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.     

Selective uptake of cholesteryl esters from various classes of lipoproteins by HepG2 cells.

Selective uptake of cholesteryl esters (CE) from lipoproteins by cells has been extensively studied with high density lipoproteins (HDL). It is only recently that such a mechanism has been attributed to intermediate and low density lipoproteins (IDL and LDL). Here, we compare the association of proteins and CE from very low density lipoproteins (VLDL), IDL, LDL and HDL3 to HepG2 cells. These lipoproteins were either labelled in proteins with 125I or in CE with 3H-cholesteryl oleate. We show that, at any lipoprotein concentration, protein association to the cells is significantly smaller for IDL, LDL, and HDL3 than CE association, but not for VLDL. At a concentration of 20 microg lipoprotein/mL, these associations reveal CE-selective uptake in the order of 2-, 4-, and 11-fold for IDL, LDL, and HDL3, respectively. These studies reveal that LDL and HDL3 are good selective donors of CE to HepG2 cells, while IDL is a poor donor and VLDL is not a donor. A significant inverse correlation (r2 = 0.973) was found between the total lipid/protein ratios of the four classes of lipoproteins and the extent of CE-selective uptake by HepG2 cells. The fate of 3H-CE of the two best CE donors (LDL and HDL3) was followed in HepG2 cells after 3 h of incubation. Cells were shown to hydrolyze approximately 25% of the 3H-CE of both lipoproteins. However, when the cells were treated with 100 microM of chloroquine, a lysosomotropic agent, 85 and 40% of 3H-CE hydrolysis was lost for LDL and HDL3, respectively. The fate of LDL and HDL3-CE in HepG2 cells deficient in LDL-receptor was found to be the same, indicating that the portion of CE hydrolysis sensitive to chloroquine is not significantly linked to LDL-receptor activity. Thus, in HepG2 cells, the magnitude of CE-selective uptake is inversely correlated with the total lipid/protein ratios of the lipoproteins and CE-selective uptake from the two best CE donors (LDL and HDL3) appears to follow different pathways.     

Dissociation of high density lipoprotein precursors from apolipoprotein B-containing lipoproteins in the presence of unesterified fatty acids and a source of apolipoprotein A-I

Incubation of low (LDL), intermediate (IDL), or very low density lipoproteins (VLDL) with palmitic acid and either high density lipoproteins (HDL), delipidated HDL, or purified apolipoprotein (apo) A-I resulted in the formation of lipoprotein particles with discoidal structure and mean particle diameters ranging from 146 to 254 A by electron microscopy. Discs produced from IDL or LDL averaged 26% protein, 42% phospholipid, 5% cholesteryl esters, 24% free cholesterol, and 3% triglycerides; preparations derived from VLDL contained up to 21% triglycerides. ApoA-I was the predominant protein present, with smaller amounts of apoA-II. Crosslinking studies of discs derived from LDL or IDL indicated the presence of four apoA-I molecules per particle, while those derived from large VLDL varied more in size and contained as many as six apoA-I molecules per particle. Incubation of discs derived from IDL or LDL with purified lecithin:cholesterol acyltransferase (LCAT), albumin, and a source of free cholesterol produced core-containing particles with size and composition similar to HDL2b. VLDL-derived discs behaved similarly, although the HDL products were somewhat larger and more variable in size. When discs were incubated with plasma d greater than 1.21 g/ml fraction rather than LCAT, core-containing particles in the size range of normal HDL2a and HDL3a were also produced. A variety of other purified free fatty acids were shown to promote disc formation. In addition, some mono and polyunsaturated fatty acids facilitated the formation of smaller, spherical particles in the size range of HDL3c. Both discoidal and small spherical apoA-I-containing lipoproteins were generated when native VLDL was incubated with lipoprotein lipase in the presence of delipidated HDL. We conclude that lipolysis product-mediated dissociation of lipid-apoA-I complexes from VLDL, IDL, or LDL may be a mechanism for formation of HDL subclasses during lipolysis, and that the availability of different lipids may influence the type of HDL-precursors formed by this mechanism.     

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.     

Characterization and quantification of serum lipoprotein subfractions by capillary isotachophoresis: relationships with lipid, apolipoprotein, and lipoprotein levels.

Human serum lipoproteins are currently defined according to their density as well as according to their electrophoretic mobility. They can be fractionated into discrete subspecies which exhibit variations in their structure and function. Capillary electrophoresis has been suggested to be a potential analytical strategy in understanding metabolic lipoprotein heterogeneity. In a sample of 35 normolipidemic subjects, we analyzed ceramide-labeled serum lipoproteins by capillary isotachophoresis linked to laser-induced fluorescent detection. Capillary isotachophoresis showed advantage to be an automated, rapid (6 min) and reproducible (CV < 7%) separation mode, on-line monitoring lipoprotein subfractions according to net charge. HDL were separated into three subfractions: i) the fast migrating HDL correlated positively with serum apoA-I (P < 0.05) and negatively with triglyceride (P < 0.01) concentrations, ii) the intermediate migrating HDL involved in HDL-cholesterol delivery and inversely related to LDL particles concentration (P < 0.001), and iii) the slow migrating prebeta(1)HDL. Triglyceride level was significantly associated with two fractions: i) the VLDL fraction correlated positively with apoE serum concentration (P < 0.01), and ii) the IDL fraction closely and positively associated with apoC-III-containing lipoprotein level (P < 0.001). Two LDL subfractions were positively related to LDL-cholesterol (0.05 相似文献   

Acute inflammation and infection maintain circulating phospholipid levels and enhance lipopolysaccharide binding to plasma lipoproteins

Circulating lipoproteins are thought to play an important role in the detoxification of lipopolysaccharide (LPS) by binding the bioactive lipid A portion of LPS to the lipoprotein surface. It has been assumed that hypocholesterolemia contributes to inflammation during critical illness by impairing LPS neutralization. We tested whether critical illness impaired LPS binding to lipoproteins and found, to the contrary, that LPS binding was enhanced and that LPS binding to the lipoprotein classes correlated with their phospholipid content. Whereas low serum cholesterol was almost entirely due to the loss of esterified cholesterol (a lipoprotein core component), phospholipids (the major lipoprotein surface lipid) were maintained at near normal levels and were increased in a hypertriglyceridemic subset of septic patients. The levels of phospholipids found in the LDL and VLDL fractions varied inversely with those in the HDL fraction, and LPS bound predominantly to lipoproteins in the LDL and VLDL fractions when HDL levels were low. Lipoproteins isolated from the serum of septic patients neutralized the bioactivity of the LPS that had bound to them. Our results show that the host response to acute inflammation and infection tends to maintain lipoprotein phospholipid levels and that, despite hypocholesterolemia and reduced HDL levels, circulating lipoproteins maintain their ability to bind and neutralize an important bacterial agonist, LPS.     

Allotypes associated with B apolipoproteins in rabbits

Western blot analysis of the alloantisera (i.e., anti-Lpq1, anti-Lpq2, anti-Lpq3, and anti-Lpq4) which defined the three lpq genes of rabbit linkage group VIII showed that they reacted strongly with an apolipoprotein of molecular weight 320,000. They also cross-reacted with an apolipoprotein of molecular weight 220,000. The two apolipoproteins that reacted with the alloantisera were found by SDS-polyacrylamide gel electrophoresis to be present in very low density (VLDL), intermediate density (IDL), and low density (LDL) lipoprotein fractions and by Western blot analysis to react with an anti-apolipoprotein B antiserum. These results support the conclusion that the alloantisera react with allotypes associated with the B apolipoproteins. The distribution of the four allotypes among different lipoprotein fractions, however, differed. The quantitative competitive Enzyme Linked Immunosorbant Assay (ELISA) showed that the Lpq1, Lpq2, and Lpq4 allotypes were found in the highest concentration in VLDL, IDL, and LDL, and in significantly lower concentrations in plasma chylomicrons. The concentrations of these allotypes in high density lipoproteins (HDL) as measured in the ELISA were about 1% of the concentrations found in LDL. The Lpq3 allotype, on the other hand, was present in the highest concentrations only in IDL and LDL and in significantly lower concentrations in VLDL and plasma chylomicrons. Surprisingly, the concentration of the Lpq3 allotype in HDL was 20% of the level found in LDL.     

Structural heterogeneity of apoB-containing serum lipoproteins visualized using cryo-electron microscopy.

Cryo-electron microscopy was used to analyze the structure of lipoprotein particles in density gradient subfractions of human very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL). Lipoproteins from a normolipidemic subject with relatively large and buoyant LDL (pattern A) and from a subject with a predominance of small dense LDL (pattern B) were compared. Projections of VLDL in vitreous ice were heterogeneous in size, but all were circular with a relatively even distribution of contrast. Selected projections of LDL, on the other hand, were circular with a high density ring or rectangular with two high density bands. Both circular and rectangular LDL projections decreased in average size with increasing subfraction density, but were found in all of 10 density gradient subfractions, both in pattern A and in pattern B profiles. Preparations of total IDL contained particles with the structural features of VLDL as well as particles resembling LDL. IDL particles resembling LDL were observed in specific density gradient subfractions in the denser region of the VLDL;-IDL density range. Within the group of IDL particles resembling LDL considerable heterogeneity was observed, but no structural features specific for the pattern A or pattern B lipoprotein profile were recognized.The observed structural heterogeneity of the apolipoprotein B-containing serum lipoproteins may reflect differences in the composition of these particles that may also influence their metabolic and pathologic properties.     

Effects of 1,2-cyclohexanedione modification on the metabolism of very low density lipoprotein apolipoprotein B: potential role of receptors in intermediate density lipoprotein catabolism

The conversion of very low density (VLDL) to low density lipoproteins (LDL) is a two-step process. The first step is mediated by lipoprotein lipase, but the mechanism responsible for the second is obscure. In this study we examined the possible involvement of receptors at this stage. Apolipoprotein B (apoB)-containing lipoproteins were separated into three fractions, VLDL (Sf 100-400), an intermediate fraction IDL (Sf 12-100), and LDL (Sf 0-12). Autologous 125I-labeled VLDL and 131I-labeled 1,2-cyclohexanedione-modified VLDL were injected into the plasma of four normal subjects and the rate of transfer of apoB radioactivity was followed through IDL to LDL. Modification did not affect VLDL to IDL conversion. Thereafter, however, the catabolism of modified apoB in IDL was retarded and its appearance in LDL was delayed. Hence, functional arginine residues (and by implication, receptors) are required in this process. Confirmation of this was obtained by injecting 125I-labeled IDL and 131I-labeled cyclohexanedione-treated IDL into two additional subjects. Again, IDL metabolism was delayed by approximately 50% as a result of the modification. These data are consistent with the view that receptors are involved in the metabolism of intermediate density lipoprotein.     

Lipoprotein secretion in lean and obese Zucker female rats in vivo and in a single-pass-perfused liver preparation

The plasma lipoprotein composition as well as lipoprotein synthesis and secretion were studied in vivo and in a single-pass-perfused liver preparation in lean and obese Zucker rats. Compared with their lean littermates the levels in the plasma of very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) + low density lipoprotein (LDL) and high density lipoprotein (HDL) were increased 4-, 2- and 2.5 fold, respectively, in obese rats. In these rats both VLDL and IDL + LDL were enriched in triglycerides, while the HDL were enriched in cholesterol. Although the VLDL and IDL + LDL protein concentrations were the same in lean and obese rats, the HDL protein concentration was 3-fold greater in the obese rats. Both the lean and obese rats incorporated similar amounts of [14C]leucine into total liver protein. However, obese rats incorporated 2.5-fold and 6-fold more [14C]leucine into VLDL and HDL in vivo, 2.7-fold and 1.7 fold more [35S]methionine in VLDL and HDL present in the perfusate, than did lean rats. The perfusate [35S]S-labelled apoproteins (apo-B100, B48; apo-E, apo-AI, apo-AIV and apo-C) were separated by gel electrophoresis and identified by autoradiography. Incorporation of [3H]glycerol into liver, VLDL, IDL + LDL and HDL triglycerides was 2-, 48-, 13- and 1.5-fold higher in obese than in lean rats, respectively. The [3H]-labelled triglycerides in VLDL and IDL + LDL present in the perfusate was 5.4-fold and 4.4-fold more in obese rat. There was no difference in the incorporation of [3H]glycerol into triglycerides of perfusate HDL between the two genotypes of rats. Thus, the hypertriglyceridaemia observed in obese Zucker rats results from very high synthetic rates of both the lipid and protein moieties of plasma lipoproteins. Before this study, no report of the simultaneous triglycerides and protein synthesis in vivo and in a single-pass-perfused liver preparations had been reported.     

Effects of fasting on serum lipids and lipoprotein profiles in the egg-laying hen (Gallus domesticus)

The effects of a 24-h fast on serum lipids and lipoprotein profiles in commercial laying hens were investigated. Blood was analyzed at 34 and 46 weeks of age from Single Comb White Leghorn hens that had been either fed ad libitum or had been fasted for 24 h prior to collection. At 12 weeks, birds were divided into 16 biological isolation units, with 8 replicate units assigned to each treatment group. Four birds out of 10 in each unit were tagged for bleeding. Parameters evaluated included total serum cholesterol and triglycerides, mean diameters of very low density lipoproteins (VLDLs) for the 10th, 50th, and 90th percentiles of serum total VLDL, mean total population VLDL particle diameter (MPD), and percentage serum cholesterol recovered in VLDL, low density lipoprotein (LDL), and high density lipoprotein (HDL) fractions. Fasting led to decreases in total serum cholesterol and triglycerides, and a decrease in mean serum VLDL particle diameter in the 90th population percentile. At Week 34, percentage serum cholesterol recovered from LDL was increased, whereas percentage serum cholesterol recovered from HDL was decreased due to fasting. At Week 46, MPD and percentage serum cholesterol recovered from VLDL were decreased, whereas percentage serum cholesterol recovered from HDL was increased due to fasting. It was concluded that a 24-h fast decreased serum lipids (cholesterol and triglycerides) and the size of VLDL particles in the 90th population percentile in commercial laying hens. Furthermore, bird age influenced the effects of a 24-h fast on MPD and the redistribution of serum cholesterol among VLDL, LDL, and HDL particles.     

Modifications of plasma lipoproteins after lipase activation in patients with chylomicronemia

Lipoprotein lipase (LPL) and hepatic lipase (HL) are enzymatic activities involved in lipoprotein metabolism. The purpose of this study was to analyze the physicochemical modifications of plasma lipoproteins produced by LPL activation in two patients with apoC-II deficiency syndrome and by HL activation in two patients with LPL deficiency. LPL activation was achieved by the infusion of normal plasma containing apoC-II and HL was released by the injection of heparin. Lipoproteins were analyzed by ultracentrifugation in a zonal rotor under rate flotation conditions before and after lipase activation. The LPL activation resulted in: a reduction of plasma triglycerides; a reduction of fast-floating very low density lipoprotein (VLDL) concentration; an increase of intermediate density lipoprotein (IDL), which maintained unaltered flotation properties; an increase of low density lipoproteins (LDL) accompanied by modifications of their flotation rates and composition; no significant variations of high density lipoprotein (HDL) levels; and an increase of the HDL flotation rate. The HL activation resulted in: a slight reduction of plasma triglycerides; a reduction of the relative triglyceride content of slow-floating VLDL, IDL, LDL2, and HDL3 accompanied by an increase of phospholipid in VLDL and by an increase of cholesteryl ester in IDL; and a reduction of the HDL flotation rate. These experiments in chylomicronemic patients provide in vivo evidence that LPL and HL are responsible for plasma triglyceride hydrolysis of different lipoproteins, and that LPL is particularly involved in determining the levels and physicochemical properties of LDL. Moreover, in these patients, the LPL activation does not directly change the HDL levels, and LPL or HL does not produce a step-wise conversion of HDL3 to HDL2 (or vice versa) but rather modifies the flotation rates of all the HDL molecules present in plasma.     

Triacylglycerol and phospholipid hydrolysis in human plasma lipoproteins: role of lipoprotein and hepatic lipase.

To explore the interactions of triacylglycerol and phospholipid hydrolysis in lipoprotein conversions and remodeling, we compared the activities of lipoprotein and hepatic lipases on human VLDL, IDL, LDL, and HDL2. Triacylglycerol and phospholipid hydrolysis by each enzyme were measured concomitantly in each lipoprotein class by measuring hydrolysis of [14C]triolein and [3H]dipalmitoylphosphatidylcholine incorporated into each lipoprotein by lipid transfer processes. Hepatic lipase was 2-3 times more efficient than lipoprotein lipase at hydrolyzing phospholipid both in absolute terms and in relation to triacylglycerol hydrolysis in all lipoproteins. The relationship between phospholipid hydrolysis and triacylglycerol hydrolysis was generally linear until half of particle triacylglycerol was hydrolyzed. For either enzyme acting on a single lipoprotein fraction, the degree of phosphohydrolysis closely correlated with triacylglycerol hydrolysis and was largely independent of the kinetics of hydrolysis, suggesting that triacylglycerol removed from a lipoprotein core is an important determinant of phospholipid removal via hydrolysis by the lipase. Phospholipid hydrolysis relative to triacylglycerol hydrolysis was most efficient in VLDL followed in descending order by IDL, HDL, and LDL. Even with hepatic lipase, phospholipid hydrolysis could not deplete VLDL and IDL of sufficient phospholipid molecules to account for the loss of surface phospholipid that accompanies triacylglycerol hydrolysis and decreasing core volume as LDL is formed (or for conversion of HDL2 to HDL3). Thus, shedding of whole phospholipid molecules, presumably in liposomal-like particles, must be a major mechanism for losing excess surface lipid as large lipoprotein particles are converted to smaller particles. Also, this shedding phenomenon, like phospholipid hydrolysis, is closely related to the hydrolysis of lipoprotein triacylglycerol.     

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.     

Evaluation of rat and rabbit sera lipoproteins in experimentally induced hyperlipidemia by analytical ultracentrifugation

Animals of various species are widely used as models with which to study atherosclerosis and the lipoprotein metabolism. The objective of this study was to investigate the lipoprotein profiles in Wistar rats and New Zealand white rabbits with experimentally induced hyperlipidemia by means of ultracentrifugation. The Schlieren curves were utilized to compare suckling and adult rat sera to determine whether aging causes alterations in lipoprotein profiles. A striking feature of the data is the high concentration of low-density lipoproteins (LDL), (>5.2 mmol/l cholesterol) in the 2-week old rat serum pool which was greatly decreased in the 3-weeks rat serum pool (<1.3 mmol/l cholesterol). Additional experiments were performed to permit a direct comparison of the amounts of lipoprotein present in rat sera in experimental hyperlipidemia post-Triton WR 1339 administration. Rapid changes in concentrations in very low-density lipoproteins (VLDL), LDL and high-density lipoproteins (HDL) were observed after Triton injection. The administration of Triton WR 1339 to fasted rats resulted in an elevation of serum cholesterol levels. Triton physically alters VLDL, rendering them refractive to the action of lipolytic enzymes in the blood and tissues, preventing or delaying their removal from the blood. Whereas the VLDL concentration was increased markedly, those of LDL and HDL were decreased at 20 h after Triton treatment. Rabbits were fed a diet containing 2% cholesterol for 60 days to develop hyperlipidemia and atheromatous aortic plaques. A combination of preparative and analytical ultracentrifugation was used to investigate of LDL aliquots, to prepare radioactive-labeled lipoproteins and to study induced hyperlipidemia in rabbits. Analytical ultracentrifugation was applied to investigate the LDL flotation peaks before and after cholesterol feeding of rabbits. Modified forms of LDL were detected in the plasma of rabbits with experimentally induced atherosclerosis. ApoB-containing particles, migrating as LDL, intermediate density lipoproteins and VLDL were the most abundant lipoproteins. Gamma camera in vivo scintigraphy on rabbits with radiolabeled lipoproteins revealed visible signals corresponding to atherosclerotic plaques of the aorta and carotid arteries.     

Density distribution, characterization, and comparative aspects of the major serum lipoproteins in the common marmoset (Callithrix jacchus), a New World primate with potential use in lipoprotein research.

Qualitative, quantitative, and comparative aspects of the serum lipoprotein profile in the Common marmoset (Callithrix jacchus), a New World primate, are described. Density gradient ultracentrifugation was used to evaluate lipoprotein distribution and to establish criteria for isolation of discrete molecular fractions. The major lipoprotein classes banded isopycnically on the gradient with the following hydrated densities: VLDL, d less than 1.017 g/mL; LDL, d = 1.027--1.055 g/mL; HDL fraction I, d = 1.070--1.127 g/mL; and HDL fraction II, d = 1.127--1.156 g/mL. Electrophoretic, immunological, and electron microscopic analyses attested to the purity of these fractions: the characteristics of each were assessed by chemical analysis, electron microscopy, immunological techniques, and polyacrylamide gel electrophoresis of their protein moieties. Marmoset VLDL and LDL were closely akin to those of man in size and chemical composition, although the former were richer in triglyceride; electrophoretic and immunological data showed the major protein component of VLDL and LDL to be a counterpart to human apo-B. The two HDL subfractions, i.e., HDL-I and HDL-II, corresponded in size and chemical composition to human HDL2 and HDL3, respectively, although slight differences in neutral lipid content were detected. By immunological and electrophoretic criteria, the major apolipoprotein of marmoset HDL was analogous to human apo-AI. In contrast, marked dissimilarities were evident in the complements of low molecular weight, tetramethylurea-soluble polypeptides of marmoset and human lipoproteins. Quantitatively, the human and marmoset lipoprotein profiles were not dissimilar, although HDL was the major class (approximately 50%); in fasting animals, serum concentrations of VLDL, LDL, and HDL were 50--90, 170--280, and 338--408 mg/dL, respectively. C. jacchus was distinct from man in displaying a greater proportion of its total HDL in the less dense (HDL-II) subfraction (marmoset HDL-I/HDL-II = approximately 4:1; human HDL2/HDL3 = approximately 1:3). These data indicate that, as an experimental animal for lipoprotein research, the Common marmoset combines the advantages of ready availability and maintenance with a serum lipoprotein profile which resembles, in many qualitative and quantitative aspects, that found in man.     

In vivo evidence of a role for hepatic lipase in human apoB-containing lipoprotein metabolism, independent of its lipolytic activity

Hepatic lipase (HL) is a key player in lipoprotein metabolism by modulating, through its lipolytic activity, the triglyceride (TG) and phospholipid content of apolipoprotein B (apoB)-containing lipoproteins and of high density lipoproteins (HDL), thereby affecting their size and density. A new and separate role has been suggested for HL in cellular lipoprotein metabolism, in which it serves as a ligand promoting cellular uptake of apoB-containing remnant lipoproteins and HDL. We tested the hypothesis that HL has both a lipolytic and a nonlipolytic role in human lipoprotein metabolism, by measuring lipid plasma concentrations, lipoprotein density distribution by density gradient ultracentrifugation, and lipoprotein composition, in three subjects with HL deficiency: two of the patients (S-1 and S-3) were characterized as having neither plasma HL activity nor detectable HL protein; the third subject (S-2) had no plasma HL activity but a detectable amount (35.5 ng/ml) of HL protein. All HL-deficient subjects showed a severalfold increase in lipoprotein TG content across the lipoprotein density spectrum [very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL), and HDL] as compared with control subjects. They also had remarkably more buoyant LDL particles (LDL-R(f) = 0.342;-0.394) as compared with the control subjects (LDL-R(f) = 0.303). Subjects S-1 and S-3 (no HL activity or protein) presented with a distinct increase in cholesterol and apoB levels in the IDL and VLDL density range as compared with patient S-2, with detectable HL protein, and the control subjects.This study provides evidence in humans that HL indeed plays an important role in lipoprotein metabolism independent of its enzymatic activity: in particular, inactive HL protein appears to affect VLDL and IDL particle concentration, whereas HL enzymatic activity seems to influence VLDL-, IDL-, LDL-, and HDL-TG content and their physical properties.     

Apolipoprotein and lipid distribution between vesicles and HDL-like particles formed during lipolysis of human very low density lipoproteins by perfused rat heart

A study was undertaken to determine the relative association of lipid and apolipoproteins among lipoproteins produced during lipolysis of very low density lipoproteins (VLDL) in perfused rat heart. Human VLDL was perfused through beating rat hearts along with various combinations of albumin (0.5%), HDL2, the infranatant of d greater than 1.08 g/ml of serum, and labeled sucrose. The products were resolved by gel filtration, ultracentrifugation, and hydroxylapatite chromatography. The composition of the lipoprotein products was assessed by analysis of total lipid profiles by gas-liquid chromatography and immunoassay of apolipoproteins. A vesicle particle, which trapped and retained 1-2% of medium sucrose, co-isolated with VLDL and VLDL remnants by gel filtration chromatography but primarily with the low density lipoprotein (LDL) fraction when isolated by ultracentrifugation. The vesicle was resolved from apoB-containing LDL lipolysis products by hydroxylapatite chromatography of the lipoproteins. The vesicle lipoprotein contained unesterified cholesterol (34%), phosphatidylcholine and sphingomyelin (50%), cholesteryl ester (6%), triacylglycerol (5%), and apolipoprotein (5%). The apolipoprotein consisted of apoC-II (7%), apoC-III (93%), and trace amounts of apoE (1%). When viewed by electron microscopy the vesicles appeared as rouleaux structures with a diameter of 453 A, and a periodicity of 51.7 A. The mass represented by the vesicle particle in terms of the initial amount in VLDL was: cholesterol (5%), phosphatidylcholine and sphingomyelin (3%), apoC-II (0.5%), apoC-III (2.2%). The majority of the apoC and E released from apoB-containing lipoproteins was associated with neutral-lipid core lipoproteins proteins which possessed size characteristics of HDL. The vesicles were also formed in the presence of HDL and serum and were not disrupted by serum HDL. It is concluded that lipolysis of VLDL in vitro results in the production of VLDL remnants and LDL apoB-containing lipoproteins, as well as HDL-like lipoproteins. A vesicular lipoprotein which has many characteristics of lipoprotein X found in cholestasis, lecithin: cholesterol acyltransferase deficiency, and during Intralipid infusion is also formed. The majority of apolipoprotein C and E released from apoB-containing lipoproteins is associated with the HDL-like lipoprotein. It is suggested that the formation and stability of the vesicle lipoprotein may be related to the high ratio of cholesterol/phospholipid in this particle.     

Preparative free-solution isotachophoresis for separation of human plasma lipoproteins: apolipoprotein and lipid composition of HDL subfractions

We have previously shown that plasma lipoproteins can be separated by analytical capillary isotachophoresis (ITP) according to their electrophoretic mobility in a defined buffer system. As in lipoprotein electrophoresis, HDL show the highest mobility followed by VLDL, IDL, and LDL. Chylomicrons migrate according to their net-charge between HDL and VLDL, because ITP has negligible molecular sieve effects. Three HDL subfractions were obtained which were designated fast-, intermediate-, and slow-migrating HDL. To further characterize these HDL subfractions, a newly developed free-solution ITP (FS-ITP)-system was used, that allows micro-preparative separation of human lipoproteins directly from whole plasma (B?ttcher, A. et al. 1998. Electrophoresis. 19: 1110-1116). The fractions obtained by FS-ITP were analyzed for their lipid and apolipoprotein composition and by two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (2D-GGE) with subsequent immunoblotting. fHDL are characterized by the highest proportion of esterified cholesterol of all three subfractions and are relatively enriched in LpA-I. Together with iHDL they contain the majority of plasma apoA-I, while sHDL contain the majority of plasma apoA-IV, apoD, apoE, and apoJ. Pre-beta-HDL were found in separate fractions together with triglyceride-rich fractions between sHDL and LDL. In summary, ITP can separate the bulk of HDL into lipoprotein subfractions, which differ in apolipoprotein composition and electrophoretic mobility. While analytical ITP permits rapid separation and quantitation for diagnostic purposes, FS-ITP can be used to obtain these lipoprotein subfractions on a preparative scale for functional analysis. As FS-ITP is much better suited for preparative purposes than gel electrophoresis, it represents an important novel tool for the functional analysis of lipoprotein subclasses.     

Impaired plasma cholesteryl ester transfer with accumulation of larger high density lipoproteins in some families of baboons (Papio sp.)

Baboons from some families have a higher concentration of plasma high density lipoproteins (HDL) on a chow diet and accumulate large HDL (HDL1) when challenged with a high cholesterol and high saturated fat (HCHF) diet. HDL1 from high HDL1 animals contained more (1.5-fold) cholesteryl ester than HDL (HDL2 + HDL3) from high or low HDL1 animals. HDL from high HDL1 baboons had lower triglyceride content than that from low HDL1 baboons. HDL3 or HDL labeled with [3H]cholesteryl linoleate was incubated with entire lipoprotein fraction (d less than 1.21 g/ml) or very low density lipoprotein + low density lipoprotein (VLDL + LDL) (d less than 1.045 g/ml) and with lipoprotein-deficient serum (LPDS), and the radioactive cholesteryl ester and mass floating at d 1.045 g/ml (VLDL + LDL) after the incubation was measured. The transfer of cholesteryl esters from either HDL or HDL3, prepared from plasma of high HDL1 animals fed chow or the HCHF diet, was slower than the transfer from either HDL or HDL3 of low HDL1 animals, regardless of the source of transfer activity or the ratio of LDL:HDL-protein used in the assay. Addition of HDL from high HDL1 baboons into an assay mixture of plasma components from low HDL1 baboons decreased the transfer of cholesteryl ester radioactivity and mass from HDL to VLDL and LDL. In addition to HDL, a fraction of intermediate density lipoprotein (IDL) and denser HDL were also effective in inhibiting the transfer. These observations suggest that accumulation of HDL1 in high HDL1 baboons fed an HCHF diet is associated with a slower transfer of cholesteryl esters from HDL to LDL.(ABSTRACT TRUNCATED AT 250 WORDS)