Abetalipoproteinemia with an ApoB-100-lipoprotein(a) glycoprotein complex in plasma. Indication for an assembly defect

Patients with autosomal recessive abetalipoproteinemia (ABL) lack in their plasma all lipoproteins containing apolipoprotein (apo)B-100 or B-48. Previous studies have suggested that this is due to the complete absence of apoB. We have investigated whether such patients (n = 10) are able to secrete the lipoprotein(a) (Lp(a] glycoprotein (apo(a] which, in normal plasma, exists as a complex with low density lipoproteins containing apoB-100 (Lp(a) lipoprotein). All 10 patients had reduced but detectable apo(a) levels in plasma (mean, 0.49 mg/dl; range, 0.2-2.03 mg/dl) but no Lp(a) lipoprotein. However, we also detected small amounts (0.2-2.8 mg/dl) of apoB in all patients with ABL. The apoB in the ABL patients had the size of apoB-100 and occurred as a lipid-poor complex with the Lp(a) glycoprotein in a fraction of density 1.22 g/ml. This material may represent partially assembled Lp(a) lipoprotein. There was also uncomplexed apo(a) and apoB-100 in the ABL plasma. The distribution and relative concentration of both proteins in the density fraction greater than 1.06 g/ml varied among patients. The data suggest that in ABL, the assembly of apoB-containing lipoproteins is defective and that apoB-100 may be secreted without its full lipid complement when complexed with apo(a).     

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)     

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)     

Human lymphedema fluid lipoproteins: particle size, cholesterol and apolipoprotein distributions, and electron microscopic structure

The concentration of cholesterol, apolipoproteins A-I, B, and E has been determined in lymphedema fluid from nine patients with chronic primary lymphedema. The concentrations were: 38.14 +/- 21.06 mg/dl for cholesterol, 15.6 +/- 6.17 mg/dl for apolipoprotein A-I, 7.5 +/- 2.8 mg/dl for apolipoprotein B, and 1.87 +/- 0.50 mg/dl for apolipoprotein E. These values represent 23%, 12%, 6%, and 38% of plasma concentrations, respectively. The ratio of esterified to unesterified cholesterol in lymphedema fluid was 1.46 +/- 0.45. Lipoproteins of lymphedema fluid were fractionated according to particle size by gradient gel electrophoresis and by exclusion chromatography. Gradient gel electrophoresis showed that a majority of high density lipoproteins (HDL) of lymphedema fluid were larger than ferritin (mol wt 440,000) and smaller than low density lipoproteins (LDL); several discrete subpopulations could be seen with the large HDL region. Fractionation by exclusion chromatography showed that more than 25% of apolipoprotein A-I and all of apolipoprotein E in lymphedema fluid was associated with particles larger than plasma HDL2. Apolipoprotein A-I also eluted in fractions that contained particles the size of or smaller than albumin. Isolation of lipoproteins by sequential ultracentrifugation showed that less than 25% of lymphedema fluid cholesterol was associated with apolipoprotein B. The majority of apolipoprotein A-containing lipoproteins of lymphedema fluid were less dense than those in plasma. Ultracentrifugally separated fractions of lipoproteins were examined by electron microscopy. The fraction d less than 1.019 g/ml contained little material, while fraction d 1.019-1.063 g/ml contained two types of particles: round particles 17-26 nm in diameter and square-packing particles 13-17 nm on a side. Fractions d 1.063-1.085 g/ml had extensive arrays of square-packing particles 13-14 nm in size. Fractions d 1.085-1.11 g/ml and fractions d 1.11-1.21 g/ml contained round HDL, 12-13 nm diameter and 10 nm diameter, respectively. Discoidal particles were observed infrequently.     

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

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

Evidence that lipid hydroperoxides inhibit plasma lecithin:cholesterol acyltransferase activity

The oxidation of low density lipoproteins (LDL) has been implicated in the development of atherosclerosis. Recently, we found that polar lipids isolated from minimally oxidized LDL produced a dramatic inhibition of lecithin: cholesterol acyltransferase (LCAT) activity, suggesting that HDL-cholesterol transport may be impaired during early atherogenesis. In this study, we have identified molecular species of oxidized lipids that are potent inhibitors of LCAT activity. Treatment of LDL with soybean lipoxygenase generated small quantities of lipid hydroperoxides (20 +/- 4 nmol/mg LDL protein, n = 3); but when lipoxygenase-treated LDL (1 mg protein/ml) was recombined with the d > 1.063 g/ml fraction of human plasma, LCAT activity was rapidly inhibited (25 +/- 4 and 65 +/- 16% reductions by 1 and 3 h, respectively). As phospholipid hydroperoxides (PL-OOH) are the principal oxidation products associated with lipoxygenase-treated LDL, we directly tested whether PL-OOH inhibited plasma LCAT activity. Detailed dose-response curves revealed that as little as 0.2 and 1.0 mole % enrichment of plasma with PL-OOH produced 20 and 50% reductions in LCAT activity by 2 h, respectively. To gain insight into the mechanism of LCAT impairment, the enzyme's free cysteines (Cys31 and Cys184) and active site residues were "capped" with the reversible sulfhydryl compound, DTNB, during exposure to either minimally oxidized LDL or PL-OOH. Reversal of the DTNB "cap" after such exposures revealed that the enzyme was completely protected from both sources of peroxidized phospholipids. We, therefore, conclude that PL-OOH inhibited plasma LCAT activity by modifying the enzyme's free cysteine and/or catalytic residues. These studies are the first to suggest that PL-OOH may accelerate the atherogenic process by impairing LCAT activity.     

Catabolism of the apoprotein of low density lipoproteins by the isolated perfused rat liver.

Isolated rat livers were perfused for 4 hours in a recirculating system containing washed rat erythrocytes. Biologically screened, radioiodinated low density lipoproteins (1.030 < d < 1.055 g/ml) were added to the perfusate with different amounts of whole serum to supply unlabeled rat low density lipoproteins. Apolipoprotein B contained 90% of the bound (131)I, other apolipoproteins contained 4%, and lipids contained the remainder. The fraction of apolipoprotein mass degraded during the perfusion was quantified by the linear increment of non-protein-bound radioiodine in the perfusate, corrected for the increment observed during recirculation of the perfusate in the absence of a liver. The fractional catabolic rate ranged from 0.3 to 1.7%/hr in seven experiments and was inversely related to the size of perfusate pool of low density apolipoprotein. The catabolic rate of low density apolipoprotein (fractional catabolic rate x pool size) in four livers, in which the concentration of rat low density lipoproteins was 50-100% of that present in intact rats, was 5.3 +/- 2.7 micro g hr(-1) (mean +/- SD). Similar results were obtained with human low density lipoproteins. These rates were compared with catabolic rates for the apoprotein of rat low density lipoproteins in intact animals. Fractional catabolic rate in vivo, obtained by multi-compartmental analysis of the disappearance curve of (131)I-labeled low density apolipoprotein from blood plasma, was 15.2 +/- 3.1% hr(-1) (mean +/- SD). Total catabolic rate in vivo (fractional catabolic rate x intravascular pool of low density apolipoprotein) was 76 +/- 14 micro g hr(-1) (mean +/- SD). The results suggest that only a small fraction of low density apolipoprotein mass in rats is degraded by the liver.     

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.     

Studies of chemical components of Angora goat seminal plasma

Ejaculates were collected by artificial vagina from 11 Angora goats, once or twice weekly, between April and July in two successive years. The mean +/- SEM ejaculate volumes each year were 0.8 +/- 0.30 and 0.98 +/- 0.52 ml; the sperm concentrations were 3.33 +/- 0.49 and 2.94 +/- 0.45 x 10(9)/ml, and the pH values were 7.01 +/- 0.34 and 7.20 +/- 0.17. The concentrations (mg/100ml) of fructose (875 +/- 97) and lactic acid (73 +/- 17) in goat seminal plasma were sufficiently high to be important substrates for maintenance of sperm motility. Only trace amounts of glucose were present in seminal plasma. The glycerylphosphorylcholine (GPC) concentration of seminal plasma (809 +/- 154 mg 100 ml ) was correlated with whole semen sperm concentration (P < 0.001), indicating that GPC is of epididymal origin. Goat sperm are not likely to utilize GPC as a substrate and its metabolizable derivatives, glycerophosphate (3.3 +/- 1.1 mg 100 ml ) and glycerol (1.8 +/- 1.0 mg 100 ml ), were not present in sufficiently high concentrations to be significant as energy sources for the sperm. The mean concentration of citric acid was 331 mg 100 ml seminal plasma. Colored semen was consistently produced by eight bucks, and in yellow, light yellow and white ejaculates, the seminal plasma riboflavin (mug/ml) concentrations were 5.38 +/- 2.89, 3.09 +/- 0.85 and 1.73 +/- 0.88, respectively. This suggests that the color is due to riboflavin, which is probably produced by the vesicular glands since the concentration of riboflavin in the seminal plasma was correlated with fructose and citric acid levels.     

Intestinal lipids and lipoproteins in the human fetus: modulation by epidermal growth factor.

The aim of the present investigation was first, to examine the ability of human fetal intestine (17-20 wk) to incorporate fatty acid into esterified lipids; and second, to study in vitro lipoprotein synthesis and secretion by fetal explants, as well as the effect of epidermal growth factor (EGF) on these processes. Cultured fetal jejunal explants were incubated in Leibovitz medium for 42 h with [14C]oleate. Both triglycerides (TG) and phospholipids (PL) were the major labeled products. Whereas TG were predominant (80%) in the culture medium, PL accounted for more than 50% of total tissue lipids. More than 60% of the radioactivity in PL was associated with phosphatidylcholine. Some labeling (< 5%) was also recovered in the cholesteryl ester fraction. Active exocytosis was demonstrated by the accumulation of newly synthesized esterified lipids in the medium and the presence of lipoproteins in the basolateral membrane region and intercellular spaces. Most of the newly synthesized lipids were found in lipoproteins of d < 0.97 g/ml (51.2%) and d < 1.21 g/ml (39.3%), whereas the rest were recovered in d < 1.006 g/ml (9.8%) and 1.063 g/ml (5.6%). A similar trend characterized the lipoprotein secretion. The synthesis of the d < 0.97 g/ml fraction (30,653 +/- 4,122 dpm/mg protein) was significantly greater than the 1.006 g/ml fraction (5,897 +/- 1,734), P < 0.005. The secretion of d < 0.97 g/ml particles into the medium was also five fold higher than that of the d < 1.006 g/ml fraction (P < 0.01). The addition of EGF to the culture medium (25, 50, and 100 ng/ml) significantly enhanced the d < 0.97 g/ml lipoprotein secretion (25-40%) and decreased the d 1.006 g/ml and 1.063 g/ml fraction output. The lipid composition of these lipoprotein fractions was never altered by the presence of EGF, suggesting that the number of lipoprotein particles, rather than size, was modified by the growth factor. The present findings provide the first evidence that the human fetal intestine has the capacity to elaborate lipoprotein fractions for the transport of newly synthesized lipids. Furthermore, our data suggest that EGF, present in significant quantity in saliva, amniotic fluid, and bile, can modulate the release of TG-rich lipoproteins by fetal intestinal explants.     

Transferrin in fetal sheep cerebrospinal fluid and plasma during gestation

1. Transferrin concentrations in fetal sheep CSF and plasma have been estimated between 31 and 125 days gestation and in the adult, using a radial immunodiffusion assay. 2. The plasma concentration was lowest (183 +/- 35 mg/100 ml) in the earliest fetuses examined (31 days). It increased to over 350 mg/100 ml by 35 days; thereafter it was around the adult value (580 mg/100 ml). 3. In CSF the transferrin concentration increased from 43 +/- 10 mg/100 ml at 31 days to a maximum of 163 +/- 14 mg/100 ml at 40 days gestation after which it decreased considerably to 6.1 +/- 0.7 mg/100 ml at 125 days and was even lower in the adult (1.1 +/- 0.2 mg/100 ml). 4. CSF: plasma ratios for transferrin especially when compared with those of other plasma proteins, are not compatible with passive leakage of protein from blood to CSF in the developing brain. The results may be explained by specific transfer of proteins into CSF but synthesis by the choroid plexus or brain has not been excluded.     

Structural and functional properties of human plasma high density-sized lipoprotein containing only apoE particles

To investigate the metabolism of HDL-apolipoprotein E (apoE) particles in human plasma, we isolated a fraction of plasma HDL-apoEs that lack apoA-I (HDL-LpE) from subjects with apoE3/3 phenotype by immunoaffinity. Plasma HDL-LpE had a particle size ranging from 9 nm to 18.5 nm in diameter and was characterized by two-dimensional nondenaturing gradient gel electrophoresis as having either gamma-, prebeta1-, prebeta2-, or alpha-electrophoretic mobility. HDL-LpE was also present in the medium of cultured human hepatoma cell lines and monocyte-derived macrophages. The majority of apoE3 was found as a monomeric form in HDL-LpE and floated at density d > 1.21 g/ml. Plasma levels of HDL-LpE in normolipidemic, CETP-deficient, and ABCA1-deficient subjects were 0.72 +/- 0.15 mg/dl (n = 12), 1.77 +/- 0.75 mg/dl (n = 3), and 0.55 +/- 0.11 mg/dl (n = 3), respectively. The ratio of HDL-apoE containing apoA-I to HDL-LpE was significantly higher 4 h after a fat load, representing a 35 +/- 9% increase (n = 3). Isolated plasma HDL-LpE3 was as effective as apoE3, reconstituted HDL particles, or apoA-I in promoting cellular cholesterol efflux. These results demonstrate that 1) plasma HDL-LpE may have hepatogenous and macrophagic origins; 2) HDL-LpE was preserved even with large reductions in apoA-I-containing lipoproteins; 3) HDL-LpE was active in the transfer of apoE to triglyceride-rich lipoproteins, and 4) HDL-LpEs efficiently take up cell-derived cholesterol.     

Concentrations and compositions of plasma lipoprotein subfractions of Lpb5-Lpu1 homozygous and heterozygous swine with hypercholesterolemia

Pigs with two mutant epitopes, Lpb5 of apolipoprotein B (apoB) and Lpu1 of a yet undefined apolipoprotein, specified by a haplotype Lpb5-Lpu1 and fed a cholesterol-free low fat diet show hypercholesterolemia. The purpose of this study was to establish whether a direct relationship exists between the swine lipoprotein concentration/composition and the genotype for the Lpb5-Lpu1 haplotype; i.e., homozygote versus heterozygote. Lipoproteins of fasted plasma from hypercholesterolemic swine, homozygous (HmHC) and heterozygous (HtHC) for Lpb5-Lpu1, and from normolipidemic (NL) pigs of other Lpb-Lpu haplotypes were separated into five layers by density gradient ultracentrifugation. Layer 1 contained particles of d less than 1.019 g/ml and layer 5 particles of d greater than 1.073 g/ml. Layers 2, 3, and 4 represented subfractions of low density lipoproteins (LDL). The plasma total cholesterol (TC) of the HmHC group (300 +/- 84 mg/dl) was different (P less than 0.05) from the HtHC group (200 +/- 80 mg/dl) and in both HmHC and HtHC, TC was significantly higher (P less than 0.0005 and P less than 0.005, respectively) than that of the NL group (69 +/- 14 mg/dl). The elevation in plasma TC was due to the increased TC in layers 2 and 3: a 13- and 7-fold increase in HmHC and a 7- and 4-fold increase in HtHC in layers 2 and 3, respectively. Parallel increases in unesterified cholesterol were observed in these two layers. Marked increases in apoB were also observed in layers 2 and 3 of HmHC and intermediate increases in apoB in the same two layers of HtHC.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro effect of Triton WR-1339 on canine plasma high density lipoproteins

We studied the effect in vitro of various concentrations of Triton WR-1339 on normolipidemic canine plasma and on the high density lipoproteins (HDL) isolated from this plasma by ultracentrifugation. As a preamble to this study, we established that Triton WR-1339 has a unimer molecular weight of 4,500, a micellar molecular weight of 180,000, and a critical micellar concentration (CMC) of 0.018 mM or 0.008 g/dl. Above its CMC, Triton WR-1339 in concentrations between 2 and 10 mg/ml induced concentration-dependent structural changes in HDL which were characterized by a progressive displacement of apoA-I from the HDL surface without loss of lipids. The addition of Triton WR-1339 to the HDL particles modified their electrophoresis mobility and caused an increase in size (95 +/- 5 A to 114 +/- 7 A). At the extreme Triton WR-1339 concentrations utilized in these studies (10 mg/ml) disruption of the HDL particles occurred; at this stage, the original, relatively homogeneous, spherical HDL particles were replaced by a heterogeneous population ranging in size between 50 and 250 A, representing complexes of Triton WR-1339 with lipids essentially free of apoA-I which could be sedimented by ultracentrifugation. The effects of Triton WR-1339 on whole plasma or isolated HDL were comparable. These studies indicate that Triton WR-1339 in vitro alters HDL in a concentration-dependent manner and that these changes vary from a displacement of apoA-I from the HDL surface to a state where all lipids are solubilized into the Triton WR-1339 micellar phase and are driven away from the protein moiety.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel cell line (Caco-2) for the study of intestinal lipoprotein synthesis

Lipoprotein synthesis by the colonic adenocarcinoma cell line Caco-2 was investigated to assess the utility of this cell line as a model for the in vitro study of human intestinal lipid metabolism. Electron micrographic analysis of conditioned medium revealed that under basal conditions of culture post-confluent Caco-2 cells synthesize and secrete lipoprotein particles. Lipoproteins of density (d) less than 1.063 g/ml consist of a heterogeneous population of particles (diameter from 10 to 90 nm). This fraction consists of very low density lipoproteins (d less than 1.006 g/ml) and low density lipoproteins (d = 1.019-1.063 g/ml). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled Caco-2 lipoproteins revealed that very low density lipoproteins contain apolipoprotein E (apoE) and C apolipoproteins, while low density lipoproteins contained apoB-100, apoE, apoA-I, and C apolipoproteins. The 1.063-1.21 g/ml density fraction contained two morphological entities, discoidal (diameter 15.6 +/- 3.9 nm) and round high density lipoprotein particles (diameter 10.2 +/- 2.3 nm). The high density lipoproteins contained apoA-I, apoB-100, apoB-48, apoE, and the C apolipoproteins. Using isoelectric focusing polyacrylamide gel electrophoresis newly secreted apoA-I was identified as pro-apoA-I. ApoE and apoC-III released by Caco-2 cells were highly sialylated. mRNA species for apoA-I, apoC-III, and apoE, but not apoA-IV were identified by Northern blot analysis. ApoA-I, apoB, and apoE were visualized in Caco-2 cells by immunolocalization analysis. This intestinal cell line may be useful for in vitro studies of nutritional and hormonal regulation of lipoprotein synthesis.     

Cholesterol metabolism in the genetically hypercholesterolemic (RICO) rat. II. A study of plasma lipoproteins and effect of dietary cholesterol

The high plasma cholesterol concentration of the genetically hypercholesterolemic RICO rats fed a low cholesterol base diet (1.28 mg/ml) compared to that of SW rats (0.73 mg/ml) results from an increase in the cholesterol content of the d greater than or equal to 1.006 lipoproteins. Since the composition of each type of lipoprotein is similar in the two groups of rats, the RICO rat, therefore, is hyperlipoproteinemic with an increase in the number of lipoprotein particles, except VLDL and chylomicrons. Furthermore, the apolipoprotein E (apoE) content in the d less than or equal to 1.063 lipoproteins is higher in RICO than in SW rats, while that of apoA-I in HDL is lower. In rats fed 0.5% cholesterol base diet, cholesterolemia doubles in the two groups (SWCH, 1.32 +/- 0.10 mg/ml; RICOCH, 2.10 +/- 0.09 mg/ml). This hypercholesterolemia is due to an increased cholesterol content in VLDL and chylomicrons. These lipoproteins carry 60% (in SWCH) and 45% (in RICOCH) of the plasma cholesterol and are cholesterol-enriched compared with the lipoproteins observed in rats fed the base diet. In RICOCH, 24% of the plasma cholesterol is found in apoE-rich LDL2 (1.040 less than or equal to d less than or equal to 1.063), whereas in SWCH, this fraction contains only 11% of the plasma cholesterol. Finally, as before with the base diet, RICOCH shows an apoE enrichment of the d less than or equal to 1.063 lipoproteins and an apoA-I depletion of HDL compared to SWCH. These data suggest that hypercholesterolemia of the RICO rats results from a modification in the turnover of apoE-containing lipoproteins.     

Substitution in vitro of lecithin-cholesterol acyltransferase. Analysis of changes in plasma lipoproteins

Lecithin-cholesterol acyltransferase (EC 2.3.1.43) was purified 15 000-fold from human plasma. The active material was homogeneous in different gel electrophoretic systems but separated into three major bands with apparent pI values of 4.28, 4.33 and 4.37 in isoelectrofocusing. The apparent Mr of the enzyme is 67 000 +/- 2000. An antiserum prepared against the purified enzyme specifically inhibited the activity of lecithin-cholesterol acyltransferase in whole serum. Serum from a patient with familial deficiency of lecithin-cholesterol acyltransferase was substituted in vitro with the highly purified enzyme. The serum from this patient did not contain immunochemically detectable enzyme protein. Substitution of enzyme resulted in the following major changes. 1. Cholesteryl ester content in serum increased by 36-89 mg/100 ml depending on the experimental conditions. The enzyme-mediated formation of cholesteryl ester led to an increase of cholesteryl ester content in high-density and very-low-density lipoproteins and in low-density lipoproteins containing apoprotein-B. No increase occurred in fractions containing very large flattened structures and the abnormal lipoprotein-X and in lipoprotein-E. Incubation of isolated fractions with lecithin-cholesterol acyltransferase led to significant cholesterol esterification only in high-density lipoproteins. 2. The characteristic disc-shaped rouleaux-forming high-density lipoproteins of enzyme-deficient serum disappeared. Instead a single homogeneous population of high-density lipoproteins formed. The particles generated were spherical and had the electrophoretic properties, density (1.080 g/ml), diameter (12.5 nm) and apoprotein composition of normal high-density lipoproteins-2. 3. The concentration of spherical particles containing apolipoprotein E (density 1.040-1.080 g/ml) and the lamellar lipoprotein-X-like structures in the low-density lipoprotein fraction were not affected by the enzyme substitution. 4. A single homogeneous population of spherical lipoprotein-B particles of 26.5-nm diameter occurred at density 1.029 g/ml. The data suggest that the discoidal high-density lipoproteins are the major site of cholesteryl ester formation that apolipoprotein-E is not involved in an undirectional transport of newly formed cholesteryl ester from high-density lipoproteins to other lipoproteins and that lipoprotein-X and lipoprotein-E are not preferential substrates for the acyltransferase.     

Interaction of cremophor EL with human plasma

1. Interaction of cremophor EL (CRM) with human plasma lipoproteins and nonlipoproteins has been investigated by ultracentrifugation. 2. VLDL has only a low or negligible capacity to bind CRM, i.e. there is little or no change in the optical absorption at 280 nm of VLDL when CRM is added. 3. A low density subfraction of low density lipoproteins seems to associate substantially with CRM at relatively low CRM concentrations (1-3 mg/ml), but such association is not evident for CRM concentrations in the region 12-116 mg/ml. 4. Low density lipoproteins (LDL) may act as a carrier for CRM-emulsions, yet there seems to be no concomitant change in the 280 nm optical absorption of the proteins of LDL. 5. The position in the gradient (i.e. in the centrifugation tube after centrifugation) of high density lipoproteins (HDL) is shifted towards lower density in the presence of 1-4 mg CRM/ml. For higher concentrations of CRM, a destruction of HDL can be observed: the HDL distribution is converted into a bimodal distribution of respectively lighter and heavier "HDL"-particles than the normal ones; the densities at the peaks of these distributions are approximately 1.07 g/ml (light), 1.20 g/ml (heavy) and 1.11 g/ml (normal HDL). The optical extinction coefficient is apparently the same for the proteins of normal--and modified HDL. 6. Even high CRM concentrations (less than or equal to 116 mg/ml) have no perceptible effect on the gradient positions and profile of human serum albumin (HSA) and/or other heavy proteins. 7. The possible biological significance of these findings is briefly touched upon.     

Heterogeneity of human plasma lipoprotein (a). Isolation and characterization of the lipoprotein subspecies and their apoproteins

Lipoprotein (a) (Lp(a] from the plasma of normolipidemic human donors was isolated by rate zonal and isopycnic density gradient ultracentrifugation. The final preparations usually contained varying amounts of isopycnic low-density lipoproteins (LDL), which were totally removed either by heparin-Sepharose column chromatography or by chromatofocusing. The Lp(a) preparations exhibited both inter- and intraindividual density heterogeneity which was accounted for by the differences in their protein and lipid composition. In addition, there was heterogeneity in the size of apoprotein (a) (apo(a] which was found to be linked to apoprotein B (apo-B) through disulfide bonds. Three different apo(a) species were obtained; they had a size either smaller, equal to, or larger than apo-B-100, the protein moiety of LDL. The apo(a) that was smaller than apo-B resided in a low-density Lp(a) particle whose peak was in the 1.019-1.063 g/ml density range. The larger apo(a) was a component of the dense Lp(a) particle and was responsible for the increased density in this Lp(a) species. The third apo(a) which was equivalent in size to apo-B resided in a density range intermediate between the other two Lp(a)s. It is concluded that Lp(a) may differ not only from one individual to another, but also within the same individual who may have more than one Lp(a) species. Part of this heterogeneity may be accounted for by differences in the (a) polypeptide.     

Nascent lipoproteins from recirculating and nonrecirculating liver perfusions and from the hepatic Golgi apparatus of African green monkeys

Perfusate apoB-100-containing lipoproteins from the isolated, perfused livers of African green monkeys consist of significant amounts of d greater than 1.006 g/ml particles in addition to very low density lipoproteins (VLDL). Distinguishing characteristics of these perfusate lipoproteins are the relative abundance of surface lipids and deficiency of core lipids. The present studies were performed to determine the likelihood that the d greater than 1.006 g/ml perfusate lipoproteins are secretion products instead of products of post-secretory modification (e.g., lipolysis) of secreted VLDL. [14C]Leucine from the perfusate became incorporated into the apoB of each of the perfusate lipoprotein classes to a similar extent in both recirculating and nonrecirculating perfusions. When endogenously radiolabeled perfusate VLDL from one liver was recirculated through a second liver, only about 15% of the radiolabeled protein appeared in the d greater than 1.006 g/ml fraction. The particle morphology and the cholesterol and apoB distribution between VLDL and d greater than 1.006 g/ml fractions were similar in recirculating and nonrecirculating perfusions. A Golgi apparatus-rich fraction was isolated from the homogenates of fresh liver samples and the isolated Golgi VLDL and d greater than 1.006 g/ml lipoproteins exhibited morphologic evidence of extra surface material analogous to that seen in perfusate. Taken together, these data support the possibility that significant amounts of d greater than 1.006 g/ml lipoproteins, many with surface-rich properties, are nascent, secretory products of the primate liver. The low level of lecithin:cholesterol acyltransferase (LCAT) in this perfusion system appears to permit detection of these secretion products and it is significant to note that the perfusate lipoprotein profile, which is unlike that of normal plasma, is similar to that of LCAT-deficient patients.