Isolation and properties of the mixed lipid micelles present in intestinal content during fat digestion in man.

To evaluate better the physicochemical characteristics of human fat digestion, a method was developed which allowed characterization of the bile acid-lipid mixed micelles of the aqueous phase of post-prandial duodenal fluid. Duodenal fluid was collected after a 36-g fat breakfast for two 90-min periods and for 60 min after i.v. cholecystokinin and was ultracentrifuged at 15,400,000 g-min. The aqueous phase was isolated, passed through a 200-nm filter, and the mixed micelles were concentrated by an ultrafiltration procedure using a 1.5-nm filter. The 1.5-nm retentate was eluted from Sepharose 6B columns with 1.5-nm filtrate for both preequilibration fluid and eluent. 1.5-nm filtrate approximated the monomer concentrations. Each sample was assayed for bile acid, fatty acid, lecithin, lysolecithin, protein, cholesterol, and counterions (pH, Na+, K+, Ca2+). Constituents were concentrated only on the 1.5-nm filter. On gel permeation chromatography, coincident peaks were observed for bile acid, fatty acid, lysolecithin, and cholesterol; and were eluted with a Kav range of 0.50-0.68 (corresponding to a Stokes radius of 2.3-3.5 nm). An average density of 1.25 and coincident peaks of bile acid and fatty acid were found for the mixed micelles on sucrose density gradients. The regression lines of micellar fatty acid, lysolecithin, and cholesterol vs. bile acid gave a stoichiometry of 1.4 mol fatty acid, 0.15 mol lysolecithin, and 0.06 mol cholesterol for each mole of bile acid. Mixed micelles were homogeneous in composition. These results provide direct evidence for the existence of the postprandial mixed micelle and describe several of its physicochemical properties.

The mechanism whereby bile acid micelles increase the rate of fatty acid and cholesterol uptake into the intestinal mucosal cell.

Studies were undertaken to define the mechanism whereby bile acid facilitates fatty acid and cholesterol uptake into the intestinal mucosal cell. Initial studies showed that the rate of uptake (Jd) of several fatty acids and cholesterol was a linear function of the concentration of these molecules in the bulk phase if the concentration of bile acid was kept constant. In contrast, Jd decreased markedly when the concentration of bile acid was increased relative to that of the probe molecule but remained essentially constant when the concentration of both the bile acid and probe molecule was increased in parallel. In other studies Jd for lauric acid measured from solutions containing either 0 or 20 mM taurodeoxycholate and saturated with the fatty acid equaled 79.8+/-5.2 and 120.8+/-9.4 nmol.min(-1).100 mg(-1), respectively: after correction for unstirred layer resistance, however, the former value equaled 113.5+/-7.1 nmol.min(-1).100 mg(-1). Maximum values of Jd for the saturated fatty acids with 12, 16, and 18 carbons equaled 120.8+/-9.4, 24.1+/-3.2, and 13.6+/-1.1 nmol.min(-1).100 mg(-1), respectively. These values essentially equaled those derived by multiplying the maximum solubility times the passive permeability coefficients appropriate for each of these compounds. The theoretical equations were then derived that define the expected behavior of Jd for the various lipids under these different experimental circumstances where the mechanism of absorption was assumed to occur either by uptake of the whole micelle, during interaction of the micelle with an infinite number of sites on the microvillus membrane or through a monomer phase of lipid molecules in equilibrium with the micelle. The experimental results were consistent both qualitatively and quantitatively with the third model indicating that the principle role of the micelle in facilitating lipid absorption is to overcome unstirred layer resistance while the actual process of fatty acid and cholesterol absorption occurs through a monomer phase in equilibrium with the micelle.

On the formation of a ternary complex between lipase, colipase and micelles of amphipathic compounds.

Pancreatic lipase, colipase and 1-glyceryl octyl ether or lysolecithin micelles were shown to form a ternary complex probably resulting from the fixation of the enzyme to the binary colipase-micelle association already well documented in the case of bile salt micelles. Kinetic assays show that the colipase favours the lipase activity on tributyrin emulsions in presence of 1-glyceryl octyl ether. The UV spectrum of a derivative of lipase in which two 2-hydroxy 5-nitrobenzyl groups are attached to tryptophan, is distinctly modified by addition of 1-glyceryl octyl ether micelles in the presence of colipase.

Further studies on the mechanism of adrenaline-induced lipolysis in lipid micelles.

Lipase [EC 3.1.1.3] depleted lipid micelles, in which lipolysis was not elicited by adrenaline, were prepared from lipid micelles. When these lipase-depleted lipid micelles incubated with adipose tissue extract containing lipase activity, adrenaline-induced lipolysis was restored to almost the same level as that of native lipid micelles. Adrenaline-induced lipolysis was not restored when the lipase-depleted lipid micelles were homogenized or sonicated. Various tissue extracts from kidney, lung, liver, and pancreas, and post-heparin plasma, which contained lipase activity, restored adrenaline-induced lipolysis in lipase-depleted lipid micelles.

Stereospecific morphine adsorption to phosphatidyl serine and other membranous components of brain.

In the course of investigating various membranous components for morphine binding, it was found that the major substance responsible was phosphatidyl serine. Other acidic lipids, such as phosphatidic acid and phosphoinositides bind to a considerably lesser extent, while neutral lipids, glycolipids and other phosphatides bind slightly or not at all. Total lipid extracts from a number of regions of rat brain exhibited different degrees of binding to (-)-morphine, such regions as the cerebral cortex and thalamus being the greatest. From an examination of the pH curve for binding and the effect of ionic strength, it was concluded that the binging was largely electrostatic. The method employed was the radioactive measurement of 14C-morphine adsorption to surface films of phosphatidyl serine. When the phosphatide was dispersed in a nonionic detergent near the critical micelle concentration, adsorption was maximal, attaining a value of 1 molecule of morphine adsorbed per molecule of phosphatidyl serine in the surface micelle. The relation between binding affinity and biological potency was not consistent. Morphine adsorption occurred with films prepared from a dispersion of the phosphatide and a hydrophobic protein from synaptic membranes. With the use of levorphanol and dextrorphan it could be shown that binding of morphine was stereospecific.

Choleresis and hepatic transport mechanisms. IV. Influence of bile salt choleresis on the hepatic transport of the organic cations, D-tubocurarine and N4 -acetyl procainamide ethobromide.

The influence of the bile salts taurocholate and dehydrocholate on the hepatic transport of two quaternary ammonium compounds, D-tubocurarine (dTc) and N4 -acetylprocainamide ethobromide (APAEB) was investigated in rats. The biliary excretion of APAEB and dTc in vivo was not enhanced by 106 mumoles/h of taurocholate or dehydrocholate. Infusion of 268 mumoles/h dehydrocholate caused an inhibition of the plasma disappearance and hepatic transport of dTc. This inhibition, which presumably occurred at the hepatic uptake level, was also observed in isolated perfused rat liver experiments. In animals with an intact renal function, the high dose of dehydrocholate caused a decreased biliary excretion and an increased renal excretion of dTc. The observed concentration gradients, plasma/liver cytosol and bile/liver cytosol 20 min after injection of both drugs were 1.6 and 23 for APAEB and 2.2 and 190 for dTc. These concentration ratios were based on free drug concentrations; corrections were made for plasma protein binding, intracellular binding and bilary micelle binding. No substantial binding of both compounds to ligandin and Z proteins was found. From the amount in the liver 20 min after injection of both drugs 70% of APAEB and 90% of dTc was bound to cellular particles. The rate limiting step in hepatic transport of APAEB from plasma into bile was concluded to be the hepatic uptake, which may explain the lack of effect of bile salt induced choleresis on its biliary excretion.

Choleresis and hepatic transport mechanisms. II. Influence of bile salt choleresis and biliary micelle binding on biliary excretion of various organic anions.

To investigate, whether binding to micelles has a function in hepatic transport, biliary excretion of three organic anions, phenolphthalein-beta-D-glucuronide (PG), dibromosulphthalein (DBSP) and indocyanine green (ICG) was studied in rats during saline, taurocholate or dehydrocholate administration. Taurocholate causes a weak choleresis with formation of biliary micelles, dehydrocholate a strong choleresis with little micelle formation. The two bile salts did not uniformly influence biliary excretion of the organic anions: biliary excretion of ICG (12.9 mumoles/kg) and DBSP (75.0 mumoles/kg) was stimulated by both bile salts: ICG excretion most pronounced by taurocholate and DBSP excretion most strongly by dehydrocholate. Biliary output of PG (25.8 and 200 mumoles/kg) was not stimulated by bile salt administration. Binding of PG, DBSP and ICG to biliary micelles was studied in sedimentation experiments by ultracentrifugation. PG, DBSP and ICG in bile showed a similar sedimentation pattern as 3H-taurocholate in bile, which indicates an association of all three anions with biliary micelles. Thus, the influence of bile salts on biliary transport of organic anions varies with the compound studied and the bile salt used, effects which cannot be explained by differences in binding to biliary micelles.

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.

The secretory characteristics of dehydrocholate in the dog: comparison with the natural bile salts.

1. During dehydrocholate administration in the taurine replete dog, the maximum excretory rate of total bile salt (almost entirely dehydrocholate derivative, mostly conjugated) was 3-84 +/- 0-53 (S.D.) mumole/min. kg body wt. (eleven experiments). This was much less than the excretory maximum previously obtained for taurocholate (8-64 +/- 1-31 (S.D.) mumole/min. kg total cholate, mostly conjugated). 2. The superimposition of taurocholate infusion did not cause any significant change in the 'dehydrocholate' maximum but taurocholate itself was excreted into bile at no more than about half its normal maximum. When taurocholate maximum excretion was established first, it was reduced by dehydrocholate administration. In both types of experiment the joint bile salt excretory maximum was of the same order as that of taurocholate alone, provided taurocholate made up at least 40-50% of the total bile salt. 3. When taurocholate administration was stopped, the maximum excretory rate of 'dehydrocholate' rose to values up to 63% above the initially determined excretory maximum; the enhanced 'dehydrocholate' excretory maximum, when calculated for optimal conditions, approached that of actively conjugated vholate, even though the effective 'dehydrocholate' concentration in bile was ten to twenty times the critical micellar concentration of taurocholate. This suggests that the effective bile salt concentration in bile is not an important determinant of the secretory performance of a bile salt. 4. To explain findings (2) and (3) it is necessary to postulate that taurocholate has both a facilitatory and an inhibitory action on 'dehydrocholate' excretion. The facilitatory action, which persists after taurocholate has left the animal, may consist either of an increase in the maximum rate at which modification of dehydrocholate takes place within the liver cell, or an increase in the number of functioning 'carriers' for 'dehydrocholate' transfer. The data suggest that the inhibitory effect is due to the competitive interaction that also appears to exist between the two bile salts. 5. The increase in bile flow rate per unit increase in 'dehydrocholate' excretion (15 ml./m-mole) was about twice that obtained for taurocholate. There was no significant formation of micellar aggregates during 'dehydrocholate' excretion, as judged from the total electrolyte concentration of bile and its osmalality. 6. During the excretion of 'dehydrocholate'-taurocholate mixtures (approximately 1:1) at submaximal rates the associated bile flow rate was not less than the sum of the separate components, thus suggesting that 'dehydrocholate' was not being incorporated in taurocholate mixed micelles.

Abnormal micelle formation in various malabsorption syndromes.

In order to clarify the abnormal micelle formation in various malabsorption syndromes, the analysis of upper intestinal contents after administering a test meal of Borgström et al. was performed. It was demonstrated that the percentage of micellar fat to total fat and lipase concentrations decreased markedly from the normal controls despite of normal concentrations of bile salts and pH in cases of pancreatitis and pancreatectomy. In contrast, it was shown that the percentage of micellar fat was markedly low and the concentrations of conjugated bile salts were reduced in cases of ileal diseases. Unconjugated bile salts were detected although the concentrations of total bile salts were normal in blind-loop syndrome. The correlation between the percentage of micellar fat and lipase concentrations or bile salts concentrations in normal controls and various malabsorption syndromes was also reported.

The effect of jejunoileal bypass on bile composition and the formation of billiary calculi.

In our series of 101 patients with small bowel bypass for morbid obesity, nine developed biliary calculi postoperatively during a mean follow-up of 29.6 months. The development of gallstones depends in part on biliary cholesterol saturation and on the zeta potential of bile. In eight consecutive patients, the lithogenicity of bile was assessed by the methods of Small, Swell and Isaksson, which are dependent on cholesterol saturation. Postoperatively, the lithogenic score decreased in six and increased in two patients, one of which developed gallstones. Taurine bile salt conjugation tends to prevent aggregation of micelles by increasing the zeta potential. The biliary glycine/taurine ratio increased (p less than 0.05) from 4.6 to 5.9 postoperatively. These results suggest that the increased incidence of cholelithiasis following small bowel bypass is not only due to a relative change in bile composition but is probably more significantly due to an increase in the biliary glycine/taurine ratio and a consequent decrease in the biliary zeta potential.

The interaction of phospholipase A2 with micellar interfaces. The role of the N-terminal region.

The localization of the previously postulated interface recognition site (IRS) in porcine pancreatic phospholipase A2, required for a specific interaction between the enzyme and organized lipid-water interfaces, was investigated by ultraviolet difference spectroscopy, by measurements of the intrinsic fluorescence of the unique Trp residue, and by protection experiments against specific tryptic hydrolysis. Using the enzymically nondegradable substrate analogues: CnH(2n+1)(0-)OOCH2CH2N+(CH3)3-(H,OH), it is shown that the rather hydrophobic N-terminal sequence of the enzyme, viz., Ala-Leu-Trp-Gln-Phe-Arg, is directly involved in the interaction with the lipid-water interface. Besides hydrophobic probably also polar interactions contribute to the binding process. At neutral or acidic pH the presence of a salt bridge between the N-terminal alpha-NH3+ group and a negatively charged side chain stablizes the interface recognition site and allows the enzyme to penetrate micellar surfaces, even in the absence of metal ion. At alkaline pH, interaction of the enzyme with micellar interfaces requires the presence of Ca2+ (Ba2+) ions.

Dual role of interfacial phospholipid in phospholipase A2 catalysis.

The results of crosslinking experiments with dimethyl suberimidate and gel filtration binding studies are used to delineate a detailed model for phospholipase A2(phosphatide 2-acyl-hydrolase, EC 3.1.1.4) action in the presence of Ca2+ on mixed micelles of Triton X-100 and phospholipid. Important features of the "dual-phospholipid" model are: (i) the use of the nonionic surfactant as an inert matrix that may influence lipid conformation but does not interact with the enzyme; (ii) the involvement of two lipid molecules in a single cycle of catalysis as an explanation for the "surface dilution" phenomenon; (iii) the requirement of an ordered reaction whereby divalent metal ion binds prior to phospholipid binding; and (iv) the induction by lipid substrate of an asymmetric dimer structure for the enzyme.

Relationship between disturbances of micellar formation and steatorrhea.

In order to clarify the correlation between decrease in micellar formation and degree of steatorrhea, upper intestinal content was analyzed after administering a test meal of Borgström et al. Fecal fat was also measured. It was demonstrated that micellar phase was composed chiefly of fatty acid and monoglyceride, with low concentrations of diglyceride and triglyceride. The fat composition of micellar phase was nearly constant in normal subjects and in cases of malabsorption syndrome. The correlation between the ratio of micellar fat to the whole ingested fat in the intestinal content and daily excretion of fecal fat was very marked.

Ribosome structure and chylomicron formation in rat intestinal mucosa.

Sucrose gradient sedimentation and electron micrographic studies were made on the ribosomes of the cells of intestinal mucosa isolated from control, bile fistula, and puromycin-treated rats. In comparison to controls, there was a 40 to 60 per cent decrease in polysome content of the cells following administration of puromycin or deprivation of luminal choline by creation of a bile fistula. Feeding of lysolecithin of choline to the bile fistula rats or addition to the isolated cells in vitro resulted in a complete restoration of the polysome profile along with lipprotein synthesis and chylomicron release. Addition of lysolecithin to isolated cells treated with puromycin in vitro also brought about a reaggregation of the ribosomes and a reactivation of phospholipid biosynthesis. Choline had no detectable effect on phospholipid synthesis or ribsosme aggregation when fed to ppuromycin-treated rats or when added to puromycin-treated cells in vitro. The results suggest that chylomicron formation and the release by the muscosal cells depend upon intact rough endoplasmic reticulum and an active protein adn phospholipid biosynthesis. The role of lysolecithin in this process is ratonalized on the basis of its ability to supply a precursor of lecithin as well as a surfactant which affects the aggregation, or membrane rebinding of ribosomes, or both.

Effects of anionic surfactants on hamster small intestinal membrane structure and function: relationship to surface activity.

The relationship of the surface properties of a group of anionic surfactants to their effects on intestinal water transport was studied. Dose-response inhibition of water transport in everted hamster jejunal segments was obtained with two long chain detergents (sodium dodecyl sulfate and dioctyl sodium sulfocuccinate), a fatty acid (ricinoleate), and dihydroxy bile salts (deoxycholate, chenodeoxycholate, and taurodeoxycholate), whereas no activity was seen with trihydroxy (cholate, glycocholate, and taurocholate) and tri-keto (dehydrocholate) bile salts. The relative effects on water transport were paralleled by their abilities to lyse the erythrocyte, a membrane model. These two biological effects were related to the surface properties of the agents, as determined by critical micelle concentration and surface tension reduction. We further characterized the action of deoxycholate on hamster small intestine, in vivo. Net water secretion was accompanied by increases in permeability of the mucosa to inulin, dextran, and albumin. These secretory and permeability changes were accompanied by both biochemical and histological alterations: exfoliation (DNA release), membrane effects (sucrase release), and shortened villi. Electron microscopy revealed extensive alteration of the brush border membrane with a decrease in binding of lanthanum and the development of permeability to tracer in villus tip cells. In contrast, taurocholate, which did not alter water transport, did not affect intestinal permeability or the brush border membrane. We believe that the surface properties of anionic surfactants cause changes in absorptive cell membranes which result in intestinal secretion.