Uptake of [3H]vitamin D3 from low and high density lipoproteins by cultured human fibroblasts

The plasma distribution and cellular uptake of [3H]vitamin D3 was studied in vitro using cultured human fibroblasts. Incubation of [3H]vitamin D3 (cholecalciferol) with plasma followed by sequential ultracentrifugal fractionation of the lipoproteins indicated that 2-4% of the radioactivity associated with the very low density lipoprotein (VLDL), 12% with low density lipoprotein (LDL), and approximately 60% with the high density lipoprotein (HDL). The remaining radioactivity, 25%, was associated with the sedimented plasma fractions. By comparison, an average of 86% of the radioactivity from [3H]1,25-dihydroxycholecalciferol associated with the sedimented plasma fractions. The uptake of [3H]vitamin D3 from plasma, LDL, or HDL was studied in cultured human cells; uptake by normal fibroblasts was greatest from LDL and least from plasma. The cellular association of vitamin D3 was time, concentration, and temperature dependent. At a concentration of 50 micrograms LDL/ml of medium, the uptake of [3H]vitamin D3 from LDL at 37 degrees C was rapid and reached a maximum at approximately 4 hr; it was slower from HDL but continued to increase slowly up to 24 hr. The significance of these in vitro findings is uncertain since much of the vitamin D3 absorbed from the intestine reportedly associates with chylomicrons and is rapidly taken up by the liver.     

High-carbohydrate diets affect the size and composition of plasma lipoproteins in hamsters (Mesocricetus auratus)

High-carbohydrate diets reduce plasma low-density lipoprotein (LDL)-cholesterol but also provoke the appearance of an atherogenic lipoprotein profile (ALP). Characterized by high plasma triglyceride, small dense LDL, and reduced high-density lipoprotein (HDL) cholesterol, an ALP is associated with insulin resistance. Despite extensive use of the fructose-fed hamster as a model of insulin resistance, little is known about changes that occur in the physical properties of circulating lipoproteins. Therefore, we investigated the metabolic and physical properties of lipoproteins in hamsters fed high-carbohydrate diets of varying complexity (60% carbohydrate as chow, cornstarch, or fructose) for 2 wk. Hamsters fed the high-fructose diet showed significantly increased very- low-density lipoprotein (VLDL)-triglyceride (92.3%), free cholesterol (68.6%), and phospholipid (95%), whereas apolipoprotein B levels remained unchanged. Median diameter of circulating VLDL was larger in fructose-fed hamsters (63 nm) than in cornstarch-fed hamsters. Fructose feeding induced a 42.5% increase LDL-triglyceride concurrent with a 20% reduction in LDL-cholesteryl ester. Compositional changes were associated with reduced LDL diameter. In contrast, fructose feeding caused elevations in all HDL fractions. The physical properties of apolipoprotein-B-containing lipoprotein fractions are similar between fructose-fed hamsters and humans with ALP. However, metabolism of high-density lipoprotein appears to differ in the 2 species.     

Studies on the metabolism of rat serum very low density apolipoprotein.

There was a rapid transfer of radioactive peptides to other lipoprotein fractions during the first 30 min after the intravenous injection of 125I-labeled rat very low density lipoprotein (VLDL) into rats. After this initial redistribution of radioactivity, label disappeared slowly from all lipoprotein fractions. The disappearance of 125I-labeled human VLDL injected into rats was the same as that of rat VLDL. Most of the radioactivity transferred from VLDL to low density (LDL) and high density (HDL) lipoproteins was associated with two peptides, identified in these studies by polyacrylamide gel electrophoresis as zone IVa and IVb peptides (fast-migrating peptides, possibly analogous to some human C apolipoproteins), although radioactivity initially associated with zone I (analogous to human apolipoprotein B) and zone III (not characterized) was also transferred to LDL and HDL. That the transfer of label from VLDL to LDL and HDL primarily involved small molecular weight peptides was confirmed in studies using VLDL predominantly labeled in these peptides by in vitro transfer from 125I-labeled HDL. Both zone I and zone IV radioactivity was rapidly removed from VLDL during the first 5 min after injection. However, although most of the zone IV radioactivity was recovered in LDL and HDL, only 12% of the label lost from zone I of VLDL was recovered in other lipoproteins, with the remainder presumably having been cleared from the plasma compartment. We have concluded that, during catabolism of rat VLDL apoprotein, there is a rapid transfer of small molecular weight peptides to both LDL and HDL. During the catabolic process, most of the VLDL is rapidly removed from the circulation, with only a small portion being transformed into LDL molecules.     

Effects of estrogen administration on the lipoproteins and apoproteins of the chicken.

The plasma lipoproteins of estrogen-treated and untreated sexually immature hens have been compared with respect to their concentration in plasma, protein and lipid composition, particle size, and and apoprotein composition. Administration of diethylstilbestrol resulted in a 400-fold rise in the concentration of very low density lipoprotein (VLDL), a 70-fold rise in low density lipoprotein (LDL), and a marked reduction in high density lipoprotein (HDL) protein. It also resulted in the production of LDL and HDL which were enriched in triacylglycerol, while the proportion of cholesterol in all three lipoprotein fractions decreased. In contrast to the lipoproteins from untreated birds, lipoproteins of density less than 1.06 g/ml from estrogen-treated birds were not clearly separable into discrete VLDL and LDL fractions, but appeared to be a single ultracentrifugal class. The apoprotein composition of VLDL and LDL from untreated birds differed from each other; however, the apoprotein patterns of VLDL and LDL from estrogen-treated birds were indistinguishable: both contained a large amount of low molecular weight protein in addition to the high molecular weight component that predominates in the untreated state. The apoprotein composition of HDL was also markedly altered by estrogen administration: the 28,000 mol. wt. protein (apo A-I) decreased in amount from 65% to less than 5% of the total, while a low molecular weight (Mr = 14,000) protein and as yet poorly defined high molecular weight components became predominant. These observations indicate that the hyperlipidemia induced by estrogen administration is accompanied by marked alterations, both qualitative and quantitative, in the plasma lipoproteins.     

The rabbit as an animal model of hepatic lipase deficiency

A natural deficiency of hepatic lipase in rabbits has been exploited to gain insights into the physiological role of this enzyme in the metabolism of plasma lipoproteins. A comparison of human and rabbit lipoproteins revealed obvious species differences in both low-density lipoproteins (LDL) and high-density lipoproteins (HDL), with the rabbit lipoproteins being relatively enlarged, enriched in triacylglycerol and depleted of cholesteryl ester. To test whether these differences related to the low level of hepatic lipase in rabbits, whole plasma or the total lipoprotein fraction from rabbits was either kept at 4 degrees C or incubated at 37 degrees C for 7 h in (i) the absence of lipase, (ii) the presence of hepatic lipase and (iii) the presence of lipoprotein lipase. Following incubation, the lipoproteins were recovered and subjected to gel permeation chromatography to determine the distribution of lipoprotein components across the entire lipoprotein spectrum. An aliquot of the lipoproteins was subjected also to gradient gel electrophoresis to determine the particle size distribution of the LDL and HDL. Both hepatic lipase and lipoprotein lipase hydrolysed lipoprotein triacylglycerol and to a much lesser extent, also phospholipid. There were, however, obvious differences between the enzymes in terms of substrate specificity. In incubations containing hepatic lipase, there was a preferential hydrolysis of HDL triacylglycerol and a lesser hydrolysis of VLDL triacylglycerol. By contrast, lipoprotein lipase acted primarily on VLDL triacylglycerol. When more enzyme was added, both lipases also acted on LDL triacylglycerol, but in no experiment did lipoprotein lipase hydrolyse the triacylglycerol in HDL. Coincident with the hepatic lipase-induced hydrolysis of LDL and HDL triacylglycerol, there were marked reductions in the particle size of both lipoprotein fractions, which were now comparable to those of human LDL and HDL3, respectively.     

Lipid profiling of FPLC-separated lipoprotein fractions by electrospray ionization tandem mass spectrometry

Glycerophospholipid and sphingolipid species and their bioactive metabolites are important regulators of lipoprotein and cell function. The aim of the study was to develop a method for lipid species profiling of separated lipoprotein classes. Human serum lipoproteins VLDL, LDL, and HDL of 21 healthy fasting blood donors were separated by fast performance liquid chromatography (FPLC) from 50 microl serum. Subsequently, phosphatidylcholine (PC), lysophosphatidylcholine, sphingomyelin (SM), ceramide (CER), phosphatidylethanolamine (PE), PE-based plasmalogen (PE-pl), cholesterol, and cholesteryl ester (CE) content of the separated lipoproteins was quantified by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Analysis of FPLC fractions with PAGE demonstrated that albumin partially coelutes with HDL fractions. However, analysis of an HDL deficient serum (Tangier disease) showed that only lysophosphatidylcholine, but none of the other lipids analyzed, exhibited a significant coelution with the albumin containing fractions. Approximately 60% of lipoprotein CER were found in LDL fractions and 60% of PC, PE, and plasmalogens in HDL fractions. VLDL, LDL, and HDL displayed characteristic lipid class and species pattern. The developed method provides a detailed lipid class and species composition of lipoprotein fractions and may serve as a valuable tool to identify alterations of lipoprotein lipid species profiles in disease with a reasonable experimental effort.     

Lipoprotein-associated alpha-tocopheryl-succinate inhibits cell growth and induces apoptosis in human MCF-7 and HBL-100 breast cancer cells

alpha-Tocopheryl succinate (alpha-TS) is a potent inhibitor of tumor cell proliferation. The goal of the present study was to investigate whether and to what extent alpha-TS associates with plasma lipoproteins and if alpha-TS-enriched lipoproteins inhibit breast cancer cell growth in a manner comparable to the free drug. In vitro enrichment of human plasma revealed that alpha-TS readily associated with the main lipoprotein classes, findings confirmed in vivo in mice. At the highest alpha-TS concentrations, lipoproteins carrying 50000 (VLDL), 5000 (LDL) and 700 (HDL) alpha-TS molecules per lipoprotein particle were generated. alpha-TS enrichment generated lipoprotein particles with slightly decreased density and increased particle radius. To study whether the level of LDL-receptor (LDL-R) expression affects alpha-TS uptake from apoB/E containing lipoprotein particles human breast cancer cells with low (MCF-7) and normal (HBL-100) LDL-R expression were used. The uptake of free, VLDL- and (apoE-free) HDL(3)-associated alpha-TS was nearly identical for both cell lines. In contrast, uptake of LDL-associated alpha-TS by HBL-100 cells (normal LDL-R expression) was about twice as high as compared to MCF-7 cells (low LDL-R expression). VLDL and LDL-associated alpha-TS inhibited proliferation most effectively at the highest concentration of alpha-TS used (100% inhibition of MCF-7 growth with 20 microg/ml of lipoprotein-associated alpha-TS). However, also alpha-TS-free VLDL and LDL inhibited HBL-100 cell proliferation up to 55%. In both cell lines, alpha-TS-enriched HDL(3) inhibited cell growth by 40-60%. Incubation of both cell lines in the presence of free or lipoprotein-associated alpha-TS resulted in DNA fragmentation indicative of apoptosis. Collectively, the present findings demonstrate that: (1) alpha-TS readily associates with lipoproteins in vitro and in vivo; (2) the lipoprotein-enrichment efficacy was dependent on the particle size and/or the triglyceride content of the lipoprotein; (3) uptake of LDL-associated alpha-TS was apparently dependent on the level of LDL-R expression; and (4) lipoproteins were efficient alpha-TS carriers inducing reduced cell proliferation rates and apoptosis in human breast cancer cells as observed for the free drug.     

Characterization of equine plasma lipoproteins after separation by density gradient

1. Plasma lipoproteins from six thoroughbred horses were separated by density gradient ultracentrifugation. For each sample, lipoprotein bands were visualized by means of a prestained plasma control and characterized by electrophoretic, chemical and morphological analysis. 2. Very low density lipoproteins (VLDL) were isolated at d less than 1.018 g/ml. 3. Two clearly resolved bands were detected in the low density lipoprotein fraction (LDL). The density limits were evaluated as follows: LDL1(1.028 less than d less than 1.045 g/ml) and LDL2(1.045 less than d less than 1.070 g/ml). Marked differences were observed in the chemical composition and particle size of LDL1 and LDL2 fractions. 4. High density lipoprotein fraction (HDL) was usually isolated as a single band, distributed over the range 1.075 less than d less than 1.180 g/ml. However, chemical composition and particle size revealed heterogeneity in HDL subfractions. 5. The density limit of LDL and HDL bands varied in each animal, indicating differences in equine lipoprotein distribution.     

Studies on the substrate specificity of human and pig lecithin: cholesterol acyltransferase: role of low-density lipoproteins

The substrate properties of low-density lipoprotein (LDL) fractions from human and pig plasma and of lipoprotein a [Lp(a)] upon incubation with either pig or human lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) were investigated and compared with those of pig high-density lipoproteins (HDL) or human HDL-3. The cholesterol esterification using purified native pig LDL-1, human LDL, or Lp(a) as a substrate was approximately 36-42% that of pig HDL or human HDL-3, while cholesteryl ester formation with pig LDL-2 was 41-47%. No significant difference was found in the substrate activity between pig HDL and human HDL-3, and between human LDL and Lp(a), respectively. After depletion of pig LDL-1, pig LDL-2, and human LDL from apolipoprotein A-I (apoA-I), cholesteryl ester formation decreased to about 22-28% of the value found with pig HDL. Depletion of human LDL from apolipoprotein E (apoE) did not result in significantly different esterification rates in comparison to native LDL. Total removal of non-apoB proteins from human LDL resulted in esterification rates of approximately 10-15% that of HDL. Readdition of apoA-I to all these LDL fractions produced solely in apoA-I-depleted LDL fractions an increase of cholesteryl ester formation, whereas in those LDL fractions that were additionally depleted from apoE and/or from apoC polypeptides, a further decrease in the esterification rate occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactivity of human lipoproteins with purified lecithin: cholesterol acyltransferase during incubations in vitro

Studies have been performed to determine the proportion of the esterified cholesterol in high-density lipoproteins (HDL), low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) that is attributable to a direct action of lecithin: cholesterol acyltransferase on each lipoprotein fraction. Esterification of [3H]cholesterol was examined in 37 degrees C incubations of either: (a) unseparated whole plasma, (b) plasma reconstituted after prior ultracentrifugation to separate the 1.21 g/ml supernatant, (c) a mixture comprising the 1.21 g/ml supernatant of plasma and purified lecithin: cholesterol acyltransferase or (d) the same mixture as (c) after supplementation with a preparation of partially purified lipid transfer protein. Each of these incubations was performed using samples collected from four different subjects, two of whom had normal and two of whom had elevated concentrations of plasma triacylglycerol. At the completion of 3-h incubations, the lipoproteins were separated into multiple fractions by gel filtration to obtain a continuous profile of esterified [3H]cholesterol across the whole spectrum of lipoproteins. There was an appearance of esterified [3H]cholesterol in each of the major lipoprotein fractions in all incubations. In unseparated plasma, 56% of the total (mean of four experiments) was in HDL, 33% in LDL and 11% in VLDL. A comparable distribution was observed in the incubations of reconstituted plasma and in the samples to which partially purified lipid transfer protein had been added. In the absence of lipid transfer protein activity in incubations containing purified lecithin: cholesterol acyltransferase, 73% of the esterified [3H]cholesterol was in HDL, 25% in LDL and only 1% in VLDL. It has been concluded that at physiological concentrations of lipoproteins, 70-80% of the cholesterol esterifying action of lecithin: cholesterol acyltransferase is confined to the HDL fraction, with most of the remainder involving the LDL fraction. Of the newly formed esterified cholesterol incorporated into LDL during incubations of unseparated plasma, it was apparent that more than 70% was independent of activity of the lipid transfer protein. Of that incorporated into VLDL in unseparated plasma, in contrast, almost 90% was derived as a transfer from other fractions as a consequence of activity of the lipid transfer protein.     

Plasma very-low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein oxidative modification induces procoagulant profiles in endogenous hypertriglyceridemia

This study was to investigate whether oxidatively modified lipoproteins were associated with changes of pro- and anticoagulant profiles in hypertriglyceridemic subjects. Plasma VLDL, LDL, and HDL were isolated with the one-step density gradient ultracentrifugation method. The oxidation of the lipoproteins was identified. Prothrombin time (PT) and activated partial thrombplastin time (APTT), tissue plasminogen activator and plasminogen activator inhibitor-1, and platelet aggregation rate were determined with a reaction system consisting of mixed fresh normal plasma, in endogenous hypertriglyceridemic (HTG) patients, in in vitro modified lipoproteins from a normolipidemic donor, and in experimental rats. The results indicated that oxVLDL, oxLDL, and oxHDL occurred in the plasma of HTG patients. Compared with the control group, PT and APTT, incubated with plasma VLDL, LDL, or HDL from HTG patients, respectively, were significantly reduced, while platelet maximal aggregation rates were significantly higher (P < 0.05-0.01). Similar procoagulant profiles were observed in in vitro modified lipoprotein components and in rats with intrinsic hypertriglyceridemia as well. These results support our previous finding that LDL, VLDL, and HDL were all oxidatively modified in vivo in the subjects with HTG, and suggest that procoagulation state may result from the abnormal plasma lipoprotein oxidative modification in vivo.     

Separation and selective detection of lipoprotein particles of patients with coronary artery disease by frit-inlet asymmetrical flow field-flow fractionation

An analytical method to improve the characterization of lipoprotein fractions is presented. Human plasma samples were treated with Sudan Black B to stain the lipid component in lipoproteins, then the stained lipoproteins were separated by frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF), according to the lipoprotein particle sizes, with the selective detection of eluting lipoprotein fractions, high-density lipoproteins (HDL), low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL), at 610 nm. The capability of this technique has been evaluated with plasma samples obtained from patients with coronary artery disease (CAD), and it showed that the retention profile of patients' lipoprotein samples was clearly distinct from those of healthy persons. The potential of this technique comes with the direct injection of a stained lipoprotein sample without a prior procedure such as ultracentrifugation for sample preparation, and the size calculation of lipoprotein particles from the experimental retention time by theory. Since sample relaxation was achieved hydrodynamically in an FI-AFlFFF channel, sample injection and separation processes were continuously made without stopping the separation flow. This study demonstrated the potential of the FI-AFlFFF technique to be utilized as a powerful tool for the determination of the LDL profiles of patients with CAD.     

1H NMR investigation on interaction between ibuprofen and lipoproteins

A large number of studies indicate that oxidative modification of plasma lipoproteins, especially low-density lipoprotein (LDL), is a critical factor in initiation and progression of atherosclerosis. We have previously found that ibuprofen (IBP), a potential antioxidant drug to inhibit LDL oxidation, interacted with lipoproteins in intact human plasma. In the present study, we compare the binding affinities of IBP to LDL and HDL (high-density lipoprotein) by (1)H NMR spectroscopy. When IBP is added into the HDL and LDL samples, the - N(+)(CH(3))(3) moieties of phosphatidylcholine (PC) and sphingomyelin (SM) in lipoprotein particles experience the chemical shift up-field drift. Intermolecular cross-peaks observed in NOESY spectra imply that there are direct interactions between ibuprofen and lipoproteins at both hydrophobic and hydrophilic (ionic) regions. These interactions are likely to be important in the solubility of ibuprofen into lipoprotein particles. Ibuprofen has higher impact on the PC and SM head group ( - N(+)(CH(3))(3)) and - (CH(2))(n) - group in HDL than that in LDL. This could be explained by either IBP has higher binding affinity to HDL than to LDL, or IBP induces orientation of the phospholipid head group at the surface of the lipoprotein particles.     

Comparison of gel permeation chromatography, density gradient ultracentrifugation and precipitation methods for quantitation of very-low-, low- and high-density lipoprotein cholesterol

Human VLDL, LDL and HDL (very-low-, low- and high-density lipoproteins) were isolated from plasma by gel permeation chromatography with one pre-ultracentrifugation step. The column effluent was monitored at 280 nm. The cholesterol content of the fractions correlated well with fractions from sequential ultracentrifugation (VLDL, r = 0.839; LDL, r = 0.924; HDL, r = 0.766) or precipitation (LDL, r = 0.975; HDL, r = 0.972) methods. The average triglyceride, phospholipid and protein compositions of the separated lipoprotein fractions were close to those of the ultracentrifugally isolated fractions reported previously. Apolipoproteins A1 and B were determined from fractions to confirm the right distribution between different lipoproteins.     

Alterations of the plasma lipoproteins and apoproteins following cholesterol feeding in the rat.

The feeding of cholesterol to rats resulted in marked alterations in the type and distribution of the plasma lipoproteins and their apoproteins. The hyperlipoproteinemia was characterized by an increase in the d < 1.006 lipoproteins (B-VLDL and VLDL), an increase in the intermediate and low density lipoproteins (LDL), and the appearance of HDL(c). Associated with these lipoproteins was a prominence of the arginine-rich apoprotein. The high density lipoproteins (HDL) were decreased. A two-dimensional immunoelectrophoretic procedure was adapted to quantitate the changes in distribution of the arginine-rich apoprotein in the plasma and various ultracentrifugal fractions obtained from control and cholesterol-fed rats. In rats fed the cholesterol diet, the total plasma arginine-rich apoprotein increased from a control value of approximately 29 mg/dl to 47 mg/dl. The method of ultracentrifugation, however, was found to markedly alter the quantitative results. When the 60 Ti rotor was used at maximum speed to isolate the ultracentrifugal fractions, less than 50% of the total plasma arginine-rich apoprotein was associated with the lipoproteins in the d < 1.006 or the d 1.006-1.02, 1.02-1.063, or 1.063-1.21 ultracentrifugal fractions. By contrast, after limited ultracentrifugation with the 40 rotor, much less arginine-rich apoprotein was lost, with approximately 20% of the arginine-rich apoprotein in control rats and 10% in cholesterol-fed rats found in the d > 1.21 fraction. Significant alterations in the arginine-rich apoprotein quantitation notwithstanding, the observations of increased arginine-rich apoprotein in the B-VLDL, intermediate fraction, and HDL(c) following cholesterol feeding remained valid. However, precise quantitation awaits refinements in lipoprotein isolation techniques.     

Lipoprotein metabolism in the suckling rat: characterization of plasma and lymphatic lipoproteins

Suckling rat plasma contains (in mg/dl): chylomicrons (85 +/- 12); VLDL (50 +/- 6); LDL (200 +/- 23); HDL1 (125 +/- 20); and HDL2 (220 +/- 10), while lymph contains (in mg/dl): chylomicrons (9650 +/- 850) and VLDL (4570 +/- 435) and smaller amounts of LDL and HDL. The lipid composition of plasma and lymph lipoproteins are similar to those reported for adults, except that LDL and HDL1 have a somewhat higher lipid content. The apoprotein compositions of plasma lipoproteins are similar to those of adult lipoproteins except for the LDL fraction, which contains appreciable quantities of apoproteins other than apoB. Although the LDL fraction was homogeneous by analytical ultracentrifugation and electrophoresis, the apoprotein composition suggests the presence of another class of lipoproteins, perhaps a lipid-rich HDL1. The lipoproteins of lymph showed low levels of apoproteins E and C. The triacylglycerols in chylomicrons and VLDL of both lymph and plasma are rich in medium-chain-length fatty acids, whereas those in LDL and HDL have little or none. Phospholipids in all lipoproteins lack medium-chain-length fatty acids. The cholesteryl esters of the high density lipoproteins are enriched in arachidonic acid, whereas those in chylomicrons, VLDL, and LDL are enriched in linoleic acid, suggesting little or no exchange of cholesteryl esters between these classes of lipoproteins. The fatty acid composition of phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine were relatively constant in all lipoprotein fractions, suggesting ready exchange of these phospholipids. However, the fatty acid composition of phosphatidylethanolamine in plasma chylomicrons and VLDL differed from that in plasma LDL, HDL1, and HDL2. LDL, HDL1, and HDL2 were characterized by analytical ultracentrifugation and shown to have properties similar to that reported for adult lipoproteins. The much higher concentration of triacylglycerol-rich lipoproteins in lymph, compared to plasma, suggests rapid clearance of these lipoproteins from the circulation.     

Study of the components of reverse cholesterol transport in lecithin:cholesterol acyltransferase deficiency

Enzymatic and lipid transfer reactions involved in reverse cholesterol transport were studied in healthy and lecithin:cholesterol acyltransferase (LCAT), deficient subjects. Fasting plasma samples obtained from each individual were labeled with [3H]cholesterol and subsequently fractionated by gel chromatography. The radioactivity patterns obtained corresponded to the elution volumes of the three major ultracentrifugally isolated lipoprotein classes (very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL)). In healthy subjects, the LCAT activity was consistently found in association with the higher molecular weight portion of HDL. Similar observations were made when exogenous purified LCAT was added to the LCAT-deficient plasma prior to chromatography. Incubation of the plasma samples at 37 degrees C resulted in significant reduction of unesterified cholesterol (FC) and an increase in esterified cholesterol (CE). Comparison of the data of FC and CE mass measurements of the lipoprotein fractions from normal and LCAT-deficient plasma indicates that: (i) In normal plasma, most of the FC for the LCAT reaction originates from LDL even when large amounts of FC are available from VLDL. (ii) The LCAT reaction takes place on the surface of HDL. (iii) The product of the LCAT reaction (CE) may be transferred to either VLDL or LDL although VLDL appears to be the preferred acceptor when present in sufficient amounts. (iv) CE transfer from HDL to lower density lipoproteins is at least partially impaired in LCAT-deficient patients. Additional studies using triglyceride-rich lipoproteins indicated that neither the capacity to accept CE from HDL nor the lower CE transfer activity were responsible for the decreased amount of CE transferred to VLDL and chylomicrons in LCAT-deficient plasma.     

Effects of insulin deficiency on lipoproteins and their hepatic receptors in rico rats

The present study was designed to examine the effect of streptozotocin (STZ)-induced diabetes on the plasma lipoprotein profile and hepatic expression of the LDL receptor and HDL binding protein (HB2) in hypercholesterolemic Rico rats. The plasma level of HDL1 (density range 1.040–1.063), which is particularly high in this rat strain, decreased (−25 %) 28 d after STZ administration (50 mg/kg). In contrast, the treatment increased (+54 %) the plasma concentration of HDL2 (density range 1.063–1.210). These variations in the lipoprotein concentrations were associated with inverse changes in the hepatic protein levels of the LDL receptor (+118 %) and HB2 (−46 %). These results suggest that the hepatic expression of HB2, a putative HDL receptor, can influence the plasma level of apo Al-rich HDL as has already been shown for the LDL receptor for apo B/E containing lipoproteins.     

Lipoprotein abnormalities associated with lipopolysaccharide-induced lecithin: cholesterol acyltransferase and lipase deficiency

Density gradient ultracentrifugation was used to isolate and characterize the plasma lipoproteins from African green monkeys before and 24 and 48 h after subcutaneous injection of 300 micrograms/kg lipopolysaccharide (LPS) to induce an acute phase response. Compared with 0 h values, reductions occurred in plasma cholesterol (39%), high density lipoprotein (HDL) cholesterol (54%), lecithin:cholesterol acyltransferase (LCAT) activity (55%), and post-heparin plasma lipase activity (68%) 48 h after LPS injection while plasma triglyceride concentrations increased 700%. Cholesterol distribution among lipoproteins shifted from 7 to 41% in very low density lipoproteins (VLDL), 65 to 38% in low density lipoproteins (LDL), and 28 to 21% in HDL after LPS injection. At 48 h after LPS injection, all lipoprotein classes were relatively enriched in phospholipid and triglyceride and depleted of cholesteryl ester. The plasma concentration of all chemical constituents in VLDL was increased 3-9-fold within 48 h after LPS injection. By negative stain electron microscopy, HDL were discoidal in shape while VLDL and LDL appeared to have excess surface material present. Even though total HDL protein concentration in plasma was unaffected, the plasma mass of the smallest HDL subfractions (HDL3b,c) doubled while the mass of intermediate-sized subfractions (HDL3a) was dramatically decreased within 24 h after treatment. HDL became enriched in apoE, acquired apoSAA, and became depleted of apoA-I, A-II, and Cs by 48 h after LPS injection while apoB-100 remained the major apoprotein of VLDL and LDL. We conclude that administration of LPS to monkeys prevents normal intravascular metabolism of lipoproteins and results in the accumulation of relatively nascent forms of lipoproteins in plasma. These immature lipoproteins resemble those isolated from the recirculating perfusion of African green monkey livers, which are relatively deficient of LCAT activity and those isolated from the plasma of patients with familial LCAT deficiency.     

Lipid and fatty acid composition of canine lipoproteins

Lipid classes and their fatty acids were studied in the major lipoprotein fractions from canine, in comparison with human, plasma. In dogs, high-density-lipoprotein (HDL), the main carrier of plasma phospholipid (PL), cholesterol ester (CE) and free cholesterol, was the most abundant lipoprotein, followed by low and very-low density lipoproteins (LDL and VLDL). Notably, LDL and VLDL contributed similarly to the total dog plasma triacylglycerol (TG). The PL composition was similar in all three lipoproteins, dominated by phosphatidylcholine (PC). Even though the content and composition of lipids within and among lipoproteins differed markedly between dog and man, the total amount of circulating lipid was similar. All canine lipoproteins were relatively richer than those from humans in long-chain (C20-C22) n-6 and n-3 polyunsaturated fatty acids (PUFA) but had comparable proportions of total saturated and monoenoic fatty acids, with 18:2n-6 being the main PUFA in both mammals. The fatty acid profile of canine and human lipoproteins differed because they had distinct proportions of their major lipids. There were more n-3 and n-6 long-chain PUFA in canine than in human plasma, because dogs had more HDL, their HDL had more PC and CE, and both these lipids were richer in such PUFA.