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Atherogenic Lipoprotein Particle Size and Concentrations and the Effect of Pravastatin in Children with Familial Hypercholesterolemia
Atherogenic Lipoprotein Particle Size and Concentrations and the Effect of Pravastatin in Children with Familial Hypercholesterolemia ANOUK VAN DER GRAAF, MD, JESSICA RODENBURG, MD, PHD, MAUD N. VISSERS, PHD, BARBARA A. HUTTEN, PHD, ALBERT WIEGMAN, MD, PHD, MIEKE D. TRIP, MD, PHD, ERIK S. G. STROES, MD, PHD, FRITS A. WIJBURG, MD, PHD, JAMES D. OTVOS, PHD, AND JOHN J. P. KASTELEIN, MD, PHD
Objective To determine lipoprotein particle concentrations and size in children with familial hypercholesterolemia (FH) and investigate the effect of pravastatin therapy on these measures. Study design Lipoprotein particle concentrations and sizes were examined by nuclear magnetic resonance (NMR) spectroscopy in 144 children with FH and 45 unaffected siblings. The effect of pravastatin therapy (20 to 40 mg) on lipoprotein particle concentration and size were compared with placebo after 1 year of treatment, using analysis of covariance. Results Compared with the unaffected siblings, the children with FH had significantly higher concentrations of very-low-density lipoprotein (VLDL) particles (115.6 nmol/L vs 51.2 nmol/L; P < .001) and low-density lipoprotein (LDL) particles (1726.8 nmol/L vs 955.3 nmol/L; P < .001), and lower concentrations of high-density lipoprotein (HDL) particles (23.2 mol/L vs 26.9 mol/L; P < .001). Compared with placebo, pravastatin therapy decreased the concentration of VLDL particles by 35.9 nmol/L (P < .001), of total LDL particles by 342.7 nmol/L (P < .001), of large LDL particles by 189.5 nmol/L (P < .001), and of small LDL particles by 156.2 nmol/L (P ⴝ .152), but increased the concentration of total HDL particles by 2.2 mol/L (P < .001), of large HDL particles by 1.0 mol/L (P ⴝ .006), and of medium HDL particles by 1.1 mol/L (P ⴝ .003). VLDL particle size increased by 1.0 nm (P ⴝ .032). Conclusions Compared with their healthy siblings, children with FH have an atherogenic lipoprotein profile based on their lipoprotein distribution and lipoprotein particle diameter. Pravastatin therapy can improve, but not fully restore, these lipoprotein abnormalities toward normal levels in these children. (J Pediatr 2008;152:873-8) hildren with familial hypercholesterolemia (FH) are characterized by elevated levels of plasma low-density lipoprotein cholesterol (LDL-C) as a consequence of mutations in the LDL receptor gene.1 These elevated levels of LDL-C are present from birth onward and predispose for premature cardiovascular disease (CVD) later in life. Studies in adults have shown that in addition to elevated LDL-C levels, LDL subclass phenotypes with elevated small, dense lipoproteins also are associated with an increased risk for CVD.2,3 These small, dense LDL particles have a lower affinity for the LDL receptor4,5 and thus are less readily cleared from the circulation. This has untoward consequences for the atherogenicity of these particles; in particular, the increased plasma residence time contributes to proatherogenic changes at the level of the endothelium.4 In addition, small dense LDL particles have an increased susceptibility toward oxidative modification,6,7 a process directly linked to foam cell formation. Up to now, no data have been available regarding lipoprotein subclasses and sizes in children with FH. Such knowledge might assist in selecting the most effective therapy to reduce the particularly high CVD risk in these children. Hydroxy-methyl-glutaryl co-enzyme A (HMG-CoA) reductase inhibitors, or statins, decrease lipid and lipoprotein levels and thus reduce CVD risk.8,9 The effect of
C
CETP CVD FH HDL HDL-C HMG-CoA IDL
Cholesteryl ester transfer protein Cardiovascular disease Familial hypercholesterolemia High-density lipoprotein High-density lipoprotein cholesterol Hydroxy-methyl-glutaryl co-enzyme A Intermediate-density lipoprotein
LDL LDL-C NMR TG VLDL IDL
Low-density lipoprotein Low-density lipoprotein cholesterol Nuclear magnetic resonance Triglyceride Very-low-density lipoprotein Intermediate-density lipoprotein
From the Departments of Vascular Medicine (A.vdG., J.R., M.V., E.S., J.K.), Clinical Epidemiology and Biostatistics (B.H.), Pediatrics (A.W., F.W.), and Cardiology (M.T.), Academic Medical Centre, University of Amsterdam, the Netherlands and LipoScience Inc, Raleigh, NC (J.O.). A.vdG. and J.R. contributed equally to this work and thus share first authorship. These 2 authors wrote the first draft of the manuscript. None of the authors received any payment for producing the manuscript. The original study was funded by BristolMyers Squibb. The sponsor played no role in the study design, data collection, data analysis, data interpretation, or writing of the report. Submitted for publication Aug 3, 2007; last revision received Oct 25, 2007; accepted Nov 29, 2007. Reprint requests: John J. P. Kastelein, MD, PhD, Department of Vascular Medicine, Academic Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: [email protected]. 0022-3476/$ - see front matter Copyright © 2008 Mosby Inc. All rights reserved. 10.1016/j.jpeds.2007.11.043
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these agents on lipoprotein subclasses and particle sizes has been evaluated in adults with FH, but the results are equivocal; some studies found that statins increase LDL size and reduce small, dense LDL particle concentrations,10,11 whereas other studies found no effect of statins on these measures.12-14 Notwithstanding these conflicting results, all studies demonstrated a reduction in absolute LDL particle concentration.1012,15 Pravastatin safely reduced LDL-C levels in children with FH,16 but the extent to which this statin alters lipoprotein distribution remains unknown. The purpose of the present study was to compare lipoprotein subclass concentrations and particle sizes in children with FH and their healthy siblings, who have not been examined to date. In addition, we evaluated the effect of pravastatin therapy on these variables in children with FH.
METHODS We previously performed a double-blind, randomized, placebo-controlled trial to determine the 2-year efficacy and safety of pravastatin therapy in 214 children age 8 to 18 years with heterozygous FH.16 In brief, children were eligible to participate in this study based on a documented LDL-receptor mutation or plasma levels above the 95th percentile for age and sex in a family with a history of premature CVD in conjunction with hypercholesterolemia and tendon xanthomas. Reasons for exclusion were homozygous FH or a secondary cause for hypercholesterolemia.16 Consenting children were randomly assigned to receive placebo or pravastatin, 20 to 40 mg/day. The dose depended on age; children under age 14 years received 20 mg/day, whereas those age 14 and older received 40 mg/day. The children were instructed to continue a low-fat diet and to maintain regular physical activity during the intervention period. In addition, relevant information was assembled for 80 unaffected siblings in whom FH was definitely excluded by DNA analysis. The study protocol was approved by the Academic Medical Centre’s Institutional Review Board. Written informed consent was obtained from all children and their parents. Efficacy and safety data from the original trial were reported previously.16 From the original cohort, blood samples from 144 FH children at baseline and 1 year after treatment and single samples from 45 unaffected siblings were available for lipoprotein subclass analysis. These children comprise the current study population. Venous blood samples were obtained after a fasting period of at least 12 hours. Plasma levels of total cholesterol and triglycerides (TGs) were determined by standardized enzymatic procedures (Boehringer, Mannheim, Germany), and high-density lipoprotein cholesterol (HDL-C) concentrations in plasma were measured by an automated method (Roche Diagnostics, Basel, Switzerland). LDL-C levels were calculated using Friedewald’s equation.17 Plasma samples for lipoprotein subclass analysis were stored at ⫺80oC. Lipoprotein subclass particle concentrations and mean lipoprotein particle sizes were measured by nuclear magnetic resonance (NMR) spectroscopy at LipoScience, Inc 874
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(Raleigh, NC) as described previously18,19 and modified recently.20 In brief, the particle concentrations of lipoprotein subclasses of different sizes were derived from the measured amplitudes of the spectroscopically distinct lipid methyl group NMR signals that they emit. In addition to the overall lipoprotein particle concentrations, concentrations of the following subclasses were measured: large (⬎60 nm) VLDL, medium (35.0 to 60.0 nm) VLDL, small (27.0 to 35.0 nm) VLDL, intermediate-density lipoprotein (IDL) (23.0 to 27.0 nm), large (21.2 to 23.0 nm) LDL, small (18.0 to 21.2 nm) LDL, large (8.8 to 13.0 nm) LDL, medium (8.2 to 8.8 nm) HDL, and small (7.3 to 8.2 nm) HDL. The VLDL and LDL subclass particle concentrations are given in nmol/L; those of HDL subclasses in mol/L. Weighted-average lipoprotein particle sizes (nm) were calculated from the subclass levels and the diameters assigned to each subclass. The reproducibility of the NMR-measured lipoprotein particle variables was determined by replicate analyses of plasma pools. The coefficients of variation were ⬍4% for overall VLDL, LDL, and HDL particle concentrations; ⬍2% for VLDL size; ⬍0.5% for LDL and HDL size; ⬍10% for large, medium, and small VLDL subclasses; ⬍8% for large and small LDL subclasses; and ⬍5% for large and small HDL subclasses. The coefficients of variation for the IDL (⬍20%) and medium HDL (⬍35%) subclasses were higher, due in part to their low concentrations. Differences in baseline characteristics, lipids, lipoprotein subclasses, and particle sizes between the children with FH and their healthy siblings were analyzed using linear or logistic regression analyses. Because some of these children were related, analyses were performed with the generalized estimating equations (GEE) method using the SAS procedure GENMOD (SAS Institute, Cary, NC) to account for correlations within families. In the FH group, baseline values between the pravastatin group and the placebo group were compared using independent t-tests. Distributions of dichotomous data between these groups were compared using 2 tests. The effects of pravastatin on lipoprotein lipid levels as well as subclass particle concentrations and particle size were studied by comparing the difference in the change between the pravastatin group and the placebo group after 1 year of treatment by analysis of covariance (ANCOVA), with treatment and baseline values as independent variables. Variables with a skewed distribution were log-transformed before the analysis. A P value of .05 was considered statistically significant (2-sided test). Statistical analyses were performed using SAS version 9.1 (SAS Institute).
RESULTS The children with FH in our cohort did not differ from their unaffected siblings in terms of baseline characteristics, except for lipid and lipoprotein values (Table I). The children with FH had significantly higher concentrations of VLDL and LDL particles and lower concentrations of HDL particles compared with the unaffected siblings (Table II). The higher concentration of VLDL particles in the children with FH was The Journal of Pediatrics • June 2008
Table I. Characteristics of children with FH and unaffected siblings
Age, years Male sex, number (%) Body mass index, kg/m2 Height, cm Weight, kg Mean arterial blood pressure, mmHg Total cholesterol, mmol/L HDL-C, mmol/L LDL-C, mmol/L TGs, mmol/L, median (IQR)
Pravastatin (n ⴝ 68)
Placebo (n ⴝ 76)
P value*
Siblings (n ⴝ 45)
P value†
12.8 ⫾ 2.9 33 (49) 19.7 ⫾ 3.4 157.1 ⫾ 15.7 49.9 ⫾ 15.0 77.8 ⫾ 9.3 7.83 ⫾ 1.49 1.23 ⫾ 0.28 6.16 ⫾ 1.46 0.77 (0.57 to 1.28)
13.0 ⫾ 3.0 40 (53) 19.5 ⫾ 3.6 155.6 ⫾ 13.5 48.5 ⫾ 14.9 77.3 ⫾ 8.4 7.83 ⫾ 1.19 1.29 ⫾ 0.28 6.19 ⫾ 1.19 0.66 (0.45 to 0.98)
.712 .623 .727 .549 .580 .749 .988 .199 .886 .011
13.3 ⫾ 2.6 21 (45) 18.6 ⫾ 3.7 154.1 ⫾ 13.9 45.2 ⫾ 15.5 75.8 ⫾ 7.9 4.27 ⫾ 0.65 1.46 ⫾ 0.36 2.49 ⫾ 0.55 0.54 (0.41 to 0.89)
.239 .645 .239 .364 .122 .183 ⬍.001 .001 ⬍.001 .007
Results are reported as mean ⫾ standard deviation unless noted otherwise. Mean arterial blood pressure is calculated as (systolic blood pressure ⫹ 2 ⫻ diastolic blood pressure)/3. IQR, interquartile range. *P value for pravastatin versus placebo. †P value for FH versus siblings adjusted for family relations.
Table II. Baseline values of lipoprotein subclasses and particle sizes of children with FH and unaffected siblings FH (n ⴝ 144) Particle concentration VLDL particles (nmol/L) Large VLDL, median (IQR) Medium VLDL, median (IQR) Small VLDL LDL particles (nmol/L) IDL, median (IQR) Large LDL Small LDL HDL particles (mol/L) Large HDL Medium HDL, median (IQR) Small HDL Particle size (nm) VLDL, median (IQR) LDL† HDL
Siblings (n ⴝ 45)
P value*
115.6 ⫾ 38.2 0.6 (0.2 to 1.9) 18.9 (8.5 to 35.0) 89.8 ⫾ 29.3 1726.8 ⫾ 391.3 24.6 (9.4 to 49.7) 850.1 ⫾ 254.5 845.3 ⫾ 397.8 23.2 ⫾ 4.0 6.3 ⫾ 2.9 0.4 (0.0 to 1.8) 15.5 ⫾ 3.0
51.2 ⫾ 19.8 0.4 (0.2 to 1.4) 17.4 (7.5 to 20.9) 33.8 ⫾ 14.8 955.3 ⫾ 191.2 1.9 (0.0 to 10.5) 416.9 ⫾ 131.8 533.3 ⫾ 217.9 26.9 ⫾ 3.8 8.5 ⫾ 2.4 3.1 (1.6 to 5.0) 14.9 ⫾ 3.4
⬍.001 .392 .057 ⬍.001 ⬍.001 ⬍.001 ⬍.001 ⬍.001 ⬍.001 ⬍.001 ⬍.001 .254
40.4 (38.2 to 43.4) 21.4 ⫾ 0.5 9.5 ⫾ 0.4
47.4 (44.2 to 52.1) 21.3 ⫾ 0.5 9.5 ⫾ 0.4
⬍001 .053 .771
Values are given as mean ⫾ standard deviation unless noted otherwise. IQR, interquartile range. *P values adjusted for family relations. †Includes IDL size.
due entirely to an elevated concentration of small VLDL particles, whereas the higher concentration of LDL particles was due to elevations of IDL and large and small LDL particles. The lower HDL particle concentration in the children with FH was caused by reduced concentrations of large and medium HDL particles. Mean VLDL particle size was significantly smaller in the children with FH compared with the healthy siblings, whereas LDL and HDL particle sizes did not differ significantly between the 2 groups. The children with FH in the pravastatin group did not differ from those in the placebo group in terms of baseline characteristics, except TG levels (Table I). In the pravastatin group, mean LDL-C levels decreased by 23.5% compared
with placebo. The mean difference in the change in VLDL particle concentration between the pravastatin and placebo groups was ⫺35.9 nmol/L, due to a reduction in medium and small VLDL particles (Table III). Between the 2 groups, the mean difference in the change in LDL particle concentration was ⫺342.7 nmol/L, with ⫺189.5 nmol/L for large LDL particles and ⫺156.2 nmol/L for small LDL particles. The latter decrease is not statistically significant. The mean differences in the changes in total HDL, large HDL, and medium HDL particle concentrations were 2.2 mol/L, 1.0 mol/L, and 1.1 mol/L, respectively, all of which are statistically significant. The mean difference in the change in VLDL particle size between the pravastatin and placebo
Atherogenic Lipoprotein Particle Size and Concentrations and the Effect of Pravastatin in Children with Familial Hypercholesterolemia
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Table III. Baseline and 1-year treatment values of lipoprotein subclasses and particle sizes in children with FH Pravastatin (n ⴝ 68) Baseline Particle concentration VLDL particles, nmol/L Large VLDL, median (IQR) Medium VLDL, median (IQR) Small VLDL LDL particles, nmol/L IDL, median (IQR) Large LDL Small LDL HDL particles, mol/L Large HDL Medium HDL, median (IQR) Small HDL Particle size, nm VLDL, median (IQR) LDL HDL
Mean change
Placebo (n ⴝ 76) Baseline
118.8 ⫾ 40.7 0.7 (0.2 to 2.0) 23.5 (9.5 to 39.3) 89.2 ⫾ 31.1 1782.6 ⫾ 463.9 23.9 (10.1 to 54.1) 814.2 ⫾ 266.3 934.5 ⫾ 437.7 22.9 ⫾ 3.4 6.1 ⫾ 2.7 0.4 (0.0 to 1.8) 15.4 ⫾ 2.6
⫺37.5 ⫾ 33.2 ⫺0.77 ⫾ 3.6 ⫺9.5 ⫾ 20.3 ⫺27.3 ⫾ 25.6 ⫺274.9 ⫾ 299.2 ⫺6.4 ⫾ 30.1 ⫺128.5 ⫾ 209.7 ⫺140.0 ⫾ 334.1 2.6 ⫾ 3.3 1.3 ⫾ 2.0 0.9 ⫾ 2.3 0.4 ⫾ 3.0
112.7 ⫾ 35.8 0.6 (0.2 to 1.7) 13.9 (7.1 to 32.0) 90.4 ⫾ 27.9 1676.9 ⫾ 307.1 25.1 (8.7 to 43.2) 882.3 ⫾ 240.7 765.5 ⫾ 341.9 23.6 ⫾ 4.4 6.5 ⫾ 3.0 0.6 (0.0 to 1.7) 15.7 ⫾ 3.3
41.2 (38.3 to 43.9) 21.3 ⫾ 0.5 9.5 ⫾ 0.4
1.49 ⫾ 6.81 0.03 ⫾0.46 ⫺0.04 ⫾ 0.28
40.2 (37.8 to 42.8) 21.5 ⫾ 0.5 9.6 ⫾ 0.4
The Journal of Pediatrics • June 2008
Values are reported as mean ⫾ standard deviation, unless noted otherwise. IQR, interquartile range. *P value for difference in mean absolute change between groups.
Mean change
95% confidence interval
⫺1.6 ⫾ 32.0 ⫺0.1 ⫾ 2.3 1.8 ⫾ 20.6 ⫺3.3 ⫾ 21.3 67.8 ⫾ 354.0 ⫺9.4 ⫾ 32.1 61.0 ⫾ 177.2 16.3 ⫾ 387.4 0.4 ⫾ 3.3 0.3 ⫾ 2.2 ⫺0.2 ⫾ 2.1 0.4 ⫾ 3.1
⫺46.7 to ⫺25.2 ⫺1.6 to 0.3 ⫺18.0 to ⫺4.5 ⫺31.7 to ⫺16.3 ⫺451.4 to ⫺234.0 ⫺7.28 to 13.3 ⫺253.2 to ⫺125.7 ⫺276.1 to ⫺36.4 1.1 to 3.3 0.3 to 1.7 0.4 to 1.9 ⫺1.0 to 1.1
0.70 ⫾ 4.98 0.05 ⫾ 0.45 ⫺0.02 ⫾ 0.31
⫺1.2 to 2.8 ⫺0.2 to 0.13 ⫺0.1 to 0.8
P value* ⬍.001 .455 .003 ⬍.001 ⬍.001 .262 ⬍.001 .152 ⬍.001 .006 .003 .850 .032 .136 .455
groups was significant (0.8 nm), but HDL and LDL particle sizes did not change significantly.
DISCUSSION We have shown that children with FH have higher VLDL and LDL particle concentrations and lower HDL particle concentrations compared with healthy controls. Pravastatin treatment significantly reduced total LDL and large LDL particle concentrations, but the reduction in the atherogenic fraction of small LDL particles was not statistically significant. Pravastatin also decreased total VLDL particle concentrations and increased total HDL particle concentrations. In addition to severely increased LDL-C levels, children with FH also are characterized by atherogenic changes in lipoprotein subclass distribution and size. The higher LDL particle concentration was due to increases not only in IDL particles, but also in large and small LDL particles. The latter in particular are associated with increased CVD risk in adults. Recent guidelines for drug therapy for high-risk lipid abnormalities in children recommend starting statin therapy for FH after age 10 years in boys and after menarche in girls.21 In the current study, pravastatin did not significantly decrease the concentration of small LDL particles, in contrast to the clear reduction of large LDL particles. Pravastatin’s lack of effect on small LDL particles is in line with the findings of 2 earlier studies in adults with FH.12,14 However, in adults with FH, more potent statins, such as atorvastatin and simvastatin, have been shown to decrease the concentration of small, dense LDL particles with a concomitant increase in LDL particle size.10,11,22-24 Although these findings suggest that reduction of atherogenic fractions occurs with more potent statins, whether more aggressive strategies have similar effects in children with FH remains unknown. The children with FH had significantly higher concentrations of small VLDL particles and lower concentrations of large and medium HDL particles compared with the healthy siblings. This higher small VLDL particle concentration is consistent with increased apolipoprotein B levels without significantly increased TG levels. Because high levels of small VLDL and IDL particles also have been associated with an increased risk of angiographic progression and clinical events,25 these small VLDL and IDL particles also likely contribute to the atherogenic pressure in these children. Pravastatin significantly increased total HDL particles and decreased total LDL and VLDL particles. The decrease in VLDL particle concentration was due mainly to a decrease in medium and small VLDL particles; consequently, mean VLDL size increased. The latter finding is compatible with a direct effect of pravastatin on the concentration of cholesteryl ester transfer protein (CETP).26 Because statins inhibit cholesterol biosynthesis, as a consequence, they also may reduce hepatic CETP expression27 by reducing intracellular oxysterol concentrations. This in turn inhibits transcriptional activation of the CETP gene by the liver X receptor.28 Although Cheung et al12 reported that pravastatin has no affect on the CETP mass in adult patients with primary hypercholesterolemia, unpublished
data from our group demonstrated that a 3-month course of pravastatin (n ⫽ 93) did decrease the CETP mass by approximately 12% (mean change, ⫺0.35 g; 95% confidence interval ⫽ ⫺0.57 to ⫺0.14 g) in children with FH compared with placebo (n ⫽ 95). This effect also may explain the increase in total HDL particles seen in the pravastatin group. Several methodological issues merit discussion. Because our hypothesis was conceived retrospectively, not all of the blood samples from the original cohort were available for NMR analysis. Because the baseline data of those patients with samples available did not differ significantly from that of the entire cohort, it is unlikely that this deficit introduced a selection bias. Moreover, we analyzed samples after 1 year of treatment instead of 2 years as performed in the original trial; however, the 23.5% decrease in LDL-C level after 1 year of pravastatin therapy was in line with the decrease after 2 years of pravastatin therapy,16 indicating similar treatment effects from 1 year and 2 years of treatment. Children with FH have a highly atherogenic lipid profile, with increased IDL, small LDL, and small VLDL particle concentrations and concomitantly lower HDL particle concentrations compared with healthy siblings. Whereas pravastatin treatment reduced large LDL and small VLDL particle concentrations, it had no effect on the most atherogenic subfraction, small LDL particles.
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22. O’Keefe JH Jr, Captain BK, Jones PG, Harris WS. Atorvastatin reduces remnant lipoproteins and small, dense low-density lipoproteins regardless of the baseline lipid pattern. Prev Cardiol 2004;7:154-60. 23. van Tits LJ, Smilde TJ, van Wissen S, de Graaf J, Kastelein JJ, Stalenhoef AF. Effects of atorvastatin and simvastatin on low-density lipoprotein subfraction profile, low-density lipoprotein oxidizability, and antibodies to oxidized low-density lipoprotein in relation to carotid intima media thickness in familial hypercholesterolemia. J Investig Med 2004; 52:177-84. 24. Guerin M, Egger P, Soudant C, Le Goff W, van Tol A, Dupuis R, et al. Dose-dependent action of atorvastatin in type IIB hyperlipidemia: preferential and progressive reduction of atherogenic apoB-containing lipoprotein subclasses (VLDL-2, IDL, small dense LDL) and stimulation of cellular cholesterol efflux. Atherosclerosis 2002; 163:287-96. 25. Tornvall P, Bavenholm P, Landou C, de Faire U, Hamsten A. Relation of plasma levels and composition of apolipoprotein B– containing lipoproteins to angiographically defined coronary artery disease in young patients with myocardial infarction. Circulation 1993;88:2180-9. 26. Guerin M, Dolphin PJ, Talussot C, Gardette J, Berthezene F, Chapman MJ. Pravastatin modulates cholesteryl ester transfer from HDL to apoB-containing lipoproteins and lipoprotein subspecies profile in familial hypercholesterolemia. Arterioscler Thromb Vasc Biol 1995;15:1359-68. 27. Guerin M, Lassel TS, Le GW, Farnier M, Chapman MJ. Action of atorvastatin in combined hyperlipidemia: preferential reduction of cholesteryl ester transfer from HDL to VLDL1 particles. Arterioscler Thromb Vasc Biol 2000;20:189-97. 28. Luo Y, Tall AR. Sterol upregulation of human CETP expression in vitro and in transgenic mice by an LXR element. J Clin Invest 2000;105:513-20.
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Objective To determine lipoprotein particle concentrations and size in children with familial hypercholesterolemia (FH) and investigate the effect of pravastatin therapy on these measures. Study design Lipoprotein particle concentrations and sizes were examined by nuclear magnetic resonance (NMR) spectroscopy in 144 children with FH and 45 unaffected siblings. The effect of pravastatin therapy (20 to 40 mg) on lipoprotein particle concentration and size were compared with placebo after 1 year of treatment, using analysis of covariance. Results Compared with the unaffected siblings, the children with FH had significantly higher concentrations of very-low-density lipoprotein (VLDL) particles (115.6 nmol/L vs 51.2 nmol/L; P < .001) and low-density lipoprotein (LDL) particles (1726.8 nmol/L vs 955.3 nmol/L; P < .001), and lower concentrations of high-density lipoprotein (HDL) particles (23.2 mol/L vs 26.9 mol/L; P < .001). Compared with placebo, pravastatin therapy decreased the concentration of VLDL particles by 35.9 nmol/L (P < .001), of total LDL particles by 342.7 nmol/L (P < .001), of large LDL particles by 189.5 nmol/L (P < .001), and of small LDL particles by 156.2 nmol/L (P ⴝ .152), but increased the concentration of total HDL particles by 2.2 mol/L (P < .001), of large HDL particles by 1.0 mol/L (P ⴝ .006), and of medium HDL particles by 1.1 mol/L (P ⴝ .003). VLDL particle size increased by 1.0 nm (P ⴝ .032). Conclusions Compared with their healthy siblings, children with FH have an atherogenic lipoprotein profile based on their lipoprotein distribution and lipoprotein particle diameter. Pravastatin therapy can improve, but not fully restore, these lipoprotein abnormalities toward normal levels in these children. (J Pediatr 2008;152:873-8) hildren with familial hypercholesterolemia (FH) are characterized by elevated levels of plasma low-density lipoprotein cholesterol (LDL-C) as a consequence of mutations in the LDL receptor gene.1 These elevated levels of LDL-C are present from birth onward and predispose for premature cardiovascular disease (CVD) later in life. Studies in adults have shown that in addition to elevated LDL-C levels, LDL subclass phenotypes with elevated small, dense lipoproteins also are associated with an increased risk for CVD.2,3 These small, dense LDL particles have a lower affinity for the LDL receptor4,5 and thus are less readily cleared from the circulation. This has untoward consequences for the atherogenicity of these particles; in particular, the increased plasma residence time contributes to proatherogenic changes at the level of the endothelium.4 In addition, small dense LDL particles have an increased susceptibility toward oxidative modification,6,7 a process directly linked to foam cell formation. Up to now, no data have been available regarding lipoprotein subclasses and sizes in children with FH. Such knowledge might assist in selecting the most effective therapy to reduce the particularly high CVD risk in these children. Hydroxy-methyl-glutaryl co-enzyme A (HMG-CoA) reductase inhibitors, or statins, decrease lipid and lipoprotein levels and thus reduce CVD risk.8,9 The effect of
C
CETP CVD FH HDL HDL-C HMG-CoA IDL
Cholesteryl ester transfer protein Cardiovascular disease Familial hypercholesterolemia High-density lipoprotein High-density lipoprotein cholesterol Hydroxy-methyl-glutaryl co-enzyme A Intermediate-density lipoprotein
LDL LDL-C NMR TG VLDL IDL
Low-density lipoprotein Low-density lipoprotein cholesterol Nuclear magnetic resonance Triglyceride Very-low-density lipoprotein Intermediate-density lipoprotein
From the Departments of Vascular Medicine (A.vdG., J.R., M.V., E.S., J.K.), Clinical Epidemiology and Biostatistics (B.H.), Pediatrics (A.W., F.W.), and Cardiology (M.T.), Academic Medical Centre, University of Amsterdam, the Netherlands and LipoScience Inc, Raleigh, NC (J.O.). A.vdG. and J.R. contributed equally to this work and thus share first authorship. These 2 authors wrote the first draft of the manuscript. None of the authors received any payment for producing the manuscript. The original study was funded by BristolMyers Squibb. The sponsor played no role in the study design, data collection, data analysis, data interpretation, or writing of the report. Submitted for publication Aug 3, 2007; last revision received Oct 25, 2007; accepted Nov 29, 2007. Reprint requests: John J. P. Kastelein, MD, PhD, Department of Vascular Medicine, Academic Medical Centre, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: [email protected]. 0022-3476/$ - see front matter Copyright © 2008 Mosby Inc. All rights reserved. 10.1016/j.jpeds.2007.11.043
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these agents on lipoprotein subclasses and particle sizes has been evaluated in adults with FH, but the results are equivocal; some studies found that statins increase LDL size and reduce small, dense LDL particle concentrations,10,11 whereas other studies found no effect of statins on these measures.12-14 Notwithstanding these conflicting results, all studies demonstrated a reduction in absolute LDL particle concentration.1012,15 Pravastatin safely reduced LDL-C levels in children with FH,16 but the extent to which this statin alters lipoprotein distribution remains unknown. The purpose of the present study was to compare lipoprotein subclass concentrations and particle sizes in children with FH and their healthy siblings, who have not been examined to date. In addition, we evaluated the effect of pravastatin therapy on these variables in children with FH.
METHODS We previously performed a double-blind, randomized, placebo-controlled trial to determine the 2-year efficacy and safety of pravastatin therapy in 214 children age 8 to 18 years with heterozygous FH.16 In brief, children were eligible to participate in this study based on a documented LDL-receptor mutation or plasma levels above the 95th percentile for age and sex in a family with a history of premature CVD in conjunction with hypercholesterolemia and tendon xanthomas. Reasons for exclusion were homozygous FH or a secondary cause for hypercholesterolemia.16 Consenting children were randomly assigned to receive placebo or pravastatin, 20 to 40 mg/day. The dose depended on age; children under age 14 years received 20 mg/day, whereas those age 14 and older received 40 mg/day. The children were instructed to continue a low-fat diet and to maintain regular physical activity during the intervention period. In addition, relevant information was assembled for 80 unaffected siblings in whom FH was definitely excluded by DNA analysis. The study protocol was approved by the Academic Medical Centre’s Institutional Review Board. Written informed consent was obtained from all children and their parents. Efficacy and safety data from the original trial were reported previously.16 From the original cohort, blood samples from 144 FH children at baseline and 1 year after treatment and single samples from 45 unaffected siblings were available for lipoprotein subclass analysis. These children comprise the current study population. Venous blood samples were obtained after a fasting period of at least 12 hours. Plasma levels of total cholesterol and triglycerides (TGs) were determined by standardized enzymatic procedures (Boehringer, Mannheim, Germany), and high-density lipoprotein cholesterol (HDL-C) concentrations in plasma were measured by an automated method (Roche Diagnostics, Basel, Switzerland). LDL-C levels were calculated using Friedewald’s equation.17 Plasma samples for lipoprotein subclass analysis were stored at ⫺80oC. Lipoprotein subclass particle concentrations and mean lipoprotein particle sizes were measured by nuclear magnetic resonance (NMR) spectroscopy at LipoScience, Inc 874
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(Raleigh, NC) as described previously18,19 and modified recently.20 In brief, the particle concentrations of lipoprotein subclasses of different sizes were derived from the measured amplitudes of the spectroscopically distinct lipid methyl group NMR signals that they emit. In addition to the overall lipoprotein particle concentrations, concentrations of the following subclasses were measured: large (⬎60 nm) VLDL, medium (35.0 to 60.0 nm) VLDL, small (27.0 to 35.0 nm) VLDL, intermediate-density lipoprotein (IDL) (23.0 to 27.0 nm), large (21.2 to 23.0 nm) LDL, small (18.0 to 21.2 nm) LDL, large (8.8 to 13.0 nm) LDL, medium (8.2 to 8.8 nm) HDL, and small (7.3 to 8.2 nm) HDL. The VLDL and LDL subclass particle concentrations are given in nmol/L; those of HDL subclasses in mol/L. Weighted-average lipoprotein particle sizes (nm) were calculated from the subclass levels and the diameters assigned to each subclass. The reproducibility of the NMR-measured lipoprotein particle variables was determined by replicate analyses of plasma pools. The coefficients of variation were ⬍4% for overall VLDL, LDL, and HDL particle concentrations; ⬍2% for VLDL size; ⬍0.5% for LDL and HDL size; ⬍10% for large, medium, and small VLDL subclasses; ⬍8% for large and small LDL subclasses; and ⬍5% for large and small HDL subclasses. The coefficients of variation for the IDL (⬍20%) and medium HDL (⬍35%) subclasses were higher, due in part to their low concentrations. Differences in baseline characteristics, lipids, lipoprotein subclasses, and particle sizes between the children with FH and their healthy siblings were analyzed using linear or logistic regression analyses. Because some of these children were related, analyses were performed with the generalized estimating equations (GEE) method using the SAS procedure GENMOD (SAS Institute, Cary, NC) to account for correlations within families. In the FH group, baseline values between the pravastatin group and the placebo group were compared using independent t-tests. Distributions of dichotomous data between these groups were compared using 2 tests. The effects of pravastatin on lipoprotein lipid levels as well as subclass particle concentrations and particle size were studied by comparing the difference in the change between the pravastatin group and the placebo group after 1 year of treatment by analysis of covariance (ANCOVA), with treatment and baseline values as independent variables. Variables with a skewed distribution were log-transformed before the analysis. A P value of .05 was considered statistically significant (2-sided test). Statistical analyses were performed using SAS version 9.1 (SAS Institute).
RESULTS The children with FH in our cohort did not differ from their unaffected siblings in terms of baseline characteristics, except for lipid and lipoprotein values (Table I). The children with FH had significantly higher concentrations of VLDL and LDL particles and lower concentrations of HDL particles compared with the unaffected siblings (Table II). The higher concentration of VLDL particles in the children with FH was The Journal of Pediatrics • June 2008
Table I. Characteristics of children with FH and unaffected siblings
Age, years Male sex, number (%) Body mass index, kg/m2 Height, cm Weight, kg Mean arterial blood pressure, mmHg Total cholesterol, mmol/L HDL-C, mmol/L LDL-C, mmol/L TGs, mmol/L, median (IQR)
Pravastatin (n ⴝ 68)
Placebo (n ⴝ 76)
P value*
Siblings (n ⴝ 45)
P value†
12.8 ⫾ 2.9 33 (49) 19.7 ⫾ 3.4 157.1 ⫾ 15.7 49.9 ⫾ 15.0 77.8 ⫾ 9.3 7.83 ⫾ 1.49 1.23 ⫾ 0.28 6.16 ⫾ 1.46 0.77 (0.57 to 1.28)
13.0 ⫾ 3.0 40 (53) 19.5 ⫾ 3.6 155.6 ⫾ 13.5 48.5 ⫾ 14.9 77.3 ⫾ 8.4 7.83 ⫾ 1.19 1.29 ⫾ 0.28 6.19 ⫾ 1.19 0.66 (0.45 to 0.98)
.712 .623 .727 .549 .580 .749 .988 .199 .886 .011
13.3 ⫾ 2.6 21 (45) 18.6 ⫾ 3.7 154.1 ⫾ 13.9 45.2 ⫾ 15.5 75.8 ⫾ 7.9 4.27 ⫾ 0.65 1.46 ⫾ 0.36 2.49 ⫾ 0.55 0.54 (0.41 to 0.89)
.239 .645 .239 .364 .122 .183 ⬍.001 .001 ⬍.001 .007
Results are reported as mean ⫾ standard deviation unless noted otherwise. Mean arterial blood pressure is calculated as (systolic blood pressure ⫹ 2 ⫻ diastolic blood pressure)/3. IQR, interquartile range. *P value for pravastatin versus placebo. †P value for FH versus siblings adjusted for family relations.
Table II. Baseline values of lipoprotein subclasses and particle sizes of children with FH and unaffected siblings FH (n ⴝ 144) Particle concentration VLDL particles (nmol/L) Large VLDL, median (IQR) Medium VLDL, median (IQR) Small VLDL LDL particles (nmol/L) IDL, median (IQR) Large LDL Small LDL HDL particles (mol/L) Large HDL Medium HDL, median (IQR) Small HDL Particle size (nm) VLDL, median (IQR) LDL† HDL
Siblings (n ⴝ 45)
P value*
115.6 ⫾ 38.2 0.6 (0.2 to 1.9) 18.9 (8.5 to 35.0) 89.8 ⫾ 29.3 1726.8 ⫾ 391.3 24.6 (9.4 to 49.7) 850.1 ⫾ 254.5 845.3 ⫾ 397.8 23.2 ⫾ 4.0 6.3 ⫾ 2.9 0.4 (0.0 to 1.8) 15.5 ⫾ 3.0
51.2 ⫾ 19.8 0.4 (0.2 to 1.4) 17.4 (7.5 to 20.9) 33.8 ⫾ 14.8 955.3 ⫾ 191.2 1.9 (0.0 to 10.5) 416.9 ⫾ 131.8 533.3 ⫾ 217.9 26.9 ⫾ 3.8 8.5 ⫾ 2.4 3.1 (1.6 to 5.0) 14.9 ⫾ 3.4
⬍.001 .392 .057 ⬍.001 ⬍.001 ⬍.001 ⬍.001 ⬍.001 ⬍.001 ⬍.001 ⬍.001 .254
40.4 (38.2 to 43.4) 21.4 ⫾ 0.5 9.5 ⫾ 0.4
47.4 (44.2 to 52.1) 21.3 ⫾ 0.5 9.5 ⫾ 0.4
⬍001 .053 .771
Values are given as mean ⫾ standard deviation unless noted otherwise. IQR, interquartile range. *P values adjusted for family relations. †Includes IDL size.
due entirely to an elevated concentration of small VLDL particles, whereas the higher concentration of LDL particles was due to elevations of IDL and large and small LDL particles. The lower HDL particle concentration in the children with FH was caused by reduced concentrations of large and medium HDL particles. Mean VLDL particle size was significantly smaller in the children with FH compared with the healthy siblings, whereas LDL and HDL particle sizes did not differ significantly between the 2 groups. The children with FH in the pravastatin group did not differ from those in the placebo group in terms of baseline characteristics, except TG levels (Table I). In the pravastatin group, mean LDL-C levels decreased by 23.5% compared
with placebo. The mean difference in the change in VLDL particle concentration between the pravastatin and placebo groups was ⫺35.9 nmol/L, due to a reduction in medium and small VLDL particles (Table III). Between the 2 groups, the mean difference in the change in LDL particle concentration was ⫺342.7 nmol/L, with ⫺189.5 nmol/L for large LDL particles and ⫺156.2 nmol/L for small LDL particles. The latter decrease is not statistically significant. The mean differences in the changes in total HDL, large HDL, and medium HDL particle concentrations were 2.2 mol/L, 1.0 mol/L, and 1.1 mol/L, respectively, all of which are statistically significant. The mean difference in the change in VLDL particle size between the pravastatin and placebo
Atherogenic Lipoprotein Particle Size and Concentrations and the Effect of Pravastatin in Children with Familial Hypercholesterolemia
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Table III. Baseline and 1-year treatment values of lipoprotein subclasses and particle sizes in children with FH Pravastatin (n ⴝ 68) Baseline Particle concentration VLDL particles, nmol/L Large VLDL, median (IQR) Medium VLDL, median (IQR) Small VLDL LDL particles, nmol/L IDL, median (IQR) Large LDL Small LDL HDL particles, mol/L Large HDL Medium HDL, median (IQR) Small HDL Particle size, nm VLDL, median (IQR) LDL HDL
Mean change
Placebo (n ⴝ 76) Baseline
118.8 ⫾ 40.7 0.7 (0.2 to 2.0) 23.5 (9.5 to 39.3) 89.2 ⫾ 31.1 1782.6 ⫾ 463.9 23.9 (10.1 to 54.1) 814.2 ⫾ 266.3 934.5 ⫾ 437.7 22.9 ⫾ 3.4 6.1 ⫾ 2.7 0.4 (0.0 to 1.8) 15.4 ⫾ 2.6
⫺37.5 ⫾ 33.2 ⫺0.77 ⫾ 3.6 ⫺9.5 ⫾ 20.3 ⫺27.3 ⫾ 25.6 ⫺274.9 ⫾ 299.2 ⫺6.4 ⫾ 30.1 ⫺128.5 ⫾ 209.7 ⫺140.0 ⫾ 334.1 2.6 ⫾ 3.3 1.3 ⫾ 2.0 0.9 ⫾ 2.3 0.4 ⫾ 3.0
112.7 ⫾ 35.8 0.6 (0.2 to 1.7) 13.9 (7.1 to 32.0) 90.4 ⫾ 27.9 1676.9 ⫾ 307.1 25.1 (8.7 to 43.2) 882.3 ⫾ 240.7 765.5 ⫾ 341.9 23.6 ⫾ 4.4 6.5 ⫾ 3.0 0.6 (0.0 to 1.7) 15.7 ⫾ 3.3
41.2 (38.3 to 43.9) 21.3 ⫾ 0.5 9.5 ⫾ 0.4
1.49 ⫾ 6.81 0.03 ⫾0.46 ⫺0.04 ⫾ 0.28
40.2 (37.8 to 42.8) 21.5 ⫾ 0.5 9.6 ⫾ 0.4
The Journal of Pediatrics • June 2008
Values are reported as mean ⫾ standard deviation, unless noted otherwise. IQR, interquartile range. *P value for difference in mean absolute change between groups.
Mean change
95% confidence interval
⫺1.6 ⫾ 32.0 ⫺0.1 ⫾ 2.3 1.8 ⫾ 20.6 ⫺3.3 ⫾ 21.3 67.8 ⫾ 354.0 ⫺9.4 ⫾ 32.1 61.0 ⫾ 177.2 16.3 ⫾ 387.4 0.4 ⫾ 3.3 0.3 ⫾ 2.2 ⫺0.2 ⫾ 2.1 0.4 ⫾ 3.1
⫺46.7 to ⫺25.2 ⫺1.6 to 0.3 ⫺18.0 to ⫺4.5 ⫺31.7 to ⫺16.3 ⫺451.4 to ⫺234.0 ⫺7.28 to 13.3 ⫺253.2 to ⫺125.7 ⫺276.1 to ⫺36.4 1.1 to 3.3 0.3 to 1.7 0.4 to 1.9 ⫺1.0 to 1.1
0.70 ⫾ 4.98 0.05 ⫾ 0.45 ⫺0.02 ⫾ 0.31
⫺1.2 to 2.8 ⫺0.2 to 0.13 ⫺0.1 to 0.8
P value* ⬍.001 .455 .003 ⬍.001 ⬍.001 .262 ⬍.001 .152 ⬍.001 .006 .003 .850 .032 .136 .455
groups was significant (0.8 nm), but HDL and LDL particle sizes did not change significantly.
DISCUSSION We have shown that children with FH have higher VLDL and LDL particle concentrations and lower HDL particle concentrations compared with healthy controls. Pravastatin treatment significantly reduced total LDL and large LDL particle concentrations, but the reduction in the atherogenic fraction of small LDL particles was not statistically significant. Pravastatin also decreased total VLDL particle concentrations and increased total HDL particle concentrations. In addition to severely increased LDL-C levels, children with FH also are characterized by atherogenic changes in lipoprotein subclass distribution and size. The higher LDL particle concentration was due to increases not only in IDL particles, but also in large and small LDL particles. The latter in particular are associated with increased CVD risk in adults. Recent guidelines for drug therapy for high-risk lipid abnormalities in children recommend starting statin therapy for FH after age 10 years in boys and after menarche in girls.21 In the current study, pravastatin did not significantly decrease the concentration of small LDL particles, in contrast to the clear reduction of large LDL particles. Pravastatin’s lack of effect on small LDL particles is in line with the findings of 2 earlier studies in adults with FH.12,14 However, in adults with FH, more potent statins, such as atorvastatin and simvastatin, have been shown to decrease the concentration of small, dense LDL particles with a concomitant increase in LDL particle size.10,11,22-24 Although these findings suggest that reduction of atherogenic fractions occurs with more potent statins, whether more aggressive strategies have similar effects in children with FH remains unknown. The children with FH had significantly higher concentrations of small VLDL particles and lower concentrations of large and medium HDL particles compared with the healthy siblings. This higher small VLDL particle concentration is consistent with increased apolipoprotein B levels without significantly increased TG levels. Because high levels of small VLDL and IDL particles also have been associated with an increased risk of angiographic progression and clinical events,25 these small VLDL and IDL particles also likely contribute to the atherogenic pressure in these children. Pravastatin significantly increased total HDL particles and decreased total LDL and VLDL particles. The decrease in VLDL particle concentration was due mainly to a decrease in medium and small VLDL particles; consequently, mean VLDL size increased. The latter finding is compatible with a direct effect of pravastatin on the concentration of cholesteryl ester transfer protein (CETP).26 Because statins inhibit cholesterol biosynthesis, as a consequence, they also may reduce hepatic CETP expression27 by reducing intracellular oxysterol concentrations. This in turn inhibits transcriptional activation of the CETP gene by the liver X receptor.28 Although Cheung et al12 reported that pravastatin has no affect on the CETP mass in adult patients with primary hypercholesterolemia, unpublished
data from our group demonstrated that a 3-month course of pravastatin (n ⫽ 93) did decrease the CETP mass by approximately 12% (mean change, ⫺0.35 g; 95% confidence interval ⫽ ⫺0.57 to ⫺0.14 g) in children with FH compared with placebo (n ⫽ 95). This effect also may explain the increase in total HDL particles seen in the pravastatin group. Several methodological issues merit discussion. Because our hypothesis was conceived retrospectively, not all of the blood samples from the original cohort were available for NMR analysis. Because the baseline data of those patients with samples available did not differ significantly from that of the entire cohort, it is unlikely that this deficit introduced a selection bias. Moreover, we analyzed samples after 1 year of treatment instead of 2 years as performed in the original trial; however, the 23.5% decrease in LDL-C level after 1 year of pravastatin therapy was in line with the decrease after 2 years of pravastatin therapy,16 indicating similar treatment effects from 1 year and 2 years of treatment. Children with FH have a highly atherogenic lipid profile, with increased IDL, small LDL, and small VLDL particle concentrations and concomitantly lower HDL particle concentrations compared with healthy siblings. Whereas pravastatin treatment reduced large LDL and small VLDL particle concentrations, it had no effect on the most atherogenic subfraction, small LDL particles.
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13. Superko HR, Krauss RM, DiRicco C. Effect of fluvastatin on low-density lipoprotein peak particle diameter. Am J Cardiol 1997;80:78-81. 14. Vega GL, Krauss RM, Grundy SM. Pravastatin therapy in primary moderate hypercholesterolaemia: changes in metabolism of apolipoprotein B– containing lipoproteins. J Intern Med 1990;227:81-94. 15. Otvos JD, Shalaurova I, Freedman DS, Rosenson RS. Effects of pravastatin treatment on lipoprotein subclass profiles and particle size in the PLAC-I trial. Atherosclerosis 2002;160:41-8. 16. Wiegman A, Hutten BA, de Groot E, Rodenburg J, Bakker HD, Buller HR, et al. Efficacy and safety of statin therapy in children with familial hypercholesterolemia: a randomized controlled trial. Jama 2004;292:331-7. 17. Friedewald WT, Levy RI, Frederickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without the use of the preparative ultracentrifuge. Clin Chem 1972;18:499-502. 18. Otvos JD. Measurement of lipoprotein subclass profiles by nuclear magnetic resonance spectroscopy. Clin Lab 2002;48:171-80. 19. Freedman DS, Otvos JD, Jeyarajah EJ, Shalaurova I, Cupples LA, Parise H, et al. Sex and age differences in lipoprotein subclasses measured by nuclear magnetic resonance spectroscopy: the Framingham Study. Clin Chem 2004;50:1189-200. 20. Festa A, Williams K, Hanley AJ, Otvos JD, Goff DC, Wagenknecht LE, et al. Nuclear magnetic resonance lipoprotein abnormalities in prediabetic subjects in the Insulin Resistance Atherosclerosis Study. Circulation 2005;111:3465-72. 21. McCrindle BW, Urbina EM, Dennison BA, Jacobson MS, Steinberger J, Rocchini AP, et al. Drug therapy of high-risk lipid abnormalities in children and adolescents: a scientific statement from the American Heart Association Atherosclerosis, Hypertension, and Obesity in Youth Committee, Council of Cardiovascular Disease in the Young, with the Council on Cardiovascular Nursing. Circulation 2007;115:1948-67.
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22. O’Keefe JH Jr, Captain BK, Jones PG, Harris WS. Atorvastatin reduces remnant lipoproteins and small, dense low-density lipoproteins regardless of the baseline lipid pattern. Prev Cardiol 2004;7:154-60. 23. van Tits LJ, Smilde TJ, van Wissen S, de Graaf J, Kastelein JJ, Stalenhoef AF. Effects of atorvastatin and simvastatin on low-density lipoprotein subfraction profile, low-density lipoprotein oxidizability, and antibodies to oxidized low-density lipoprotein in relation to carotid intima media thickness in familial hypercholesterolemia. J Investig Med 2004; 52:177-84. 24. Guerin M, Egger P, Soudant C, Le Goff W, van Tol A, Dupuis R, et al. Dose-dependent action of atorvastatin in type IIB hyperlipidemia: preferential and progressive reduction of atherogenic apoB-containing lipoprotein subclasses (VLDL-2, IDL, small dense LDL) and stimulation of cellular cholesterol efflux. Atherosclerosis 2002; 163:287-96. 25. Tornvall P, Bavenholm P, Landou C, de Faire U, Hamsten A. Relation of plasma levels and composition of apolipoprotein B– containing lipoproteins to angiographically defined coronary artery disease in young patients with myocardial infarction. Circulation 1993;88:2180-9. 26. Guerin M, Dolphin PJ, Talussot C, Gardette J, Berthezene F, Chapman MJ. Pravastatin modulates cholesteryl ester transfer from HDL to apoB-containing lipoproteins and lipoprotein subspecies profile in familial hypercholesterolemia. Arterioscler Thromb Vasc Biol 1995;15:1359-68. 27. Guerin M, Lassel TS, Le GW, Farnier M, Chapman MJ. Action of atorvastatin in combined hyperlipidemia: preferential reduction of cholesteryl ester transfer from HDL to VLDL1 particles. Arterioscler Thromb Vasc Biol 2000;20:189-97. 28. Luo Y, Tall AR. Sterol upregulation of human CETP expression in vitro and in transgenic mice by an LXR element. J Clin Invest 2000;105:513-20.
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