© 1997 American Heart Association, Inc.
Articles |
A Familial Combined Hyperlipidemic Kindred With Impaired Apolipoprotein B Catabolism
Kinetics of Apolipoprotein B During Placebo and Pravastatin Therapy
the Departamento de Diabetes y Metabolismo de Lipidos, Instituto Nacional de la Nutricion "Salvador Zubiran," Mexico DF, Mexico (C.A.A.-S.); Resource Facility for Kinetic Analysis, University of Washington, Seattle (P.H.R.B.); and Division of Atherosclerosis and Lipid Research, Washington University School of Medicine, St Louis, Mo (J.P., X.L.Z., G.S.).
Correspondence to Gustav Schonfeld, MD, Washington University School of Medicine, 660 S Euclid, Box 8046, St Louis, MO 63110.
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Abstract |
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Familial combined hyperlipidemia (FCHL) is a heterogeneous disorder characterized by multiple lipoprotein phenotypes, a high risk for coronary heart disease, and predominance among the LDL fraction of smaller and denser particles. We report on an FCHL kindred (the M-kindred) in which decreased VLDL- and LDL-apoB elimination rates rather than enhanced production rates were the main kinetic abnormalities. Lipoprotein levels and metabolic parameters of all apoB-containing lipoproteins (including light and dense LDLs) were determined during placebo and pravastatin treatment periods. ApoB metabolism was studied by endogenous labeling with stable isotopes and a multicompartmental model. Five members of the M-kindred participated. The study was doubly blinded, randomized, and placebo controlled. Treatment periods of 6 weeks were separated by 2-week washout periods. All subjects had high apoB levels, 2 had a mixed lipemia, 1 had hypercholesterolemia, and 2 had hypertriglyceridemia. Familial dysbetalipoproteinemia, hypercholesterolemia, and defective apoB-100 were excluded by genetic testing. Kinetic parameters were remarkably similar in the five study subjects during the placebo period, despite their diverse plasma lipid profiles. Compared with nine normolipidemic control subjects, low VLDL-apoB fractional catabolic rates (FCRs) (3.6±.1 versus 9.3±2.9 pools per day) and low LDL-apoB FCRs (0.19±0.05 versus 0.41±0.13 pool per day) were observed in every case. The majority of the LDL particles were identified in the denser fraction (d=1.036 to 1.063 g/mL). A clear precursor-product relationship was observed from VLDL to IDL to light LDL to dense LDL, ie, there was no "metabolic channeling." Light LDL had significantly higher FCR than dense LDL (0.82±0.21 versus 0.22±0.08 pool per day). VLDL-apoB production rates were normal (19.7±6.0 versus 21.6±6.1 mg/kg per day for control subjects). In contrast, in two subjects drawn from two other FCHL kindreds (the C- and K-kindreds), VLDL-apoB production rates were increased (35.6 and 32.1 mg/kg per day, respectively). In these two, more "typical" FCHL subjects, FCRs of LDL-apoB were near normal (0.351 and 0.311 pool per day, respectively). Pravastatin (20 mg/d) resulted in significantly lower plasma cholesterol (265±30 to 218±16 mg/dL, P<.01), LDL cholesterol (186±31 to 145±15 mg/dL, P<.03), and apoB levels (168±14 to 125±16 mg/dL, P<.01) in the five FCHL subjects of the M-kindred. No changes were observed in plasma HDL cholesterol, apoA-I, or lipoprotein(a). Pravastatin significantly increased the LDL-apoB FCR (from 0.19±0.05 to 0.34±0.04 pool per day). The FCRs of both LDL subclasses increased with treatment. No pravastatin-induced changes were seen in apoB production rates.
Key Words: familial combined hyperlipidemia • small dense LDL • apoprotein B • apoprotein A-I • stable isotopes • pravastatin
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Introduction |
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Familial combined hyperlipidemia is a common and heterogeneous disorder characterized by the presence of multiple lipoprotein phenotypes and high risk for coronary heart disease within kindreds. A feature of the disorder is the abnormal distribution of the LDL, with a predominance of smaller and denser particles. The presence of small dense LDL is a positive risk factor for coronary heart disease in subjects with or without FCHL.1 2 To diagnose FCHL with certainty, family studies are required because the lipoprotein phenotype can be variable, not just among affected members of the same family, but also at different times in the same affected individual, and no identifying genetic marker is available.
In some subjects, the FCHL phenotype is associated with low activities of lipoprotein lipase.3 In other kindreds there are associations between FCHL and genetic variants of the apoB gene4 or the apoA-I/C-III/A-IV gene complex.5 In vitro studies have demonstrated abnormalities in the function of basic proteins I and II,6 although the metabolic consequences of these defects remain to be established. Overproduction of apoB-100, the main structural apolipoprotein of VLDL and LDL, is a common feature of the syndrome.7 8 9 10 11 However, normal VLDL-apoB production rates have been reported in some patients.8 12 13
The HMG-CoA reductase inhibitors provide a potent therapeutic option in the treatment of FCHL.14 Two physiological mechanisms have been proposed by which the HMG-CoA reductase inhibitors may achieve their effect of lowering LDL-C. First, catabolism could increase from the drug-induced suppression of cholesterol synthesis and the consequent upregulation of LDL receptors.15 This mechanism seems to operate in heterozygotes for FH.16 Alternately, decreased rates of LDL production could occur. Lower LDL PRs in turn could result from two different physiological mechanisms: either decreased PRs of VLDL (including apoB), the LDL precursor, or increased removal of VLDL (and IDL) from plasma by upregulated LDL receptors, resulting in decreased conversion of VLDL to LDL. Changes in apoB production were noted in other forms of hypercholesterolemia, in which no effect was observed on LDL clearance.17 Recently, our group analyzed the mechanism of action of an HMG-CoA reductase inhibitor in an apoB overproduction state, the nephrotic syndrome,18 and found that lovastatin uniformly decreased LDL PRs by increasing the removal of VLDL (and IDL) from plasma, in effect directing precursors away from the LDL production pathway and hence reducing LDL production. We also studied the effects of HMG-CoA inhibitor therapy in a group of patients with mixed lipemia who were overproducing VLDL-apoB19 and found that VLDL- and LDL-apoB production rates were unaffected. In contrast, VLDL- and LDL-apoB FCRs were increased.
In the majority of relevant kinetic studies performed to date, autologous exogenously labeled VLDL and/or LDL was injected. This method has the significant limitations of infusion of radioactive materials and a low probability of detecting rapid-turnover pools (eg, nascent VLDL and other LDL precursors). Our group and others7 18 19 20 21 22 23 24 have successfully employed the combined use of endogenous labeling with multicompartmental analysis to analyze lipoprotein kinetics in several clinical situations.18 19 20 21 24 Because of its safety, this approach allows the performance of several studies in the same subject. In addition, a better representation of all subpopulations that constitute the apoB-containing lipoproteins may be achieved.
Our purpose was to study the kinetic abnormalities of apoB-containing lipoproteins in several members of a well-characterized FCHL kindred, anticipating that they would be apoB overproducers and that HMG-CoA reductase inhibition would decrease VLDL-apoB PRs. We performed apoB metabolism studies using endogenous labeling with [13C]leucine while subjects were on placebo and again while they were taking the HMG-CoA reductase inhibitor pravastatin. Surprisingly, in contrast to most reported FCHL kindreds, the metabolic defects in this kindred were not increased PRs for VLDL-apoB but decreased FCRs for VLDL- and LDL-apoB. Pravastatin did not affect PRs but rather increased the FCRs for IDL- and LDL-apoB.
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Methods |
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Subjects
Five members of the M-kindred with a family history of coronary heart disease, exhibiting several differing lipid patterns, and elevated plasma apoB levels agreed to participate in this study. The subjects were recruited from the Lipid Center of the Washington University School of Medicine, St Louis, Mo. None received drug therapy for hyperlipidemia, ß-blockers, or steroids for 6 weeks before the study. Two subjects from two other FCHL kindreds (the C- and K-kindreds) were also studied to compare apoB kinetic parameters in a wider sample of FCHL subjects. Eight persons who were the subjects of a previous report21 and one normolipidemic person who had not been the subject of a published report were used as a normal comparison group. The latter subject was a 57-year-old white woman with a body mass index of 22.3 and total cholesterol and TG levels of 200 and 134 mg/dL, respectively. The clinical characteristics of the other normolipidemic subjects have been described.21
The study protocol was approved by the Human Studies Committee of Washington University School of Medicine, and all patients gave written consent. The clinical characteristics of the subjects and the control group are shown in Tables 1 and 2
. Fig 1 contains the pedigree of the FCHL M-kindred under study. The details of the comparisons of the FCHL C- and K-kindreds are given in Tables 3 and 4, and their pedigrees are shown in Figs 2 and 3.
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Protocol
A double-blind, randomized, placebo-controlled crossover design was used. Patients were randomly assigned to receive placebo or pravastatin first (20 mg at night). Each drug was given for 6 weeks, separated by a 2-week washout period. All patients consumed an American Heart Association phase I diet 6 weeks before and during the entire study. Adherence to the diet was confirmed with the use of dietary records of 3 different days per 6-week period. A physical examination, lipid profile, and blood chemistries of liver, kidney, and thyroid function were performed at the beginning of each phase of the study. At the end of each phase, a primed constant 8-hour [13C]leucine infusion was administered to evaluate the kinetics of the apoB-containing lipoproteins.20 21 24 Pravastatin or placebo was continued during the course of the kinetic studies. For the [13C]leucine infusion, patients were admitted to the Washington University School of Medicine General Clinical Research Center at 6 PM after fasting 12 hours. A bolus of [13C]leucine (0.85 mg/kg) was administered, immediately followed by 0.85 mg/kg per hour as a constant infusion over 8 hours; when the tracer infusion was stopped the patients remained fasting for another 8 hours. The 12-hour fast before the infusion was necessary to avoid possible contamination of apoB-100 with apoB-48 during the precipitation of apoB. The 16-hour fast during and after the infusion was necessary to maintain a steady state, which is a requirement of the kinetic model. The fasting was well tolerated. Noncaloric liquids were allowed. The duration of the fasting did not affect the conclusions of the study because control subjects and patients during both the placebo and pravastatin phases were studied under the same conditions. The methods used to obtain data in the study are similar to previously reported methods.24 Previous work by our group using the same protocol demonstrated an increased VLDL-apoB PR in patients with nephrotic syndrome and mixed lipemia.18 19 Thus, the model is capable of detecting increased PRs when present (see also the VLDL-apoB PRs for members of the C- and K-kindreds below). For 4 days after the infusion, subjects continued their phase I AHA diets and drugs. A total of 37 samples were drawn for determination of plasma [13C]leucine enrichments. Twenty-eight of the samples were used for determination of the enrichment of VLDL-, IDL-, and LDL-apoB in [13C]leucine. ApoB and lipoprotein lipid concentrations were measured in five samples during each infusion to evaluate whether steady states were present.
Analytical Methods
Isolation of Lipoproteins
Blood was collected in EDTA-containing tubes, and plasma was separated by low-speed centrifugation. VLDL (d<1.006 g·mL-1), IDL (d=1.006 to 1.019 g·mL-1), LDL-1 (d=1.019 to 1.035 g·mL-1), LDL-2 (d=1.036 to 1.063 g·mL-1), and HDL (d=1.064 to 1.21 g·mL-1) were isolated from 4 mL of plasma by sequential ultracentrifugation25 and dialyzed against ammonium bicarbonate (5 mmol/L) for 24 hours.
Measurements of Lipids, ApoB, and ApoA-I
ApoB concentrations were measured in plasma, VLDL, IDL, LDL-1, and LDL-2 by immunoturbidometry (Behring Diagnostics, Inc). Cholesterol and TG were measured by commercially available tests (WAKO Pure Chemical Industries, Ltd). VLDL, IDL, LDL-1, and LDL-2 apoB pool sizes were determined by multiplying the measured apoB concentration by plasma volume (body weight multiplied by 0.045).
Genotyping
Genotypes for apoE were determined by PCR gene amplification and cleavage with HhaI as described by Hixson and Vernier.26 Genotypes of apoB were determined by PCR amplification of the 3' variable number of tandem repeats27 and by use of a closely linked DNA microsatellite marker on chromosome 2.28 The apoB-3500 mutation was sought by allele-specific asymmetrical PCR.29 The LDL receptor marker was a closely linked DNA microsatellite sequence on chromosome 19.28
Isolation of Plasma Amino Acids
Plasma amino acids were isolated from 0.3 mL of plasma by cation exchange chromatography.30
Isolation and Hydrolysis of ApoB
Apo B was isolated from each lipoprotein fraction by precipitation with butanol-isopropyl ether.30 Precipitated apoB was dried under nitrogen and hydrolyzed in 12N HCl for 16 hours at 110°C. The HCl was subsequently evaporated.
Determination of Enrichment and Calculation of Tracer-Tracee Ratio
Amino acids obtained from plasma samples or from the hydrolyzed apoB by cation exchange chromatography30 were derivatized to heptafluoropropyl esters. Leucine enrichment was determined by gas chromatography–mass spectrometry with 1.5x2.0-mm glass columns (Supelco) packed with coated material (Amino Acid Packing, Alltech Assoc) and a Finnigan 3300 quadruple mass spectrometer, as described previously.32 Isotope enrichment and tracer-tracee ratios were calculated from the observed ion current ratios m/z 371/370. The enrichment of the infused [13C]leucine was 99% (Cambridge Isotope Laboratories). Because of the nonnegligible mass associated with stable isotope tracers, it was necessary to transform enrichment data to tracer-tracee ratios.33
Model of ApoB Metabolism and Calculation of Kinetic Parameters
A multicompartmental model (Fig 4) was used to describe VLDL-, IDL-, and LDL-apoB leucine tracer-tracee ratios. Each compartment or pool represents a group of kinetically homogeneous particles. In this study the CONSAM/SAAM programs34 were used to fit the model to the observed tracer data. Metabolic parameters are subsequently derived from the model parameters giving the best fit. We previously presented a model that describes the kinetics of apoB in VLDL, IDL, and LDL fractions in hyperlipidemic individuals.19 This model has been applied successfully in subjects with mixed hyperlipidemia and nephrotic syndrome.18 19 We added one LDL compartment to include the data obtained from both subclasses of LDL and one VLDL compartment to account for the more complicated kinetics of apoB seen in these patients (Fig 4). The model consists of a precursor compartment of amino acids (compartment 1) and a delay compartment (compartment 2), which accounts for the time required for synthesis and secretion of VLDL-apoB into plasma. Plasma leucine tracer-tracee data were fit with a triexponential function as described by Parhofer et al.24 This triexponential function was then used as a forcing function to drive the appearance of tracer (amino acid) in the compartmental model. The purpose of the forcing function is to decouple components of the system under investigation. We used the triexponential function to decouple the kinetics of the amino acid from that of the tracer in apoB. Mathematically, the forcing function replaces the amount of tracer in compartment 1 with the value of the triexponential forcing function (FF) at the same time. In other words, the value of q1(t), the amount of material or tracer in compartment 1 at time t, is replaced by the term q1·FF(t). Consequently, use of the forcing function takes into account the recycling of the tracer and minimizes its effects on the slow-turnover compartments. The model assumes that all apoB enters plasma through compartment 10. Compartments 10 through 15 are used to describe the kinetics of apoB in VLDL subfractions. Compartments 10 through 14 represent a delipidation cascade, as originally described by Phair et al.35 It is assumed that the residence time of particles in each compartment of the cascade is the same. In addition, the fraction of each compartment in the cascade converted to the slow-turnover VLDL compartment (compartment 15) is the same. Particles in compartments 11 through 14 can be transferred directly to compartment 15, a slow-turnover VLDL compartment. VLDL particles in compartment 14 can be converted to IDL or can be removed directly from plasma. The IDL section of the model includes compartments 20 and 21, a rapid- and slow-turnover pool of IDL particles, respectively. Particles in compartment 20 can be converted to the slow IDL compartment, to LDL, or can be removed directly from plasma. In this model, LDL-apoB kinetics were described by two compartments, 30 and 31. Compartment 30 represents LDL-I (d=1.019 to 1.035 g·mL-1), and compartment 31 represents LDL-2 (d=1.036 to 1.063 g·mL-1). All LDL-apoB was derived from IDL, compartment 20. The particles reach the LDL section of the model through compartment 30, in which it can be converted to the slow-turnover LDL-2 compartment (compartment 31) or can be removed directly from plasma. After we fit the model to the tracer-tracee data, apoB FCRs, PRs, and conversion rates were determined. The FCR for VLDL-apoB was calculated by dividing the VLDL PR, ie, the PR of apoB, into compartment 10 by the mass of apoB in the VLDL fraction. The FCR for IDL was determined by dividing the apoB transport rate from compartments 14 through 20 by the mass of apoB in the IDL fraction. The FCR for LDL-1 was determined by dividing the apoB transport rate from compartment 20 by the apoB mass in the LDL-1 fraction. The FCR for LDL-2 is equivalent to the value of the rate constant L(0,31). In this report, the terms transport rate and PR are used synonymously, as are FCR and fractional turnover rate. The FCR for the whole LDL fraction was determined by dividing the apoB transport rate from compartment 20 by the total mass of the two LDL fractions.
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The model used for the control subjects was similar, although simpler, to that used in this study.21 The more complex model could have been fit to the normal data. Had this been done, the parameter differences that are apparent between the control and FCHL subjects would have persisted. In the present study two LDL subfractions were isolated, and therefore the model required two LDL compartments. In our study of the control subjects, only one LDL compartment was required. With respect to the VLDL section of the model, we expanded the delipidation cascade present in the normal model and included one additional compartment to account for a slow-turnover pool of VLDL particles seen in FCHL subjects. Had the present model been fit to the control subjects, the rate constants of the model would be different and some of the pathways would have not existed. However, the metabolic parameters for the control subjects would not have changed.
Statistical Analysis
Results are presented as mean±SD. Wilcoxon signed rank tests were used to compare results obtained during pravastatin and placebo therapy. Paired t tests corrected for multiple comparisons provided similar results. Least significant differences based on P=.05 are also provided. The Pearson correlation coefficient was used to analyze associations between variables. All statistical analyses were calculated with Statgraphics (STSC, Inc).
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Lipoprotein Lipids and Lipid Diagnosis
Five study subjects were members of the M-kindred (Fig 1
), in which multiple phenotypes segregated most likely as an autosomal dominant trait.36 Several members of the kindred had histories of or had died of coronary heart disease. Unfortunately, a crucial subject for genetic analysis, I-7, died at 45 years with a myocardial infarction several years before our study commenced. The assignments of symbols in the pedigree are based on 10th and 90th sex-, race-, and age-based percentiles,32 as indicated. In terms of Cholesterol Education Program Adult Treatment Panel II guidelines,38 three subjects had LDL-C >160 mg/dL, and two of these also had TG >200 mg/dL. The other two subjects had LDL-C <140 mg/dL and TG >200 mg/dL. None had either an apoE-2 genotype or VLDL-C/TG ratios >0.22, which ruled out familial dysbetalipoproteinemia.39 Levels of LDL-C and patterns of segregation were atypical for FH.40 Genetic analysis of apoB gene based on a variable number of tandem repeats demonstrated different alleles among the five subjects, ie, there was no cosegregation of the hyperlipidemic phenotype with any given apoB allele. Specific testing for the apoB-3500 mutation29 showed the absence of the defective allele. These results ruled out familial defective apoB.41 42 FH was also considered unlikely by the absence of any given LDL receptor allele cosegregating with the FCHL phenotype (Table 2). Demographic and lipoprotein values for the nine nonstudied members of the kindred are also given in Tables 1 and 2 to further to document the lipoprotein disorder segregating the kindred. Analogous demographic, lipoprotein, and genetic data are provided for the C- and K-kindreds, which we also believe to have FCHL (Tables 3 and 4 and Figs 2 and 3). Interestingly, a subject with the FH phenotype married into the C-kindred, and the union produced a son with probable FH (subjects I-2 and II-1, Tables 3 and 4 and Fig 2). Mean demographic and lipoprotein values for the normolipidemic comparison group are also given in Tables 1 and 2.
As is characteristic of patients with FCHL,3 36 the lipid levels of the M-kindred members during the placebo phase (Table 4) differed from the values on which original assignments were made (Table 2). Nevertheless, overall patterns of lipids were retained, and pravastatin therapy was instituted according to protocol, resulting in significant drops in total cholesterol, LDL-C, and apoB (by 18%, 22%, and 26%, respectively), but not in TG, HDL-C, or apoA-I (Table 5). Similar pravastatin-induced drops were seen in the members of the C- and K-kindreds (data not shown).
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Lipoprotein Kinetics
A set of representative curves for subject 5 (II-26, M-kindred) is shown during placebo and pravastatin periods in Fig 5; model fitted lines and experimental points showed close agreement. Individual values of kinetic parameters for the study group and mean values for the normolipidemic comparison group are shown in Table 6.
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During the placebo period, contrary to expectations, mean values of VLDL-apoB PRs for the FCHL members of the M-kindred and normal subjects were almost identical, while the VLDL-apoB PRs of the two subjects from the C- and K-kindreds were higher (Table 6). Furthermore, IDL- and LDL-apoB PRs of the M-kindred also did not significantly differ from control subjects, while the analogous PRs tended to be higher in the C- and K-kindred patients. FCRs for VLDL- and LDL-apoB in the M-kindred were less than in the control subjects and in the two subjects from the C- and K-kindreds (Tables 1 and 3).19 Pravastatin caused significant increases in the FCRs for LDL-apoB but not in PRs or percent conversions.
The body mass indexes of the subjects from the C- and K-kindreds were similar to those of the M-kindred and the control subjects (Tables 1 and 3), suggesting that greater degrees of obesity did not account for their increased apoB PRs.
LDL Subfractions
To assess which subfraction of LDL may have been more affected by pravastatin therapy, LDLs of the M-kindred members were subfractionated by ultracentrifugation into LDL-1 (d=1.019 to 1.035 g·mL-1) and LDL-2 (d=1.036 to 1.063 g·mL-1). The components of LDL-1 and LDL-2 were quantified, and the kinetics of their apoBs were analyzed (Table 7). During the placebo phase, most of the LDLs were found in the LDL-2 subclass in all five subjects. As expected, the lighter LDL-1 contained larger amounts of cholesterol per particle than the denser LDL-2 (cholesterol-apoB ratio, 1.54±0.1 versus 1.2±0.07; P<.05).
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Both LDL-1 and LDL-2 cholesterol and apoB were lowered by pravastatin, but therapy did not shift the distribution of the LDLs between the subclasses (percent apoB in LDL-1 subclass during placebo and pravastatin, 24.3±2.4% versus 26.6±5.03%, respectively). The drug decreased cholesterol content per LDL-1 particle but did not modify LDL-2 composition.
Fig 6 shows the observed tracer-tracee ratios and the best fit obtained by the model for LDL-1 and LDL-2 in all five M-kindred subjects. On placebo, in every case, the LDL-1 curve had a faster appearance of the tracer and an earlier plateau than the LDL-2 curve. Thus, a precursor-product relationship of LDL-1
LDL-2 was observed. There was no need to invoke other sources of LDL-2 precursors, different from LDL-1, to fit the data. The kinetic parameters obtained with the multicompartmental model demonstrated that LDL-1 particles had a significantly higher FCR (0.82±0.21 versus 0.22±0.08 pool per day, P<.01) and a slightly higher PR (11.9±3.3 versus 9.8±3.2 mg/kg per day, P=.13) than LDL-2. Pravastatin increased the FCRs of both LDL subclasses (61% for LDL-1 apoB and 68% for LDL-2 apoB). Small, nonsignificant increases in mean PRs were observed for both LDL subclasses (LDL-1 apoB, 11.9±3.3 versus 15.7±2.4, P=.1; LDL-2 apoB, 9.8±3.2 versus 12.2±1.9 mg/kg per day, P=.17).
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Discussion |
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We have studied the effects of HMG-CoA reductase inhibitors on apoB kinetics in apoB overproduction states18 19 to test the hypothesis that these agents could be acting not only by enhancing FCRs of apoB-containing lipoproteins, ie, by upregulating LDL receptors, as has been reported in FH,16 but also in part by inhibiting the PRs of apoB-containing lipoproteins, as has been reported in FCHL.17 In previous studies we were unable to document inhibition of PRs in two types of patients with apoB overproduction, those with mixed lipemia19 and those with focal segmental glomerulosclerosis.18 In the present set of studies we aimed to test the hypothesis in members of a kindred with FCHL, a condition widely reported to be accompanied by increased FCRs of apoB.8 10 11 12
The M-kindred chosen for study met the common criteria for the condition3 36 : various lipoprotein phenotypes in different members of the kindred segregating as an autosomal dominant condition (Fig 1, Tables 2 and 3), predominance of dense LDLs (Table 5), and the presence of coronary heart disease. Familial dysbetalipoproteinemia seems to be very unlikely due to the presence of apoE-3 or apoE-4 genotypes, the absence of apoE-2 genotypes, and the normal ratios of VLDL-C and TG (Table 1). The lipoprotein profiles also would be atypical for FH or familial defective apoB-100.41 Furthermore, neither the apoB nor the LDL receptor alleles examined cosegregated with the FCHL phenotype, virtually excluding either familial defective apoB-100 or FH (Table 2).
To our surprise, rather than having increased PRs, the affected members had apoB PRs indistinguishable from those of previously studied normal subjects21 (Table 4) when we used a methodology with which it is possible to readily detect increased PRs in human subjects.18 19 In one of the previous reports,19 we presented mean apoB kinetic data on four subjects with "mixed hyperlipoproteinemia." Subsequently, the kindreds of two of those subjects have been studied (Tables 3 and 4, Figs 2 and 3), and we now know that those subjects are members of kindreds harboring FCHL diagnosed by the same criteria applied to the M-kindred. However, both of these subjects had high PRs of VLDL- and LDL-apoB (Table 6), as expected from previous reports.7 8 9 10 12 13 In the M-kindred, instead of the expected high PRs, FCRs for VLDL-apoB and LDL-apoB were lower than normal, and PRs for LDL-apoB were lower than in the C- and K- kindred members (even in those M-kindred FCHL subjects with <75th percentile LDL-C levels). The percentage of conversion of VLDL to LDL was normal.
The kinetics of apoB-containing lipoproteins in FCHL have been previously described in six reports including 49 subjects.7 8 9 10 12 13 The inclusion criteria and the experimental methodology were different among these studies. Nevertheless, in the vast majority of the cases (41 of 49 subjects), an overproduction of VLDL and/or LDL particles was observed. However, normal VLDL-apoB PRs were observed in 2 of 7 patients reported by Chait et al,12 3 of 11 by Janus et al,13 and 1 of 9 by Kissebah et al.8 Most of these subjects had normal VLDL-apoB concentrations. Our results showing normal VLDL-apoB PRs confirm and extend the findings in References 8, 12, and 13 and reinforce the heterogeneous nature of the kinetic of apoB in FCHL across different kindreds.
Two kinetic abnormalities were observed in our study subjects: low VLDL-apoB FCRs and low LDL-apoB FCRs (Table 6). Remarkably, the same kinetic abnormalities were observed in every case despite the presence of different lipid profiles (Tables 2 and 5).
A decreased VLDL-apoB FCR has been a frequent finding in FCHL and hyperapobetalipoproteinemia and was also present in the two members of the C- and K- kindreds.11 Possible explanations for this abnormality include decreased lipoprotein lipase activity,43 44 abnormal VLDL and LDL composition,44 and abnormal function of the recently described VLDL receptor.45 Approximately 30% of FCHL cases have decreased lipoprotein lipase activity.44 Abnormal VLDL compositions frequently result in a lower affinity of these particles for lipoprotein lipase.46 However, the mechanism that explains the decreased VLDL-apoB FCR remains to be elucidated.
The LDL-apoB FCR was reported as normal in most FCHL patients and was close to normal in the two members of the C- and K-kindreds. The low LDL-apoB FCRs in the M-kindred could be explained by the predominance among the LDL subclass of the denser particles, which manifest low affinities for the LDL receptor,47 analogous to that found in a familial defective apoB homozygous subject, in whom a predominance of small dense particles was found in the LDL fraction.48 Alternatively, downregulated or defective LDL receptors could inhibit clearance of LDLs, facilitating conversion of LDL-1 to LDL-2 and the accumulation of LDL-2.
To distinguish between the above possibilities and to elucidate the mechanism by which the small dense LDLs were formed in our subjects, the two LDL subfractions were separated and their kinetic properties determined. The distributions of apoB and the cholesterol-apoB ratios in the LDL subfractions were very similar to those reported by Teng et al.11 A clear relationship between precursor and product was observed between light and dense LDL, suggesting that LDL-2 particles represented the final products of the metabolism of the apoB-containing lipoproteins secreted from the liver. Kinetically, the light LDL-1 had a significantly higher FCR than the dense LDL-2 (0.82±0.21 versus 0.22±0.08 pool per day), suggesting that LDL-1 was cleared adequately, perhaps due to greater contents of apoE,48 which confer higher affinities of binding to the LDL receptor. Hence, LDL-2s accumulate more than LDL-1s because of their relatively lower FCRs.
As expected, pravastatin resulted in significantly decreased total cholesterol, LDL-C, LDL-1, LDL-2, and apoB concentrations. Two mechanisms by which HMG-CoA reductase inhibitors achieve their effects of lowering LDL-C have been proposed. The first is increased LDL-apoB catabolism due to suppression of hepatic cholesterol synthesis and the subsequent upregulation of LDL receptors.15 This mechanism seems to account for the drug-induced falls in LDL in heterozygotes for FH16 On the other hand, HMG-CoA reductase inhibitors decreased apoB production in FCHL patients with increased apoB PRs.17 In the M-kindred, in which decreased VLDL- apoB and LDL-apoB FCRs were the main kinetic abnormalities, pravastatin at the dose used decreased LDL-C by increasing LDL-apoB catabolism. It is possible that effects on VLDL production could be observed at higher doses.
In conclusion, our results reinforce the heterogeneous nature of FCHL, provide new information regarding the metabolism of the LDL subfractions in FCHL, and document the physiological mechanisms of action of HMG-CoA reductase inhibitors in this atypical "normal apoB PR" variant of FCHL.
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Acknowledgments |
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This study was supported in part by General Clinical Research Center (GCRC) grant (US Public Health Service) MO1 RR00036, National Institutes of Health (NIH) National Center for Reseach Resources (NCRR) grant RR02176, NIH grant HL-42460, and by a grant from Bristol-Myers Squibb Co. Dr Aguilar-Salinas was supported by a fellowship from the Programa Universitario de Investigacion en Salud of the Universidad Nacional Autonoma de Mexico. The authors thank the study subjects. The personnel of the Core Laboratory of the Lipid Research Center were expert in their techniques, and the personnel of the Lipid Research Clinic and the General Clinical Research Center of Washington University were very helpful with the metabolic studies. We express our gratitude to Tish Kettler and Tom Kitchens for superior technical help and to Mary Lou Rheinheimer for typing the manuscript. We are grateful to Dr Helen Hobbs for discussions about the genetics of FH.
Received October 12, 1995;
revision received May 10, 1996;
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1. Campos H, Genest JJ, Blijlevens E, McNamara JR, Jenner K, Ordovas JM, Wilson PWF, Schaefer EJ. Low density lipoprotein size and coronary artery disease. Arterioscler Thromb. 1992;12:187-195.
2.
Austin MA, Breslow K, Hennekens CA, Buring JE, Willett WC, Krauss RM. Low density lipoprotein subclass pattern and risk of myocardial infarction. JAMA. 1988;260:1917-1921.
3. Grundy SM, Chait A, Brunzell JD. Familial Combined Hyperlipidemia Workshop. Arteriosclerosis. 1987;7:203-207.
4. Pairitz G, Davignon J, Maillous H, Sing CF. Sources of interindividual variation in the quantitative levels of apolipoprotein B in pedigrees ascertained through a lipid clinic. Am J Hum Genet. 1986;38:492.[Medline] [Order article via Infotrieve]
5. Xu C-F, Talmud P, Schuster H, Houlston R, Miller G, Humphries S. Association between genetic variation at the APO AI-CIII-AIV gene cluster and familial combined hyperlipidaemia. Clin Genet. 1994;46:385-397.[Medline] [Order article via Infotrieve]
6. Cianflone KM, Maslowska MH, Sniderman AD. Impaired response of fibroblasts from patients with hyperapobetalipoproteinemia to acylation-stimulating protein. J Clin Invest. 1990;85:722-730.
7. Cortner JA, Bennett MJ, Le NA, Coates PM. Familial combined hyperlipidemia: use of stable isotopes to demonstrate overproduction of very low density lipoprotein apolipoprotein B by the liver. J Inher Metab Dis. 1991;14:915-922.[Medline] [Order article via Infotrieve]
8. Kissebah AH, Alfarsi S, Adams PW. Integrated regulation of very low density lipoprotein triglycerides and apolipoprotein B kinetics in man: normolipidemic subjects, familial hypertriglyceridemia and familial combined hyperlipidemia. Metabolism. 1981;30:856-868.[Medline] [Order article via Infotrieve]
9.
Kissebah AH, Alfarsi S, Evans DJ. Low density lipoprotein metabolism in familial combined hyperlipidemia. Arteriosclerosis. 1984;4:614-624.
10.
Venkatesan S, Cullen P, Pacy P, Halliday D, Scott J. Stable isotopes show a direct relation between VLDL apoB overproduction and serum triglycerides levels and indicate a metabolically and biochemically coherent basis for familial combined hyperlipidemia. Arterioscler Thromb. 1993;13:1110-1118.
11. Teng B, Sniderman AD, Soutar AK, Thompson GR. Metabolic basis of hyperapobetalipoproteinemia: turnover of apolipoprotein B in low density lipoprotein and its precursors and subfractions compared with normal and familial hypercholesterolemia. J Clin Invest. 1986;77:663-672.
12. Chait A, Albers JJ, Brunzell JD. Very low density overproduction in genetic forms of hypertriglyceridemia. Eur J Clin Invest. 1980;10:161-172.[Medline] [Order article via Infotrieve]
13. Janus ED, Nicoli AM, Turner PR, Majili P, Lewis B. Kinetic bases of the primary hyperlipidemia: studies of apolipoprotein B turnover in genetically defined subjects. Eur J Clin Invest. 1980;10:161-172.
14. Schonfeld G. HMG CoA reductase inhibitors. In: LaRosa JC, ed. Practical Management of Lipid Disorders. Fort Lee, NJ: Health Care Communications, Inc; 1992:75-90.
15. Brown MS, Goldstein JL. Multivalent feedback regulation of HMG-CoA reductase, a control mechanism coordinating isoprenoid synthesis and cell growth. J Lipid Res. 1980;21:505-517.[Abstract]
16. Hagemenas FC, Pappu AS, Illingworth DR. The effects of simvastatin on plasma lipoproteins and cholesterol homeostasis in patients with heterozygous familial hypercholesterolaemia. Eur J Clin Invest. 1990;20:150-157.[Medline] [Order article via Infotrieve]
17. Arad Y, Ramakrishnan R, Ginsberg HN. Lovastatin therapy reduces low density lipoprotein apoB levels in subjects with combined hyperlipidemia by reducing the production of apoB-containing lipoproteins: implications for the pathophysiology of apoB production.J Lipid Res. 1990;31:567-582.[Abstract]
18. Aguilar-Salinas CA, Barrett PHR, Kelber J, Delmez J, Schonfeld G. Physiologic mechanism of action of lovastatin in nephrotic syndrome. J Lipid Res. 1995;36:188-199.[Abstract]
19. Parhofer KG, Barrett PHR, Dunn J, Schonfeld G. Effect of pravastatin on metabolic parameters of apolipoprotein B in patients with mixed hyperlipoproteinemia. Clin Invest. 1993;71:939-946.[Medline] [Order article via Infotrieve]
20. Parhofer KG, Barrett PHR, Bier DM, Schonfeld G. Lipoproteins containing the truncated apolipoprotein, apoB-89, are cleared from human plasma more rapidly than apoB-100 containing lipoproteins in vivo. J Clin Invest. 1990;89:1931-1937.
21.
Aguilar-Salinas CA, Barrett PHR, Parhofer KG, Young SG, Tessereau D, Bateman J, Quinn C, Schonfeld G. Apoprotein B-100 production is decreased in subjects heterozygous for truncations of apoprotein B. Arterioscler Thromb Vasc Biol. 1995;15:71-80.
22. Lichtenstein A, Cohn J, Hachey D, Millar J, Ordovas JM, Schaefer EJ. Comparison of deuterated leucine, valine and lysine in the measurement of human apolipoprotein A-I and B-100 kinetics. J Lipid Res. 1990;31:1693-1701.[Abstract]
23.
Schaefer JR, Rader DJ, Ikewaki K, Fairwell K, Zech LA, Kindt MR, Davignon J, Gregg RE, Brewer HB. In vivo metabolism of apolipoprotein A-I in a patient with homozygous familial hypercholesterolemia. Arterioscler Thromb. 1992;12:843-848.
24. Parhofer KG, Barrett PHR, Bier DM, Schonfeld G. Determination of the kinetic parameters of apolipoprotein B metabolism using amino acids labeled with stable isotopes. J Lipid Res. 1991;32:1311-1323.[Abstract]
25. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultra-centrifugally separated lipoproteins in human serum. J Clin Invest. 1955;34:1345-1353.
26. Hixson JE, Vernier DT. Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with Hhal. J Lipid Res. 1990;31:545-548.[Abstract]
27. Ludwig EH, Friedl W, McCarthy BJ. High resolution analysis of a hypervariable region in the human apolipoprotein B gene. Am J Hum Genet.. 1989;45:458-464.[Medline] [Order article via Infotrieve]
28. The chromosome 19 marker for the LDL receptor was D19S394. The Genome Data Base (GDB) ID number was G00-198-395. The chromosome 2 marker D2S149 had the ID number of G00-133-397.
29. Schuster H, Rauh G, Muller S, Keller C, Wolfram G, Zollner N. Allele-specific and asymmetric polymerase chain reaction amplification in combination: a one step polymerase chain reaction protocol for rapid diagnosis of familial defective apolipoprotein B-100. Anal Biochem. 1992;204-22-25.
30. Adams RF. Determination of amino acid profiles in biological samples by gas chromatography. J Chromotogr. 1974;95:189-212.
31. Klein RL, Zilversmit D. Direct determination of human and rabbit apolipoprotein B selectively precipitated with butanol-isopropyl ether. J Lipid Res. 1984;25:1380-1386.[Abstract]
32. Matthews DE, Ben-Galim E, Bier DM. Determination of stable isotopic enrichment in individual plasma amino acids by chemical ionization mass spectrometry. Ann Chem. 1979;51:80-84.
33.
Cobelli C, Toffolo G, Bier DM, Nosadini R. Models to interpret kinetic data in stable isotope tracer studies. Am J Physiol. 1987;253:E551-E564.
34. Berman M. SAAM Manual. Washington, DC: 1978. US Dept of Health and Human Services publication NIH 78-180.
35. Phair RD, Hammond MG, Bowden JA, Fried M, Fisher WR, Berman M. A preliminary model for human lipoprotein metabolism in hyperlipoproteinemia. Fed Proc. 1975;34:2263-2270.[Medline] [Order article via Infotrieve]
36. Kwiterovich PO. Genetics and molecular biology of familial combined hyperlipidemia. Curr Opin Lipidol.. 1993;4:133-143.
37.
Lipid Research Clinics Program Epidemiology Committee. Plasma lipid distributions in selected North American populations: the Lipid Research Clinics Program prevalence study. Circulation. 1979;60:427-439.
38.
National Cholesterol Education Program. Summary of the second report of the National Cholesterol Education Program (NCEP) Expert Panel on detection, evaluation, and treatment of high blood cholesterol in adults (Adult Treatment Panel II). JAMA. 1993;269:3015-3023.
39. Havel RJ. Familial dysbetalipoproteinemia: new aspects of pathogenesis and diagnosis. Med Clin North Am. 1982;66:441-454.[Medline] [Order article via Infotrieve]
40. Williams RR, Hunt SC, Schumacher C, Hegele RA, Leppert MF, Ludwig EH, Hopkins PN. Diagnosing heterozygous familial hypercholesterolemia using new practical criteria validated by molecular genetics. Am J Cardiol. 1993;72:171-176.[Medline] [Order article via Infotrieve]
41. Myant NB. Familial defective apolipoprotein B-100: a review, including some comparisons with familial hypercholesterolemia. Atherosclerosis. 1993;104:1-18.[Medline] [Order article via Infotrieve]
42. Pullinger CR, Hennessy LK, Chatterton JE, Liu W, Love JA, Mendel CM, Frost PH, Malloy MJ, Schumaker VN, Kane JP. Familial ligand-defective apolipoprotein B: identification of a new mutation that decreases LDL receptor binding affinity. J Clin Invest. 1995;95:1225-1234.
43.
Gagne E, Genest J, Zhang H, Clarke LA, Hayden MR. Analysis of DNA changes in the LPL gene in patients with familial combined hyperlipidemia. Arterioscler Thromb. 1994;14:1250-1257.
44. Babirak SP, Brown BG, Brunzell JD. Familial combined hyperlipidemia and abnormal lipoprotein lipase. Arterioscler Thromb. 1992;12:1176-1183.[Abstract]
45. Jokinen E, Sakai J, Yamamoto T, Hobbs HH. CGG triple repeat polymorphism in VLDL receptor (VLDL-4) gene. Mol Genet.. 1994;3:3.
46.
Chung BH, Cone JT, Segrest JP. Defective in vitro lipolysis of type IV hyperlipidemic human plasma by purified milk lipoprotein lipase. J Biol Chem. 1982;257:7472-7480.
47.
Galeano NF, Milne R, Marcel YL, Walsh MT, Levy E, Nguyen TD, Gleeson A, Arad Y, Witte L, Alhaideri M, Runsey SC, Deckelbaum RJ. Apolipoprotein B structure and receptor recognition of triglyceride-rich low-density lipoprotein (LDL) is modified in small LDL but not in triglyceride-rich LDL of normal size. J Biol Chem. 1994;269:511-519.
48. Marz W, Baumstark MW, Scharnagl H, Ruzicka V, Buxbaum S, Herwig J, Pohl T, Russ A, Schaaf L, Berg A, Bohles H, Usadel KH, Grob W. Accumulation of `small dense' low density lipoprotein (LDL) in a homozygous patient with familial defective apolipoprotein B-100: results from heterogenous interaction of LDL subfraction with the LDL receptor. J Clin Invest. 1993;92:2922-2933.
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