LDL受体介导的血浆低密度脂蛋白胆固醇的内吞

胆固醇是动物细胞细胞膜的重要组成成分,其做为细胞和环境之间的屏障调节细胞膜的流动性。胆固醇是体内所有的类固醇激素和胆酸合成的前体物质,参与体内代谢。同时胆固醇在神经系统的发育中也起着重要的作用。在血浆中胆固醇以低密度脂蛋白和高密度脂蛋白这两种胆固醇运载血脂蛋白的形式运输。动物细胞通过细胞表面的低密度脂蛋白受体(LDL receptor,LDLR)介导的内吞可以从血液中摄取富含胆固醇的低密度脂蛋白,当细胞表面的LDLR的功能缺陷时,可以导致高胆固醇血症,继而引起动脉粥样硬化、冠心病和中风等严重疾病。本文综述了LDL受体的概述及其通过内吞调节血液中低密度脂蛋白胆固醇水平的作用,并对LDL受体的调节进行了阐述。     

Increased uptake of methylated low-density lipoprotein induced by noradrenaline in carotid arteries of anaesthetized rabbits

Atherosclerosis is accelerated in hyperlipidaemias but, apart from the concentration of low-density lipoprotein (LDL) in the blood, very little is known about other influences on the disease process. We now provide evidence that in anaesthetized rabbits the atherogenic uptake of LDL by arterial walls is accelerated by noradrenaline at its physiological concentrations in rabbit and human blood. The principle of the experiments was to compare the uptake of intravenously injected, radioactively labelled LDL, methylated to prevent removal by high-affinity receptors, in the two carotid arteries of anaesthetized rabbits after infusing low concentrations of noradrenaline into one carotid and saline as control into the other, the volume rates of infusion being about 1% of the carotid blood flows. Human LDL, which behaves sufficiently like rabbit LDL for these purposes, was prepared, methylated and radio-iodinated by standard methods. At the end of the infusions, the arteries were excised and their radioactivities determined. Noradrenaline infused for 2 h to produce local blood concentrations of nominally 1, 10, 50 and 100 nM significantly increased the LDL radioactivities of the walls of the noradrenaline-infused carotids. Concentrations of nominally 100 nM also increased the LDL radioactivities of the walls of the saline-infused carotids; this was associated with significant increases in their blood noradrenaline concentrations. These results may contribute towards an explanation for the accelerated atherosclerosis and the increased incidence of its clinical manifestations in conditions associated with elevated blood noradrenaline concentrations, including the episodic increases associated with stress and cigarette smoking as well as the more persistent increases caused by phaeochromocytoma.     

In vivo cholesteryl ester selective uptake of mildly and standardly oxidized LDL occurs by both parenchymal and nonparenchymal mouse hepatic cells but SR-BI is only responsible for standardly oxidized LDL selective uptake by nonparenchymal cells

In blood circulation, low density lipoproteins (LDL) can undergo modification, such as oxidation, and become key factors in the development of atherosclerosis. Although the liver is the major organ involved in the elimination of oxidized LDL (oxLDL), the identity of the receptor(s) involved remains to be defined. Our work aims to clarify the role of the scavenger receptor class B type I (SR-BI) in the hepatic metabolism of mildly and standardly oxLDL as well as the relative contribution of parenchymal (hepatocytes) and nonparenchymal liver cells with a special emphasis on CE-selective uptake. The association of native LDL and mildly or standardly oxLDL labeled either in proteins or in cholesteryl esters (CE) was measured on primary cultures of mouse hepatocytes from normal and SR-BI knock-out (KO) mice. These in vitro assays demonstrated that hepatocytes are able to mediate CE-selective uptake from both LDL and oxLDL and that SR-BI KO hepatocytes have a 60% reduced ability to selectively take CE from LDL but not towards mildly or standardly oxLDL. When lipoproteins were injected in the mouse inferior vena cava, parenchymal and nonparenchymal liver cells accumulated more CE than proteins from native, mildly and standardly oxLDL, indicating that selective uptake of CE from these lipoproteins occurs in vivo in these two cell types. The parenchymal cells contribute near 90% of the LDL-CE selective uptake and SR-BI for 60% of this pathway. Nonparenchymal cells capture mainly standardly oxLDL while parenchymal and nonparenchymal cells equally take up mildly oxLDL. An 82% reduction of standardly oxLDL-CE selective uptake by the nonparenchymal cells of SR-BI KO mice allowed emphasizing the contribution of SR-BI in hepatic metabolism of standardly oxLDL. However, SR-BI is not responsible for mildly oxLDL metabolism. Thus, SR-BI is involved in LDL- and standardly oxLDL-CE selective uptake in parenchymal and nonparenchymal cells, respectively.     

Selective uptake of cholesteryl esters from low density lipoproteins in vitro and in vivo.

Evidence for the direct uptake ("selective uptake") of cholesteryl esters (CE) from low density lipoproteins (LDL) by perfused luteinized rat ovaries (Azhar, S., A. Cooper, L. Tsai, W. Maffe, and E. Reaven. 1988. J. Lipid Res. 29: 869-882) led to this examination of LDL selective uptake in cultured cells and in rats using LDL doubly labeled with intracellularly trapped tracers of the CE and apoB moieties. Studies in vitro demonstrated LDL selective uptake by human fibroblasts at a low rate relative to LDL particle uptake; the fractional rate of this selective uptake increased with decreasing LDL particle size. Mouse Y1-BS1 adrenal cortical tumor cells also selectively took up LDL CE; on ACTH treatment, LDL selective uptake increased in parallel with high density lipoproteins (HDL) selective uptake, and accounted for the majority of LDL CE uptake. Metabolism of doubly labeled LDL was examined in rats. Adrenal gland and liver selectively took up CE from rat LDL, as did lung and adipose tissue. Selective uptake from human LDL was at a lower fractional rate than from rat LDL, and could not be demonstrated in as many organs. Although selective uptake from LDL by ovaries of adult rats was not significant, ovaries of immature rats consistently exhibited LDL selective uptake; on treatment of these rats with hormones to produce superovulated, luteinized ovaries, LDL selective uptake increased in the ovaries and nowhere else. Selective uptake was also apparent in liver, where it accounted for 27% of total hepatic uptake of rat LDL CE. These studies indicate a significant contribution of selective uptake to LDL CE metabolism in rats, suggesting the possibility of a role in other animals as well.     

Autoxidation of human low density lipoprotein: loss of polyunsaturated fatty acids and vitamin E and generation of aldehydes

The alteration of structural and biological properties of human plasma low density lipoprotein (LDL) exposed to oxidative conditions is in part ascribed to lipid peroxidation. The objective of this investigation was to measure quantitatively several parameters in oxidizing LDL indicative for lipid peroxidation. Exposure of freshly prepared EDTA-free LDL to an oxygen-saturated buffer led to a complete depletion of alpha- and gamma-tocopherol within 6 hr, thereafter lipid peroxidation commenced as indicated by the kinetics of the loss of linoleic (18:2) and arachidonic (20:4) acids, the formation of aldehydic lipid peroxidation products and fluorescent apoB. Within 24 hr of oxidation, on average 79 nmol of 18:2 (initial 345) and 12.8 nmol of 20.4 (initial 25.6) were oxidized per mg of LDL and the sample contained in total 7.1 nmol of aldehydes with the following molar distribution: 36.6% malonaldehyde, 25% hexanal, 8.9% propanal, 8.2% 4-hydroxynonenal, 7.6% butanal, 4.1% 2.4-heptadienal, 3.4% pentanal, 3.4% 4-hydroxyhexenal, and 2.5% 4-hydroxyoctenal. Malonaldehyde was predominantly (93%) in the aqueous phase, whereas the other aldehydes remained mostly (34-98%) within the LDL particle, where the total aldehyde concentration was in the range of 12 mM. Oxidized LDL exhibited a 1.6-fold enhanced electrophoretic mobility. Similarily, native LDL incubated for 5 hr with aldehydes showed increased electrophoretic mobility. At equal concentrations (5 mM) 4-hydroxynonenal was most effective, followed by 2,4-heptadienal, hexanal, and malonaldehyde. This study reports for the first time the rate and extent of the change of LDL constituents occurring during lipid peroxidation.     

A Comparison of the Uptake and Translocation of Some Organic Herbicides and a Systemic Fungicide by Barley: I. ABSORPTION IN EELATION TO PHYSICO-CHEMICAL PROPERTIES

A comparative study has been made of the uptake by and translocationfrom roots of intact barley plants of six herbicides and a systemicfungicide (four triazines, diuron, 2,4-dichloro-phenoxyaceticacid (2,4-D) and ethirimol). Relationships between uptake andtranspiration rate are discussed in the light of the physico-chemicalproperties of these compounds, notably their partition coefficientsin oil/water systems and their dissociation constants. Apartfrom 2,4-D, sorption of these compounds appears to be a passiveprocess. At pH4 the uptake of 2,4-D seems to be influenced bymetabolism; not only may the concentration of this compoundin the transpiration stream be considerably greater than thatin the medium surrounding the roots but absorption by rootsis markedly reduced at low temperatures and by sodium azide. The initial rate of uptake of these compounds correlates reasonablywell with their partition coefficients in olive oil/water orn-dodecane/water systems; likewise the concentration in thetranspiration stream is greater for lipophilic than for lipophobicsubstances. Whereas the hydrogen ion and calcium concentrations of the ambientmedium appear to have no effect on the uptake of compounds withlow pK's, the uptake of those substances which protonate betweenpH4 and pH6 is affected by them. These findings are discussedfrom the viewpoint that the pathways of transport of lipophilicand lipophobic compounds across the roots may differ. Although there is some evidence that retention by roots canlimit transport to shoots, there is no simple inverse correlationbetween the total concentration of the different substancesin the roots and that in the transpiration stream. This questionis discussed in a subsequent paper.     

Gadolinium chloride-induced shifts in intrahepatic distributions of liposomes

Intravenously administered gadolinium chloride caused only a slight decrease in the rate of elimination of small unilamellar liposomes from the blood and had no influence on the total hepatic uptake of these vesicles, but did alter their intrahepatic distribution substantially. Uptake by the non-parenchymal cells was substantially decreased, whereas uptake by the parenchymal cells showed a concomitant increase. Our earlier observations (Roerdink et al. (1981) Biochim. Biophys. Acta 677, 79-89) on the effect of lanthanides on the in vivo distribution of multilamellar liposomes have been extended, in that we demonstrate, in addition to the drop in elimination rate from the blood and in the over-all hepatic uptake, a shift of liposome distribution within the Kupffer cell population. While the larger Kupffer cells, which normally take up a major fraction of an injected liposome dose, were strongly inhibited in liposome uptake, the more numerous small macrophages showed a 3-4-fold increase in uptake.     

Proteolytic enzymes of mast cell granules degrade low density lipoproteins and promote their granule-mediated uptake by macrophages in vitro

Secretory granules exocytosed from rat serosal mast cells bind low density lipoprotein (LDL), and on being phagocytosed by macrophages, carry the bound LDL into these cells (Kokkonen, J. O., and Kovanen, P. T. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2287-2291). The binding of LDL to the granules is mediated through interactions between the apolipoprotein B (apoB) component of LDL and the heparin proteoglycan component of the granules. Here we report how degradation of apoB by the neutral proteases of the granules affects the granule-mediated uptake of LDL by cultured mouse macrophages. During incubation of LDL with proteolytically inactive granules, the rate of uptake of LDL by macrophages increased by 10-fold; whereas during incubation with proteolytically active granules, it increased by 50-fold, the increase in the rate of uptake during proteolysis correlating with the degree of apoB degradation. The 5-fold greater capacity of the proteolytically active granules to enhance the uptake of LDL resulted from their greater capacity to bind LDL, and consequently, to carry it into the macrophages. Electron microscopic analysis of LDL bound to the proteolytically active granules disclosed large spherical particles of fused LDL. The diameters of the granule-bound particles ranged up to 90 nm compared with an average diameter of 22 nm for both native LDL and the LDL bound to proteolytically inactive granules. The results show that granule proteases, by inducing fusion of granule-bound LDL, increase the amount of LDL bound per unit weight of granule heparin proteoglycan. Hence, the two components of mast cell granules, the proteases and the heparin proteoglycan, act in concert to promote the uptake of LDL by macrophages in vitro.     

Cultured human hepatocytes. Evidence for metabolism of low density lipoproteins by a pathway independent of the classical low density lipoprotein receptor

Studies of low density lipoprotein (LDL) metabolism in nonhuman model systems have indicated that the mammalian liver has dual mechanisms for the uptake and regulation of the concentration of plasma LDL. Heretofore, direct evaluation of lipoprotein uptake mechanisms in human hepatocytes has not been possible. In order to compare hepatocyte LDL uptake with fibroblast LDL metabolism, human hepatocytes were isolated and cultured from small biopsy specimens obtained from normolipidemic and homozygous familial hypercholesterolemic patients. Cells cultured in serum-free culture medium retained the morphological and biochemical characteristics of hepatocytes for at least 7 days. The uptake and degradation of LDL by hepatocytes was compared to that of the cultured human fibroblasts. Like fibroblasts, hepatocytes bound, internalized, and degraded LDL. In both cell types, uptake approached saturation at a concentration of 50 micrograms of LDL protein/ml. Competition for LDL binding by LDL, high density lipoprotein, and modified LD revealed that the hepatocyte binding was specific for LDL. Cellular cholesterol loading by incubation in LDL-enriched culture medium resulted in diminished LDL uptake in both cell types. Chemical modification of LDL by acetoacetylation, acetylation, and reductive methylation abolished LDL uptake and degradation by fibroblasts. However, hepatocytes bound and degraded the modified LDL at 30-50% the level of native LDL. Homozygous familial hypercholesterolemic hepatocytes were devoid of the LDL receptor pathway but metabolized native LDL to the extent observed with modified LDL uptake by normal hepatocytes. In contrast to the classic LDL receptor pathway, the second or alternate pathway does not lead to regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity. These findings indicate the presence of two separate pathways of LDL uptake in human hepatocytes which have different effects on hepatocytic cholesterol metabolism.     

Uptake of lactosylated low-density lipoprotein by galactose-specific receptors in rat liver.

The liver contains two types of galactose receptors, specific for Kupffer and parenchymal cells respectively. These receptors are only expressed in the liver, and therefore are attractive targets for the specific delivery of drugs. We provided low-density lipoprotein (LDL), a particle with a diameter of 23 nm in which a variety of drugs can be incorporated, with terminal galactose residues by lactosylation. Radioiodinated LDL, lactosylated to various extents (60-400 mol of lactose/ mol of LDL), was injected into rats. The plasma clearance and hepatic uptake of radioactivity were correlated with the extent of lactosylation. Highly lactosylated LDL (greater than 300 lactose/LDL) is completely cleared from the blood by liver within 10 min. Pre-injection with N-acetylgalactosamine blocks liver uptake, which indicates that the hepatic recognition sites are galactose-specific. The hepatic uptake occurs mainly by parenchymal and Kupffer cells. At a low degree of lactosylation, approx. 60 lactose/LDL, the specific uptake (ng/mg of cell protein) is 28 times higher in Kupffer cells than in parenchymal cells. However, because of their much larger mass, parenchymal cells are the main site of uptake. At high degrees of lactosylation (greater than 300 lactose/LDL), the specific uptake in Kupffer cells is 70-95 times that in parenchymal cells. Under these conditions, Kupffer cells are, despite their much smaller mass, the main site of uptake. Thus not only the size but also the surface density of galactose on lactosylated LDL is important for the balance of uptake between Kupffer and parenchymal cells. This knowledge should allow us to design particulate galactose-bearing carriers for the rapid transport of various drugs to either parenchymal cells or Kupffer cells.     

Low density lipoprotein uptake: holoparticle and cholesteryl ester selective uptake.

Low density lipoproteins (LDL) contain apolipoprotein B-100 and are cholesteryl ester-rich, triglyceride-poor macromolecules, arising from the lipolysis of very low density lipoproteins. This review will describe the receptors responsible for uptake of whole LDL particles (holoparticle uptake), and the selective uptake of LDL cholesteryl ester. The LDL-receptor mediates the internalization of whole LDL through an endosomal-lysosomal pathway, leading to complete degradation of LDL. Increasing LDL-receptor expression by pharmacological intervention efficiently reduces blood LDL concentrations. The lipolysis stimulated receptor and LDL-receptor related protein may also lead to complete degradation of LDL in presence of free fatty acids and apolipoprotein E- or lipase-LDL complexes, respectively. Selective uptake of LDL cholesteryl ester has been demonstrated in the liver, especially in rodents and humans. This activity brings five times more LDL cholesteryl ester than the LDL-receptor to human hepatoma cells, suggesting that it is a physiologically significant pathway. The lipoprotein binding site of HepG2 cells mediates this process and recognizes all lipoprotein classes. Scavenger receptor class B type I and CD36, which mediate the selective uptake of high density lipoprotein cholesteryl ester, are potentially involved in LDL cholesteryl ester selective uptake, since they both bind LDL with high affinity. It is not known whether they are identical to the uncloned lipoprotein binding site and if the selective uptake of LDL cholesteryl ester produces a less atherogenic particle. If this is verified, pharmacological up-regulation of LDL cholesteryl ester selective uptake may become another therapeutic approach for reducing blood LDL-cholesterol levels and the risk of atherosclerosis.     

Glycosylation of LDL decreases its ability to interact with high-affinity receptors of human fibroblasts in vitro and decreases its clearance from rabbit plasma in vivo

Incubation of human LDL in vitro at 37 degrees C for 48 h with [14C]glucose at concentrations from 5 to 200 mM resulted in a glycosylated LDL, containing 0.4-20 mol of glucose incorporated per apolipoprotein B of 250 000 daltons. The extent of glucose incorporated was proportional to the time of incubation and concentration of glucose. Glycosylation of LDL abolished its uptake and degradation by the high-affinity process for LDL in normal human skin fibroblasts. 125I-labeled glycosylated LDL was bound, internalized and degraded by the fibroblasts via a nonspecific low-affinity process. The 125I-labeled glycosylated LDL and 125I-labeled LDL were taken up and degraded at similar rates in a non-saturable, low-affinity process by peritoneal macrophages isolated from mice. When 125I-labeled glycosylated LDL or 125I-labeled LDL were injected into rabbits, the glycosylated LDL had a delayed plasma clearance in comparison to the LDL. The mean fractional catabolic rates were 0.67 day-1 and 1.70 day-1 for 125I-labeled glycosylated LDL and 125I-labeled LDL, respectively. The uptake and degradation of 125I-labeled LDL by human skin fibroblasts was decreased as the concentration of free carbohydrate, glucose, sucrose or sorbitol, in the medium was increased from 10 mM to 1 M. It is speculated that pathologic levels of plasma glucose in vivo could result in a decrease in LDL uptake as a result of glycosylation of LDL. A decrease in uptake of native or modified LDL in vivo could contribute to hypercholesterolemia and its pathophysiology.     

The regulation of 3-hydroxy-3-methylglutaryl-CoA reductase activity, cholesterol esterification and the expression of low-density lipoprotein receptors in cultured monocyte-derived macrophages.

Human blood monocytes cultured in medium containing 20% whole serum showed the greatest activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and [14C]acetate incorporation into non-saponifiable lipids around the 7th day after seeding, the period of greatest growth. Although there was enough low-density lipoprotein (LDL) in the medium to saturate the LDL receptors that were expressed by normal cells at that time, HMG-CoA reductase activity and acetate incorporation were as high in normal cells as in cells from familial-hypercholesterolaemic (FH) patients. Both the addition of extra LDL, which interacted with the cells by non-saturable processes, and receptor-mediated uptake of acetylated LDL significantly reduced reductase activity and increased incorporation of [14C]oleate into cholesteryl esters in normal cells and cells from FH patients ('FH cells'), and reduced the expression of LDL receptors in normal cells. Pre-incubation for 20h in lipoprotein-deficient medium apparently increased the number of LDL receptors expressed by normal cells but reduced the activity of HMG-CoA reductase in both normal and FH cells. During subsequent incubations the same rate of degradation of acetylated LDL and of non-saturable degradation of LDL by FH cells was associated with the same reduction in HMG-CoA reductase activity, although LDL produced a much smaller stimulation of oleate incorporation into cholesteryl esters. In normal cells pre-incubated without lipoproteins, receptor-mediated uptake of LDL could abolish reductase activity and the expression of LDL receptors. The results suggested that in these cells, receptor-mediated uptake of LDL might have a greater effect on reductase activity and LDL receptors than the equivalent uptake of acetylated LDL. It is proposed that endogenous synthesis is an important source of cholesterol for growth of normal cells, and that the site at which cholesterol is deposited in the cells may determine the nature and extent of the metabolic events that follow.     

Lipoprotein and cholesterol metabolism in rabbit arterial endothelial cells in culture.

Like all other peripheral cells types thus far studied in culture, endothelial cells derived from the rabbit aorta bind, internalize and degrade low density lipoprotein (LDL) at a significant rate. At any given LDL concentration, the metabolism by rabbit endothelial cells was slower than that by fibroblasts or smooth muscle cells. Thus, longer incubations were required to achieve a net increment in cell cholesterol content or to suppress endogenous sterol synthesis; after 18-24 h incubation in the presence of LDL at 100 microgram LDL protein/ml inhibition was greater than 80% relative to the rate in cells incubated in the absence of lipoproteins. High density lipoproteins (HDL) were also taken up and degraded but did not inhibit sterol synthesis. Studies of LDL binding to the cell surface suggested the presence of at least two classes of binding sites; the high-affinity binding sites were fully saturated at very low LDL concentrations (about 5 microgram LDL protein/ml). However, the degree of inhibition of endogenous sterol synthesis increased progressively with increasing LDL concentrations from 5 to 100 microgram LDL/ml, suggesting that uptake from the low affinity sites in this cell line contributes to the suppression of endogenous sterol synthesis. The internalization and degradation of LDL also increased with concentrations as high as 700 microgram/ml. Thus, in vivo, where the cells are exposed to LDL concentrations far above that needed to saturate the high affinity sites, most of the LDL degradation would be attributable to LDL taken up from low affinity sites. As noted previously in swine arterial smooth muscle cells and in human skin fibroblasts, unlabeled HDL reduced the binding, internalization and degradation of labeled LDL. Cells incubated for 24 h in the presence of high concentrations of LDL alone showed a net increment in cell cholesterol content; the simultaneous presence of HDL in the medium significantly reduced this LDL-induced increment in cell cholesterol content. The possible relationship between LDL uptake and degradation by these cells in vitro is discussed in relationship to their transport function in vivo.     

Nonenzymatic galactosylation of human LDL decreases its metabolism by human skin fibroblasts

Incubation of human LDL with galactose in vitro resulted in a glycosylated-LDL containing radiolabel covalently attached to apo B. The rate of radiolabel incorporation was proportional to the time of incubation and concentration of carbohydrate. The rate of incorporation of galactose into apo B was higher than with glucose or mannose. The nonenzymatic glycosylation of LDL decreased its uptake and metabolism by the high affinity, receptor dependent process for LDL in normal human skin fibroblasts.     

Uptake of 2,3,7,8-tetrachlorodibenzo-p-dioxin from plasma lipoproteins by cultured human fibroblasts

Tritiated 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) added to human plasma in vitro associated with the plasma lipoproteins. The effects of plasma and lipoproteins on cellular uptake of dioxin were studied using normal human skin fibroblasts and mutant fibroblasts from a patient with homozygous familial hypercholesterolemia. The latter cells lack the normal cell membrane receptor for low density lipoprotein (LDL). The time- and temperature-dependent cellular uptake of [3H]dioxin was greatest from LDL, intermediate from high density lipoprotein (HDL) and least from serum. A significantly greater uptake from LDL by the normal cells compared to the mutant cells indicated the involvement of the LDL receptor-mediated pathway. Concentration-dependent studies indicated that the cellular uptake at 37 degrees C of [3H]dioxin varied linearly with dioxin concentration at constant LDL concentration. Thin-layer chromatography (TLC) showed that conversion to more polar compounds may have occurred after 24-h incubation with cells. [3H]Dioxin could be removed from cells efficiently by incubation with 20% serum greater than HDL greater than LDL. Since the vehicle of delivery may influence subsequent location and metabolism of this compound in cells, it is concluded that the physiologic vehicles (either serum- or LDL-associated dioxin), rather than organic solvents, should be used in experiments with cultured cells or perfused organs.     

Coupled computational analysis of arterial LDL transport -- effects of hypertension

Hypertension, a risk factor for atherosclerosis, increases the uptake of low density lipoproteins (LDL) by the arterial wall. Our objective in this work was to use computational modeling to identify physical factors that could be partially responsible for this effect. Fluid flow and mass transfer patterns in the lumen and wall of an arterial model were computed in a coupled manner, replicating as closely as possible previous experimental studies in which LDL uptake into the artery wall was measured in straight, excised arterial segments. Under conditions of both flow and no-flow, simulations predicted an increase in concentration polarization of LDL at the artery wall when arterial pressure was increased from 120 to 160 mmHg. However, this led to only a slight increase in mean LDL concentration within the arterial wall. However, if the permeability of the endothelium to LDL was allowed to vary with intra-arterial pressure, then the simulations predicted that the uptake of LDL would be enhanced 1.9-2.6 fold at higher pressure. The magnitude of this increase was consistent with experimental data. We conclude that the concentration polarization effects, enhanced by elevated intra-arterial pressure, cannot explain the increase in LDL uptake seen under hypertensive conditions. Instead, the data are most consistent with a pressure-linked increase in endothelial permeability to LDL.     

Peroxide modification of low density lipoproteins in ischemic heart disease

It is shown that the LDL peroxide lipid products level is 1.6-4 times higher in patients with the ischemic heart diseases (IHD) than in normal subjects. At the same time the LDL uptake by macrophages was identical in case of normal subjects and of IHD patients. It is suggested that the LDL premodification in the blood flow in the IHD patients promotes the further modification of LDL particles after their penetration into the vessel wall.     

Development of headspace solid-phase microextraction with on-fiber derivatization for determination of hexanal and heptanal in human blood

Hexanal and heptanal in human blood have been regarded as potential biomarkers of lung cancer. Owing to their high volatilities and activities, it is difficult to accurately measure the two biomarkers. In the current work, headspace solid-phase microextraction (HS-SPME) with on-fiber derivatization technique was developed for quantitative analysis of hexanal and heptanal in human blood. In the proposed method, the two aldehydes in blood were headspace extracted by using a poly (dimethylsiloxane)/divinylbenzene (PDMS/DVB) fiber with O-2,3,4,5,6-(pentafluorobenzyl) hydroxylamine (PFBHA) at 60 degrees C for 8 min. The aldehyde oximes formed on the fiber were desorbed and analyzed by gas chromatography-mass spectrometry (GC-MS). The method validations including detection limit, recovery and precision were studied. It was found that the method provided low detection limits of 0.006 nM for hexanal and 0.005 nM for heptanal, recoveries from 89% to 95% and R.S.D. values less than 8.5%. The present method was applied to quantitative analysis of hexanal and heptanal in normal blood and lung cancer blood. Hexanal concentrations from 7.33 to 15.23 microM and heptanal concentrations from 2.47 to 9.23 microM were found in the lung cancer blood, while both hexanal and heptanal in the control blood were lower than 0.6 microM. This further demonstrated that hexanal and heptanal might be the biomarkers of lung cancer. The experimental results showed that GC-MS and HS-SPME with on-fiber derivatization is a simple, rapid, sensitive and solvent-free method for determination of in hexanal and heptanal human blood.     

The processing of ligands by the class A scavenger receptor is dependent on signal information located in the cytoplasmic domain

The mechanisms that regulate the transport of the macrophage class A scavenger receptor during ligand uptake were investigated. Kinetic analysis of the changes in receptor phosphorylation demonstrated that serine phosphorylation increased during the internalization of acetyl-low density lipoproteins (LDL) by macrophages. The increase was maximal at about 2.5 min after the initiation of ligand uptake. Oxidized LDL also stimulated serine phosphorylation, but the relative increase was smaller and the time to maximum was shorter. Receptor mutants expressed in Chinese hamster ovary and COS cells showed that elimination of the potential phosphorylation site at Ser(21) increased acetyl-LDL metabolism, whereas inactivation of the site at Ser(49) reduced acetyl-LDL uptake. The increase in uptake by the Ser(21) mutant was due to an increase in surface receptor expression. In contrast, elimination of the site at Ser(49) did not affect receptor expression but slowed receptor internalization. To identify potential internalization signal sequences, beta-turn structure in the cytosolic domain was targeted for mutagenesis. Disruption of one region near Asp(25) inhibited receptor activity. The studies support a model whereby receptor internalization requires the presence of an internalization signal motif but that the rate of receptor internalization is governed by the pattern of receptor phosphorylation induced by the ligand.