Head group-independent interaction of phospholipids with bile salts. A fluorescence and EPR study

Bile salts are essential for phospholipid secretion into the bile. To study the relevance of the structure of phospholipids for their interaction with bile salts, we used spin-labeled or fluorescent phospholipid analogues of different head groups and acyl chain length. Those analogues form micelles in aqueous suspension. Their solubilization by bile salts resulting in the formation of mixed micelles was followed by the decrease of spin-spin interaction of spin-labeled analogues or by the relief of fluorescence self-quenching of (7-nitro-2-1,3-benzooxadiazol (NBD))-labeled analogues. Solubilization of analogue micelles occurred at and above the critical micellar concentration (CMC) of the bile salts. As revealed by stopped-flow technique, solubilization of NBD-analogues was very rapid with half times as low as 0.1 sec above the CMC of taurocholate. Both kinetics and extent of solubilization were independent of the phospholipid head group, but were significantly affected by the fatty acid chain length. Furthermore, using vesicles with varying phospholipid composition and different types of analogues in self-quenching concentrations, we could show that bile salt-mediated vesicle solubilization depended on the fatty acid chain length of phospholipids. In contrast, neither for phospholipids nor for analogues could an influence of the lipid head group on the solubilization process be observed. These findings support a head group-independent mechanism of bile salt-mediated enrichment of specific phospholipids in the bile fluid.     

Micelle formation of sodium chenodeoxycholate and solubilization into the micelles: comparison with other unconjugated bile salts

Micellization of sodium chenodeoxycholate (NaCDC) was studied for the critical micelle concentration (CMC), the micelle aggregation number, and the degree of counterion binding to micelle at 288.2, 298.2, 308.2, and 318.2 K. They were compared with those of three other unconjugated bile salts; sodium cholate (NaC), sodium deoxycholate (NaDC), and sodium ursodeoxycholate (NaUDC). The I(1)/I(3) ratio of pyrene fluorescence and the solubility dependence of solution pH were employed to determine the CMC values. As the results, a certain concentration range for the CMC and a stepwise molecular aggregation for micellization were found reasonable. Using a stepwise association model of the bile salt anions, the mean aggregation number (n) of NaCDC micelles was found to increase with the total anion concentration, while the n values decreased with increasing temperature; 9.1, 8.1, 7.4, and 6.3 at 288.2, 298.2, 308.2, and 318.2 K, respectively, at 50 mmol dm(-3). The results from four unconjugated bile salts indicate that the number, location, and orientation of hydroxyl groups in the steroid nucleus are quite important for growth of the micelles. Activity of the counterion (Na(+)) was determined by a sodium ion selective electrode in order to confirm the low counterion binding to micelles. The solubilized amount of cholesterol into the aqueous bile salt solutions increased in the order of NaUDC相似文献   

Surface and solution properties of steroid antibiotics: 3-acetoxylfusidic acid, cephalosporin P1 and helvolic acid.

The colloid/chemical properties of the fusidane antibiotics, 3-acetoxylfusidic acid, cephalosporin P1, and helvolic acid, and their sodium salts, were investigated. The sodium salts of 3-acetoxylfusidic acid and cephalosporin P1 were found to be detergent-like molecules with micellar properties comparable to the parent compound sodium fusidate and the bile salt sodium cholate. Critical micellar temperatures (cmt) were less than 0 degrees C except for sodium helvolate which being sparingly soluble did not form micelles between 0 and 50 degrees C. Potentiometric titrations of dilute solutions gave apparent pK values (5.2-6.5) in the range expected for carboxylated steroid detergents. The apparent pK values increased significantly once the detergent concentration exceeded the critical micellar concentration (cmc). Micellar properties were determined by surface tension, titration with a water-soluble dye (Rhodamine 6G), light scattering, and solubilization of lecithin and cholesterol. Cmc's, in the range of 1.5 to 5.6 mM, were found which varied slightly depending on the method employed and in all cases fell slightly in the presence of added NaCl. The number of monomers per micelle (aggregation number) in concentrations well above the cmc was extrapolated from Debye light scattering plots in 0.15 M NaCl. The values varied from 6 for fusidate to 14 for 3-acetoxylfusidate with sodium cephalosporin P1 having an intermediate value. Each detergent readily solubilized the phospholipid lecithin.     

Solubilization of multilamellar liposomes of egg yolk lecithin by the bile salt sodiumtaurodeoxycholate and the effect of cholesterol--a rapid-ultrafiltration study

The solubilization of multilamellar egg yolk lecithin liposomes by sodiumtaurodeoxycholate in aqueous phase was studied by ultrafiltration as a function of time, bile salt and cholesterol concentration. The corresponding equilibrium states were analysed. Complete solubilization was achieved at total bile salt/lecithin molar mixing ratios of approximately 5. The minimum ratio to start solubilization was 0.1, corresponding to a free bile salt concentration of only 5% of the critical micelle concentration (CMC). Mean equilibrium constants for the partition of bile salts between non-filterable aggregates and filterable mixed micelles and also the free bile salt concentration were determined. Sodiumtaurodeoxycholate had a higher affinity for small mixed micelles than for lamellar mixed aggregates especially in the presence of cholesterol, which reduces the degree and rate of the solubilization process. A non-homogeneous distribution of bile salts in the lipid phase was detected at low bile salt concentrations.     

Solubilization of lipids from hamster bile-canalicular and contiguous membranes and from human erythrocyte membranes by conjugated bile salts.

We have demonstrated in vitro the efficacy of the taurine-conjugated dihydroxy bile salts deoxycholate and chenodeoxycholate in solubilizing both cholesterol and phospholipid from hamster liver bile-canalicular and contiguous membranes and from human erythrocyte membrane. On the other hand, the dihydroxy bile salt ursodeoxycholate and the trihydroxy bile salt cholate solubilize much less lipid. The lipid solubilization by the four bile salts correlated well with their hydrophobicity: glycochenodeoxycolate, which is more hydrophobic than the tauro derivative, also solubilized more lipid. All the dihydroxy bile salts have a threshold concentration above which lipid solubilization increases rapidly; this correlates approximately with the critical micellar concentration. The non-micelle-forming bile salt dehydrocholate solubilized no lipid at all up to 32 mM. All the dihydroxy bile acids are much more efficient at solubilizing phospholipid than cholesterol. Cholate does not show such a pronounced discrimination. Lipid solubilization by chenodeoxycholate was essentially complete within 1 min, whereas that by cholate was linear up to 5 min. Maximal lipid solubilization with chenodeoxycholate occurred at 8-12 mM; solubilization by cholate was linear up to 32 mM. Ursodeoxycholate was the only dihydroxy bile salt which was able to solubilize phospholipid (although not cholesterol) below the critical micellar concentration. This similarity between cholate and ursodeoxycholate may reflect their ability to form a more extensive liquid-crystal system. Membrane specificity was demonstrated only inasmuch as the lower the cholesterol/phospholipid ratio in the membrane, the greater the fractional solubilization of cholesterol by bile salts, i.e. the total amount of cholesterol solubilized depended only on the bile-salt concentration. On the other hand, the total amount of phospholipid solubilized decreased with increasing cholesterol/phospholipid ratio in the membrane.     

Spectrophotometric determination of the critical micellar concentration of bile salts using bilirubin monoglucuronide as a micellar probe. Utility of derivative spectroscopy.

We have developed a simple biologically non-invasive method for determining the critical micellar concentration (CMC) of bile salts using pure naturally occurring bilirubin IX alpha monoglucuronide (BMG), an important bile pigment present in virtually all mammalian biles. This methodology employs visible absorbance spectroscopy of BMG in bile salts over a range of bile salt concentrations that include the reported CMC. Using 100 microM-BMG in 0.4 M-imidazole buffer at pH 7.8, we calculated that the CMC for sodium taurochenodeoxycholate is between 2.5 and 3.0 mM based on: (1) an abrupt change in lambda max. in this concentration range, (2) a precipitous decrease in the amplitude of the absorbance shoulder at 450 nm, (3) a sudden decrease in the second derivative absorbance of BMG at 400 nm and an increase in absorbance at 470 nm, (4) a sharp change in the 4th derivative absorbance at 375 and 395 nm. In contrast, sodium taurocholate, a bile salt that reportedly does not have a CMC but continuously self-associates over a wide concentration range, exhibited none of these changes. The use of derivative spectroscopy enhances the ability to detect the CMC changes and also indicates the number of BMG species in solution and their relative energy states.     

Micelle formation of sodium deoxycholate and sodium ursodeoxycholate (part 1)

Micellization of sodium deoxycholate (NaDC) and sodium ursodeoxycholate (NaUDC) was studied for the critical micelle concentration (CMC), the micelle aggregation number, and the degree of counterion binding to micelle, where sodium cholate (NaC) was used as a reference. The fluorescence probe technique of pyrene was employed to determine accurately the CMC values for the bile salts, which indicated that a certain concentration range of CMC and a stepwise aggregation for micellization were reasonable. The temperature dependences of micellization for NaDC and NaUDC were studied at 288.2, 298.2, 308.2, and 318.2 K by aqueous solubility change with solution pH. Aggregations of the bile salt anions were analyzed using the stepwise association model and found to grow in size with increasing concentration, which confirmed that the mass action model worked quite well. The average aggregation number was found to be 2.5 (NaUDC) and 10.5 (NaDC) at the concentration of 20 mM and at 308.2 K. The aggregation number determined by static light scattering also agreed well with those by the solubility method in the order of size: NaUDC相似文献   

Surface behaviour of bile salts and tetrahydrolipstatin at air/water and oil/water interfaces.

The surface behaviour of two bile salts, sodium deoxycholate (NaDC) and sodium taurodeoxycholate (NaTDC), as well as that of tetrahydrolipstatin (THL), a potent gastrointestinal lipase inhibitor, was studied at air/water and oil/water interfaces, using interfacial tensiometry methods. The surface behaviour of NaDC and NaTDC was comparable at both oil/water and air/water interfaces. A fairly compact interfacial monolayer of bile salts is formed well below the critical micellar concentration (CMC) and can help to explain the well-known effects of bile salts on the kinetic behaviour of pancreatic lipases. Using the Wilhelmy plate technique, the surface pressure-molecular area curves recorded with THL at the air/water interface showed a collapse point at a surface pressure of 24.5 mN.m(-1), corresponding to a molecular area of 70 A(2). Surprisingly, using the oil drop method, a limiting molecular area of 160 A(2) was found to exist at the oil/water interface. On the basis of the above data, space-filling models were proposed for bile salts and THL at air/water and oil/water interfaces.     

Physicochemical and physiological properties of 5alpha-cyprinol sulfate,the toxic bile salt of cyprinid fish

5alpha-Cyprinol sulfate was isolated from bile of the Asiatic carp, Cyprinus carpio. 5alpha-Cyprinol sulfate was surface active and formed micelles; its critical micellization concentration (CMC) in 0.15 M Na+ using the maximum bubble pressure device was 1.5 mM; by dye solubilization, its CMC was approximately 4 mM. At concentrations >1 mM, 5alpha-cyprinol sulfate solubilized monooleylglycerol efficiently (2.1 molecules per mol micellar bile salt). When infused intravenously into the anesthetized rat, 5alpha-cyprinol sulfate was hemolytic, cholestatic, and toxic. In the isolated rat liver, it underwent little biotransformation and was poorly transported (Tmax congruent with 0.5 micromol/min/kg) as compared with taurocholate. 5alpha-Cyprinol, its bile alcohol moiety, was oxidized to its corresponding C27 bile acid and to allocholic acid (the latter was then conjugated with taurine); these metabolites were efficiently transported. 5alpha-Cyprinol sulfate inhibited taurocholate uptake in COS-7 cells transfected with rat asbt, the apical bile salt transporter of the ileal enterocyte. 5alpha-Cyprinol had limited aqueous solubility (0.3 mM) and was poorly absorbed from the perfused rat jejunum or ileum. Sampling of carp intestinal content indicated that 5alpha-cyprinol sulfate was present at micellar concentrations, and that it did not undergo hydrolysis during intestinal transit. These studies indicate that 5alpha-cyprinol sulfate is an excellent digestive detergent and suggest that a micellar phase is present during digestion in cyprinid fish.     

Role of 57-72 loop in the allosteric action of bile salts on pancreatic IB phospholipase A(2): regulation of fat and cholesterol homeostasis

Mono- and biphasic kinetic effects of bile salts on the pancreatic IB phospholipase A2 (PLA2) catalyzed interfacial hydrolysis are characterized. This novel phenomenon is modeled as allosteric action of bile salts with PLA2 at the interface. The results and controls also show that these kinetic effects are not due to surface dilution or solubilization or disruption of the bilayer interface where in the mixed-micelles substrate replenishment becomes the rate-limiting step. The PLA2-catalyzed rate of hydrolysis of zwitterionic dimyristoylphosphatidylcholine (DMPC) vesicles depends on the concentration and structure of the bile salt. The sigmoidal rate increase with cholate saturates at 0.06 mole fraction and changes little at the higher mole fractions. Also, with the rate-lowering bile salts (B), such as taurochenodeoxycholate (TCDOC), the initial sigmoidal rate increase at lower mole fraction is followed by nearly complete reversal to the rate at the pre-activation level at higher mole fractions. The rate-lowering effect of TCDOC is not observed with the (62-66)-loop deleted DeltaPLA2, or with the Naja venom PLA2 that is evolutionarily devoid of the loop. The rate increase is modeled with the assumption that the binding of PLA2 to DMPC interface is cooperatively promoted by bile salt followed by allosteric k(cat)(*)-activation of the bound enzyme by the anionic interface. The rate-lowering effect of bile salts is attributed to the formation of a specific catalytically inert E(*)B complex in the interface, which is noticeably different than the 1:1 EB complex in the aqueous phase. The cholate-activated rate of hydrolysis is lowered by hypolidemic ezetimibe and guggul extract which are not interfacial competitive inhibitors of PLA2. We propose that the biphasic modulation of the pancreatic PLA2 activity by bile salts regulates gastrointestinal fat metabolism and cholesterol homeostasis.     

Bile acid solubility and precipitation in vitro and in vivo: the role of conjugation, pH, and Ca2+ ions.

The principles governing the in vitro solubility of the common natural conjugated and unconjugated bile acids and salts in relation to pH, micelle formation, and Ca2+ concentration are considered from a theoretical standpoint and then correlated first with experimental observations on model systems and second with the formation of precipitates containing bile acids in health and disease. In vitro, taurine-conjugated bile acids are soluble at strongly acidic pH; glycine-conjugated bile acids are poorly soluble at moderately acidic pH; and many of the common, natural unconjugated bile acids are insoluble at neutral pH. For both glycine-conjugated and unconjugated bile acids, solubility rises exponentially, with increasing pH, until the concentration of the anion reaches the critical micellization concentration (CMC) when micelle formation occurs and solubility becomes practically unlimited. In vivo, in health, conjugated bile acids are present in micellar form in the biliary and intestinal tract. Unconjugated bile acids formed in the large intestine remain at low monomeric concentrations because of the acidic pH of the proximal colon, binding to bacteria, and absorption across the intestinal mucosa. In diseases in which proximal small intestinal content is abnormally acidic, precipitation of glycine-conjugated bile acids (in protonated form) occurs. Increased bacterial formation of unconjugated bile acids occurs with stasis in the biliary tract and small intestine; in the intestine, unconjugated bile acids precipitate in the protonated form. If the precipitates aggregate, an enterolith may be formed. In vitro, the calcium salts of taurine conjugates are highly water soluble, whereas the calcium salts of glycine conjugates and unconjugated bile acids possess limited aqueous solubility that is strongly influenced by bile acid structure. Precipitation occurs extremely slowly from supersaturated solutions of glycine-conjugated bile acids because of metastability, whereas super-saturated solutions of unconjugated bile acids rapidly form precipitates of the calcium salt. In systems containing Ca2+ ions and unconjugated bile acids, pH is important, since it is the key determinant of the anion concentration. For bile acids with relatively soluble calcium salts (or with a low CMC), the concentration of the anion will reach the CMC and micelles will form, thus precluding formation of the insoluble calcium salt. For bile acids, with relatively insoluble calcium salts (or with a high CMC), the effect of increasing pH is to cause the anion to reach the solubility product of the calcium salt before reaching the CMC so that precipitation of the calcium salt occurs instead of micelle formation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Partition coefficients of beta-blockers in bile salt/lecithin micelles as a tool to assess the role of mixed micelles in gastrointestinal absorption

The objective of this study was to develop non-invasive spectroscopic methods to quantify the partition coefficients of two beta-blockers, atenolol and nadolol, in aqueous solutions of bile salt micelles and to assess the effect of lecithin on the partition coefficients of amphiphilic drugs in mixed bile salt/lecithin micelles, which were used as a simple model for the naturally occurring mixed micelles in the gastrointestinal tract. The partition coefficients (Kp) at 25.0 +/- 0.1degreesC and at 0.1 M NaCl ionic strength were determined by spectrofluorimetry and by derivative spectrophotometry, by fitting equations that relate molar extinction coefficients and relative fluorescence intensities to the partition constant Kp. Drug partition was controlled by the: (i) drug properties, with the more soluble drug in water (atenolol) exhibiting smaller values of Kp, and with both drugs interacting more extensively in the protonated form; and by (ii) the bile salt monomers, with the dihydroxylic salts producing larger values of Kp for the beta-blockers, and with glycine conjugation of the bile acid increasing the values of Kp for the beta-blockers. Addition of lecithin to bile salt micelles decreases the values of Kp of the beta-blockers. Mixed micelles incorporate hydrophobic compounds due to their large size and the fluidity of their core, but amphiphilic drugs, for which the interactions are predominantly polar/electrostatic, are poorly incorporated in mixed micelles of bile salts/lecithin.     

Site-directed mutagenesis of the distal basic cluster of pancreatic bile salt-dependent lipase

Previous studies have postulated the presence of two bile salt-binding sites regulating the activity of the pancreatic bile salt-dependent lipase. One of these sites, located in an N-terminal basic cluster, has been identified as the specific bile salt-binding site. Interaction of primary bile salts with this proximal site induces the formation of a micellar binding site from a pre-existing nonspecific or pre-micellar bile salt-binding site. Here we have investigated the functional significance of another basic cluster comprised of amino acid residues Arg(423), Lys(429), Arg(454), Arg(458), and Lys(462), distal from the catalytic site. For this purpose these residues were mutagenized in Ile or Ala residues. The mutagenized enzyme lost activity on both soluble and emulsified substrates in the presence of bile salts. However, in the absence of bile salts, the mutagenized enzyme displayed the same activity on soluble substrate as the wild-type recombinant enzyme. Consequently, the distal basic cluster may represent the nonspecific (or pre-micellar) bile salt-binding site susceptible to accommodate primary and secondary bile salts. According to the literature, tyrosine residue(s) should participate in this site. Therefore, two tyrosine residues, Tyr(427) and Tyr(453), associated with the distal basic cluster were also mutagenized. Each tyrosine substitution to serine did not inhibit the enzyme activity on soluble substrate, independently of the presence of primary or secondary bile salts. However, the enzyme activity on cholesteryl oleate solubilized in primary bile salt micelles was decreased by mutations substantiating that these residues are part of the nonspecific bile salt-binding site.     

Accurate separation of vesicles, micelles and cholesterol crystals in supersaturated model biles by ultracentrifugation, ultrafiltration and dialysis.

Gel filtration with bile salts at intermixed micellar/vesicular concentrations (IMC) in the eluant has been proposed to isolate vesicles and micelles from supersaturated model biles, but the presence of vesicular aggregates makes this method unreliable. We have now validated a new method for isolation of various phases. First, aggregated vesicles and - if present - cholesterol crystals are pelleted by short ultracentrifugation. Cholesterol contained in crystals and vesicular aggregates can be quantitated from the difference of cholesterol contents in the pellets before and after bile salt-induced solubilization of the vesicular aggregates. Micelles are then isolated by ultrafiltration of the supernatant through a highly selective 300 kDa filter and unilamellar vesicles by dialysis against buffer containing bile salts at IMC values. Lipids contained in unilamellar vesicles are also estimated by subtraction of lipid contents in filtered micelles from lipid contents in (unilamellar vesicle+micelle containing) supernatant ('subtraction method'). 'Ultrafiltration-dialysis' and 'subtraction' methods yielded identical lipid solubilization in unilamellar vesicles and identical vesicular cholesterol/phospholipid ratios. In contrast, gel filtration yielded much more lipids in micelles and less in unilamellar vesicles, with much higher vesicular cholesterol/phospholipid ratios. When vesicles obtained by dialysis were analyzed by gel filtration, vesicular cholesterol/phospholipid ratios increased strongly, despite correct IMC values for bile salts in the eluant. Subsequent extraction of column material showed significant amounts of lipids. In conclusion, gel filtration may underestimate vesicular lipids and overestimate vesicular cholesterol/phospholipid ratios, supposedly because of lipids remaining attached to the column. Combined ultracentrifugation-ultrafiltration-dialysis should be considered state-of-the-art methodology for quantification of cholesterol carriers in model biles.     

Transfer of orlistat through oil-water interfaces

The transfer of radiolabelled orlistat ([14C]orlistat), a potent gastrointestinal lipase inhibitor, through an oil-water interface from a single oil droplet to an aqueous phase was investigated, using an oil drop tensiometer. The absolute transfer fluxes were found to be very low, even in the presence of micellar concentrations of bile salts, which increased their values from 0.2 to 2.5 and 6.5 pmol cm(-2) min(-1) in the presence of 0, 4 and 15 mM NaTDC, respectively. Adding either a lipid emulsion or pure human pancreatic lipase (HPL) or human serum albumin or beta-lactoglobulin had no effect on the flux of transfer of orlistat. The presence of colipase or a mixture of colipase and HPL was found, however, to reduce the flux of orlistat transfer, probably because it partly covered the single oil drop surface, even in the presence of bile salts. Using a finely emulsified system, we investigated the partitioning of orlistat between the aqueous and oil phases, in the absence or presence of bile salts above their CMC (4 mM NaTDC, final concentration). Under these emulsified conditions, orlistat was found to be mostly associated with the oil phase, since more than 98.8% of the total radioactivity was recovered after decantation with the oil phase. The low transfer rates of orlistat, as well as its partitioning coefficient between the oil and the aqueous phases, should help us to better understand the inhibitory effects of orlistat on lipid digestion in humans.     

Role of 57-72 loop in the allosteric action of bile salts on pancreatic IB phospholipase A2: Regulation of fat and cholesterol homeostasis

Mono- and biphasic kinetic effects of bile salts on the pancreatic IB phospholipase A2 (PLA2) catalyzed interfacial hydrolysis are characterized. This novel phenomenon is modeled as allosteric action of bile salts with PLA2 at the interface. The results and controls also show that these kinetic effects are not due to surface dilution or solubilization or disruption of the bilayer interface where in the mixed-micelles substrate replenishment becomes the rate-limiting step. The PLA2-catalyzed rate of hydrolysis of zwitterionic dimyristoylphosphatidylcholine (DMPC) vesicles depends on the concentration and structure of the bile salt. The sigmoidal rate increase with cholate saturates at 0.06 mole fraction and changes little at the higher mole fractions. Also, with the rate-lowering bile salts (B), such as taurochenodeoxycholate (TCDOC), the initial sigmoidal rate increase at lower mole fraction is followed by nearly complete reversal to the rate at the pre-activation level at higher mole fractions. The rate-lowering effect of TCDOC is not observed with the (62-66)-loop deleted ΔPLA2, or with the Naja venom PLA2 that is evolutionarily devoid of the loop. The rate increase is modeled with the assumption that the binding of PLA2 to DMPC interface is cooperatively promoted by bile salt followed by allosteric k_cat^?-activation of the bound enzyme by the anionic interface. The rate-lowering effect of bile salts is attributed to the formation of a specific catalytically inert E^?B complex in the interface, which is noticeably different than the 1:1 EB complex in the aqueous phase. The cholate-activated rate of hydrolysis is lowered by hypolidemic ezetimibe and guggul extract which are not interfacial competitive inhibitors of PLA2. We propose that the biphasic modulation of the pancreatic PLA2 activity by bile salts regulates gastrointestinal fat metabolism and cholesterol homeostasis.     

Effects of nonionic surfactants on the solubilization and mineralization of phenanthrene in soil-water systems

The solubilization and mineralization of (14)C-phenanthrene in soil-water systems was examined with several commercially available surface-active agents, viz., an alkyl ethoxylate C(12)E(4); two alkylphenol ethoxylate surfactants: C(8)PE(9.5) and C(9)PE(10.5); two sorbitan ethoxylate surfactants: the sorbitan monolaurate (Tween 20) and the sorbitan monooleate (Tween 80); two pairs of nonionic ethoxylate surfactant mixtures: C(12)E(4)/C(12)E(23) at a 1:1 ratio, and C(12-15)E(3)/C(12-15)E(9) at a 1:3 ratio; and two surfactants possessing relatively high critical micelle concentration (CMC) values and low aggregation numbers: CHAPS and octyglucoside. Surface tension experiments were performed to evaluate surfactant sorption onto soil and the surfactant doses required to attain the CMC in the soil-water systems. Surfactant solubilization of (14)C-phenanthrene commenced with the onset of micellization. The addition of surface-active agents was observed not to be beneficial to the microbial mineralization of phenanthrene in the soil-water systems and, for supra-CMC surfactant doses, phenanthrene mineralization was completely inhibited for all the surfactants tested. A comparison of solubilization, surface tension, and mineralization data confirms that the inhibitory effect on microbial degradation of phenanthrene is related to the CMC of the surfactant in the presence of soil. Additional tests demonstrated the recovery of mineralization upon dilution of surfactant concentration to sub-CMC levels, and a relatively high exit rate for phenanthrene from micelles. These tests suggest that the inhibitory effect is probably related to a reversible physiological surfactant micelle-bacteria interaction, possibly through partial complexing or release of membrane material with disrupting membrane lamellar structure. This study indicates that nonionic surfactant solubilization of sorbed hydrophobic organic compounds from soil may not be beneficial for the concomitant enhancement of soil bioremediation. Additional work is needed to address physicochemical processes for bioavailability enhancement, and effects of solubilizing agents on microorganisms for remediation and treatment of hydrophobic organic compounds and nonaqueous phase liquids. (c) 1992 John Wiley & Sons Inc.     

Stability of mixed micellar bile models supersaturated with cholesterol.

The maximal equilibrium solubility of cholesterol in mixtures of phosphatidylcholine (PC)1 and bile salts depends on the cholesterol/PC ratio (Rc) and on the effective ratio (Re) between nonmonomeric bile salts and the sum (CT) of PC and cholesterol concentrations (Carey and Small, 1978; Lichtenberg et al., 1984). By contrast, the concentration of bile salts required for solubilization of liposomes made of PC and cholesterol does not depend on Rc (Lichtenberg et al., 1984 and 1988). Thus, for Rc greater than 0.4, solubilization of the PC-cholesterol liposomes yields PC-cholesterol-bile salts mixed micellar systems which are supersaturated with cholesterol. In these metastable systems, the mixed micelles spontaneously undergo partial revesiculation followed by crystallization of cholesterol. The rate of the latter processes depends upon Rc, Re, and CT. For any given Rc and Re, the rate of revesiculation increases dramatically with increasing the lipid concentration CT, reflecting the involvement of many mixed micelles in the formation of each vesicle. The rate also increases, for any given CT and Re, upon increasing the cholesterol to PC ratio, Rc, probably due to the increasing degree of supersaturation. Increasing the cholate to lipid effective ratio, Re, by elevation of cholate concentration at constant Rc and CT has a complex effect on the rate of the revesiculation process. As expected, cholate concentration higher than that required for complete solubilization at equilibrium yields stable mixed micellar systems which do not undergo revesiculation, but for lower cholate concentrations decreasing the degree of supersaturation (by increasing [cholate]) results in faster revesiculation. We interpret these results in terms of the structure of the mixed micelles; micelles with two or more PC molecules per one molecule of cholesterol are relatively stable but increasing the bile salt concentration may cause dissociation of such 1:2 cholesterol:PC complexes, hence reducing the stability of the mixed micellar dispersions. The instability of PC-cholesterol-cholate mixed systems with intermediary range of cholate to lipids ratio may be significant to gallbladder stone formation as: (a) biliary bile contains PC-cholesterol vesicles which may be, at least partially, solubilized by bile salts during the process of bile concentration in the gallbladder, resulting in mixtures similar to our model systems; and (b) the bile composition of cholesterol gallstone patients is within an intermediary range of bile salts to lipids ratio.     

Nonspecific high affinity binding of bile salts to carboxylester lipases

The interactions with bile salts of carboxylester lipases (EC 3.1.1.13) from human pancreatic juice and pig pancreas were characterized by physical methods. Bile salts cause a decrease in the fluorescence intensity of the proteins at the emission maximum of 333-335 nm. The concentration dependence of this decrease shows saturation behavior, is relatively nonspecific with respect to bile salt conjugation or the presence of the 7 alpha-hydroxyl group, and is consistent with a 1:1 interaction between enzyme and bile salt. Direct measurement of the binding of [3H]cholate by equilibrium dialysis supports the stoichiometry. Other detergents also bind, causing fluorescence changes, but with much lower affinities. Binding of taurocholate to the monomeric pig enzyme is enhanced by increasing ionic strength, indicating the predominance of hydrophobic interactions. In the range of pH 5.5-6.8, binding is pH-independent with dissociation constants of 3-20 microM. At higher pH, affinity is greatly reduced and the fluorescence spectrum changes, indicating the importance of a protonated group for efficient interaction. Occupancy of the bile salt binding site partially stabilizes the enzyme against inactivation by heat but not trypsin. However, circular dichroism spectra do not indicate that bile salt binding is accompanied by any change in secondary structure. The monomeric pig enzyme binds to the argon/water interface in the presence of bile salts and binding of taurocholate to diisopropylphosphoryl-enzyme is similar to that measured with native enzyme. These results suggest that surface binding and catalysis occur at sites distinct from the bile salt binding site of the enzyme. Stabilization of the monomeric pig enzyme against denaturation at high energy surfaces occurs concomitantly with occupancy of the bile salt binding site. Overall, the data suggest that an important role of bile salts in vivo is to stabilize these enzymes at lipid-water interfaces.     

Relationships between bile salts hydrophilicity and phospholipid composition in bile of various animal species

Bile salts and phospholipids from bile of chicken, dog, sheep, rat, ox, pig, guinea-pig and man were analyzed by high-performance liquid chromatography. Bile salts showed marked differences in their hydrophilic properties, owing to hydroxyl structure and type of conjugation. Phospholipids were generally similar, containing 90-95% of phosphatidylcholine which was made of molecular species containing palmitic acid in the sn-1 position. The comparative analysis of bile salts and phosphatidylcholines profile demonstrated that bile salts hydrophilicity influences the quantity of phosphatidylcholine in bile but not the quality.