Modulating effects of bile salt hydrophobicity on bile secretion of the major protein of the bile lipoprotein complex.

Bile lipids are secreted in association with a newly identified major apoprotein called anionic polypeptide fraction-calcium binding protein (APF-CBP), which is synthesized in the hepatocytes and has been detected in both bile and plasma and characterized. The secretion of the lipids in bile depends both on the concentration and the hydrophobicity of the bile salts (BS) secreted. The present study was undertaken to determine whether the synthesis and the secretion of APF-CBP are similarly regulated by BS, using two methods. The synthesis and secretion of labelled, newly synthesized APF-CBP by isolated rat hepatocytes were monitored by solid-phase immunoassay. For this purpose, hepatocytes were incubated with either glycodeoxycholate (GDC) or taurocholate (TC). The synthesis and secretion of labelled, newly synthesized APF-CBP by perfused rat liver were measured by immunological enzyme-linked assay (ELISA) upon perfusing the liver with either GDC or TC. We found that (i) the synthesis and the secretion of APF-CBP were increased during either TC or GDC perfusion, but the increase was more pronounced with TC; (ii) in GDC perfusion the APF-CBP levels measured were more closely related to the levels of bile salts and not to phospholipid levels, (iii) when the two bile salts were perfused in reverse order, i.e., first GDC and then TC, the secretion of APF-CBP in bile decreased when GDC was perfused, but increased when TC was perfused. Similar results were obtained in experiments with isolated hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cyclosporine and corticosteroids on bile secretion in the rat.

In order to study their effects on the bile secretion, cyclosporine and methylprednisolone were injected intravenously into rats at a dose of 10 mg/kg b.w. for 30 min. Methylprednisolone had no effect on bile secretion. Cyclosporine led to transient intrahepatic cholestasis characterized by decreased bile flow as well as a decrease of bile salts and cholesterol in bile. Phospholipid levels were not affected. Liver biopsy showed no particular anomaly. These findings suggest that the observed cholestatic reaction may be due to impairment of the metabolism of cholesterol into bile salts or of the conjugation of bile salts rather than to disturbances in bile secretion. After liver transplantation in humans, cholestasis associated with acute rejection or nonspecific cholestasis cannot be attributed directly to the effect of cyclosporine. Cholestasis can be offset by administering taurocholate at a dose of 10 mumol/min/kg b.w. in order to maintain bile salt and phospholipid levels high enough to ensure proper "vectorization" of cholesterol to bile.

Studies on the origin of biliary phospholipid. Effect of dehydrocholic acid and cholic acid infusions on hepatic and biliary phospholipids.

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.

Evidence for the synthesis and secretion of APF--a bile lipid associated protein--by isolated rat hepatocytes.

Bile lipids are thought to be secreted in a lipoprotein complex in which they are associated with cholesterol and a protein called the anionic polypeptidic fraction (APF). APF is present in both bile and serum HDL. The association of APF with both bile and lipoprotein strongly suggests that hepatocytes may be responsible for the synthesis and secretion of this protein. In the present work we attempted to verify this by studying the incorporation of [14C]leucine into APF in isolated rat hepatocytes and by immunolocalization in cell cultures. Results obtained showed that synthesis of APF by cells follows the same kinetic pattern as albumin and that it was the third most abundant protein in the bile secretion. Immunolocalization confirmed that APF is synthesized in the endoplasmic reticulum of hepatocytes. This protein which appears to be rapidly secreted could be of great value for the specific detection of the lipids destined for bile secretion.

Detection and characterization of anionic polypeptide fraction binding sites in rat liver plasma membranes and cultured hepatocytes.

The binding of human 125I-labeled 'anionic polypeptidic fraction' (APF) to purified rat liver plasma membranes was studied. The dissociation constant for this binding was 3.0 micrograms protein/mg membrane protein. Binding was competitively inhibited by unlabeled human APF, but not by human LDL (low density lipoproteins). When unlabeled HDL3 was added, binding of labeled APF was competitively reduced to a level between that of unlabeled APF and unlabeled LDL. Experiments with cultured rat hepatocytes confirmed those obtained with liver membranes and suggested the presence in rat liver of saturable APF-binding sites which seem to be specific for APF. The physiologic significance of these APF binding sites is discussed in relation to the fate of cholesterol in the liver.

Quantitative data on very highly diluted solutions.

Computation using a coherent system of units demonstrates simply that no significant specific biological and pharmacological effects can be expected from very highly diluted solutions.

Identification and localization of the apoprotein fraction of the bile lipoprotein complex in human gallstones.

The anionic polypeptidic fraction is the protein constituent of the bile lipoprotein complex. Double immunodiffusion and sodium dodecyl sulfate polyacrylamide gel electrophoresis studies show that the anionic polypeptidic fraction is present in human gallstones. In terms of weight percentage, this protein accounts for 0.1% +/- 0.087 (n = 6) of the total weight of gallstones. Immunolocalization studies confirm the presence of the anionic polypeptidic fraction in human gallstones and suggest that this protein is preferentially associated with pigmented layers in gallstones. A speculative role for the anionic polypeptidic fraction in cholesterol nucleation is discussed.

Effect of bile anionic polypeptidic fraction on the fate of cholesterol carried by liposomes in the rat.

[14C]Cholesterol associated with liposomes with or without anionic polypeptidic fraction was administered intravenously to the rat. The cholesterol originated from liposomes including anionic polypeptidic fraction is secreted in bile much later, is stored in liver in higher quantity, and is metabolized into bile salts in lesser quantity during the 4 hr of experimentation than the cholesterol issued from liposomes exempt of anionic polypeptidic fraction. From these results it can be postulated that the cholesterol associated with liposomes containing anionic polypeptidic fraction might be directed in a particular liver pathway.

Metabolism of low- and high-density-lipoprotein-free cholesterol in rats fed high-fat diets.

The regulating process of cholesterol in the liver was studied in relation to its exogenous contribution in the rats fed high-fat (28%) high-cholesterol (1.2%) diets rich in saturated (S) fat (lard) or polyunsaturated (PU) fat (corn oil). Accordingly, the fate of 14C free cholesterol originating from high- or low-density lipoproteins (LDL) was examined in the biliary, hepatic and plasmatic lipids, as well as the activity of two key enzymes in the metabolism of lipoproteins: lipoprotein lipase (LPL) and lecithin cholesterol acyl transferase (LCAT). The LPL activity increased in the S diet, in comparison to the PU diet or to a low fat (6%) control (C) diet and the LCAT activity increased but not significantly in the PU diet. In bile the secretion of 14C-cholesterol and 14C-bile salts originating from 14C-cholesterol-HDL increased in the S diet compared to the PU diet and a C diet [previous results]. S and PU diets increased to the same extent the hepatic storage of 14C-esterified cholesterol originating from LDL, compared to the C diet. This cholesterol would contribute to a greater extent to the hepatic synthesis of the lipoproteins destined for the plasma in the case of the S diet than that of PU diet. These results may be explained by the adaptation of hepatic acyl cholesterol acyl transferase and cholesterolesterase to both high-fat-diet enzymes acting simultaneously on the two free and esterified cholesterol compartments. It resulted in an important redistribution of the cholesterol of these two compartments between plasma, bile and liver.

Effect of pectin, wheat bran and cellulose on serum lipids and lipoproteins in rats fed on a low- or high-fat diet.

1. Four groups of adult male Sprague-Dawley rats were fed for 6 weeks on a diet with a low-fat content (50 g/kg) and another four groups were given a diet rich in fat (250 g/kg) and cholesterol (12 g/kg). In both cases, the basal diets were either fibre-depleted or supplemented with cellulose (60 g/kg), wheat bran (100 g/kg) or low-methoxyl pectin (100 g/kg). 2. Low-methoxyl pectin displayed the most hypocholesterolaemic effect and decreased the cholesterol content of the very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL), when the low-fat diet was given. When rats were fed on the high-fat diet, pectin no longer had a hypocholesterolaemic effect but still decreased the VLDL-cholesterol content. Pectin lowered serum triglyceride and VLDL-triglyceride levels only when the low-fat diet was given. 3. Wheat bran exerted no hypocholesterolaemic effect in rats fed on the low- and high-fat diets, but decreased the cholesterol content of VLDL and lowered serum triglycerides and VLDL-triglycerides when the high-fat diet was given. 4. Purified cellulose had no significant effect on plasma lipids. 5. As shown by multivariance analysis, low-methoxyl pectin and wheat bran both beneficially modified the serum triglyceride and cholesterol variables except VLDL-triglycerides. However, the magnitude of the effect of each individual type of fibre was dependent on the fat and cholesterol content of the diet, suggesting the existence of different mechanisms of action.

Combined effects of chlorpromazine and high fat diet on lipid levels and enzyme activities in bile, liver and plasma of Wistar rats.

The effects of high fat diet and injection of chlorpromazine on bile lipid secretion were studied in the rats fed a control diet (C), a saturated fat, high cholesterol diet (S) and a polyunsaturated fat, high cholesterol diet (PU). As compared to controls, injection of chlorpromazine in the S and PU diet groups caused no appreciable change in the level of bile salts and bile phospholipids. Chlorpromazine did however enhance bile cholesterol, especially in the PU group, and lower secretion of lysosomal enzyme (beta-glucuronidase) into bile. Impairment of lysosomal enzyme secretion but not of bile lipid secretion suggests that the lysosomal activity is not directly involved in the bile secretion mechanism. These data point up the risks of using chlorpromazine therapy in association with a diet high in fat and cholesterol.

Determination of the physicochemical parameters of a lipoprotein class from weight percentage data.

Determination of the physicochemical parameters of a lipoprotein class from weight percentage data in light of a recent quantitative biodynamic concept which strictly adheres to the CGS unit system (cm, g, s) and the mass/volume chemical unit, mol.cm-3.

Determination of physicochemical parameters of liposomes from concentration data.

Determination of the physicochemical parameters of unilamellar and multilamellar liposomes from concentration data in light of a recent quantitative biodynamic concept which strictly adheres to the CGS unit system (cm, g, s) and the mass/volume chemical unit, mol.cm-3.

Lipid biodynamics: new perspectives [published errtum appears in Biochimie 1987 May;69(5):558].

Active biological systems can be divided into five phases: the aqueous polar phase, the monolayer and/or bilayer interfacial phase, the apolar or hydrophobic phase, the solid or insoluble phase and the gaseous phase. The micellar phase is a special dispersed state of an interfacial phase. Molecules are distributed among these five phases according to their physicochemical properties. Herein is proposed a standardization in strict compliance with the CGS (cm, g, s) unit system and uses the mass/volume, mole per cm3 (mol X cm-3) chemical unit. This standardization requires a new set of symbols to clearly distinguish the concentrations in the different phases. The numerous implications of this standardization are discussed with respect to the quantitative classification of lipids based upon interphase partition coefficients, a new definition of micelles, simple models for the study of lipid biodynamic behavior and sites of action of lipid metabolism enzymes as well as determination of the physicochemical parameters of circulating lipoproteins. By compartmentalization in an aqueous polar phase, an interfacial phase comprising phospholipids and free cholesterol and an apolar phase comprising triglycerides and esterified cholesterol, this standardization will greatly simplify quantitative research on the factors regulating and disturbing cholesterol homeostasis. The notion of total cholesterol must be foresaken, since the biodynamic behavior of free cholesterol and esterified cholesterol are fundamentally different. Free cholesterol shares the fate of the interfacial phase of which it is a part, this fate being hinged on enzymatic biotransformations and/or ligand--receptor interactions. The proposed standardization gives rise to a new rationale using simple calculations and its advantage will be 2-fold: first, in the design of experimental protocols; and second, in allowing immediate and unambiguous comparison of experimental data based upon strictly defined parameters.

Bile lipid secretion in isolated perfused rat liver. A model for metabolic studies.

Isolated perfused rat liver was used to study the effects of constant taurocholate perfusion, with or without the addition of phosphatidylcholine unilamellar vesicles, upon both the bile salt-dependent and bile salt-independent secretion of bile. Taurocholate introduction increased bile flow and normalized the bile lipid secretion by restoring the bile salt-dependent secretion. At a flow rate of 30 ml/min, the liver was perfused by a single-pass method. The perfusion medium contained 17.5 microM taurocholate with or without 5.83 microM phosphatidylcholine. In light of a recent quantitative dynamic concept on the interphase partition of lipids, it was calculated that more than 99% of the taurocholate reaches the liver as monomers and/or dimers. It was also deduced that the lipids were secreted in bile as small discoidal lipoprotein structures rather than unilamellar lipoproteic vesicles. During the course of the experiments (2 hr), the excellent criteria of viability of this model make it highly suitable for the investigation of hepatic metabolism. Furthermore, the addition of phosphatidylcholine unilamellar vesicles to the perfusate constitutes a potential vector for various liposoluble molecular species.

The relationship between HDL-, LDL-, liposomes-free cholesterol, biliary cholesterol and bile salts in the rat.

In order to study the relationship between bile cholesterol and free cholesterol carried by high and low density lipoproteins (HDL and LDL), 10 male Wistar rats, 11 weeks old and fed with a standard diet were divided into 3 groups which received an intravenous infusion (jugular vein) of either LDL, HDL or liposomes. Liposomes were used for comparison because they are assimilated by hepatocytes, but are not recognized by specific receptors. HDL isolated from rat sera were labeled with [14C]cholesterol by molecular exchange and LDL were labeled by exchange with [14C]cholesterol incorporated into phosphatidyl choline/cholesterol liposomes. The peaks of radioactivity appeared in bile 30 min after the HDL or liposome injection and after 210 min for the LDL injection. The kinetic behavior of the cholesterol carried by the liposomes was quite similar to that of cholesterol carried by HDL. Cholesterol carried by HDL was metabolized in bile salts faster than that carried by LDL: cholesterol-HDL or cholesterol-liposomes contributed to the same extent to the secretion of bile cholesterol (15 and 11%, respectively, of the injected dose), LDL (20% of the injected dose). However, the main part of [14C]cholesterol from HDL, LDL or liposomes was metabolized in bile salts. Thus, cholesterol from an exogenous source seemed to be used mainly as a substrate for bile salts. Our study revealed a difference between the hepatic metabolism of HDL, liposomes and LDL in the rat: the kinetic difference between the secretions of the radioactive compounds in bile may be explained by differences in assimilation, intracellular pathways or bile secretion.

A quantitative dynamic concept of the interphase partition of lipids: application to bile salt-lecithin-cholesterol mixed micelles.

A system is proposed for a quantitative classification of lipids, based on interphase partition coefficients. This system enables calculation of exchanges of lipid molecules between phases. The mass/volume chemical unit mol X cm-3, strictly derived from the CGS system, is used, thus simplifying mathematical relations. Applied to bile salt-lecithin-cholesterol mixed micelles, this dynamic concept gives new insight into the variations of physico-chemical parameters. Experimental results obtained with the glycodesoxycholate and the taurocholate show a striking difference in partition coefficients between aqueous and mixed bile salt-lecithin interfacial phases. A new model applying triangular co-ordinates to a bile salt-lecithin-cholesterol mixed lipid phase is described.

Purification of the human anionic polypeptide fraction of the apo-bile lipoprotein complex by zonal ultracentrifugation.

The two main proteic constituents of the human Apo-bile lipoprotein complex (BLC), i.e., the anionic polypeptide fraction (APF) and the IgA fragments, were separated by preparative zonal ultracentrifugation using a sucrose gradient containing 1.5 mM glycodesoxycholate. The purification of the APF was verified by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and immunology, and its amino acid composition then was determined. This procedure was used to obtain a polyclonal antiserum directed solely against the APF.

Hydrolysis of intralipid by pancreatic lipase and phospholipase A2-gel filtration studies.

Intralipid was incubated with pancreatic lipase (EC 3.1.1.3) and/or phospholipase A2 (EC 3.1.1.4) at two bile salts/phosphatidylcholine molar ratios and at two different triglyceride hydrolysis rates using various amounts of lipase. Incubations were studied by gel filtration. Results show: During lipase action, three phases of lipids coexist: an emulsified phase, a micellar phase and an intermediate heavy phase sized between the two others. The equilibrium between each phase is dependent upon the bile salts concentration. Under these conditions, pancreatic lipase was at 60% bound to the emulsified phase, whereas pancreatic phospholipase A2 was bound at 94% to the micellar phase.