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.     

Behavior of biliary phospholipids in intestinal lumen during fat digestion in rat

The phospholipids present in the intestinal lumen of rats following ingestion of triglycerides are of biliary origin. They consist of lecithins accompanied by a small proportion of lysolecithins. Their behavior in comparison with the other lipid constituents of the intestinal content was studied by subjecting the latter to gel filtration on an agarose column in the presence of a solution of 6 mM sodium taurocholate in 0.1 M NaCl. Part of the phospholipids is present with the triglycerides and diglycerides in the emulsified phase excluded from the gel where pancreatic lipase and colipase also are found. The remainder is found in optically clear fractions containing fatty acids, monoglycerides, and bile salts. These fractions are eluted at 2.0 column volumes, while mixed fatty acids, monoglycerides, bile salts micelles emerge from the column at 2.4 column volumes in the same chromatographic conditions. This difference in behavior may be explained by the presence of biliary lecithins. This presence could have an important bearing upon the mucosal uptake of the lipolysis products of triglycerides.     

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 saltsphosphatidylcholines-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 billary phosphatidylcholines are absorbed independently of pancreatic secretion in an unhydrolyzed form.     

Influence of acute injection of chloroquine on the biliary secretion of lipids and lysosomal enzyme on rats

Phospholipids and cholesterol combine with a protein fraction (IgA and an acid polypeptide) in bile to form the bile lipoprotein complex. We wished to determine whether lysosomes participated only in IgA secretion or if their secretory role also involved the lipid components of the bile complex. This aspect was studied with a single acute injection of chloroquine, a lysosomotropic drug. The results show that a nonnegligible quantity of IgA travels through the lysosomes. In addition, phospholipid and cholesterol levels undergo a significant (P<0.05) decrease 1 hr after injection before increasing to normal levels. In contrast to the total inhibition of protein secretion (β-glucuronidase, acid phosphatase), a transitory decrease of the secretion of bile lipids takes place that suggest secretory mechanisms involving organelles other than lysosomes.     

Protective effect of biliary lipids on rat pancreatic lipase and colipase

The effect of biliary components on the inactivation of rat pancreatic lipase and colipase was studied. In vitro incubations of these proteins were performed at 37 C in the presence or absence of trypsin, under various conditions. The influences of bile lipoprotein complex, bile salts below or above the critical micellar concentration (CMC), or albumin were investigated. The results showed that albumin and bile salts below the CMC had no protective effect on the inactivation rate of lipase or colipase. Under both denaturating conditions, bile salts above the CMC had a very slight effect, whereas the presence of the bile lipoprotein complex maintained lipase and colipase activity. The magnitude of this effect was related to the biliary phospholipid concentration. By means of gel filtration, the protective effect of bile was found to be due to associations of bile lipoprotein complex with these proteins in presence of bile salts. A correlation between the amount of colipase and the protection of lipase in the presence of biliary phospholipids was observed. Intestinal content of rats with normal and diverted bile secretion was submitted to the same in vitro incubation, and the enzyme was more stable in the segments containing biliary phospholipids. This suggests that the interaction between the bile lipoprotein complex, colipase and lipase in the presence of bile salts could have an important role in the intestinal lumen by retaining the enzyme activity.     

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 μM taurocholate with or without 5.83 μM phosphatidylcholine. In light of a recent quantitative dynamic concept on the interphase partition of lipids, it was calculated that more than99% 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 consitutes a potential vector for various liposoluble molecular species.     

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: (i) 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. (ii) 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.     

A simple method for labeling lipids in the bile lipoprotein

A simple method is proposed for the specific radioactive labeling of phosphatidylcholines and cholesterol in the bile lipoprotein complex. It can be used for human and animal bile samples and results in labeling with the desired specific radioactivity and position. Experiments which determined the intermicellar concentration of lipid constituents suggested that incorporation of radioactive lipids could occur through small dialyzable structures termed mixed premicelles in therm odynamic equilibrium with the bile lipoprotein complex.     

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. The data suggest that the hydrophobicity of the BS used in the infusion modulates the synthesis and secretion of APF-CBP. In the liver, the pool of APF-CBP can be modified by BS and responds rapidly to BS stimulation.     

In vitro studies on interaction of rat pancreatic lipase and colipase with biliary lipids

Lipase and colipase were prepared separately from rat pancreatic juice, and their respective interaction with biliary lipids was investigated by gel filtration on agarose in the presence of a micellar solution of sodium taurocholate. It was found that the cofactor can associate with the biliary lipids, whereas the enzyme forms a high mol wt complex only in the presence of colipase. It is suggested that biliary phospholipids might participate in the in vivo formation of the enzyme-cofactor substrate complex at the triglyceride-water interface in the presence of bile salts.