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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1309.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

In Vivo Metabolism of ApoB, ApoA-I, and VLDL Triglycerides in a Form of Hypobetalipoproteinemia Not Linked to the ApoB Gene

Nizar Elias; Bruce W. Patterson; Gustav Schonfeld

From the Division of Atherosclerosis, Lipid Research, and Nutrition, and the Division of Gastroenterology (B.W.P.), Department of Internal Medicine, Washington University School of Medicine, St Louis, Mo.

Correspondence to Gustav Schonfeld, MD, Department of Internal Medicine, Washington University School of Medicine, 660 S Euclid, Box 8046, St Louis, MO 63110. E-mail gschonfe{at}im.wustl.edu

	Abstract

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Abstract—Familial hypobetalipoproteinemia (FHBL) is an autosomal codominant disorder that may result from different mutations in the apolipoprotein B (apoB) gene or chromosome 2. However, linkage of FHBL to the apoB gene was ruled out in 2 kindreds reported to date, and the genetic and metabolic bases for FHBL remain unknown. One of the reported kindreds is our 40-member F kindred, in which we found linkage of FHBL to a novel susceptibility region on chromosome 3p21.1-2. In addition to having low apoB levels, some, but not all, of the affected subjects in the F kindred also had low levels of high density lipoprotein (HDL) cholesterol and apoA-I. Our aim was to define the metabolic bases of the disorder in the F kindred. Therefore, we studied the in vivo kinetics of apoB and apoA-I and very low density lipoprotein (VLDL) triglycerides in 4 affected subjects and 5 normolipidemic relatives. Deuterated leucine and deuterated glycerol were used to label the apolipoproteins and triglycerides, respectively. Compartmental modeling was used to obtain the kinetic parameters. Affected subjects had (1) normal fractional catabolic rates (FCRs) for VLDL apoB, (2) increased FCRs for low density lipoprotein (LDL) apoB (0.050±0.009 versus 0.030±0.006 pools per hour for normal subjects, P=0.005), and (3) decreased production rates of VLDL apoB (11.4±1.7 versus 25.6±4.9 mg · kg^-1 · d^-1, P=0.003), LDL apoB (7.8±1.3 versus 12.7±3.7 mg · kg^-1 · d^-1, P=0.04), and VLDL triglycerides (8.2±4.5 versus 19.6±10.8 58 µmol · kg^-1 · h^-1, P=0.09). These data differ from those obtained in previously studied FHBL heterozygotes bearing apoB-2 and apoB-9, 2 very short truncations of apoB. Low HDL cholesterol and apoA-I levels were caused by higher apoA-I FCRs (0.035±0.005 versus 0.018±0.005 pools per hour in controls, P<0.01) without significant decrease in apoA-I production rates (18.7±2.7 versus 22.8±5.6 mg · kg^-1 · d^-1). In conclusion, decreased secretion of apoB-containing lipoproteins and hypercatabolism of LDL account for low apoB and cholesterol levels in this novel form of FHBL.

Key Words: familial hypobetalipoproteinemia • apolipoprotein B • apolipoprotein A-I • triglycerides • stable isotopes

	Introduction

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Apolipoprotein B plays a crucial role in the intracellular assembly and secretion of triglyceride-rich lipoproteins and is a ligand for the LDL receptor.¹ High plasma levels of apoB are directly correlated with the risk of atherosclerotic coronary artery disease.² ApoB exists in plasma in 2 forms, apoB-100 secreted by the liver in VLDL particles and apoB-48 secreted by the intestine in chylomicrons. Both forms are the products of a single apoB gene located on chromosome 2.¹ ApoB-48 results from a tissue-specific apoB mRNA editing process in the intestine, which allows translation of 48% of the amino terminal of apoB-100. Three inherited syndromes characterized by low plasma levels of apoB have been described: (1) Abetalipoproteinemia, in which apoB-containing lipoproteins are nearly absent from plasma, is inherited as an autosomal-recessive disorder. It is caused by mutations in the microsomal triglyceride transfer protein (MTP) gene.³ (2) Chylomicron retention disease, a disorder that selectively affects chylomicron secretion by the intestine, is also inherited as an autosomal-recessive trait, but its molecular etiology has not been identified.⁴ (3) Familial hypobetalipoproteinemia (FHBL) is inherited as an autosomal-dominant trait, with heterozygotes having apoB and LDL cholesterol levels below the fifth percentile. Although the molecular cause in most cases is unknown,^{5 6} FHBL can result from 1 of several mutations in the apoB gene that produce truncated forms of apoB.^{7 8} Low plasma levels of apoB in these cases are caused by decreased secretion rates of the truncated and the normal apoB-100 forms of apoB and in some cases by enhanced catabolism of the truncation-containing lipoproteins.^{9 10 11 12} However, in 2 reported FHBL families, such truncations are not present in plasma, and the disorder is not linked to the apoB gene.^{13 14} The genetic and metabolic bases of these cases of FHBL remain unknown.

We have reported on a 40-member FHBL kindred in which the hypobetalipoproteinemia phenotype segregates as an autosomal-dominant trait, but no apoB truncations were identified in the plasma of affected subjects. Linkage analysis studies ruled out linkage of hypobetalipoproteinemia to the apoB gene¹⁴ and MTP gene (B. Yuan, G. Schonfeld, unpublished data). Instead, linkage to a new FHBL susceptibility region on chromosome 3p21.1-2 was found.¹⁵ Some, but not all, FHBL-affected subjects of this kindred also have plasma HDL cholesterol and apoA-I levels well below the fifth percentile for age and sex, a finding not present in typical FHBL cases caused by apoB gene mutations. The aim of the present study was to ascertain the metabolic mechanisms responsible for the low plasma levels of apoA-I–containing and apoB-containing lipoproteins in this novel form of FHBL. Therefore, we studied the in vivo kinetics of apoB, VLDL triglycerides, and apoA-I simultaneously in affected and normolipidemic subjects. Deuterated leucine and deuterated glycerol were infused to label the apolipoproteins and triglycerides, respectively. Kinetic parameters were obtained by compartmental modeling.

	Methods

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Kindred and Study Subjects
The F kindred is a 3-generation 40-member kindred of German descent.^{14 15} Ten affected subjects, defined as having apoB and LDL cholesterol levels below the fifth percentile for age and sex, were found. In addition, 5 affected subjects had levels of HDL cholesterol and apoA-I below the fifth percentile; the other 5 had normal levels. None of the unaffected subjects had low HDL cholesterol or apoA-I levels. There was no family history of coronary heart disease. The number of affected subjects in this kindred was too small for formal longevity studies.

Four affected subjects from the F kindred participated in the present study. Four unaffected normolipidemic blood relatives and 1 unrelated spouse served as the control group. All subjects were asymptomatic and had no abnormal findings on physical examinations. None was taking any medication. Blood counts, liver function tests, and lipid profiles performed on enrollment were normal. All participants gave written informed consent, and the study protocol was approved by the Human Studies Committee of Washington University School of Medicine. The clinical characteristics of the study subjects are described in Table 1.

View this table: [in this window] [in a new window]   Table 1. Clinical Characteristics of Study Subjects

Study Protocol
The subjects were instructed to adhere to their regular diet for 10 days before the study. For the kinetic study, subjects were admitted to the General Clinical Research Center (GCRC) after a 10-hour fast. An intravenous catheter was inserted into the antecubital vein of one arm for the infusion of tracers, and a second catheter was inserted into a vein in the other arm for blood sampling. A bolus of [²H₅]glycerol (99% ²H, Cambridge Isotopes) at a dose of 60 µmol/kg was given, immediately followed by a primed constant infusion of deuterated leucine. [²H₃]Leucine (98% ²H, Cambridge Isotopes) was administered as a priming dose of 7 µmol/kg, followed by 7 µmol · kg^-1 · h^-1 for 8 hours through a calibrated syringe pump (Harvard Apparatus). The subjects remained fasting during the infusion period and for another 8 hours after the termination of the infusion. Noncaloric drinks were permitted throughout the study. Food was given at 16 hours and thereafter up to 84 hours, when the study was terminated. Blood was collected into EDTA-containing tubes, and plasma was separated immediately by low-speed centrifugation.

Analytical Methods
Measurements of Plasma Lipids and Apolipoprotein Levels
Total cholesterol, triglycerides, LDL cholesterol, and HDL cholesterol were determined in baseline plasma samples by the Lipid Research Center Core Laboratory at Washington University School of Medicine by use of commercially available enzymatic kits (Technicon Instruments). Plasma apoB and apoA-I were determined by immunonephelometry (Behring).

Characterization of Plasma Lipoproteins by FPLC
Fresh plasma (2 mL) was applied to two 25-mL Superose 6 columns connected in series in a fast protein liquid chromatography (FPLC) system (Pharmacia Biotech).¹⁶ The columns were equilibrated and eluted at ambient temperature with a buffer consisting of 1 mmol/L EDTA and 0.15 mol/L NaCl, pH 8.0. The column eluent fractions (0.5 mL/min) were analyzed for total cholesterol, free cholesterol, triglycerides, and phospholipids.

Nondenaturing Gradient Gel Electrophoresis
HDL particle size distribution was evaluated by nondenaturing gradient gel electrophoresis as previously described.¹⁷ Briefly, lipoprotein fraction (density [d]<1.21) was separated from plasma by ultracentrifugation and then subjected to 4% to 30% polyacrylamide gradient gel electrophoresis. For particle size calibration, a standard protein mixture (containing thyroglobulin, ferritin, catalase, lactate dehydrogenase, and BSA) was applied to the gel. Gels were fixed in 10% sulfosalicylic acid, stained in Coomassie blue, and destained in 5% acetic acid. Gels were scanned by laser densitometry, and areas under the curve for each HDL subpopulation were integrated.

Isolation of Lipoproteins
VLDL (d<1.006 g/mL), IDL (1.006<d<1.019 g/mL), and LDL (1.019<d<1.063 g/mL) were isolated by sequential ultracentrifugation at 40 000 rpm for 18 hours. HDL (1.063<d<1.21 g/mL) was isolated by ultracentrifugation at 45 000 rpm for 24 hours. Lipoprotein fractions were dialyzed against EDTA-saline for 24 hours and then stored at -70°C until further analysis. Total cholesterol, free cholesterol, triglyceride, and phospholipid concentrations in different lipoprotein fractions were determined enzymatically. ApoB concentrations in VLDL, IDL, and LDL were determined by ELISA with use of the monoclonal antibody C 1.4.¹⁸ LDL apoB concentrations were confirmed by protein assays¹⁹ as described previously.¹⁰

Isolation of Plasma Amino Acids, ApoB, and ApoA-I for Enrichment Measurements
Amino acids were isolated from 0.25 mL of plasma by cation exchange chromatography.²⁰ ApoB was isolated from VLDL, IDL, and LDL fractions by precipitation with butanol–isopropyl ether²¹ and hydrolyzed in 6N HCl for 24 hours at 110°C. ApoA-I was isolated from HDL fractions by SDS–polyacrylamide gel (12%) electrophoresis. ApoA-I bands were excised from the gels and hydrolyzed in 6N HCl for 24 hours at 110°C, and the resulting amino acids were separated by cation exchange chromatography. Blank lanes on the gel containing no protein were also excised and processed similarly to test the amount of contamination with unlabeled leucine that occurred with this procedure. Amino acids obtained from plasma, apoB hydrolysates, and apoA-I hydrolysates were derivatized to N-heptafluorobutyryl-n-propyl esters and dissolved in ethyl acetate for enrichment measurements.²² Leucine enrichment was determined by gas chromatography–mass spectrometry on a Hewlett-Packard model 5971 or 5973 MSD equipped with a DB-17 capillary column. Ions at mass-to-charge ratios of 282 (m+0) and 285 (m+3) formed by electron impact ionization were selectively monitored. The enrichment data were converted to tracer/tracee ratios by analysis of suitable [²H₃]leucine isotopic enrichment standards.

Isolation of Plasma and VLDL Glycerol for Enrichment Measurements
Aliquots of 0.25 mL of plasma were deproteinized with acetone and then mixed with hexane and water.²³ The aqueous phase containing plasma glycerol was separated by low-speed centrifugation, dried in a Speedvac evaporator (Savant), and reacted with heptafluorobutyric anhydride.²⁴ Lipids were extracted from VLDL fractions by chloroform/methanol (1:2 [vol/vol]) and separated by thin-layer chromatography on silica gel plates (Whatman Inc). Triglyceride bands were scraped from the plates, and triglycerides were extracted with CHCl₃/methanol (3:1), dried, and reacted with acetyl chloride/methanol (1:20 [vol/vol]) at 70°C for 30 minutes. After drying in a Speedvac evaporator, fatty acid methyl esters were dissolved in heptane and discarded. Free glycerol generated by this reaction was then derivatized with heptafluorobutyric anhydride. Glycerol enrichment in VLDL triglycerides and plasma was determined by gas chromatography–mass spectrometry on a Hewlett-Packard model 5971 or 5973 equipped with a DB-17 capillary column. Ions at mass-to-charge ratios of 467 (m+0) and 472 (m+5) formed by electron impact ionization were selectively monitored. The data were expressed as tracer/tracee ratio by analysis of suitable [²H₅]glycerol isotopic enrichment standards.

Kinetic Analysis
Compartmental modeling was performed by use of the SAAM II program (SAAM Institute, University of Washington, Seattle). The model for [²H₃]leucine in apoB and apoA-I was an extension of the model we have previously used for apoB; an identical 8-hour primed continuous infusion protocol²⁵ was used (Figure 1). The time course of plasma [²H₃]leucine enrichment was used as a forcing function that provided a source of tracer for an amino acid precursor pool, which served as the source for apoB and apoA-I. After a delay (d3 in Figure 1), apoB emerged as VLDL (compartment 11). As in our previous studies,²⁵ a single compartment was sufficient to describe VLDL-apoB kinetics by use of the 8-hour primed constant infusion protocol. Kinetic heterogeneity requiring 1 VLDL compartment was not evident in the data. VLDL was converted into IDL (compartment 20) and LDL (compartment 30). Direct conversion of VLDL to LDL (bypassing IDL) and direct loss of IDL were considered, but neither pathway was required for any set of data evaluated. The precursor pool (compartment 9) also gave rise to apoA-I (compartment 40) after a delay (d4 in Figure 1). Exchange of LDL apoB and apoA-I with the nonplasma space (compartments 31 and 41) was required to provide an optimal fit to the data. The precursor pool (compartment 9) was exchanged with another pool representative of tissue protein breakdown (compartment 10). This exchange mechanism was necessary to provide an optimal fit to the terminal tail of VLDL apoB-100 enrichment by allowing a prolonged appearance of tracer into apoB after the infusion was halted. Inclusion or exclusion of this feature had virtually no impact on the major features of tracer appearance and disappearance in VLDL apoB.

View larger version (21K): [in this window] [in a new window]   Figure 1. Compartmental model of apoB and apoA-I leucine as drawn by the SAAM II program. See Methods for detailed description of model. Compartment numbers are indicated as q’s. Sites where leucine isotopic enrichment was measured are indicated as s1 through s5. Rate constants k(i,j) represent the fraction of compartment j going to compartment i per unit time. Delays are represented by d3 and d4. The fraction of each delay compartment transferred to the next compartment in line is indicated by d(11,3) and d(40,4).

The model used for [²H₅]glycerol labeling in VLDL triglycerides is shown in Figure 2. The time course of plasma glycerol enrichment (compartment 1) was used as a forcing function that provided tracer for the appearance of tracer in plasma VLDL triglycerides (compartment 3). Two biosynthetic pathways between plasma glycerol and VLDL triglycerides were necessary to describe the data as previously reported by Zech et al²⁶ in studies using a bolus tracer, [³H]glycerol. A fast pathway incorporated a time delay (d4), whereas a slow pathway featured a pool of tracer (presumed to be hepatic glycerolipids, compartment 5), which turned over at a rate of 0.3/h. Inclusion or exclusion of the slow pathway affected only the terminal tail of VLDL-triglyceride enrichment and had no impact on the initial rates of tracer appearance and disappearance from VLDL triglycerides. As for apoB, a single compartment was adequate to describe VLDL triglyceride tracer kinetics; kinetic heterogeneity of VLDL triglycerides was not evident in these normolipidemic and hypolipidemic subjects.

View larger version (10K): [in this window] [in a new window]   Figure 2. Compartmental model for VLDL triglyceride glycerol. See Methods for detailed description. See Figure 1 for symbols.

Production rates of VLDL, IDL, and LDL apoB, HDL apoA-I, and VLDL triglycerides were calculated as the product of their respective fractional catabolic rates (FCRs) and pool sizes; a plasma volume of 0.045 L/kg was assumed.

Statistical Analysis
The results are presented as mean±SD. Unpaired t tests were used to compare results obtained in affected and normal subjects. A value of P<0.05 was considered to be significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Plasma Lipids and Lipoproteins
The affected subjects and the subjects in the control group were well matched for sex, age, and body mass index (Table 1). By definition, plasma levels of total cholesterol, LDL cholesterol, triglycerides, and apoB were significantly lower in FHBL heterozygotes compared with control subjects (P<0.001 for all parameters). Mean HDL cholesterol levels were also lower in affected subjects versus control subjects (0.8±0.4 versus 1.2±0.2 mmol/L, P=0.07) as was the mean plasma apoA-I level (75±31 versus 117±13 mg/dL, P=0.03). ApoA-I and HDL cholesterol levels were particularly low in subjects 1 and 3 (Table 1).

Figure 3 shows representative FPLC profiles in an affected and a control subject. The FPLC method separates plasma lipoproteins by their size. In the control subjects, lipid levels peak in fractions 8, 29, and 47, corresponding to VLDL, LDL, and HDL, as previously described.¹⁶ In the affected subjects, no peak corresponding to VLDL was demonstrated, reflecting the very low levels of plasma VLDL. LDL peaked in fraction 30, whereas the HDL peak was shifted to the right and showed a plateau at fractions 49 to 54. This finding indicates that the HDL particles in affected subjects are distributed mostly in the smaller HDL size range. Estimation of HDL particle size distribution by nondenaturing gradient gel electrophoresis showed similar results. In affected subjects with low HDL cholesterol levels, a higher proportion of HDL particles was distributed in the lower size range compared with control subjects (not shown).

View larger version (18K): [in this window] [in a new window]   Figure 3. FPLC profiles of subject 2 of the affected group (top) and subject 5 of the control group (bottom). In the normal subject, VLDL, LDL, and HDL peaks are in fractions 8, 29, and 47, respectively. In the affected subject, the HDL peak is shifted to fractions 49 to 54. CHOL indicates cholesterol; TRIG, triglycerides.

Compositional analysis of the lipoprotein particles showed that LDL particles of affected subjects were enriched with triglycerides (8.5% versus 4.4% mass, P<0.05) and diminished in cholesteryl esters (34% versus 40% mass, P<0.05) The percent compositions of the other LDL constituents were not different between the affected and control subjects. The compositions of the HDL particles did not differ significantly between the 2 groups.

Kinetic Parameters
Concentrations of VLDL and LDL apoB and of VLDL triglycerides were determined at 4 time points from each kinetic study. The coefficients of variation for all parameters were <10% between different samples, indicating that each subject remained in a steady state throughout the study. IDL apoB concentrations were too low in the affected subjects to allow reliable measurements by apoB immunoassays; thus, IDL apoB pool sizes and production rates are not reported. VLDL apoB, IDL apoB, LDL apoB, and HDL apoA-I leucine tracer/tracee ratio curves from an affected and a control subject are shown in Figure 4. As the figure shows, there was excellent agreement between the observed tracer data and the model-derived fits. The FCR of VLDL apoB was similar in affected and control subjects (Table 2), whereas the FCRs of IDL and LDL were significantly higher in affected subjects than in the control group. Compared with normal control subjects, hypobetalipoproteinemia subjects had lower production rates of VLDL apoB (11.4±4.3 versus 25.6±6.3 mg · kg^-1 · d^-1, 44% of controls, P=0.003) and LDL apoB (7.8±1.3 versus 12.7±3.7 mg · kg^-1 · d^-1, P=0.04).

View larger version (25K): [in this window] [in a new window]   Figure 4. Tracer/tracee ratios (TTRs) of leucine in VLDL apoB (A), IDL apoB (B), LDL apoB (C), and HDL apoA-I (D) in subject 2 of the affected group (•) and a control subject (). Symbols represent observed values; solid and dashed lines represent model-derived fits.

View this table: [in this window] [in a new window]   Table 2. Kinetic Parameters of ApoB and VLDL TGs

For the VLDL triglyceride glycerol tracer/tracee ratio curves, there was also excellent agreement between the observed tracer data and model-derived fits (not shown). The mean FCR of VLDL triglycerides was increased in affected compared with normal subjects, although it did not reach statistical significance (2.67±1.76 versus 1.22±0.47 pools per hour, P=0.11; Table 2). The mean production rate of VLDL triglycerides in affected subjects was 42% of that in normal control subjects (8.2±4.5 versus 19.6±10.8 µmol · kg^-1 · h^-1, P=0.09; Table 2). The similar decreases in VLDL apoB and VLDL triglyceride production rates suggest that VLDL particles of unchanged composition were being secreted. This was verified by analysis: VLDL TG/VLDL apoB mass ratios of affected and control subjects were 9.7±7.5 versus 15.1±8.7 (mean±SD, P=0.36).

In hypobetalipoproteinemia subjects with low HDL, the FCR of apoA-I was increased significantly (0.035±0.005 pools per hour) compared with control subjects (0.018±0.005 pool per hour) and with hypobetalipoproteinemia subjects with normal HDL (0.015±0.001 pools per hour, Table 3). The mean production rates of apoA-I were not significantly different in the 3 groups. Thus, the levels of apoA-I and HDL cholesterol in low HDL hypobetalipoproteinemia subjects are solely attributable to higher apoA-I FCRs.

View this table: [in this window] [in a new window]   Table 3. Kinetic Parameters of ApoA-I

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
FHBL can result from 1 of several mutations in the apoB gene on chromosome 2. This form of FHBL has been studied extensively, and the molecular and metabolic mechanisms responsible for low plasma apoB levels have been defined.^{7 8 9 10 11 12 27} We studied a kindred with FHBL in which linkage to the apoB gene was excluded¹⁴ to assess the metabolic mechanisms responsible for low levels of apoB and cholesterol in presumed heterozygotes. Previously, Fazio et al¹³ described a family with FHBL without apoB truncation in which linkage to the apoB gene was excluded, but no studies to elucidate the mechanism of the dyslipidemia were reported. Therefore, this is the first study to evaluate the metabolic basis of this form of FHBL. Our results show that low production rates of apoB-containing lipoproteins and increased catabolism of LDL account for the low apoB levels in plasma. High FCRs of apoA-I account for the low levels of apoA-I, where present.

ApoB-100 is secreted from the liver and associated with lipids in VLDL particles. Although some investigators have suggested direct production of apoB in LDL particles, most kinetic studies indicate that this pathway contributes very little to apoB production.²⁸ It was not necessary in our subjects to invoke the direct secretion of LDL to fit the present data to the kinetic model. Hence, apoB-100 in our subjects was produced exclusively in VLDL, and all LDL particles were formed by the metabolism of VLDL via the delipidation pathway. The VLDL apoB-100 production rates of the normal subjects in the present study were similar to those reported for other normal subjects.¹² The production rates of VLDL apoB-100 and VLDL triglycerides in affected subjects of the F kindred were decreased to 44% and 42% of normal, respectively. Although similar in direction, the quantitative findings in these affected individuals differ from those seen in FHBL heterozygotes with the apoB-9 and apoB-2 truncations.²⁵ In the latter subjects, VLDL apoB-100 secretion is more significantly reduced to 25% of normal. VLDL apoB-100 production rates are also reduced to 25% to 30% of control rates in heterozygotes with other apoB truncations.^{12 29} VLDL triglyceride production rates are comparably decreased to 40% of normal in both forms of FHBL.²⁵ Thus, different factors regulating VLDL apoB and VLDL triglyceride production may be present in the 2 forms of FHBL. Although it is impossible to completely rule out that rapid hepatic reuptakes may masquerade as low production rates, our protocol, which uses endogenous labeling and frequent early sampling of apoB tracer/tracee ratios, makes this less likely than if we had used exogenous labels.¹⁸

Several metabolic steps in the production of apoB-containing lipoproteins may differ between the various forms of FHBL. In cultured hepatocytes, apoB secretion is regulated by the availability of triglycerides^{30 31} and/or cholesteryl esters,³² the association of lipids with apoB in the endoplasmic reticulum,³³ the translocation of newly synthesized apoB through the endoplasmic reticulum membrane,³⁴ and its secretion via the Golgi apparatus. Genetic factors affecting any of these processes could decrease hepatic VLDL apoB production significantly. Although only defects in the apoB, apoE, and MTP genes have been defined thus far as causes of genetically determined low apoB syndromes, the results of our metabolic studies suggest that in the F kindred, other genetically controlled loci affecting apoB secretion may be affected. The strong linkage of the hypobetalipoproteinemia trait to a new candidate locus on chromosome 3p21.1-2¹⁵ strongly supports this possibility.

The catabolism of apoB-containing lipoproteins was increased in hypobetalipoproteinemia subjects of the F kindred, as reflected by higher FCRs of LDL apoB. The enhanced metabolism of these lipoproteins further contributes to lower apoB and LDL cholesterol levels in affected subjects. The mechanism(s) underlying the higher FCR of apoB-containing lipoproteins is not fully understood. High FCRs may be due to upregulation of the LDL receptor secondary to severe hypocholesterolemia. It is well established in vitro that cholesterol deprivation causes upregulation of LDL receptors in cultured cells.³⁵ In vivo kinetic studies support this finding; eg, Parhofer et al³⁶ demonstrated a 2-fold increase in LDL apoB FCRs in patients with familial hypercholesterolemia treated by LDL apheresis to lower cholesterol levels. However, in vivo studies in FHBL subjects failed to demonstrate that low plasma levels of apoB-containing lipoproteins induced increases in the FCR of LDL apoB-100.¹² The possibility that the rapid clearance of LDL particles from plasma in our affected subjects is due to an enhanced affinity of interaction of apoB-containing lipoproteins with the LDL receptor cannot be excluded. However, this is unlikely because enhanced apoB-LDL receptor interactions are more typical of cases of FHBL associated with truncated apoB forms that interact with the LDL receptor with greater affinity, eg, apoB-89, apoB-87, and apoB-75.^{8 9}

An interesting finding in the F kindred was the presence of very low levels of HDL cholesterol and apoA-I in some, but not all, affected subjects. Some affected hypobetalipoproteinemia subjects in the family described by Fazio et al¹³ have similarly low HDL cholesterol and apoA-I levels. By contrast, in typical FHBL caused by apoB gene mutations, levels of HDL and apoA-I are normal in affected subjects.^{12 25} We showed that the FCR of apoA-I was nearly trebled in those affected subjects with low HDL, whereas apoA-I production was unchanged, suggesting that hypercatabolism of apoA-I was solely responsible for the hypoalphalipoproteinemia. Kinetic studies in other low HDL conditions are compatible with ours inasmuch as they demonstrated a strong inverse correlation between HDL cholesterol levels and FCR of apoA-I.^{37 38} Catabolism of HDL apoA-I is believed to be affected by the HDL particle size and its lipid content. Factors that decrease HDL particle size increase apoA-I FCR, thus decreasing HDL cholesterol levels.³⁹ Enrichment of the HDL by triglycerides and depletion of the cholesterol ester content, usually a consequence of hypertriglyceridemia, causes an increase in apoA-I FCR and higher catabolism of the HDL particles.⁴⁰ In our affected subjects, there was no change in the composition of the HDL particles, although on nondenaturing gradient gel electrophoresis and FPLC, HDL particle sizes appeared to be smaller in affected subjects. Whether the metabolic derangements of apoB-containing and apoA-I–containing lipoproteins are related awaits further study.

In conclusion, we have demonstrated that in an FHBL kindred in which the hypobetalipoproteinemia trait is not linked to the apoB gene, decreased secretion of apoB-containing lipoproteins is a primary abnormality, perhaps because of defects in genes regulating intracellular degradation or apoB secretion. Further studies that aim to define the genetic defects in this form of FHBL may provide new insight into the genetic factors controlling the production of apoB-containing lipoproteins.

	Acknowledgments

 
This study was supported by National Institute of Health grants R37-HL-42460 and R01-HL-59515, GCRC grant USPHS MO1-RR-00036, Clinical Nutrition Research Unit P30-DK-06341, and Biomedical Mass Spectrometry Resource grant RR-00954. We wish to thank Tom Kitchens for his excellent technical help and Sherry Banez-Muth for recruiting the study subjects. We also thank the personnel of the Core Laboratory for Clinical Studies at Washington University School of Medicine for their assistance in performing lipid profile measurements and the nurses of the GCRC for their help in performing the metabolic studies.

Received November 29, 1999; accepted January 28, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Kane JP, Havel RJ, Disorders of the biogenesis and secretion of lipoproteins containing the B apolipoproteins. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The Metabolic Bases of Inherited Disease. 7th ed. New York, NY: McGraw-Hill Book Co; 1995:1853–1885.
2. Sniderman A, Shapiro S, Marpola D, Skinner B, Tang B, Kwiterovich PO. Association of coronary atherosclerosis with hyperapobetalipoproteinemia. Proc Natl Acad Sci U S A. 1980;77:604–608.[Abstract/Free Full Text]
3. Wetterau JR, Aggerbeck LP, Bouma ME, Eisenberg C, Munck A, Hermier A, Schmitz M, Gay J, Rader DJ, Gregg RE. Absence of microsomal triglyceride transfer protein in individuals with abetalipoproteinemia. Science. 1992;258:999–1001.[Abstract/Free Full Text]
4. Roy CC, Levy E, Green PHR, Sniderman A, Letarte J, Buts JP, Orguin J, Brochu P, Weber AM, Morin CL, Marcel Y, Deckelbaum RJ. Malabsorption, hypocholesterolemia, fat-filled enterocytes with increased intestinal apoprotein B: chylomicron retention disease. Gastroenterology. 1987;92:390–399.[Medline] [Order article via Infotrieve]
5. Wu J, Kim J, Li Q, Kwok P, Cole T, Cefalu B, Averna M, Schonfeld G. Known mutations of apoB account for only a small minority of hypobetalipoproteinemia. J Lipid Res. 1999;40:955–959.[Abstract/Free Full Text]
6. Welty FK, Lahoz C, Tucker KL, Ordovas JM, Wilson PW, Schaefer EJ. Frequency of apoB and apoE gene mutations as causes of hypobetalipoproteinemia in the Framingham offspring population. Arterioscler Thromb Vasc Biol. 1998;18:1745–1751.[Abstract/Free Full Text]
7. Schonfeld G. The hypobetalipoproteinemias. Annu Rev Nutr. 1995;15:23–34.[Medline] [Order article via Infotrieve]
8. Linton MF, Farese RV, Young SG. Familial hypobetalipoproteinemia. J Lipid Res. 1993;34:521–541.[Medline] [Order article via Infotrieve]
9. Parhofer KG, Barrett PHR, Bier DM, Schonfeld G. Positive linear correlation between the length of truncated apolipoprotein B and its secretion rate (in vivo studies in apoB-89, apoB-75, apoB-54.8, and apoB 31 heterozygotes). J Lipid Res. 1996;37:844–852.[Abstract]
10. 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. 1992;89:1931–1937.
11. Krul ES, Parhofer KG, Barrett PHR, Wagner RD, Schonfeld G. ApoB-75, a truncation of apolipoprotein B associated with familial hypobetalipoproteinemia: genetic and kinetic studies. J Lipid Res. 1992;33:1037–1050.[Abstract]
12. Aguilar-Salinas CA, Barrett PHR, Parhofer KG, Young SG, Tessareau D, Bateman J, Quinn C, Schonfeld G. Apolipoprotein B-100 production is decreased in subjects heterozygous for truncations of apoprotein B. Arterioscler Thromb. 1995;15:71–80.[Abstract/Free Full Text]
13. Fazio S, Sidoli A, Vivenzio A, Maletta A, Giampaoli S, Menotti A, Antonini R, Urbinati G, Baralle FE, Ricci G. A form of familial hypobetalipoproteinemia not due to a mutation in the apolipoprotein-B gene. J Intern Med. 1991;229:41–47.[Medline] [Order article via Infotrieve]
14. Pulai JI, Neuman RJ, Groenewegen AW, Wu J, Schonfeld G. Genetic heterogeneity in familial hypobetalipoproteinemia: linkage and non-linkage to the apoB gene in Caucasian families. Am J Med Genet.. 1998;76:79–86.[Medline] [Order article via Infotrieve]
15. Yuan B, Neuman R, Duan SH, Weber JL, Kwok PY, Saccone NL, WU JS, Liu KY, Schonfeld G. Linkage of a gene for familial hypobetalipoproteinemia to chromosome 3p21.1–22. Am J Hum Genet. In press.
16. Cole TG, Kitchens RT, Daugherty A, Schonfeld G. An improved method of separation of triglyceride-rich lipoproteins by FPLC. Pharm Biocommun. 1988;4:4–6.
17. Nichols AV, Krauss RM, Musliner TA. Nondenaturing polyacrylamide gradient gel electrophoresis. Methods Enzymol. 1983;128:417–431.
18. Parhofer KG, Barrett PH, Bier DM, Schonfeld G. Determination of kinetic parameters of apolipoprotein-B metabolism using amino-acids labeled with stable isotopes. J Lipid Res. 1991;32:1311–1323.[Abstract]
19. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265–275.[Free Full Text]
20. Adams RF. Determination of amino acid profiles in biological samples by gas chromatography. J Chromatogr. 1974;95:189–212.[Medline] [Order article via Infotrieve]
21. Klein RL, Zilversmit DB. Direct determination of human and rabbit apolipoprotein B selectively precipitated with butanol-isopropyl ether. J Lipid Res. 1984;25:1380–1386.[Abstract]
22. Patterson BW, Hachey DL, Cook GL, Amann JM, Klein PD. Incorporation of a stable isotopically labeled amino acid into multiple human apolipoproteins. J Lipid Res. 1991;32:1063–1072.[Abstract]
23. Patterson BW, Zhao G, Elias N., Hachey DL, Klein S. Validation of a new procedure to determine plasma fatty acid concentration and isotopic enrichment. J Lipid Res. 1999;40:2118–2124.[Abstract/Free Full Text]
24. Horowitz JF, Coppack SW, Paramore D, Cryer PE, Zhao G, Klein S. Effect of short-term fasting on lipid kinetics in lean and obese women. Am J Physiol. 1999;276:E278–E284.[Abstract/Free Full Text]
25. Elias N, Patterson BW, Schonfeld G. Decreased production rates of VLDL triglycerides and apolipoprotein B 100 in subjects heterozygotes for familial hypobetalipoproteinemia. Arterioscler Thromb Vasc Biol. 1999;19:2714–2721.[Abstract/Free Full Text]
26. Zech LA, Grundy SM, Steinberg D, Berman M. Kinetic model for production and metabolism of very low density lipoprotein triglycerides: evidence for a slow production pathway and results for normolipidemic subjects. J Clin Invest. 1979;63:1262–1273.
27. Young SG, Krul ES, McCormick S, Farese RV Jr, Linton MF. Identification and characterization of truncated forms of apolipoprotein B in hypobetalipoproteinemia. Methods Enzymol. 1996;263:120–145.[Medline] [Order article via Infotrieve]
28. Janus ED, Nicoll AM, Turner PR, Magill P, Lewis B. Kinetic basis of the primary hyperlipidemias: studies of apolipoprotein B turnover in genetically defined subjects. Eur J Clin Invest. 1980;10:161–172.[Medline] [Order article via Infotrieve]
29. Welty FK, Lichtenstein AH, Barrett PHR, Dolnikowski GG, Ordovas JM, Schaefer EJ. Decreased production and increased catabolism of apolipoprotein B-100 in apolipoprotein B-67/B-100 heterozygotes. Arterioscler Thromb Vasc Biol. 1997;17:881–888.[Abstract/Free Full Text]
30. Ginsberg HN. Synthesis and secretion of apolipoprotein B from cultured liver cells. Curr Opin Lipidol. 1995;6:275–280.[Medline] [Order article via Infotrieve]
31. Benoist F, Grand-Perret T. ApoB-100 secretion by HepG2 cells is regulated by the rate of triglyceride biosynthesis but not by intracellular lipid pools. Arterioscler Thromb Vasc Biol. 1996;16:1229–1235.[Abstract/Free Full Text]
32. Cianflone KM, Yasruel Z, Rodriguez MA, Vas D, Sniderman AD. Regulation of apoB secretion from HepG2 cells: evidence for a critical role for cholesteryl ester synthesis in the response to a fatty acid challenge. J Lipid Res. 1990;31:2045–2055.[Abstract]
33. Jamil H, Gordon DA, Eustice DC, Brooks CM, Dickson JK Jr, Chen Y, Ricci B, Chu CH, Harrity TW, Closek CP Jr, Biller SA, Gregg RE, Wetterau JR. An inhibitor of microsomal triglyceride transfer protein inhibits apoB secretion from HepG2 cells. Proc Natl Acad Sci U S A. 1996;93:11991–11995.[Abstract/Free Full Text]
34. Sakata N, Wu X, Dixon JL, Ginsberg HN. Proteolysis and lipid facilitated translocation are distinct but competitive processes which regulate secretion of apolipoprotein B in HepG2 cells. J Biol Chem. 1993;268:22967–22970.[Abstract/Free Full Text]
35. Brown MS, Goldstein JL. Receptor-mediated control of cholesterol metabolism. Science. 1976;191:151–1554.
36. Parhofer KG, Barrett PHR, Demant T, Richter WO, Schwandt P. Effect of apheresis on kinetic parameters of apoprotein B metabolism in familial hypercholesterolemia. Circulation. 1996;94:1–583.[Free Full Text]
37. Brinton EA, Eisenberg S, Breslow JL. Increased apoA-I and apoA-II fractional catabolic rates in patients with low high density lipoprotein-cholesterol levels with or without hypertriglyceridemia. J Clin Invest. 1991;87:536–544.
38. Emmerich J, Verges B, Tauveron I, Rader D, Santamarina-Fojo S, Schaefer J, Ayraultl-Jarrier M, Thieblot P, Brewer HB Jr. Familial HDL deficiency due to marked hypercatabolism of normal apoA-I. Arterioscler Thromb. 1993;13:1299–1306.[Abstract/Free Full Text]
39. Brinton EA, Eisenberg S, Breslow JL. Human HDL cholesterol levels are determined by apoA-I fractional catabolic rate, which correlates inversely with estimates of HDL particle size: effects of gender, hepatic and lipoprotein lipases, triglyceride and insulin levels, and body fat distribution. Arterioscler Thromb Vasc Biol. 1994;14:707–720.[Abstract/Free Full Text]
40. Lamarche B, Uffelman KD, Carpentier A, Cohn JS, Steiner G, Barrett PH, Lewis GF. Triglyceride enrichment of HDL enhances in vivo metabolic clearance of HDL apoA-I in healthy men. J Clin Invest. 1999;103:1191–1199.[Medline] [Order article via Infotrieve]

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