Characterization of high density lipoprotein particles in familial apolipoprotein A-I deficiency

Our aim was to characterize HDL subspecies and fat-soluble vitamin levels in a kindred with familial apolipoprotein A-I (apoA-I) deficiency. Sequencing of the APOA1 gene revealed a nonsense mutation at codon -2, Q[-2]X, with two documented homozygotes, eight heterozygotes, and two normal subjects in the kindred. Homozygotes presented markedly decreased HDL cholesterol levels, undetectable plasma apoA-1, tuboeruptive and planar xanthomas, mild corneal arcus and opacification, and severe premature coronary artery disease. In both homozygotes, analysis of HDL particles by two-dimensional gel electrophoresis revealed undetectable apoA-I, decreased amounts of small alpha-3 migrating apoA-II particles, and only modestly decreased normal amounts of slow alpha migrating apoA-IV- and apoE-containing HDL, while in the eight heterozygotes, there was loss of large alpha-1 HDL particles. There were no significant decreases in plasma fat-soluble vitamin levels noted in either homozygotes or heterozygotes compared with normal control subjects. Our data indicate that isolated apoA-I deficiency results in marked HDL deficiency with very low apoA-II alpha-3 HDL particles, modest reductions in the separate and distinct plasma apoA-IV and apoE HDL particles, tuboeruptive xanthomas, premature coronary atherosclerosis, and no evidence of fat malabsorption.     

Role of LCAT in HDL remodeling: investigation of LCAT deficiency states

To better understand the role of LCAT in HDL metabolism, we compared HDL subpopulations in subjects with homozygous (n = 11) and heterozygous (n = 11) LCAT deficiency with controls (n = 22). Distribution and concentrations of apolipoprotein A-I (apoA-I)-, apoA-II-, apoA-IV-, apoC-I-, apoC-III-, and apoE-containing HDL subpopulations were assessed. Compared with controls, homozygotes and heterozygotes had lower LCAT masses (-77% and -13%), and LCAT activities (-99% and -39%), respectively. In homozygotes, the majority of apoA-I was found in small, disc-shaped, poorly lipidated prebeta-1 and alpha-4 HDL particles, and some apoA-I was found in larger, lipid-poor, discoidal HDL particles with alpha-mobility. No apoC-I-containing HDL was noted, and all apoA-II and apoC-III was detected in lipid-poor, prebeta-mobility particles. ApoE-containing particles were more disperse than normal. ApoA-IV-containing particles were normal. Heterozygotes had profiles similar to controls, except that apoC-III was found only in small HDL with prebeta-mobility. Our data are consistent with the concepts that LCAT activity: 1) is essential for developing large, spherical, apoA-I-containing HDL and for the formation of normal-sized apoC-I and apoC-III HDL; and 2) has little affect on the conversion of prebeta-1 into alpha-4 HDL, only slight effects on apoE HDL, and no effect on apoA-IV HDL particles.     

Defective functionality of HDL particles in familial apoA-I deficiency: relevance of alterations in HDL lipidome and proteome

To evaluate functional and compositional properties of HDL in subjects from a kindred of genetic apoA-I deficiency, two homozygotes and six heterozygotes, with a nonsense mutation at APOA1 codon -2, Q[-2]X, were recruited together with age- and sex-matched healthy controls (n = 11). Homozygotes displayed undetectable plasma levels of apoA-I and reduced levels of HDL-cholesterol (HDL-C) and apoC-III (5.4% and 42.6% of controls, respectively). Heterozygotes displayed low HDL-C (21 ± 9 mg/dl), low apoA-I (79 ± 24 mg/dl), normal LDL-cholesterol (132 ± 25 mg/dl), and elevated TG (130 ± 45 mg/dl) levels. Cholesterol efflux capacity of ultracentrifugally isolated HDL subpopulations was reduced (up to −25%, P < 0.01, on a glycerophospholipid [GP] basis) in heterozygotes versus controls. Small, dense HDL3 and total HDL from heterozygotes exhibited diminished antioxidative activity (up to −48%, P < 0.001 on a total mass basis) versus controls. HDL subpopulations from both homozygotes and heterozygotes displayed altered chemical composition, with depletion in apoA-I, GP, and cholesteryl ester; enrichment in apoA-II, free cholesterol, and TG; and altered phosphosphingolipidome. The defective atheroprotective activities of HDL were correlated with altered lipid and apo composition. These data reveal that atheroprotective activities of HDL particles are impaired in homozygous and heterozygous apoA-I deficiency and are intimately related to marked alterations in protein and lipid composition.     

Apolipoprotein distribution in human lipoproteins separated by polyacrylamide gradient gel electrophoresis

The heterogeneity of serum lipoproteins (excluding very low density (VLDL) and intermediate density (IDL) lipoproteins) and that of lipoproteins secreted by HepG2 cells has been studied by immunoblot analysis of the apolipoprotein composition of the particles separated by polyacrylamide gradient gel electrophoresis (GGE) under nondenaturing conditions. The reactions of antibodies to apoA-I, apoA-II, apoE, apoB, apoD, and apoA-IV have revealed discrete bands of particles which differ widely in size and apolipoprotein composition. GGE of native serum lipoproteins demonstrated that apoA-II is present in lipoproteins of limited size heterogeneity (apparent molecular mass 345,000 to 305,000) and that apoB is present in low density lipoproteins (LDL) and absent from all smaller or denser lipoproteins. In contrast, serum apoA-I, E, D, and A-IV are present in very heterogeneous particles. Serum apoA-I is present mainly in particles of 305 to 130 kDa where it is associated with apoA-II, and in decreasing order of immunoreactivity in particles of 130-90 kDa, 56 kDa, 815-345 kDa, and finally within the size range of LDL, all regions where there is little detectable apoA-II. Serum apoE is present in three defined fractions, one within the size range of LDL, one containing heterogeneous particles between 640 and 345 kDa, and one defined fraction at 96 kDa. Serum apoD is also present in three defined fractions, one comigrating with LDL, one containing heterogeneous particles between 390 and 150 kDa, and one band on the migration front. Most of serum apoA-IV is contained in a band comigrating with albumin. GGE of centrifugally prepared LDL shows the presence of apoB, apoE, and apoD, but not that of apoA-I. However, the particles containing apoA-I, which, in serum, migrated within the LDL size range and as bands of 815 to 345 kDa, were recovered upon centrifugation in the d greater than 1.21 g/ml fraction. GGE of high density lipoproteins (HDL) indicated that most of apoA-I, A-II, and A-IV were present in lipoproteins of the same apparent molecular mass (390-152 kDa). ApoD tended to be associated with large HDL, and this was also significant for HDL apoE, which is present in lipoproteins ranging from 640 to 275 kDa. GGE of very high density lipoproteins (VHDL) presented some striking features, one of which was the occurrence of apolipoproteins in very discrete bands of different molecular mass. ApoA-II was bimodally distributed at 250-175 kDa and 175-136 kDa, the latter fraction also containing apoA-I.(ABSTRACT TRUNCATED AT 400 WORDS)     

Linkage analysis of the genetic determinants of high density lipoprotein concentrations and composition: evidence for involvement of the apolipoprotein A-II and cholesteryl ester transfer protein loci

We have tested for evidence of linkage between the genetic loci determining concentrations and composition of plasma high density lipoproteins (HDL) with the genes for the major apolipoproteins and enzymes participating in lipoprotein metabolism. These genes include those encoding various apolipoproteins (apo), including apoA-I, apoA-II, apoA-IV, apoB, apoC-I, apoC-II, apoC-III, apoE, and apo(a), cholesteryl ester transfer protein (CETP), HDL-binding protein, lipoprotein lipase, and the low density lipoprotein (LDL) receptor. Polymorphisms of these genes, and nearby highly polymorphic simple sequence repeat markers, were examined by quantitative sib-pair linkage analysis in 30 coronary artery disease families consisting of a total of 366 individuals. Evidence for linkage was observed between a marker locus D16S313 linked to the CETP locus and a locus determining plasma HDL-cholesterol concentration (P = 0.002), and the genetic locus for apoA-II and a locus determining the levels of the major apolipoproteins of HDL, apoA-I and apoA-II (P = 0.009 and 0.02, respectively). HDL level was also influenced by the variation at the apo(a) locus on chromosome 6 (P = 0.02). Thus, these data indicate the simultaneous involvement of at least two different genetic loci in the determination of the levels of HDL and its associated lipoproteins.     

Characterization and functional studies of lipoproteins, lipid transfer proteins, and lecithin:cholesterol acyltransferase in CSF of normal individuals and patients with Alzheimer's disease

We investigated the lipoprotein distribution and composition in cerebrospinal fluid (CSF) in a group of patients with Alzheimer's disease (AD) or affected by other types of dementia in comparison to non-demented controls. We found slightly decreased apolipoprotein (apo)E and cholesterol concentrations in CSF of AD patients and moderately increased apoA-I concentrations, while in patients suffering from other types of dementia the apoA-I CSF concentration was increased. ApoA-IV concentrations varied widely in human CSF, but were not associated with any clinical condition. HDL(2)-like apoE-containing lipoproteins represent the major lipoprotein fraction. In CSF of normal controls, only a minor HDL(3)-like apoA-I-containing lipoprotein fraction was observed; this fraction was more prevalent in AD patients. ApoA-II was recovered mostly in the HDL(3) density range, while apoA-IV was not associated with lipoproteins but appeared in a lipid-free form, co-localizing with LCAT immunoreactivity. Bi-dimensional analysis demonstrated pre-beta and alpha apoA-I-containing particles; apoE and apoA-II were detected only in alpha-migrating particles. ApoA-IV distributed both to pre-beta and gamma-migrating particles; the LCAT signal was co-localized in this gamma-migrating fraction. Enzymatically active LCAT was present in human CSF as well as PLTP activity and mass; no CETP mass was detected. In CSF from AD patients, LCAT activity was 50% lower than in CSF from normal controls. CSF lipoproteins induced a significant cholesterol efflux from cultured rat astrocytes, suggesting that they play an active role in maintaining the cholesterol homeostasis in brain cells.     

Synthesis, modification, and flotation properties of rat hepatocyte apolipoproteins

We have studied apolipoprotein synthesis, intracellular modification and secretion by primary adult rat hepatocyte cultures using continuous pulse or pulse chase labeling with [35S]methionine, immunoprecipitation and two-dimensional isoelectric focusing/polyacrylamide gel electrophoresis. The flotation properties of the newly secreted apolipoproteins were studied by discontinuous density gradient ultracentrifugation and one- and two-dimensional polyacrylamide gel electrophoresis. These studies showed that rat hepatocyte apoE is modified intracellularly to produce minor isoproteins that differ in size and charge. One of these minor isoproteins represents a monosialated apoE form (apoE3s1). Similarly, apoCIII is modified intracellularly to produce a disialated apoCIII form (apoCIIIs2), whereas newly synthesized apoA-I and apoA-IV are not glycosylated and overlap on two-dimensional gels with the proapoA-I and the plasma apoA-IV form, respectively. Both unmodified and modified apolipoproteins are secreted into the medium. Separation of secreted apolipoproteins by density gradient ultracentrifugation has shown that 50% of apoE, 80% of apoA-I, and more than 90% of apoA-IV and apoCIII are secreted in a lipid-poor form, whereas apoB-100 and apoB-48 are 100% associated with lipids. ApoB-100 floats in the VLDL and IDL regions, whereas apoB-48 is found in all lipoprotein fractions. ApoE and small amounts of apoA-I, apoA-IV and apoCIII float in the HDL region. Small amounts of apoE and apoCIII are also found in the VLDL and IDL regions, and apoE in the LDL region. Ultracentrifugation of nascent lipoproteins in the presence of rat serum promoted flotation of apoA-I and apoA-IV in the HDL fraction and resulted in increased flotation and distribution of apoE and apoCs in VLDL, IDL and LDL regions. These observations are consistent with the hypothesis that intracellular assembly of lipoproteins involves apoB-48 and apoB-100 forms, whereas a large portion of apoA-I, apoCIII and apoA-IV can be secreted in a lipid-poor form, which associates extracellularly with preexisting lipoproteins.     

Characterization of four lipoprotein classes in human cerebrospinal fluid.

Lipoprotein metabolism in brain has not yet been fully elucidated, although there are a few reports concerning lipids in the brain and lipoproteins and apolipoproteins in the cerebrospinal fluid (CSF). To establish normal levels of lipoproteins in human CSF, total cholesterol, phospholipids, and fatty acids as well as apolipoprotein E (apoE) and apoA-I levels were determined in CSF samples from 216 individuals. For particle characterization, lipoproteins from human CSF were isolated by affinity chromatography and analyzed for size, lipid and apolipoprotein composition. Two consecutive immunoaffinity columns with antibodies, first against apoE and subsequently against apoA-I, were used to define four distinct lipoprotein classes. The major lipoprotein fraction consisted of particles of 13;-20 nm containing apoE and apoA-I as well as apoA-IV, apoD, apoH, and apoJ. In the second particle class (13;-18 nm) mainly apoA-I and apoA-II but no apoE was detected. Third, there was a small number of large particles (18;-22 nm) containing no apoA-I but apoE associated with apoA-IV, apoD, and apoJ. In the unbound fraction we detected small particles (10;-12 nm) with low lipid content containing apoA-IV, apoD, apoH, and apoJ. In summary, we established lipid and apolipoprotein levels in CSF in a large group of individuals and described four distinct lipoprotein classes in human CSF, differing in their apolipoprotein pattern, lipid composition, and size. On the basis of our own data and previous findings from other groups, we propose a classification of CSF lipoproteins.     

Distribution of apolipoprotein A-IV among lipoprotein subclasses in rat serum

The distribution of apolipoproteins (apo) A-I, A-IV, and E in sera of fed and fasted rats was studied using various methods for the isolation of lipoproteins. Serum concentrations of apoA-I and apoA-IV decreased significantly during fasting (16 and 31%, respectively), while apoE concentrations remained essentially the same. Chromatography of sera on 6% agarose columns showed that apoA-IV is present on HDL and as so-called "free" apoA-IV. The concentration of "free" apoA-IV decreased six- to seven-fold during fasting, explaining the decrease in total serum apoA-IV. Serum apoA-I and apoE are almost exclusively associated with HDL-sized particles. When sera are centrifuged at a density of 1.21 g/ml, marked quantities of apoA-I (8-9%) and apoE (11-22%) are recovered in the "lipoprotein-deficient" infranatant, suggesting that ultracentrifugation affects the integrity of serum HDL. The nature of the chromatographically separated carriers of serum apoA-IV was investigated by quantitative immunoprecipitation. From these studies, it is concluded that apoA-IV in rat serum is present in at least three fractions: 1) particles with the size and composition of HDL, containing both apoA-I and apoA-IV and possibly minor quantities of apoE; 2) HDL-sized particles containing apoA-IV, but no apoA-I or apoE; 3) "free" apoA-IV probably containing small amounts of bound cholesterol and phospholipid.     

Apolipoprotein A-IV. A determinant for binding and uptake of high density lipoproteins by rat hepatocytes

To identify the role of a specific apoprotein other than apoE which might be responsible for the receptor-mediated uptake of high density lipoprotein (HDL) by rat hepatocytes, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) was combined with rat apoE, apoA-I, or apoA-IV to form apoprotein-phospholipid complexes and the complexes were tested for their binding and uptake by primary rat hepatocytes. Apoprotein-POPC complexes were labeled with the specific fluorescent probe, 1,1-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine to monitor their uptake by cultured rat hepatocytes at 37 degrees C using digital fluorescence imaging microscopy or were labeled with 125I to study their binding to hepatocytes at 4 degrees C. POPC, either alone or with apoA-I, was not internalized by rat hepatocytes while complexes containing apoE or apoA-IV were taken up by the cells. Specific binding at 4 degrees C was demonstrated for apoE-free HDL, apoA-IV X POPC, and apoE X POPC but not for apoA-I X POPC. The binding of apoE-free HDL was inhibited by apoA-IV X POPC, apoE-free HDL, and apoA-IV + apoA-I X POPC but not by apoA-I X POPC. Binding of apoA-IV X POPC was inhibited by apoE-free HDL, apoA-IV X POPC, and apoA-IV + apoA-I X POPC, but not by apoE X POPC or apoE-enriched HDL. These data indicate that apoA-IV is a ligand responsible for the rat HDL binding to primary rat hepatocytes and that apoA-IV binds to a receptor site distinct from apoE-dependent receptors such as the apoB,E or chylomicron-remnant receptor.     

Effect of a neutralizing monoclonal antibody to cholesteryl ester transfer protein on the redistribution of apolipoproteins A-IV and E among human lipoproteins

The effect of inhibiting cholesteryl ester transfer protein (CETP) on the in vitro redistribution of apolipoproteins(apo) A-IV and apoE among lipoproteins in whole plasma was studied in seven normal male subjects. Plasmas were incubated in the presence of a purified monoclonal antibody TP2 (Mab TP2) that neutralizes the activity of CETP. Mab TP2 had no effect on lecithin:cholesterol acyltransferase (LCAT) activity. Prior to and following a 6-h incubation at 37 degrees C in the presence of Mab TP2 or a control mouse myeloma immunoglobulin (IgG), plasmas were gel-filtered on Sephacryl S-300 and the distribution of apoA-IV and apoE among lipoproteins was determined by radioimmunoassay. Incubation (i.e., with active LCAT and CETP) increased the amount of apoA-IV associated with lipoproteins by 240%. When CETP activity was inhibited during incubation, the amount of apoA-IV that became lipoprotein-associated was significantly increased (315% of basal). Plasma incubation also caused a redistribution of apoE from high density lipoproteins (HDL) to larger lipoproteins (131% of basal); however, when CETP was inhibited, significantly greater amounts of apoE became associated with the larger particles (155% of basal). These effects were observed in all seven subjects. Increased movement of apoE from HDL to triglyceride-rich particles was not due to displacement by apoA-IV since loss of apoE from HDL was still observed when no movement of apoA-IV onto HDL occurred, such as during LCAT or combined LCAT and CETP inhibition. We speculate that low CETP activity (e.g., in species such as rats) may lead to an increased content of HDL apoA-IV and also to apoE enrichment of triglyceride-rich lipoproteins, augmenting their clearance.     

A polymorphism affecting apolipoprotein A-II translational efficiency determines high density lipoprotein size and composition

High density lipoproteins (HDL) are heterogeneous particles consisting of about equal amounts of lipid and protein that are thought to mediate the transport of cholesterol from peripheral tissues to liver. We show that a previously identified polymorphism affecting HDL electrophoretic mobility in mice is due to a monogenic variation controlling HDL size and apolipoprotein composition. Thus, the HDL particles of various inbred strains of mice exhibit a striking difference in the ratio fo the two major apolipoproteins of HDL, apoA-I and apoA-II. HDL particles in all strains examined contain an average of about five apoA-I molecules; however, whereas the strains with small HDL contain two to three apoA-II molecules per particle, the strains with large HDL contain about five apoA-II molecules per particle. This increase in the protein content of the large HDL is also accompanied by increased lipid content. The HDL size polymorphism and apoA-II levels cosegregate with the apoA-II structural gene on mouse chromosome 1, indicating that a mutation of the apoA-II gene locus is responsible. The rates of synthesis of apoA-II are increased in the strains with large HDL and high apoA-II levels as compared to the strains with small HDL and low apoA-II levels. On the other hand, the fractional catabolic rates of both apoA-I and apoA-II among the strains are very similar, confirming that apoA-II concentrations are controlled at the level of synthesis. Despite the difference in rates of apoA-II synthesis between strains, the apoA-II mRNA levels in the strains are not discernibly different, suggesting that a mutation of the apoA-II structural gene controls apoA-II translational efficiency. This was confirmed by translating apoA-II mRNA in vitro using a rabbit reticulocyte lysate system. Sequencing of apoA-II cDNA from the strains revealed a number of nucleotide substitutions, which may affect translational efficiency. We conclude that the assembly of apoA-II into HDL does not have a set stoichiometry but, rather, is controlled by the production of apoA-II. As apoA-II levels increase, the HDL particles become larger and acquire more lipid, but apoA-I content per particle remains unchanged. These studies with mice provide a model for the metabolic relationships between apoA-I, apoA-II, and HDL lipid in humans.     

Tangier disease: isolation and characterization of LpA-I,LpA-II,LpA-I:A-II and LpA-IV particles from plasma

Tangier disease (TD) is characterized by extremely low plasma levels of HDL, apoA-I and apoA-II due to very rapid catabolism. However, the risk of premature coronary heart disease (CHD) is not markedly increased in TD. In order to gain insight into reverse cholesterol transport in TD, we isolated LpA-I, LpA-I:A-II, LpA-II and LpA-IV particles from fasting plasma of 5 TD patients. LpA-I composition was similar to control LpA-I, but TD LpA-I had more LCAT and CETP activity (respectively, 0.35 ± 0.14 and 0.14 ± 0.04 μmol of cholesterol esterified/h/μg of protein, and 7 ± 2.5 and 1.4 ± 0.3 μmol of cholesteryl ester transferred/h/μg of protein). In contrast, TD LpA-I:A-II had abnormal composition, with a low molar ratio of apoA-I to apoA-II (0.2–1.33). In addition, LpA-I:A-II in TD contained a substantial amount of apoA-IV compared with control, making this particle an LpA-I:A-II:A-IV complex. LpA-I:A-II from normal plasma do not promote cholesterol efflux from adipocytes cells, whereas TD LpA-I:A-II:A-IV complexes promoted cholesterol efflux from these cells. Moreover LpA-I:A-II:A-IV complexes have more LCAT and CETP activity than control (respectively 1.2 ± 0.16 and 0.01 ± 0.01 μmol of cholesterol esterified/h/μg of protein and, 41 ± 3.7 and 1 ± 0.4 μmol of cholesteryl ester transferred /h/μg of protein). The LpA-II particle in TD represented in fact in LpA-II: A-IV complex (75% mol apoA-II and 22% mol apoA-IV). This particle did not promote cholesterol efflux, but LCAT and CETP activity were present. LpA-IV particles had the capacity to promote cholesterol efflux and had both LCAT and CETP activity. LpA-IV may contribute to maintain the reverse cholesterol transport in TD. Our results indicate the potential importance of apoA-IV in maintaining reverse cholesterol transport in TD. In spite of the low steady state HDL-cholesterol levels in TD, LpA-I, LpA-I: A-II: A-IV complex and LpA-IV appear to be active in reverse cholesterol transport and may help to prevent premature CHD in TD.     

Apolipoprotein A-II inhibits high density lipoprotein remodeling and lipid-poor apolipoprotein A-I formation

The high density lipoproteins (HDL) in human plasma are classified on the basis of apolipoprotein composition into those containing apolipoprotein (apo) A-I but not apoA-II, (A-I)HDL, and those containing both apoA-I and apoA-II, (A-I/A-II)HDL. Cholesteryl ester transfer protein (CETP) transfers core lipids between HDL and other lipoproteins. It also remodels (A-I)HDL into large and small particles in a process that generates lipid-poor, pre-beta-migrating apoA-I. Lipid-poor apoA-I is the initial acceptor of cellular cholesterol and phospholipids in reverse cholesterol transport. The aim of this study is to determine whether lipid-poor apoA-I is also formed when (A-I/A-II)rHDL are remodeled by CETP. Spherical reconstituted HDL that were identical in size had comparable lipid/apolipoprotein ratios and either contained apoA-I only, (A-I)rHDL, or (A-I/A-II)rHDL were incubated for 0-24 h with CETP and Intralipid(R). At 6 h, the apoA-I content of the (A-I)rHDL had decreased by 25% and there was a concomitant formation of lipid-poor apoA-I. By 24 h, all of the (A-I)rHDL were remodeled into large and small particles. CETP remodeled approximately 32% (A-I/A-II)rHDL into small but not large particles. Lipid-poor apoA-I did not dissociate from the (A-I/A-II)rHDL. The reasons for these differences were investigated. The binding of monoclonal antibodies to three epitopes in the C-terminal domain of apoA-I was decreased in (A-I/A-II)rHDL compared with (A-I)rHDL. When the (A-I/A-II)rHDL were incubated with Gdn-HCl at pH 8.0, the apoA-I unfolded by 15% compared with 100% for the apoA-I in (A-I)rHDL. When these incubations were repeated at pH 4.0 and 2.0, the apoA-I in the (A-I)rHDL and the (A-I/A-II)rHDL unfolded completely. These results are consistent with salt bridges between apoA-II and the C-terminal domain of apoA-I, enhancing the stability of apoA-I in (A-I/A-II)rHDL and possibly contributing to the reduced remodeling and absence of lipid poor apoA-I in the (A-I/A-II)rHDL incubations.     

Gradient gel electrophoresis-immunoblot analysis (GGEI): a sensitive method for apolipoprotein profile determinations

A method is described which will determine the distribution of individual apolipoproteins within the HDL subclasses. This method requires 1-2 microliters of plasma per determination and involves six steps: 1) electrophoresis of samples on non-denaturing 2-30% concave acrylamide gradient gels; 2) electrophoretic transfer of the lipoproteins to charge-modified nylon membranes; 3) fixation of the transferred lipoproteins with glutaraldehyde; 4) immunolocalization of the apolipoproteins with iodinated monospecific antibodies; 5) autoradiography followed by densitometry; and 6) reduction of the data to provide a plot of percent distribution versus particle size. When this method was applied to the analysis of rat apolipoproteins, differences were noted in the distribution of apoA-I, apoA-IV, and apoE. The majority of apoA-I was localized to HDL particles between 9 and 12 nm in diameter, with a median diameter of 10.0 nm, while apoE resided on substantially larger particles with a median diameter of 12.5 nm. ApoA-IV could be localized to three distinct areas: an HDL particle with a median diameter approximately 0.4 nm larger than apoA-I HDL, a particle smaller than albumin (lipoprotein-free apoA-IV), and a particle of 7.6 nm that does not appear to contain apoA-I or apoE.     

Persistence of high density lipoprotein particles in obese mice lacking apolipoprotein A-I

Obese mice without leptin (ob/ob) or the leptin receptor (db/db) have increased plasma HDL levels and accumulate a unique lipoprotein referred to as LDL/HDL1. To determine the role of apolipoprotein A-I (apoA-I) in the formation and accumulation of LDL/HDL1, both ob/ob and db/db mice were crossed onto an apoA-I-deficient (apoA-I(-/-)) background. Even though the obese apoA-I(-/-) mice had an expected dramatic decrease in HDL levels, the LDL/HDL1 particle persisted. The cholesterol in this lipoprotein range was associated with both alpha- and beta-migrating particles, confirming the presence of small LDLs and large HDLs. Moreover, in the obese apoA-I(-/-) mice, LDL particles were smaller and HDLs were more negatively charged and enriched in apoE compared with controls. This LDL/HDL1 particle was rapidly remodeled to the size of normal HDL after injection into C57BL/6 mice, but it was not catabolized in obese apoA-I(-/-) mice even though plasma hepatic lipase (HL) activity was increased significantly. The finding of decreased hepatic scavenger receptor class B type I (SR-BI) protein levels may explain the persistence of LDL/HDL1 in obese apoA-I(-/-) mice. Our studies suggest that the maturation and removal of large HDLs depends on the integrity of a functional axis of apoA-I, HL, and SR-BI. Moreover, the presence of large HDLs without apoA-I provides evidence for an apoA-I-independent pathway of cholesterol efflux, possibly sustained by apoE.     

Characterization of apoA- and apoB-containing lipoprotein particles in a variant of familial apoA-I deficiency with planar xanthoma: the metabolic significance of LP-A-II particles.

This study describes a variant of familial apoA-I deficiency associated with a moderate risk for premature coronary artery disease. The proband, a 25-year-old man of Philippine origin, and his 62-year-old maternal aunt had peripheral corneal opacification, xanthelasma, and planar xanthoma; the aunt had coronary artery bypass surgery at 61 years of age. Proband's parents and three brothers were asymptomatic and apparently healthy. The characteristic apolipoprotein features of affected patients were the immunochemically and chemically undetectable apoA-I, reduced levels of apoA-II, apoC-II, apoC-III, and apoD, and normal levels of apoB and apoE; except for negligible levels of high density lipoprotein (HDL)-cholesterol (2-3 mg/dl), their plasma lipid profile was normal. The apoA-I levels in all five unaffected relatives were more than one SD below the normal mean values for their age and sex; the HDL-cholesterol levels of proband's unaffected brothers were below the 10th percentile of normal control values. Patient's very low density lipoprotein (VLDL), low density lipoprotein (LDL), and HDL contained 1.4, 80.4, and 18.1%, whereas those of control subjects contained 2.7, 28.8, and 68.1% of the total apolipoprotein mass, respectively. In unaffected relatives, the levels of LP-A-I, but not LP-A-I:A-II, were significantly lower than in controls. Neither of the two patients had detectable concentrations of LP-A-I or LP-A-I:A-II. Their HDL only consisted of LP-A-II particles, the levels of which (7-13 mg/dl) were similar to those of unaffected relatives or controls. There was no difference in the lipid composition of LP-A-II between patients and their relatives. However, LP-A-II from patients contained substantial amounts of apoC-peptides and apoE (0.40-0.98 mg/mg apoA-II), whereas those from unaffected relatives were free of these minor apolipoproteins. In patients, among all four major apoB-containing lipoproteins, only the levels of LP-B and LP-B:C were slightly higher than those in controls. Results of this study suggest a genetic cause for this variant of apoA-I deficiency characterized most probably by autosomal recessive inheritance. It appears that patients are likely to be homozygous for a gene present in single dose in the parents and brothers of the affected proband.(ABSTRACT TRUNCATED AT 400 WORDS)     

Composition and ultrastructure of size subclasses of normal human peripheral lymph lipoproteins: quantification of cholesterol uptake by HDL in tissue fluids

Peripheral lymph lipoproteins have been characterized in animals, but there is little information about their composition, and none about their ultrastructure, in normal humans. Therefore, we collected afferent leg lymph from 16 healthy males and quantified lipids and apolipoproteins in fractions separated by high performance-size exclusion chromatography. Apolipoprotein B (apoB) was found almost exclusively in low density lipoproteins. The distribution of apoA-I, particularly in lipoprotein A-I (LpA-I) without A-II particles, was shifted toward larger particles relative to plasma. The fractions containing these particles were also enriched in apoA-II, apoE, total cholesterol, and phospholipids and had greater unesterified cholesterol-to-cholesteryl ester ratios than their counterparts in plasma. Fractions containing smaller apoA-I particles were enriched in phospholipid. Most apoA-IV was lipid poor or lipid free. Most apoC-III coeluted with large apoA-I-containing particles. Electron microscopy showed that lymph contained discoidal particles not seen in plasma. These findings support other evidence that high density lipoproteins (HDL) undergo extensive remodeling in human tissue fluid. Total cholesterol concentration in lymph HDL was 30% greater (P < 0.05) than could be explained by the transendothelial transfer of HDL from plasma, providing direct confirmation that HDL acquire cholesterol in the extravascular compartment. Net transport rates of new HDL cholesterol in the cannulated vessels corresponded to a mean whole body reverse cholesterol transport rate via lymph of 0.89 mmol (344 mg)/day.     

In vivo metabolism of apolipoproteins A-IV and A-I associated with high density lipoprotein in normolipidemic subjects

The kinetics of apolipoprotein A-IV associated with high density lipoproteins (HDL) of plasma from fasting human subjects was followed for 15 days in five healthy normolipidemic volunteers. Purified apoA-IV and apoA-I were radioiodinated, respectively, with 125I and 131I, incubated in vitro with normal HDL, isolated at density 1.250 g/ml, and finally reinjected intravenously as HDL-125I-labeled apoA-IV and HDL-131I-labeled apoA-I. Blood samples were withdrawn at regular intervals for 15 days, and 24-h urine samples were collected. More than 93% (93.5 +/- 0.9%) of apoA-IV was recovered in apoA-I-containing lipoprotein particles after affinity chromatography on an anti-apoA-I column and 69.7 +/- 4.8% was bound to apoA-II in apoA-I:A-II particles separated on an anti-apoA-II column. 125I-labeled apoA-IV showed a much faster decay than 131I-labeled apoA-I for the first 5 days and thereafter the curves became parallel. Urinary/plasma ratios (U/P) for the 125I-labeled parallel. Urinary/plasma ratios (U/P) for the 125I-labeled apoA-IV were much higher than those for 131I-labeled apoA-I for the first days, but the U/P curves became parallel for the last 7 days, suggesting heterogeneity of apoA-IV metabolism. A heterogeneous multicompartmental model was constructed to describe the metabolism of lipoprotein particles containing apoA-IV and apoA-I and to calculate the kinetic parameters, fitting simultaneously all plasma and urine data for both tracers.(ABSTRACT TRUNCATED AT 250 WORDS)