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Lecithin Protects against Plasma Membrane Disruption by Bile Salts
JOURNAL OF SURGICAL RESEARCH ARTICLE NO.
78, 131–136 (1998)
JR985364
Lecithin Protects against Plasma Membrane Disruption by Bile Salts P. K. Narain, M.D., E. J. DeMaria, M.D., and D. M. Heuman, M.D. Departments of Surgery and Medicine, Medical College of Virginia of Virginia Commonwealth University, Richmond, Virginia and McGuire Department of Veterans Affairs Medical Center, Richmond, Virginia 23298-0711 Presented at the Annual Meeting of the Association for Academic Surgery, Dallas, Texas, November 6 – 8, 1997
Introduction. Detergent disruption of epithelial plasma membranes by bile salts may contribute to pathogenesis of cholestasis and gastroesophageal reflux disease. Bile, despite containing high concentrations of bile salts, normally is not toxic to biliary or intestinal epithelia. We hypothesize that lecithin in bile may protect cell membranes from disruption by bile salts. Methods. We studied the interactions of taurine conjugates of ursodeoxycholate (TUDCA), cholate (TCA), chenodeoxycholate (TCDCA), and deoxycholate (TDCA) with erythrocyte plasma membranes with or without large unilamellar egg lecithin vesicles for various times at 23°C. Release of hemoglobin was quantified spectrophotometrically. The concentration of bile salt monomers and simple micelles in the intermixed micellar aqueous phase (IMMC) was determined by centrifugal ultrafiltration. Results. The degree of hemolysis depended on the hydrophobicity of the bile salts and was progressive over time. Addition of lecithin reduced the hemolytic effects of 20 mM TCA or 2 mM TDCA in a concentrationdependent manner at both 30 min and 4 h. Increasing the concentration of lecithin progressively reduced the IMMC of TDCA. Hemolysis following addition of lecithin to 2 mM TDCA was comparable to hemolysis produced by lecithin-free TDCA solutions when diluted to similar IMMC values. Conclusion. We conclude that lecithin reduces plasma membrane disruption by hydrophobic bile salts. This protection may be attributable to association of bile salts with vesicles and mixed micelles, reducing the concentration of bile salt monomers and simple micelles available to interact with cell membranes. Lecithin may play a key role in preventing bile salt injury of biliary and gastrointestinal epithelia. © 1998 Academic Press Key Words: lecithin; phospholipid; phosphatidylcholine; bile salt; toxicity; erythrocyte; membrane.
INTRODUCTION
Bile salts are organic detergents, formed in the liver by the oxidation of cholesterol [1]. Their detergency derives from their amphiphilic structure, with a hydrophobic hydrocarbon surface and a hydrophilic surface
produced by polar hydroxyl and carboxy groups. Different bile salts vary in their hydrophilic– hydrophobic balance, and detergency tends to increase with increasing relative hydrophobicity [2]. Bile salts are present in bile in high concentrations and are critically important for the absorption of lipids from the intestine. Because they also can solubilize the lipids that make up cell membranes, bile salts are potentially toxic. Bile salt toxicity has been implicated in pathogenesis of cholestatic liver disease, gastroesophageal reflux disease, cholecystitis, pancreatitis, and colon cancer [3– 6]. Bile salts normally do not damage biliary membranes, despite their presence in bile at high concentrations. Recent data suggest that the explanation for this may lie with the other major biliary lipids, such as lecithin (phosphatidylcholine) and cholesterol, that are secreted by the hepatic canaliculus as large unilamellar vesicles [7]. A protective effect of biliary vesicles is suggested by the mdr2 knockout mouse model, in which a key phospholipid transport protein required for vesicle assembly is lacking and biliary lecithin secretion is negligible [8]. The mdr2 knockout mouse develops severe progressive cholestatic liver disease that is aggravated by feeding of hydrophobic bile salts [9, 10]. Interaction of bile salts with vesicles in bile is complex. At low concentrations, bile salts are present as monomers. Above a critical micellar concentration they self-aggregate to form simple micelles. Bile salt monomers also partition into lipid bilayers such as vesicles or membranes. A phase transition occurs when the vesicle becomes saturated with bile salt, leading to formation of rod-like mixed micelles [11]. The concentration of bile salts in monomers and simple micelles, also termed the intervesicular, intermixed micellar concentration (IMMC),1 drives the concentration of bile salts in lipid bilayers and their transition into mixed micelles [12]. We hypothesize that biliary lecithin, which constitutes over 95% of the biliary phospholipids, may be of 1 Abbreviations used: TUDCA, tauroursodeoxycholic acid; TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; TDCA, taurodeoxycholic acid; IMMC, intermixed micellar intervesicular bile salt concentration.
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key importance in protecting cells against disruption by bile salts, and this protective effect of lecithin may derive from the partitioning of bile salts into vesicles and mixed micelles, thereby reducing the bile salt IMMC. This is the first study to address the role of the IMMC in bile salt-induced cytotoxicity. As a model plasma membrane system, we chose isolated human erythrocytes. Erythrocytes lack nuclei, do not synthesize proteins, have a relatively simple metabolic profile, and do not actively transport bile salts. Previous studies have shown that erythrocytes can be used as a general model system to study disruptive interactions of surfactants with plasma membranes [13–15].
filtration using centricon 10 cartridges (Amicon, Beverly, MA). Ultrafiltration allows free passage of monomers and simple micelles, but completely retains bile salts associated with lecithin in larger physical aggregates such as mixed micelles and vesicles. Bile salt binding to nonfiltrable lecithin-containing particles was quantified by comparing the concentration of the unbound radiolabeled bile salts in the filtrate to the total concentration of radioactivity in the unfiltered sample. After incubation of red cells, vesicles and radiolabeled bile salts ([3H]TCA or [3H]TDCA) for 30 min and 4 h, 1-ml aliquots were subjected to gentle centrifugation (1000g) for 10 min in the centricon 10 tubes. Radioactivity in 0.1 ml of the prefiltration sample and 0.1 ml of the filtrate was determined on a liquid scintillation counter (Beckman LS 6000IC, Fullerton, CA). Correction for Gibbs–Donnan effects was not required as these are negligible at the low bound bile salt concentrations used in these studies [12].
Statistical Analysis MATERIALS AND METHODS
Materials Egg yolk lecithin (.99% phosphatidylcholine) was purchased from Avanti Polar Lipids (Alabaster, AL). Sodium salts of the taurine conjugates of cholic (TCA), deoxycholic (TDCA), chenodeoxycholic (TCDCA), and ursodeoxycholic acids (TUDCA) were purchased from Calbiochem (La Jolla, CA). [3H]Taurocholate was purchased from NEN DuPont (Boston, MA). [3H]- and [14C]taurine conjugates of deoxycholate were synthesized using a modification of the method of Tserng et al., [16] and purified by thin-layer chromatography. Preparation of vesicles. Egg yolk lecithin (average mol wt 760.09) was dissolved in chloroform and large unilamellar vesicles of 100 nm mean diameter (comparable in size to biliary vesicles) were prepared by the method of Hope et al. [17]. Briefly, the lipids were dried under nitrogen, dissolved in warm terbutanol, frozen at 270°C, and lyophilized overnight. They were then suspended in Tris-buffered saline, vortexed vigorously for 20 min, followed by five freeze and thaw cycles using liquid nitrogen, and extruded under nitrogen (100 to 400 psi) through paired 0.1-mm polycarbonate filters (Poretics Corp. Livermore, CA) using a Thermobarrel extruder (Lipex Biomembranes, Vancouver, BC). The extruded vesicles were stored under nitrogen at 4°C and used within a week. Vesicles formed this way are uniform, large, and unilamellar. They have a uniform surface available for surface interaction, and they do not show the strain anomalies related to curvature, typically seen in small vesicles prepared by sonication. Preparation of human erythrocytes. Human erythrocytes were collected fresh from a single volunteer (P.K.N.). Blood collected in ethylenediaminetetraacetic acid (EDTA) tubes was spun at 1000g (IEC Model CU 5000, Needham Heights, MA) at 23°C for 10 min. Discarding the supernatant and the buffy coat, the red blood cells were washed three times with Tris-buffered saline (0.14 M NaCl, 0.01 M Tris–HCl, pH 7.4). The washed red blood cells were reconstituted to a hematocrit of 20% (v/v) in this buffer, stored at 4°C, and used within a week for hemolysis studies.
Experiment Design Freshly prepared erythrocytes were used for all the experiments at a final hematocrit of 4%. Red cells were incubated in 0.14 M saline with 0.01 M Tris (pH 7.4), along with increasing concentrations of taurocholic (TCA), taurodeoxycholic (TDCA), taurochenodeoxycholic (TCDCA), and tauroursodeoxycholic acid (TUDCA) for up to 24 h at 23°C. To assess cell disruption (release of hemoglobin), we precipitated erythrocytes by rapid centrifugation (1000g for 10 min) and determined optical density of the supernatant at 540 nm on a Beckman DU 50 spectrophotometer (Fullerton, CA). Percentage hemolysis was calculated by comparing the measured absorption with that of an equal volume of erythrocytes lysed in distilled water. In some studies, following addition of lecithin to bile salt solutions, the concentration of bile salts as monomers and simple micelles in the intermixed micellar aqueous phase (termed the IMMC) was quantified as described by Donovan et al. [12] by centrifugal ultra-
All experiments were run in quadruplicate. Comparisons between two groups were made with Student’s paired t test and a P value of 0.05 or less was taken as significant. For sigmoidal curve fitting we employed Sigmaplot 4.0 for Windows 95 (Jandel Scientific, San Rafael, CA).
RESULTS
In initial studies, the extent of hemolysis was assessed as a function of time and bile salt concentration. Figure 1 depicts the relative toxicity of TUDCA, TCA, TCDCA, and TDCA. TUDCA, a very hydrophilic bile salt and weak detergent, caused little hemolysis at concentrations of 40, 60, or 80 mM. TCA, a primary bile acid of intermediate hydrophobicity, caused increasingly rapid and severe hemolysis at concentrations of 5, 10, 15, and 20 mM. TCDCA, a more hydrophobic primary bile acid, produced hemolysis at a concentration of 2.5 mM which progressed at 4 or 5 mM. TDCA, the most hydrophobic of the bile salts studies, produced measurable hemolysis at 1.25 mM, and complete hemolysis was produced at 2 mM concentration within 30 min. The relative toxicity of TDCA was about 10-fold that of TCA. In the next set of studies, lecithin vesicles were added to fixed concentrations of bile salts, after which red cells were added and hemolysis was determined as a function of time. Results with 2 mM TDCA and 20 mM TCA are shown in Fig. 2. Increasing the concentration of lecithin remarkably reduced hemolysis. This was true for both TDCA and TCA and for both 30 min and 4 h. Lecithin appeared more potent in protecting against TDCA than TCA. At 4 h, 50% reduction of TDCA-induced hemolysis was noted with only 1 mM lecithin; in contrast, 50% reduction of TCA-induced hemolysis required .2 mM lecithin. Similarly, complete suppression of TDCA-induced hemolysis at 4 h required 5 mM lecithin, whereas complete protection against TCA-induced hemolysis required 10 mM lecithin. Protection was statistically significant (P , 0.05) for TDCA at lecithin concentrations $1 mM and for TCA at lecithin concentrations $2 mM. The effect of increasing lecithin concentration on the IMMC of TDCA or TCA is expressed in Fig. 3. The free bile salt concentration, consisting of monomers and simple micelles, declined progressively with addition of
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FIG. 1. Hemolytic effects of bile salts as a function of time, concentration and hydrophobicity. Time is shown on the horizontal axis and fractional hemolysis on the vertical axis. Data are shown for four bile acid taurine conjugates, in order of increasing hydrophobicity (TUDCA , TCA , TCDCA , TDCA).
lecithin. This decline is attributable to sequestering of bile salts in large, lecithin-containing particles (vesicles and mixed micelles). This fall was more dramatic for the more hydrophobic TDCA than for the relative hydrophilic TCA. To determine whether this lecithin-induced decrease in the IMMC accounts for its protective effect, we determined hemolysis as a function of time for a wide range of lecithin-free TDCA solutions. From these studies we were able to produce a standard curve relating fractional hemolysis to bile salt IMMC (Fig. 4, solid line) in the absence of lecithin. On the same axes we have plotted fractional hemolysis produced by 2 mM TDCA following addition of various concentrations of lecithin (Fig. 4, symbols). For both lecithin-free and lecithin-containing TDCA samples, similar degrees of hemolysis were observed at similar IMMC values. This finding indicates that the protective effect of lecithin is attributable to its ability to bind TDCA and lower its IMMC to nontoxic levels. DISCUSSION
By virtue of their detergency, concentrated solutions of hydrophobic bile salts can cause injury to the cells with which they come in contact. Apical membranes of hepatocytes, biliary and intestinal epithelia normally are exposed to millimolar concentrations of bile salts. Epithelia of esophagus, stomach, colon, and pancreatic ducts also may be exposed to concentrated bile salts under some circumstances. Bile salts in the lumen of
FIG. 2. The effect of lecithin vesicles (horizontal axis) on bile salt-induced fractional hemolysis (vertical axis). All samples were exposed to 2 mM TDCA (top) or 20 mM TCA (bottom). In the absence of lecithin, hemolysis was partial by 30 min and near complete at 4 h. The addition of lecithin vesicles progressively reduced or eliminated hemolysis.
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FIG. 3. Bile salt intervesicular intermixed micellar bile salt concentration (IMMC, vertical axis) as a function of increasing lecithin concentration (horizontal axis). Studies were performed with 2 mM TDC (top) and 20 mM TC (bottom). IMMC was determined by rapid centrifugal ultrafiltration. The decline in IMMC with increasing lecithin concentration results from sequestration of bile salt in large, lecithin-associated particles (vesicles, mixed micelles).
the biliary canaliculus may damage the hepatocyte. Bile salts at high infusion rates produce hepatocellular necrosis and cessation of bile flow in bile fistula rats [18 –20]. These effects are accompanied by release into bile of structural canalicular membrane lipids such as sphingomyelin, and ectoenzymes such as alkaline phosphatase [20 –22]. The threshold infusion rate at which these toxic effects are observed generally is inversely related to the hydrophobicity of the bile salt infused. In the gastrointestinal tract, bile salts refluxing into the stomach and esophagus may cause mucosal disruption and aggravate damage produced by acid and pepsin [23]. The absence of bile salt toxicity in the biliary tract and proximal intestine under normal conditions has not been explained. Previous studies have suggested that lecithin, a major constituent of bile, may attenuate bile salt disruption of epithelia. O’Leary showed that bile salts cause release of mucus from gallbladder epithelium, which was diminished by inclusion of lecithin in the medium [24]. In vitro studies of Velardi and associates [25] found that lecithin prevented bile salt injury in several cultured cell lines. Heuman [26] noted that the extraction of alkaline phosphatase from isolated canalicular membranes by hydrophobic bile salts was attenuated by addition of lecithin vesicles. Duane and Wiegand demonstrated that the damaging effect of bile salts on the permeability of gastric epithelium was
attenuated by inclusion of lecithin [27]. The protective effect of lecithin was also suggested in the studies of Sagawa and associates [28] employing erythrocytes. The current studies are the first to systematically examine the effect of lecithin on the bile salt IMMC as a determinant of lecithin’s protective effect in preventing bile salt disruption of plasma membranes. Previous studies from our laboratory and others have demonstrated that bile salts partition into lecithin bilayers as a function of their relative hydrophobicity [29 –32]. The concentration of bile salts associated with lecithin in vesicles and the transition from vesicles to mixed micelles increase with increasing concentration of free, non-lecithin-associate bile salts in the intervesicular, intermixed micellar aqueous phase (IMMC). Inclusion of cholesterol in a lecithin bilayer or membrane reduces the affinity of bile salts for the membrane and increases the bile salt IMMC required to produce membrane disruption and mixed micelle formation [30, 32]. Biliary vesicles, having a relatively hydrophilic molecular species of phosphatidylcholine and a low cholesterol content, undergo dissolution by bile salts at relatively low IMMC [33]. In contrast, plasma membranes have a high cholesterol content, contain more hydrophobic molecular species of phosphatidylcholine, and other phospholipids such as sphingomyelin, phosphatidylethanolamine, and phosphatidlylserine. Because of these properties, plasma membranes have low affinity for bile salts and require relatively high bile salt IMMC before disruption and dissolution can occur. In the current studies, the protective effect of lecithin against toxicity of TDCA was found to be explainable largely by its effect on the IMMC. The close parallel between the standard toxicity curve and the toxicity of TDCA when the concentration is attenuated by the addition of lecithin vesicles indicates that the protective effect of lecithin under these circumstances is largely attributable to this reduction of the IMMC
FIG. 4. Fractional hemolysis (vertical axis) as a function of IMMC (horizontal axis); studies with TDCA. Curve shown was determined for varying concentrations of TDCA in the absence of lecithin; closed circles are IMMC resulting from addition of lecithin to 2 mM TDCA.
NARAIN, DEMARIA, AND HEUMAN: LECITHIN PROTECTS AGAINST BILE SALT TOXICITY
through preferential uptake of TDCA into lecithincontaining vesicles and mixed micelles. At each IMMC value examine, fractional hemolysis in the presence of lecithin was slightly lower than in the absence of lecithin. This finding suggests that lecithin may have a second, minor protective effect that may be unrelated to reduction of the IMMC. The IMMC consists of both monomers and simple micelles. Bile salt simple micelles can incorporate small amounts of lecithin and cholesterol [34], and it is possible that lecithin may cause a shift of bile salts from monomers to simple micelles, thereby reducing the bile salt monomer activity to a proportionally greater extent than the IMMC. Alternatively, bile salts may facilitate transfer of lecithin from vesicles to cell plasma membranes [35] and may prevent membrane disruption through changes in cell membrane composition. Bile salts have been shown to be injurious to gastric and esophageal epithelium [27, 36]. Recent studies have demonstrated that gastric epithelium contains cells that secrete lecithin into the gastric lumen providing a protective hydrophobic barrier to ionic diffusion [37]. Our results, consistent with those of Romero and Lichtenberger [38], suggest that gastric lecithin secretion may also serve to provide a secondary defense against bile salts that may regurgitate into the stomach from the duodenum by adsorbing bile salts and reducing the IMMC to nontoxic levels. However, care should be taken in extrapolating the current findings to gastric or esophageal injury because in the current studies we employed exclusively bile salt taurine conjugates. Human biliary bile acids are a mixture of taurine and glycine conjugates at ratios of approximately 1:2. When fully ionized, the detergent properties of taurine or glycine conjugates of a bile salt are similar. The taurine conjugates, with pKa less than 1, remain ionized throughout the range of physiological pH and their detergent properties are insensitive to acid. In contrast glycine conjugates (pKa 3.9) become protonated as pH falls below 7, in the process losing charge, becoming poorly water soluble, and partitioning with increased affinity into vesicles or micelles. The effect of acid pH on membrane disruptive properties of mixed solutions of taurine and glycine-conjugated bile salts is thus quite complex and to date has not been carefully studied. Biliary vesicles contain cholesterol as well as lecithin. Because nascent mixed micelles have a lower capacity for cholesterol than for lecithin, the cholesterol:lecithin ratio of residual vesicles increases gradually with progressive vesicle dissolution as bile is concentrated in the gall bladder [11]. High cholesterol vesicles have reduced affinity for bile salts; thus we have proposed that bile with a high cholesterol:phospholipid ratio, as occurs in gallstone disease, may be inherently toxic [3]. In cholesterol-fed prairie dogs, onset of cholesterol supersaturation of bile is followed by a marked increase in release of mucin from gallbladder epithelium, prior to appearance of cholesterol crystals [39]. Mucin secretion in vivo appears to be a manifes-
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tation of bile salt cytotoxicity and is attenuated by lecithin [24]. Studies to definitively test this hypothesis currently are in progress in our laboratories. ACKNOWLEDGMENTS These studies were supported in part by grants from the Department of Veterans Affairs and the National Institutes of Health.
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78, 131–136 (1998)
JR985364
Lecithin Protects against Plasma Membrane Disruption by Bile Salts P. K. Narain, M.D., E. J. DeMaria, M.D., and D. M. Heuman, M.D. Departments of Surgery and Medicine, Medical College of Virginia of Virginia Commonwealth University, Richmond, Virginia and McGuire Department of Veterans Affairs Medical Center, Richmond, Virginia 23298-0711 Presented at the Annual Meeting of the Association for Academic Surgery, Dallas, Texas, November 6 – 8, 1997
Introduction. Detergent disruption of epithelial plasma membranes by bile salts may contribute to pathogenesis of cholestasis and gastroesophageal reflux disease. Bile, despite containing high concentrations of bile salts, normally is not toxic to biliary or intestinal epithelia. We hypothesize that lecithin in bile may protect cell membranes from disruption by bile salts. Methods. We studied the interactions of taurine conjugates of ursodeoxycholate (TUDCA), cholate (TCA), chenodeoxycholate (TCDCA), and deoxycholate (TDCA) with erythrocyte plasma membranes with or without large unilamellar egg lecithin vesicles for various times at 23°C. Release of hemoglobin was quantified spectrophotometrically. The concentration of bile salt monomers and simple micelles in the intermixed micellar aqueous phase (IMMC) was determined by centrifugal ultrafiltration. Results. The degree of hemolysis depended on the hydrophobicity of the bile salts and was progressive over time. Addition of lecithin reduced the hemolytic effects of 20 mM TCA or 2 mM TDCA in a concentrationdependent manner at both 30 min and 4 h. Increasing the concentration of lecithin progressively reduced the IMMC of TDCA. Hemolysis following addition of lecithin to 2 mM TDCA was comparable to hemolysis produced by lecithin-free TDCA solutions when diluted to similar IMMC values. Conclusion. We conclude that lecithin reduces plasma membrane disruption by hydrophobic bile salts. This protection may be attributable to association of bile salts with vesicles and mixed micelles, reducing the concentration of bile salt monomers and simple micelles available to interact with cell membranes. Lecithin may play a key role in preventing bile salt injury of biliary and gastrointestinal epithelia. © 1998 Academic Press Key Words: lecithin; phospholipid; phosphatidylcholine; bile salt; toxicity; erythrocyte; membrane.
INTRODUCTION
Bile salts are organic detergents, formed in the liver by the oxidation of cholesterol [1]. Their detergency derives from their amphiphilic structure, with a hydrophobic hydrocarbon surface and a hydrophilic surface
produced by polar hydroxyl and carboxy groups. Different bile salts vary in their hydrophilic– hydrophobic balance, and detergency tends to increase with increasing relative hydrophobicity [2]. Bile salts are present in bile in high concentrations and are critically important for the absorption of lipids from the intestine. Because they also can solubilize the lipids that make up cell membranes, bile salts are potentially toxic. Bile salt toxicity has been implicated in pathogenesis of cholestatic liver disease, gastroesophageal reflux disease, cholecystitis, pancreatitis, and colon cancer [3– 6]. Bile salts normally do not damage biliary membranes, despite their presence in bile at high concentrations. Recent data suggest that the explanation for this may lie with the other major biliary lipids, such as lecithin (phosphatidylcholine) and cholesterol, that are secreted by the hepatic canaliculus as large unilamellar vesicles [7]. A protective effect of biliary vesicles is suggested by the mdr2 knockout mouse model, in which a key phospholipid transport protein required for vesicle assembly is lacking and biliary lecithin secretion is negligible [8]. The mdr2 knockout mouse develops severe progressive cholestatic liver disease that is aggravated by feeding of hydrophobic bile salts [9, 10]. Interaction of bile salts with vesicles in bile is complex. At low concentrations, bile salts are present as monomers. Above a critical micellar concentration they self-aggregate to form simple micelles. Bile salt monomers also partition into lipid bilayers such as vesicles or membranes. A phase transition occurs when the vesicle becomes saturated with bile salt, leading to formation of rod-like mixed micelles [11]. The concentration of bile salts in monomers and simple micelles, also termed the intervesicular, intermixed micellar concentration (IMMC),1 drives the concentration of bile salts in lipid bilayers and their transition into mixed micelles [12]. We hypothesize that biliary lecithin, which constitutes over 95% of the biliary phospholipids, may be of 1 Abbreviations used: TUDCA, tauroursodeoxycholic acid; TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; TDCA, taurodeoxycholic acid; IMMC, intermixed micellar intervesicular bile salt concentration.
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key importance in protecting cells against disruption by bile salts, and this protective effect of lecithin may derive from the partitioning of bile salts into vesicles and mixed micelles, thereby reducing the bile salt IMMC. This is the first study to address the role of the IMMC in bile salt-induced cytotoxicity. As a model plasma membrane system, we chose isolated human erythrocytes. Erythrocytes lack nuclei, do not synthesize proteins, have a relatively simple metabolic profile, and do not actively transport bile salts. Previous studies have shown that erythrocytes can be used as a general model system to study disruptive interactions of surfactants with plasma membranes [13–15].
filtration using centricon 10 cartridges (Amicon, Beverly, MA). Ultrafiltration allows free passage of monomers and simple micelles, but completely retains bile salts associated with lecithin in larger physical aggregates such as mixed micelles and vesicles. Bile salt binding to nonfiltrable lecithin-containing particles was quantified by comparing the concentration of the unbound radiolabeled bile salts in the filtrate to the total concentration of radioactivity in the unfiltered sample. After incubation of red cells, vesicles and radiolabeled bile salts ([3H]TCA or [3H]TDCA) for 30 min and 4 h, 1-ml aliquots were subjected to gentle centrifugation (1000g) for 10 min in the centricon 10 tubes. Radioactivity in 0.1 ml of the prefiltration sample and 0.1 ml of the filtrate was determined on a liquid scintillation counter (Beckman LS 6000IC, Fullerton, CA). Correction for Gibbs–Donnan effects was not required as these are negligible at the low bound bile salt concentrations used in these studies [12].
Statistical Analysis MATERIALS AND METHODS
Materials Egg yolk lecithin (.99% phosphatidylcholine) was purchased from Avanti Polar Lipids (Alabaster, AL). Sodium salts of the taurine conjugates of cholic (TCA), deoxycholic (TDCA), chenodeoxycholic (TCDCA), and ursodeoxycholic acids (TUDCA) were purchased from Calbiochem (La Jolla, CA). [3H]Taurocholate was purchased from NEN DuPont (Boston, MA). [3H]- and [14C]taurine conjugates of deoxycholate were synthesized using a modification of the method of Tserng et al., [16] and purified by thin-layer chromatography. Preparation of vesicles. Egg yolk lecithin (average mol wt 760.09) was dissolved in chloroform and large unilamellar vesicles of 100 nm mean diameter (comparable in size to biliary vesicles) were prepared by the method of Hope et al. [17]. Briefly, the lipids were dried under nitrogen, dissolved in warm terbutanol, frozen at 270°C, and lyophilized overnight. They were then suspended in Tris-buffered saline, vortexed vigorously for 20 min, followed by five freeze and thaw cycles using liquid nitrogen, and extruded under nitrogen (100 to 400 psi) through paired 0.1-mm polycarbonate filters (Poretics Corp. Livermore, CA) using a Thermobarrel extruder (Lipex Biomembranes, Vancouver, BC). The extruded vesicles were stored under nitrogen at 4°C and used within a week. Vesicles formed this way are uniform, large, and unilamellar. They have a uniform surface available for surface interaction, and they do not show the strain anomalies related to curvature, typically seen in small vesicles prepared by sonication. Preparation of human erythrocytes. Human erythrocytes were collected fresh from a single volunteer (P.K.N.). Blood collected in ethylenediaminetetraacetic acid (EDTA) tubes was spun at 1000g (IEC Model CU 5000, Needham Heights, MA) at 23°C for 10 min. Discarding the supernatant and the buffy coat, the red blood cells were washed three times with Tris-buffered saline (0.14 M NaCl, 0.01 M Tris–HCl, pH 7.4). The washed red blood cells were reconstituted to a hematocrit of 20% (v/v) in this buffer, stored at 4°C, and used within a week for hemolysis studies.
Experiment Design Freshly prepared erythrocytes were used for all the experiments at a final hematocrit of 4%. Red cells were incubated in 0.14 M saline with 0.01 M Tris (pH 7.4), along with increasing concentrations of taurocholic (TCA), taurodeoxycholic (TDCA), taurochenodeoxycholic (TCDCA), and tauroursodeoxycholic acid (TUDCA) for up to 24 h at 23°C. To assess cell disruption (release of hemoglobin), we precipitated erythrocytes by rapid centrifugation (1000g for 10 min) and determined optical density of the supernatant at 540 nm on a Beckman DU 50 spectrophotometer (Fullerton, CA). Percentage hemolysis was calculated by comparing the measured absorption with that of an equal volume of erythrocytes lysed in distilled water. In some studies, following addition of lecithin to bile salt solutions, the concentration of bile salts as monomers and simple micelles in the intermixed micellar aqueous phase (termed the IMMC) was quantified as described by Donovan et al. [12] by centrifugal ultra-
All experiments were run in quadruplicate. Comparisons between two groups were made with Student’s paired t test and a P value of 0.05 or less was taken as significant. For sigmoidal curve fitting we employed Sigmaplot 4.0 for Windows 95 (Jandel Scientific, San Rafael, CA).
RESULTS
In initial studies, the extent of hemolysis was assessed as a function of time and bile salt concentration. Figure 1 depicts the relative toxicity of TUDCA, TCA, TCDCA, and TDCA. TUDCA, a very hydrophilic bile salt and weak detergent, caused little hemolysis at concentrations of 40, 60, or 80 mM. TCA, a primary bile acid of intermediate hydrophobicity, caused increasingly rapid and severe hemolysis at concentrations of 5, 10, 15, and 20 mM. TCDCA, a more hydrophobic primary bile acid, produced hemolysis at a concentration of 2.5 mM which progressed at 4 or 5 mM. TDCA, the most hydrophobic of the bile salts studies, produced measurable hemolysis at 1.25 mM, and complete hemolysis was produced at 2 mM concentration within 30 min. The relative toxicity of TDCA was about 10-fold that of TCA. In the next set of studies, lecithin vesicles were added to fixed concentrations of bile salts, after which red cells were added and hemolysis was determined as a function of time. Results with 2 mM TDCA and 20 mM TCA are shown in Fig. 2. Increasing the concentration of lecithin remarkably reduced hemolysis. This was true for both TDCA and TCA and for both 30 min and 4 h. Lecithin appeared more potent in protecting against TDCA than TCA. At 4 h, 50% reduction of TDCA-induced hemolysis was noted with only 1 mM lecithin; in contrast, 50% reduction of TCA-induced hemolysis required .2 mM lecithin. Similarly, complete suppression of TDCA-induced hemolysis at 4 h required 5 mM lecithin, whereas complete protection against TCA-induced hemolysis required 10 mM lecithin. Protection was statistically significant (P , 0.05) for TDCA at lecithin concentrations $1 mM and for TCA at lecithin concentrations $2 mM. The effect of increasing lecithin concentration on the IMMC of TDCA or TCA is expressed in Fig. 3. The free bile salt concentration, consisting of monomers and simple micelles, declined progressively with addition of
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FIG. 1. Hemolytic effects of bile salts as a function of time, concentration and hydrophobicity. Time is shown on the horizontal axis and fractional hemolysis on the vertical axis. Data are shown for four bile acid taurine conjugates, in order of increasing hydrophobicity (TUDCA , TCA , TCDCA , TDCA).
lecithin. This decline is attributable to sequestering of bile salts in large, lecithin-containing particles (vesicles and mixed micelles). This fall was more dramatic for the more hydrophobic TDCA than for the relative hydrophilic TCA. To determine whether this lecithin-induced decrease in the IMMC accounts for its protective effect, we determined hemolysis as a function of time for a wide range of lecithin-free TDCA solutions. From these studies we were able to produce a standard curve relating fractional hemolysis to bile salt IMMC (Fig. 4, solid line) in the absence of lecithin. On the same axes we have plotted fractional hemolysis produced by 2 mM TDCA following addition of various concentrations of lecithin (Fig. 4, symbols). For both lecithin-free and lecithin-containing TDCA samples, similar degrees of hemolysis were observed at similar IMMC values. This finding indicates that the protective effect of lecithin is attributable to its ability to bind TDCA and lower its IMMC to nontoxic levels. DISCUSSION
By virtue of their detergency, concentrated solutions of hydrophobic bile salts can cause injury to the cells with which they come in contact. Apical membranes of hepatocytes, biliary and intestinal epithelia normally are exposed to millimolar concentrations of bile salts. Epithelia of esophagus, stomach, colon, and pancreatic ducts also may be exposed to concentrated bile salts under some circumstances. Bile salts in the lumen of
FIG. 2. The effect of lecithin vesicles (horizontal axis) on bile salt-induced fractional hemolysis (vertical axis). All samples were exposed to 2 mM TDCA (top) or 20 mM TCA (bottom). In the absence of lecithin, hemolysis was partial by 30 min and near complete at 4 h. The addition of lecithin vesicles progressively reduced or eliminated hemolysis.
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FIG. 3. Bile salt intervesicular intermixed micellar bile salt concentration (IMMC, vertical axis) as a function of increasing lecithin concentration (horizontal axis). Studies were performed with 2 mM TDC (top) and 20 mM TC (bottom). IMMC was determined by rapid centrifugal ultrafiltration. The decline in IMMC with increasing lecithin concentration results from sequestration of bile salt in large, lecithin-associated particles (vesicles, mixed micelles).
the biliary canaliculus may damage the hepatocyte. Bile salts at high infusion rates produce hepatocellular necrosis and cessation of bile flow in bile fistula rats [18 –20]. These effects are accompanied by release into bile of structural canalicular membrane lipids such as sphingomyelin, and ectoenzymes such as alkaline phosphatase [20 –22]. The threshold infusion rate at which these toxic effects are observed generally is inversely related to the hydrophobicity of the bile salt infused. In the gastrointestinal tract, bile salts refluxing into the stomach and esophagus may cause mucosal disruption and aggravate damage produced by acid and pepsin [23]. The absence of bile salt toxicity in the biliary tract and proximal intestine under normal conditions has not been explained. Previous studies have suggested that lecithin, a major constituent of bile, may attenuate bile salt disruption of epithelia. O’Leary showed that bile salts cause release of mucus from gallbladder epithelium, which was diminished by inclusion of lecithin in the medium [24]. In vitro studies of Velardi and associates [25] found that lecithin prevented bile salt injury in several cultured cell lines. Heuman [26] noted that the extraction of alkaline phosphatase from isolated canalicular membranes by hydrophobic bile salts was attenuated by addition of lecithin vesicles. Duane and Wiegand demonstrated that the damaging effect of bile salts on the permeability of gastric epithelium was
attenuated by inclusion of lecithin [27]. The protective effect of lecithin was also suggested in the studies of Sagawa and associates [28] employing erythrocytes. The current studies are the first to systematically examine the effect of lecithin on the bile salt IMMC as a determinant of lecithin’s protective effect in preventing bile salt disruption of plasma membranes. Previous studies from our laboratory and others have demonstrated that bile salts partition into lecithin bilayers as a function of their relative hydrophobicity [29 –32]. The concentration of bile salts associated with lecithin in vesicles and the transition from vesicles to mixed micelles increase with increasing concentration of free, non-lecithin-associate bile salts in the intervesicular, intermixed micellar aqueous phase (IMMC). Inclusion of cholesterol in a lecithin bilayer or membrane reduces the affinity of bile salts for the membrane and increases the bile salt IMMC required to produce membrane disruption and mixed micelle formation [30, 32]. Biliary vesicles, having a relatively hydrophilic molecular species of phosphatidylcholine and a low cholesterol content, undergo dissolution by bile salts at relatively low IMMC [33]. In contrast, plasma membranes have a high cholesterol content, contain more hydrophobic molecular species of phosphatidylcholine, and other phospholipids such as sphingomyelin, phosphatidylethanolamine, and phosphatidlylserine. Because of these properties, plasma membranes have low affinity for bile salts and require relatively high bile salt IMMC before disruption and dissolution can occur. In the current studies, the protective effect of lecithin against toxicity of TDCA was found to be explainable largely by its effect on the IMMC. The close parallel between the standard toxicity curve and the toxicity of TDCA when the concentration is attenuated by the addition of lecithin vesicles indicates that the protective effect of lecithin under these circumstances is largely attributable to this reduction of the IMMC
FIG. 4. Fractional hemolysis (vertical axis) as a function of IMMC (horizontal axis); studies with TDCA. Curve shown was determined for varying concentrations of TDCA in the absence of lecithin; closed circles are IMMC resulting from addition of lecithin to 2 mM TDCA.
NARAIN, DEMARIA, AND HEUMAN: LECITHIN PROTECTS AGAINST BILE SALT TOXICITY
through preferential uptake of TDCA into lecithincontaining vesicles and mixed micelles. At each IMMC value examine, fractional hemolysis in the presence of lecithin was slightly lower than in the absence of lecithin. This finding suggests that lecithin may have a second, minor protective effect that may be unrelated to reduction of the IMMC. The IMMC consists of both monomers and simple micelles. Bile salt simple micelles can incorporate small amounts of lecithin and cholesterol [34], and it is possible that lecithin may cause a shift of bile salts from monomers to simple micelles, thereby reducing the bile salt monomer activity to a proportionally greater extent than the IMMC. Alternatively, bile salts may facilitate transfer of lecithin from vesicles to cell plasma membranes [35] and may prevent membrane disruption through changes in cell membrane composition. Bile salts have been shown to be injurious to gastric and esophageal epithelium [27, 36]. Recent studies have demonstrated that gastric epithelium contains cells that secrete lecithin into the gastric lumen providing a protective hydrophobic barrier to ionic diffusion [37]. Our results, consistent with those of Romero and Lichtenberger [38], suggest that gastric lecithin secretion may also serve to provide a secondary defense against bile salts that may regurgitate into the stomach from the duodenum by adsorbing bile salts and reducing the IMMC to nontoxic levels. However, care should be taken in extrapolating the current findings to gastric or esophageal injury because in the current studies we employed exclusively bile salt taurine conjugates. Human biliary bile acids are a mixture of taurine and glycine conjugates at ratios of approximately 1:2. When fully ionized, the detergent properties of taurine or glycine conjugates of a bile salt are similar. The taurine conjugates, with pKa less than 1, remain ionized throughout the range of physiological pH and their detergent properties are insensitive to acid. In contrast glycine conjugates (pKa 3.9) become protonated as pH falls below 7, in the process losing charge, becoming poorly water soluble, and partitioning with increased affinity into vesicles or micelles. The effect of acid pH on membrane disruptive properties of mixed solutions of taurine and glycine-conjugated bile salts is thus quite complex and to date has not been carefully studied. Biliary vesicles contain cholesterol as well as lecithin. Because nascent mixed micelles have a lower capacity for cholesterol than for lecithin, the cholesterol:lecithin ratio of residual vesicles increases gradually with progressive vesicle dissolution as bile is concentrated in the gall bladder [11]. High cholesterol vesicles have reduced affinity for bile salts; thus we have proposed that bile with a high cholesterol:phospholipid ratio, as occurs in gallstone disease, may be inherently toxic [3]. In cholesterol-fed prairie dogs, onset of cholesterol supersaturation of bile is followed by a marked increase in release of mucin from gallbladder epithelium, prior to appearance of cholesterol crystals [39]. Mucin secretion in vivo appears to be a manifes-
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tation of bile salt cytotoxicity and is attenuated by lecithin [24]. Studies to definitively test this hypothesis currently are in progress in our laboratories. ACKNOWLEDGMENTS These studies were supported in part by grants from the Department of Veterans Affairs and the National Institutes of Health.
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