The anionic peptide fraction is present on the gallbladder apical epithelium and favours biliary cholesterol absorption.

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.

Composition and immunofluorescence studies of biliary "sludge" in patients with cholesterol or mixed gallstones.

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 (100000 g/l 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.

Chronically gorging v. nibbling fat and cholesterol increases postprandial lipaemia and atheroma deposition in the New Zealand white rabbit.

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 gorging v. nibbling a fat and cholesterol-enriched diet exacerbates postprandial lipaemia by reducing the rate of TCRL clearance and favours atheroma deposition.

Cholesterol crystallization in gall-bladder bile of pigs given cholesterol-beta-cyclodextrin-enriched diets with either casein or soyabean concentrate as protein sources.

Cholesterol precipitation from supersaturated bile is the earliest and determinant step in the formation of cholesterol gallstones, which is thought to be diet-dependent. Bile composition, appearance and growth of cholesterol crystals were studied in fresh gall-bladder biles from pigs adapted to four different protein-containing diets over 3 weeks: 160 g dietary protein/kg as casein (C16; n 6), or as soyabean-protein concentrate (S16; n 6), or a mixture of both protein sources (casein-soyabean protein, 70:30, w/w) (CS16; n 6), or 320 g of the mixed protein/kg (CS32; n 6). Moreover, all four diets contained 3 g cholesterol/kg and 50 g beta-cyclodextrin/kg as modifiers of bile composition towards cholesterol pro-crystallization. Cholesterol precipitation was most active after the high-protein diet, CS32, and the casein diet, C16, and lowest after the soyabean-protein diet, S16. It was intermediate after the mixed diet, CS16, but still much lower than in the former two groups. These diet-induced variations were suggested to be mediated through modifications in the biliary profile of bile acids, whereas all other biliary constituents studied were essentially unchanged. The fasting level of plasma cholesterol was lowest in both 160 g protein/kg diets containing soyabean protein (S16 and CS16), highest for the high-protein diet CS32, and intermediate for the C16 diet. These results should encourage clinical studies on the effect of soyabean protein, or other vegetable proteins, for primary or recurrence prevention of cholelithiasis at its earliest stage.

Effects of dexamethasone and linoleic acid on hepatic secretion of biliary lipids and anionic polypeptide factor: In vivo and in vitro studies.

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 microg/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.

Differential regulation by IL-4 and IL-10 of radiation-induced IL-6 and IL-8 production and ICAM-1 expression by human endothelial cells.

Radiation exposure results in an inflammatory reaction with acute as well as subacute consequences. Leukocyte infiltration is one of the predominant early histological changes and involves both cytokines and adhesion molecules. Endothelial cells play a key role in this reaction. We have previously shown the increased production of interleukin 6 (IL-6) and IL-8 and the upregulation in intercellular adhesion molecule 1 (ICAM-1) expression by HUVEC following gamma ray exposure. In the present study, we used the cytokines IL-4 and IL-10 to regulate these radiation-induced manifestations. Human umbilical vascular endothelial cells (HUVEC) were treated with IL-4 and IL-10 (50 pg/ml) either before or after 10- Gy irradiation. Three and seven days after irradiation, IL-6 and IL-8 production by HUVEC (either treated or non-treated) was assessed by enzyme-linked immunosorbent assay (ELISA). Our results show that IL-4, when added after irradiation, reversed the radiation-induced increase in IL-8 production, although slightly increased IL-6 production. IL-10 decreased both IL-8 and IL-6 production when added after irradiation. ICAM-1 expression was evaluated 3 days after irradiation by flow cytometry. The radiation-induced upregulation in ICAM-1 expression remained unaffected by the use of IL-4. Altogether, our results show that radiation-induced endothelial cell activation may be ameliorated by IL-4 and/or IL-10, which is of significance in designing strategies for cytokine-mediated intervention and/or therapy of radiation damage.

HMG CoA reductase and LDL receptor genes are regulated differently by 15-ketosterols in Hep G2 cells.

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.

Output of liver fatty acid-binding protein (L-FABP) in bile.

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.

Beneficial effects of gamma linolenic acid supplementation on nerve conduction velocity, Na+, K+ ATPase activity, and membrane fatty acid composition in sciatic nerve of diabetic rats.

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.

Acute ingestion of different dietary fatty acid species modulates postprandial lipid responses in New Zealand white rabbits.

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.

Effect of beta-cyclodextrin dietary supplementation on biliary proteins and their resulting cholesterol nucleating activity in pigs.

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.

Specific interaction of HIV-1 and HIV-2 surface envelope glycoproteins with monolayers of galactosylceramide and ganglioside GM3.

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.

Effects of lowering fat and increasing dietary fiber on fasting and postprandial plasma lipids in hypercholesterolemic subjects consuming a mixed Mediterranean-Western diet.

The aim of this study was to evaluate the cholesterol-lowering effects of reducing fat and increasing or not increasing dietary fiber in subjects consuming a mixed Mediterranean-Western diet. Thirty-one free-living, mildly hypercholesterolemic subjects were randomly allocated to two groups. Subjects in both groups first shifted for 4 wk to a low-fat, low-fiber diet (LFLFD). For an additional 4-wk period, subjects in group 1 continued consuming the LFLFD whereas subjects in group 2 consumed a low-fat, high-fiber diet (LFHFD). Most dietary fatty acids were monounsaturated (38-41%) and fibers, when provided (up to 35 g/d), came from unrefined cereals, legumes, and soluble-fiber-enriched ready-to-eat cereals. After period 1 of the LFLFD, mean serum and low-density-lipoprotein (LDL)-cholesterol concentrations of subjects in groups 1 (-12.5% and -15.5%, respectively) and 2 (-10.5% and -15.5%, respectively) decreased significantly from baseline (P < 0.05). After period 2, mean serum and LDL-cholesterol concentrations of subjects consuming the LFLFD (group 1) were still lower (by 8.8% and 9.2%, respectively, from baseline) whereas in subjects consuming the LFHFD (group 2) these values decreased further to significantly lower values (14.2% and 17.6% from baseline, respectively). Fasting high-density-lipoprotein (HDL) cholesterol, apolipoprotein A-I, glycemia, and insulinemia did not change significantly. In seven men, postprandial lipemia transiently increased more after a breakfast test meal at the completion of the LFHFD period than after the LFLFD period. In conclusion, an LFHFD more comparable with the traditional Mediterranean diet may improve the dietary management of moderate hypercholesterolemia.

Calcium and the anionic polypeptide fraction (APF) have opposing effects on cholesterol crystallization in model bile.

BACKGROUND/AIMS: Cholesterol gallstones contain both calcium and biliary proteins, but their respective roles in gallstone 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.

Postprandial lipaemia is exacerbated in fat-cholesterol-fed rabbits: relationship to atheroma deposition.

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 24 h post-absorptively and 10, 24, 34 and 48 h 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 24-34 h after meal intake. The 0-48 h areas under the curves of triacylglycerol and triacylglycerol-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 (EC 3.1.1.34) and hepatic lipase (EC 3.1.1.3) 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 cholesterol in HF rabbits. In conclusion, chronic ingestion of a HF diet led to marked increases in postprandial lipaemia and TRL particles, and atheroma deposition.

APF/CBP, the small, amphipathic, anionic protein(s) in bile and gallstones, consists of lipid-binding and calcium-binding forms.

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.

[Affinity of 3-beta-(2-hydroxyethoxy)-5-alpha-cholest-8(14)-ene-15-one to oxysterol binding protein and its metabolism in HepG2 hepatoma cells]. / Srodstvo 3beta-(2-gidroksiétoksi)-5alpha-kholest-8(14)-en-15-ona k oksisterinsviazyvaiushchemu belku i ego metabolizm v kletkakh gepatomy HepG2.

beta-(2-Hydroxyethoxy)-5 alpha-cholest-8(14)-en-15-one, a synthetic inhibitor of cholesterol biosynthesis, was shown to exhibit a high affinity to oxysterol binding protein. This was proved by ultracentrifugation of the protein fraction from rabbit liver in the presence of the 3H-labeled inhibitor, 3 beta-(2-hydroxy-2-[3H]ethoxy)-5 alpha-cholest-8(14)-en-15-one, or by the substitution of the [3H]-25-hydroxycholesterol in its complex with the oxysterol binding protein. In human hepatoma Hep G2 cells, the inhibitor decreased activity of 3-hydroxy-3-methylglutaryl CoA reductase [ID50 (2.7 +/- 0.7) x 10(-5) M] and was transformed into 3 beta-[2-(9-Z-octadecenoyloxy)ethoxy]-5 alpha-cholest-8(14)-en-15-one.

Inducing cholesterol precipitation from pig bile with beta-cyclodextrin and cholesterol dietary supplementation.

BACKGROUND/METHODS: 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. RESULTS: 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: 0<<10<5% 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: 0<5<10% 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. CONCLUSIONS: 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.

Reduced cholesterol absorption in hamsters by crilvastatin, a new 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor.

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).

Hypocholesterolemic action of beta-cyclodextrin and its effects on cholesterol metabolism in pigs fed a cholesterol-enriched diet.

To examine the effects of beta-cyclodextrin (BCD), a non-absorbable carbohydrate, on lipid metabolism, growing pigs were fed a 0.3% cholesterol-enriched diet for 4 weeks or this diet containing 5% or 10% BCD. Pigs fed a basal diet without added cholesterol or BCD were used as controls. The cholesterol-rich diet induced hypercholesterolemia (1.75 vs. 0.84 g/l plasma) due to increased LDL concentration, delayed the plasma clearance of vitamin A, enhanced liver cholesterol storage, lowered the hepatic activities of LDL-receptors (by 47%) and HMG-CoA reductase (by 62%), stimulated cholesterol 7alpha-hydroxylase (x3), and accelerated the fecal output of neutral sterols (x4). Addition of BCD to the cholesterol-rich diet prevented the elevation of plasma cholesterol due to dietary cholesterol excess. Moreover, BCD produced a dose-dependent effect in reducing liver cholesterol storage, stimulating hepatic cholesterogenesis, increasing the proportion of primary bile acids in bile and in feces, and the fecal loss of neutral sterols and bile acids. Pigs receiving 10% BCD thus differed markedly from controls, especially for HMG-CoA reductase and cholesterol 7alpha-hydroxylase hepatic activities (x5), and fecal output of total bile acids (x3) and hyocholic acid (x20), and their overall cholesterol synthesis was higher (+50%), despite the abundant dietary cholesterol. Owing to the property of BCD to bind cholesterol and bile acids in vitro, these results suggest that this resistant carbohydrate accelerates body cholesterol turnover by reducing cholesterol absorption, increasing cholesterol and bile acid synthesis, and altering the action of the intestinal microflora.