Very fast ultracentrifugation of serum lipoproteins: influence on lipoprotein separation and composition

A very short run time and small sample volumes in the separation of lipoproteins by preparative ultracentrifugation are needed for several investigations. Recently, a very fast sequential separation method was described that needs only 100 min for one run in a centrifugal field of 625 000 × g. We studied the influence of centrifugal fields of this dimension on lipoprotein separation and lipoprotein particle integrity using a Beckman Optima^TM TLX ultracentrifuge with a TLA-120.2 rotor. Rotor speed (120/90/60/30 · 10³ rev./min) and run time (100 min/3 h/6.7 h/27 h) were selected in such a way that the product of centrifugal field and run time remained constant. The first conditions correspond to the very fast ultracentrifugation (VFU) procedure with a centrifugal field of 625 000 × g. Thirty different plasma samples covering a wide range of lipid and protein concentrations were separated in the course of two centrifugal runs at densities of 1.006 and 1.063 kg/l which yielded very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and the subnatant of low-density lipoproteins, including high-density lipoproteins (HDL) and concomitant sedimented plasma proteins. The major lipid components of the lipoproteins, triacylglycerols, free and esterified cholesterol, phospholipids and the apolipoproteins B and A-I, were estimated considering the masses of the tube contents after a slicing procedure. Measurements of lipids and proteins showed a very good recovery of better than 94% and 91%, respectively, and precision-within-series (coefficient of variation) of better than 4.2% and 6.5%, respectively. The effects of the rotor speed on the lipoprotein structure appeared to be weak. With increasing rotor speed, VLDL and LDL lipid constituents principally tended to decrease, whereas they increased in the subnatant of the LDL-run. The mean lipoprotein mass composition, considering the mass percentage of each measured particle constituent, did not show significant alterations. Total protein decreased in VLDL and in LDL and increased in the subnatant of the LDL-run. As checked by an enzyme-linked immunosorbent assay (ELISA) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein effects were due to nearly complete disappearence of contaminating plasma proteins, especially albumin as the major contamination of VLDL and LDL. The apolipoproteins (apo) B-100, A-I, E and C-I to C-III remained nearly unaffected. The main advantages of VFU were the very short run time (cumulative flotation time is 3.4 h) and the elimination of albumin without repeated runs. The procedure was suitable for the assessment of lipid and protein constituents in lipoproteins from very small plasma samples (500 μl).     

Effect of rat plasma lipoproteins on aldosterone production by rat zona glomerulosa cells in vitro

The possible role of plasma lipoproteins in steroidogenesis by zona glomerulosa cells was studied by examining the effect of rat plasma lipoproteins on aldosterone production by isolated rat zona glomerulosa cells. Neither very low density lipoprotein nor high density lipoprotein caused any increase in either basal or K⁺-stimulated aldosterone production. Low density lipoprotein (LDL) had no effect on basal aldosterone production but had a biphasic effect on K⁺-stimulated aldosterone production, causing a consistent enhancement of steroidogenesis at LDL cholesterol concentrations of 30–60 μg/ml. These data suggest that LDL may play a role in vivo in the supply of cholesterol for steroidogenesis by the rat zona glomerulosa cell.     

Time-related changes in the synthesis and secretion of very low density,low density and high density lipoproteins by cultured rat hepatocytes

Lipoproteins in the three major density classes were isolated from the medium of cultured rat hepatocytes incubated in the absence of serum for periods ranging from 1 to 48 h. De novo synthesis was suggested by the cyclo-heximide-sensitive incorporation of [³H]leucine into the apolipoproteins of the secreted lipoproteins.Hepatocyte d < 1.006 and d 1.006−1.063 g/ml lipoproteins were similar to plasma very low density lipoprotein (VLDL) and low density lipoprotein (LDL), respectively, in chemical composition, morphology and apolipoprotein distribution. The isolation of plasma-like d 1.006−1.063 g/ml particles is evidence for the hepatic origin of rat LDL; however, whether these particles are synthesized directly or result from catabolism of secreted VLDL has not been determined. Spherical d 1.063−1.21 g/ml particles containing predominantly apolipoprotein A-I were isolated from the media. In contrast to plasma high density lipoprotein (HDL) the hepatocyte particles contained significant concentrations of triacylglycerol and apolipoproteins of M_r > 100000 and lacked apolipoprotein A-IV.The pattern of lipoprotein secretion was related to the time of incubation. After incubation for 1, 3 and 6.5 h, VLDL comprised approx. 56% of the total lipoprotein mass, LDL 20% and HDL 24%. After 17 and 48 h the VLDL concentration was greatly reduced (approx. 20% of the total mass) while LDL and HDL concentrations were increased (33 and 47% of the total, respectively). Exogenous sodium oleate resulted in a concentration-dependent stimulation of VLDL synthesis at longer incubation periods. The triacylglycerol content of the secreted LDL fraction was also significantly increased following sodium oleate addition and there was an increased number of 425–650 Å particles present, which may represent catabolic products of VLDL. Hepatocyte mono-layers which can be maintained in serum-free media for extended periods should be useful for studying regulation of hepatic metabolism of the three major lipoprotein classes.     

Free cholesterol is a potent regulator of lipid transfer protein function

This study investigates the effect of altered lipoprotein free cholesterol (FC) content on the transfer of cholesteryl ester (CE) and triglyceride (TG) from very low- (VLDL), low- (LDL), and high-(HDL) density lipoproteins by the plasma-derived lipid transfer protein (LTP). The FC content of VLDL and HDL was selectively altered by incubating these lipoproteins with FC/phospholipid dispersions of varying composition. FC-modified lipoproteins were then equilibrated with [3H] TG, [14C]CE-labeled lipoproteins of another class to facilitate the subsequent modification of the radiolabeled donor lipoproteins. LTP was added and the extent of radiolabeled TG and CE transfer determined after 1 h. With either LDL or VLDL as lipid donor, an increase in the FC content of these lipoproteins caused a concentration-dependent inhibition (up to 50%) of CE transfer from these particles, without any significant effect on TG transfer. In contrast, with HDL as donor, increasing the HDL FC content had little effect on CE transfer from HDL, but markedly stimulated (up to 2.5-fold) the transfer of TG. This differential effect of FC on the unidirectional transfer of radiolabeled lipids from VLDL and HDL led to marked effects on LTP-facilitated net mass transfer of lipids. During long-term incubation of a constant amount of LTP with FC-modified VLDL and HDL, the extent of net mass transfer was linearly related to lipoprotein FC content; a 4-fold increase in FC content resulted in a 3-fold stimulation of the CE mass transferred to VLDL, which was coupled to an equimolar, reciprocal transfer of TG mass to HDL. Since lipid transfer between lipoproteins is integral to the process of reverse cholesterol transport, we conclude that lipoprotein FC levels are a potent, positive regulator of the pathways involved in sterol clearance. FC may modulate lipid transfer by altering the availability of CE and TG to LTP at the lipoprotein surface.     

Distribution of oleoyl-estrone in rat plasma lipoproteins.

Pooled adult normal rat plasma was used for the separation of lipoprotein fractions: VLDL, LDL and HDL, from which a total lipids extract was obtained. The presence of fragments with the MW of estrone and oleoyl-estrone in the lipoprotein fractions was analyzed by HPLC-MS. The results show that oleoyl-estrone is the major estrone component in lipoproteins; this molecular species was present in all three lipoprotein lipid extracts. The lipoprotein fractions were used for the analysis of protein and lipid classes: triacylglycerols, total and esterified cholesterol and phospholipids as well as acyl-estrone. About half of the total acyl-estrone was in the HDL fraction and only about 10% in the VLDL fraction. HDLs contained about one molecule in 50 particles, LDLs one molecule per particle and VLDLs 15 molecules per particle, i.e. given their size, the larger lipoproteins contained more oleoyl-estrone than the HDLs. The distribution of this hormone suggests that oleoyl-estrone is lost with other lipids as the lipoproteins shrink. The results presented show that oleoyl-estrone is a molecule found naturally in rat lipoproteins in low concentrations - the lowest in HDLs - that are consistent with its postulated role in the control of body weight.     

Oxidation of lipoprotein Lp(a). A comparison with low-density lipoproteins

Aimed at identifying possible mechanisms of the suggested high atherogenicity of Lp(a), its susceptibility for Cu(II)-induced oxidation was studied and compared with that of LDL. Since the content of antioxidants as well as the fatty acid pattern of a lipoprotein greatly affects its oxidizability, Lp(a) and LDL were characterized first with respect to these substances. Paired samples of low-density lipoproteins (LDL) and Lp(a) were isolated from seven individual donors and compared with each other. This study showed that LDL and Lp(a) are very similar with respect to their fatty acid and antioxidant composition. LDL contains approx. 1132 nmol of total fatty acids/mg lipoprotein and LDL 1466 nmol total fatty acids/mg lipoprotein. Analysis of the fatty acid composition of individual lipid classes (cholesteryl esters, phospholipids and triacylglycerols) revealed also a high similarity in the composition of these lipid classes between the two lipoproteins. A comparison of the antioxidant composition showed that Lp(a) contains less α-tocopherol than LDL (1.6 ± 0.35 nmol/mg vs. 2.1 ± 0.25 nmol/mg LDL). In copper(II)-induced lipid peroxidation experiments we found a striking difference in the susceptibility of individual lipoprotein classes between all donors. In addition, Lp(a) exhibited a 1.2 to 2.4 longer lag-phase than the corresponding LDL preparation from the same blood donor. Treatment of Lp(a) with neuraminidase resulted in a drastic decrease of thelag-phase of Lp(a). Neuraminidase treatment of LDL on the other hand had no significant effects on its susceptibility to oxidation. Supplementation of neuraminidase-treated Lp(a) with N-acetylneuraminic acid (NANA) at concentrations comparable to the naturally occurring amounts of NANA in the Lp(a) protein moiety led to an increase of the lag-phase yielding values which were comparable to those observed with native Lp(a). These results demonstrate that the fatty acid composition as well as the antioxidant concentrations of Lp(a) and LDL are quite similar; despite this fact, Cu²⁺-mediated oxidation of Lp(a) is retarded in comparison to LDL which might be due to the higher content of NANA in Lp(a).     

The effect of reduction of lipoprotein (a) on cellular cholesterol synthesis in non-diabetic and Type 2 diabetic subjects

This study investigates the effect of Lipoprotein (a) (Lp(a)) on cellular cholesterol synthesis in non-diabetic (n = 7) and Type 2 (non-insulin-dependent) diabetic subjects (n = 7) with elevated levels of Lp(a) (> 20 mg/dl). N-Acetylcysteine was used to lower Lp(a) in the control subjects and their lipoproteins were re-examined after 7 days of treatment. Low-density lipoprotein (LDL) was isolated and separated from Lp(a) by sequential ultracentrifugation. Regulation of cellular cholesterol synthesis was assessed by measuring incorporation of [¹⁴C]acetate into mononuclear leucocytes in the presence of LDL and Lp(a). Cellular cholesterol content was determined by a fluorometric assay. Delivery of cholesterol to the cell was examined using [³H]cholesteryl oleate-labelled LDL or Lp(a). LDL (5 μg/ml) from non-diabetic subjects suppressed cellular cholesterol synthesis by 66.2%, while Lp(a) at a similar concentration only suppressed cholesterol synthesis by 5.8% (P < 0.001). At a concentration of 20 μg/ml, Lp(a) suppressed cholesterol synthesis by 31.7%. The situation was similar in the diabetic subjects. Serum LDL cholesterol in non-diabetic subjects was 4.2 ± 0.5 mmol/1 and the LDL esterified/free cholesterol ratio was 2.6 ± 0.2. Following treatment with N-acetylcysteine, LDL cholesterol did not change, while Lp(a) decreased significantly by 24% (P < 0.05). The LDL esterified/free cholesterol ratio decreased to 2.2 ± 0.2 (P < 0.05) and there was a significant increase in the ability of the subjects LDL to inhibit cellular cholesterol synthesis (P < 0.05). There was a significant negative correlation between plasma Lp(a) and the ability of the patients' LDL to inhibit cellular cholesterol synthesis (r = − 0.68, P < 0.01). [³H]Cholesteryl-oleate-LDL (5 μg/ml) delivered 266 ± 13 ng cholesteryl oleate/mg cell protein, while it took 20 μg of [³H]cholesteryl oleate-labelled-Lp(a) to deliver a similar concentration (315 ± 21 ng cholesteryl oleate/mg cell protein). In conclusion it appears possible that the atherogenicity of Lp(a) may be associated with its effect on the LDL receptor which alters LDL receptor uptake, LDL composition and cellular cholesterol synthesis.     

Distribution of cell-derived cholesterol among plasma lipoproteins: a comparison of three techniques

Three fractionation procedures (immunoaffinity chromatography, two-dimensional nondenaturing electrophoresis, and heparin-agarose affinity chromatography) have been compared in determining the kinetics of free and ester cholesterol transfer in normolipemic native plasma. Similar results were obtained in each case. Cell-derived free cholesterol is initially enriched in high density lipoproteins (HDL) (mainly HDL without apoE); at longer time periods (greater than 10 min) greater proportions are observed in very low density lipoproteins (VLDL) and low density lipoproteins (LDL). The major part of cholesteryl ester (about 90%) was retained in HDL, while VLDL and LDL, which contained about 75% of total cholesteryl ester mass, received only about 10% of cell-derived cholesteryl ester. Within HDL, almost all cholesteryl ester was in the apoE-free fraction. These data provide evidence that lipoprotein free and esterified cholesterol are not at chemical equilibrium in normal plasma, and that cell-derived cholesterol is preferentially directed to HDL. The techniques used had a comparable effectiveness for the rapid fractionation of labile lipoprotein lipid radioactivity.     

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.     

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.     

Evidence for a Trypanosoma brucei lipoprotein scavenger receptor

African trypanosomes are lipid auxotrophs that live in the bloodstream of their human and animal hosts. Trypanosomes require lipoproteins in addition to other serum components in order to multiply under axenic culture conditions. Delipidation of the lipoproteins abrogates their capacity to support trypanosome growth. Both major classes of serum lipoproteins, LDL and HDL, are primary sources of lipids, delivering cholesterol esters, cholesterol, and phospholipids to trypanosomes. We show evidence for the existence of a trypanosome lipoprotein scavenger receptor, which facilitates the endocytosis of both native and modified lipoproteins, including HDL and LDL. This lipoprotein scavenger receptor also exhibits selective lipid uptake, whereby the uptake of the lipid components of the lipoprotein exceeds that of the protein components. Trypanosome lytic factor (TLF1), an unusual HDL found in human serum that protects from infection by lysing Trypanosoma brucei brucei, is also bound and endocytosed by this lipoprotein scavenger receptor. HDL and LDL compete for the binding and uptake of TLF1 and thereby attenuate the trypanosome lysis mediated by TLF1. We also show that a mammalian scavenger receptor facilitates lipid uptake from TLF1 in a manner similar to the trypanosome scavenger receptor. Based on these results we propose that HDL, LDL, and TLF1 are all bound and taken up by a lipoprotein scavenger receptor, which may constitute the parasite's major pathway mediating the uptake of essential lipids.     

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.     

Specific elimination of albumin in serum lipoprotein preparations.

In the preparation of pure serum lipoproteins by means of ultracentrifugation techniques, it is necessary to perform consecutive centrifugations in order to reduce the content of contaminating serum albumin (1) impairing apolipoprotein determination and purification and turnover studies with ¹²⁵I-labeled lipoproteins. Extensive centrifugation, however, may cause in vitro changes in the composition of the lipoprotein particles (1,2). The present report describes the use of matrix-bound anti-albumin for specific elimination of albumin in very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) preparations from human serum.     

Mass spectrometry-based analytical tools for the molecular protein characterization of human plasma lipoproteins

Lipoproteins are a heterogeneous population of blood plasma particles composed of apolipoproteins and lipids. Lipoproteins transport exogenous and endogenous triglycerides and cholesterol from sites of absorption and formation to sites of storage and usage. Three major classes of lipoproteins are distinguished according to their density: high-density (HDL), low-density (LDL) and very low-density lipoproteins (VLDL). While HDLs contain mainly apolipoproteins of lower molecular weight, the two other classes contain apolipoprotein B and apolipoprotein (a) together with triglycerides and cholesterol. HDL concentrations were found to be inversely related to coronary heart disease and LDL/VLDL concentrations directly related. Although many studies have been published in this area, few have concentrated on the exact protein composition of lipoprotein particles. Lipoproteins were separated by density gradient ultracentrifugation into different subclasses. Native gel electrophoresis revealed different gel migration behaviour of the particles, with less dense particles having higher apparent hydrodynamic radii than denser particles. Apolipoprotein composition profiles were measured by matrix-assisted laser desorption/ionization-mass spectrometry on a macromizer instrument, equipped with the recently introduced cryodetector technology, and revealed differences in apolipoprotein composition between HDL subclasses. By combining these profiles with protein identifications from native and denaturing polyacrylamide gels by liquid chromatography-tandem mass spectrometry, we characterized comprehensively the exact protein composition of different lipoprotein particles. We concluded that the differential display of protein weight information acquired by macromizer mass spectrometry is an excellent tool for revealing structural variations of different lipoprotein particles, and hence the foundation is laid for the screening of cardiovascular disease risk factors associated with lipoproteins.     

Lipid class and molecular species interrelationships among plasma lipoproteins of type III and type IV hyperlipemic subjects

As a further appraisal of lipoprotein interconversion and equilibration of lipid components a detailed examination was made of the chemical class and molecular species interrelationships among the major fasting plasma lipoprotein fractions within each of six male Type III and Type IV hyperlipemic subjects subsisting on free choice diets. The lipoprotein fractions were prepared by conventional ultracentrifugation and the lipid class and molecular species composition of the corresponding lipoprotein fractions were determined by gas chromatography of the intact glycerol esters and ceramides. In general, each lipoprotein fraction possessed a well defined lipid class composition, which was characterized by a dramatically decreasing triacylglycerol and increasing phospholipid and cholesteryl ester content, when progressing from the very low (VLDL) to the low (LDL) and high (HDL) density lipoproteins, as already established for normolipemic subjects. Likewise, the LDL, and LDL₂ of the hyperlipemic subjects contained about two times higher proportion of total phospholipid as sphingomyelin than VLDL and HDL. Furthermore, the sphingomyelins of the HDL fraction contained about 30% more of the higher and 30% less of the lower molecular weight species than the sphingomyelins of the VLDL. Smaller differences were seen in the molecular species composition of the phosphatidylcholines, cholesteryl esters and triacylglycerols among the corresponding lipoproteins. In comparison to normolipemic subjects analyzed previously, the hyperlipemic subjects showed greater individual variability. Despite this variability the lipid class and molecular species composition in the hyperlipemic subjects was again incompatible with the hypothesis which postulates direct VLDL conversion into LDL and HDL under the influence of lipoprotein lipase and lecithin: cholesterol acyltransferase. The main differences between normolipemic and hyperlipemic plasma were found to reside in the number of the VLDL and LDL, lipoprotein particles and not in their chemical composition or physical structure, or in the apparent mechanism of their metabolic interconversion.     

Synthesis and secretion of lipoproteins by human hepatocytes in culture

Summary Confluent monolayers of normal human hepatocytes obtained by collagenase perfusion of liver pragments were incubated in a serum-free medium. Intracellular apolipoproteins apo AI, apo C, apo B, and apo E were detected between Day 1 and Day 6 of the culture by immunoenzymatic staining using polyclonal antibodies directed against these apoproteins and monoclonal antibodies directed against both forms of apo B (B100 and B48). Translation of mRNA isolated from these hepatocytes in an acellular system revealed that apo AI and apo E were synthesized as the precusor forms of mature plasma apo AI and apo E. Three lipoprotein fractions corresponding to the density of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) were isolated from the medium at Day 5 of culture and examined by electron microscopy after negative staining. VLDL and LDL particles are similar in size and shape to plasma lipoproteins; spherical HDL are larger than normal plasma particles isolated at the same density. Their protein represented 44, 19.5, and 36.5% respectively, of the total lipoprotein protein. The secretion rate of VLDL protein corresponded to that measured in primary cultures of rat hepatocytes. After incorporation of [³H]glycerol, more than 92% of the [³H]triglyceride secreted into the medium was recovered in the VLDL fraction. These results demonstrate that primary cultures of normal human hepatocytes are able to synthesize and secrete lipoproteins and thus could be a useful model to study lipoprotein metabolism in human liver.     

Is the atherosclerotic phenotype of preeclamptic placentas due to altered lipoprotein concentrations and placental lipoprotein receptors? Role of a small-for-gestational-age phenotype

Atherosis of spiral arteries in uteroplacental beds from preeclamptic women resemble those of atherosclerosis, characterized by increased plasma lipids and lipoproteins. We hypothesized that: 1) lipoprotein receptors/transporters in the placenta would be upregulated in preeclampsia, associated with increased maternal and fetal lipoprotein concentrations; and 2) expression of these would be reduced in preeclamptic placentae from women delivering small-for-gestational-age (SGA) infants. Placental biopsies and maternal and umbilical serum samples were taken from 27 normotensive and 24 preeclamptic women. Maternal/umbilical cord serum LDL, HDL, total cholesterol, and triglycerides were measured. Placental mRNA expression of lipoprotein receptors/transporters were quantified using quantitative RT-PCR. Protein localization/expression of LDL receptor-related protein 1 (LRP-1) in the preeclamptic placentae with/without SGA was measured by immunohistochemistry. Placental mRNA expression of all genes except paraoxonase-1 (PON-1), microsomal triglyceride transfer protein (MTTP), and protein disulfide isomerase family A member 2 (PDIA2) were observed. No differences for any lipoprotein receptors/transporters were found between groups; however, in the preeclamptic group placental LRP-1 expression was lower in SGA delivering mothers (n = 7; P = 0.036). LRP-1 protein was localized around fetal vessels and Hofbauer cells. This is the first detailed study of maternal/fetal lipoprotein concentrations and placental lipoprotein receptor mRNA expression in normotensive and preeclamptic pregnancies. These findings do not support a role of altered lipid metabolism in preeclampsia, but may be involved in fetal growth.     

Binding of low-density lipoprotein and chylomicron remnants to the hepatic low-density lipoprotein receptor of dogs, rats and rabbits demonstrated by ligand blotting. Failure to detect a distinct chylomicron-remnant-binding protein by ligand blotting

In this paper, human low-density lipoprotein (LDL), rat chylomicron remnants and very-low-density lipoproteins of beta-mobility from cholesterol-fed rabbits (beta VLDL) have been shown to bind strongly to a protein present in solubilised liver membranes of rats, rabbits and dogs by ligand blotting with biotin-modified lipoproteins. This binding protein was identified as the LDL-receptor on several criteria. First, binding of the lipoproteins to the receptor was saturable and Ca2+-dependent; secondly, the apparent relative molecular mass of the binding protein (ranging from 128,000 in the rabbit, 145,000 in the rat to 147,000 in the dog) was similar to that of the purified bovine LDL receptor. Finally, binding activity was greatly increased in the livers of rats treated with oestrogen in pharmacological doses and absent from the liver of Watanabe heritable hyperlipidaemic (WHHL) rabbits that have a genetic defect in the LDL receptor. Some binding was also observed to a high-molecular-mass protein present in solubilised liver membranes of rats and rabbits, which, in rabbits at least, shared antigenic determinants with rabbit apoB and was not likely to be related to the LDL receptor as it was present in equal amounts in normal and WHHL rabbits. No evidence was obtained for a specific chylomicron remnant binding protein, distinct from the LDL receptor, whose activity could be detected in solubilised liver membranes by ligand blotting although a variety of solubilisation and fractionation conditions were employed.     

Remnant lipoproteins induced proliferation of human prostate cancer cell,PC-3 but not LNCaP,via low density lipoprotein receptor

Background: Hypertriglyceridemia has been shown to be one of the risk factors for prostate cancer. In this study, we investigated the effect of remnant lipoproteins on cell growth in prostate cancer cell lines. Methods: Remnant lipoproteins were isolated as remnant like particles (RLP) from human plasma. We used RLP for TG-rich lipoproteins and low density lipoproteins (LDL) for cholesterol-rich lipoproteins respectively and examined the effect of lipoproteins on proliferation of PC-3 and LNCaP cells using MTS assays. Moreover, we studied the effect of RLP and LDL treatment on the regulation of lipoprotein receptors in prostate cancer cells to investigate the relationship between lipoprotein-induced cell proliferation and lipoprotein receptor expression using real-time PCR, Western blotting assays and siRNA. Results: RLP effectively induced PC-3 cell proliferation more than LDL, whereas both RLP and LDL could not induce LNCaP cell proliferation except at a higher concentration of RLP. LDL receptor (LDLr) was expressed in both prostate cancer cells but there was a sharp difference of sterol regulation between two cells. In PC-3 cells, LDL decreased the LDLr expression in some degree, but RLP did not. Meanwhile LDLr expression in LNCaP was easily downregulated by RLP and LDL. Blocking LDLr function significantly inhibited both RLP- and LDL-induced PC-3 cell proliferation. Conclusions: This study demonstrated that RLP-induced PC-3 cell proliferation more than LDL; however, both RLP and LDL hardly induced LNCaP cell proliferation. The differences of proliferation by lipoproteins might be involved in the regulation of LDLr expression.     

Incorporation of cholesterol into serum high and low density lipoproteins

Excess cholesterol (CHL) was incorporated into serum lipoproteins by incubation with Celite^®-dispersed CHL or CHL crystals. The initial rates of uptake were 15 and 7 mol CHL/mol lipoprotein × h^? for low (LDL) and high (HDL) density lipoprotein, respectively. Saturation values were obtained after 48 h and were 90 and 65 mol CHL/mol LDL, and 42 and 32 mol/mol HDL using CHL on Celite and CHL cyrstals, respectively. Characterization of the lipo-proteins showed a small change in electrophoretic mobility and an increase in molecular masses, especially after incubation with CHL on Celite. Spectroscopic methods showed only minor effects on the protein moieties.