Identification of defective binding of low density lipoprotein by the U937 proliferation assay in German patients with familial defective apolipoprotein B-100

Abstract. Familial defective apolipoprotein B-100 (FDB) is a dominantly inherited disorder characterized by decreased binding of low density lipoprotein (LDL) to the LDL receptor due to a substitution of glutamine for arginine in residue 3500 of apolipoprotein B-100. We present the results of the U937 cell proliferation assay for the detection of familial defective apo B100 in 13 German FDB patients. Due to a defect in the pathway of cholesterol synthesis the human myelomonocytic tumour cell line U937 lacks the ability to synthesize cholesterol which makes proliferation of these cells dependent on the presence of exogenous LDL-cholesterol. U937 cells were incubated with LDL from 13 FDB-patients, 10 healthy normocholesterolaemic individuals (NC) and 26 patients with familial hypercholesterolaemia due to a defective LDL-receptor (FH). At LDL-cholesterol concentrations below 1 μg ml^-1 no proliferation occurred. In the presence of LDL from FDB patients at concentrations between 2·5 μg ml^-1 and 15·0 μg ml^-1, the proliferation was significantly reduced compared to LDL from FH-patients and normocholesterolaemic controls. At 5 μg ml^-1 the reduction was 31–80% regardless of age, sex, apo E genotype, Lp (a)- and lipid levels. At concentrations above 25·0 μg ml^-1 no further differences were observed. The present results indicate that the U937 proliferation assay is a reliable test for the detection of defective LDL-binding due to the 3500 mutation in FDB patients. It may be useful for the detection of defective binding of LDL due to other mutations in the apo B-100 gene.     

Evidence for a dominant gene that suppresses hypercholesterolemia in a family with defective low density lipoprotein receptors.

This paper describes an unusual kindred with familial hypercholesterolemia in which one-third of the relatives with a mutant LDL receptor gene have normal plasma cholesterol concentrations. The proband, a 9-yr-old boy with a plasma cholesterol value greater than 500 mg/dl, is homozygous for a point mutation that changes Ser156 to Leu in the LDL receptor. This substitution in the fourth repeat of the ligand binding domain slows the transport of the protein to the cell surface. The defective receptor cannot bind LDL, which contains apo B-100, but it does bind beta-migrating VLDL, which contains apo E in addition to apo B-100. Although the mother is heterozygous for this mutation, her LDL-cholesterol concentration is consistently in the 28th percentile for the population. Through direct examination of genomic DNA, we identified the mutant gene in heterozygous form in 17 of the mother's relatives, five of whom had normal LDL-cholesterol values. The pedigree was consistent with dominant transmission of a single gene that ameliorates or suppresses the hypercholesterolemic effect of the LDL receptor mutation. Through linkage analysis, we excluded the possibility that this suppressor gene was an allele at the LDL receptor locus. We also excluded the genes for the two ligands for the LDL receptor, apo B-100 and apo E. The existence of this putative suppressor gene may explain the occasional observation of normal LDL-cholesterol concentrations in heterozygotes for LDL receptor mutations.     

Compound heterozygous familial hypercholesterolemia and familial defective apolipoprotein B-100 produce exaggerated hypercholesterolemia

BACKGROUND: Familial hypercholesterolemia (FH) and familial defective apolipoprotein B-100 (FDB) represent ligand-receptor disorders that are complementary. Individuals with both FH and FDB are unusual. We report a family with both disorders and the impact of the mutations on the phenotypes of the family members. METHODS: We used single strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) for genetic analysis of all 18 exons and the promoter region of the LDL receptor and DGGE for genetic analysis of the apolipoprotein B-100 (apo B-100) gene. The functional significance of the apo B-100 mutation was studied using a U937 cell proliferation assay. Fasting serum lipid profiles were determined for the index case and seven first-degree relatives. RESULTS: One of the patient's sisters had a missense mutation (Asp(407)-->Lys) in exon 9 of the LDL receptor and a serum LDL-cholesterol concentration of 4.07 mmol/L. Four other first-degree relatives had hyperlipidemia but no LDL-receptor mutation. However, these subjects had a mutation of the apo B-100 gene (Arg(3500)-->Trp). The cell proliferation rate of U937 cells fed with LDL from other subjects with the same mutation was fourfold less than that of controls. The index case had both FH- and FDB-related mutations. Her serum LDL-cholesterol (9.47 mmol/L) was higher than all other relatives tested. CONCLUSIONS: Existence of both FH and FDB should be considered in families with LDL-receptor mutations in some but not all individuals with hypercholesterolemia or when some individuals in families with FH exhibit exaggerated hypercholesterolemia.     

Familial hypobetalipoproteinemia caused by a mutation in the apolipoprotein B gene that results in a truncated species of apolipoprotein B (B-31). A unique mutation that helps to define the portion of the apolipoprotein B molecule required for the formation of buoyant, triglyceride-rich lipoproteins.

Apolipoprotein B-100 has a crucial structural role in the formation of VLDL and LDL. Familial hypobetalipoproteinemia, a syndrome in which the concentration of LDL cholesterol in plasma is abnormally low, can be caused by mutations in the apo B gene that prevent the translation of a full-length apo B-100 molecule. Prior studies have revealed that truncated species of apo B [e.g., apo B-37 (1728 amino acids), apo B-46 (2057 amino acids)] can occasionally be identified in the plasma of subjects with familial hypobetalipoproteinemia; in each of these cases, the truncated apo B species has been a prominent protein component of VLDL. In this report, we describe a kindred with hypobetalipoproteinemia in which the plasma of four affected heterozygotes contained a unique truncated apo B species, apo B-31. Apolipoprotein B-31 is caused by the deletion of a single nucleotide in the apo B gene, and it is predicted to contain 1425 amino acids. Apolipoprotein B-31 is the shortest of the mutant apo B species to be identified in the plasma of a subject with hypobetalipoproteinemia. In contrast to longer truncated apo B species, apo B-31 was undetectable in the VLDL and the LDL; however, it was present in the HDL fraction and the lipoprotein-deficient fraction of plasma. The density distribution of apo B-31 in the plasma suggests the possibility that the amino-terminal 1425 amino acids of apo B-100 are sufficient to permit the formation and secretion of small, dense lipoproteins but are inadequate to support the formation of the more lipid-rich VLDL and LDL particles.     

Familial defective apo B-100, characterization of an Italian family

Abstract. Familial defective apolipoprotein (apo) B-100 is a genetic disorder presenting with hypercholes-terolaemia and abnormal low-density lipoprotein (LDL) that binds poorly to LDL receptors. This disease appears to be caused by a mutation in the apo B-100 gene. In the present study thirteen members of a family with moderate hypercholesterolaemia (250–350 mg dl^-1) were investigated. Biochemical studies on cultured skin fibroblasts ruled out classical familial hypercholesterolaemia (FH, receptor deficiency). We then studied the interaction between LDL and their receptors by an in vitro cell binding assay. LDL from nine affected members displayed a reduced affinity (2·5-fold) for the receptor, and were less effective than LDL from control and unaffected members in suppressing LDL receptor expression and in stimulating cholesterol esterification. LDL from the affected members had normal electrophoretic mobility, size and chemical composition. Partial delipidation did not modify the LDL binding defect. The disorder is transmitted over three generations as an autosomal codominant trait and all the affected members are heterozygotes and hypercholesterolaemics. Analysis of DNA from family members showed a point mutation leading to an Arg to Gln substitution at amino acid 3500 of the mature protein that segregated with hypercholesterolaemia and LDL defective binding. We conclude that this family is affected by familial defective apolipoprotein B-100 (FDB).     

Lipoproteins containing the truncated apolipoprotein, Apo B-89, are cleared from human plasma more rapidly than Apo B-100-containing lipoproteins in vivo.

We have reported previously on two truncations of apolipoprotein B (apo B-40 and apo B-89) in a kindred with hypobetalipoproteinemia. Premature stop codons were found to be responsible for both apo B-40 and apo B-89, but the physiologic mechanisms accounting for the reduced plasma concentrations of these proteins have not been determined in vivo. This study investigates the metabolism of apo B-89 in two subjects heterozygous for apo B-89/apo B-100 and in one apo B-40/apo B-89 compound heterozygote. In both heterozygotes total apo B concentration is approximately 30% of normal and apo B-89 is present in lower concentrations in plasma than apo B-100. After the administration of [1-13C]leucine as a primed constant infusion over 8 h, 13C enrichments of plasma leucine as well as enrichments of VLDL-, IDL-, and LDL-apo B-89 leucine and VLDL-, IDL-, and LDL-apo B-100 leucine were measured over 110 h. Enrichment values were subsequently converted to tracer/tracee ratios and a multicompartmental model was used to estimate metabolic parameters. In both apo B-89/apo B-100 heterozygotes apo B-89 and apo B-100 were produced at similar rates. Respective transport rates of apo B-89 and apo B-100 for subject 1 were 2.13 +/- 0.18 and 2.56 +/- 0.13 mg.kg-1.d-1, and for subject 2, 6.59 +/- 0.18 and 8.23 +/- 0.39 mg.kg-1.d-1. However, fractional catabolic rates of VLDL, IDL, and LDL particles containing apo B-89 were 1.4-3 times higher than the rates for corresponding apo B-100-containing particles. Metabolic parameters of apo B-89 in the apo B-40/apo B-89 compound heterozygote compared favorably with those established for apo B-89 in apo B-89/apo B-100 heterozygotes. Thus, the enhanced catabolism of VLDL, IDL, and LDL particles containing the truncated apolipoprotein is responsible for the relatively low levels of apo B-89 seen in these subjects.     

Mutations in the apolipoprotein (apo) B-100 receptor-binding region: detection of apo B-100 (Arg3500-->Trp) associated with two new haplotypes and evidence that apo B-100 (Glu3405-->Gln) diminishes receptor-mediated uptake of LDL.

BACKGROUND: Ligand-defective apolipoprotein (apo) B-100 is a major cause of hypercholesterolemia. For many years, apo B-100 (Arg3500-->Gln) has been the only mutation known to cause ligand-defective apo B-100. METHODS: Using temperature gradient gel electrophoresis, we screened 297 unrelated individuals with LDL-cholesterol >1.55 g/L and triglycerides <2.0 g/L for sequence variants of the putative LDL receptor-binding domain of apo B-100. RESULTS: We found apo B-100 (Arg3500-->Gln) in 21 individuals (7.1%). When extrapolated to the general population, this corresponds to the highest prevalence of apo B-100 (Arg3500-->Gln) reported to date. Furthermore, we identified three unrelated carriers (1%) of a silent substitution (CTG-->CTA) affecting the codon for leucine3350, four carriers (1.3%) of apo B-100 (Glu3405-->Gln), and two subjects (0.7%) with apo B-100 (Arg3500-->Trp). apo B-100 (Arg3500-->Trp) was assigned to two different, previously unknown haplotypes. The binding, uptake, and degradation of apo B-100 (Arg3500-->Trp) was lower than that of normal LDL, but higher than with apo B-100 (Arg3500-->Gln), implying that the substitution of Trp3500 for Arg may cause less severe reduction of binding than the substitution of Gln. LDL from individuals heterozygous for apo B-100 (Glu3405-->Gln) bound to LDL receptors at the same rate as normal LDL, but was taken up and degraded at significantly reduced rates, suggesting that domains of apo B-100 involved in binding and uptake do not completely overlap. CONCLUSIONS: In Germany, apo B-100 (Arg3500-->Gln) may be more frequent than previously assumed. Both apo B-100 (Arg3500-->Trp) and apo B-100 (Glu3405-->Gln) may contribute to the phenotype of ligand-defective LDL. These variants will be missed if screening is confined to apo B-100 (Arg3500-->Gln) only.     

A monoclonal-antibody-based enzyme-linked immunosorbent assay of lipoprotein(a)

Lipoprotein(a) [Lp(a)] is a low-density lipoprotein (LDL)-like lipoprotein particle recently described as a risk factor for premature coronary heart disease, stroke, and atherosclerosis. Structurally, Lp(a) is similar to LDL in that it has comparable lipid composition and contains apolipoprotein B-100 (apo B-100). In addition, Lp(a) contains the glycoprotein apolipoprotein(a) [apo(a)], which is disulfide-linked to apo B-100. The recent awareness of a striking correlation between atherosclerosis and concentrations of Lp(a) in plasma prompted our development of an accurate quantitative assay for plasma Lp(a), a monoclonal-antibody-based enzyme-linked immunosorbent assay for Lp(a) that is shown to be sensitive, precise, and highly specific. The response to several isoforms of Lp(a) is linear, and as many as 80 samples can be quantified on one plate. This easily performed assay is suitable for use in the clinical laboratory and for screening large populations.     

Flow cytometric assessment of LDL ligand function for detection of heterozygous familial defective apolipoprotein B-100

BACKGROUND: Familial defective apolipoprotein (apo) B-100 (FDB) is caused by a mutation in the apoB gene and characterized by decreased binding of LDL to LDL receptors because of reduced function of the apoB-100 ligand. FDB may be associated with severe hypercholesterolemia and cannot always be distinguished from familial hypercholesterolemia phenotypically. METHODS: We used a fluorescence flow cytometry assay with Epstein-Barr virus-transformed lymphocytes to detect reduced LDL ligand function by competitive binding with fluorescently conjugated LDL (DiI-LDL). The assay was tested and validated using LDL from patients heterozygous for the Arg(3500)-Gln mutation and their first-degree relatives. Knowing the actual apoB genotype of patients and relatives allowed us to assess the ability of the assay to predict the results of DNA analysis. The results were compared to measurements of LDL ligand function in unrelated healthy control subjects to characterize functionally the Arg(3500)-Gln mutation. RESULTS: Fluorescence was significantly increased in cells incubated with DiI-LDL in competition with unlabeled LDL from FDB(R3500Q) heterozygotes compared with cells incubated with DiI-LDL in competition with unlabeled LDL from relatives or unrelated healthy control subjects. Thus, patients heterozygous for the Arg(3500)-Gln mutation had significantly reduced LDL ligand function. The binding affinity of LDL from FDB(R3500Q) heterozygotes was 32% of that in non-FDB relatives and healthy controls. The assay had a diagnostic sensitivity of 0.95 and diagnostic specificity of 0.89. CONCLUSIONS: The diagnostic accuracy of the assay was too low to allow reliable diagnosis of individual cases of heterozygous FDB(R3500Q). However, fluorescence flow cytometry may supplement genetic identification of FDB and functionally characterize gene mutations associated with major reductions in LDL ligand function.     

Truncated apo B-70.5–containing lipoproteins bind to megalin but not the LDL receptor

Apo B-100 of LDL can bind to both the LDL receptor and megalin, but the molecular interactions of apo B-100 with these 2 receptors are not completely understood. Naturally occurring mutant forms of apo B may be a source of valuable information on these interactions. Apo B-70.5 is uniquely useful because it contains the NH2-terminal portion of apo B-100, that includes only one of the two putative LDL receptor-binding sites (site A). The lipoprotein containing apo B-70. 5 (Lp B-70.5) was purified from apo B-100/apo B-70.5 heterozygotes by sequential ultracentrifugation combined with immunoaffinity chromatography. Cell culture experiments, ligand blot analysis, and in vivo studies all consistently showed that Lp B-70.5 is not recognized by the LDL receptor. The kidney was identified as a major organ in catabolism of Lp B-70.5 in New Zealand white rabbits. Autoradiographic analysis revealed that renal proximal tubular cells selectively removed Lp B-70.5. On ligand blotting of renal cortical membranes, Lp B-70.5 bound only to megalin. The ability of megalin to mediate cellular endocytosis of Lp B-70.5 was confirmed using retinoic acid/dibutyryl cAMP-treated F9 cells. This study suggests that the putative LDL receptor-binding site A on apo B-100 might not by itself be a functional binding domain and that the apo B-binding sites recognized by the LDL receptor and by megalin may be different. Moreover, megalin may play an important role in renal catabolism of apo B truncations, including apo B-70.5.     

Effect of low density lipoprotein receptor deficiency on the metabolism of apolipoprotein B-100 in blood plasma. Kinetic studies in normal and Watanabe heritable hyperlipidemic rabbits

The kinetics of apolipoprotein (apo) B-100 in particles containing apo E (B,E particles) or lacking apo E (B particles) were studied in Watanabe heritable hyperlipidemic (WHHL) rabbits deficient in low density lipoprotein (LDL) receptors, and compared with those of normal rabbits after injection of radioiodinated very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and LDL. In both groups results of kinetic modeling were consistent with the hypothesis that all apo B enters the plasma in VLDL, mainly as B,E particles, followed by delipidation and partial conversion to IDL and LDL, with concomitant conversion of some B,E particles to B particles. In WHHL rabbits, production of VLDL apo B was reduced by 40%, but LDL production was increased threefold. Defective removal of B,E and B particles in all three lipoprotein classes, coupled with preserved processes of delipidation, can account for the observed increases in the concentration of apo B (threefold in VLDL, fivefold in IDL, and twenty-twofold in LDL) in WHHL rabbits.     

Characterization of an abnormal species of apolipoprotein B, apolipoprotein B-37, associated with familial hypobetalipoproteinemia.

Steinberg and colleagues have previously described a unique kindred with normotriglyceridemic hypobetalipoproteinemia (1979. J. Clin. Invest. 64:292-301). In a reexamination of this kindred, we found an abnormal apolipoprotein (apo) B species, apo B-37 (203,000 mol wt), in the plasma lipoproteins of multiple members of the kindred. In affected individuals apo B-37 was found in very low density lipoproteins, along with the normal apo B species, apo B-100 and apo B-48. High density lipoproteins (HDL) also contained apo B-37, but no other apo B species. The first 13 amino-terminal amino acids of apo B-37 were identical to those of normal apo B-100. We utilized a panel of 18 different apo B-specific monoclonal antibodies and polyclonal antisera specific for apo B-37 and the thrombin cleavage products of apo B-100 to map apo B-37 in relation to apo B-100, apo B-48, and the thrombin cleavage products of apo B-100. The results of those immunochemical studies indicated that apo B-37 contains only amino-terminal domains of apo B-100. In affected individuals, the majority of apo B-37 in plasma was contained in the HDL density fraction. Within that fraction apo B-37 was found on discrete lipoprotein particles, termed Lp-B37, that had properties distinct from normal HDL particles containing apo A-I. This report documents for the first time the existence of an abnormal apo B species in humans. Further study of apo B-37 and lipoprotein particles containing apo B-37 should lead to an improved understanding of apo B structure and function.     

Plasma triglycerides determine low density lipoprotein composition, physical properties, and cell-specific binding in cultured cells.

The relationship between the plasma triglycerides and the LDL triglycerides of 30 normal and 48 hypertriglyceridemic subjects has been quantified; the data fit a simple adsorption isotherm, LDL triglyceride/(LDL triglyceride+LDL cholesterol ester) = 0.65 plasma triglyceride/(464 + plasma triglyceride). In vitro transfer of triglyceride from concentrated VLDL to VLDL-depleted plasma produced triglyceride-rich LDL that had similar properties. LDL uptake by HepG2 cells increased with LDL triglyceride content whereas the reverse was found with skin fibroblasts. At 37 degrees C, the cores of both normal and hypertriglyceridemic LDL were isotropic liquids. Circular dichroic spectra revealed no difference in the secondary structure of normal and triglyceride-rich LDL. The affinity of monoclonal antibody MB47, which binds to the receptor ligand of apo B-100 was independent of LDL triglyceride content. MB3, which binds near residue 1022 of apo B-100, showed a triglyceride-dependent decrease in affinity for LDL from hypertriglyceridemic subjects and from in vitro incubations. LDL with an elevated triglyceride content formed in vitro had reduced proteolytic cleavage of apo B-100 by Staphylococcus aureus V8 protease. From these data, we infer that (a) LDL triglyceride is a predictable function of plasma triglyceride, (b) triglyceride induces subtle changes in apo B-100 structure at a site that is remote from the putative receptor binding ligand, and (c) the triglyceride-dependent receptor-binding determinants of apo B-100 are recognized differently by fibroblasts and HepG2 cells.     

Overexpression of human low density lipoprotein receptors leads to accelerated catabolism of Lp(a) lipoprotein in transgenic mice.

Lp(a) lipoprotein purified from human plasma bound with high affinity to isolated bovine LDL receptors on nitrocellulose blots and in a solid-phase assay. Lp(a) also competed with 125I-LDL for binding to human LDL receptors in intact fibroblasts. Binding led to cellular uptake of Lp(a) with subsequent stimulation of cholesterol esterification. After intravenous injection, human Lp(a) was cleared slowly from the plasma of normal mice. The clearance was markedly accelerated in transgenic mice that expressed large amounts of LDL receptors. We conclude that the covalent attachment of apo(a) to apo B-100 in Lp(a) does not interfere markedly with the ability of apo B-100 to bind to the LDL receptor and that this receptor has the potential to play a major role in clearance of Lp(a) from the circulation of intact humans.     

Evaluation of the Roche Diagnostics LightCycler-Apo B 3500 Mutation Detection Kit.

Familial defective apolipoprotein (apo) B-100 is an autosomal codominant disorder associated with hypercholesterolemia and an increased risk of coronary artery disease. Two independent mutations affecting the codon 3500 (Arg3500-->Gln and Arg3500-->Trp) have been shown to cause ligand-defective apo B-100. Identification of carriers of these mutations is an important step in the risk stratification of individuals and families with hypercholesterolemia. We evaluated a homogeneous assay for detection of mutations at codon 3500 that combines rapid-cycle PCR with allele-specific fluorescent probe melting profiles for product genotyping. This single-tube analysis is performed on the LightCycler, a microvolume fluorimeter integrated with a thermal cycler. Continuous acquisition of fluorescence data during a melting curve analysis at completion of PCR allows the detection of mutations, as loss of fluorescence occurs in an allele-specific manner. By plotting melting peaks, the three apo B-100 alleles were readily distinguishable. Using this method, genotyping of 32 samples is completed within 40 min without the need for any post-PCR sample manipulation, thereby eliminating the risks of end-product contamination and sample tracking errors. The specific detection of mutations at codon 3500 of the apo B gene on the LightCycler is a rapid and reliable method that is ideally suitable for typing both small and large numbers of samples.     

Significance of acidic sugar chains of apolipoprotein B-100 in cellular metabolism of low-density lipoproteins

We have elucidated the carbohydrate structures of the N-linked sugar chains of human and rabbit apolipoprotein B-100 (apo B-100), which is similar in composition to oligosaccharides (Arch Biochem Biophys 1989;273:197-205, Arteriosclerosis 1990; 10:386-93). We have also shown the negative correlation of the ratio of acidic sugar chains of apo B-100 to the serum cholesterol levels in Watanabe heritable hyperlipidemic rabbits (Atherosclerosis 1992;93:229-35). The acidity of sugar chains is determined by the existence of sialic acid residues at the terminal of oligosaccharides. In the present study we investigated N-linked sugar chains of apo B-100 from patients with coronary artery disease (CAD) who had moderate hypercholesterolemia (less than 400 mg/dL). There was no difference in the structure of their oligosaccharides and the ratio of acidic sugar chains of apo B-100 from CAD patients as compared with that from healthy individuals reported previously. To clarify the role of sialic acid residues in apo B-100 for lipoprotein metabolism, we studied cellular uptake of low-density lipoproteins (LDLs) treated with sialidase (desialylated LDL). Desialylated LDLs were taken up and degraded to a 2-fold greater degree than control LDL by human monocyte-derived macrophages and stimulated cholesterol esterification in these cells. These results indicate that sialic acid residues of apo B- 100 play an important role in cellular uptake and degradation of LDL.     

Analysis of the role of microsomal triglyceride transfer protein in the liver of tissue-specific knockout mice

A deficiency in microsomal triglyceride transfer protein (MTP) causes the human lipoprotein deficiency syndrome abetalipoproteinemia. However, the role of MTP in the assembly and secretion of VLDL in the liver is not precisely understood. It is not clear, for instance, whether MTP is required to move the bulk of triglycerides into the lumen of the endoplasmic reticulum (ER) during the assembly of VLDL particles. To define MTP's role in hepatic lipoprotein assembly, we recently knocked out the mouse MTP gene (Mttp). Unfortunately, achieving our objective was thwarted by a lethal embryonic phenotype. In this study, we produced mice harboring a "floxed" Mttp allele and then used Cre-mediated recombination to generate liver-specific Mttp knockout mice. Inactivating the Mttp gene in the liver caused a striking reduction in VLDL triglycerides and large reductions in both VLDL/LDL and HDL cholesterol levels. The Mttp inactivation lowered apo B-100 levels in the plasma by >95% but reduced plasma apo B-48 levels by only approximately 20%. Histologic studies in liver-specific knockout mice revealed moderate hepatic steatosis. Ultrastructural studies of wild-type mouse livers revealed numerous VLDL-sized lipid-staining particles within membrane-bound compartments of the secretory pathway (ER and Golgi apparatus) and few cytosolic lipid droplets. In contrast, VLDL-sized lipid-staining particles were not observed in MTP-deficient hepatocytes, either in the ER or in the Golgi apparatus, and there were numerous cytosolic fat droplets. We conclude that MTP is essential for transferring the bulk of triglycerides into the lumen of the ER for VLDL assembly and is required for the secretion of apo B-100 from the liver.     

Apolipoprotein B-100 deficiency. Intestinal steatosis despite apolipoprotein B-48 synthesis

We describe a child, the issue of phenotypically normal parents, who had fat malabsorption, both intestinal and hepatic steatosis, and serum cholesterol and triglyceride concentrations of 38 and 63 mg/dl, respectively. Lipoprotein electrophoresis, Ouchterlony double diffusion, and electron microscopy demonstrated that normal low density lipoproteins (LDL: 1.006 less than rho less than 1.063 g/ml) were absent. Lipoprotein particles in the rho less than 1.006-g/ml fraction were triglyceride rich, very large (93.2 +/- 35.1 nm), and contained the B-48 but not the B-100 apoprotein; both species of apolipoprotein (apo) B were found in the parents' lipoproteins. These chylomicrons and chylomicron remnants were present even in the patient's fasting plasma, which suggested prolonged dietary fat absorption. Plasma levels of high density lipoprotein lipids and proteins were low, and the phosphatidylcholine/sphingomyelin ratio was reduced as in typical abetalipoproteinemia. The monosialylated form of apo C-III was not identified on polyacrylamide gel electrophoresis, which suggested that this protein was elaborated only with very low density lipoproteins (VLDL). A radioimmunoassay for apo B employing a polyclonal antisera to plasma LDL gave apparent plasma apo B levels of 0.6, 66, and 57 mg/dl in the patient and his father and mother, respectively. The displacement curve generated by the parents' VLDL and LDL did not did not differ from control lipoproteins. The patient's chylomicron-chylomicron remnant fraction displaced normal LDL over the entire radioimmunoassay range, but the efficiency of displacement was strikingly less than with B-100 containing lipoproteins. If the patient's B-48 protein is not qualitatively abnormal, these results confirm very limited immunochemical cross-reactivity between at least one major epitope on B-100 and the epitopes expressed on B-48. The apo B defect in this patient appears to be recessive. It abolishes B-100 production and may additionally limit the formation of B-48.     

Isolation and characterization of apolipoprotein B-48 and B-100 very low density lipoproteins from type III hyperlipoproteinemic subjects.

Two major species of human apolipoprotein (apo) B have been identified, apo B-48 and apo B-100, which are the predominant forms in chylomicrons and very low density lipoproteins (VLDL), respectively. Due to defective hepatic clearance, apo B-48 containing lipoproteins accumulate in the plasma of subjects with type III hyperlipoproteinemia. In the present study, we have used immunoaffinity chromatography to separate type III VLDL into a nonretained (apo B-48 VLDL) and a retained (apo B-100 VLDL) fraction. To achieve complete separation, as determined by electrophoresis and radioimmunoassay, it was necessary to employ two different insolubilized anti-apo B-100 monoclonal antibodies because of immunochemical heterogeneity within the apo B-100 VLDL fraction. The ability to separate apo B-100 VLDL from apo B-48 VLDL shows that the two apo B species are found on different particles. The apo B-48 VLDL had an electrophoretic mobility similar to chylomicrons, whereas the apo B-100 VLDL migrated similarly to total type III VLDL. Both fractions showed a concentration of particles with diameters approximately 100 nm, with apo B-48 VLDL being somewhat more heterogeneous in particle size. The two fractions were qualitatively similar in apolipoprotein composition but apo B-48 VLDL was enriched in apo E, relative to apo B-100 VLDL. Apo B-48 VLDL was enriched in cholesterol esters and deficient in triglycerides and phospholipids when compared with apo B-100 VLDL. The existence of immunochemical heterogeneity in the apo B-100 VLDL may reflect different functional subpopulations of particles within this fraction.     

Measurement of very low density and low density lipoprotein apolipoprotein (Apo) B-100 and high density lipoprotein Apo A-I production in human subjects using deuterated leucine. Effect of fasting and feeding.

Six normolipidemic male subjects, after an 8-h overnight fast, were given a bolus injection and then a 15-h constant intravenous infusion of [D3]L-leucine. Subjects were studied in the fasted state and on a second occasion in the fed state (small, physiological meals were given every hour for 15 h). Apolipoproteins were isolated by preparative gradient gel electrophoresis from plasma lipoproteins separated by sequential ultracentrifugation. Incorporation of [D3]L-leucine into apolipoproteins was monitored by negative ionization, gas chromatography-mass spectrometry. Production rates were determined by multiplying plasma apolipoprotein pool sizes by fractional production rates (calculated as the rate of isotopic enrichment [IE] of each protein as a fraction of IE achieved by VLDL (d less than 1.006 g/ml) apo B-100 at plateau. VLDL apo B-100 production was greater, and LDL (1.019 less than d less than 1.063 g/ml) apo B-100 production was less in the fed compared with the fasted state (9.9 +/- 1.7 vs. 6.4 +/- 1.7 mg/kg per d, P less than 0.01, and 8.9 +/- 1.2 vs. 13.1 +/- 1.2 mg/kg per d, P less than 0.05, respectively). No mean change was observed in high density lipoprotein apo A-I production. We conclude that: (a) this stable isotope, endogenous-labeling technique, for the first time allows for the in vivo measurement of apolipoprotein production in the fasted and fed state; and (b) since LDL apo B-100 production was greater than VLDL apo B-100 production in the fasted state, this study provides in vivo evidence that LDL apo B-100 can be produced independently of VLDL apo B-100 in normolipidemic subjects.