by pravastatin administration. However, the rate of CE transfer from HDL to LDL was reduced by 24% by pravastatin (61±17 μg CE · h−1 · mL−1 plasma; P<.0005), although intermediate and dense LDL subfractions again accounted for the majority (71%) of the total CE transferred to LDL. Thus, pravastatin induced reduction of plasma CETP activity without change in the preferential targeting of the transfer of HDL-CE towards the denser LDL subfractions. In conclusion, pravastatin reduces the elevated flux of CE from HDL to apo B–containing lipoproteins in subjects with heterozygous FH as a result of a reduction in the LDL particle acceptor concentration.
- Received May 24, 1995.
- Accepted June 20, 1995.
In plasma, CETP mediates the redistribution of CEs between HDL and apo B–containing lipoproteins, ie, VLDL, IDL, and LDL.1 CETP is a major component of the reverse cholesterol transport pathway, whose activity may be influenced by several factors. Among them, the composition and the relative proportions of both donor and acceptor lipoprotein particles appear to play an important role in the modulation of CETP activity.2 3 4 Moreover, CETP has also been shown to influence the particle distribution within the LDL and HDL density intervals.5 6
Previous studies have demonstrated a potential antiatherogenic role of CETP in normolipidemic subjects by promotion of the preferential redistribution of CE to LDL subfractions of intermediate density (d=1.023 to 1.050 g/mL),7 8 which are known to possess an elevated affinity for the cellular LDL receptor.9 Both CETP concentration and activity are, however, elevated in the plasma of hypercholesterolemic subjects.4 10 11 We have recently demonstrated that CETP promotes a preferential transfer of HDL-CE towards the intermediate and dense LDL subfractions (d=1.029 to 1.063 g/mL) in patients with FH.11 By this mechanism, CETP may contribute to the formation of the CE-enriched LDL subspecies observed in these subjects and may enhance the atherogenic potential of LDL particles in FH. Under these conditions, CETP may play an important proatherogenic role in FH.
To date, the potential effects of several HMG-CoA reductase inhibitors on plasma CETP activity have been studied. Initially, Bagdade et al12 reported that lovastatin did not modify the net mass of CE transferred from HDL to the apo B–containing lipoproteins in hypercholesterolemia. More recently, simvastatin has been shown to decrease CE transfer between HDL and the apo B–containing lipoproteins in normolipidemic subjects.13 Furthermore, it has been reported that pravastatin is without effect both on plasma CETP mass14 and activity15 in hypercholesterolemic patients. The substantial inconsistencies between these findings may be explained at least in part by the marked contrast between the different assay procedures used. Nevertheless, these data attest to the elevation in CETP activity in hypercholesterolemia.
In the present study, we have investigated whether the known LDL-lowering effects of the HMG-CoA reductase inhibitor pravastatin14 15 16 17 18 19 20 21 22 23 24 25 are accompanied on the one hand by a normalization of the transfer of HDL-CE to LDL subfractions and on the other by a normalization of the qualitative abnormalities of LDL particles in patients with heterozygous FH. For that purpose, CE transfer from HDL to apo B–containing lipoproteins, as well as the quantitative and qualitative features of the apo B– and the apo AI–containing lipoprotein subspecies, has been determined in plasma from patients with FH before and after pravastatin treatment. Our data reveal that pravastatin significantly reduced CE transfer from HDL to apo B–containing lipoproteins; such reduction involved an indirect mechanism in which LDL particle acceptor number was diminished in the absence of an effect on plasma CETP itself.
Subjects and Blood Samples
The control group, which has been described previously,11 consisted of five normolipidemic subjects (four men and one woman) with a plasma cholesterol level of less than 250 mg/dL. Six heterozygous patients (four men and two women) with plasma cholesterol level of more than 300 mg/dL were selected and taken off all lipid-modifying drugs for 6 weeks before the initial blood sampling. A second blood sample was taken after 12 weeks of pravastatin treatment (40 mg/d). Two of the men were 19 and 24 years old and had BMIs of 21.6 and 21.4 kg/m2, respectively. The remaining 4 patients were between 53 and 58 years of age and had BMIs between 21.7 and 25.4 kg/m2. The mean BMI for all subjects with heterozygous FH was 22.4±1.7. The two women were 54 and 58 years old and were postmenopausal. It is worthy of note that both total HDL concentrations and subclass distribution (HDL2 and HDL3) in male and female patients with FH resembled each other closely (data not shown). The plasma LDL subfraction concentrations and CETP activity of four of the six patients with FH reported here at baseline have been previously studied (see Reference 1111 ). No modification of the individual BMIs was observed after pravastatin treatment.
Blood samples were obtained from the subjects by venipuncture after an overnight fast and placed in sterile EDTA-containing tubes (final concentration, 1 mg/mL). Plasma was separated by low-speed centrifugation at 4°C and maintained at this temperature until its use on the same day. All subjects in this study had low levels of lipoprotein (a) (<20 mg/dL) and were nonsmokers. Approval by the Human Subjects Review Committee of the hospital and informed consent from all participants was obtained before the start of the study.
Isolation of Plasma Lipoproteins
Lipoproteins were isolated from plasma by density gradient ultracentrifugation with a Beckman SW41 Ti rotor centrifuged at 40 000 rpm for 44 hours in a Beckman XL70 at 15°C and by a slight modification of the method of Chapman et al.26 In brief, plasma density was increased to 1.21 g/mL by addition of dry solid KBr. A discontinuous density gradient was then constructed at ambient temperature in a 12-mL tube by addition of 2 mL of an NaCl-KBr solution of density 1.24 g/mL. The following solutions were then overlayered: 3 mL of plasma at 1.21 g/mL; 2 mL of NaCl-KBr solution of d=1.063 g/mL; 2.5 mL of d=1.019 g/mL; and 2.5 mL of NaCl solution of d=1.006 g/mL. All density solutions contained sodium azide (0.01%), EDTA (0.01%), and gentamicin (0.005%) at pH 7.4. After centrifugation, gradients were collected from the top of the tubes with an Eppendorf precision pipette in aliquots of 0.4 mL. A total of 30 fractions were collected. The first 23 fractions were analyzed for their lipid and protein content.
Lipid and Protein Analysis
Lipids in plasma or in isolated lipoprotein fractions were quantified enzymatically by use of Bio-Merieux kits for TC, FC, TG, and PL.26 CE mass was calculated as 1.67 times (TC-FC) mass. Protein was measured with the method of Lowry et al.27 Lipoprotein mass corresponded to the sum of the mass of the individual components for each lipoprotein fraction.
Electrophoretic Analysis of HDL
Native HDL fractions were submitted to gradient polyacrylamide gel electrophoresis (ISOLAB polyacrylamide 4% to 25%) under nondenaturing conditions.28 This procedure separated HDL into two subfractions that could be respectively considered as HDL2 and HDL3. After the gels were stained with Coomassie Brilliant Blue, the Stokes’ diameters of the HDL particles were estimated from the densitometric scans of the gels.
Measurement of LCAT Activity
LCAT activity was measured by use of the endogenous substrate method of Albers et al,29 in which the decrease in plasma FC content after incubation at 37°C was determined.
Measurement of CE Transfer From HDL to Apo B–Containing Lipoproteins
CE net mass transfer was determined in whole plasma from individual subjects by use of an in vitro method previously described by us.7 In brief, identical aliquots of individual subjects’ plasma were incubated for various time periods at 37°C in the presence of iodoacetamide (final concentration, 1.5 mmol/L) to inhibit LCAT activity; after the incubation the lipoprotein fractions were isolated by density gradient ultracentrifugation as described above. The lipid and protein compositions of individual density fractions were then determined. The recovery of cholesterol from the gradient was 98%, while that for TG and PL was 90% to 95%. Net mass transfer of CE represents the augmentation or decrease in CE content of the individual lipoprotein subfractions. Total transfer of CE to LDL represents the summation of the transfer to the individual LDL subfractions after 6 hours of incubation, after which net mass transfer no longer occurs.7 Data reported in Figs 1⇓ and 2⇓ represent the 6-hour values corresponding to total CE mass transferred from HDL to apo B–containing lipoproteins expressed in micrograms of CE transferred per milliliter of plasma. The rate of net mass transfer is calculated from the linear portion of the curve (Fig 3⇓) in the first 4 hours of incubation and expressed in micrograms of CE transferred per hour per milliliter of plasma.
CETP-Dependent Esterified Cholesterol Transfer Assay
For estimation of plasma CETP mass, the CETP-dependent esterified cholesterol assay of Ahnadi et al13 was used. In brief, the density of 1 mL of each plasma sample was adjusted to 1.21 g/mL by addition of dry solid KBr. After centrifugation at 541 000g/maxmin at 10°C, plasma lipoproteins were removed and the bottom fraction of 1 mL was extensively dialyzed in Spectrapor membrane tubing at 4°C against a buffer containing (in mmol/L) NaCl 150, Tris base 10, EDTA 1, and sodium azide 1, pH 7.4. This bottom fraction was used as a source of CETP. Labeled HDL-CE (200 nmol) was mixed with 800 nmol of VLDL/LDL-CE in the presence or absence of 400 μL of the CETP-containing bottom fraction in a final volume of 1 mL. After 0, 0.5, 1, 2, and 3 hours of incubation at 37°C, 150 μL of sample was withdrawn and immediately chilled to 0°C. Lipoproteins were then precipitated by the phosphotungstate/magnesium chloride procedure. The VLDL/LDL pellets were dissolved in 20 μL of 0.5 mol/L Na2CO3, and their content of radioactivity 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.
HDL-Dependent CE Transfer Assay
To evaluate whether pravastatin treatment resulted in an altered capacity of plasma HDL to participate in CE transfer, an HDL-dependent CE transfer assay was performed as previously described.30 In this procedure, unidirectional mass transfers of CE were measured from native HDL to acceptor lipid emulsions in the presence of partially purified CETP.31 The lipid emulsions used as acceptors of CE contained triolein, egg lecithin, FC (mass ratio, 80:17:3) and traces of tritiated triolein (360 cpm/nmol)32 and permitted a simultaneous evaluation of the reciprocal transfer of triolein from the lipid emulsion to HDL. For the transfer assay, HDL (250 nmol CE) was mixed with emulsions (1 mg triolein) in the presence or absence of 0.3 mg partially purified CETP in a final volume of 0.5 mL. Samples contained 1.5 mmol/L iodoacetate to inhibit LCAT activity. Incubations were performed at 37°C with gentle stirring. After 0, 30, 60, 90, or 120 minutes, the transfer reaction was stopped by cooling of the tubes in an ice bath. Aliquots of 0.2 mL were adjusted to a density of 1.07 g/mL with solid NaBr and layered with 600 μL of water before ultracentrifugation at 4°C for 30 minutes at 50 000 rpm in a TLS-55 swinging bucket rotor of the Beckman TL-100 ultracentrifuge. The emulsions were discarded, and HDL was recovered for CE determination by enzymatic assay of TC and FC and for TG determination by liquid scintillation spectrometry. The amounts of CE and TG lost and gained, respectively, by HDL during the transfer reaction were calculated by difference with respect to their contents at time zero. CETP-facilitated HDL-dependent CE transfer was calculated as the difference between the values obtained in the presence of CETP and those obtained in its absence.
The effects of pravastatin on the plasma concentrations and chemical compositions of lipoprotein subspecies and CE transfer were determined by comparison of these parameters at inclusion time and at 12 weeks after therapy by Student’s paired t test. Comparison of values (mean±SD) in FH patients before or after treatment with those in control subjects was performed using Student’s unpaired t test.
Plasma Lipid and Apolipoprotein Distribution
Plasma lipid levels before and after pravastatin treatment in patients with FH are shown in Table 1⇓. At inclusion, the patients with FH displayed significantly elevated plasma cholesterol, LDL cholesterol, and apo B levels compared with normolipidemic subjects.11 The plasma concentrations of TG were elevated in patients with FH in comparison with our control group11 but were within the normal range. After pravastatin administration, we observed significant reductions of plasma cholesterol (31%; P=.0001), LDL cholesterol (35%; P<.0001), and apo B (30%; P=.0001) levels. Moreover, no significant changes in VLDL cholesterol or HDL cholesterol were induced by pravastatin.
In this study, plasma lipoprotein particles containing apo B or apo AI were separated by density gradient ultracentrifugation yielding multiple LDL and HDL subfractions. We divided the LDL fraction into five LDL subfractions, LDL-1 to LDL-5, and the HDL fraction into two major subfractions, HDL2 and HDL3. The distribution of lipoprotein mass among density gradient subfractions from the plasma of patients with FH before and after pravastatin treatment is shown in Fig 4⇓. After pravastatin administration, the mean plasma LDL concentration was reduced by 36% (P=.0001) in patients with FH (745±52 mg/dL and 474±51 mg/dL before and after treatment, respectively), as shown in Table 2⇓. Plasma lipoprotein analysis showed a net asymmetry in the LDL profile towards 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), at baseline in patients with FH in comparison with the symmetrical LDL profile observed in the control group, as shown in Fig 4⇓. Indeed, the mean plasma concentrations of LDL-4 and LDL-5 (166.6±99.3 mg/dL and 53.8±26.9 mg/dL, respectively) in patients with FH were significantly greater than those of the corresponding subspecies in the control group (LDL-4, 51.9±18.1 mg/dL; LDL-5, 27.4±6.0 mg/dL; P<.02 and P<.03, respectively). Furthermore, whereas pravastatin therapy significantly reduced the concentration of each plasma LDL subfraction (P<.0001), it did not normalize the LDL profile of FH patients (Fig 4⇓).
Within the hydrated density range of HDL (d=1.063 to 1.179 g/mL), no significant differences in total HDL plasma concentrations were noted before and after pravastatin treatment (Table 2⇑). Comparison of HDL cholesterol values for the subjects with FH before and after pravastatin treatment on an individual basis showed increases in three subjects (51 to 59, 55 to 68, and 66 to 68 mg/dL), decreases in two subjects (39 to 31 and 49 to 44 mg/dL), and no change in one subject (52 to 52 mg/dL) after treatment. The relative proportion of the HDL3 subfraction (d=1.112 to 1.179 g/mL) was increased by 12.3% in patients with FH before treatment in comparison with normolipidemic subjects (59.2±5.3% and 46.9±6.6%, respectively; P<.01). After pravastatin administration, the HDL profile in patients with FH was normalized and the HDL density peak shifted towards the HDL2 subfraction (d=1.063 to 1.112 g/mL) (Fig 4⇑). Indeed, the relative proportion of the HDL2 subfraction was increased by 11.8% (40.8±5.3% to 52.6±3.6% in patients with FH before and after treatment; P=.0001). At the same time, pravastatin administration significantly reduced (by 11.8%) the relative proportion of plasma HDL3 (59.2±5.3% and 47.4±3.6% before and after treatment, respectively; P=.0001). Moreover, the HDL2/HDL3 ratio was significantly reduced in patients with FH before treatment (Table 2⇑) and normalized by pravastatin therapy (HDL2/HDL3 ratio in control subjects, 1.12±0.3). These observations could not be attributed to any difference in plasma LCAT activities; indeed, no significant change in LCAT activity was induced by pravastatin administration in patients with FH (83±35 versus 75±20 nmol CE formed · h−1 · mL−1 plasma in patients with FH before and after pravastatin therapy, respectively).
Interestingly, particle diameters for both HDL2 and HDL3 were significantly decreased after pravastatin treatment. The mean HDL2 particle diameter in the patients with FH before treatment was 11.6±0.3 nm and decreased to 11.3±0.4 nm after treatment (P<.003). Similar results were obtained for HDL3 particles (10.1±0.3 nm and 9±0.5 nm before and after pravastatin treatment, respectively; P<.002). By use of nondenaturing polyacrylamide gradient gel electrophoresis, Blanche et al28 identified five distinct subfractions of HDL with different mean apparent diameters: HDL2a, HDL2b, HDL3a, HDL3b, and HDL3c. To compare our HDL subspecies with those previously described by these authors, we analyzed each HDL gradient subfraction on nondenaturing polyacrylamide gradient gels and determined their mean diameters (data not shown). Under these conditions, density gradient fractions 13 to 15 (d=1.063 to 1.091 g/mL) appeared to be equivalent to HDL2b and density gradient fractions 16 and 17 (d=1.091 to 1.112 g/mL) to HDL2a. The HDL3a, HDL3b, and HDL3c were equivalent to HDL density gradient fractions 18 and 19 (d=1.112 to 1.133 g/mL), fractions 20 and 21 (d=1.133 to 1.156 g/mL), and fractions 22 and 23 (d=1.156 to 1.179 g/mL), respectively. After pravastatin treatment, we observed a reduction of 30% in net mass transfer of HDL-CE to apo B–containing lipoproteins concomitant with an increase in HDL2a levels and a reduction in those of HDL3b. However, only the diminution of plasma HDL3b levels was correlated with the decrease in CE transfer (r=.65; P=.04). These results are in good agreement with earlier reports in which an increase in specific HDL subspecies, Lp(AI with AII) and Lp(AI without AII), was induced by pravastatin therapy.20 As proposed by Lagrost et al5 for normolipidemic subjects, plasma HDL3b level may represent a specific marker of a relatively high CETP activity in hypercholesterolemia.
The mean weight percent chemical compositions of native LDL density gradient subfractions (LDL-1 to LDL-5) in patients with FH before and after 12 weeks of pravastatin administration and in control subjects are presented in Table 3⇓⇓. LDL subfractions (LDL-3 to LDL-5) from patients with FH displayed a significant increase in their relative proportion of CE (LDL-3, 40.6±2.9%; LDL-4, 39.8±2.4%; and LDL-5, 40.3±3.5%) compared with the corresponding subfractions in subjects from the normolipidemic group (LDL-3, 36.3±1.0%, P=.006; LDL-4, 36.2±1.9%, P=.006; LDL-5, 37.2±1.6%, P=.0036). After pravastatin therapy, no significant change in the relative proportions of CE in LDL subfractions was observed, and LDL subspecies LDL-3, LDL-4, and LDL-5 were still CE enriched in comparison with those of control subjects (LDL-3, 39.6±1.8%, P=.0026; LDL-4, 39.3±2.5%, P=.0026; and LDL-5, 40.9±5.1%, P=.027). No significant change in Lp(a) level was induced by pravastatin therapy (12±0.6 and 10±0.4 mg/dL before and after treatment, respectively).
CE Transfer From HDL to Apo B–Containing Lipoproteins
Fig 3A⇑, 3B⇑, and 3C⇑ present the kinetics for the time-dependent mass transfer of CE from HDL to the apo B–containing lipoproteins of patients with FH before and after pravastatin treatment; data for control subjects are presented for comparison. In all cases, net mass transfer ceased after 6 hours of incubation, a consistent finding in all of our studies to date.7 11 Compared with control values, the total transfer of HDL-CE to apo B–containing lipoproteins in subjects with FH is greatly elevated (488 μg/mL plasma compared with 263 μg/mL plasma in control subjects). Pravastatin treatment, which resulted in a 36% reduction in LDL levels, was associated with a marked reduction in the total HDL-CE transferred to this fraction (342 μg/mL plasma; −29.9%) in patients with FH after 6 hours of incubation. Analysis of the initial exponential rates of transfer of HDL-CE to the apo B–containing lipoproteins showed an elevated rate of transfer in untreated subjects with FH (93.5±27.8 μg CE transferred · h−1 · mL−1 plasma; Fig 3B⇑) compared with control subjects (56.7±12.3 μg CE transferred · h−1 · mL−1 plasma; Fig 3A⇑). After pravastatin treatment, the transfer rates were reduced (76.3±11.9 μg CE transferred · h−1 · mL−1 plasma; Fig 3C⇑), but remained elevated compared with those of the control subjects. Analysis of data for the rates of transfer of HDL-CE to LDL alone showed a similar pattern (43±7 μg CE transferred · h−1 · mL−1 plasma for control subjects; 80±16 μg CE transferred · h−1 · mL−1 plasma for subjects with FH before treatment; 61±17 μg CE transferred · h−1 · mL−1 plasma for subjects with FH after treatment), with a net reduction in transfer rate of 24% (P=.0005) after pravastatin treatment. These data are consistent with the observation that LDL represents the major apo B–containing species in plasma and that pravastatin treatment resulted in a 36% reduction in LDL levels and a 24% decrease in CE transfer rate to LDL.
The net mass transfer of CE from HDL to apo B–containing lipoproteins in each group after 6 hours of incubation is shown in Fig 1A⇑. For each subject group, no significant difference was observed in the total CE mass transferred from HDL to VLDL (control subjects, 64.8±8.7 μg CE transferred/mL plasma; patients with FH before pravastatin treatment, 73.9±41.6 μg CE transferred/mL plasma; and patients with FH after pravastatin treatment, 57.5±5.7 μg CE transferred/mL plasma). However, in each case the LDL fraction (d=1.019 to 1.063 g/mL) represented the major quantitative CE acceptor among the apo B–containing lipoproteins. The total transfer of CE to this fraction was significantly elevated (twofold) in untreated subjects with FH and, although reduced after pravastatin treatment, was still significantly higher than values in control subjects (Fig 1A⇑). In normolipidemic subjects, the LDL-2 to LDL-4 subspecies accounted for 75% of the total CE mass transferred to LDL.11 By contrast, subfractions LDL-3 to LDL-5 (d=1.029 to 1.063 g/mL) represented the principal CE acceptors on a quantitative basis among LDL subspecies in patients with FH (Fig 1B⇑). Indeed, these LDL subfractions accounted for 73% of the total CE mass transferred from HDL to LDL in the latter subjects. Pravastatin induced a marked reduction of CE mass transferred from HDL to each LDL subfraction (35% to 54%; P=.0001). However, subfractions LDL-3 to LDL-5 again accounted for the majority (71%) of the total CE mass transferred to LDL.
When the total CE mass transferred from HDL to VLDL and LDL is expressed relative to the unit mass of each lipoprotein acceptor, the ability of each apo B–containing lipoprotein subspecies to accept CE from HDL can be estimated (Fig 2⇑). Thus, in normolipidemic subjects, the VLDL and LDL-1 subfractions represented the preferential CE acceptors among apo B–containing lipoproteins (VLDL/LDL, 2:1; LDL-1/LDL-2 to LDL-5, 1.6:1). However, VLDL, LDL-1, and LDL-2 displayed a superior affinity for CE transferred from HDL (VLDL/LDL, 2.25:1; LDL1-2/LDL-3 to LDL-5, 2.1:1) in FH patients. As we have previously demonstrated,11 the ability of LDL subspecies to accept CE from HDL is positively correlated with their relative TG content in both normolipidemic subjects and patients with FH (r=.47; P=.0001). Pravastatin treatment did not modify the relative ability of TG-enriched lipoprotein particles to accept CE from HDL (VLDL/LDL, 2.1:1; LDL1-2/LDL-3 to LDL-5, 1.9:1). Moreover, a similar correlation between the capacity of these LDL subspecies to accept CE from HDL and their relative TG content was observed (r=.49; P=.0001).
To evaluate whether variations in the plasma levels of CETP protein mass were responsible for the observed differences in CE transfer from HDL to apo B–containing lipoproteins after pravastatin treatment, we used an exogenous assay of CETP activity as described in “Methods.”13 This assay has been previously shown to accurately reflect plasma CETP mass.10 13 CETP activity in the plasma of each patient with FH was assessed before and after treatment by use of an exogenous system containing HDL donor and VLDL/LDL acceptor particles isolated from control plasmas (see “Methods”). The results are shown in Fig 5⇓. After 3 hours of incubation, the mean resulting transfer of CE in patients with FH before treatment was 7.8±0.4%; pravastatin administration did not induce any modification of the CETP-dependent transfer (7.9±0.4%). Furthermore, Fig 5⇓ shows that the rates of CE transfer over the 3-hour period were indistinguishable. Thus, we conclude that pravastatin treatment induced no modification in plasma CETP levels.
To assess whether any subtle structural variation in HDL after pravastatin treatment was responsible for the observed reduction in CETP activity, we evaluated the ability of HDL from subjects with FH to transfer CE to a triacylglycerol-rich acceptor emulsion and receive triacylglycerols therefrom. The kinetics of the CETP-facilitated HDL-dependent CE transfer were first-order reactions, which permitted calculation of a rate constant (pool · h−1). Comparison of the amount of CE lost and the amount of TG gained by HDL clearly indicated that such neutral lipid exchange was equimolar (r=.86, P<.00001). After treatment with pravastatin, the rate constant was not statistically different from that in plasmas from patients with FH before treatment (0.064±0.008 pool · h−1 and 0.066±0.014 pool · h−1, respectively). Thus, pravastatin did not affect HDL-dependent CE transfer. Moreover, despite the effect of pravastatin, the rate constant of CE transfer was positively correlated with the FC/PL ratio in HDL (r=.98, P<.0002).
For the first time, we report the effects of pravastatin on plasma CETP activity in subjects with FH in studies to determine the potential role of CETP in this atherogenic dyslipoproteinemia. Pravastatin therapy for 12 weeks at 40 mg/d induced reductions of 31% and 35% in plasma TC and LDL cholesterol levels respectively. In addition, pravastatin significantly reduced plasma LDL levels but did not appear to affect the LDL subspecies profile or particle composition. By contrast, plasma HDL concentrations were not modified by pravastatin therapy, but the HDL particle profile was normalized. Indeed, we observed an increase in the relative proportion of HDL2 (11.4%), which was associated with a decrease in the relative proportion of HDL3 (11.6%). Furthermore, a marked reduction (42%) in mass transfer of HDL-CE to LDL in patients with FH was detected after pravastatin administration. Moreover, in these patients, intermediate and dense LDL subspecies (LDL-3, LDL-4, and LDL-5) accounted for the majority of CE transferred from HDL to LDL. This potentially atherogenic redistribution of CE to the denser LDL subspecies was not modified by pravastatin therapy.
We have observed that pravastatin clearly induced a decrease in CE transfer from HDL to the apo B–containing lipoproteins in plasma from hypercholesterolemic patients. In an attempt to determine which criteria could explain this reduction—ie, changes in lipoprotein composition, concentration, and/or a decrease of plasma CETP activity—we performed two series of CE transfer assays. First, the CETP-dependent transfer was not affected by pravastatin, indicating that this drug did not affect plasma CETP protein mass. These results are consistent with those of an earlier study, in which plasma CETP protein mass was unaffected by pravastatin therapy.14 In contrast, McPherson et al,10 using a solid-phase radioimmunoassay, reported an elevation of CETP mass in hypercholesterolemia and a correlation between CETP mass and total plasma cholesterol (r=.52). On the basis of this correlation, the pravastatin-mediated reduction of plasma cholesterol in our patients with FH might be expected to result in reduction in plasma CETP mass. If this is the case, then the data in Fig 2⇑ would suggest that CETP is not, in itself, a rate-limiting factor in CE transfer in these subjects.
Bagdade et al12 evaluated the kinetics of transfer of HDL-CE to the apo B–containing lipoproteins in subjects with FH before and after treatment with lovastatin using precipitation techniques. Despite a 35.5% reduction in plasma LDL-CE after drug therapy, they observed no reduction in total CE mass transfer to LDL after lovastatin treatment and transfer kinetics almost identical to those observed before drug therapy. Under their assay conditions, transfer was only linear for the first hour of incubation, and had virtually ceased after 2 hours in the subjects with FH but not in the control subjects. These workers did, however, observe a markedly increased rate of HDL-CE transfer to apo B–containing lipoproteins in subjects with FH for the first 2 hours of incubation compared with control subjects. After 6 hours of incubation, the quantity of HDL-CE transferred was almost the same in the control subjects and the subjects with FH. We have no ready explanation for these discrepant findings other than to suggest that they may be methodologically related. In the present study, however, the actual transfer and accumulation of CE mass within the VLDL and LDL fractions was quantitated. In contrast, Bagdade et al12 measured the loss of HDL-CE mass after precipitation of the apo B–containing lipoproteins. On the basis of the data presented here, we suggest that because the concentration of LDL, the major plasma acceptor of HDL-CE, is reduced by pravastatin treatment, this may be the major determinant in the observed reduction in the rate and total mass transfer of CE after drug therapy. In the same way, the HDL-dependent transfer was not modified by treatment, although it varied according to the lipid surface composition of HDL. The correlation between HDL-dependent transfer and the FC/PL ratio in HDL is in agreement with previous work showing that the FC concentration of donor particles was a positive regulator of the mass transfer of CE.33 Our data demonstrate that pravastatin induced a lower rate of CE transfer as a result of modifications in the acceptors of CE, ie, the apo B–containing lipoproteins. Thus, a significant positive correlation was observed between the net mass transfer of CE from HDL to the apo B–containing lipoproteins and the mass of each lipoprotein acceptor particle in both normolipidemic and hypercholesterolemic subjects (r=.89; P=.0001). This correlation was not modified by pravastatin treatment, and a similar relationship between CE mass transferred and apo B–containing lipoprotein mass was observed in all subject groups (r=.88; P=.0001). These observations suggest that pravastatin exerted neither a direct nor an indirect inhibitory effect on CETP activity.
Several studies have investigated the effects of pravastatin on plasma lipid levels.16 17 18 Our present data on the cholesterol-lowering effect of pravastatin are consistent with those previously described by others. Cheung et al14 described only a 15% decrease in plasma TC in patients with primary hypercholesterolemia after 8 weeks of low-dose pravastatin treatment (10 mg/d). The more significant effect on plasma cholesterol levels that we observed was probably due to the high dose (40 mg/d) and prolonged administration period (12 weeks) of pravastatin used.16
It is of interest that Cheung et al14 observed low-dose pravastatin (10 mg/d) to be apparently without effect on both the LDL and HDL particle profiles in a population of hypercholesterolemic subjects displaying a wide range of TG levels (49 to 259 mg/dL). In contrast, the present study of patients with heterozygous FH involving treatment with pravastatin at a higher dose (40 mg/d) has revealed minor but significant modifications in the HDL subspecies profile, whereas the qualitative features of the LDL subspecies profile were not detectably affected. Thus, we observed a distinct shift in the mass distribution of HDL subpopulations from the dense (HDL3) to the light (HDL2) subclass. Indeed, this shift corresponded to a significant degree of normalization of HDL subclass pattern (HDL2/HDL3, 1.11±0.16 and 0.69±0.14 after and before pravastatin treatment, respectively); more specifically, an increase in the mass of light HDL2a particles occurred at the expense of the HDL3b subspecies. It is of interest to compare these observations with those of Johansson et al,34 who studied hypercholesterolemic patients receiving simvastatin (10 mg/d) for 6 weeks. These authors detected a selective elevation of both HDL2b and HDL3a concentrations with a concomitant diminution in those of the HDL3b subspecies. Thus, the overall simvastatin-induced trend in the HDL subspecies profile seen in these patients closely resembled that effected by pravastatin in the present investigation: ie, a shift from dense, small particles (HDL3a or HDL3b) to light (HDL2a or HDL2b) subspecies of larger size.
Several interactive molecular mechanisms may underlie the modulation of the HDL size subspecies profile seen in both of these studies. Principal among them may be (1) the reduction in CE acceptor particle number and thus in CE transfer rate from HDL to apo B–containing lipoproteins, thereby favoring an increase in the HDL-CE pool and thus in HDL particles of larger size enriched in CE, and (2) a possible reduction in the rate of HDL particles’ cycling from large to small as a result of reduction in CE transfer activity. The lack of effect of pravastatin on the low VLDL-TG levels in our patients tends to suggest that a lipolytic mechanism is not a key feature of the modulation that we have detected; equally, the roles of the hepatic lipase and of hepatic LDL receptor regulation in the shift of HDL profile remain indeterminate.
In the present study, we did not observe any significant modifications in plasma HDL cholesterol levels in hypercholesterolemic patients after 12 weeks of pravastatin treatment (40 mg/d). However, the effect of pravastatin on HDL cholesterol was heterogeneous in the six patients studied. Indeed, three of them had increases in HDL cholesterol level of 3% to 23% after treatment; two subjects showed reductions of 10% and 20%; and one had similar HDL cholesterol levels before and after pravastatin. This heterogeneity of the effect of pravastatin on HDL cholesterol in patients with FH is entirely consistent with the contradictory data reported on this aspect by several authors. Thus, Wiklund et al35 demonstrated a moderate (9%) increase of HDL cholesterol levels in patients with FH after 12 weeks of pravastatin treatment (40 mg/d). Similarly, Mabuchi et al17 described a 15% increase in plasma HDL cholesterol concentration after CS-514 (pravastatin) therapy. Moreover, in a recent study, Malacco et al25 detected an increase of 10% in HDL cholesterol after pravastatin treatment. In contrast, however, several studies on the effect of pravastatin in FH have failed to reveal any significant modification in HDL cholesterol levels by pravastatin.14 19 23
Conflicting data have been published concerning the effect of pravastatin on plasma Lp(a) levels. Klausen et al36 have demonstrated an apo(a) isoform-dependent increase in Lp(a) concentration in patients with FH treated with pravastatin for 24 weeks (40 mg/d). However, Wiklund et al37 reported no change in the concentrations of Lp(a) in patients with FH treated with pravastatin at the same dose for 3 months. These latter results were consistent with the present study, in which we did not observe any significant modification in plasma Lp(a) level in patients with FH after drug therapy.
We conclude that the reduction of plasma CETP activity induced by pravastatin in patients with FH results from a reduction in LDL particle acceptor concentration. Similar conclusions were reached by Mann et al38 with respect to VLDL in hypertriglyceridemic patients treated with bezafibrate. Furthermore, our data suggest that CE transfer from HDL to apo B–containing lipoproteins markedly influences the LDL and HDL subspecies profiles and enhances the formation of the CE-enriched dense LDL subspecies and HDL3b in patients with FH. However, in this study we observed that the reduction of denser LDL particle number from before to after pravastatin treatment (LDL-4, from 43 700×1010 to 26 400×1010 particles/mL of plasma; LDL-5, from 14 100×1010 to 8640×1010 particles/mL of plasma) did not modify the characteristic redistribution of CE mediated by CETP among these particles. Consequently, the redistribution of CE between plasma lipoproteins mediated by CETP appears to play a determinant role in the formation of atherogenic LDL particles in FH.
Selected Abbreviations and Acronyms
|BMI||=||body mass index|
|CETP||=||cholesteryl ester transfer protein|
We are indebted to the Spécia Division (Drs B. Stehle and N. Bertholom) of Rhône-Poulenc Rorer (Contrat de Valorisation, INSERM No. 91015), to the Medical Research Council of Canada (MT-5999 to Dr Dolphin), and to INSERM for generous support of these studies. Dr Guérin was the recipient of a Research Fellowship from the Association Claude Bernard. It is a pleasure to acknowledge the collaboration of Drs F. Dairou, M. Farnier, and L. Perrot in these studies.
Eisenberg S. Preferential enrichment of large sized very low density lipoprotein populations with transferred cholesteryl esters. J Lipid Res. 1985;26:487-493.
Bagdade JD, Ritter MC, Subbaiah PV. Accelerated cholesteryl ester transfer in plasma of patients with hypercholesterolemia. J Clin Invest. 1991;87:1259-1265.
Lagrost L, Gandjini H, Athias A, Guyard-Dangremont V, Lallemant C, Gambert P. Influence of plasma cholesteryl ester transfer activity on the LDL and HDL distribution profiles in normolipidemic subjects. Arterioscler Thromb. 1993;13:815-825.
Guérin M, Dolphin PJ, Chapman MJ. A new in vitro method for the simultaneous evaluation of cholesteryl ester exchange and mass transfer between HDL and apoB-containing lipoprotein subspecies: identification of preferential cholesteryl ester acceptors in human plasma. Arterioscler Thromb. 1993;14:199-206.
Marzetta CA, Meyers TJ, Albers JJ. Lipid transfer protein–mediated distribution of HDL-derived cholesteryl esters among plasma apo B–containing lipoprotein subpopulations. Arterioscler Thromb. 1993;13:834-841.
Nigon F, Lesnik P, Rouis M, Chapman MJ. Discrete subspecies of human low-density lipoproteins are heterogeneous in their interaction with the cellular LDL receptor. J Lipid Res. 1991;32:1741-1754.
McPherson R, Mann CJ, Tall AR, Hogue M, Martin L, Milne RW, Marcel YL. Plasma concentrations of cholesteryl ester transfer protein in hyperlipoproteinemia: relation to cholesteryl ester transfer protein activity and other lipoprotein variables. Arterioscler Thromb. 1991;11:797-804.
Guérin M, Dolphin PJ, Chapman MJ. Preferential cholesteryl ester acceptors among the LDL subspecies of subjects with familial hypercholesterolemia. Arterioscler Thromb. 1994;14:679-685.
Bagdade JD, Lane JT, Stone N, Ritter MC, Subbaiah PV. Persistent abnormalities in lipoprotein composition and cholesteryl ester transfer following lovastatin treatment. J Lipid Res. 1990;31:1263-1269.
Cheung MC, Austin MA, Moulin P, Wolf AC, Cryer D, Knopp RH. Effects of pravastatin on apolipoprotein-specific high density lipoprotein subpopulations and low density lipoprotein subclass phenotypes in patients with primary hypercholesterolemia. Atherosclerosis. 1993;102:107-119.
Nishiwaki M, Ishikawa T, Homma Y, Tada N, Kagami A, Ohshima K, Ito T, Tomiyasu K, Shige H, Miyajima E, Nakamura H. Effects of pravastatin on plasma lipids, apolipoproteins, lp(a), remnant-like particle, cholesteryl ester transfer protein, and lecithin:cholesterol acyltransferase: 12-month study. Nutr Metab Cardiovasc Dis. 1994;4:10-15.
Bard JM, Parra HJ, Douste-Blazy P, Fruchart JC. Effect of pravastatin, an HMG CoA reductase inhibitor, and cholestyramine, a bile sequestrant, on lipoprotein particles defined by their apolipoprotein composition. Metabolism. 1990;30:269-273.
Volpe R, Arca M, Ginnetti MG, Antonini R, Ricci G, Urbinati G. The efficacity and safety of pravastatin and simvastatin in patients with primary hypercholesterolemia. Curr Ther Res. 1992;51:422-430.
Malacco E, Magni A, Scandiani L, Casini A, Albano S, Ansuini R, Biasion T, Biffi E, Bilardo G, Boccuzzi G, et al. Pravastatin vs gemfibrozil in the treatment of primary hypercholesterolaemia. Drug Invest. 1994;7:331-339.
Chapman MJ, Goldstein S, Lagrange D, Laplaud PM. A density gradient ultracentrifugal procedure for the isolation of the major lipoprotein classes from human serum. J Lipid Res. 1981;22:339-358.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem. 1951;193:265-275.
Derksen A, Small DM. Interaction of apo AI and apo E-3 with triglyceride-phospholipid emulsions containing increasing cholesterol concentrations: model of triglyceride-rich nascent and remnant lipoproteins. Biochemistry. 1989;28:90-96.
Morton RE. Free cholesterol is a potent regulator of lipid transfer protein function. J Biol Chem. 1988;263:12235-12241.
Wiklund O, Angelin B, Fager G, Ericksson M, Olofsson SO, Berglund L, Linden T, Sjoberg A, Bondjers G. Treatment of familial hypercholesterolemia: a controlled trial of the effects of pravastatin or cholestyramine therapy on lipoprotein and apolipoprotein levels. J Intern Med. 1990;228:241-247.
Mann CJ, Yen FT, Grant AM, Bihain BE. Mechanism of plasma cholesteryl ester transfer in hypertriglyceridemia. J Clin Invest. 1991;88:2059-2066.