The effects of apolipoprotein B depletion on HDL subspecies composition and function.

HDL cholesterol (HDL-C) efflux function may be a more robust biomarker of coronary artery disease risk than HDL-C. To study HDL function, apoB-containing lipoproteins are precipitated from serum. Whether apoB precipitation affects HDL subspecies composition and function has not been thoroughly investigated. We studied the effects of four common apoB precipitation methods [polyethylene glycol (PEG), dextran sulfate/magnesium chloride (MgCl2), heparin sodium/manganese chloride (MnCl2), and LipoSep immunoprecipitation (IP)] on HDL subspecies composition, apolipoproteins, and function (cholesterol efflux and reduction of LDL oxidation). PEG dramatically shifted the size distribution of HDL and apolipoproteins (assessed by two independent methods), while leaving substantial amounts of reagent in the sample. PEG also changed the distribution of cholesterol efflux and LDL oxidation across size fractions, but not overall efflux across the HDL range. Dextran sulfate/MgCl2, heparin sodium/MnCl2, and LipoSep IP did not change the size distribution of HDL subspecies, but altered the quantity of a subset of apolipoproteins. Thus, each of the apoB precipitation methods affected HDL composition and/or size distribution. We conclude that careful evaluation is needed when selecting apoB depletion methods for existing and future bioassays of HDL function.

Lipid apheresis, indications, and principles.

Low-density lipoprotein apheresis (LDL apheresis) is a term that describes a group of apheresis techniques and devices that selectively remove apolipoprotein B containing lipoproteins. A number of different devices are available worldwide, which all effectively remove low-density lipoprotein cholesterol while sparing other important plasma components. LDL apheresis is used to treat familial hypercholesterolemia (FH), an inherited condition of accelerated atherosclerosis and severe coronary artery disease resulting in premature death. It has also been used to treat other disorders, although the evidence for its use is limited. This review describes the underlying pathophysiology of FH, the mechanism of action of the various LDL apheresis devices available, and how LDL apheresis is used to treat this uncommon metabolic condition.

Dual size-exclusion chromatography for efficient isolation of extracellular vesicles from bone marrow derived human plasma.

Isolation of pure extracellular vesicles (EVs), especially from blood, has been a major challenge in the field of EV research. The presence of lipoproteins and soluble proteins often hinders the isolation of high purity EVs upon utilization of conventional separation methods. To circumvent such problems, we designed a single-step dual size-exclusion chromatography (dSEC) column for effective isolation of highly pure EVs from bone marrow derived human plasma. With an aim to select appropriate column design parameters, we analyzed the physiochemical properties of the major substances in bone marrow derived plasma, which include EVs, lipoproteins, and soluble proteins. Based on these findings, we devised a novel dSEC column with two different types of porous beads sequentially stacked each other for efficient separation of EVs from other contaminants. The newly developed dSEC columns exhibited better performance in isolating highly pure EVs from AML plasma in comparison to conventional isolation methods.

Characterization of metabolic interrelationships and in silico phenotyping of lipoprotein particles using self-organizing maps.

Plasma lipid concentrations cannot properly account for the complex interactions prevailing in lipoprotein (patho)physiology. Sequential ultracentrifugation (UCF) is the gold standard for physical lipoprotein isolations allowing for subsequent analyses of the molecular composition of the particles. Due to labor and cost issues, however, the UCF-based isolations are usually done only for VLDL, LDL, and HDL fractions; sometimes with the addition of intermediate density lipoprotein (IDL) particles and the fractionation of HDL into HDL(2) and HDL(3) (as done here; n = 302). We demonstrate via these data, with the lipoprotein lipid concentration and composition information combined, that the self-organizing map (SOM) analysis reveals a novel data-driven in silico phenotyping of lipoprotein metabolism beyond the experimentally available classifications. The SOM-based findings are biologically consistent with several well-known metabolic characteristics and also explain some apparent contradictions. The novelty is the inherent emergence of complex lipoprotein associations; e.g., the metabolic subgrouping of the associations between plasma LDL cholesterol concentrations and the structural subtypes of LDL particles. Importantly, lipoprotein concentrations cannot pinpoint lipoprotein phenotypes. It would generally be beneficial to computationally enhance the UCF-based lipoprotein data as illustrated here. Particularly, the compositional variations within the lipoprotein particles appear to be a fundamental issue with metabolic and clinical corollaries.

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.

Effects of nonketotic streptozotocin diabetes on apolipoprotein B synthesis and secretion by primary cultures of rat hepatocytes.

The effects of hypoinsulinemic nonketotic streptozotocin diabetes on hepatic apo B synthesis and secretion was studied in primary cultures of rat hepatocytes. Diabetic rats were characterized by their significantly elevated serum glucose, apo B, and triglyceride levels, while serum insulin levels were less than a third of normal. Serum transminase activities of diabetic rats were significantly elevated when compared with control rats, which was attributed to an increase in liver transaminase activity in diabetic rats. The pattern of enzyme activities of hepatocytes reflected that observed in livers of donor rats and the pattern was retained by primary cultures of hepatocytes over the culture period. Hepatocytes from diabetic rats secreted only one third of the apo B secreted by hepatocytes from control rats, which was determined by monoclonal immunoassay of rat total apo B. Decreases in secretion were confirmed by measurement of secretory [35S]methionine-labeled lipoprotein apo B radioactivity. The decreased apo B content of media of hepatocytes from diabetic rats was not due to increased apo B catabolism since hepatocytes from diabetic rats were shown to degrade less lipoprotein-apo B than hepatocytes from normal rats in control experiments. In addition, the apo B content of detergent-solubilized hepatocytes from diabetic rats was significantly less than that of hepatocytes from control rats. These results suggest that insulin is necessary for normal hepatic apo B synthesis and secretion and that the hyperlipidemia associated with hypoinsulinemia in vivo is primarily of intestinal origin.

Transcriptional regulation of the apolipoprotein B100 gene: purification and characterization of trans-acting factor BRF-2.

Apolipoprotein B100 (apoB), the only protein of low-density lipoprotein, is produced primarily in the liver and serves as a ligand for the low-density lipoprotein receptor. Hepatic cell-specific expression of the human apoB gene is controlled by at least two cis-acting positive elements located between positions-128 and -70 (H. K. Das, T. Leff, and J.L. Breslow, J. Biol. Chem. 263:11452-11458, 1988). The distal element (-128 to -85) appears to be liver specific since it shows positive activity in HepG2 cells and negative activity in HeLa cells. The proximal element (-84 to -70) acts as a positive element in both these cell lines, and two rat liver nuclear proteins, BRF-1 and C/EBP, bind to two overlapping sites (-84 to -60 and -70 to -50, respectively). By gel mobility shift assay, we have identified a rat liver nuclear protein (BRF-2) which binds to the distal element (-128 to -85) of the apoB gene. This putative trans-acting factor has been purified to apparent homogeneity by DEAE-cellulose, heparin-agarose, and DNA-specific affinity chromatography. The purified BRF-2 has an apparent molecular mass of 120 kDa and was found to specifically recognize sequence -128 to -85; BRF-2 also produced a strong hypersensitive site at nucleotide position -95 with copper-orthophenanthroline reagent. A double-stranded oligonucleotide (-128 to -85) containing a 3-nucleotide (TTC) insertion between position -95 and -94 was found to abolish DNA binding by BRF-2. This result suggests that the region surrounding the hypersensitive site -95 is important for protein-DNA interaction. By using apoB promoter fragments containing various internal deletions as templates for gel mobility shift assay, the region between -104 and -85 was identified to be crucial for binding by BRF-2. We propose that BRF-2 may play an important role in the tissue-specific regulation of apoB gene transcription.

A newly discovered apolipoprotein B-containing high-density lipoprotein produced by human hepatoma cells.

Apolipoprotein B-containing high-density lipoprotein was detected in the high-density lipoprotein fraction of the concentrated conditioned medium of human hepatoma HuH-7 cells (Apo-B-containing HDLHuH-7). Electrophoretically, the Apo-B-containing HDLHuH-7 migrated more slowly and broadly than beta-lipoprotein on agarose gel. The Apo-B-containing HDLHuH-7 was not precipitated by heparin-Mn2+ treatment. High-performance gel filtration chromatography indicated that the peak of Apo-B-containing HDLHuH-7 was eluted faster than that of plasma LDL and electron microscopic analysis demonstrated that the particles of Apo-B-containing HDLHuH-7 ranged from 140 A to about 450 A, indicating the heterogeneity of Apo-B-containing HDLHuH-7. The protein/lipid ratio of HDLHuH-7 (1.35) is higher than that of plasma HDL3 (1.2). The lipids of HDLHuH-7 are composed of phospholipids (170 micrograms/mg protein), free cholesterol (410 micrograms/mg protein), cholesterol ester (20 micrograms/mg protein) and triacylglycerols (140 micrograms/mg protein). The results described above indicated that Apo-B-containing HDLHuH-7 was a novel lipoprotein discovered for the first time.

Apolipoproteins B, C-III and E in two major subpopulations of low-density lipoproteins.

To determine the concentration and distribution of apolipoproteins C-III and E in low density lipoproteins (LDL) of d 1.025-1.043 g/ml, fresh human plasma was fractionated by single-spin density gradient ultracentrifugation into five layers. Two major subpopulations including layer 2 (d 1.025-1.029 g/ml) and layer 3 (d 1.032-1.043 g/ml) were isolated and characterized by determination of flotation coefficient, neutral lipids and apolipoproteins B, C-III and E. The apolipoprotein B/C-III/E ratio of layer 2 was 100/(3.3 +/- 2.0)/(5.1 +/- 2.9) (wt/wt) and that of layer 3 was 100/(0.61 +/- 0.32)/(0.58 +/- 0.29) (wt/wt). These weight ratios corresponded to molar ratios of 1.0/(1.90 +/- 1.16)/(0.74 +/- 0.42) and 1.0/(0.34 +/- 0.18)/(0.08 +/- 0.04), respectively. Layer 2 contained 6-23% of the total plasma apolipoprotein B or 7-27% of total LDL2 (d 1.019-1.063 g/ml) apolipoprotein B. Layer 3 contained 41-65% of plasma apolipoprotein B or 62-86% of LDL2 apolipoprotein B. About 5-17% of apolipoprotein C-III and 8-30% of apolipoprotein E in plasma are distributed in layers 2 and 3 with the majority present in layer 2. These results show an evident apolipoprotein heterogeneity of LDL2 isolated from normolipidemic subjects. Moreover, they show that the relatively small amounts of apolipoprotein C-III and apolipoprotein E in lower-density segments of LDL2 take on a greater significance when presented in molar rather than weight concentrations. The existence of different ratios of apolipoprotein C-III/apolipoprotein E in layer 2 and layer 3 suggest the presence in LDL2 of varying amounts of several discrete apolipoprotein B- and/or apolipoprotein C-III- and apolipoprotein E-containing lipoprotein particles.

Isolation and characterization of human Lp-B lipoprotein containing apolipoprotein B as the sole apolipoprotein.

A sequential immunoaffinity chromatography procedure was developed to isolate from whole normolipidemic human plasma a subpopulation of apoB containing particles (Lp-B) which is virtually free of non apoB protein. The absence of non apoB protein in Lp-B was assessed by enzyme immunoassay against apolipoproteins A-I, A-II, A-IV, E, C-III and (a). Electron microscopy and fractionation of the isolated particles by gel filtration demonstrated that these particles were heterogeneous in size. However, most of them had diameters between 18 and 26 nm. These particles were found to be rich in cholesterol (molar ratio cholesterol/apoB = 2246 +/- 995) poor in triacylglycerol (molar ratio triacylglycerol/apoB = 555 +/- 518) and had a phospholipids/apoB molar ratio of 713 +/- 348. Most of the cholesterol was esterified (66% +/- 5%). Lp-B particles bound to the apoB, E receptor of HeLa cells with a lower affinity than LDL prepared by ultracentrifugation (1.030 kg/l less than d less than 1.053 kg/l). (KD = 18.9 vs 10.5 nmol/l).

Characterization of a monoclonal antibody that binds equally to all apolipoprotein and lipoprotein forms of human plasma apolipoprotein B. II. Isolation of apolipoprotein B-containing lipoproteins from human plasma.

Monoclonal antibody ('Pan B' antibody) that binds equally to all major forms of human plasma apolipoprotein B was used in an immunoaffinity chromatography procedure to isolate apolipoprotein B-containing lipoproteins from hyperlipidemic human plasma. These lipoproteins were compared with lipoproteins in native plasma, with lipoproteins isolated by polyclonal antibodies and with lipoproteins isolated by the conventional ultracentrifugational method. Judged by the apolipoprotein and lipid composition, lipoproteins isolated with 'Pan B' antibody were virtually identical to those isolated by ultracentrifugation or polyclonal antibodies. Lipoproteins isolated by 'Pan B' antibody were comparable in size and shape to the lipoproteins in native plasma and to the lipoproteins isolated by polyclonal antibodies or ultracentrifugation. The immunoaffinity column with monoclonal 'Pan B' antibody retained all apolipoprotein B-containing lipoproteins and showed significantly higher capacity than polyclonal immunoaffinity column. The column with the highest capacity allowed the isolation from whole plasma of 0.144 mg of apolipoprotein B per ml of gel in less than 2 h.

Oleic acid stimulation of apolipoprotein B secretion from HepG2 and Caco-2 cells occurs post-transcriptionally.

HepG2 and Caco-2 cells were studied to compare the regulation of liver and intestinal apolipoprotein (apo) biosynthesis and secretion by fatty acids. Incubation with fatty acid consistently stimulated apoB production by both HepG2 and Caco-2 cells. Media concentrations of apoB, determined by radioimmunoassay, were approx. 3-fold greater for cells incubated for 24 h in serum-free medium containing oleic acid bound to albumin than for cells incubated with albumin alone. Oleic acid also resulted in a 2-3-fold accumulation of cellular triacylglycerol for HepG2 cells and Caco-2 cells. Cellular apoB and media and cellular apoA-I concentrations were not affected by oleic acid. Immunoblotting with a monoclonal antibody against human apoB confirmed a greater mass of apoB in media from HepG2 and Caco-2 cells incubated with oleic acid. Radiolabeled apoB-100 was also increased in media from HepG2 and Caco-2 cells incubated with [35S]methionine for 24 h in the presence of oleic acid, suggesting enhanced apoB synthesis. However, apoB mRNA concentrations were unchanged in response to oleic acid. Gel filtration of media by fast protein liquid chromatography (FPLC) revealed a redistribution of apoB from LDL-sized particles to VLDL or chylomicrons in media from Caco-2 cells incubated with oleic acid, whereas apoB remained in LDL for HepG2 cells. ApoA-I in media from HepG2 and Caco-2 cells eluted as free or lipid-poor apoA-I, and the apoA-I distribution was unaltered by incubation with oleic acid. These data demonstrate that HepG2 and Caco-2 cells maintained in supplemented serum-free medium respond to oleic acid by a similar post-transcriptional increase in apoB synthesis, but different packaging of apoB into triacylglycerol-rich lipoproteins.

Alterations in the structure of apolipoprotein B-100 determine the behaviour of LDL towards thromboplastin.

Apolipoprotein B-100 acts as an inhibitor of thromboplastin activity independently of the tissue factor pathway inhibitor (TFPI) associated with plasma lipoproteins. Analysis of the primary structure of Apo B-100 showed a higher than expected occurrence of lysine groups in the receptor-binding region. In order to demonstrate the participation of lysine groups of Apo B-100 in the inhibition of thromboplastin, thromboplastin and Apo B-100 were incubated together in the presence of poly-L-lysine, poly-L-arginine, lysine and arginine monomers. The inhibition of thromboplastin by Apo B-100 was completely suppressed in the presence of poly-L-lysine. Poly-L-arginine was found to be less effective and neither lysine or arginine monomers had any significant effect on the inhibitory effect of Apo B-100. Alterations in the structure of Apo B-100 reconstituted in lipid vesicles resembling LDL, brought about by lipid peroxidation and lipid loading were examined by means of Fourier transform infra-red spectroscopy. It was found that, upon oxidation without the addition of cupric ions, the apolipoprotein attains a more exposed conformation with an increase in alpha-helical structure. This increase occurred at the expense of beta-structure. On lipid loading, an increase in beta-structure at the expense of the alpha-helix, was demonstrated. It is therefore proposed that the variable action of LDL towards thromboplastin derives from alterations in the secondary structure of the Apo B-100, particularly the receptor-binding region.

Ubiquitin-proteasome-dependent degradation of apolipoprotein B100 in vitro.

Apolipoprotein B100 (apoB) is a large secretory protein that forms very low density lipoprotein in liver. An in vitro degradation assay was developed using rabbit reticulocyte (RR) lysate in order to investigate the mechanism of intracellular degradation of newly synthesized apoB by the ubiquitin-proteasome pathway. [3H]apoB, isolated from [3H]leucine pulsed/chased Hep G2 cells, was degraded 51% when incubated for 2 h at 37 degreesC in an assay mixture that included RR lysate (source of the ubiquitin conjugation system and proteasome) and an exogenous ATP regenerating system. ApoB degradation was ATP-dependent and degradation fragments were not observed suggesting that the very large apoB molecule was extensively degraded. ApoB degradation was decreased to 50% when potent proteasome inhibitors, clasto-lactacystin beta-lactone (10 microM) or MG-132 (50 microM), were added to the reaction mixture, but was not affected by the cysteine protease inhibitor, E-64, or the serine protease inhibitor, phenylmethylsulfonyl fluoride. ApoB degradation was inhibited by the mutant ubiquitin protein K48R and by ubiquitin aldehyde, an inhibitor of ubiquitin-protein isopeptidases. During incubation ubiquitination of apoB increased even as apoB was being degraded. These results suggest that in vitro degradation of apoB, a large secretory protein that is normally found in the endoplasmic reticulum (ER) lumen or associated with the ER membrane, was proteasome-dependent and involved both ubiquitination and deubiquitination steps.

The visceral yolk sac--an important site of synthesis and secretion of apolipoprotein B containing lipoproteins in the feto-placental unit of the rat.

Rat fetuses exhibit a high serum LDL concentration at term. Delivery caused a marked decrease of the LDL apolipoprotein (apo) B concentration independent of whether this occurred on days 21, 22 or 23 of gestation. The interruption of the yolk sac circulation by a ligature in situ for 6 h led to the same alterations of the LDL-apo B concentration as Caesarean section. Immunoelectronmicroscopic studies provided evidence that the epithelial cells of the visceral yolk sac exhibited electron dense LDL-sized and apo B containing particles which were localized over the compartments of the Golgi complexes, endoplasmatic reticulum, secretory vesicles and intercellular spaces, but not over the cell nuclei, mitochondria or lysosomes. ApoB containing LDL-sized particles could be obtained by ultracentrifugation from the disrupted material of the microsomal fraction of yolk sac homogenates. Isolated segments of the yolk sac membranes were capable to secrete apoB containing lipoproteins floating in the d less than 1.020 g/ml as well as in the d = 1.020-1.064 g/ml fraction with a 10-fold higher amount of apoB in the higher density class. Incorporation experiments with [35S] methionine gave evidence that these lipoproteins were at least partially provided with newly synthesized apoB predominantly found in the LDL fraction. The size of the negatively stained particles in the d = 1.020-1.064 g/ml fraction secreted from yolk sac segments corresponded to that of LDL from fetal rat serum. In contrast their acylglycerol content was significantly higher, whereas the percentage contribution of total cholesterol and protein was markedly reduced in comparison with serum LDL of the fetus. In summary, biochemical and ultrastructural studies provide clear cut evidence that the rat yolk sac is able to synthesize and to deliver apo B containing lipoproteins in the density ranges of VLDL, IDL and particular of LDL thus contributing to the supply of serum lipoproteins in the rat fetus. By recalculation of recent tracer kinetic data (Plonné et al. (1990) J. Lipid Res. 31, 747) using a mathematical step function model it was possible to assess the contribution of the rat yolk sac to the LDL influx into the fetal serum.

Growth-stimulating effect of lipoproteins on human arterial smooth-muscle cells and lung fibroblasts is due to apo B-containing lipoproteins, type LDL and VLDL, and requires LDL receptors.

Excessive growth of the arterial smooth muscle is essential for the development of atherosclerosis and leads to arterial insufficiency in several other conditions. It is therefore important to elucidate the mechanisms that regulate the growth of the human arterial smooth-muscle cell, SMC. Like other untransformed cells, SMC require plasma for sustained growth in vitro. As found in an earlier study most of the material in plasma which stimulates SMC growth is related to the lipoproteins (LP), and is widespread among LP of different density classes. In the present study we investigated whether the growth-stimulating activity might be more specifically related to certain lipoproteins defined by criteria other than density or particle size. Activity was assayed using human SMC and human lung fibroblasts as both a change of culture size and DNA synthesis. The growth-stimulating activity was confined to apo B-containing LP, as defined by their strong affinity to heparin-Sepharose, electrophoretic beta-mobility, the presence of apo B and the absolute requirement of low density lipoprotein (LDL) receptors for the growth-stimulating effect to appear. It was strongly potentiated by PDGF-BB. A much higher level of LDL was required to initiate synthesis of DNA in SMC than in fibroblasts but at optimal LDL concentration the degree of activation was similar for both cell types. Apo B-containing LP are very powerfully related to atherosclerosis. As intimal thickening is a primary change in atherogenesis, the growth-stimulating effect of them may be of direct pathogenetic importance.

Apolipoprotein B is a major perforin inhibitor protein in human serum.

We purified the high-molecular-weight perforin inhibitor protein from normal human serum using DEAE-cellulose, HPLC-gel filtration and hydroxylapatite chromatography. This protein was shown to be identical to the serum apolipoprotein B-100 in terms of amino acid composition and the sequence of the digested peptides. This inhibitor protein not only inhibits the membrane binding activity of perforin but also the pore insertion activity of membrane-bound perforin.

The chromogenicity and quantitation of apoB-100 and apoB-48 of human plasma lipoproteins on analytical SDS gel electrophoresis.

ApoB-100 and apoB-48 may be readily resolved in 3.3% sodium dodecyl sulphate-polyacrylamide gels. This study has characterized the relative chromogenicities (staining intensity/micrograms protein) of human apoB-100 and apoB-48 in various lipoprotein classes with Coomassie Brilliant Blue (R250) upon SDS-PAGE. The relation between dye uptake and the mass of each apoB species in any lipoprotein preparation, was linear at least within the concentration range of total apoprotein B which is optimally resolved in these gels (20-50 micrograms total apoprotein B), and was a function of the density of the particular lipoprotein fraction under investigation. There was a constant and characteristic difference between the chromogenicity for apoB-100 and that for apoB-48 as determined from the slopes of their respective chromogenicity curves. The slope of the lines describing staining intensity vs. protein mass for both apoB-100 and apoB-48 decreased as the density of the lipoprotein fraction increased. The slope of the line for apoB-100 was steeper than that for apoB-48 (i.e. chromogenicity apoB-100 greater than apoB-48) in all lipoprotein fractions where both were present. The relationship between the slopes of the lines for apoB-100 and apoB-48 was constant regardless of the density of the lipoprotein fraction. The chromogenicity curves for apoB-100 and for apoB-48 obtained when lipoprotein samples were applied to gels in concentrations conventionally used for this technique (i.e. 20-100 micrograms total apoB/gel) did not extrapolate to the same point on the ordinate, which precludes the use of a simple ratio or "chromogenicity factor" to describe their relative chromogenicities over this concentration range, Hence, a novel approach was developed to determine the relative mass of apoB-100/apoB-48 in lipoprotein samples, based on their staining characteristics in SDS-PAGE.

Extraction of lipoprotein(a), apo B, and apo E from fresh human arterial wall and atherosclerotic plaques.

Several studies have analysed apo(a) quantitatively in arterial wall tissue derived from post mortem samples. The purpose of this study was a qualitative analysis of Lp(a) in fresh human arterial wall tissue. It was evaluated whether Lp(a) exists as an intact lipoprotein or whether it is degraded. Additionally it was analysed whether there are differences in the apolipoprotein composition between lesion-free and diseased human arterial wall tissue. Serum and intimal tissue samples taken from the abdominal aorta and the inferior caval vein of 18 organ donors were analysed for lipids, Lp(a), and apolipoproteins apo B and apo E. Serum and tissue parameters were correlated. In the aortic tissue, higher Lp(a) and apolipoprotein levels were observed in the diseased samples. The total amount of Lp(a) recovered during three different extraction procedures was 5 micrograms/g wet weight in tissue free of plaque and 11.8 micrograms/g wet weight in atherosclerotic tissue. The corresponding values for apo B and apo E were 4.3 and 6.1 micrograms/g wet weight vs. 5.0 and 9.1 micrograms/g wet weight. After density gradient centrifugation of the aortic tissue extracts, it was shown that the major parts of apo(a) and apo B detected in the lesion-free vessel wall were present as Lp(a)-like particles. In the diseased tissue Lp(a) was partly dissociated into LDL-like particles and free apo(a). With this study we confirm that Lp(a) accumulates in the arterial wall, preferentially in diseased tissue, and that Lp(a) particles, deposited in atherosclerotic plaques, are partly degraded to LDL-like particles and free apo(a) in atherosclerotic plaques.

Beta-very low density lipoproteins in cholesterol-fed rabbits are of hepatic origin.

When rabbits are fed a cholesterol-rich diet they accumulate beta-migrating very low density lipoprotein (beta-VLDL) in their plasma. beta-VLDL are cholesteryl ester-rich lipoproteins which contain apolipoproteins B and E. There are 2 forms of apolipoprotein B in beta-VLDL. About 90% of apolipoprotein B is present as a 320 000-dalton protein and the remainder is present as a 210 000-dalton protein. These apolipoproteins are tissue specific. Lipoproteins secreted by perfused rabbit livers contain only the 320 000-dalton apolipoprotein B while lipoproteins secreted by the intestine contain only the 210 000-dalton apolipoprotein B. The tissue specificity of apolipoprotein B shows that beta-VLDL is largely of hepatic origin and that only a small fraction is of intestinal origin. The composition of VLDL secreted from the livers of cholesterol-fed rabbits is similar to that of plasma beta-VLDL. Both are cholesteryl ester-rich, in contrast to plasma and perfusate VLDL from normal rabbits which are both triglyceride-rich. This indicates that the cholesteryl ester-rich hepatic VLDL is a direct precursor for plasma beta-VLDL.