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)     

Testing the role of apoA-I,HDL, and cholesterol efflux in the atheroprotective action of low-level apoE expression

Low levels of transgenic mouse apolipoprotein E (apoE) suppress atherosclerosis in apoE knockout (apoE-/-) mice without normalizing plasma cholesterol. To test whether this is due to facilitation of cholesterol efflux from the vessel wall, we produced apoA-I-/-/apoE-/- mice with or without the transgene. Even without apoA-I and HDL, apoA-I-/-/apoE-/- mice had the same amount of aorta cholesteryl ester as apoE-/- mice. Low apoE in the apoA-I-/-/apoE-/- transgenic mice reduced aortic lesions by 70% versus their apoA-I-/-/apoE-/- siblings. To define the free cholesterol (FC) efflux capacity of lipoproteins from the various genotypes, sera were assayed on macrophages expressing ATP-binding cassette transporter A1 (ABCA1). Surprisingly, ABCA1 FC efflux was twice as high to sera from the apoA-I-/-/apoE-/- or apoE-/- mice compared with wild-type mice, and this activity correlated with serum apoA-IV. Immunodepletion of apoA-IV from apoA-I-/-/apoE-/- serum abolished ABCA1 FC efflux, indicating that apoAI-V serves as a potent acceptor for FC efflux via ABCA1. With increasing apoE expression, apoA-IV and FC acceptor capacity decreased, indicating a reciprocal relationship between plasma apoE and apoA-IV. Low plasma apoE (1-3 x 10(-8) M) suppresses atherosclerosis by as yet undefined mechanisms, not dependent on the presence of apoA-I or HDL or an increased capacity of serum acceptors for FC efflux.     

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.     

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)     

Distribution of apolipoprotein A-IV in human plasma

Human apoA-IV was purified from delipidated urinary chylomicrons. Monospecific antibodies were raised in rabbits and used to develop a double antibody radioimmunoassay (RIA). Displacement of 125I-labeled apoA-IV by plasma or purified chylomicron apoA-IV resulted in parallel displacement curves, indicating that apoA-IV from both sources share common antigenic determinants. The apoA-IV level in plasma from normal healthy fasting male subjects (n = 5) was 37.4 +/- 4.0 mg/dl, while fat-feeding increased the level to 49.1 +/- 7.9 mg/dl (P less than 0.05) at 4 hr. The apoA-IV level in plasma from abetalipoproteinemic fasting subjects was 13.7 +/- 3.1 mg/dl (n = 5). Plasma from a single fasting Tangier subject showed a reduced apoA-IV level of 21.1 mg/dl. The distribution of apoA-IV in fasting and postprandial plasma was determined by 6% agarose gel chromatography. Fifteen to 25% of plasma apoA-IV eluted in the region of plasma high density lipoprotein (HDL), with the remainder eluting in subsequent column fractions. In abetalipoproteinemic plasma this HDL fraction is reduced and lacks apoA-IV, suggesting that at least some of the apoA-IV on these particles is normally derived from triglyceride-rich lipoproteins. Lipemic plasma from a fat-fed subject showed a small rise (3%) in chylomicron-associated apoA-IV. Gel-filtered HDL and subsequent apoA-IV-containing fractions were subjected to 4-30% polyacrylamide gradient gel electrophoresis (4/30 GGE), and apoA-IV was identified by immunolocalization following transfer of proteins to nitrocellulose paper. In normal plasma apoA-IV was localized throughout all HDL fractions. In addition, normal plasma contained apoA-IV localized in a small particle (diameter 7.8-8.0 nm). This particle also contained apoA-I and lipid. A markedly elevated saturated to unsaturated cholesteryl ester ratio was present in gel-filtered plasma fractions containing small HDL, suggesting an intracellular origin of these particles. In abetalipoproteinemic plasma apoA-IV was absent from all HDL fractions except for the small HDL particles, suggesting that they are not derived from the surface of triglyceride-rich particles. All plasmas contained free apoA-IV. In contrast to gel-filtered plasma, lipoprotein subfractions of fasted normal plasma prepared in the ultracentrifuge primarily contained apoA-IV in the d greater than 1.26 g/ml fraction, suggesting an artifactual redistribution of the apolipoprotein during centrifugation. Overall, these data suggest that apoA-IV secretion into plasma is increased with fat feeding, and that apoA-IV normally exists as both a free apolipoprotein and in association with HDL particles.     

Apolipoprotein composition of HDL in cholesteryl ester transfer protein deficiency

Our purpose was to compare HDL subpopulations, as determined by nondenaturing two-dimensional gel electrophoresis followed by immunoblotting for apolipoprotein A-I (apoA-I), apoA-II, apoA-IV, apoCs, and apoE in heterozygous, compound heterozygous, and homozygous subjects for cholesteryl ester transfer protein (CETP) deficiency and controls. Heterozygotes, compound heterozygotes, and homozygotes had CETP masses that were 30, 63, and more than 90% lower and HDL-cholesterol values that were 64, 168, and 203% higher than those in controls, respectively. Heterozygotes had approximately 50% lower pre-beta-1 and more than 2-fold higher levels of alpha-1 and pre-alpha-1 particles than controls. Three of the five heterozygotes' alpha-1 particles also contained apoA-II, which was not seen in controls. Compound heterozygotes and homozygotes had very large particles not observed in controls and heterozygotes. These particles contained apoA-I, apoA-II, apoCs, and apoE. However, these subjects did not have decreased pre-beta-1 levels. Our data indicate that CETP deficiency results in the formation of very large HDL particles containing all of the major HDL apolipoproteins except for apoA-IV. We hypothesize that the HDL subpopulation profile of heterozygous CETP-deficient patients, especially those with high levels of alpha-1 containing apoA-I but no apoA-II, represent an improved anti-atherogenic state, although this might not be the case for compound heterozygotes and homozygotes with very large, undifferentiated HDL particles.     

Triiodothyronine increases rat apolipoprotein A-I synthesis and alters high-density lipoprotein composition in vivo

The effects of altered serum 3,3',5-triiodothyronine levels on rat lipoprotein metabolism were examined. Daily injections of the hormone (50 micrograms/100 g body mass) over a period of six days led to an increase of 6.4-fold in the hepatic mRNA level for apolipoprotein(apo)A-I, and a 21% increase in serum apoA-I levels. 12h after a single injection of 3,3',5-triiodothyronine the rate of [14C]leucine incorporation into apoA-I increased 2.1 fold. Conversely, in hypothyroid rats there was a decrease in hepatic mRNA levels for apoA-I and a decreased rate of [14C]leucine incorporation into apoA-I. The increase in hepatic apoA-I mRNA levels following 3,3',5-triiodothyronine treatment occurred prior to significant changes in serum triacylglycerol levels. High-density lipoprotein (HDL) particles isolated from the serum of hyperthyroid rats were smaller and enriched in apoA-I compared to apoA-IV and apoE. Similar changes in HDL composition were observed following in vitro incubations of normal rat serum with purified rat apoA-I. The results suggest that during altered thyroid status, changes in serum HDL size and composition occur in association with significant changes in apoA-I gene expression.     

GPI-specific phospholipase D associates with an apoA-I- and apoA-IV-containing complex

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in serum and associates with high density lipoproteins (HDL). We have characterized the distribution of GPI-PLD among lipoproteins in human plasma. Apolipoprotein (apo)-specific lipoproteins containing apoB (Lp[B]), apoA-I and A-II (Lp[A-I, A-II]), or apoA-I only (Lp[A-I]) were isolated using dextran sulfate and immunoaffinity chromatography. In six human plasma samples with HDL cholesterol ranging from 39 to 129 mg/dl, 79 +/- 14% (mean +/- SD) of the total plasma GPI-PLD activity was associated with Lp[A-I], 9 +/- 12% with Lp[A-I, A-II], and 1 +/- 1% with Lp[B]; and 11 +/- 10% was present in plasma devoid of these lipoproteins. Further characterization of the GPI-PLD-containing lipoproteins by gel-filtration chromatography and nondenaturing polyacrylamide and agarose gel electrophoresis revealed that these apoA-I-containing particles/complexes were small (8 nm) and migrated with pre-beta particles on agarose electrophoresis. Immunoprecipitation of GPI-PLD with a monoclonal antibody to GPI-PLD co-precipitated apoA-I and apoA-IV but little or no apoA-II, apoC-II, apoC-III, apoD, or apoE. In vitro, apoA-I but not apoA-IV or bovine serum albumin interacted directly with GPI-PLD, but did not stimulate GPI-PLD-mediated cleavage of a cell surface GPI-anchored protein. Thus, the majority of plasma GPI-PLD appears to be specifically associated with a small, discrete, and minor fraction of lipoproteins containing apoA-I and apoA-IV. -- Deeg, M. A., E. L. Bierman, and M. C. Cheung. GPI-specific phospholipase D associates with an apoA-I- and apoA-IV-containing complex. J. Lipid Res. 2001. 42: 442--451.     

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.     

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.     

Identification and characterization of rat serum lipoprotein subclasses. Isolation by chromatography on agarose columns and sequential immunoprecipitation

Lipoproteins, present in serum of chow-fed rats, were fractionated according to size by chromatography of serum on 6% agarose columns. The distributions of apolipoprotein (apo) A-I, E, and A-IV within the high density lipoprotein (HDL) size range (i.e., lipoprotein complexes smaller than low density lipoproteins) showed the existence of lipoprotein subclasses with different size and chemical composition. Sequential immunoprecipitations were performed on these fractions obtained by agarose column chromatography, using specific antisera against apoA-I, apoE, and apoA-IV. The resulting precipitates and supernatants were analyzed for cholesteryl esters, unesterified cholesterol, phospholipids, triglycerides, and specific lipoproteins. The following conclusions were drawn from these experiments. Sixty-three +/- 3% of apoE in the total HDL size range is present on a large particle (mol wt 750,000). This lipoprotein contains apoE as its sole protein constituent and is called LpE. Thirty-nine +/- 4% of the cholesterol found in the HDL size range is present in this fraction. The cholesterol:phospholipid ratio is 1:1.1. Sixty-nine +/- 8% of apoA-I in the total HDL size range is present on a smaller particle (mol wt 250,000). This apoA-I-HDL has apoA-I as its major protein component and possibly contains minor amounts of C apoproteins and A-II, but neither apoE nor apoA-IV. It contains 39 +/- 8% of the total cholesterol found in the HDL size range and the cholesterol:phospholipid ratio is 1:1.6.(ABSTRACT TRUNCATED AT 250 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.     

Effect of lecithin:cholesterol acyltransferase on distribution of apolipoprotein A-IV among lipoproteins of human plasma

The effect of cholesterol esterification on the distribution of apoA-IV in human plasma was investigated. Human plasma was incubated in the presence or absence of the lecithin:cholesterol acyltransferase (LCAT) inhibitor 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and immediately fractionated by 6% agarose column chromatography. Fractions were monitored for apoA-IV, apoE, and apoA-I by radioimmunoassay (RIA). Incubation resulted in an elevated plasma concentration of cholesteryl ester and in an altered distribution of apoA-IV. After incubation apoA-IV eluted in the ordinarily apoA-IV-poor fractions of plasma that contain small VLDL particles, LDL, and HDL2. Inclusion of DTNB during the incubation resulted in some enlargement of HDL; however, both cholesterol esterification and lipoprotein binding of apoA-IV were inhibited. Addition of DTNB to plasma after incubation and prior to gel filtration had no effect on the apoA-IV distribution when the lipoproteins were immediately fractionated. Fasting plasma apoE was distributed in two or three peaks; in some plasmas there was a small peak that eluted with the column void volume, and, in all plasmas, there were larger peaks that eluted with the VLDL-LDL region and HDL2. Incubation resulted in displacement of HDL apoE to larger lipoproteins and this effect was observed in the presence or absence of DTNB. ApoA-I was distributed in a single broad peak that eluted in the region of HDL and the gel-filtered distribution was unaffected by incubation either in the presence or absence of DTNB. Incubation of plasma that was previously heated to 56 degrees C to inactivate LCAT resulted in no additional movement of apoA-IV onto lipoproteins, unless purified LCAT was present during incubation. The addition of heat-inactivated LCAT to the incubation, had no effect on movement of apoA-IV. These data suggest that human apoA-IV redistribution from the lipoprotein-free fraction to lipoprotein particles appears to be dependent on LCAT action. The mechanism responsible for the increased binding of apoA-IV to the surface of lipoproteins when LCAT acts may involve the generation of "gaps" in the lipoprotein surface due to the consumption of substrate from the surface and additional enlargement of the core. ApoA-IV may bind to these "gaps," where the packing density of the phospholipid head groups is reduced.     

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.     

Characterization of lipoprotein produced by the perfused rhesus monkey liver

Isolated livers from rhesus monkeys (Macaca mulatta) were perfused in order to asses the nature of newly synthesized hepatic lipoprotein. Perfusate containing [3H]leucine was recirculated for 1.5 hr, followed by an additional 2.5-hr perfusion with fresh perfusate. Equilibrium density gradient ultracentrifugation clearly separated VLDL from LDL. The apoprotein composition of VLDL secreted by the liver was similar to that of serum VLDL. The perfusate LDL contained some poorly radiolabeled, apoB-rich material, which appeared to be contaminating serum LDL. There was also some material of an LDL-like density, which was rich in radiolabeled apoE. Rate zonal density gradient ultracentrifugation fractionated HDL. All perfusate HDL fractions had a decreased cholesteryl ester/unesterified cholesterol ratio, compared to serum HDL. Serum HDL distributed in one symmetric peak near the middle of the gradient, with coincident peaks of apoA-I and apoA-II. The least dense fractions of the perfusate gradient were rich in radiolabeled apoE. The middle of the perfusate gradient contained particles rich in radiolabeled apoA-I and apoA-II. The peak of apoA-I was offset from the apoA-II peak towards the denser end of the gradient. The dense end of the HDL gradient contained lipoprotein-free apoA-I, apoE, and small amounts of apoA-II, probably resulting from the relative instability of nascent lipoprotein compared to serum lipoprotein. Perfusate HDL apoA-I isoforms were more basic than serum apoA-I isoforms. Preliminary experiments, using noncentrifugal methods, suggest that some hepatic apoA-I is secreted in a lipoprotein-free form. In conclusion, the isolated rhesus monkey liver produces VLDL similar to serum VLDL, but produces LDL and HDL which differ in several important aspects from serum LDL and HDL.     

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.     

Macrophage interaction of HDL subclasses separated by free flow isotachophoresis

Preparative isotachophoresis (ITP) was used for the fractionation of fasting and postprandial high density lipoproteins (HDL) according to their net charge in the absence of molecular sieve effects. Three major HDL subpopulations with fast, intermediate, and slow mobility have been recognized. Particle size analysis by gradient gel electrophoresis has shown that in the fast-migrating subpopulation particles dominate with a size of HDL3a and HDL2b. The subpopulation with intermediate mobility contains particles with a size between HDL2a and HDL3b, while in the slow migrating subpopulation particles dominate with a size of HDL2b, HDL3a, and HDL3c. The fast-migrating subpopulation is rich in apoA-I and phosphatidylcholine. The particles of this fraction bind at 4 degrees C to HDL receptors on macrophages with high affinity (KD = 7.71 micrograms/ml; Bmax = 245.6 ng). The subpopulations with intermediate mobility is rich in apoA-II, apoE, C apolipoproteins, cholesteryl esters, and sphingomyelin. Its affinity to HDL receptors (KD = 17.7 micrograms/ml; Bmax = 198.4 ng) is lower than that of the HDL particles in the fast-migrating subfraction. The slow-migrating subpopulation consists of particles rich in apoA-IV and is associated with a high LCAT activity. This fraction expresses the highest nonspecific binding to mouse peritoneal macrophages compared to the other HDL fractions and contains only a small amount of particles that interact with HDL receptors by high affinity binding (KD = 7.3 micrograms/ml; Bmax = 95.9 ng). In 37 degrees C binding experiments the fast-migrating subfraction reveals the highest total cell-associated activity. 72% of which is trypsin-resistant. The other subfractions express a lower total cell-associated activity and 45% of the activity of the intermediate- and 43% of the activity of the slow-migrating fraction is trypsin-sensitive. When the HDL fractions are isolated from postprandial sera of the same donor, the fast-migrating particles bind at 4 degrees C with a higher affinity (KD = 4.6 micrograms/ml) while no significant changes are observed in the intermediate- and slow-migrating subpopulations. The slow- and the fast-migrating HDL subpopulations isolated from fasting serum have a high capacity to promote cholesterol removal from macrophages. We hypothesize that the HDL subpopulations rich in apoA-I promote cholesterol removal predominantly via the interaction with HDL receptors, while apoA-IV-rich HDL particles receive their driving force for cholesterol efflux from the concomitant action of LCAT via a predominantly nonspecific interaction of the particles with the cell surface.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Human apolipoprotein A-IV: displacement from the surface of triglyceride-rich particles by HDL2-associated C-apoproteins

Human apolipoprotein A-IV rapidly dissociates from the surface of lymph chylomicrons following their entry into circulation by an unknown mechanism. We have therefore investigated the binding of human apoA-IV to triglyceride-rich particles and the interaction of these apoA-IV/lipid complexes with human HDL2. Human apoA-IV was purified from lipoprotein depleted serum (J. Lipid Res. 1983. 24:52-59). Triglyceride-rich particles of well-defined properties were isolated from Intralipid, a commercially available phospholipid-triglyceride emulsion. Various concentrations of radiolabeled human apoA-IV were incubated at 24 degrees C with a fixed quantity of lipid particles; the particles were reisolated by centrifugation, and bound and free apoA-IV were quantitated. In 50 mM Tris, pH 7.4, apoA-IV bound to the triglyceride-rich particles in a non-cooperative manner, with a Kd of 2.0 microM. The calculated maximal binding was 4.96 X 10(-4) mol of apoA-IV bound per mol of phospholipid. The addition of increasing amounts of human HDL2 to the incubations caused the progressive dissociation of apoA-IV from the triglyceride-rich particles. Analysis of the reisolated particles by isoelectric focusing demonstrated the presence of C-apoproteins, suggesting their transfer from HDL2. Addition of purified apoC-III-1 to the incubations at concentrations equivalent to those present in HDL2 caused a similar dissociation of apoA-IV. HDL2 was modified to selectively remove C-apoproteins, without alteration of other physical characteristics. This modified HDL2 was four times less effective in causing apoA-IV dissociation. These results demonstrate that the lipid binding properties of human apoA-IV may be quantitatively examined using triglyceride-rich particles as model chylomicrons. This approach reproduces in vitro the dissociation of apoA-IV that occurs in vivo when mesenteric lymph chylomicrons enter the circulation, and suggests that the primary mechanism for this phenomenon is the transfer of C-apoproteins from high density lipoproteins to the triglyceride-rich particle surface. We hypothesize that this mechanism may play an important role in the modulation of chylomicron apoA-IV content in man.