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Assimilation of lipid nutrients depends on the efficiency of emulsified fat hydrolysis by digestive lipases. As shown in vitro, the activity of preduodenal and pancreatic lipases is governed by the physicochemical properties of emulsions. Thus the aim of this study was to evaluate in the rat how emulsions are digested and assimilated depending on their droplet size or solute composition. Fasted rats were intragastrically tube fed emulsions with different median droplet sizes (0.6 microns, fine; 22 microns, coarse) or solute composition (0.8 microns, complex fine) containing 14C-triolein and 3H-cholesterol. Two and 5 hours after feeding, fat-droplet size was measured in gastric and duodenal contents, and lipids were radioactively quantified in different compartments. In the stomach, the droplet size of the fine emulsions significantly increased to values (13 microns to 24 microns) comparable with those of the coarse emulsion (35 microns to 36 microns). In the duodenum, the droplet sizes of the three emulsions were in the range of 14 microns to 33 microns. After 2 hours, gastric triglyceride hydrolysis was significantly higher with the fine than with the coarse emulsion and was lower with the complex fine emulsion. Gastric emptying of fat was significantly different, with the following decreasing order: coarse, fine, and complex fine emulsion. In the small intestine, the fine and coarse emulsions were processed comparably, whereas the assimilation of the fine complex emulsion was significantly delayed. Calculations indicate that ingested fatty acids were distributed in the peripheral tissues at different rates with the same decreasing order. The fate of a lipophilic nutrient, cholesterol, was also markedly altered by the type of emulsion. These data support the concept that tube-fed emulsions with different droplet sizes and solute composition are digested differently and thus are metabolized differently.

Cholesterol constitutes the major component of most gallstones. It was identified and determined in gallstones by thermal analysis technique (DSC and TG-DTA), mainly by the use of the melting temperature (Tonset=145°C and Tmax=149°C) and by DTG peak decomposition (Tmax=364°C). Cholesterol anhydrous (ChA), which showed endothermic polymorphic peak, Tmax=40°C, without mass loss, was differentiated from cholesterol monohydrate (ChH), which showed a broad endothermic peak, Tmax=59°C, attributed to loss of water of crystallization (theoretical 4.45%). Morphological studies of gallstones were performed by optical microscopy and scanning electron microscopy (SEM). The stones consisted of a pigmented core with a variably-sized irregular central cavity, surrounded by a radially arranged deposits of plate-like ChH. The outer part of the stones showed ChA crystal arborescences. X-ray microanalysis gave a typical spectrum rich in C and O, and in some instances the presence of P, which was attributed to the presence of phospholipids. CaCO3 was easily characterized by TG with the use of DTG decomposition peak at 674°C.

Cholesterol gallstones affect approximately 10-15% of the adult population in North America. Phosphatidylcholine (PC) is considered to be the main cholesterol solubilizer in bile. This study examined the effect of a PC-enriched diet on gallstone incidence in mice susceptible to cholelithiasis. The result obtained showed that the feeding of a lithogenic (LG) diet for 4 weeks or 8 weeks resulted in cholesterol gallstone incidences of 47% and 89%, respectively. These gallstone incidences were either reduced or prevented when the LG diet was enriched with 2% or 6% PC, respectively. The cholesterol saturation index (CSI) was reduced only in mice fed with LG + 6% PC diet as compared with mice fed the LG diet alone. However, in all groups, the CSI was significantly higher than in mice fed Purina chow diet. The biliary anionic polypeptide fraction (APF) was significantly increased in mice fed the LG + 2% PC diet and was reduced in those fed with LG + 6% PC diet. In conclusion, prevention or delay of gallstone formation was not due to a consistent effect on biliary lipid composition, suggesting a direct effect of PC on cholesterol solubilization and/or the effect of an additional nonlipid biliary component such as APF.

Fatty acid bile acid conjugates (FABACs) are a new family of synthetic molecules designed to solubilize biliary cholesterol. They were shown to prevent and dissolve cholesterol gallstones in inbred C57L/J mice fed a lithogenic, high-fat diet (HFD). In these mice, fatty liver was observed in the controls but not in the FABAC-treated ones. The present study was designed to study the effect of FABAC (arachidyl-amido-cholanoic acid) on diet-induced fatty liver in rats, hamsters, and mice. The fatty liver score (on a scale of 0-4 by light microscopy) was 4.0 in control hamsters and 0.3 in the FABAC-fed hamsters (P < .001). In mice it was 1.5 and 0.4, respectively (P < .01). The lipid/protein ratio in the liver was 1.3 ± 0.44 (mg lipid/mg protein) in control rats and 0.66 ± 0.04 in the FABAC group (P = .001) after 14 days. In hamsters it was 1.41 ± 0.27 and 1.11 ± 0.20, respectively (P = .03), after 21 days. In Imperial Charles River (ICR) mice the ratio was 0.34 ± 0.10 and 0.17 ± 0.07 (P = .03), respectively, after 24 days. Liver fat concentration, measured as mg lipid/g liver tissue, decreased similarly by FABAC feeding. The decrease in liver fat affected mainly the triglyceride levels. FABAC-fed animals gained weight similarly to the controls. Triglyceride absorption was unaffected by FABAC supplementation. In conclusion, oral FABAC therapy prevents/reduces the development of fatty liver in animals consuming a HFD. (Hepatology 2003;38:436-442.)

Cholesterol constitutes the major component of most gallstones. It was identified and determined, in gallstones, issued from eleven patients, by thermal analysis: differential scanning calorimetry (DSC), with the use of the melting temperature and enthalpy, thermogravimetry (TG), with the mass loss of water. Anhydrous cholesterol (ChA) was characterized by two endothermic peaks (polymorphic, melting) and cholesterol monohydrate (ChH) by two endothermic peaks (dehydration, melting), too. Cha needle and Chh plate crystals were observed under polarizing light microscopy. The numerous stones obtained from nine patients were cholesterol stones: the ChA was higher 45 and lower 96%. ChH was present in stones of three patients.

Evidence is now in favor of protein-facilitated mechanisms for the intestinal cholesterol absorption. Here we report that the unesterified cholesterol uptake by rat jejunal brush border membrane vesicles (BBMVs) is efficient, saturable, and protein-mediated. The human apolipoproteins biliary anionic peptide factor (APF) and A-I (apoA-I) up-regulate micellar cholesterol uptake in a dose-dependent manner, but for all tested concentrations (0.1–20 μM), the lipid-free APF was more efficient than apoA-I. This uptake stimulation was suppressed after addition of Pabs directed to the external lipid-binding domain of the CLA-1/SR-BI and reduced by Pabs directed to the external loop of CD36. Thus, CLA-1/SR-BI and to a lesser extent CD36 are involved in the regulation of intestinal cholesterol uptake. APF, the main protein bound to biliary lipids, is likely one of their physiological effectors. As APF is an unesterified cholesterol carrier, it could facilitate the intestinal absorption of biliary cholesterol.

Background/Aims: Gallbladder bile from patients with cholesterol or mixed gallstones frequently contains biliary "sludge", a suspension of cholesterol monohydrate crystals and pigment granules embedded in mucin and proteins. The composition of biliary "sludge" and the preferential localization of mucin and proteins could be an indicator for its potential role in gallstone formation. Methods: Ultracentrifugation (100 000 g/1 h) was used to precipitate "sludge" from bile, and the concentration difference of its main components between native bile and ultracentrifuged bile samples was calculated. After purification of the sediment, immunolocalization was performed for the detection of mucin, IgA, albumin, aminopeptidase, and anionic polypeptide fraction using polyclonal and monoclonal antibodies. Results: The amount of sludge in gallbladder bile was 4.26 mg/ml±0.78 (mean±SEM) in patients with cholesterol and 2.51 mg/ml±0.39 in patients with mixed stones and cholesterol was the main component (48.9±4.6% and 44.4±7.1%). The sediment appeared as a mixture of vesicular aggregates and pigment particles which were linked by a gel matrix of mucin containing cholesterol crystals. While anionic polypeptide fraction and aminopeptidase were associated to pigments, IgA was uniformly spread in the crystalline parts of "core-like" structures, and albumin, when it was present, appeared as randomly located small spots. Conclusions: Our study demonstrates that the cholesterol content and the distribution pattern of mucin and different proteins is similar in the sediments of biliary "sludge" to that previously shown in cholesterol and mixed gallstones. This suggests that biliary "sludge" represents an early stage of gallstone formation in these patients.

Although several investigations have linked the degree of fatty acid saturation to plasma lipid responses in the postprandial state, further evaluation is necessary. In this study, we compared the effect of saturated (SFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acids on postprandial lipid metabolism using complementary in vivo and in vitro approaches. Fat (10 g) cholesterol (0.5 g) test meals that provided either lard (SFA), olive oil (MUFA), or sunflower oil (PUFA) were ingested by chow-fed New Zealand white rabbits (n = 8). In addition, hepatic uptake of triglyceride-cholesterol-rich lipoproteins (TCRL) isolated from rabbits chronically ingesting SFA, MUFA, or PUFA diets was measured using freshly isolated chow-fed rabbit hepatocytes. Whatever dietary fatty acids ingested, postprandial triglyceridemia and occurrence of radiolabelled dietary lipids in plasma were not markedly different. Conversely, SFA induced higher postprandial cholesterolemia and phospholipemia than MUFA (P < 0.05) whereas PUFA prevented postprandial cholesterol increase. TCRL disappearance from cultured liver cell media was delayed with SFA-rich TCRL and faster with PUFA whereas MUFA-rich TCRL showed an intermediate figure. From these data, we conclude that SFA, MUFA, and PUFA elicited different postprandial plasma and lipoprotein lipid responses. The fatty acid composition of TCRL had a major impact on their subsequent metabolism, especially uptake by cultured hepatocytes. The SFA-induced hypercholesterolemia could be related to an altered hepatic uptake whereas a faster clearance and hepatic uptake could explain the cholesterol-lowering effect of PUFA in rabbits. MUFA, like PUFA, accelerate uptake by hepatocytes but favor cholesterol ester enrichment of TCRL.

An overview on the applications and the contributions of electrophoretic techniques to the analysis of biliary proteins is presented. The electrophoretic techniques include free-, paper-, agar-, acetate cellulose film- and starch gel-electrophoresis, immunoelectrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis and high resolution two-dimensional gel electrophoresis. These techniques were applied in the separation and characterization of hepatic bile proteins, gallbladder bile proteins, gallstone proteins, and finally vesicular and micellar proteins. The chief contribution of electrophoresis to research in the field of cholesterol crystal and gallstone formation has been in the characterization of pro- and anti-nucleating proteins.

Synthetic glucocorticoids, such as dexamethasone, and diets enriched with unsaturated fatty acids have been shown to stimulate hepatic bile salt synthesis. This fact led us to investigate the effects of dexamethasone and linoleic acid supplementation on bile secretion. Cholesterol (Ch) and phospholipid secretions are bile acid dependent. Ch and phospholipid in bile are also highly bound to a small apoprotein, the anionic polypeptide factor (APF). In bile, APF may play a physiological role in stabilizing cholesterol:phospholipid vesicles and might also be important in the regulatory process of bile lipid secretion. In order to study the factors influencing bile secretion, the biliary secretion rates of bile lipids and APF were experimentally modulated in perfused rat liver (PRL) and HepG2 cells. As expected, dexamethasone induced an increase in the biliary secretion rate of bile salts (BS) in the two models (PRL: 34 up to 67 nmol/l/min/g liver; HepG2 cells: 234% vs. 100% in controls). The bile secretion rates for phospholipids (PRL: from 5 down to 1.5 nmol/l/min/g liver; HepG2 cells: 93 vs. 100% in controls) and APF (PRL: from 0.34 down to 0.12 µg/l/min/g liver; cells: 86 vs. 100% in controls) rapidly decreased independently from those of BS. The data from experimental cell models supplemented with linoleic acid indicated a correlation between the BS and APF levels (APF: 71 and 63%; BS: 161 and 197% vs. 100% in controls). The phospholipid level was regulated independently from that of APF and BS and increased (106 and 111% vs. 100% in controls), while Ch remained nevertheless unchanged. Our data showed that dexamethasone induced changes in bile and that linoleic acid clearly impaired the regulation exerted by the dexamethasone on bile lipids.

The aim of this work was to assess, the effect of a dietary supplement of soyabean lecithin on the apoprotein (apo) AI–high density lipoprotein system in humans. Adult outpatients (three women and seven men, aged 52 ± 5 years) were selected on the basis of a major type IIA hypercholesterolemia (>6.5 mmol/L). For each subject, a previous control period of 6 weeks consisted in a daily dietary supplement of 3 g soyabean oil (SO) as placebo distributed in two capsules at three main meals. Subjects then were given a daily dietary supplement of 3 g purified soyabean phosphatidylcholine (SPC) for 6 weeks, also distributed in two capsules at the three main meals. The usual basal diet of each subject was monitored every 4 weeks by a 3-day food recall. Between the end of the SO period and the end of the SPC period, plasma level of apo AI significantly increased (from 1.01 to 1.23 g/L), as did unesterified cholesterol (from 1.80 to 2.00 mmol/L). The esterified cholesterol to total cholesterol ratio and apo E level significantly decreased between the beginning and the end of the experiment. Esterified cholesterol and apo B levels remained unchanged. The levels of apo AI or unesterified cholesterol were not correlated to the alcoholic calories or to total or lipid energy. Dietary soyabean lecithin contributed to stimulation of the reverse cholesterol transport by increasing apo AI system and decreasing apo AII and apo E systems. Thus, soyabean lecithin could be considered an effective nutrient useful in the dietary treatment of mild hypercholesterolemia.

We explored the possibility that the biliary protein fraction may support part of the variation in the nucleating activity previously measured in gallbladder biles of pigs. Eighteen gallbladder aspirates freshly obtained from three dietary groups (0, 5, or 10% beta-cyclodextrin) of six pigs were chromatographed to purify their total protein fraction. Proteins were quantified, and analysed through electrophoresis and immunoblotting or enzyme-linked immunosorbent assay for albumin, and five putative effectors of cholesterol crystallisation, mucins, immunoglobulin A, 130 kDa, apolipoprotein A-I, and anionic polypeptide fraction. Each total protein fraction was also assayed for its ability to influence cholesterol precipitation, when added to supersaturated model bile. The current data provided evidence that the cholesterol crystallisation-promoting activity of biliary proteins in model biles increased with the beta-cyclodextrin dietary content. This occurred without any significant change in the total biliary protein content, but was associated with a significant decrease in the concentration of albumin and apolipoprotein A-I, resulting in changes in the overall balance of proteins in bile. Comparison of these results with the crystallisation figures previously obtained from the corresponding native biles led us to conclude that biliary proteins might influence the outcome of the crystallisation process, namely the final crystal concentration at equilibrium, but would not systematically represent a major driving force for determining the velocity of crystal formation in native bile of pigs.

Cellular glycosphingolipids mediate the fusion between some viruses and the plasma membrane of target cells. In the present study, we have analyzed the interaction of human immunodeficiency virus (HIV)-1 and HIV-2 surface envelope glycoproteins from distinct viral isolates with monolayers of various glycosphingolipids at the air-water interface. The penetration of the viral glycoproteins into glycosphingolipid monolayers was detected as an increase in the surface pressure. We found that HIV-1 recombinant gp120 (IIIB isolate) could penetrate into a monomolecular film of alpha-hydroxylated galactosylceramide (GalCer-HFA), while ceramides, GluCer, and nonhydroxylated GalCer were totally inactive. The glycoproteins isolated from HIV-1 isolates LAI and NDK and from HIV-2(ROD) could also interact with a GalCer-HFA monolayer, whereas gp120 from HIV-1(SEN) and HIV-1(89.6) did not react. These data correlated with the ability of the corresponding viruses to gain entry into the CD4(-)/GalCer+ cell line HT-29, demonstrating the determinant role of GalCer-HFA in this CD4-independent pathway of HIV-1 and HIV-2 infection. In contrast, all HIV-1 and HIV-2 glycoproteins tested were found to interact with a monolayer of GM3, a ganglioside abundantly expressed in the plasma membrane of CD4(+) lymphocytes and macrophages. A V3 loop-derived synthetic peptide inhibitor of HIV-1 and HIV-2 infection in both CD4(-) and CD4(+) cells could penetrate into various glycosphingolipid monolayers, including GalCer-HFA and GM3. Taken together, these data suggest that the adsorption of human immunodeficiency viruses to the surface of target cells involves an interaction between the V3 domain of the surface envelope glycoprotein and specific glycosphingolipids, i.e. GalCer-HFA for CD4(-) cells and GM3 for CD4(+) cells.

In this study, pigs fed for 3 weeks a well-balanced semi-purified diet enriched with 0.3% cholesterol and 0, 5 or 10% beta-cyclodextrin were proposed as new animal donors of gallbladder bile exhibiting different rates of cholesterol crystallization, in order to gain insight into the early mechanisms underlying cholesterol precipitation in vivo. The appearance and growth of cholesterol crystals were monitored in the incubated freshly collected gallbladder biles through light microscopy and concomitant time-sequential determination of crystallized cholesterol concentration, and interpreted in terms of the composition of the bile.Although the concentration of total lipids and proteins and the relative proportions of bile acids, phospholipids, and cholesterol remained unchanged under beta-cyclodextrin, the cholesterol crystallization increased in the following order: 0105% beta-cyclodextrin. Concomitantly, the proportion of chenodeoxycholic acid in bile, and the hydrophobicity index of the biliary bile acid mixture increased in the following order: 0510% beta-cyclodextrin (the same as reported elsewhere for the decrease in the antinucleating ApoA1), while sn-2 arachidonoyl biliary lecithins were specifically increased with 5% beta-cyclodextrin in the diet.We hypothesized that lecithin molecular species may be the determinant factor in modulating high cholesterol crystallization rates in biles otherwise enriched with hydrophobic bile acids.

Two very similar small anionic, amphipathic proteins, a phospholipid-binding apoprotein (anionic polypeptide fraction [APF]) and a calcium-binding polypeptide (CBP), are found abundantly in bile and all types of gallstones. The often disparate properties among various preparations of APF/CBP could reflect different sources and separation procedures, leading to partly degraded and/or denatured protein and varied association of bile salts, lipids, bile pigments, and detergents. The present study presents new methods for isolation and purification of APF/CBP, and characterizes the preparations thus obtained. It was found that isolation by selective precipitation of proteins from fresh T-tube bile by added calcium chloride, followed by demineralization with ethylenediaminetetraacetic acid (EDTA), removal of salts, lipids, and some pigment by Sephadex LH-20, and serial ultrafiltration yields the purest preparations. Though free of lipids, bile salts, detergents, and most pigments, these new preparations all show the same 7-kd and 12-kd bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the same major peaks on hydrophobic high-performance liquid chromatography (HPLC), and retain the self-associative, lipid- and calcium-binding functions, typical of older preparations obtained by potentially denaturative procedures. The varied properties among APF/CBP preparations are thus apparently related mainly to their content of different proportions of two major components, lipid-binding APF and calcium-binding CBP. Immunologic cross-reactions indicate common epitopes, and amino acid analyses are also similar, suggesting that APF and CBP may have the same polypeptide backbone, but differ because of posttranslational modification(s). Sufficiently pure APF and CBP have now been obtained to permit possible structural identification by sequencing and molecular biological techniques, though such attempts have thus far been unsuccessful.

We present here a computational method based on the analysis of amino acid composition for performing comparisons between proteins. This user-friendly and reliable test is aimed at rapidly identifying, from data-base subsets, sequences--if necessary, partial sequences--which share similar amino acid compositions to the input composition (deduced from experimental results). Apparent molecular weight (as determined by SDS-PAGE) and artefactual modifications due to the experimental determination of the amino acid composition are taken into account to perform the comparison. This program thus constitutes a useful tool in searching for the probable identification of either non-sequenced proteins or peptides from hydrolysed proteins.

This study evaluates the possible interaction between chronic oat bran intake and the postmeal metabolic response. Six normolipidemic men consumed three different diets for 14 d, at the end of which they consumed a test meal. The diets were C (control), basal low-fiber diet (15.6 g fiber/d) and a low-fiber (2.8 g fiber) test meal; OB (oat bran), basal low-fiber diet and a 40-g oat bran-enriched test meal (12.8 g fiber); and OB-A (oat bran-adaptation), 14-d oat bran (40 g/d) supplemented diet (23.8 g fiber/d) and an oat bran test meal (12.8 g fiber). The diets were fed in a random order. Fasting and postmeal blood samples were obtained for 7 h and lipoproteins were isolated. Adding oat bran to the test meals markedly reduced the postmeal insulin rise (P < 0.05). Compared with the low-fiber control diet, the effects elicited postprandially by adding oat bran to a single meal were enhanced after 14 d of oat bran feeding, ie, increased plasma triglycerides, phospholipids, and free cholesterol; decreased plasma esterified cholesterol; increased chylomicron and small-sized triglyceride-rich lipoprotein triglycerides; increased LDL and HDL free cholesterol; and decreased HDL esterified cholesterol. Thus, chronic oat bran feeding alters the postmeal response in human subjects.

Our aim was to determine the effects of increasing amounts of dietary cholesterol (0-710 mg) on the postprandial plasma lipid responses and lipoprotein changes in normolipidemic human subjects. Ten subjects were fed five different test meals in a random order: one meal did not contain fat or cholesterol while the four others contained a fixed amount of lipids (45 g) and 0, 140, 280, and 710 mg cholesterol, respectively. Fasting and post-meal blood samples were obtained for 7 h. Large and small triglyceride-rich lipoproteins (TRL), low density (LDL), and high density (HDL) lipoproteins were isolated. Compared to the no-fat, no-cholesterol meal, the fat-enriched meals raised (P < 0.05) plasma triglycerides, phospholipids, and free cholesterol and lowered cholesteryl esters postprandially. The meals containing zero or 140 mg cholesterol generally elicited comparable postprandial plasma and lipoprotein lipid responses. The meals providing 280 or 710 mg cholesterol significantly increased postprandial plasma phospholipids and large TRL triglycerides and decreased plasma esterified cholesterol. The lipid composition of the large TRLs and the concentrations of the small TRL lipid components were not altered postprandially by cholesterol intake. On the other hand, LDL free cholesterol increased after 3 h, LDL cholesteryl esters dropped after 3 and 7 h, HDL cholesteryl esters dropped after 3 h, and HDL phospholipids increased 7 h after ingesting meals highly enriched in cholesterol. Blood insulin, apoA-I and apoB were not altered postprandially by cholesterol intake. Thus, the data show that ingesting more than 140 mg cholesterol per meal significantly alters the postprandial lipoprotein response in healthy subjects.

The purpose of this study was to investigate in rats the effects of three anthracyclines, pirarubicin, doxorubicin and epirubicin on gastric prostaglandin E 2 (PGE 2 ) metabolism and phospholipase A 2 (PLA 2 , EC 3.1.1.4) activity. The level of the membrane precursor, arachidonic acid, and the stability of the membrane were investigated by analysis of the composition of fatty acids. Enzymatic activities involved in the turnover of membrane phospholipids such as lysophospholipase (LPase, EC 3.1.1.5) and acyl-CoA lysophosphatidylcholine: acyltransferase (ACLAT, EC 2.3.1.23), and in the detoxification of lipid hydroperoxides, selenium-dependent glutathione-peroxidase (GSH-PX, EC 1.11.1.9) were measured after injection of the drugs for 4 consecutive days. Pirarubicin does not give rise to any changes in these activities but doxorubicin and epirubicin decreased PGE 2 production and the activities of PLA 2 , LPase and ACLAT. GSH-PX activity was not changed by any of the drugs. The decrease in PLA 2 activity does not seem to be related to variations in membrane lipid composition because the total phospholipids content was unchanged. The P/S (polyunsaturated/ saturated) ratio increased in the doxorubicin group and decreased in the epirubicin group, and the unsaturation index was moderately modified. Arachidonic acid was increased only in the doxorubicin group. In vitro , PLA 2 activity was not inhibited by the three drugs in the micromolar range. A marked inhibition was observed at 2.5 mM for pirarubicin and at 1.0 mM for doxorubicin and epirubicin. The Lineweaver-Burk representation showed that these inhibitions were of an uncompetitive type. Pirarubicin may therefore be considered to be an anthracycline without marked side-effects on gastric mucosa. However, the in vitro inhibition of PLA 2 activity by anthracyclines does not fully explain the in vitro decrease in PLA 2 specific activity observed after doxorubicin and epirubicin treatment, and in this context membrane structure modifications unconnected with the lipid composition can not be excluded. In vivo these phenomena may affect PGE 2 synthesis, whose level was lower in the doxorubicin and epirubicin groups than in control group.

Crilvastatin is a drug from the pyrrolidone family that had been shown to induce non-competitive inhibition of rat hydroxymethylglutaryl-coenzyme A reductase activity in vitro. The aim of this study was to evaluate the activity of crilvastatin on the hepatic metabolism of cholesterol in rats. Crilvastatin increased low density lipoprotein (LDL)-cholesterol uptake by the liver more than high density lipoprotein (HDL) uptake, thus increasing by up 30% the clearance of excess plasma cholesterol. In normolipidemic rats, crilvastatin significantly enhanced acyl coenzyme A: cholesterol acyl transferase and cholesterol 7α-hydroxylase activity. In rats with a previous high cholesterolemia, crilvastatin also enhanced cholesterol 7α-hydroxylase activity and did not increase liver acyl coenzyme A: cholesterol acyl transferase activity. These findings suggest that a drug such as crilvastatin could have a hypocholesterolemic effect by a mechanism other than the sole inhibition of cholesterol synthesis, possibly by stimulating cholesterol and bile salt secretion via the biliary tract in previously hypercholesterolemic rats.

Normolipidemic males (six) ingested on separate days a low-fiber (2·8 g) testmeal (70 g fat, 0·75 g cholesterol), enriched or not with 10 g dietary fiber in the form of either oat bran, rice bran or wheat fiber or 4·2 g fiber as wheat germ. Fasting and post-meal blood samples were obtained for 7 h. Chylomicrons, VLDL, LDL and HDL were isolated from the baseline samples and the samples of the 2–3 h triglyceride peak. Chylomicron triglycerides were significantly ( p ≤ 0·05) reduced with wheat fiber. All fiber sources reduced chylomicron cholesterol. VLDL lipid components were not markedly changed. LDL cholesterol decreased post-prandially and this was reversed after adding oat bran and wheat germ. HDL triglycerides were unchanged but the post-prandial HDL cholesterol decrease was significantly amplified after adding oat bran and wheat germ. In conclusion, dietary fiber-rich fractions from cereals can alter post-prandial lipoproteins in humans to a variable extent.

In a previous study (Latron et al. 1991. Arterioscler. Thromb. 11: 1821-1829) we have shown that oxidized low density lipoproteins (ox-LDL) stimulated the synthesis and secretion of plasminogen activator inhibitor-1 (PAI-1) by human umbilical vein endothelial cells (HUVEC) in culture. The present study is intended to give insight into the intracellular process responsible for this stimulation. The HUVEC lipids were labeled for 16 h with [3H]arachidonate and incubated either with native LDL (n-LDL) or ox-LDL for various times (15, 30, 60 min). Compared with unstimulated cells (no LDL added), ox-LDL induced a significant increase in the intracellular level of unesterified [3H]arachidonate, concomitantly with a significant decrease of the phosphatidylinositol fraction. The most marked effect was observed at 30 min and was significantly much less with n-LDL. Phospholipase inhibitors (4-bromophenacylbromide and mepacrine) added to the culture medium completely prevented the ox-LDL-induced stimulation of phosphatidylinositol degradation, [3H]arachidonate release, and PAI-1 secretion. HUVEC possess both phospholipase C and A activities and a high lysophospholipase activity, the phospholipase A pathway being in vitro more sensitive to inhibition by 4-bromophenacylbromide than the phospholipase C pathway. These results suggest that the stimulation of PAI-1 secretion by ox-LDL is mediated by the hydrolysis of membrane phosphatidylinositol through the activation of phospholipase A.

Biliary amphipathic anionic polypeptide (APF) the major protein of the pigment-lipoprotein complex in bile, and calcium-binding protein (CBP) from gallstones are both small (less than 10 kDa), highly acidic, amphipathic proteins present in bile and closely associated also with pigmented areas in human gallstones. Polyclonal antibodies against APF have shown cross-reactivity with plasma high density lipoproteins (HDL). This study examines the hypothesis that APF and CBP might be closely related or even identical, and might also share common epitopes with the larger apoA-I (23 kDa). To assess this, immunoreactivity of the three delipidated, highly purified proteins was determined against a panel of 12 monoclonal antibodies (MAbs) prepared against APF and a panel of 4 MAbs against apoA-I. APF was isolated from bile by zonal ultracentrifugation. CBP was isolated from proteins precipitated from bile by CaCl2, as well as from the calcium bilirubinate shells of cholesterol gallstones, by extraction successively with methyl-t-butyl ether, methanol, and Na2EDTA, followed by Sephadex G-25 chromatography and two-stage preparative SDS-PAGE. ApoA-I was prepared by two types of chromatography: Sephacryl S200 chromatography and heparin-chromatographic immunoaffinity. Specific polyclonal antibodies to APF and apoA-I were prepared from immunized rabbits. MAbs to APF and apoA-I were prepared by immunization of mice, using standard hybridoma technique. Western blotting of APF and CBP in 15% SDS-PAGE yielded one band with an apparent molecular weight of 6.5 kDa, which, along with apoA-I, was immunostained by polyclonal antibodies to APF and apoA-I. Using 12 MAbs against APF with three types of ELISA (direct antigen binding, competitive antigen displacement, and epitope competition between antibodies), it was shown that APF and delipidated apoA-I shared six epitopes, three of which were detected also on the surface of intact HDL particles. Six other epitopes were present in APF but not apoA-I, four of which were exposed on the surface of HDL. Four MAbs against apoA-I reacted with APF and CBP. Amino acid analyses of APF and CBP were similar with 20-23% acidic and 7-11% basic amino acids and low contents of cysteine, methionine, and tyrosine; both differed from apoA-I in containing isoleucine and cysteine. Using ELISA and one MAb (no. 32) against APF, this polypeptide was detected in human plasma HDL, the pigment-lipoprotein complex in the bile of humans, dogs, and rats, and in both pigment and cholesterol gallstones.(ABSTRACT TRUNCATED AT 400 WORDS)

The purpose of this study was to investigate in the rat heart and liver the effects of an acute administration of three anthracyclines, doxorubicin, epirubicin and pirarubicin, and an anthracenedione, mitoxantrone, on the membrane peroxidative status, which was estimated by the composition of polyunsaturated fatty acids (PUFA), and on the activities of the enzymes involved in membrane repair processes and lipid hydroperoxide detoxification. Rats were injected for four consecutive days with the drugs or saline (control) and killed 24 hr after the last injection. All the drugs induced an increase in plasma thiobarbituric reactive substances and alpha-tocopherol concentrations, both expressed per milligram of plasma lipids. Plasma vitamin A was decreased by about a factor of two by all the drugs. The fatty acid profile in the heart lipids showed that the polyunsaturated species (20:4 n-6, 22:6 n-3) remained at the same or even higher levels after anthracycline treatment. This can be explained by the fact that the activities of the enzymes involved in either the recycling of membrane phospholipids, such as phospholipases A1 and A2 (EC 3.1.1.4 and EC 3.1.1.32), lysophospholipases (EC 3.1.1.5) and acylCoA:lysophosphatidylcholine acyltransferases (EC 2.3.1.23), or hydroperoxide detoxification, such as selenium-dependent glutathione peroxidase (GSH-PX, EC 1.11.1.9) and glutathione S-transferases (GSH-T, EC 2.1.5.18), were maintained at the same level of activity after the antitumoral treatment. In liver, membrane phospholipid levels of PUFA were maintained as well as the activities of phospholipid-metabolizing enzymes. GSH-PX activity was not affected whereas that of GSH-T was slightly lowered by the drugs. These results suggest that during acute antitumoral-induced lipid peroxidation of membranes, the multi-enzymatic complex of the immediate processes of repair and detoxification is fully operational, allowing the membrane to rapidly recover its functional status. The results are discussed in the context of the equivocal relationships between antitumoral-induced lipid peroxidation and cardiac disturbances.

The inhibition of pancreatic lipase activity by extracts of raw, milled or processed cereals was measured in vitro. The inhibitory activity was high in durum wheat, soft wheat and millet, moderate in barley and white sorghum and very low in red sorghum. Milling whole-grain into flours markedly decreased the lipase inhibitory activity in all species. In durum wheat, the germ and the aleurone-layer fraction exhibited the highest inhibitory activity. Processing of soft wheat by bread making, popping, flaking, drum-drying and extrusion-cooking, or durum wheat by making pasta markedly decreased the lipase inhibitory capacity. Extractible proteins were implicated in the inhibition process. These effects also had some nutritional implications.

In previous short-term studies in rats and humans, the ingestion of raw wheat germ lowered plasma triglycerides and cholesterol. Thus, the present study was designed to investigate the possible long-term effects of wheat germ intake. Diet supplementation with raw wheat germ or partially defatted wheat germ was tested in two separate groups of 10 and 9 free-living human subjects, respectively. They all exhibited hypercholesterolemia (6.14-9.67 mmol/L cholesterol) and 11 had hypertriglyceridemia. None was diabetic. Fasting blood samples were taken at the beginning of the study, after 4 wk of 20 g/d wheat germ intake, after 14 additional weeks of 30 g/d wheat germ intake and after 12 wk without any supplementation. Dietary records were kept for seven and three consecutive days, before and during the wheat germ intake periods, respectively. Raw wheat germ intake significantly decreased plasma cholesterol (-8.7%) and tended to reduce VLDL cholesterol (-19.6%) after 4 wk. After 14 additional weeks, plasma cholesterol (-7.2%) and LDL cholesterol (-15.4%) remained lower and plasma triglycerides (-11.3%) tended to be lower. The apo B:apo A1 ratio significantly decreased after both periods. Partially defatted wheat germ transiently decreased plasma triglycerides and cholesterol after a 4-wk intake. The present data indicate that wheat germ reduces cholesterolemia in the long term and could play a beneficial role in the dietary management of type IIa and IIb hyperlipidemia.

The present study was performed to investigate the possible effects of wheat germ supplementation on lipid metabolism in humans. Ten free-living adult subjects participated in the study. None was obese or diabetic. They all presented an hypercholesterolemia (from 6.58 to 9.50 mM), associated in 6 over 10 cases to an hypertriglyceridemia (from 1.70 to 5.00 mM). The subjects were studied in three consecutive periods, during which they first were on their usual diet (first week), they then ingested a daily supplement of 30 g wheat germ (4 weeks) and then they returned to their usual basal diet (4 weeks follow-up). Dietary records were obtained for 7 and 3 consecutive days before and during wheat germ supplementation, respectively. Fasting blood samples were taken at the end of each period. After 4 weeks of wheat germ intake, glycemia did not change while total plasma cholesterol significantly decreased (paired Student's t test, p less than or equal to 0.05) from 7.80 to 7.15 mM. LDL and HDL cholesterol values did not show marked changes, but VLDL cholesterol significantly dropped by 40.6%. Thus, the plasma/HDL total cholesterol ratio was significantly lower. Apoprotein B and A1 decreased. In the hypertriglyceridemic subjects, this was accompanied by a significant reduction of plasma triglycerides (1.64 vs. 2.68 mM) and a marked drop of VLDL triglycerides (-51%). Taken as a whole, the present results obtained in humans are very close to those previously obtained in the rat and point out that wheat germ may play a beneficial role in the dietary management of hyperlipidemia.

In the present study, we have performed experiments to gain some insight into the subcellular localization and biochemical properties of gastric mucosal phospholipase A2. After classical subcellular fractionation of whole glandular stomach mucosa, we found that gastric phospholipase A2 was essentially enriched in the 105000 × g pellet that contains microsomes and plasma membranes. Except for the cytosol, all the subcellular fractions exhibited similar phospholipase A2 activity (i.e., optimum of pH, calcium dependence, apparent Km and positional specificity). The high-speed pellet was further characterized by ultracentrifugation on a sucrose gradient. Data showed that the sedimentation profile of phospholipase A2 was quite similar to those of plasma membrane markers and more specifically to an apical membrane marker. These results, taken together, showed that a gastric phospholipase A2 is distributed among the various subcellular fractions (as a result of cross-contamination) together with the membrane fraction on which it is associated. It is proposed that this fraction is the apical plasma membrane which would be the main site of phospholipase A2 action for arachidonic acid release. Lysophospholipase showed the same sedimentation profile as phospholipase A2, whereas acyl CoA-lysophosphatidylcholine; acyltransferase mainly sedimented with heavy microsomes. The substrate specificity of the enzyme was assessed by endogenous hydrolysis of gastric mucosal phospholipids. We were able to show that the enzyme acts at nearly the same rate on two major gastric membrane phospholipids, namely phosphatidylcholine and phosphatidylethanolamine.

The effect of dietary n-6/n-3 fatty acid ratio on alpha-tocopherol homeostasis was investigated in rats. Animals were fed diets containing fat (17% w/w) in which the n-6/n-3 ratio varied from 50 to 0.8. This was achieved by combining corn oil, fish oil, and lard. The polyunsaturated to saturated ratio and total alpha-tocopherol remained constant in all diets. Results showed that enrichment of n-3 polyunsaturated fatty acids in the diet, even at a low amount (3.9% w/w), resulted in a dramatic reduction of blood alpha-tocopherol concentration, which, in fact, is the result of a decrease in plasma lipids, since the alpha-tocopherol to total lipids ratio was not significantly altered. The most striking effect observed was a considerable alpha-tocopherol enrichment (x 4) of the heart as its membranes became enriched with n-3 polyunsaturated fatty acids. This process appeared even with a low amount of fish oil (3.9% w/w) added to the diet. Accordingly, a strong positive correlation was found between heart alpha-tocopherol and docosahexaenoic acid (r = 0.86) or docosahexaenoic acid plus eicosapentaenoic acid levels (r = 0.84). Conversely, the liver alpha-tocopherol level dropped dramatically when n-3 polyunsaturated fatty acids were gradually added to the diet. It is concluded that fish oil intake dramatically alters the alpha-tocopherol homeostasis in rats.

The correlation between the secretion of biliary phospholipid (PL) and bile acid suggests a regulatory effect of bile acid on PL secretion. Bile acids may influence PL synthesis and/or the mobilization of a preformed PL pool. The objective of this study was to determine the contribution of these two sources to biliary PL, by using an experimental protocol in which dehydrocholic acid (DHCA) and cholic acid (CA) were infused to manipulate biliary PL secretion. In control rats, there was a steady state in bile flow. PL secretion and the biliary secretion of newly synthesized phosphatidylcholine (PC). The specific radioactivity of PC in bile was significantly higher than in plasma, microsomes and canalicular membranes. DHCA infusion decreased biliary PC secretion rate by 80%, and secretion returned to normal values at the transport maximum of CA. The specific radioactivity of biliary PC was decreased by 30% by DHCA infusion and reached normal values during CA infusion. There were no significant changes in the specific radioactivity of PC in plasma or cellular organelles during infusion of bile acids. These data indicate that: (1) newly synthesized PC contributes a small percentage to biliary PC; thus a preformed pool (microsomal and extrahepatic) is a major source of biliary PL; (2) the contribution of the extrahepatic pool to the biliary PL may be more important than the microsomal pool.

Fatty acid composition of membrane phospholipids of cultured cardiomyocytes can be modified by the type of polyunsaturated fatty acids (n-3 or n-6 PUFA) constituting the culture medium. In this study, we investigated the effect of fatty acid modification on the activities of the key enzymes involved in the deacylation-reacylation cycle of membrane phospholipids. Results showed that cardiomyocytes grown in the presence of n-6 PUFA exhibited a higher specific alkaline phospholipase A (mainly A2) activity (+34%) and a moderately lower lysophospholipase activity (-17%) than when incubated with n-3 PUFA. AcylCoA:lysophosphatidylcholine acyltransferase, acid lysosomal phospholipase A1 and acylCoA synthetase activities were not significantly altered by changes in cellular PUFA composition. It was demonstrated that the differences between phospholipase A activities of the two types of cultured cells were linked neither to a differential leakage of enzyme nor to oxidative injury to the enzyme through blockage of essential sulfhydryl groups. One likely explanation is that the PUFA-induced changes in membrane composition alter membrane physical properties which, in turn, affect membrane-bound phospholipase A activity. Possible beneficial effects of the n-3 PUFA-induced changes on membrane stability are discussed.

Rats were fed on diets more or less enriched with n-3 and n-6 unsaturated fatty acids, before removal of the small intestine. The global protein, cholesterol and phospholipid contents of enterocyte microsomes were measured. Fatty acids of the total lipid extracts were determined. Acyl coenzyme A: cholesterol acyl transferase (ACAT) was chosen as the enzyme whose activity reflects metabolic changes induced by lipid diets. Fluorescence measurements using diphenylhexatriene as the membrane probe were performed. As dietary fat may change the fatty acid composition of membranes, the order parameter S calculated from fluorescence measurements was studied with regard to dietary fatty acid composition. The S values, distributed over a large range, were not different between rat groups. They were positively correlated with the ratios of cholesterol and proteins to phospholipids and the molar percentage of saturated fatty acids. ACAT activity was negatively correlated with S. Variations in S values among rats, whatever the diet, could in part be attributed to individual factors.

Background/aims We investigated (a) in vitro and in vivo the changes of biliary mass of the anionic peptide fraction, apolipoproteinA-I, immunoglobulin-A, albumin and cholesterol over time in the excluded gallbladder and (b) in vivo the localization in the gallbladder epithelium of the anionic peptide fraction and cholesterol absorbed from bile. Methods Native bile was substituted with pig bile containing radiolabeled cholesterol in the in vitro isolated intra-arterially perfused pig gallbladder (n = 9) and in vivo in anestethized pigs with excluded gallbladders (n = 6). The amount of cholesterol (scintillation counting) and proteins (enzyme-linked immunosorbent assay) in gallbladder bile were measured over time. The localization of the anionic peptide fraction and cholesterol absorbed from bile in the gallbladder epithelium was studied in vivo by immunohistochemistry and fluoro-phospho-imager analysis. Results The rate of biliary cholesterol disappeared from bile was a function of the initial concentration and of the biliary mass changes over time of the anionic peptide fraction, but not of that of the other biliary proteins. The anionic peptide fraction colocalized with biliary cholesterol absorbed by the gallbladder on the apical side of gallbladder epithelial cells. Conclusions These data indirectly suggest that biliary anionic peptide fraction could favour biliary cholesterol absorption by the gallbladder epithelium.

With the demonstration of pronucleating and antinucleating proteins, the role of biliary proteins became of considerable research interest. Anionic polypeptide fraction is the third most abundant biliary protein; it is found in association with biliary lipids, has antinucleating properties for calcium and is found in gallstones. Its levels in various human biles have not been studied as of this writing. In this investigation the concentration of anionic polypeptide fraction in gallbladder bile was measured in 16 subjects without gallstones, 19 subjects with cholesterol stones and 15 subjects with pigment stones in Tel Aviv. Anionic polypeptide fraction concentrations in bile (mean +/- S.D.) were 0.76 +/- 0.09 gm/L in controls and 0.81 +/- 0.25 gm/L (which was not significant) in patients with cholesterol gallstones. They were significantly higher 1.03 +/- 0.23 (p < 0.05) in patients with pigment gallstones. The anionic polypeptide fraction/phospholipid ratio and the anionic polypeptide fraction/total lipid ratio were significantly higher in patients with pigment gallstones (p < 0.005 and 0.05, respectively). The anionic polypeptide fraction lipid ratios were insignificantly elevated in biles of patients with cholesterol stones compared with the ratios in biles of controls. Only the anionic polypeptide fraction/phospholipid ratio was significantly higher in biles of patients with pigment stones compared with those with cholesterol gallstones. The values were similar although higher in a small group of gallstone patients from Marseilles. The role of anionic polypeptide fraction in the pathogenesis of gallstones, particularly pigment gallstones, requires further study.

In the present study, we compared the effects of nibbling and gorging on postprandial lipaemia and lipoproteins, hepatic lipid uptake and atheroma deposition. New Zealand White rabbits were fed on a low-fat (LF) control diet or a peanut oil- (10 g/d) and cholesterol- (0·5 g/d) enriched (HF) diet with the fat and cholesterol components given either by nibbling (HF-N) or gorging (HF-G). After 4 and 8 weeks, rabbits were given a test meal, which was either nibbled or taken as a bolus. The LF diet did not noticeably alter postprantial lipid variables. Triacylglycerol levels, 0–35 h lipid responses and plasma accumulation of dietary lipids were significantly higher in the HF-G group than in the HF-N group, despite higher post-heparin plasma lipase activities. Furthermore, as studied on cultured isolated hepatocytes, the higher the rate of supply of triacylglycerol- and cholesterol-rich lipoproteins (TCRL), the lower the rate of lipid uptake and bile salt secretion. Atheroma deposition was significantly increased by gorging the HF diet and was correlated with levels of most postprandial lipid variables. We conclude that gorgingv. nibbling a fat and cholesterol-enriched diet exacerbates postprandial lipaemia by reducing the rate of TCRL clearance and favours atheroma deposition.

The ability of two 15-ketosubstituted sterols, 5alpha-cholest-8(14)-en-3beta-ol-15-one and 3beta-(2-hydroxyethoxy)-5alpha-cholest-8(14)-en-15-one, to alter the mRNA levels of 3-hydroxy-3-methylglutaryl-CoA reductase, low density lipoprotein receptor, and oxysterol binding protein was studied and compared with the effects of 25-hydroxycholesterol in Hep G2 cells. All three oxysterols decreased the level of HMG CoA reductase mRNA at concentrations of 10-30 microM, although 25-hydroxycholesterol was effective at concentrations of 1-3 microM. 25-Hydroxycholesterol lowered the level of LDL receptor mRNA more efficiently after 8 hours than after 24 hours of incubation, whereas 15-ketosterols did not decrease the mRNA level of the LDL receptor. The transcriptions of HMG CoA reductase and LDL receptor genes are therefore independently regulated by 15-ketosterols in Hep G2 cells. In addition, the level of the oxysterol binding protein mRNA is not affected by oxysterols in Hep G2 cells.

International audience; Liver fatty acid-binding protein (L-FABP) is a small cytoplasmic molecule highly expressed in the liver. Since L-FABP exhibits affinities for several biliary components, its presence in bile was explored by Western blotting and competitive ELISA in various mammalian species. A L-FABP-like immunoreactivity was consistently found in both hepatic and gallbladder bile. A close molecular identity between this 14 kDa biliary protein and the purified L-FABP was assessed by immunological analyses and high performance capillary electrophoresis. Pharmacological induction of hepatic L-FABP biosynthesis led to a similar increase in biliary L-FABP levels showing a close relationships between the cytosolic and biliary contents of this protein. Finally, a correlation between the presence of L-FABP in bile and both bile flow and bile acid release was found. These data suggest an output of L-FABP in bile in normal conditions which might be coupled with the physiological release of biliary components.

Metabolic and vascular abnormalities are implicated in the pathogenesis of diabetic neuropathy. Two principal metabolic defects are altered lipid metabolism resulting from the impairment of delta-6-desaturase, which converts linoleic acid (LA) into gamma linolenic acid (GLA), and reduced nerve Na+, K+ ATPase activity. This reduction may be caused by a lack of incorporation of (n-6) fatty acids in membrane phospholipids. Because this ubiquitous enzyme maintains the membrane electrical potential and allows repolarization, disturbances in its activity can alter the process of nerve conduction velocity (NCV). We studied the effects of supplementation with GLA (260 mg per day) on NCV, fatty acid phospholipid composition, and Na+, K+ ATPase activity in streptozotocin-diabetic rats. Six groups of 10 rats were studied. Two groups served as controls supplemented with GLA or sunflower oil (GLA free). Two groups with different durations of diabetes were studied: 6 weeks with no supplementation and 12 weeks supplemented with sunflower oil. To test the ability of GLA to prevent or reverse the effects of diabetes, two groups of diabetic rats were supplemented with GLA, one group for 12 weeks and one group for 6 weeks, starting 6 weeks after diabetes induction. Diabetes resulted in a 25% decrease in NCV (P < 0.0001), a 45% decrease in Na+, K+ ATPase activity (P < 0.0001), and an abnormal phospholipid fatty acid composition. GLA restored NCV both in the prevention and reversal studies and partially restored Na+, K+ ATPase activity in the preventive treatment group (P < 0.0001). These effects were accompanied by a modification of phospholipid fatty acid composition in nerve membranes. Overall, the results suggest that membrane fatty acid composition plays a direct role in NCV and confirm the beneficial effect of GLA supplementation in diabetic neuropathy.

Background/Aims: Cholesterol gallstones contain both calcium and biliary proteins, but their respective roles in gallstones pathogenesis are unknown. We have studied the effects of calcium and a major biliary protein, anionic polypeptide fraction, on the process of cholesterol crystallization in bile. Methods: Anionic polypeptide fraction was purified from human bile. Model bile composed of cholesterol, egg yolk lecithin and sodium taurocholate was prepared in a lipid concentration (18 mM, 37 mM, and 120 mM, respectively) simulating lithogenic human gallbladder bile. The crystallization process was observed by phase contrast light microscopy, and sequential separation of precipitable cholesterol structures by sucrose density gradient ultracentrifugation. Results: Addition of calcium, or anionic polypeptide fraction alone, or both together did not influence the crystal observation time of bile (the time which elapsed from initiation of supersaturation to the first appearance of crystals). However, the rate and quantity of cholesterol precipitation and crystal formation were affected by both. Calcium increased in a dose-dependent manner the cholesterol monohydrate crystal mass before apparent equilibrium was reached. This effect was inhibited by anionic polypeptide fraction, which increased the amount of cholesterol within precipitable phospholipid vesicles, and decreased the rate of crystal formation. Fluorescence-labeled anionic polypeptide fraction revealed that anionic polypeptide fraction (with and without calcium) was primarily associated with vesicle aggregates. Conclusions: Our data demonstrate that calcium and anionic polypeptide fraction have opposing effects on the process of cholesterol crystallization and the resultant crystal mass without influencing the crystal observation time of bile. These findings suggest that biliary proteins, in addition to being crystallization effectors by themselves, may further influence cholesterol crystallization and gallstone formation by interacting with calcium and possibly other elements that coexist in bile.

The aim of the present study was to evaluate the links between chronic fat-cholesterol intake, postprandial lipaemia and atherogenesis in New Zealand White rabbits. Adult rabbits were fed on either a low-fat control diet (LF) or a high-fat, high-cholesterol diet (HF). Rabbits received a test meal containing [3H]cholesterol and [14C]triolein on days 0 and 63 for the LF group and days 14,28, 42,63 and 84 for the HF group. Blood was collected 24h post-absorptively and 10,24,34 and 48h after test-meal intake. Post-absorptive as well as postprandial lipoproteins and lipaemia were not modified in the LF rabbits, who did not show any atheroma deposition on day 119. In HF rabbits, postprandial plasma triacylglycerols peaked 24–34h after meal intake. The 0-48 h areas under the curves of triacylglycerol andtriacylglycerol-rich lipoproteins (TRL) steadily increased with time of chronic lipid feeding with values significantly higher than those in the LF rabbits. The postprandial plasma and TRL concentrations of dietary radiolabelled lipids were significantly higher in HF than LF rabbits. Post-heparin lipoprotein lipase (EC3.1.1.34) and hepatic lipase (EC3.1.1.3) activities were twofold higher in HF rabbits than in LF rabbits at day 105. In HF rabbits, a marked atheroma plaque deposition in the aorta was observed (30·9 (SE 3·9) % of total surface). The extent of atheroma deposition was positively correlated to the postprandial responses of plasma total triacylglycerols and dietary-derived lipids as well as total cholesterol and dietary-derived cholesterolin HF rabbits. In conclusion, chronic ingestion of a HF diet led to marked increases in postprandial lipaemia and TRL particles, and atheroma deposition.

Crilvastatin, a new drug from the pyrrolidone family, has been previously shown to inhibit the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, in vitro and in vivo, to reduce the absorption of dietary cholesterol and to stimulate the activity of cholesterol 7 alpha-hydroxylase in the rat. The aim of this study was to evaluate the effects of crilvastatin on cholesterol and bile acid metabolism in the hamster. In hamsters fed on a lithogenic diet for 8 weeks, crilvastatin treatment (200 mg/day per kg body weight) did not change plasma lipid levels, failed to improve bile parameters and did not prevent gallstone formation. In hamsters fed on a basal cholesterol-rich (0.2%) diet for 8 weeks, crilvastatin at the same dose reduced the cholesterol level in the plasma by 20%, with a decrease of both low-density and high-density lipoprotein cholesterol. The drug did not significantly stimulate the biliary secretion of bile acids but significantly decreased the activity of acyl coenzyme A:cholesterol acyltransferase in the small intestine by 64%. This effect was enhanced when cholestyramine, a bile acid-sequestering resin, was given in combination with crilvastatin. Crilvastatin alone did not change the activity of cholesterol 7 alpha-hydroxylase in the liver, despite the marked reduction in both hepatic cholesterogenesis and intestinal absorption of dietary cholesterol (the absorption coefficient was 44 +/- 2% in treated hamsters vs. 61 +/- 7% in controls).

Background/Aims: Cholesterol gallstones consist of cholesterol crystals and smaller amounts of pigments and calcium salts, arrayed on a mucin plus protein matrix. The localization of the various biliary proteins in the stones has not been characterized. We aimed to localize several biliary proteins in gallstones in order to determine their possible role in stone formation and growth. Methods: The distribution of several matrix proteins and their relationships to the minerals were determined using immunostaining and EDAX microanalysis on hemisected cholesterol gallstones. Results: Pigment areas were rich in calcium and contained Cu, P and S. These elements were absent in cholesterol regions. Mucin was identified in a three-dimensional network intercalated between cholesterol crystals and as septa between deposits of pigments and cholesterol; APF/CBP and ApN coated only the pigment deposits. No specific topographical localization was found for albumin or IgA. Conclusions: This suggests a role for mucin, APF/CBP and ApN in the formation of cholesterol gallstones. We propose that cholesterol crystals bind directly to mucin, whereas calcium salts and pigments deposits on APF/CBP and ApN bind to the mucin.

1. The objective of this study was to compare in cultured human hepatocytes or Hep G2 cells, changes in the fate of unesterified low density lipoprotein (LDL)-cholesterol induced by crilvastatin, a new cholesterol lowering drug and a reference statin, simvastatin. 2. The experiments were carried out for 20 h, each well contained 4.2 x 10(5)/cm2 Hep G2 cells or 0.5 x 10(5)/Cm2 human hepatocytes, 130 microM ursodeoxycholate, 0.68 microCi or 1.59 microCi unesterified human [14C]-LDL-cholesterol, crilvastatin or simvastatin at 0 or 50 microM (both cell types) or 300 microM (Hep-G2 cells). Incubation with the two drugs resulted in increased amounts of unesterified [14C]-LDL-cholesterol taken by the two cell types, compared to control. 3. Crilvastatin 50 microM led to significantly higher quantities of [14C]-glyco-tauro-conjugated bile salts, compared to simvastatin. Statins reduced the apo B100 level secreted by the two cell types (simvastatin) or human hepatocytes (crilvastatin). Crilvastatin enhanced both the level of apo A1 secreted by the Hep G2 cells and the level of APF, a high density lipoprotein (HDL) and biliary apoprotein. 4. Crilvastatin not only acts by stimulating LDL-cholesterol uptake by hepatocytes, but also by enhancing the catabolism of LDL-cholesterol in bile salts and probably by stimulating HDL and/or bile component secretion. Such a mechanism was not previously described for HMG CoA reductase inhibitors. Our results on APF show that this apoprotein could be considered also as an indicator of changes in bile and/or HDL compartments. 5. The human hepatocyte model appeared to be a suitable and relevant model in the pharmacological-metabolic experiments carried out in this study. It led to more consistent data than those obtained with Hep G2 cells.

This in vitro study was designed to test the hypothesis that soluble dietary fibres can alter the process of intragastric lipid emulsification and possibly subsequent triacylglycerol lipolysis. Three guar gums, two pectins and gum arabic were dissolved in acidic gastric medium in the concentration range 0.3–2.0% (w/v). Viscosities of fibre solutions were measured and apparent viscosities varied over a wide range (0.7–77 mPa·s). Emulsification of a lipid mixture (triolein/phosphatidylcholine/cholesterol) was performed under mild conditions in the presence of increasing concentrations of soluble fibres. The amount of emulsified lipid was not affected whereas the size of the emulsified droplets was increased by raising the concentration of viscous fibres only. The droplet size (r=0.75, P=0.006) and overall droplet surface area (r=-0.69, P=0.009) were strongly correlated with the medium viscosity in the range 0–20 mPa·s. The addition of solutions of viscous fibres to a preformed standard emulsion did not change the initial velocity of human gastric lipase reaction. Conversely, when emulsions prepared in the presence of fibres (i.e. with different droplet sizes) were incubated with excess gastric enzyme for 2 h, the high-viscosity guar gum significantly reduced the extent of triacylglycerol lipolysis, as compared with control and low- or medium-viscosity fibres. In conclusion, the data obtained show that reducing emulsification of dietary lipids in the mildly acid medium found in the stomach is a mechanism by which soluble viscous fibres can alter lipid assimilation.

Previous studies have shown an increase in the intracellular free arachidonic acid content associated with a disturbance in phospholipid metabolism in P815 tumor cells exposed to subtoxic concentration of tert-butyl hydroperoxide. The present study was to determine the respective contribution of the major phospholipid-metabolizing enzymes that could be involved in this process. The enzymes (phospholipase A, lysophospholipase, acylCoA:lysophosphatidylcholine acyltransferase and acylCoA synthetase) were studied under their respective optimal conditions. When P815 cells were treated with 50 microM of tert-butyl hydroperoxide, a significant stimulation (x 2.5) of phospholipase A was observed after 15 min of treatment. The activity of the acyltranferase tended to be higher in cells treated by tert-butyl hydroperoxide while the other enzyme activities (lysophospholipase and acyl CoA synthetase) were not affected. t-BHP did not significantly induce higher levels of lipid peroxides in P815 cells. These results show that, in the tumor cell line P815, the disturbance of phospholipid and arachidonate metabolism induced by t-BHP is linked to phospholipase A, the activation of which seems independent of oxidative stress.

Benfluorex and its three main metabolites at 30 μM have been shown to inhibit Acyl CoA cholesterol acyl transferase activity in rat liver microsome preparations and to fluidize these membranes, as reflected by a decrease in the lipid order parameter. When drug concentrations were higher (60–200 μM), the compounds differed in their enzymatic inhibition properties but retained the same fluidizing effects. Only the parent compound had a dose-dependent inhibiting effect. These results are discussed with regard to the chemical properties of compounds, in particular their electric charges and their lipophilic characters.

A calcium-binding protein of 68 kDa was isolated from human bile that precipitates upon addition of 5 mM CaCl2. This protein was recognized by an immunoaffinity purified anti-annexin VI antibody and it had a similar aminoacid composition as annexin VI. Phospholipase A2 activity was inhibited in vitro in a dose-dependent manner as reported for annexin VI. It is the first time that an annexin secretion is demonstrated in bile.