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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:348-353.)
© 1997 American Heart Association, Inc.

Articles

Homozygous Familial Defective Apolipoprotein B-100

Enhanced Removal of Apolipoprotein E–Containing VLDLs and Decreased Production of LDLs

Juergen R. Schaefer; Hubert Scharnagl; Manfred W. Baumstark; Horst Schweer; Loren A. Zech¹; Hansjorg Seyberth; Karl Winkler; Armin Steinmetz; Winfried Marz

the Division of Cardiology, Department of Medicine, Philipps-University Marburg, (J.R.S., H. Schweer, H. Seyberth, A.S.), the Division of Clinical Chemistry (H. Scharnagl, K.W., W.M.), and the Division of Sports Medicine (M.W.B.), Department of Medicine, Albert Ludwigs-University, Freiburg, Germany, and the Molecular Disease Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md (L.A.Z.).

	Abstract

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Familial defective apolipoprotein B-100 (FDB) is a frequently inherited disorder of lipoprotein metabolism. The glutamine-for-arginine substitution at position 3500 of apolipoprotein (apo) B-100 leads to defective binding of apo B-100 to the low density lipoprotein (LDL) receptor and accumulation of LDL in the plasma. We recently identified a patient homozygous for this mutation. His LDL cholesterol and apo B concentrations were approximately twice normal, whereas his apo E plasma level was low. Using a stable-isotope labeling technique ([²H₃]leucine–primed constant infusion), we studied lipoprotein turnover in vivo in the fasting state in this patient and three clinically healthy, normolipidemic individuals not carrying the FDB mutation. The residence time of LDL apo B-100 was prolonged 3.6-fold in the FDB homozygote (8.3 vs 2.3 days). The production rate of LDL apo B-100 was decreased (7.4 vs 15 mg per kg per day). In FDB the residence time of very low density lipoprotein (VLDL) apo B-100 was longer (2.6 vs 1.3 hours), whereas the residence time of VLDL apo E was shorter (2.6 vs 4.5 hours) than normal. These data show that the in vivo metabolism of apo B-100–containing lipoproteins in FDB is different from that in familial hypercholesterolemia, in which LDL receptors are defective. In both conditions the residence times of LDL apo B-100 appear to be increased to approximately the same degree. This contrasts with the LDL apo B-100 synthetic rate, which is increased in familial hypercholesterolemia and decreased in FDB. The decreased production of LDL apo B-100 in FDB may originate from enhanced removal of apo E–containing LDL precursors by LDL receptors, which may be upregulated in response to the decreased flux of LDL-derived cholesterol into hepatocytes.

Key Words: hypercholesterolemia • apolipoprotein B-100 • atherosclerosis • genetic disease • stable-isotope tracer kinetics

	Introduction

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Apo B-100 is a major constituent of VLDL, IDL, and LDL. The interaction of apo B-100 with LDL receptors is responsible for the transfer of LDL-C from the blood to the liver and most other cells in the body.^{1 2 3 4} FDB was discovered by the observation that radiolabeled LDLs from patients with primary hyperlipoproteinemia were cleared at a slower rate than in normal individuals.^{5 6} Soria et al⁷ identified the underlying molecular defect as a point mutation resulting in the replacement of Arg by Gln at position 3500 of the apo B-100 amino acid sequence. The frequency of heterozygous FDB is .01 to .03 in hypercholesterolemic subjects, and thus, FDB is probably one of the most common monogenetic causes of hypercholesterolemia.^{8 9 10 11 12}

Recently homozygous FDB patients were identified.^{13 14 15 16} Hypercholesterolemia was less severe in these subjects than in patients homozygous for FH, in which the LDL receptor is defective. We studied the receptor-mediated endocytosis of LDL from a homozygous FDB patient in normal cultured human skin fibroblasts.¹⁴ Binding, internalization, and degradation of FDB-LDL (d=1.019 to 1.063 kg/L) was diminished but not completely abolished. We noticed that this phenomenon was due to the presence of multiple LDL subfractions, all of which differed markedly with regard to their receptor binding. The small, dense LDL subfractions (d>1.040 kg/L) of our homozygous patient were completely defective in binding. In contrast, cellular uptake of buoyant LDL (d=1.019 to 1.034 kg/L) was virtually normal owing to the presence apo E on the surface of these particles. Consistently only the binding-defective, small, dense LDL but not the buoyant LDL accumulated in the plasma of this patient. Total apo E plasma concentration was decreased. We hypothesized from these observations that apo E may partially compensate for the defective binding of apo B-100 in FDB, thus assuming a crucial role in the metabolism of apo B-100–containing lipoproteins. To evaluate this concept further, we studied the in vivo metabolism of apo B-100 and apo E in this homozygous FDB patient and three normolipidemic individuals by using endogenous labeling with stable isotopes, an approach that has become well established for investigating the in vivo kinetics of apolipoproteins.¹⁷

	Methods

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Study Subjects
We studied the homozygous FDB patient (F.B.) described previously.^{13 14} F.B. is a 54-year-old white male without serious symptoms of disease. He had multiple xanthelasmas and a discrete arcus corneae. F.B. had no history of coronary artery disease, and his exercise electrocardiogram was normal. His apo E phenotype was E3/3. Routine blood biochemistry measurements were all normal except for a slight increase in -glutamine transferase activity (36 U/L; upper limit of the reference range, 27 U/L).

At the time of the study, F.B. was consuming a normal diet and not taking any lipid-lowering drugs. As control subjects, three healthy, normolipidemic, male individuals were included in the study. They were also consuming a normal diet and did not smoke or take medications. Routine blood biochemistry and hematology findings were normal. They all had a normal body mass index and normal fasting plasma lipid and lipoprotein levels. They were all of apo E phenotype E3/3 and were negative for the ArgGln substitution at position 3500 of apoB-100, as shown by a modification of the method of Ruzicka et al¹⁸ of the Msp I restriction-typing method described by Hansen et al.¹⁹

Study Protocol
The in vivo turnover study followed a recently described protocol,²⁰ which had been approved by the Ethics Review Committee of the University of Marburg. Informed, written consent was obtained from each subject; all procedures were in accordance with the Helsinki Declaration of 1975 and revised in 1983.

Three days before the study, all study subjects received a standardized, isocaloric diet. The stable-isotope turnover study was performed under fasting conditions in all subjects and was started at 8 AM after an overnight fast. Two indwelling plastic catheters were placed intravenously on contralateral arm veins; one was used for tracer infusion and the other for blood sampling. Deuterium-labeled (three times) L-Leu (L-Leu methyl-D3 99%; MSD Isotopes) was administered as a priming bolus of 1.34 mg/kg immediately followed by a constant infusion of 0.022 mg/kg per minute for 12 hours. Blood samples (10 to 15 mL) were drawn into tubes containing EDTA at a final concentration of 1 g/L before the tracer injection; after 10, 20, 30, 40, 60, 90, and 120 minutes; and then hourly for 12 hours. The blood was kept on ice and the plasma immediately separated by centrifugation (3000g, 20 minutes at 4°C). Sodium azide and aprotinin were added to the plasma at final concentrations of 0.5 g/L and 200 000 KIU/L, respectively.

Isolation of Lipoproteins and Apolipoproteins
VLDL (d<1.006 kg/L) and LDL (d=1.019 to 1.063 kg/L) were isolated by sequential ultracentrifugation starting from 5 mL plasma (rotor 50.3 Ti and centrifuge L8-55M, Beckman Instruments). Isolated lipoproteins were not recentrifuged, dialyzed against 10 mmol/L NH₄HCO₃, lyophilized, and delipidated. VLDL apo B-100, VLDL apo E, and LDL apo B-100 were isolated by preparative gradient (5% to 15%) SDS–polyacrylamide gel electrophoresis.²¹

Determination of Isotopic Enrichment
Samples were prepared for GC-MS as reported previously.²⁰ Apolipoprotein bands were excised from the gels and hydrolyzed in 6 mol/L HCl (Ultrapure grade, Merck) at 110°C for 24 hours under N₂. The protein hydrolysates were lyophilized in a Speed Vac evaporator (Savant Instruments). Free amino acids were purified from plasma or protein hydrolysates by cation exchange chromatography (AG-50W-X8, Bio-Rad Laboratories), then derivatized to N-heptafluorobutyrylisobutyl esters, and analyzed by GC-MS-MS on a tandem mass spectrometer (Finnigan MAT) in the chemical ionization and selected-ion monitoring modes. The ions monitored were 363 m/z for unlabeled and 366 m/z for the three times–labeled Leu. Enrichment was calculated from the isotope ratio by the method of Cobelli et al.²² Enrichment was converted to the tracer-to-tracee ratio at a given time point t according to the equation A_t=e_t/(e_i-e_t), where e_t is the enrichment of each sample at time t, and e_i is the enrichment of the infusate, which was 0.99.

Analysis of Kinetic Data
A monoexponential function was fitted to the tracer-to-tracee ratio curves of VLDL apo B-100, VLDL apo E, and LDL apo B-100 using SAAM 30 as previously described.²³ In brief the function was defined as A_t=A_p(1-e^-k(t-d)). A_t is the tracer-to-tracee ratio at time t and A_p the tracer-to-tracee ratio of the precursor pool of the apolipoprotein of interest, as determined by the respective plateau enrichment; d is the delay time and k the FSR. At steady state, the FSR reflects the FCR. The precursor enrichment for apo B-100 was assumed to be reflected by the plateau enrichment of VLDL apo B-100, as described earlier.^{24 25} The plateau enrichment of VLDL apo E was used as its own precursor. Absolute PRs were calculated as the product of the FSR and the pool size (mean concentration of samples collected during the study multiplied by 4.0% of body weight). Student's t test was used to determine whether the kinetic parameters obtained in the FDB homozgygote differed from those derived for the normolipidemic individuals.

Analytical Methods
C and triglycerides were measured by enzymatic techniques (Boehringer Mannheim). HDL-C was determined after precipitation of apo B–containing lipoproteins with phosphotungstic acid and MgCl₂ (Boehringer Mannheim). Apo A-I and apo B levels were measured by immunonephelometry (Behringwerke).²⁶ Apo E content was determined with a microplate enzyme immunoassay,¹⁴ which uses polyclonal and monoclonal antibodies as capturing and detecting antibodies, respectively, specific for apo E.

	Results

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The lipoprotein profiles and ages of the study subjects are summarized in Table 1. Total-C, LDL-C and apo B levels were elevated in the FDB patient compared with normal control subjects, whereas HDL-C and apo A-I levels were normal. Plasma lipid and apolipoprotein concentrations were determined in each plasma sample obtained during the course of the infusion studies and remained stable throughout.

View this table: [in this window] [in a new window]   Table 1. Clinical Data, Lipids, Lipoproteins, and Apolipoproteins in an FDB Homozygote (F.B.) and Three Healthy Men Studied by the Endogenous-Labeling Stable-Isotope Technique

Within 30 minutes of infusion, the tracer-to-tracee ratios of plasma free [²H₃]Leu reached steady state in all study subjects, and this was sustained throughout the entire infusion period. The tracer-to-tracee ratio of VLDL apo B-100 and VLDL apo E increased rapidly and reached plateaulike enrichment during the infusion period in all subjects. In the FDB patient, VLDL apo B-100 reached plateaulike enrichment slightly later than it did in normolipidemic individuals, suggesting that fractional catabolism of VLDL apo B-100 was delayed. In contrast, the tracer-to-tracee curve of VLDL apo E indicated accelerated metabolism in the patient compared with control subjects.

In all of the healthy control subjects, the tracer-to-tracee curve of VLDL apo B-100 was steeper than that of VLDL apo E, demonstrating that the fractional catabolism of VLDL apo B-100 was faster than that of VLDL apo E in normal subjects (Fig 1). In FDB, however, the tracer-to-tracee curve of VLDL apo B-100 was almost identical to that of VLDL apo E, suggesting very similar fractional catabolism of the two proteins (Fig 2).

View larger version (13K): [in this window] [in a new window]   Figure 1. In vivo metabolism of VLDL apo B-100 and VLDL apo E in normal individuals. Deuterium-labeled (three times) L-Leu was administered as a priming bolus (1.34 mg/kg) followed by constant infusion of 0.022 mg/kg per minute for 12 hours. Blood samples were obtained at time points indicated on the x axis, and the tracer-to-tracee ratio was measured as described in "Methods." indicate VLDL apo B-100; , VLDL apo E. Data are mean values of three study subjects. To facilitate direct comparison of the two VLDL proteins in the study subjects, tracer-to-tracee curves were normalized to a tracer-to-tracee plateau enrichment of 0.10.

View larger version (13K): [in this window] [in a new window]   Figure 2. In vivo metabolism of VLDL apo B-100 and VLDL apo E in a patient homozygous for FDB. Deuterium-labeled (three times) L-Leu was administered as a priming bolus followed by constant infusion for 12 hours. Blood samples were obtained at the time points indicated on the x axis. The tracer-to-tracee ratio was measured as described in "Methods." indicate VLDL apo B-100; , VLDL apo E. Tracer-to-tracee curves were normalized to a plateau enrichment of 0.10.

Owing to the slow turnover of LDL apo B-100, the tracer-to-tracee curve of LDL apo B-100 did not reach plateaulike enrichment in any of the study subjects. However, in the FDB patient the tracer-to-tracee ratio of LDL apo B-100 increased at a clearly slower rate than that in the normal subjects.

Using monoexponential regression, we calculated the kinetic parameters of VLDL apo E, VLDL apo B-100, and LDL apo B-100 in the FDB patient and the normal individuals. These results are shown in Table 2. The PR of VLDL apo B-100 was slightly lower in the FDB patient than in the normal individuals, but this difference did not reach statistical significance. The FCR of VLDL apo B-100 was decreased by twofold in FDB. As expected, the VLDL apo E PR in the FDB patient was similar to that in normal subjects. VLDL apo E fractional catabolism was two times faster in FDB than in normal individuals. In the FDB patient, the PR of LDL apo B-100 was approximately half the rate of healthy subjects. Finally, the FCR of LDL apo B-100 was decreased to approximately one fourth of the normal rate in the FDB patient (Fig 3).

View this table: [in this window] [in a new window]   Table 2. In Vivo Kinetics of VLDL and LDL Apolipoproteins in a Homozygous Patient With FDB and Three Healthy Normolipidemic Individuals

View larger version (14K): [in this window] [in a new window]   Figure 3. In vivo metabolism of LDL apo B-100 in a patient homozygous for FDB () compared with a normal control subject (). Deuterium-labeled (three times) L-Leu was administered as a priming bolus followed by constant infusion for 12 hours. Blood samples were obtained at the time points indicated on the x-axis, and the tracer-to-tracee ratio was measured as described in "Methods."

	Discussion

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We report the first study of the in vivo metabolism of apo B-100–containing lipoproteins in a patient homozygous for FDB. In this disorder, LDL clearance is reduced because the LDLs are defective in binding to LDL receptors.^{5 7} Recently, other patients homozygous for FDB have been described.¹³ Interestingly, total-C and LDL-C levels in those patients and our FDB homozygotes^{15 16} were significantly lower than those in subjects with homozygous FH, in which LDL clearance is disrupted owing to defective LDL receptors.²⁷ In an attempt to explain the unexpectedly low concentration of LDL in homozygous FDB, we examined the binding of FDB-LDL to fibroblasts in culture.¹⁴ From these in vitro studies, we concluded that the ArgGln substitution in FDB mainly affects receptor recognition of those LDL particles that completely lack apo E. In contrast, interaction of lipoproteins containing both apo E and defective apo B-100 with LDL receptors appeared normal, suggesting that hepatic uptake in vivo of lipoprotein precursors of LDL containing apo E as a ligand for cell surface receptors was not significantly altered. Beyond this, we hypothesized that the decreased apo B-100–mediated delivery of C to liver cells in FDB might stimulate expression of LDL receptors. We predicted that this would result in enhanced clearance of LDL precursors via apo E and a decrease in the fraction of VLDL apo B-100 converted to LDL. To examine whether these mechanisms were in fact operating in vivo, we studied the kinetics of VLDL and LDL apolipoproteins by using endogenous labeling with stable isotopes.

As expected, the FCR of LDL apo B-100 in the FDB homozygote was reduced to approximately one fourth of the normal rate. This concurs well with the relative decrease in the FCR of LDL apo B-100 in homozygous FH. Although estimates of the FCR of LDL apo B-100 vary considerably among laboratories, most reports agree that the FCR in homozygous FH is reduced to one fourth to one third that of normal subjects.^{28 29 30} Studies using simultaneous intravenous administration of ¹²⁵I-labeled LDL and cyclohexanedione-modified ¹³¹I-labeled LDL have demonstrated that LDLs are catabolized exclusively by LDL receptor–independent routes in homozygous FH and that no catabolism occurs via the LDL receptor route.^{29 31} Because the changes in the catabolism of LDL apo B-100 in homozygous FH and homozygous FDB are roughly equivalent (an approximately fourfold decrease in the FCR in both conditions), the LDL receptor pathway appears to make little if any contribution to the catabolism of LDL in homozygous FDB.

The LDL receptor mediates the catabolism not only of LDL itself but also of its precursor particles. As shown in hyperlipidemic humans and Watanabe heritable rabbits, an inbred strain of rabbit with defective LDL receptors, the accumulation of LDL in FH results from a combination of decreased catabolism and increased production of LDL due to impaired hepatic removal of LDL precursors.^{32 33 34 35 36} As LDL precursors are taken up into cells by virtue of their apo E moiety,³⁷ increased production of LDL would not be expected in FDB, in which LDL receptor function is normal. Our in vivo kinetic data are consistent with this concept. The formation of LDL in FDB is even markedly reduced compared with that in normolipidemic individuals. As discussed above, the reduced production of LDL in FDB may be explained by upregulation of hepatic LDL receptors in response to disruption of the apo B-100–mediated flux of C into hepatocytes. This concept is supported by the low apo E concentration in our patient and the increased catabolism of VLDL apo E. The metabolism of apo B–containing lipoproteins in FDB is thus in a sense the reverse of that in individuals with dysbetalipoproteinemia and type III hyperlipidemia. In the latter, apo E is defective in binding to lipoprotein receptors, resulting in C starvation of hepatocytes, upregulation of LDL receptors, and enhanced clearance of LDL via apo B-100.³⁸

In normal individuals, the FCR of VLDL apo B-100 is more than three times higher than that of VLDL apo E, presumably because of an exchangeable apo E pool on HDL particles. In FDB, the FCRs of the two VLDL apolipoproteins were identical, due to both a decrease in VLDL apo B-100 clearance and an increase in VLDL apo E clearance. Because 1 VLDL particle contains 1 molecule of apo B-100, the delayed FCR of VLDL apo B-100 in FDB is at first glance difficult to reconcile with the concept of accelerated apo E–mediated catabolism of VLDL. However, the reduction in VLDL apo B-100 metabolism (half the normal FCR) occurred to a lesser extent than in LDL (one fourth the normal FCR), indicating that VLDL apo B-100 catabolism must be driven by a force other than apo B-100 (ie, apo E). Furthermore, there is ample evidence that VLDL is physicochemically and metabolically inhomogeneous. Packard et al³⁹ suggested that the liver secretes VLDL particles of different size, the larger ones giving rise to remnants and being removed from the circulation and the smaller ones serving as precursors for LDL. Apart from their size and density, these particles may also differ with respect to their complement of apo E. Yamada et al⁴⁰ have shown that rabbit VLDL, IDL, and LDL contain particles endowed with both apo B and apo E and particles with apo B but devoid of apo E; many of the apo E–and apo B–containing particles are removed rapidly from the circulation without being converted to LDL. It is tempting to speculate that the decrease in VLDL apo B-100 metabolism in FDB that we observed in our study was due to a subpopulation of VLDL particles that lacked apo E. Because such a subpopulation would also be able to acquire apo E in the circulation, the delay in VLDL apo B-100 clearance would be only twofold rather than fourfold, as in the case of FDB-LDL. However, firm experimental evidence for this attractive possibility is presently unavailable.

Another interesting finding of our study is the slightly reduced PR of VLDL apo B-100. The reduction in VLDL apo B-100 synthesis, however, approximated only 15% and was thus unlikely to account for the 50% decrease in LDL production.

In conclusion, both FDB and FH are genetic disorders that lead to elevated LDL-C levels. The hypercholesterolemia and the clinical course in FDB appear less severe than in FH. As we have demonstrated herein, the in vivo metabolism of apo B–containing lipoproteins in these two diseases is also considerably different. In homozygous FH, the increased concentration of LDL is due to delayed LDL catabolism in combination with overproduction of LDL, the latter resulting from ineffective clearance of LDL precursors from the circulation.^{34 35 36} In homozygous FDB, LDL apo B-100 production proceeds at half the normal rate. If one considers that LDL production is increased approximately twofold over normal in homozygous FH,²⁹ then the LDL PR would be roughly fourfold in FH compared with that of FDB. Therefore, it is not surprising that LDL-C levels are twofold to threefold higher in FH than in FDB, although the FCRs of LDL are very much alike in both conditions. The fractional catabolism of apo E is low in FH,³⁰ whereas it is increased in FDB. This kinetic difference between FH and FDB underlines the importance of apo E in compensating for defective apo B-100. Apo E might be considered a "backup system" of lipoprotein metabolism in FDB. The crucial role of apo E to the metabolism of apo B-100 is well documented.^{38 41 42 43} Patients with type III hyperlipidemia^{44 45} and mice that lack apo E develop severe arteriosclerosis.^{46 47} Our in vivo observations demonstrate the significance of apo E in driving the metabolism of VLDL apo B-100. On the basis of our data, we predict that simultaneous homozygosity for both FDB and apo E2 would result in a more severe clinical phenotype closely resembling that of homozygous FH.

Note added in proof
While this manuscript was being prepared for publication, the kinetics in vivo of apoB-100–containing lipoproteins in five individuals heterozygous for FDB in comparison with six normalipidemic individuals was reported by Pietsch et al (J Lipid Res. 1996;37:2074-2087). Their data were consistent with the concept presented herein that enhanced removal of apo E–containing precursors of LDLs resulted in decreased production of LDLs in patients with FDB.

	Selected Abbreviations and Acronyms

 

C = cholesterol FCR = fractional catabolic rate FDB = familial defective apolipoprotein B-100 FH = familial hypercholesterolemia FSR = fractional synthetic rate GC-MS = gas chromatography-mass spectrometry m/z = mass-to-ion ratio PR = production rate

	Acknowledgments

 
Part of this study was supported by research grants from Bristol/Myers Squibb (Munich, Germany) to J.R.S. and W.M. The authors thank Sabine Black (deceased), Bettina Donnerhak, and Sabine Motzny for technical assistance.

	Footnotes

 
Reprint requests to Dr Winfried Marz, Division of Clinical Chemistry, Department of Medicine, Albert Ludwigs-University, Hugstetter Straße 55, 79106 Freiburg, FRG.

¹ Deceased.

Received January 17, 1996; accepted March 26, 1996.

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