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.     

Coupling of photoactivatable glycolipid probes to apolipoproteins A-I and A-II in human high density lipoproteins 2 and 3

High density lipoprotein (HDL) from human serum was subfractionated into HDL2 and HDL3 by rate-zonal density gradient ultracentrifugation. The orientation of apoproteins (apo) A-I and A-II in these subfractions was investigated by use of the photosensitive glycolipid probes, 2-(4-azido-2-nitrophenoxy)-palmitoyl[1-14C]glucosamine (compound A) and 12-(4-azido-2-nitrophenoxy)-stearoyl[1-14C]glucosamine (compound B). Both probes were added to the HDL-structures in a ratio of two or three probe molecules per particle and were photoactivated by irradiation at a wavelength above 340 nm. After delipidation the probe-apoprotein adducts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the "shallow" probe (compound A) and the "depth" probe (compound B) were coupled for 10-14% (of the label added) to apoA-I and apoA-II from HDL3 and for about 6% to apoA-I and apoA-II from HDL2. By taking into account the relative amounts of apoA-I and apoA-II, it was estimated that the "shallow" probe labeled apoA-I 40% more effectively than apoA-II in both HDL2 and HDL3; the "depth" probe labeled apoA-I and apoA-II equally well in both subfractions. The data suggest that towards the surface HDL2 and HDL3 contain a relatively larger portion of apoA-I than apoA-II, whilst towards the core both subfractions are occupied by an equal portion of apoA-I and apoA-II. Application of these photolabels has failed to point out differences in the structural organization of HDL2 and HDL3.     

Spur cells in patients with alcoholic liver cirrhosis are associated with reduced plasma levels of apoA-II, HDL3, and LDL

The precise nature and origin(s) of the abnormalities in lipoprotein and apolipoprotein profile associated with severe hepatic dysfunction and the presence of spur cells remain poorly defined. To shed light on this question, we have analyzed the plasma lipoprotein and apolipoprotein profiles in five patients with alcoholic cirrhosis and spur cells, and compared them with those of a group with similar hepatocellular dysfunction, but lacking spur cells, and with that of a control group. Lipoproteins were subfractionated by density gradient ultracentrifugation and their physicochemical properties were determined; apolipoprotein A-I, A-II, and B contents in plasma and the respective subfractions were quantitated by radial immunodiffusion, while the complement of low molecular weight apolipoproteins in each subfraction was analyzed by isoelectric focusing and electrophoresis in alkaline-urea polyacrylamide gels. Spur cell plasma was distinguished by reduced levels of apoA-II and elevated ratios of apoA-I/apoA-II (approximately 13:1 as compared to 3.3-3.9:1 in the other two groups), and by reduced concentrations of HDL3. Gradient fractionation showed the apoA-II content of HDL3 to be dramatically and significantly diminished in spur cell plasma; in addition, apoA-II content was reduced relative to apoA-I in this subclass (4.7:1 as compared to 1:1 in cirrhotics lacking spur cells and 1.9:1 in controls). Spur cell HDL2 was similarly deficient in apoA-II, with elevated ratios of apoA-I:apoA-II (9.8:1 in comparison with 1.9-2.5:1 in the two other groups). Nonetheless, high HDL2 concentrations were seen in both series of cirrhotic patients, irrespective of red cell morphology. Spur cell HDL2 thus appears to consist primarily of particles possessing only apoA-I, with a minor population containing both apoA-I and apoA-II. The free cholesterol content of all lipoprotein subfractions from spur cell plasma was increased, as indeed was the molar ratio of free cholesterol to phospholipid, in comparison with that of corresponding fractions from alcoholic cirrhotics lacking spur cells and of control subjects. LDL levels were reduced in spur cell plasma, thereby distinguishing this group from the cirrhotics without spur cells who displayed elevated LDL levels. Markedly reduced plasma levels of apoA-II, HDL3, and LDL appear characteristic of alcoholic cirrhotics presenting with spur cells. Our findings suggest that apoA-II may be essential to the normal function and metabolism of HDL, one aspect of which may be the transport of free cholesterol and thereby the direct or indirect maintenance of red cell morphology.     

Dissociation of apolipoprotein A-I from porcine and bovine high density lipoproteins by guanidine hydrochloride.

Dissociation of apolipoprotein A-I from pig and steer high density lipoproteins (HDL) deficient in apoA-II was determined by exposing native HDL fractions to 6 M guanidine hydrochloride (Gdn-HCl) at 37 degrees C for periods from 5 min to 18 h. Bovine high density lipoprotein (HDL-B) was isolated at d 1.063--1.100 g/ml while porcine high density lipoprotein (HDL-P) was isolated at d 1.125--1.21 g/ml. Incubation for 5 min with Gdn-HCl resulted in a 45 and 3% loss of apo-A-I from HDL-P and HDL-B, respectively. Exposure to the denaturant for 3 h resulted in a 75% loss of apoA-I from HDL-P and a 30% loss from HDL-B. Analytic ultracentrifugation, patterns paralleled the degree of apoA-I dissociation from each HDL species. The initial flotation peak for HDL-P shifted from F degrees 1.20 2.68 to F degrees 1.20 10.75 after 3 h exposure while HDL-B showed only a small shift from F degrees 1.20 8.30 to F degrees 1.20 8.96 after 3 h exposure. HDL-P particle diameter increased 25% after 5 min of Gdn-HCl treatment and large, flattened structures predominated after 3 h. There was no changes in the size of HDL-B after 5 min exposure and only 16% increase in particle diameter after 3 h. The difference in behavior of HDL-B and HDL-P to Gdn-HCl exposure is discussed in terms of differences in apolipoprotein A-I amino acid composition, interaction of apolipoprotein A-I with phospholipids and the possible involvement of the cholesteryl ester core.     

Interaction of free apolipoproteins with macrophages. Formation of high density lipoprotein-like lipoproteins and reduction of cellular cholesterol

Mouse peritoneal macrophages, loaded with cholesteryl ester by incubating with acetylated human low density lipoprotein containing [3H]cholesteryl oleate, were exposed to purified human apolipoproteins (apo) A-I, A-II, C-III, or E in aqueous solutions. Unesterified cholesterol was released into the medium in the presence of apoA-I, -A-II, or -E, accompanied by the decrease in intracellular cholesteryl ester. ApoC-III had no such effects. Apparent Km values of the cholesterol release were estimated as 0.11, 0.14, and 0.24 microM, and Vmax values 35, 11, and 14 micrograms of cholesterol/mg of cell protein/6 h, for apoA-I, -A-II, and -E, respectively. The products formed with apoA-I, -A-II, or -E in the media were analyzed by density gradient ultracentrifugation when the cells were preloaded with [3H]cholesteryl oleate-acetylated low density lipoproteins and [3H]choline. Free [3H]cholesterol, [3H]phosphatidylcholine, and [3H]sphingomyelin were detected coincidentally as a symmetric peak at the density of 1.1 in each case. In the complex of lipids and apoA-I or apoA-II, the weight ratios of apolipoprotein/cholesterol/phosphatidylcholine/sphingomyelin/lysophosphatidyl- choline were estimated as 2.2:1:0.6:0.2:0.07 and 4.0:1:0.5:0.3:0.07, respectively. Both of the products formed with apoA-I and -A-II migrated slower than plasma high density lipoprotein in electrophoresis on agarose gel. Because the Km values are as low as 1:340-400, 1:140-160, and 1:6-8 of plasma concentrations of apoA-I, -A-II, and -E, respectively, the results have physiological relevance for a function of the free apolipoproteins in interstitial fluid to form high density lipoprotein and to reduce cellular cholesterol.     

Differential tissue-specific expression of human apoA-I and apoA-II.

To evaluate the sources of high density lipoprotein (HDL) particles containing only apolipoprotein A-I (apoA-I), the synthesis of apoA-I and apolipoprotein A-II (apoA-II) was examined in human liver and small intestine as well as the human intestinally derived cell line, Caco-2. Human liver contained apoA-I, apoA-II as well as apolipoprotein B (apoB) mRNA. In contrast, human adult small intestine total and polyA+ RNA had little or no apoA-II despite the presence of apoA-I and apoB. Intestinal biopsies from normal individuals failed to show de novo apoA-II protein synthesis in the media of organ cultures during [35S]methionine pulse-chase labeling, whereas apoA-I could be readily detected. Caco-2 cells contained apoA-II mRNA and secreted apoA-II protein into the tissue culture media. These data indicate that the primary site of human apoA-II synthesis is in the liver and that the small intestine secretes apoA-I-containing high density lipoproteins.     

Serum opacity factor, a streptococcal virulence factor that binds to apolipoproteins A-I and A-II and disrupts high density lipoprotein structure

Serum opacity factor (SOF) is a virulence determinant of group A streptococci that opacifies mammalian sera. We analyzed the specificity and mechanism of the opacity reaction using a recombinant form of the amino-terminal opacification domain of SOF, rSOF. Our data indicate that rSOF is neither a protease nor a lipase, but rather it is the binding of rSOF to high density lipoprotein (HDL) that triggers the opacity reaction. rSOF did not opacify plasma from apoA-I(-/-) mice or purified low or very low density lipoproteins but readily opacified HDL. rSOF binding to HDL was characterized by two high affinity binding sites; it bound to apoA-I (K(d) = 6 nm) and apoA-II (K(d) = 30 nm), and both apoA-I and apoA-II blocked the binding of rSOF to HDL. Electron microscopic examination and biochemical analyses of HDL treated with rSOF revealed the formation of lipid droplets devoid of apolipoproteins. Thus, SOF interacts with HDL in human blood by binding to apoA-I and apoA-II and causing the release of HDL lipid cargo, which coalesces to form lipid droplets, resulting in opacification. The disruption of HDL may attenuate its anti-inflammatory functions and contribute to the pathogenesis of group A streptococcal infections.     

In vivo interactions of apoA-II, apoA-I, and hepatic lipase contributing to HDL structure and antiatherogenic functions

Studies with mice have revealed that increased expression of apolipoprotein A-II (apoA-II) results in elevations in high density lipoprotein (HDL), the formation of larger HDL, and the development of early atherosclerosis. We now show that the increased size of HDL results in part from an inhibition of the ability of hepatic lipase (HL) to hydrolyze phospholipids and triglycerides in the HDL and that the ratio of apoA-I to apoA-II determines HDL functional and antiatherogenic properties. HDL from apoA-II transgenic mice was relatively resistant to the action of HL in vitro. To test whether HL and apoA-II influence HDL size independently, combined apoA-II transgenic/HL knockout (HLko) mice were examined. These mice had HDL similar in size to apoA-II transgenic mice and HLko mice, suggesting that they do not increase HDL side by independent mechanisms. Overexpression of apoA-I from a transgene reversed many of the effects of apoA-II overexpression, including the ability of HDL to serve as a substrate for HL. Combined apoA-I/apoA-II transgenic mice exhibited significantly less atherosclerotic lesion formation than did apoA-II transgenic mice. These results were paralleled by the effects of the transgenes on the ability of HDL to protect against the proinflammatory effects of oxidized low density lipoprotein (LDL). Whereas nontransgenic HDL protected against oxidized LDL induction of adhesion molecules in endothelial cells, HDL from apoA-II transgenic mice was proinflammatory. HDL from combined apoA-I/apoA-II transgenic mice was equally as protective as HDL from nontransgenic mice. Our data suggest that as the ratio of apoA-II to apoA-I is increased, the HDL become larger because of inhibition of HL, and lose their antiatherogenic properties.     

High affinity, stability, and lactonase activity of serum paraoxonase PON1 anchored on HDL with ApoA-I

Serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated enzyme exhibiting antiatherogenic properties. This study examined the interaction of recombinant PON1 with reconstituted HDL comprised of PC, cholesterol, and various apolipoproteins (apoA-I, -II, and -IV). The affinity, stability, and lactonase activity were strongly correlated, with apoA-I exhibiting the strongest effects, apoA-IV exhibiting weaker yet significant effects, and apoA-II having a negative effect relative to protein-free particles. We found that PON1 binds apoA-I HDL with sub-nanomolar affinities (K(d) < 10(-)(9) M) and slow dissociation rates (t(1/2) > 80 min), while binding affinity for other particles was dramatically lower. A truncated form of PON1 lacking the N-terminal helix maintains considerable binding to apoA-I HDL (K(d) = 1.2 x 10(-)(7) M), validating the structural model which indicates additional parts of the enzyme involved in HDL binding. Kinetic inactivation assays revealed the existence of an equilibrium between two forms of PON1 differing in their stability by a factor of 100. Various lipoproteins and detergent preparations shift this equilibrium toward the more stable conformation. Consistent with its highest affinity, only apoA-I HDL is capable of totally shifting the equilibrium toward the stable form. The paraoxonase and arylesterase activities were stimulated by HDL by 2-5-fold as previously reported, almost independently of the apoliporotein content. In contrast, only apoA-I is capable of stimulating the lactonase activity by 相似文献   

Human apolipoprotein A-I liberated from high-density lipoprotein without denaturation.

Apolipoprotein A-I (apoA-I) was liberated from human high-density lipoprotein (HDL) without exposure to organic solvents or chaotropic salts by the action of isolated insect hemolymph lipid transfer particle (LTP). LTP-catalyzed lipid redistribution results in transformation of HDL into larger, less dense particles accompanied by an overall decrease in HDL particle surface area:core volume ratio, giving rise to an excess of amphiphilic surface components. Preferential dissociation of apolipoprotein versus phospholipid and unesterified cholesterol from the particle surface results in apolipoprotein recovery in the bottom fraction following ultracentrifugation at a density = 1.23 g/mL. ApoA-I was then isolated from other contaminating HDL apolipoproteins by incubation with additional HDL in the absence of LTP, whereupon apolipoprotein A-II and the C apolipoproteins reassociate with the HDL surface by displacement of apoA-I. After a second density gradient ultracentrifugation, electrophoretically pure apoA-I was obtained. Sedimentation equilibrium experiments revealed that apoA-I isolated via this method exhibits a tendency to self-associate in an aqueous solution while its circular dichroism spectrum was indicative of a significant amount of alpha-helix. Both measurements are consistent with that observed on material prepared by denaturation/renaturation. The ability of apoA-I to activate lecithin:cholesterol acyltransferase was found to be similar to that of apoA-I isolated by conventional methods. The present results illustrate that LTP-mediated alteration in lipoprotein particle surface area leads to dissociation of substantial amounts of surface active apoprotein components, thus providing the opportunity to isolate apoA-I without the denaturation/renaturation steps common to all previous isolation procedures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis, transport, and processing of apolipoproteins of high density lipoproteins

Cell biology methods have greatly influenced the elucidation of the biosynthetic pathways of apolipoproteins. In vitro and tissue culture systems allow the study, to a large extent, of the process of synthesis, intracellular processing, secretion, and extracellular processing of the major high density lipoprotein apoproteins apoA-I and A-II and also of a minor component, apoA-IV. Whereas the latter apoprotein is equipped only with a signal sequence, the primary translation products of apoA-I and apoA-II carry N-terminal extensions of preprosequence of 24 amino acids for apoA-I and 23 amino acid residues for apoA-II. The pro-form of apoA-I characterized by a hexapeptide extension is completely stable intracellularly and is secreted as such. The pro-form is further processed by a serum protease specific for an unusual -Gln-Gln-Asp-Glu-sequence site. Pro-apoA-II, a pentapeptide sequence, is partially processed intracellularly to its mature form and secreted together with the residual pro-form. The cleavage site of pro-apoA-II is characterized by two basic amino acid residues Arg-Arg, present also in other known pro-proteins. The biological function of the N-terminal pro-sequences and details of their final processing by the serum protease(s) have yet to be established.     

Plasma lipoproteins of free-ranging howling monkeys (Alouatta palliata)

1. Plasma lipids and lipoproteins of free-ranging howling monkeys from Costa Rica (Alouatta palliata), aged 5 months to 23 years, were characterized. 2. High density lipoproteins were lipid-rich, similar to HDL2 of human plasma. 3. Fatty acid compositions of major lipid classes of very low, low and high density lipoproteins differed among social groups, possibly due to both dietary and genetic factors. 4. Low and high density lipoprotein phospholipids were enriched in phosphatidylethanolamine. 5. Howler plasma cross reacted with antihuman apoA-I antibodies but not with antihuman LDL antibodies. 6. No dimeric form of apoA-II was present, unlike human apoA-II.     

Prevention of low density lipoprotein aggregation by high density lipoprotein or apolipoprotein A-I

We have shown previously that low density lipoprotein (LDL) subjected to vortexing forms self-aggregates that are avidly phagocytosed by macrophages. That phagocytic uptake is mediated by the LDL receptor. We now show that LDL self-aggregation is strongly inhibited (80-95%) by the presence of high density lipoprotein (HDL) or apolipoprotein (apo) A-I. Another type of LDL aggregation, namely that induced by incubation of LDL with phospholipase C, was also markedly inhibited by HDL or apoA-I. The aggregation of LDL induced by vortexing was not inhibited by 2.5 M NaCl, and apoA-I was still able to block LDL aggregation at this high salt concentration, strongly suggesting hydrophobic interactions as the basis for the effect of apoA-I. The fact that apoA-I protected against LDL aggregation induced by two apparently quite different procedures suggests that the aggregation in these two cases has common features. We propose that these forms of LDL aggregation result from the exposure of hydrophobic domains normally masked in LDL and that the LDL-LDL association occurs when these domains interact. ApoA-I, because of its amphipathic character, is able to interact with the exposed hydrophobic domains of LDL and thus block the intermolecular interactions that cause aggregation.     

Monoclonal antibodies to plasma high density lipoprotein (HDL) of eel (Anguilla japonica)

Six week-old female mice (Balb/c) injected intraperitonealy with 50 μg of eel high density lipoprotein (HDL) emulsified with equal volume of adjuvant three times every two weeks. Three weeks after the third injection, hyperimmunized mice were boosted by injection of 100 μg of HDL. After 5 days, the best responding mouse to injected HDL was sacrificed, and spleen cells were fused with mouse myeloma cells (Sp2/O–Ag14), and hybridomas were cultured in a selection medium. Monoclonal antibodies specific to apolipoprotein A-I or A-II (apoA-I or apoA-II) of HDL were obtained by cloning and recloning the hybridomas. Eighteen monoclonal antibodies specific to apoA-I and/or apoApII were isolated. Antibodies in the culture medium were purified by a HiTrap Protein G or an eel-HDL column. These purified antibodies belong to the subclass IgG1. The monoclonal antibodies specific to eel apoA-I and apoA-II secreted by clone 10D12 and 2G3, respectively, interact with serum proteins of some fish species such as red-sea bream and carp. The anti-eel apoA-I antibody of 10D12 did not bind to serum proteins of rat, rabbit, and chicken, while the anti-eel apoA-II of 2G3 antibody did.     

Studies on the lipid and apolipoprotein compositions of two species of apoA-I-containing lipoproteins in normolipidemic males and females

Two species of apoA-I-containing lipoproteins (A-ILp), lipoprotein containing apoA-I and apoA-II (LpA-I/A-II) and lipoprotein containing apoA-I but no apoA-II (LpA-I), have been isolated from 20 normolipidemic adults (10 males and 10 females) by immunoaffinity chromatography. We have characterized the lipid and apolipoprotein compositions in these lipoproteins, and found sex differences. In A-ILp, the levels of lipids, except triglyceride, and the level of apoE were significantly higher in females than in males. In LpA-I/A-II, sex differences were found only in the levels of apoA-I and apoE. In LpA-I, the levels of all lipids, except triglyceride, and the level of apoA-I were significantly higher in females than in males. Therefore, sex differences observed in A-ILp appear to be due primarily to the differences found in LpA-I. Of considerable significance is our finding that the ratio of cholesteryl ester to total cholesterol in LpA-I was significantly lower than that in LpA-I/A-II in both males and females. This might suggest that LpA-I could be a carrier of free cholesterol.     

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)     

Alteration of high density lipoprotein subfraction distribution with induction of serum amyloid A protein (SAA) in the nonhuman primate

Overnight chair restraint results in a dramatic increase in serum amyloid A protein (apoSAA) of nonhuman primate high density lipoprotein (HDL). To determine whether apoSAA induction resulted in a displacement of indigenous HDL protein or a change in the subfraction distribution of HDL, we analyzed the characteristics of HDL subfractions in eight vervet monkeys before and 24 hr after apoSAA induction. Blood was taken from each animal before and after chair restraint to induce apoSAA. HDL was isolated from the plasma by ultracentrifugation and agarose column chromatography. The isolated HDL was subfractionated by density gradient centrifugation and five resulting subfractions were analyzed for protein and lipid content. With apoSAA induction there was a significant increase in d less than 1.09 g/ml protein, phospholipid, and free and esterified cholesterol which resulted in a 44% increase in the total mass of this subfraction. Concomitantly, there was a significant decrease in d 1.10-1.11 g/ml protein, total cholesterol, and cholesteryl ester, which resulted in a 16% decrease in the total mass of the subfraction. The response of the d 1.10-1.11 and d greater than 1.12 g/ml subfraction protein, cholesterol, and phospholipid concentrations to chair restraint for individual animals was directly proportional to their plasma HDL concentrations. Although there was a change in the HDL subfraction concentrations after chair restraint, there was no change in the lipid composition of the HDL subfractions nor in the total amount of HDL protein. However, the apoSAA/A-I ratio was significantly increased with induction while the apoA-II + C's/A-I ratio remained unchanged. The apoSAA/A-I ratio progressively increased with the density of the HDL subfraction. The protein composition of the d greater than 1.12 g/ml subfraction was changed from an average of three apoA-I and two apoA-II (or C's) molecules per particle to an average of two apoA-I, one apoA-II (or C's), and three or four apoSAA molecules per particle after chair restraint. Thus, apoSAA was predominantly associated with the denser HDL subfractions even though the lighter HDL subfractions were the most responsive in terms of changes in concentration. These data suggest that chair restraint of nonhuman primates induces apoSAA which displaces apoA-I and apoA-II or C's from HDL without altering the overall lipid and protein composition of the particle. In addition, chair restraint alters the concentration of HDL subfractions in ways that may be independent of apoSAA induction.     

Lipoprotein composition of human suction-blister interstitial fluid

Interstitial fluid (IF) was obtained in 27 apparently healthy subjects (12 males, 15 females) by applying mild suction (200-250 mm Hg) on the skin either on the midvolar forearm or on the paraumbilical region of the abdomen. The IF concentrations of lipids and apolipoproteins (apo) were studied and compared with those of serum (S). The mean ratio between interstitial fluid and serum (IF/S ratio) varied from 0.14 for forearm apoE to 0.29 for apoA-II on the abdomen. This ratio was consistently lower for apoE, C-II, C-III, and B than for apoA-I and A-II, and significantly lower on the arm than on the abdomen for all apolipoproteins studied. The IF/S ratios showed marked variations among individuals. However, interstitial fluid apolipoprotein concentrations at different blister sites were highly correlated within each individual. Studies with agarose gel electrophoresis and density gradient ultracentrifugation revealed that large triglyceride-rich particles were virtually lacking in interstitial fluid and that the relation between the low density lipoproteins (LDL) and high density lipoproteins (HDL) was shifted towards a greater proportion of HDL. The lipoprotein distribution in the HDL range of interstitial fluid differed from that of serum showing one maximum at a density of about 1.070 g/ml (serum HDL2 about 1.090 g/ml) and one at a density of 1.130-1.140 g/ml (serum HDL3, 1.110-1.120 g/ml). The former subfraction contained most of the lipoprotein-bound apoE while the latter contained the major part of apoA-I and apoA-II. Studies of the lipoproteins of interstitial fluid may add to our understanding of the development of atherosclerosis and xanthomatosis and may also provide valuable information on the permeability of the capillary membrane in normo- and pathophysiological states.     

Homologues of the human C and A apolipoproteins in the Macaca fascicularis (cynomolgus) monkey

We used antisera to human A and C apolipoproteins to identify homologues of these proteins among the high-density lipoprotein apoproteins of Macaca fascicularis (cynomolgus) monkeys, and NH2-terminal analysis was used to verify the homology. The NH2-terminal sequence of the M. fascicularis apoA-I is identical with that of another Old World species, Erythrocebus patas, and differs from human apoA-I at only 4 of the first 24 residues. M. fascicularis apoA-II contains a serine for cysteine replacement at position 6 and is therefore monomeric like the apoA-II from all species below apes. Human and monkey apoA-II are not otherwise different through their first 25 residues. About 20% of M. fascicularis apoC-I aligns with human apoC-I through residue 22, and 80% lacks an NH2-terminal dipeptide. Otherwise, the monkey apoC-I differs from the human protein at only 2 of 25 positions. Two forms of M. fascicularis apoC-II were identified. ApoC-II1 is highly homologous with human apoC-II, whereas an NH2-terminal hexapeptide is absent from apoC-II2. ApoC-II2 was the predominant species, and apoC-II1 appears to represent a propeptide from which a hexapeptide prosegment is cleaved at a Gln-Asp bond. Both forms of monkey apoC-II are potent activators of lipoprotein lipase. There are two polymorphic forms of M. fascicularis apoC-III, and their electrophoretic mobilities become identical after treatment with neuraminidase. Except for a glycine for serine substitution at position 10, the first 15 NH2-terminal residues of M. fascicularis and human apoC-III are the same.     

Rapid quantitative apolipoprotein analysis by gradient ultracentrifugation and reversed-phase high performance liquid chromatography

A new methodology for the analysis of lipoprotein composition using a combination of gradient ultracentrifugation and high performance liquid chromatography was used to determine the differences in lipoprotein composition between non-hyperlipidemic men and women. Lipoproteins from each subject were separated into six subfractions: VLDL, IDL, LDL, and three subfractions of HDL by a single gradient ultracentrifugation spin of less than 5 hr. The HDL subfractions were designated HDL-L (the lightest density subfraction, rich in apoCs and poor in apoA-II), HDL-M (the middle subfraction, rich in apoA-II), and HDL-D (the most dense, relatively poor in both the apoCs and apoA-II). The concentrations of the water-soluble apolipoproteins in each subfraction were determined using reversed-phase HPLC. The concentrations of apoB and the lipid components of the lipoproteins were determined by chemical and enzymatic methods. This methodology proved to be highly reproducible when performed on fresh plasma samples and we were able to identify many sex-associated differences in lipoprotein composition. This methodology is the only nonimmunological technique available for analyzing lipoprotein composition that offers such a combination of accuracy, speed, and completeness.