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.

Pancreatic phospholipase A2 hydrolysis of phosphatidylcholines in various physicochemical states.

Bile salts-phosphatidylcholines-cholesterol mixed micelles, native bile, egg yolk and intralipid emulsions were used as pancreatic phospholipase A2 (EC 3.1.1.4) substrates. The enzyme activity depends on the bile salt/phosphatidylcholine molar ratio. The enzyme had a low specific activity on bile phosphatidylcholines, because of the existence in native bile of a high bile salt/phosphatidylcholine molar ratio generating unfavorable conditions of hydrolysis, as demonstrated with mixed micelles. However when the bile salt/phosphatidylcholine molar ratio from bile was decreased to 2 : 1, enzyme activity increases up to an optimum. This optimal activity was about one third that observed when the substrate was mixed micelles. Under these optimal conditions a simultaneous hydrolysis of intralipid and bile phosphatidylcholine mixture shows comparable initial hydrolysis rates. During an extended incubation, however, nearly all intralipid phosphatidylcholines and only half the bile phosphatidylcholines were hydrolyzed by pancreatic phospholipase A2. Bile salts mixture or native bile desorb a portion of the phosphatidylcholines from the intralipid emulsion in optimally hydrolysable bile salts phosphatidylcholines mixed micelles. These micelles bind about 85% of the enzyme indicating that hydrolysis occurs primarily in the micellar phase. These results are discussed in terms of fat lipolysis in vivo.

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.

The influence of bile salts and bile lipoprotein complex on pancreatic lipase hydrolysis of monomolecular films.

We report a new technique which allows us to follow the lipolysis of monomolecular films in the presence of bile salts by using a 'zero-order' trough (Verger, R. and de Haas, G.H. (1973) Chem. Phys. Lipids 10, 127). The effects of bile salts, the bile lipoprotein complex and colipase on pancreatic lipase hydrolysis of rac-1,2-didodecanoylglycerol films were studied at different surface pressures. Taking into account previous studies, lipase activity was interpreted as a function of its degree of binding to the bile lipoprotein complex.

Effect of bile lipids on the adsorption and activity of pancreatic lipase on triacylglycerol emulsions.

Emulsions of natural triacylglycerols obtained with different shear forces were used to study lipase adsorption and lipolysis. The influence of the bile lipoprotein complex on these two processes was determined. Optimal lipase activity was observed to occur with a given phospholipid : triacylglycerol ratio. This ratio depended on the degree of triacylglycerol emulsification and was accompanied by maximal adsorption of the bile lipoprotein complex. These results support our previous model for pancreatic lipolysis under physiological conditions, according to which colipase controls lipase binding to the bile lipoprotein complex and the resulting association directs enzyme adsorption to the acylglycerol particle (Lairon, D., Nalbone, G., Lafont, H., Léonardi, J., Domingo, N., Hauton, J.C. and Verger, R. (1978) Biochemistry 17, 5263--5269).

Effects of dietary fibers and cholestyramine on the activity of pancreatic lipase in vitro.

Most experiments were conducted in the presence of human gallbladder bile; colipase and pancreatic lipase were purified using porcine pancreas. The adsorption of bile salts, phospholipids and cholesterol from the bile, together with that of pancreatic lipase was measured on wheat bran, cellulose, hemicellulose (xylan), slightly methylated pectin (42%) and cholestyramine. In contrast to cholestyramine which intensively binds biliary lipids (61.7-81.7%) and pancreatic lipase (47.5%), the fibers studied only had a low adsorbent power. The direct influence of these fibers and of cholestyramine at concentrations ranging from 0-5% on lipase activity was measured at constant pH, using two conventional assay systems, long chain triglycerides and tributyrin. In the presence of human bile and colipase, a drastic reduction in triglyceride hydrolysis by lipase was observed with cholestyramine (loss of 66-82%) and wheat bran (loss of 77-94%) at 1% concentration. The other fibers did not have any marked effects on enzyme activity. The use of a radio labeled lipase made it possible to demonstrate that the inhibitory effect of bran on enzyme activity was independent of adsorption phenomena on bran. The fraction of bran that can be solubilized in the aqueous phase, in fact, induced this reduction in activity. The presence of protein inhibitor in bran may be responsible for the reduction in pancreatic lipase activity.

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.

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.

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.

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.

Zone electrophoresis study of the bile lipoprotein complex.

Sucrose gradient column electrophoresis was performed with human hepatic and gallbladder bile. It is shown that bile phosphatidylcholines exhibit a more rapid anodic mobility than do bile salts and serum albumin. This high mobility of bile phosphatidylcholines is not due to the negatively charged lipids which are present in bile, i.e. bile salts or free fatty acids. It is demonstrated that phosphatidylcholines are associated with anionic polypeptides. Electrophoresis of reassociations between these purified polypeptides and dilaurylphosphatidylcholine showed that these anionic polypeptides are primarily responsible for the high anodic mobility of the bile lipoprotein complex. This work describes a procedure for the purification of the bile lipoprotein complex which can be useful for the study of other kinds of lipid-polypeptide associations.

Interaction of immunoglobulins and lipids in human gallbladder bile.

The lipoprotein complex from human gallbladder bile was challenged with anti-IgA and anti-IgG antisera in order to determine whether the apolipoprotein complex isolated from the detergent-free form of bile lipoprotein contains IgA and IgG. The apolipoprotein complex indeed crossreacts with anti-IgA and anti-IgG. In addition, the interactions of IgA and IgG with lipids were studied by ultracentrifugation and gel chromatography to determine whether these interactions occur in human bile.

The apoprotein fraction of the bile lipoprotein complex: isolation, partial characterization and phospholipid binding properties.

A bile apoprotein fraction (Apo BLC) was isolated by preparative isoelectric focusing (I.E.F.) from the detergent-free form of the bile lipoprotein complex (BLC). Analytical I.E.F. of Apo BLC yields a characteristic and reproducible pattern of two narrow acidic bands (pI 4,8-5,0). This apoprotein presents a strong tendency to undergo self-aggregation in aqueous buffer. A low molecular weight constituent of Apo BLC has been isolated after gel filtration, its mean Mw is estimated by SDS-PAGE at 7,500 daltons. The binding capacity of Apo BLC for phospholipids was investigated on dimyristoylphosphatidylcholine liposomes by gel filtration and zone electrophoresis. The resulting structures, larger than the original single-shelled vesicles, acquire and anodic electrophoretic mobility. Apo BLC has a weaker affinity for lysophosphatidylcholines: these phospholipids decrease the degree of aggregation of the apoprotein. These studies contribute additional data concerning the high affinity of Apo BLC for phosphatidylcholines, which are the major phospholipid constituents of bile. The discussion deals with the fact that association of Apo BLC with bile phosphatidylcholines may present some implications in the pathogeny of LpX and in the process of intestinal fat absorption.

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.

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.

A comparative in vivo study of intestinal absorption of biliary phosphatidylcholines and micellar phosphatidylcholines in the rat.

An in vivo study was performed using rats with the purpose of comparing the absorption of native biliary and purified phosphatidylcholines. The latter were purified from bile and solubilized in the form of mixed micelles of bile salts-phosphatidylcholines-cholesterol. The animals all bore bile duct diversions, and were divided into two groups: one had a normal pancreatic secretion while in the other group the pancreatic duct was ligated. Animals with normal pancreatic secretion showed comparable rates of absorption of micellar and biliary phosphatidylcholines. In the absence of normal pancreatic secretion, the rate of absorption of biliary phosphatidylcholines was unchanged, whereas that of micellar phosphatidylcholines markedly decreased. The results are consistent with the concept that some biliary phosphatidylcholines are absorbed independently of pancreatic secretion in an unhydrolyzed form.

Glycodihydrofusidate: biliary excretion and its effect on biliary secretion of the rat.

Glycodihydrofusidate, which has the same detergent properties a bile salts, is excreted almost exclusively by the bile duct after intravenous injection in the rat. As with bile salts, it leads to a significant (P less tthan or equal to 0.05) increase in excretion of lecithins and cholesterol (0.15 mumol lecithin and 0.026 mumol cholesterol per 1 mumol of glycodihydrofusidate excreted). In addition, this drug simulataneously inhibits excretion of both endogenous bile salts and bile pigments.

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.