[Publication of biological samples collections catalogues by tumor banks]. / La publication de catalogues de collections d'échantillons biologiques par les tumorothèques.

Biobanks in general, and specifically tumour banks, are considered as essential tools for the development of translational and clinical research in biology and oncology. Biobank tasks include the collection and preservation of biological samples, and their association with information that will be essential for further scientific use ("annotations" that allow for the "qualification" of biological samples in biological resource). A collection is made of a series of biological resource that are representative of a homogeneous group of individuals or patients that are defined on the basis of clinical or biological information. Collections are used by scientists that are aware of their existence. In the absence of a published catalogue, this awareness is most often limited to research teams that are geographically close, or to investigators who already established collaborative projects with medical teams within the hospital that operates the tumour bank. Publications of catalogues, especially digitalized and online catalogues, should foster the development of high-level, large-scale and multicentric scientific projects. In addition, tumour banks will formalize rules that allow publication of collections, and upstream, rules that are used to qualify biological samples in biological resource: this should translate in an improved overall quality of samples and annotations. Tumour bank catalogues remain relatively few; however, some recent achievements established the "proof of concept" and already raise questions regarding rules for publication. It will be important to demonstrate that these high expectations translate into measurable benefits.

Apolipoprotein A-II/A-I ratio is a key determinant in vivo of HDL concentration and formation of pre-beta HDL containing apolipoprotein A-II.

Overexpression of human apolipoprotein A-II (apo A-II) in mice induced postprandial hypertriglyceridemia and marked reduction in plasma HDL concentration and particle size [Boisfer et al. (1999) J. Biol. Chem. 274, 11564-11572]. We presently compared lipoprotein metabolism in three transgenic lines displaying plasma concentrations of human apo A-II ranging from normal to 4 times higher, under ad libitum feeding and after an overnight fast. Fasting dramatically decreased VLDL and lowered circulating human apo A-II in transgenic mice; conversely, plasma HDL levels increased in all genotypes. The apo A-I content of HDL was inversely related to the expression of human apo A-II, probably reflecting displacement of apo A-I by an excess of apo A-II. Thus, the molar ratios of apo A-II/A-I in HDL were significantly higher in fed as compared with fasted animals of the same transgenic line, while endogenous LCAT activity concomitantly decreased. The number and size of HDL particles decreased in direct proportion to the level of human apo A-II expression. Apo A-II was abundantly present in all HDL particles, in contrast to apo A-I mainly present in large ones. Two novel findings were the presence of pre-beta migrating HDL transporting only human apo A-II in the higher-expressing mice and the increase of plasma HDL concentrations by fasting in control and transgenic mice. These findings highlight the reciprocal modifications of VLDL and HDL induced by the feeding-fasting transition and the key role of the molar ratio of apo A-II/A-I as a determinant of HDL particle metabolism and pre-beta HDL formation.

Analysis of the relationship between triglyceridemia and HDL-phospholipid concentrations: consequences on the efflux capacity of serum in the Fu5AH system.

The high triglyceride/low HDL-cholesterol trait is a common finding in the general population. The aim of the present study was to analyze and interpret the relationships between triglycerides (TG), HDL-related parameters and serum cholesterol efflux potential in an asymptomatic population including both normo- and hyperlipidemic individuals. In a large sample (n = 1143) of this population, there was a negative correlation between TG and HDL-cholesterol (HDL-C) (r = -0.49, P<0.0001) whereas the negative correlation between TG and HDL-phospholipid (HDL-PL) (r = -0.29, P<0.0001) was weaker, leading to a strong positive correlation between TG and HDL-PL/C ratio (r = 0.58, P<0.0001). Thus, increased TG concentrations were associated with an enrichment of HDL with PL. Since we have demonstrated previously that HDL-PL is the major determinant for cholesterol efflux potential from Fu5AH rat hepatoma cells, we determined the effect of the variations in HDL lipid composition on the cholesterol efflux capacity in a subsample of 198 subjects. Compared with normolipidemic subjects (NLP) (TG< or = 1.7 mmol/l; LDL-C< or = 4.1 mmol/l, n=58), hypertriglyceridemic subjects (HTG) (TG>1.7 mmol/l, n=63) exhibited lower HDL-C levels (1.08+/-0.21 vs. 1.25+/-0.32, P=0.0003) whereas they showed similar HDL-PL concentrations (1.25+/-0.21 vs. 1.25+/-2.7) and, thus, higher HDL-PL/C ratio (1.17+/-0.15 vs. 1.02+/-0.14, P=0.0001). The relative efflux capacity of serum measured in the Fu5AH system (5% serum, 4 h incubation at 37 degrees C) was on average identical in the HTG and NLP groups. Thus, this study provides evidence that despite decreased HDL concentrations, as determined routinely by the HDL-C assay, some HTG subjects maintained serum cholesterol efflux capacity thanks to the enrichment of HDL with PL.

A new DNA polymorphism in the 5' untranslated region of the human SREBP-1a is related to development of atherosclerosis in high cardiovascular risk population.

Sterol-regulatory element binding proteins (SREBPs) are ubiquitous transcription factors that regulate the genes encoding key proteins in the control of cholesterol homeostasis. We looked for mutations or polymorphisms within the sequences of the SREBP-1a gene critical for the synthesis and/or activity of the protein in 204 asymptomatic men. A single G deletion at base pair -36 of the translation initiation site (designated G-) was found using single-strand conformation polymorphism (SSCP), in addition to three rare variants. This new marker was then assessed for its influence on the lipid parameters of 812 men at high cardiovascular risk, and on the presence of echographic atherosclerotic plaque in their peripheral arteries. The allelic frequency of the -36delG polymorphism was 0.58. At least one plaque was found in the carotid in 24% of subjects, in the femoral arteries of 48%, and in the aorta of 25%. There were significant associations between the -36delG polymorphism and mean total cholesterol (p=0.02) and LDL-cholesterol (P=0.02). There was a graded relationship between the G- allele and the presence of carotid plaque (r=0.084, P=0.02). In addition, there was a statistically significant interaction between the -36delG genotype and the apoE phenotype for plasma LDL-cholesterol (P=0.04) and apoB (P=0.05), suggesting a gene-gene interaction. Stepwise multiple regression analysis for lipid traits, risk factors, and apoE phenotype showed an independent association between carotid plaque and the -36delG polymorphism (beta=0.311, P=0.03). Thus, we have identified a new polymorphism in the 5' untranslated region of the SREBP-1a gene, and demonstrated its association with an atherogenic lipid profile and echographic plaques.

Human ApoA-IV overexpression in transgenic mice induces cAMP-stimulated cholesterol efflux from J774 macrophages to whole serum.

The role of apolipoprotein A-IV (apoA-IV) in lipoprotein metabolism has not been established. The aim of the present study was to investigate the role of apoA-IV in reverse cholesterol transport by comparing cellular cholesterol efflux to serum or serum fractions from control mice and from mice transgenic for human apoA-IV (HuA-IVTg mice). When Fu5AH hepatoma cells were used, the cholesterol efflux to serum from either control or transgenic mice was similar. When control J774 macrophage cells were used, a comparison of efflux to serum or lipoprotein-deficient serum (LPDS) failed to demonstrate any differences between control and transgenic mice. In contrast, when the J774 cells were pretreated with cAMP, there was a stimulation of efflux to whole serum or LPDS from HuA-IVTg mice. cAMP treatment had no effect on efflux to serum or LPDS from control mice. Pretreatment of the cells with cAMP did not enhance the efflux response to high density lipoprotein isolated from HuA-IVTg mouse serum. Our results suggest that apoA-IV, unassociated with high density lipoprotein particles, is responsible for enhanced cholesterol efflux. This study illustrates the role of lipid-free apolipoproteins in mediating cellular cholesterol efflux with use of a biological fluid and is potentially of physiological relevance, especially in apolipoprotein-rich extravascular fluids.

Study of vitamin E net mass transfer between alpha-tocopherol-enriched HDL and erythrocytes: application to asymptomatic hypercholesterolemic men.

We previously showed that hypercholesterolemic asymptomatic men had lower erythrocyte vitamin E content, despite normal plasma concentrations compared to normocholesterolemic men. We hypothesized that the reduced erythrocyte vitamin E concentration could be due to an impairment of transfer of vitamin E from plasma lipoproteins. We first developed a model for testing the ability of erythrocytes to accept vitamin E from high-density lipoproteins (HDL) pre-enriched in vitamin E, which allows to measure a net mass transfer of vitamin E from HDL to erythrocytes. Vitamin E-enriched HDL were obtained in controlled conditions of concentration and incubation time with a good reproducibility (CV

Fractional efflux and net change in cellular cholesterol content mediated by sera from mice expressing both human apolipoprotein AI and human lecithin:cholesterol acyltransferase genes.

Human lecithin:cholesterol acyltransferase (LCAT) is a key enzyme in the metabolism of cholesterol and is postulated to participate in the physiological process called reverse cholesterol transport. We have used transgenic mice (Tgm) expressing either both human apolipoprotein AI (apo AI) and human LCAT genes or only the human apo AI gene (HuAILCAT or HuAI Tgm, respectively) to assess the consequences of LCAT overexpression on serum lipid and lipoprotein profiles and on the ability of each serum to promote bidirectional flux of cholesterol between serum and Fu5AH hepatoma cells. Mean serum LCAT activity of HuAILCAT Tgm was 2-fold increased compared to the HuAI group (48+/-9 vs. 24+/-5 nmol/ml per h, P<0.01 for HuAILCAT and HuAI Tgm, respectively) and the cholesterol esterification rates were not significantly different between the two groups of animals (66+/-11 vs. 74+/-18 nmol/ml per h for HuAILCAT and HuAI Tgm, respectively). HuAILCAT Tgm exhibited higher total cholesterol serum values (2.3-fold) due to an increase in both HDL-cholesterol (1. 9-fold) and non-HDL-cholesterol (3-fold). The HDL particles from HuAILCAT Tgm were relatively phospholipid depleted and cholesterol enriched compared to HuAI mice. When cells were incubated for six hours with the mouse serum, the fractional efflux of radiolabeled cholesterol was slightly increased with the HuAILCAT Tgm (1.2-fold) but the increase in intracellular cholesterol content was also 2-fold higher than with the HuAI Tgm. Fu5AH can be viewed as a model for the evaluation of bidirectional flux of cholesterol in SR-BI-rich cells. In this model LCAT overexpression in mice, by increasing both HDL and non-HDL-cholesterol, mostly enhances the uptake of cholesterol by the cells, which would be of benefit for the last step of reverse cholesterol transport in hepatocytes.

Homocysteine decreases endothelin-1 production by cultured human endothelial cells.

Hyperhomocysteinemia is believed to be responsible for the development of vascular disease via several mechanisms, including the impairment of endothelial-cell functionality. In-vitro studies have demonstrated that homocysteine decreases the production or bioavailability of vasodilator autacoids, such as prostacyclin and NO. Here, we show that the treatment of human endothelial cells with noncytotoxic homocysteine concentrations leads to a dose-dependent decrease in both the secretion of the vasoconstrictor agent endothelin-1 (ET-1) and the level of its mRNA. Homocysteine had an inhibitory effect at pathophysiological (0.1 and 0.5 mmol.L(-1)) and pharmacological noncytotoxic (1.0 and 2.0 mmol.L(-1)) concentrations. Mean percentage variation from control for ET-1 production was -36. 2 +/- 18.9% for 0.5 mmol.L(-1) homocysteine and -41.5 +/- 26.8% for 1.0 mmol.L(-1) homocysteine, after incubation for 8 h. Mean percentage variation from control for steady-state mRNA was -17.3 +/- 7.1% for 0.5 mmol.L(-1) homocysteine and -46.0 +/- 10.1 for 1.0 mmol.L(-1) homocysteine, after an incubation time of 2 h. ET-1 production was also reduced by incubation with various other thiol compounds containing free thiol groups, but not by incubation with thiol compounds with no free thiol group. Co-incubation of cells with homocysteine and the sulfhydryl inhibitor N-ethylmaleimide prevented the effect of homocysteine on ET-1 production, confirming a sulfhydryl-dependent mechanism. Based on the reciprocal feedback mechanism controlling the synthesis of vasoactive mediators, these preliminary data suggest a mechanism by which homocysteine may selectively impair endothelium-dependent vasodilation by primary inhibition of ET-1 production.

Cell cholesterol efflux: integration of old and new observations provides new insights.

Numerous studies using a variety of cell/acceptor combinations have demonstrated differences in cholesterol efflux among cells. These studies also show that different acceptors, ranging from simple molecules like cyclodextrins to serum, stimulate efflux through a variety of mechanisms. By combining early observations with data derived from recent studies, it is now possible to formulate a model for cell cholesterol efflux which proposes that an array of different mechanisms, including aqueous diffusion, lipid-free apolipoprotein membrane microsolubilization, and SR-BI-mediated cholesterol exchange contribute to cholesterol flux. In this model the relative importance of each mechanism would be determined both by the cell type and the nature of the extracellular cholesterol acceptor.

Overexpression of human apolipoprotein A-II in mice induces hypertriglyceridemia due to defective very low density lipoprotein hydrolysis.

Two lines of transgenic mice, hAIItg-delta and hAIItg-lambda, expressing human apolipoprotein (apo)A-II at 2 and 4 times the normal concentration, respectively, displayed on standard chow postprandial chylomicronemia, large quantities of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) but greatly reduced high density lipoprotein (HDL). Hypertriglyceridemia may result from increased VLDL production, decreased VLDL catabolism, or both. Post-Triton VLDL production was comparable in transgenic and control mice. Postheparin lipoprotein lipase (LPL) and hepatic lipase activities decreased at most by 30% in transgenic mice, whereas adipose tissue and muscle LPL activities were unaffected, indicating normal LPL synthesis. However, VLDL-triglyceride hydrolysis by exogenous LPL was considerably slower in transgenic compared with control mice, with the apparent Vmax of the reaction decreasing proportionately to human apoA-II expression. Human apoA-II was present in appreciable amounts in the VLDL of transgenic mice, which also carried apoC-II. The addition of purified apoA-II in postheparin plasma from control mice induced a dose-dependent decrease in LPL and hepatic lipase activities. In conclusion, overexpression of human apoA-II in transgenic mice induced the proatherogenic lipoprotein profile of low plasma HDL and postprandial hypertriglyceridemia because of decreased VLDL catabolism by LPL.

Cholesterol efflux from Fu5AH cells to the serum of patients with Alagille syndrome. Importance of the hdl-phospholipids/free cholesterol ratio and of the hdl size distribution.

We have previously described the lipoprotein abnormalities in cholestatic children with paucity of interlobular bile ducts (PILBD), and we have shown that two different profiles emerged among these patients, depending on the level of lecithin:cholesterol acyltransferase (LCAT) activity. Reduced LCAT activity was associated with hypo-alpha-lipoproteinemia (group I) whereas normal LCAT activity was associated with hyper-alpha-lipoproteinemia (group II). In both groups, high density lipoproteins (HDL) were enriched with phospholipids and LpA-I particles were predominant. Here, we have investigated the ability of serum and of isolated HDL, obtained from PILBD and control subjects, to promote cellular cholesterol efflux, from Fu5AH rat hepatoma cells. The mean fractional efflux to 5% serum in each group was, on average, following the differences in HDL concentrations (control: 30.1 +/- 4.2%; group I: 23.7 +/- 7.9%, ns; group II: 44.2 +/- 6.5%, P < 0.001). The variations in efflux values in group II were positively correlated to the variations in HDL-PL concentrations (P < 0.0001) and in HDL-PL to serum apo-AI ratio (P < 0.003). By contrast, the variation in efflux in group I was only positively related to the large range of HDL-PL to free cholesterol (FC) ratio values (P < 0.0004). Fractional efflux to isolated HDL, measured at a constant HDL-PL amount, confirmed this relationship (P < 0.0001). Two-dimensional gel electrophoresis of the HDL size and apo A-I distribution in serum, revealed that small size HDL(3) and pre-beta HDL were predominant in the serum of patients from group I, especially those exhibiting low HDL-PL to FC ratio, whereas in the serum of patients from group II, both small HDL(3) and large HDL2 were present. These results suggest that a combination of an imbalance between phospholipids and free cholesterol in the HDL particles and a deficit in large acceptors of cholesterol will be responsible for an impairment of cellular cholesterol efflux in PILBD patients with reduced lecithin:cholesterol acyltransferase activity.-Davit-Spraul, A., V. Atger, M. L. Pourci, M. Hadchouel, A. Legrand, and N. Moatti. Cholesterol efflux from Fu5AH cells in the serum of patients with Alagille syndrome: importance of the HDL-phospholipids/free cholesterol ratio and of the HDL size distribution.

Oxidative modification of high-density lipoprotein 3 induced by human polymorphonuclear neutrophils. Protective effect of pentoxifylline.

The function of high-density lipoproteins (HDLs) in reverse cholesterol transport is impaired if HDLs are subjected to oxidative stress. Polymorphonuclear neutrophils (PMNs), which have been detected in the earliest stages of atherosclerotic lesions, are one of the most likely sources of the reactive oxygen species that cause such stress. In this study, we investigated the effect of a PMN oxidative burst on HDL3. We also studied the impact on these events of pentoxifylline, a drug that regulates granulocyte function. HDL3 (370 nmol.mL-1 cholesterol-HDL) was incubated with PMNs (2 x 106. mL-1) in NaCl/Pi in the presence or absence of an iron chelate complex (10 microm Fe-nitrilotriacetic acid) at 37 degreesC for 60 min or 24 h. Phorbol myristate acetate (PMA) or formyl-methionylleucyphenylalanine (fMetLeuPhe) was used to stimulate PMNs. In iron-free NaCl/Pi medium, PMA-stimulated PMNs had a 40% lower HDL3 alpha-tocopherol content, whatever the incubation time. In NaCl/Pi medium containing iron, there was 80% less HDL3 alpha-tocopherol at 60 min, and HDL3 alpha-tocopherol had almost disappeared after 24 h. In this latter condition, the amount of thiobarbituric acid-reactive substances was significantly higher than the respective control HDL3 (P < 0.05) and oxidation of HDL3 by PMA-stimulated PMNs was associated with cross-linking of apoprotein AI, which was detected by SDS/PAGE. Similar results were obtained with fMetLeuPhe-stimulated PMN except that HDL3 alpha-tocopherol was consumed much more slowly during the first 60 min. Pretreatment of PMNs with various concentrations of pentoxifylline (0.001-20 mm) led to the concentration-dependent inhibition of oxidative modification of HDL3 induced by stimulated PMNs. The addition of 20 mm pentoxifylline in the most extreme oxidative stress conditions resulted in 70% of HDL3 alpha-tocopherol being maintained, with no formation of thiobarbituric acid-reactive substances and a lower level of apoprotein AI cross-linking. Thus HDL3 is susceptible to oxidative modifications induced by stimulated PMNs, in the presence of an exogenous source of iron. Pentoxifylline inhibited the oxidative modification of HDL3 by PMNs.

Effects of postprandial hyperlipemia on the vitamin E content of lipoproteins. GERBAP section Lipoprotéines. Groupe d'Evaluation et de Recherche de l'Assistance Publique des Hôpitaux de Paris.

Delayed postprandial clearance of triglyceride-rich lipoproteins (TGRL) could induce a decrease of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) vitamin E content through its transfer into TGRL. We thus studied lipoprotein vitamin E content during postprandial hypertriglyceridemia induced by a high fat meal without vitamin E supplement. Venous blood was drawn from five healthy male volunteers following a 12 h fast at (t0) then 2 h (t2), 4 h (t4), 6 h (t6) and 8 h (t8) after a 80 g fat meal. In plasma, only TG significantly varied during the postprandial period with a large interindividual variability. Mean composition of lipoproteins in terms of mass was not significantly modified. The amount of vitamin E significantly increased in TGRL and decreased in LDL plus HDL at t4 and t6 relative to t0. Vitamin E content of TGRL and LDL but not of HDL decreased significantly at t4. The mean decrease was 20% (range 5%-54%) for the LDL. LDL- and HDL vitamin E content correlated inversely with plasma TGRL levels. Our data suggest that LDL from subjects with delayed chylomicron clearance could be less protected against oxidation.

Erythrocyte antioxidant status in asymptomatic hypercholesterolemic men.

An imbalance between antioxidant and oxidant-generating systems leading to an oxidative stress has already been proposed in the pathogenesis of atherosclerosis. In the present study we investigated the antioxidant status in 60 asymptomatic hypercholesterolemic (HC) men compared with 48 normocholesterolemic (NC) men. Hypercholesterolemic subjects had a significantly lower red blood cell vitamin E (vit E-RBC) content in spite of their normal total plasma and HDL vitamin E concentrations. Activities of erythrocyte superoxide dismutase and glutathione peroxidase were not significantly different between groups. We also determined the resistance of RBCs to an oxidative stress by determining the extent of hemolysis induced by a water-soluble azo-compound. This resistance was significantly decreased in HC men compared with NC subjects. These results demonstrate an altered antioxidant status of RBC in asymptomatic HC men associated with an increased erythrocyte susceptibility to an oxidative stress. The measure of the vitamin E content in RBC might be the most sensitive parameter for evidencing early oxidative stress which does not need an adaptation of enzymatic protective systems.

Plasma insulin and ankle on brachial systolic blood pressure ratio in overweight men with hypertension.

BACKGROUND: Hypertension is often associated with multiple metabolic abnormalities included in the insulin resistance syndrome. In hypertensive individuals, the ratio between ankle and brachial systolic blood pressure (ABI) is considered to be an independent cardiovascular risk factor. Insulin resistance has not been studied in relation to ABI ratio in men with essential hypertension and who are moderately overweight. OBJECTIVE: To identify whether a decrease in the ABI ratio is associated with the degree of abdominal obesity and, hence, with the biochemical characteristics of resistance to insulin. METHODS: In 166 overweight men with mild-to-moderate essential hypertension, insulinaemia was measured using radioimmunoassay. The ABI ratio was measured by using a pressure cuff of appropriate diameter, a standard mercury sphygmomanometer and a Doppler probe. Patients with diabetes or arteriosclerosis obliterans of the lower limbs, or both, were excluded from the study. RESULTS: The ABI ratio was significantly associated with the degree of abdominal obesity, but also with plasma triglycerides and cholesterol, low high-density lipoprotein cholesterol, plasma glucose and insulin. In a multiple regression analysis, the ABI ratio was significantly and negatively associated with only two variables: age and plasma insulin. This result was independent of age and drug treatment of hypertension. CONCLUSION: Because alterations in the ABI ratio may be considered markers of the changes in the structure and function of the arteries of lower limbs, the study provides evidence that plasma insulin, independently of atherosclerotic occlusive lesions, can significantly influence the status of conduit arteries of the lower limbs.

HDL phospholipid content and composition as a major factor determining cholesterol efflux capacity from Fu5AH cells to human serum.

The relationships of cell cholesterol efflux to HDL phospholipid (PL) content and composition in human serum were analyzed in two groups of subjects selected on the basis of their HDL cholesterol (HDL-C) levels: a norm-HDL group (1.10 mmol/L < HDL-C < 1.50 mmol/L) and a high-HDL group (HDL-C > 1.75 mmol/L). In the high-HDL group, the relative fractional efflux was significantly higher than in the norm-HDL group, and in both groups, fractional efflux was correlated with a number of lipoprotein parameters, the best correlation and the only one that remained significant after multivariate analysis being with HDL phospholipid (HDL-PL). Analysis of the HDL-PL subclasses revealed that HDL in the high-HDL sera was enriched with phosphatidylethanolamine (HDL-PE) and relatively deficient in sphingomyelin (HDL-SM) compared with norm-HDL sera. Moreover, the fractional efflux values in the high-HDL group were negatively correlated with the proportion of HDL-PE (r = -.64, P < .0001) and positively correlated with the proportion of HDL-SM (r = .43, P < .01). Thus, this study provides evidence that HDL-PL concentration can be used to predict the capacity of serum to accept cellular cholesterol. Among the differences described between norm-HDL and high-HDL sera, the variability in PE to SM ratio might reflect changes in serum cholesterol acceptors that modulate the first step of reverse cholesterol transport.

Structural and functional comparison of HDL from homologous human plasma and follicular fluid. A model for extravascular fluid.

In the preovulatory period, follicular fluid contains only HDL. Biochemical characterization of such lipoproteins showed that follicular fluid HDLs were cholesterol-poor particles compared with serum HDLs, whereas the amount of phospholipids, expressed as percent weight, was significantly higher in follicular fluid HDLs (28.5%) than in serum HDLs (25.0%, P < .05). The amount of apolipoprotein (apo) A-IV per apo A-I was significantly higher in follicular fluid than in serum (0.77 versus 0.58 mg/g apo A-I, P < .02). To explore the role of HDLs as cholesterol acceptors in physiological media, we compared the ability of either whole human follicular fluids or homologous sera to promote cellular cholesterol efflux using Fu5AH rat hepatoma cells. At equivalent concentrations of HDL cholesterol in follicular fluid and in serum, t1/2 values for cholesterol efflux were in the same range. In addition, estimated maximal efflux values were not significantly different in follicular fluid and serum (45.9% and 49.6%, respectively), as were K(m) values (0.064 and 0.071 mmol/L HDL cholesterol respectively). In addition, isolated HDLs displayed the same capacity to promote cellular cholesterol efflux in both media. Thus, the kinetics and dose-response data between these two physiological media showed that HDLs play the major role in cellular cholesterol efflux. The rate of cholesterol esterification, as measured in the presence of cells, was significantly higher in follicular fluid than in serum at constant HDL cholesterol concentrations, whereas the rate of esterified cholesterol transfer toward added LDL was lower. In contrast, in a cell-free system, lecithin:cholesterol acyltransferase activity represented only 26% of that in serum HDL, whereas cholesterol ester transfer protein activities were comparable. In summary, in this particular model, we confirmed the essential role of HDLs as physiological acceptors in the removal of cellular cholesterol.

Cyclodextrins as catalysts for the removal of cholesterol from macrophage foam cells.

Low concentrations of cyclodextrins (< 1.0 mM) added to serum act catalytically, accelerating the exchange of cholesterol between cells and lipoproteins. J774 macrophages incubated with serum and 2-hydroxypropyl-beta-cyclodextrin (< or = 1 mM) released fivefold more labeled cholesterol than with serum alone. Increased efflux was not accompanied by a change in cell cholesterol mass; thus, cyclodextrin functioned as a cholesterol shuttle, enhancing cholesterol bidirectional flux without changing the equilibrium cholesterol distribution between cells and medium. The addition of phospholipid vesicles to serum and cyclodextrin shifted the equilibrium distribution to favor the medium, producing rapid and extensive depletion of cell cholesterol mass. The combination of serum, phospholipid vesicles, and cyclodextrin also stimulated the rapid clearance of both free and esterified cholesterol from mouse peritoneal macrophages loaded with free and esterified cholesterol. This study: (a) demonstrates that a compound can function as a catalyst to enhance the movement of cholesterol between cells and serum, (b) illustrates the difference between cholesterol exchange and net transport in a cell/serum system, (c) demonstrates how net movement of cholesterol is linked to concentration gradients established by phospholipids, (d) provides a basis for the development of the shuttle/sink model for the first steps in reverse cholesterol transport, (e) validates the model using artificial shuttles (cyclodextrins) and sinks (large unilamellar vesicles), and (f) suggests that cyclodextrin-like cholesterol shuttles might be of pharmacological significance in treating unstable atherosclerotic plaques.

Abnormal lipoprotein pattern in patients with Alagille syndrome depends on Icterus severity.

BACKGROUND & AIMS: Children with Alagille syndrome have lipid abnormalities that differ according to the severity of icteric periods. The lipoprotein profiles of 22 patients with Alagille syndrome were determined and the findings were compared with the severity of jaundice. METHODS: Plasma lipids and apolipoproteins (apos), isolated lipoprotein composition, and lecithin/ cholesterol acyltransferase (LCAT) activity were analyzed in patients. Patients were classified into two groups according to their bilirubin levels; patients in group I had total bilirubin levels of > 100 mumol/L, and patients in group II had total bilirubin levels of < 100 mumol/L. RESULTS: In patients from group II, hypercholesterolemia was associated with increased levels of high-density lipoprotein and high concentrations of apoAI and apoAII; in a few cases, an abnormal lipoprotein with a slow alpha migration was observed. In contrast, in patients from group I, the levels of high-density lipoprotein cholesterol and apoAI and apoAII were very low, and the abnormal lipoprotein X was in many cases responsible for hypercholesterolemia. In group I, the decreased LCAT activity was consistent with the very high level of unesterified cholesterol and the emergence of lipoprotein X. In both groups of patients, the levels of apoE, apoCII, and apoCIII were high, and all the lipoprotein fractions were enriched in phospholipids. CONCLUSIONS: The variations of LCAT activity caused by the degree of jaundice in patients with Alagille syndrome are implicated in the abnormal lipid profiles.

Role of HDL phospholipid in efflux of cell cholesterol to whole serum: studies with human apoA-I transgenic rats.

Sera of transgenic rats expressing human apoA-I were tested for their ability to stimulate efflux of radiolabeled cholesterol from Fu5AH rat hepatoma cells. Expression of human apoA-I resulted in a dose-dependent increase in HDL, as measured by both HDL-cholesterol and HDL-phospholipid, and produced a decrease in rat apoA-I. In rats expressing high concentrations of human apoA-I (TgR[hAI]high, human apoA-I > 250 mg/dl), the increase in HDL-phospholipid was not proportional to the increase in human apoA-I, as illustrated by a HDL-PL/total apoA-I ratio of 0.84 +/- 0.19 compared to a ratio of 1.28 +/- 0.29 for control rats and of 1.28 +/- 0.39 for rats expressing low levels of human apoA-I (TgR[hAI]low, human apoA-I < 250 mg/dl). Compared to sera from control animals, efflux of cell cholesterol was increased by 26% in the sera from TgR[hAI]low, and by 76% in the TgR[hAI]high. An examination of the relationships between efflux and HDL-related parameters demonstrated a hyperbolic relationship between efflux and either HDL-cholesterol or HDL-apoA-I. In contrast, there was a strong linear association (r2 = 0.84) between cholesterol efflux and HDL-phospholipid, indicating that this parameter is the component of HDL that best reflects the serum's efflux efficiency. The importance of phospholipids in modulating cholesterol efflux was further explored by measuring the effect of supplementation of serum with dimyristoylphosphatidylcholine (DMPC) vesicles, apoA-I, or both DMPC vesicles and apoA-I. Whereas addition of human apoA-I had no effect on efflux, supplementation with DMPC vesicles produced a substantial increase in efflux that was further stimulated by the combination of DMPC vesicles and apoA-I. These results demonstrate that a major component of HDL that modulates cell cholesterol efflux is phospholipid.