Oxidized cholesterol in the diet is a source of oxidized lipoproteins in human serum

The aim of this study was to determine in humans whether oxidized cholesterol in the diet is absorbed and contributes to the pool of oxidized lipids in circulating lipoproteins. When a meal containing 400 mg cholestan-5alpha,6alpha-epoxy-3beta-ol (alpha-epoxy cholesterol) was fed to six controls and three subjects with Type III hyperlipoproteinemia, alpha-epoxy cholesterol in serum was found in chylomicron/chylomicron remnants (CM/RM) and endogenous (VLDL, LDL, and HDL) lipoproteins. In controls, alpha-epoxy cholesterol in CM/RM was decreased by 10 h, whereas in endogenous lipoproteins it remained in the circulation for 72 h. In subjects with Type III hyperlipoproteinemia, alpha-epoxy cholesterol was mainly in CM/RM. In vitro incubation of the CM/RM fraction containing alpha-epoxy cholesterol with human LDL and HDL that did not contain alpha-epoxy cholesterol resulted in a rapid transfer of oxidized cholesterol from CM/RM to both LDL and HDL. In contrast, no transfer was observed when human serum was substituted with rat serum, suggesting that cholesteryl ester transfer protein is mediating the transfer. Thus, alpha-epoxy cholesterol in the diet is incorporated into the CM/RM fraction and then transferred to LDL and HDL, contributing to lipoprotein oxidation. Moreover, LDL containing alpha-epoxy cholesterol displayed increased susceptibility to further copper oxidation in vitro. It is possible that oxidized cholesterol in the diet accelerates atherosclerosis by increasing oxidized cholesterol levels in circulating LDL and chylomicron remnants.     

The lipid composition of serum lipoproteins in the harbour seal, Phoca vitulina

The density profile of serum lipoproteins and their lipid composition was studied in 12 adult, female harbour seals. The animals were sampled after an approximate 20 hr fast. The density profile of lipoproteins showed that the harbour seals displayed a distinct VLDL (density less than 1.006 g/ml) and HDL band (density about 1.125 g/ml), but no clear LDL band. There was a rather diffuse population of lipoproteins in the density range of 1.019-1.100 g/ml. Mean serum total cholesterol concentration was 5.7 mmol/l; about 60% of this cholesterol was located in the HDL fraction (density greater than 1.063 g/ml). The fasted seals were found to carry 4% of serum total lipids in chylomicrons. These lipoproteins consisted of 51% of triaclyglycerols (on the basis of total chylomicron lipids). The LDL (defined as heparin-manganese precipitable lipoproteins in VLDL and chylomicron-deficient serum) contained 49% of cholesterol and 43% of phospholipids (on the basis of total LDL lipids). The HDL (defined as heparin-manganese soluble lipoproteins in VLDL and chylomicron-deficient serum) contained 36% of cholesterol and 58% of phospholipids (on the basis of total HDL lipids).     

Evaluation of the use of serum lathosterol concentration to assess whole-body cholesterol synthesis in rabbits.

Serum lathosterol concentration in rabbits was assessed as a possible indicator of whole-body cholesterol synthesis. In random-bred New Zealand White (NZW) rabbits fed a control diet or a diet containing either cholesterol, simvastatin, or cholestyramine, neither serum lathosterol concentration nor the serum lathosterol:total cholesterol ratio systematically corresponded with the anticipated rate of cholesterol synthesis. In control rabbits and those fed simvastatin or cholestyramine, whole-body cholesterol synthesis, which was calculated from the sterol balance, was correlated with serum lathosterol concentration when expressed relative to cholesterol in very low, intermediate, and low density lipoproteins (VLDL + IDL + LDL) (r = 0.61; n = 23; P = 0.002). The low correlation coefficient indicates that the predictive value of the lathosterol: (VLDL + IDL + LDL) cholesterol ratio is limited when applied to individual rabbits. Cholesterol and simvastatin feeding reduced the group mean serum lathosterol:(VLDL + IDL + LDL) cholesterol ratio, whereas cholestyramine in the diet raised the group mean ratio in the NZW rabbits. We conclude that the serum lathosterol:(VLDL + IDL + LDL) cholesterol ratio may be an indicator of group mean rates of whole-body cholesterol synthesis in rabbits but may not yield reliable information on individual rabbits. The lathosterol:(VLDL + IDL + LDL) cholesterol ratio predicted that in hyperresponsive inbred rabbits, showing an excessive hypercholesterolemia after cholesterol feeding, baseline whole-body cholesterol synthesis is lower than in hyporesponsive rabbits. Addition of cholesterol to the diet caused a reduction of predicted cholesterol synthesis in hypo- but not in hyper-responsive rabbits.     

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.     

Method for quantitating cholesterol in subfractions of serum lipoproteins separated by gradient gel electrophoresis

Extensive heterogeneity in particle size distribution of serum lipoproteins of baboons was resolved by a procedure that combined Sudan black B prestaining, polyacrylamide gradient gel electrophoresis (GGE), and quantitative densitometry. Each densitometric scan represented a continuous distribution of the relative amount of cholesterol in a serum sample, as a function of the lipoprotein particle size. For analytical purposes, each scan was divided into 12 fractions, representing 12 particle size ranges. The relationship between the estimated cholesterol concentrations in the summed GGE/densitometric fractions corresponding to very low-density lipoproteins (VLDL) + low-density lipoproteins (LDL) and those corresponding to high-density lipoproteins (HDL) and concentrations measured by the heparin-Mn2+ precipitation/enzymatic procedure was linear over a broad range. However, a systematic overestimation of HDL cholesterol concentration and an underestimation of VLDL + LDL cholesterol concentration was apparent. Therefore, correction factors were developed for adjusting the estimates of VLDL + LDL and HDL cholesterol concentrations obtained by the GGE/densitometric method. This analytical method is rapid, repeatable, economical, and useful for genetic and dietary research in which cholesterol concentrations in multiple particle size ranges of lipoproteins must be measured in large numbers of samples. It also is adaptable to immunoblotting procedures for detecting the distribution of specific apolipoproteins among the size-resolved lipoproteins.     

Effects of acyl-CoA: cholesterol O-acyltransferase inhibition on cholesterol absorption and plasma lipoprotein composition in hamsters.

1. The ACAT inhibitors, CL 277082 and SA 58-035 were administered for 7 days to hamsters fed diets containing 0.5% cholesterol. 2. Both agents inhibited cholesterol absorption, decreased hepatic. VLDL and IDL cholesterol esters, plasma HDL and HDL apoE and A-I. 3. In addition, CL 277082 treatment produced significant decreases in plasma cholesterol, VLDL apoB and plasma IDL. 4. The cholesteryl esters in VLDL and LDL but not HDL were more polyunsaturated in CL 277082 treated animals. 5. These results support the hypothesis that ACAT inhibition in the cholesterol fed hamster results in an inhibition of dietary cholesterol absorption, thus limiting the cholesterol supply required for the hepatic production of triglyceride-rich lipoproteins.     

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)     

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.     

Concentration and composition of serum lipoproteins in the fetal rat]

1. Concentration and composition of the "very low density lipoproteins" (VLDL), "low density lipoproteins" (LDL) and "high density lipoproteins" (HDL) and of non-floatable lipids of fetal rat serum (day 22 of pregnancy) were determined by ultracentrifugation, thin-layer chromatographic separation of the floated lipids and quantitation of the lipid and protein moiety. 2. The concentration of VLDL is in the fetal rat by one order of magnitude lower, and that of LDL, 5fold higher than in the adult animal; the concentration of HDL in fetal serum amounts to 60% of the value of adult animals. 3. The composition of LDL and HDL of fetal serum does not differ from that in the serum of adult animals; in contrast, the fetal VLDL have a higher proportion of protein and cholesterol and a lower proportion of triglycerides than the VLDL of adult serum. The electrophoretic mobility of the fetal VLDL is lower than that of adult VLDL.     

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 chylomicron remnants and beta-VLDL on the class and composition of newly secreted lipoproteins by HepG2 cells

The regulation of lipoprotein secretion in the cell line HepG2 was studied. HepG2 cells were preincubated with chylomicron remnants (triglyceride- and cholesterol-rich) or with beta very low density lipoproteins (beta-VLDL) (cholesterol-rich). The medium was removed and the cells were incubated for and additional 24 hr in a lipoprotein-free medium that contained either [2-3H]glycerol or DL-[2-3H]mevalonate. Cells and media were harvested, and lipoproteins were separated and fractionated. The mass and radioactivity of the lipids in cells and in the lipoproteins were measured. The activities of cellular acyl-CoA:cholesterol acyltransferase (ACAT) and 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase were also determined. Preincubation with chylomicron remnants induced an increase in cellular triglyceride and stimulated both HMG-CoA reductase and ACAT. Preincubation with beta-VLDL induced an increase in cellular free and esterified cholesterol, inhibited HMG-CoA reductase and stimulated ACAT. Although the absolute amount of VLDL is small, chylomicron remnants induced large relative increases in the amount of triglyceride and phospholipid secreted in VLDL and decreases in the amount of triglyceride secreted in low density (LDL) and high density (HDL) lipoproteins as well as a decrease in the amount of phospholipid secreted in HDL. In contrast, preincubation with beta-VLDL did not affect triglyceride secretion, but markedly stimulated the amount of phospholipid secreted in HDL. Comparison of the mass of glycerolipid actually secreted with that calculated from the cellular specific activity suggested that glycerolipids are secreted from single, rapidly equilibrating pools. Cholesterol and cholesteryl ester secretion were affected differently. Preincubation with chylomicron remnants increased the amount of free cholesterol secreted in both VLDL and LDL, but did not alter cholesteryl ester secretion. Preincubation with beta-VLDL increased free cholesterol secretion in all lipoprotein fractions and increased cholesteryl ester secretion in VLDL and LDL, but not HDL. Comparison of isotope and mass data suggested that the cholesteryl ester secreted came primarily from a preformed, rather than an newly synthesized, pool. In summary, these data provide insight to the mechanism whereby a liver cell regulates the deposition of exogenous lipid.     

The lecithin-cholesterol acyl transferase activity of rat intestinal lymph.

The lecithin-cholesterol acyl transferase (LCAT) activity in rat mesenteric lymph was examined as a possible source of chylomicron cholesteryl ester. Lymph activity was only 2-3% of rat serum activity. Removal of d less than 1.006 lipoproteins increased lymph LCAT activity, but only to 6-8% of that of serum. Relative to total cholesterol in the d greater than 1.08 g/ml fractions, lymph LCAT activity in lymph from fasting rats was less than serum, but in lymph from nonfasting rats the ratio LCAT/HDL-cholesterol reached levels greater than serum, suggesting a contribution of enzyme from the gut. Both LCAT activity and HDL concentration in mesenteric lymph increased during feeding. Subfractions of lymph that inhibited serum LCAT were: chylomicrons, VLDL, chylomicron lipid, VLDL apoprotein, and HDL apoprotein. In the rat, the low LCAT activity of mesenteric lymph was in part due to the low enzyme concentration present, and the activity was apparently lowered further by lipid-rich lipoproteins that inhibited the reaction. Enzyme inhibition due to the apoprotein fractions of lipoproteins is probably minor in the rat in vivo.     

High-performance liquid chromatography of human serum lipoproteins : Selective detection of choline-containing phospholipids by enzymatic reaction

A convenient method for the quantitation of choline-containing phospholipids in each lipoprotein fraction has been developed by combining separation by high-performance liquid chromatography with gel permeation columns and selective detection by enzymatic reaction in the post-column effluent.The elution patterns monitored by choline-containing phospholipids were compared with those monitored by cholesterol. The elution patterns of choline-containing phospholipids were found to give much more information about the distribution of lipoproteins according to their particle-size differentiation than analyses done by cholesterol.This choline-containing phospholipid monitoring method not only resolves lipoprotein peaks of the major classes (chylomicron + VLDL, LDL, HDL₂ and HDL₃) quantitatively, but also detects the presence of abnormal lipoproteins containing a large amount of choline-containing phospholipids. We could detect these abnormal lipoproteins using a small amount of whole serum (10–20 μl) from patients with various liver diseases. Our examination of HDL subclasses using this technique showed that the HDL fraction was composed of several subfractions due to their particle-size differentiation.     

Effect of human plasmalipoproteins on erythropoietic progenitor cells in serum-free cultures

The purpose of the present study was to investigate the influence of human lipoproteins on CFU-e and BFU-e proliferation from human bone marrow in a serum-free system. In our previously described miniaturized agar system the main lipoprotein-density-classes from human plasma, namely very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), high density lipoproteins2 (HDL2) and HDL3 and a mixture of all the five lipoproteins were added in rising concentrations (from 1/10 to normal human plasma concentration) to serum-free medium containing delipidated and deionized bovine serum albumin (BSA), iron saturated transferrin and erythropoietin. The results demonstrate that all lipoproteins markedly increased the CFU-e and BFU-e proliferation after 7 and 14 days of incubation, respectively. Moreover, the lipoproteins induced a shift towards a lower threshold concentration of erythropoietin. Serumlike conditions were obtained if LDL and the mixture of lipoproteins were added to serum-free medium. Furthermore, in the serum-free cultures a maturation to the mature erythrocyte could be found.     

The effect of L-carnitine on the apolipoprotein pattern of rats fed a cholesterol-rich diet

1. L-Carnitine was administered to hypercholesterolemic rats for eight days, after which the blood was collected by intracardiac puncture and lipoproteins were collected by preparative ultracentrifuge. 2. The chemical composition of lipoproteins of normal rats and hypercholesterolemic rats before and after L-carnitine treatment was determined. 3. The total serum B and E apoproteins were assayed by rocket electrophoresis, while apoproteins in VLDL, IDL, LDL and HDL were determined by densitometric scanning of SDS and TMU polyacrylamide disc gel electrophoresis. 4. The results obtained showed that the L-carnitine treatment tends to restore to normal values both the chemical composition of lipoproteins and the apoproteins pattern of rats fed on a diet enriched with cholesterol.     

Disparities in the interaction of rat and human lipoproteins with cultured rat fibroblasts and smooth muscle cells. Requirements for homology for receptor binding activity

This study characterizes the interactions of various rat and human lipoproteins with the lipoprotein cell surface receptors of rat and human cells. Iodinated rat very low density lipoproteins (VLDL), rat chylomicron remnants, rat low density lipoproteins (LDL), and rat high density lipoproteins containing predominantly apoprotein E (HDL1) bound to high affinity cell surface receptors of cultured rat fibroblasts and smooth muscle cells. Rat VLDL and chylomicron remnants were most avidly bound; the B-containing LDL and the E-containing HDL1 displayed lesser but similar binding. Rat HDL (d = 1.125 to 1.21) exhibited weak receptor binding; however, after recentrifugation to remove apoprotein E, they were devoid of binding activity. Competitive binding studies at 4 degrees C confirmed these results for normal lipoproteins and indicated that VLDL (B-VLDL), LDL, and HDLc (cholesterol-rich HDL1) isolated from hypercholesterolemic rats had increased affinity for the rat receptors compared with their normal counterparts, the most pronounced change being in the LDL. The cell surface receptor pathway in rat fibroblasts and smooth muscle cells resembled the system described for human fibroblasts as follows: 1) lipoproteins containing either the B or E apoproteins interacted with the receptors; 2) receptor binding activity was abolished by acetoacetylation or reductive methylation of a limited number of lysine residues of the lipoproteins; 3) receptor binding initiated the process of internalization and degradation of the apo-B- and apo-E-containing lipoproteins; 4) the lipoprotein cholesterol was re-esterified as determined by [14C]oleate incorporation into the cellular cholesteryl esters; and 5) receptor-mediated uptake (receptor number) was lipoprotein cholesterol. An important difference between rat and human fibroblasts was the inability of human LDL to interact with the cell surface receptors of rat fibroblasts. Rat lipoproteins did, however, react with human fibroblasts. Furthermore, the rat VLDL were the most avidly bound of the rat lipoproteins to rat fibroblasts. When the direct binding of 125I-VLDL was subjected to Scatchard analysis, the very high affinity of rat VLDL was apparent (Kd = 1 X 10(-11) M). Moreover, compared with data for rat LDL, the data suggested each VLDL particle bound to four to nine lipoprotein receptors. This multiple receptor binding could explain the enhanced binding affinity of the rat VLDL. The Scatchard plot of rat 125I-VLDL revealed a biphasic binding curve in rat and human fibroblast cells and in rat smooth muscle cells, suggesting two populations of rat VLDL. These results indicate that rat cells have a receptor pathway similar to, but not identical with, the LDL pathway of human cells. Since human LDL bind poorly to rat cell receptors on cultured rat fibroblasts and smooth muscle cells, metabolic studies using human lipoproteins in rats must be interpreted cautiously.     

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.     

Metabolomic analysis of polar metabolites in lipoprotein fractions identifies lipoprotein-specific metabolic profiles and their association with insulin resistance

While the molecular lipid composition of lipoproteins has been investigated in detail, little is known about associations of small polar metabolites with specific lipoproteins. The aim of the present study was to investigate the profiles of polar metabolites in different lipoprotein fractions, i.e., very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL) and two sub-fractions of the high-density lipoprotein (HDL). The VLDL, IDL, LDL, HDL(2), and HDL(3) fractions were isolated from serum of sixteen individuals having a broad range of insulin sensitivity and characterized using comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GC×GC-TOFMS). The lipoprotein fractions had clearly different metabolite profiles, which correlated with the particle size and surface charge. Lipoprotein-specific associations of individual metabolites with insulin resistance were identified, particularly in VLDL and IDL fractions, even in the absence of such associations in serum. The results indicate that the polar molecules are strongly attached to the surface of the lipoproteins. Furthermore, strong lipoprotein-specific associations of metabolites with insulin resistance, as compared to their serum profiles, indicate that lipoproteins may be a rich source of tissue-specific metabolic biomarkers.     

Lipoprotein cholesteryl ester production, transfer, and output in vivo in humans

Our aim was to identify and quantify the major in vivo pathways of lipoprotein cholesteryl ester transport in humans. Normal (n = 7), bile fistula (n = 5), and familial hypercholesterolemia (FH; n = 1) subjects were studied. Each received isotopic free cholesterol in HDL, LDL, or particulate form, along with another isotope of free or esterified cholesterol or mevalonic acid. VLDL, intermediate density lipoprotein (IDL), LDL, HDL, blood cells, and bile were collected for up to 6 days for analysis of radioactivity and mass of free and esterified cholesterol. These raw data were subjected to compartmental analysis using the SAAM program. Results in all groups corroborated net transport of free cholesterol to the liver from HDL, shown previously in fistula subjects. New findings revealed that 70% of ester was produced from free cholesterol in HDL and 30% from free cholesterol in LDL, IDL, and VLDL. No evidence was found for tissue-produced ester in plasma. There was net transfer of cholesteryl ester to VLDL and IDL from HDL and considerable exchange between LDL and HDL. Irreversible ester output was from VLDL, IDL, and LDL, but very little was from HDL, suggesting that selective and holoparticle uptakes of HDL ester are minor pathways in humans. It follows that 1) they contribute little to reverse transport, 2) very high HDL would not result from defects thereof, and 3) the clinical benefit of high HDL is likely explained by other mechanisms. Reverse transport in the subjects with bile fistula and FH was facilitated by ester output to the liver from VLDL plus IDL.     

Characterization of monoclonal anti-rabbit apolipoprotein E antibodies and chemical composition of lipoproteins separated by anti-apolipoprotein E immuno-affinity chromatography

Six mouse monoclonal antibodies against rabbit apolipoprotein E (apo E) have been developed. Of these monoclonal antibodies, clone 5 revealed a high affinity for purified apo E, very low density lipoprotein (VLDL) and beta-VLDL. This monoclonal antibody was used to prepare an immunoaffinity column. Coupled to Sepharose 4B, this antibody allowed complete removal of lipoproteins containing apo E from plasma of New Zealand white (NZW) rabbits; 62, 46, 14, and 3% of VLDL-, IDL-, LDL-, and HDL-protein, respectively, were bound to the anti-apo E affinity column. The bound VLDL was significantly rich in free cholesterol (FC) and cholesteryl esters (CE) relative to the unbound VLDL, whereas bound IDL, LDL and HDL were significantly rich in FC only. All of the bound fractions were characterized by significantly increased ratios of FC/phospholipids (PL). These results indicate that the two lipoprotein populations with and without apo E have different lipid compositions. The relatively high content of cholesterol in lipoproteins containing apo E suggests a contribution of apo E to plasma cholesterol transport.