Lipoproteins and apolipoproteins in intestinal lymph of the preruminant calf, Bos spp., at peak lipid absorption

We have recently evaluated the in vivo role of the liver in lipoprotein homeostasis in the preruminant calf (Bauchart, D., D. Durand, P. M. Laplaud, P. Forgez, S. Goulinet, and M. J. Chapman, 1989. J. Lipid Res. 30: 1499-1514). We now present the partial characterization of lipoprotein particles in postprandial intestinal lymph at peak lipid absorption (i.e., 10 h after a meal) in the preruminant calf fed a curdled milk replacer. Intestinal lymph from four male preruminant calves was analyzed for its content of lipids and fractionated by sequential and density gradient ultracentrifugation into chylomicrons (Sf greater than 400), very low density lipoproteins (VLDL) (Sf less than 400; d less than 1.006 g/ml), and a series of lipoprotein subfractions with d greater than 1.006 g/ml. Postprandial lymph contained predominantly triglycerides (1099 +/- 611 mg/100 ml), with lesser amounts of phospholipids (197 +/- 107 mg/100 ml) and cholesterol (52 +/- 30 mg/100 ml). The most abundant particles were triglyceride-rich chylomicrons and VLDL which accounted for approximately 76% and approximately 19%, respectively, of total d less than 1.21 g/ml lipoproteins. As judged by negative stain electron microscopy, chylomicron particle diameters ranged from 650 to 2400 A, while VLDL were smaller and distributed over a distinct size range (340-860 A). These two lipoprotein classes each presented protein components with Mr comparable to those of human apoB-48, apoA-I, and C apoproteins, together with an Mr 52,000 protein resembling human beta 2-glycoprotein-I. In addition, VLDL exhibited a polypeptide with Mr approximately 61,000. Lymph lipoproteins with d greater than 1.006 g/ml consisted primarily (approximately 81% of total) of particles distributed over the 1.053-1.119 g/ml density range. Electrophoretic analysis of the latter lipoprotein fraction showed it to be heterogeneous, including particles with the migration characteristics of low and of high density lipoproteins, respectively. Subfractions in the d 1.053-1.076 g/ml range were dominated by particles with Stokes diameters typical of high density lipoproteins (HDL), but also contained three different populations of low density lipoprotein-like particles. The high molecular weight apolipoproteins in these same cholesteryl ester-rich (greater than 30% of lipoprotein mass) subfractions comprised components with Mr resembling those of human apoB-100 and apoB-48, respectively, and with the latter protein predominating to a varying degree. A counterpart to human apoA-I was the major protein component over the entire density range from d 1.053 to 1.119 g/ml.(ABSTRACT TRUNCATED AT 400 WORDS)     

Subfractionation of 1.006-1.063 g/ml components of badger plasma lipoproteins by using heparin-Sepharose affinity chromatography: relevance to the endocrine regulation of lipoprotein metabolism

Badger plasma lipoproteins with density 1.006-1.063 g/ml have been subfractionated by means of affinity chromatography on a heparin-Sepharose column, using a modification of the method reported by Weisgraber and Mahley (1980. J. Lipid Res. 21: 316-325). These experiments have provided evidence for the presence of three lipoprotein subfractions hereinafter termed fractions I, II, and III. Fraction I was cholesteryl ester- and phospholipid-rich (ca. 35% and 30% of lipoprotein mass, respectively), and contained apoA-I as its prominent apolipoprotein constituent. In contrast, triglyceride-rich fractions II and III both exhibited a complex apolipoprotein pattern, including apoB-100, apoA-I, and apoE whose amino acid composition and NH2-terminal sequence in the badger are reported. However, fraction III appeared markedly enriched in apoE when compared to fraction II. On polyacrylamide gel electrophoresis, fraction I presented as a spectrum of particles with diameters in the 140-190 A range. In contrast, fraction II migrated as a single band with a diameter of approximately 200 A, and fraction III presented as a single band or a doublet with a diameter of 195-200 A. The respective plasma concentrations and chemical compositions of the three chromatographic fractions were determined at four different dates of the year (i.e., April, August, November, and January), each of which corresponded to a different endocrine status in the badger. Thus hypothyroidism appeared to be associated with an increase in the concentration of fraction I, while the lowering in summer of the plasma level of testosterone correlated well with an increase in the concentration of fraction II. At the same time, the respective proportions of hydrophobic lipids in this latter material modified with an increase of triglycerides. Finally, both the apolipoprotein pattern of fraction III, and the chronologic profile of the successive variations of its concentration, suggest that it could represent a metabolic precursor of fraction II. These results suggest that the respective metabolism of the lipoproteins constituting the three chromatographic fractions could be under control by thyroid and testis secretions, operating via a complex combined regulation of the activities of the enzymes and receptors involved in these metabolic processes.     

Plasma lipoproteins and apolipoproteins in the preruminant calf, Bos spp: density distribution, physicochemical properties, and the in vivo evaluation of the contribution of the liver to lipoprotein homeostasis

The in vivo role of the liver in lipoprotein homeostasis in the preruminant calf, a functional monogastric, has been evaluated. To this end, the hydrodynamic and physicochemical properties, density distribution, apolipoprotein content, and flow rates of the various lipoprotein particle species were determined in the hepatic afferent (portal vein and hepatic artery) and efferent (hepatic vein) vessels in fasting, 3-week-old male preruminant calves. Plasma lipoprotein profiles were established by physicochemical analyses of a series of subfractions isolated by isopycnic density gradient ultracentrifugation. Triglyceride-rich very low density lipoproteins (VLDL) (d less than 1.018 g/ml) were minor plasma constituents (approximately 1% or less of total d less than 1.180 g/ml lipoproteins). The major apolipoproteins of VLDL were apoB-like species, while the complement of minor components included bovine apoA-I and apoC-like peptides. Particles with diameters (193-207 A) typical of low density lipoproteins (LDL) were present over the density interval 1.026-1.076 g/ml; however, only LDL of d 1.026-1.046 g/ml were present as a unique and homogeneous size subspecies, containing the two apoB-like species as major protein components in addition to elevated cholesteryl ester contents. LDL represented approximately 10% of total d less than 1.180 g/ml lipoproteins in fasting plasma from all three hepatic vessels. Overlap in the density distribution of particles with the diameters of LDL and of high density lipoproteins (HDL) occurred in the density range from 1.046 to 1.076 g/ml; these HDL particles were 130-150 A in diameter. HDL were the major plasma particles (approximately 90% of total d less than 1.180 g/ml substances) and presented as two distinct populations which we have termed light (HDLL) and heavy (HDLH) HDL. Light HDL (d 1.060-1.091 g/ml) ranged in size from 120 to 140 A, and were distinguished by their high cholesteryl ester (29-33%) and low triglyceride (1-3%) contents; apoA-I was the principal apolipoprotein. Small amounts of apolipoproteins with Mr less than 60,000, including apoC-like peptides, were also present. Heavy HDL (d 1.091-1.180 g/ml) accounted for almost half (47%) of total calf HDL, and like HDLL, were also enriched in cholesteryl ester and apoA-I; they ranged in size from 93 to 120 A. The protein moiety of HDLH was distinct in its possession of an apoA-IV-like protein (Mr 42,000). Blood flow rates were determined by electromagnetic flowmetry, thereby permitting determination of net lipoprotein balance across the liver. VLDL were efficiently removed during passage through the liver (net uptake 1.06 mg/min per kg body weight).(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Prebeta-migrating high density lipoprotein: quantitation in normal and hyperlipidemic plasma by solid phase radioimmunoassay following electrophoretic transfer

A quantitative solid phase immunoassay has been developed for the determination of the mass of electrophoretically separated prebeta apolipoprotein A-I (apoA-I) in human plasma. Conditions have been identified for the quantitative transfer and immunoblotting of the apolipoprotein in the absence of organic solvents or detergents. In normolipidemic plasma, the prebeta-migrating fraction of apoA-I represented 4.2 +/- 1.8% of total apoA-I (61 +/- 26 micrograms of apoA-I per ml of plasma). Significantly higher levels were found in hypercholesterolemia of genetic origin, in primary and secondary hypertriglyceridemia, and in congenital lecithin:cholesterol acyltransferase deficiency. In all cases prebeta-migrating apoA-I consisted in large part of low molecular weight lipoprotein species, compared to the size of the major, alpha-migrating apoA-I fraction.     

Distribution and functions of lecithin:cholesterol acyltransferase and cholesteryl ester transfer protein in plasma lipoproteins. Evidence for a functional unit containing these activities together with apolipoproteins A-I and D that catalyzes the esterification and transfer of cell-derived cholesterol

The distribution of apolipoprotein A-I, apolipoprotein D, lecithin:cholesterol acyltransferase, and cholesteryl ester transfer protein in fasting normal human plasma was determined by two-dimensional electrophoresis followed by immunoblotting. The synthesis and transfer of labeled cholesteryl esters generated in plasma briefly incubated with [3H]cholesterol-labeled fibroblasts was followed in terms of the lipoprotein species containing these antigens. Following the early appearance of labeled free cholesterol in two pre beta-migrating apolipoprotein A-I species (Castro, G. R., and Fielding, C. J. (1988) Biochemistry 27, 25-29), labeled esters were first detected, after a 2-min delay, in a third pre beta-migrating species which also contained apolipoprotein D, lecithin:cholesterol acyltransferase, and cholesteryl ester transfer protein. Pulse-chase experiments determined that label generated in this fraction was the precursor of at least a major part of labeled cholesteryl esters in the bulk of alpha-migrating high density lipoprotein. Over the maximum time course of these experiments (15 min, 37 degrees C), less than 10% of labeled cholesteryl esters were recovered in low or very low density lipoproteins separated by electrophoresis, immunoaffinity, or heparin-agarose chromatography. These data suggest channeling of cell-derived cholesterol and cholesteryl esters derived from it through a preferred pathway involving several minor pre beta-migrating lipoproteins to alpha-migrating high density lipoprotein.     

Characterization of apolipoprotein A-IV complexes and A-IV isoforms in human lymph and plasma lipoproteins

We have isolated and characterised A-IV apolipoprotein (apo-A-IV) from human lymph and plasma by immunoabsorbance chromatography and two-dimensional electrophoresis. Two different apo-A-IV-containing lipoproteins were isolated from four different sources, human lymph triglyceride-rich fraction (TRL), lymph lipoprotein-deficient fraction (LDF), plasma high-density lipoprotein (HDL), and plasma lipoprotein-deficient fraction (LDF). The lipoprotein complexes obtained from lymph TRL and plasma HDL were similar and contained apo-A-IV, apo-A-I, and small molecular weight peptides (apo-C or -A-II). The second lipoprotein complex was isolated from lymph LDF and plasma LDF, and contained apo-A-IV, apo-A-I, and a peptide of Mr = 59,000. The lipid composition of the lipoprotein complexes varied according to the source: triglyceride predominating in lymph TRL and phospholipid and cholesteryl ester from the other sources. Free cholesterol was conspicuously present in very small amounts. Using two-dimensional electrophoresis and immunoblotting techniques, eleven isoproteins of apo-A-IV were identified (pI-4.98, 5.06, 5.10, 5.15, 5.20, 5.22, 5.25, 5.30, 5.34, 5.42, and 5.48). The isoprotein pattern of lymph TRL and plasma HDL was similar, but that of lymph and plasma LDF were different patterns. These results suggest that apo-A-IV associated with d less than 1.21 lipoproteins and apo-A-IV present in LDF may be in metabolically separate lipoproteins and may have different physiological roles.     

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)     

Apolipoprotein E is the major physiological activator of lecithin-cholesterol acyltransferase (LCAT) on apolipoprotein B lipoproteins

Our previous studies have indicated that lecithin-cholesterol acyltransferase (LCAT) contributes significantly to the apoB lipoprotein cholesteryl ester (CE) pool. Cholesterol esterification rate (CER) in apoA-I(-)(/)(-) apoE(-)(/)(-) mouse plasma was <7% that of C57Bl/6 (B6) mouse plasma, even though apoA-I(-)(/)(-) apoE(-)(/)(-) plasma retained (1)/(3) the amount of B6 LCAT activity. This suggested that lack of LCAT enzyme did not explain the low CER in apoA-I(-)(/)(-) apoE(-)(/)(-) mice and indicated that apoE and apoA-I are the only major activators of LCAT in mouse plasma. Deleting apoE on low-density lipoprotein (LDL) reduced CER (1% free cholesterol (FC) esterified/h) compared to B6 (6% FC esterified/h) and apoA-I(-)(/)(-) (11% FC esterified/h) LDL. Similar sized LDL particles from all four genotypes were isolated by fast protein liquid chromatography (FPLC) after radiolabeling with [(3)H]-free cholesterol (FC). LDLs (1 microg FC) from each genotype were incubated with purified recombinant mouse LCAT; LDL particles from B6 and apoA-I(-)(/)(-) plasma were much better substrates for CE formation (5.7% and 6.3% CE formed/30 min, respectively) than those from apoE(-)(/)(-) and apoE(-)(/)(-) apoA-I(-)(/)(-) plasma (1.2% and 1.1% CE formed/30 min). Western blot analysis showed that the amount of apoA-I on apoE(-)(/)(-) LDLs was higher compared to B6 LDL. Adding apoE to incubations of apoA-I(-)(/)(-) apoE(-)(/)(-) very low density lipoprotein (VLDL) resulted in a 3-fold increase in LCAT CER, whereas addition of apoA-I resulted in a more modest 80% increase. We conclude that apoE is a more significant activator of LCAT than apoA-I on mouse apoB lipoproteins.     

Characterization of two lipoproteins containing apolipoproteins B and E from lesion-free human aortic intima

Lesion-free areas of aortic intimas from seven men, 30 to 49 years old, were extracted with aqueous buffer within a few hours after an accidental or sudden death. Two lipoprotein fractions could be isolated by density gradient ultracentrifugation from all cases. The mean composition of fraction I (d less than 1.012 g/ml) resembled that reported for the cholesteryl ester-rich, beta-migrating very low density lipoprotein (beta-VLDL); the composition of fraction II (d 1.021-1.046 g/ml) resembled that of plasma low density lipoprotein (LDL). Mean diameter of the particles was 35 +/- 8 nm in fraction I and 25 +/- 5 nm in fraction II (22 +/- 2 nm in plasma LDL). Both fractions contained apolipoproteins B (apoB) and E (apoE), and had increased electrophoretic mobilities and reduced contents of linoleic acid. The immunoreactivity of apoB to a polyclonal and two monoclonal antibodies in both fractions was not different from that of plasma lipoproteins. The apoE isoform patterns in both fractions were similar to those obtained from the respective postmortem plasmas. When incubated with mouse peritoneal macrophages, fractions I and II enhanced the incorporation of radioactive oleate into cholesteryl esters by 10- to 20-fold and 3- to 4-fold, respectively, in comparison to plasma LDL. In conclusion, our results indicate that lesion-free human aortic intima contains two types of apoB- and apoE-containing lipoprotein particles, both of which might be potentially atherogenic.     

Plasma lipoprotein changes attending the intravenous administration of Triton WR-1339 in normolipidemic dogs: preferential effect on high density lipoproteins

The nonionic detergent Triton WR-1339 was injected intravenously into normolipidemic dogs in a single dose of 150 mg/kg body weight followed by three other injections (75 mg/kg) on days 2, 6, and 12. The Triton produced a significant elevation of the plasma cholesterol of these animals, but not of their triglyceride levels, and profound changes of their plasma lipoproteins, particularly of the high density lipoprotein class. These changes were dependent on the concentration of Triton attained in plasma; when the levels were above 1.5 mg/ml, density gradient ultracentrifugation, electrophoretic, and chemical analyses indicated that an interaction between Triton and HDL had occurred. This interaction was attended by a gradual loss of the surface components of HDL, namely apoA-I, phospholipids, and unesterified cholesterol, and by the appearance of two cholesteryl ester-rich lipoproteins of d 1.019-1.024 g/ml and d 1.038-1.058 g/ml containing apoA-I and proteins with electrophoretic mobilities of apoB, apoE, and apoA-IV. At the time that these changes had occurred, the activities of the enzymes lecithin: cholesterol acyltransferase and post-heparin lipase were unaffected. When 125I-labeled apoA-I was injected intravenously into animals receiving Triton, the residence time of the radiolabeled protein in plasma increased from a control value of 3.1 days to 7.2 days. However, the apparent half-times of the radiolabeled apoA-I varied among the lipoprotein fractions it was associated with: d 1.119-1.159 g/ml, 5.28 days; d 1.019-1.024 g/ml, 7.55 days, and d 1.038-1.058 g/ml, 5.39 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Conjugated Plasma Proteins of the American Cockroach : I. The normal state

Five protein fractions have been separated by paper electrophoresis from the plasma of the American cockroach. With the utilization of various staining procedures several of the plasma fractions were shown to be conjugated proteins. Two of these (fractions II and IV) are readily identifiable by their phospholipid, carbohydrate, and protein composition. A third conjugated protein, fraction III, is characterized by its high neutral lipid and sterol content. This lipoprotein is also sex-specific. Another fraction (I) contains neutral lipid, sterol, and protein but electrophoretically is more mobile than fraction III. Fraction V, the last and least mobile of the normally occurring proteins, possesses electrophoretic properties similar to human fibrinogen.     

Synthetic high density lipoprotein particles. Application to studies of the apoprotein specificity for selective uptake of cholesterol esters

Particles closely resembling rat high density lipoproteins (HDL) in terms of equilibrium density profile and particle size were prepared by sonication of apoA-I with a microemulsion made with egg lecithin and cholesterol oleate. These particles, like authentic HDL, allowed selective uptake of their cholesterol ester moieties by cultured cells without parallel uptake of the particle itself. That uptake was saturable and competed by HDL. In rats, the plasma decay kinetics and sites of uptake of a cholesteryl ether tracer were similar whether that tracer was incorporated into synthetic or authentic HDL. Synthetic particles containing other apoproteins were made by generally the same method, but using in place of apoA-I either a mixture of rat apoCs or apoE that was either competent or reductively methylated to prevent interaction with the B/E receptor. These particles, of lower density and larger Stokes radius than those made with apoA-I, also allowed selective uptake of cholesterol esters, albeit with a lower degree of selectivity than in the case of apoA-I. Thus a specific apoprotein component in the subject lipoprotein particle is not required for selective uptake. However, selective uptake was shown to be a function of particle density or size, and part of the difference in rates of selective uptake from the particles made with various apoproteins was explained by their differences in density or size.     

Heterogeneity of dog interstitial fluid (peripheral lymph) high density lipoproteins: implications for a role in reverse cholesterol transport

The heterogeneity of dog interstitial fluid (peripheral lymph) high density lipoprotein (HDL) was investigated and compared to plasma HDL. Interstitial fluid and plasma HDL of normal and cholesterol-fed dogs was subfractionated by ultracentrifugation and affinity and molecular weight sieving chromatography. Both plasma (P) and interstitial fluid (L) HDL can be subfractionated into a larger fraction (P-I and L-I) and a smaller one (P-II and L-II). Cholesterol feeding induces a large increase in the P-I and L-I component of HDL, but the increase in L-I is far greater in proportion than that of P-I. Furthermore, L-I of cholesterol-fed dogs appears to be almost exclusively discoid in shape, while only approximately 15% of particles in P-I are discoidal. The discoid HDL of L-I is reflected in its chemical composition: 28% unesterified cholesterol, 6% cholesteryl ester, 45% phospholipid, and 21% protein. It contains large amounts of apoE in addition to apoA-I and apoA-IV. We found that the association of apoE with discoid particles is frequent, but not necessary. Calculations based on known protein mass and quantitation of discoid particles on electron micrographs suggest that the concentration of discoid particles in the peripheral lymph of cholesterol-fed dogs is about fourfold that of the plasma of the same animal. These findings provide strong circumstantial evidence for the peripheral formation of discoid HDL, perhaps as an early event in reverse cholesterol transport.     

Cellular cholesterol substrate pools for adenosine-triphosphate cassette transporter A1-dependent high-density lipoprotein formation

PURPOSE OF REVIEW: The removal of cellular cholesterol and phospholipids to apolipoprotein A-I (apoA-I), facilitated by the membrane transporter ATP-binding cassette transporter A1 (ABCA1), is the rate-limiting step in the formation of high density lipoprotein particles. This review summarizes recent literature concerning the relative contributions of different cellular pools of cholesterol used by ABCA1 in the initial lipidation of apoA-I for high density lipoprotein particle formation. RECENT FINDINGS: Cell culture studies have shown that apart from lipidating apoA-I directly, ABCA1 can also mediate cholesterol delivery indirectly to apoA-I in the plasma membrane. Moreover, it is now clear that the late endosome/lysosome pool of cholesterol is a critical part of the total cholesterol substrate pool for ABCA1. Internalization of ABCA1 appears to be a requirement for maximum ABCA1-mediated cholesterol mobilization for high density lipoprotein formation. SUMMARY: Current evidence suggests that ABCA1-mediated cholesterol efflux to apoA-I involves mobilization of cholesterol from plasma membrane, endoplasmic reticulum, trans-Golgi network, late endocytic and lysosomal compartments, and cholesteryl ester droplets. Apart from lipidating apoA-I directly, ABCA1 has also been found to efflux cholesterol indirectly to apoA-I in plasma membranes.     

Influence of phospholipid depletion on the size, structure, and remodeling of reconstituted high density lipoproteins

This study shows that phospholipid depletion has a major impact on the size and structure of spherical, reconstituted high density lipoproteins (rHDL) and their remodeling by cholesteryl ester transfer protein (CETP). Spherical rHDL, 9.2 nm in diameter with a phospholipid/cholesteryl ester/unesterified cholesterol/apolipoprotein A-I (apoA-I) (PL/CE/UC/A-I) molar ratio of 37.3/24.5/4.1/1.0, were depleted progressively of phospholipids by incubation with phospholipase A(2). After 30 min of incubation the PL/CE/UC/A-I molar ratio of the rHDL was 8.0/31.2/4.4/1.0 and their diameter had decreased to 8.0 nm. Comparable changes in rHDL size and composition were also apparent when the incubations were carried out in the presence of other lipoprotein classes and lipoprotein-deficient plasma. The changes in size and composition were not accompanied by the dissociation of apoA-I from the rHDL. Phospholipid depletion did not affect rHDL surface charge or the structure and stability of apoA-I. The remodeling of unmodified and phospholipid-depleted rHDL by CETP was also investigated. When the rHDL were incubated for 3 h with CETP and Intralipid, transfers of core lipids between the phospholipid-depleted rHDL and Intralipid were decreased relative to unmodified rHDL. This difference was no longer apparent when the incubations were extended beyond 3 h. In these incubations apoA-I dissociated from the phospholipid-depleted and unmodified rHDL at 3 and 12 h, respectively. At 24 h the respective diameters of the unmodified rHDL and phospholipid-depleted rHDL were 8.0 and 7.8 nm. In conclusion, phospholipid depletion has a major impact on rHDL size and their remodeling by CETP.     

Apoprotein composition and turnover in rat intestinal lymph during steady-state triglyceride absorption.

Apoproteins of chylomicrons, very low density lipoprotein (VLDL), and a low density + high density fraction secreted by proximal and distal rat small intestine into mesenteric lymph were examined during triglyceride (TG) absorption. Apoprotein output and composition were determined and the turnover rates of labeled non-apoB (soluble) apoproteins in lipoprotein fractions were measured after an intraluminal [(3)H]leucine pulse during stable TG transport into lymph. The output of VLDL apoproteins exceeded that of chylomicrons during the absorption of 45 micro mol of TG per hour. More [(3)H]leucine was incorporated into VLDL than into chylomicrons and the decay of newly synthesized VLDL apoproteins was more rapid than that of chylomicrons, in part due to higher concentrations of apoA-I and apoA-IV with a rapid turnover rate. Chylomicrons from proximal intestine contained more apoA-I and less C peptides than chylomicrons from distal intestine. Ninety percent of [(3)H]leucine incorporated into soluble apoproteins was in apoA-I and apoA-IV, but little apoARP was labeled. The turnover rate of apoA-I and apoA-IV differed significantly in the lymph lipoproteins examined. Although total C peptide labeling was small, evidence for intestinal apoC-II formation and differing patterns of apoC-III subunit labeling was obtained. [(3)H]Leucine incorporation and apoprotein turnover rates in lipoprotein secreted by proximal and distal intestine were similar. The different turnover rates of apoA-I and apoA-IV in individual lipoproteins suggest that these A apoproteins are synthesized independently in the intestine.-Holt, P. R., A-L. Wu, and S. Bennett Clark. Apoprotein composition and turnover in rat intestinal lymph during steady-state triglyceride absorption.     

Density distribution and physicochemical properties of plasma lipoproteins and apolipoproteins in the goose, Anser anser, a potential model of liver steatosis

The fractionation and physicochemical characterization of the complex molecular components composing the plasma lipoprotein spectrum in the goose, a potential model of liver steatosis, are described. Twenty lipoprotein subfractions (d less than 1.222 g/ml) were separated by isopycnic density gradient ultracentrifugation, and characterized according to their chemical composition, particle size and particle heterogeneity, electrophoretic mobility, and apolipoprotein content. Analytical ultracentrifugal analyses showed high density lipoproteins (HDL) to predominate (approximately 450 mg/dl plasma), the peak of its distribution occurring at d approximately 1.090 g/ml (F1.21 approximately 2.5). The HDL class displayed marked density heterogeneity, HDL1-like particles being detected up to a lower density limit of approximately 1.020 g/ml, particle size decreasing progressively from 17-19 nm at d 1.024-1.028 g/ml to 10.5-12 nm (d 1.055-1.065 g/ml), and then remaining constant (approximately 9 nm) at densities greater than 1.065 g/ml. HDL subfractions displayed multiple size species; five subspecies were present over the range d 1.103-1.183 g/ml with diameters of 10.5, 9.9, 9.0, 8.2, and 7.5 nm, four in the range d 1.090-1.103 g/ml (diameters 10.5, 9.9, 9.0, and 8.2 nm) and three over the range d 1.076-1.090 g/ml (diameters 10.5, 9.9, and 9.0 nm). ApoA-I (Mr 25,000-27,000) was the major apolipoprotein in all goose HDL subfractions, while the minor components (apparent Mr 100,000, 91,000, 64,000, 58,000, approximately 42,000, 18,000 and apoC-like proteins) showed marked quantitative and qualitative variation across this density range (i.e., 1.055-1.165 g/ml). The d 1.063 g/ml boundary for separation of goose low density lipoproteins (LDL) from HDL was inappropriate, since HDL-like particles were present in the density interval 1.024-1.063 g/ml, while particles enriched in apoB (Mr approximately 540,000) and resembling LDL in size (approximately 20.5 nm) were detected up to a density of approximately 1.076 g/ml. Goose LDL itself was a major component of the profile (90-172 mg/dl) with a single peak of high flotation rate (Sf approximately 10.5). The physicochemical properties and apolipoprotein content of intermediate density lipoproteins (IDL) and LDL varied but little over the range d 1.013-1.040 g/ml, presenting as two particle species (diameters 20.5 and 21 nm) of essentially constant chemical composition; LDL (d 1.019-1.040 g/ml) were separated from HDL1 by gel filtration chromatography and appeared to contain primarily apoB with lesser amounts of apoA-I.(ABSTRACT TRUNCATED AT 400 WORDS)     

Apolipoprotein A-I charge and conformation regulate the clearance of reconstituted high density lipoprotein in vivo

While low apolipoprotein A-I (apoA-I) levels are primarily associated with increased high density lipoprotein (HDL) fractional catabolic rate (FCR), the factors that regulate the clearance of HDL from the plasma are unclear. In this study, the effect of lipid composition of reconstituted HDL particles (LpA-I) on their rate of clearance from rabbit plasma has been investigated. Sonicated LpA-I containing 1 to 2 molecules of purified human apoA-I and 5 to 120 molecules of palmitoyl-oleoyl phosphatidylcholine (POPC) exhibit similar charge and plasma FCR to that for lipid free apoA-I, 2.8 pools/day. Inclusion of 1 molecule of apoA-II to an LpA-I complex increases the FCR to 3.5 pools/day, a value similar to that observed for exchanged-labeled HDL3. In contrast, addition of 40 molecules of triglyceride, diglyceride, or cholesteryl ester to a sonicated LpA-I containing 120 moles of POPC and 2 molecules of apoA-I increases the negative charge of the particle and reduces the FCR to 1.8 pools/day. Discoidal LpA-I are the most positively charged lipoprotein particles and also have the fastest clearance rates, 4.5 pools/day. Immunochemical characterization of the different LpA-I particles shows that the exposure of an epitope at residues 98 to 121 of the apoA-I molecule is associated with an increased negative particle charge and a slower clearance from the plasma.We conclude that the charge and conformation of apoA-I are sensitive to the lipid composition of LpA-I and play a central role in regulating the clearance of these lipoproteins from plasma. conformation regulate the clearance of reconstituted high density lipoprotein in vivo.