A Thermodynamic Study of Bile Salt Interactions with

We have compared the partitioning of bile salts into sphingomyelin (SM) and phosphatidylcholine (PC) bilayers. The partitioning of the sodium salts of...
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Langmuir 2001, 17, 2835-2840

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A Thermodynamic Study of Bile Salt Interactions with Phosphatidylcholine and Sphingomyelin Unilamellar Vesicles Fredrik Ollila and J. Peter Slotte* Department of Biochemistry and Pharmacy, Åbo Akademi University, P.O. Box 66, FIN 20521 Turku, Finland Received October 9, 2000. In Final Form: January 31, 2001 We have used isothermal titration calorimetry to study how the membrane lipid composition affects the partitioning of bile salts into membranes. We have compared the partitioning of bile salts into sphingomyelin (SM) and phosphatidylcholine (PC) bilayers. The partitioning of the sodium salts of cholate and deoxycholate, at concentrations below the critical micelle concentration, into egg yolk phosphatidylcholine (EPC), hydrogenated EPC (HEPC), and egg yolk sphingomyelin (ESM) membranes was studied at 25 °C. Deoxycholate (0.3 mM) partitioned into ESM membranes with a K value of 2200 ( 100 M-1, whereas the K was 520 ( 30 M-1 with EPC membranes under identical conditions. At concentrations above 0.6 mM, deoxycholate solubilized the ESM but not the EPC membranes. The partition enthalpy for deoxycholate was 13.2 ( 2.8 and 10.8 ( 1.3 kJ mol-1 in ESM and EPC membranes, respectively. The partitioning of cholate at 0.3 mM into ESM and EPC membranes did not give measurable heats. At 1 mM, cholate partitioning into EPC membranes was characterized by a K of 123 ( 1 M-1 and a ∆H of 11 ( 0.5 kJ mol-1. Deoxycholate (at 1 mM) partitioned into hydrogenated EPC (K of 275 ( 5 M-1, ∆H of 7 ( 0.5 kJ mol-1) to a lower extent than into EPC membranes (K of 410 ( 10 M-1, ∆H of 9 ( 0.5 kJ mol-1). These results show that bile salt partitioning into membranes is influenced both by the phospholipid type (SM versus PC) and by the acyl chain composition (unsaturated versus saturated).

Introduction Detergent resistant membrane (DRM) fragments may represent the putative microdomains in cell membranes that have been suggested to serve as relay stations for several proteins and to be involved in intracellular signaling.1-4 Yu and co-workers found that sphingolipids were a major constituent of the detergent insoluble fraction and they proposed that the tight interaction between the sphingolipid molecules was the origin of its insolubility.5,6 Natural sphingomyelins differ from most biological phospholipids in containing long, mostly saturated acyl chains, which enable them to pack tighter than natural glycerophospholipids in membranes.7 This packing property gives natural sphingolipids much higher melting temperatures (Tm) than membrane glycerophospholipids, which are rich in unsaturated acyl chains.8 Although natural sphingolipids have a higher Tm compared to natural glycerophospholipids, they have been shown to be metastable resulting in local instabilities in the bilayer.9 Bile salts are important biological amphiphiles. They have a steroid structure and are synthesized from cholesterol by the hepatocytes (Chart 1). The carboxyl and hydroxyl moieties that bile salts possess are on the R-side of the steroid structure. This gives them an

Chart 1. Chemical Structure of Cholesterol and the Bile Salts Cholate, Deoxycholate, Glycocholate, and Taurocholate and an Illustration of the Hydrophilic-Hydrophobic Balance of Cholate

* Corresponding author: Fax: +358 2 2654745; E-mail: jpslotte@ abo.fi. (1) Simons, K.; Ikonen, E. Nature 1997, 387, 569. (2) Hooper, N. M. Mol. Membr. Biol. 1999, 16, 145. (3) Kasahara, K.; Sanai, Y. Biophys. Chem. 1999, 82, 121. (4) Dobrowsky, R. T. Cell Signal 2000, 12, 81. (5) Yu, J.; Fischman, D. A.; Steck, T. L. J. Supramol. Struct. 1973, 1, 233. (6) Brown, D. A.; Rose, J. K. Cell 1992, 68, 533. (7) Smaby, J. M.; Momsen, M. M.; Brockman, H. L.; Brown, R. E. Biophys. J. 1997, 73, 1492. (8) Barenholz, Y. In Physiology of membrane Fluidity; Shinitzky, M., Ed.; CRC Press: Boca Raton, FL, 1984; p 131. (9) Estep, T. N.; Calhoun, W. I.; Barenholz, Y.; Biltonen, R. L.; Shipley, G. G.; Thompson, T. E. Biochemistry 1980, 19, 20.

10.1021/la0014196 CCC: $20.00 © 2001 American Chemical Society Published on Web 03/29/2001

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amphipathic character with a hydrophobic side (β-side) and hydrophilic side (R-side), which makes them much more water-soluble than the cholesterol from which they are synthesized.10 Bile salts have for a long time been known to partition into lipid bilayers and alter membrane properties such as fluidity and permeability to small ions.11-19 Bile salt/membrane interactions have recently received much attention due to the hepatocellular damage that amphipathic bile salts induce.20,21 The extent of the damage has been shown to correlate with the hydrophobic-hydrophilic balance of the bile salts, with more hydrophobic species inducing more cell damage.22,23 Isothermal titration calorimetry (ITC) has proved to be a valuable tool for studying the thermodynamics of solute and detergent partitioning into lipid bilayers.24-26 Results from partitioning of different solutes and detergents into lipid membranes have been presented in several recent publications,26-31 but the molecular and thermodynamical aspect of bile salt partitioning into lipid membranes has still not been presented. Since sphingomyelins are, together with cholesterol, one of the major constituents in raft formations6,32-34 and exhibit a protective roll in cell membranes toward the cytotoxic effect of bile salts,20,21 we wanted to compare the partitioning of bile salts into bilayers composed of sphingomyelin and phosphatidylcholine. Our study presents a thermodynamic description of the interactions of four distinct bile salts with egg yolk phosphatidylcholine (EPC), hydrogenated egg yolk phosphatidylcholine (HEPC), and egg yolk sphingomyelin (ESM) membranes. (10) Small, D. M. In The Bile Acids; Nair, P. P., Kritchevsky, D., Eds.; Plenum Press: New York, 1971. (11) Billington, D.; Coleman, R. Biochim. Biophys. Acta 1978, 509, 33. (12) Lowe, P. J.; Coleman, R. Biochim. Biophys. Acta 1981, 640, 55. (13) Ulmius, J.; Lindblom, G.; Wennerstrom, H.; Johansson, L. B.; Fontell, K.; Soderman, O.; Arvidson, G. Biochemistry 1982, 21, 1553. (14) Lichtenberg, D.; Robson, R. J.; Dennis, E. A. Biochim. Biophys. Acta 1983, 737, 285. (15) Lichtenberg, D. Biochim. Biophys. Acta. 1985, 821, 470. (16) O’Connor, C. J.; Wallace, R. G.; Iwamoto, K.; Taguchi, T.; Sunamoto, J. Biochim. Biophys. Acta 1985, 817, 95. (17) Bayerl, T. M.; Werner, G. D.; Sackmann, E. Biochim. Biophys. Acta 1989, 984, 214. (18) Zimniak, P.; Little, J. M.; Radominska, A.; Oelberg, D. G.; Anwer, M. S.; Lester, R. Biochemistry 1991, 30, 8598. (19) Donovan, J. M.; Leonard, M. R.; Batta, A. K.; Carey, M. C. Gastroenterology 1994, 107, 831. (20) Amigo, L.; Mendoza, H.; Zanlungo, S.; Miquel, J. F.; Rigotti, A.; Gonzalez, S.; Nervi, F. J. Lipid Res. 1999, 40, 533. (21) Moschetta, A.; vanBerge-Henegouwen, G. P.; Portincasa, P.; Palasciano, G.; Groen, A. K.; van Erpecum, K. J. J. Lipid Res. 2000, 41, 916. (22) Coleman, R.; Iqbal, S.; Godfrey, P. P.; Billington, D. Biochem. J. 1979, 178, 201. (23) Barnwell, S. G.; Lowe, P. J.; Coleman, R. Biochem. J. 1983, 216, 107. (24) Wimley, W. C.; White, S. H. Biochemistry 1993, 32, 6307. (25) Heerklotz, H. H. In Biocalorimetry: Applications of Calorimetry in the Biological Sciences; Ladbury, J. E., Chowdhry, B. Z., Eds.; John Wiley & Sons: New York, 1998; p 89. (26) Heerklotz, H. H.; Binder, H.; Epand, R. M. Biophys. J. 1999, 76, 2606. (27) Wenk, M. R.; Seelig, J. Biophys. J. 1997, 73, 2565. (28) Wenk, M. R.; Alt, T.; Seelig, A.; Seelig, J. Biophys. J. 1997, 72, 1719. (29) Rowe, E. S.; Zhang, F.; Leung, T. W.; Parr, J. S.; Guy, P. T. Biochemistry 1998, 37, 2430. (30) Heerklotz, H.; Seelig, J. Biophys. J. 2000, 78, 2435. (31) Heerklotz, H.; Seelig, J. Biochim. Biophys. Acta 2000, 1508, 69. (32) Ahmed, S. N.; Brown, D. A.; London, E. Biochemistry 1997, 36, 10944. (33) Brown, D. A.; London, E. Biochem. Biophys. Res. Commun. 1997, 240, 1. (34) Brown, D. A.; London, E. Annu. Rev. Cell. Dev. Biol. 1998, 14, 111.

Ollila and Slotte

Experimental Procedures Material. The sodium salt of cholate (3R,7R,12R-trihydroxy5β-cholanoic acid), deoxycholate (3R,12R-dihydroxy-5β-cholanoic acid), glycocholate (N-[3R,7R,12R-trihydroxy-24-oxocholan-24yl]glycine), and taurocholate (2-[(3R,7R,12R-trihydroxy-24-oxo5β-cholan-24-yl) amino]ethanesulfonic acid) were all obtained from Sigma Chemicals (USA) and used as provided. Egg phosphatidylcholine (EPC), hydrogenated egg phosphatidylcholine (HEPC), and egg sphingomyelin (ESM) were purchased from Avanti Polar Lipids (Birmingham, AL). The phospholipids were at least 99% pure, as determined by HPTLC. All chemicals were used without further purification. 1,6-Diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniophenyl)-6-phenyl-1,3,5hexatriene (TMA-DPH) were obtained from Molecular Probes (Leiden, The Netherlands). The water used for the isothermal titration calorimetry (ITC) and fluorometric experiments was purified by reverse osmosis followed by a passage through a Millipore UF Plus water purification system, to yield a product with a resistivity of 18.2 MΩ cm. If not otherwise specifically denoted, the buffer composition used was 10 mM Tris, 140 mM NaCl, pH 7.4. Isothermal Titration Calorimetry. Isothermal titration calorimetry was performed using a high-sensitivity isothermal titration calorimeter 4200 from Calorimetry Sciences Corp. (USA). The isothermal titration calorimeter was calibrated electrically. The data were acquired by computer software provided by Calorimetry Sciences Corp. All experiments were performed with constant stirring (300 rpm) driven by a stepping motor coupled to the isothermal titration calorimeter. The sample cell volume was 1335 µL in all experiments. Bile salt solutions were applied at concentrations of 0.3, 0.6, or 1 mM, which are below their critical micelle concentration (cmc). The experiments were carried out as described by Wenk and co-workers.28 In control experiments 10 µL aliquots of phospholipid vesicle solution were injected into the sample cell containing buffer without detergent. The obtained dilution heat was small (below -50 µJ/injection) compared to the heat of reaction acquired from the partitioning experiments. The dilution heat was corrected for when analyzing the results. Preparation of Large Unilamellar Vesicles. These large unilamellar vesicles (LUVs) were prepared from either EPC, HEPC, or ESM using a Lipextruder (Lipex Biomembranes, Vancouver, BC) and the extrusion technique described by Hope and co-workers.35 In brief, lipids were suspended and briefly sonicated in buffer to yield a lipid dispersion to be used for the extrusion. The lipids were then extruded at 55 °C through 100nm polycarbonate filters (Costar Corp., Cambridge, MA) for 10 cycles to yield a homogeneous solution with unilamellar vesicles.36 All vesicle types were size analyzed by means of light scattering. The experiments were performed with a Malvern 4700 (Malvern Instruments Ltd, Malvern, Worcestershire, U.K.) using an argon laser (514.5 nm) at a 90° angle. The average vesicle size was found to be 110 ( 30 nm for all phospholipids used. The lipid concentrations were determined with phosphorus analysis37 and the LUV phospholipid concentration was adjusted with buffer to 10, 30, or 60 mM. The LUV and bile salt solutions were prepared with the same buffer to minimize the dilution heat. Determination of Possible Vesicle Solubilization by Bile Salts. To test whether bile salts solubilized the vesicles, lightscattering experiments were performed with a Quantamaster 1 spectrofluorimeter operating in the T-format (Photon Technology International, Monmouth Junction, NJ) with both excitation and emission wavelengths set at 300 nm. The slit width was adjusted to 0.5 nm. Experiments were carried out by titrating 10 µL aliquots of lipid vesicles (10 or 30 mM) into the cuvette containing detergent (1335 µL) at 25 °C. The light-scatter intensity was monitored after every injection. Control experiments were performed as described but using buffer instead of detergent. Determination of Steady-State Fluorescence Anisotropy of DPH in Unilamellar Vesicles. In brief, 400 nmol of the (35) Hope, M. J.; Najar, R.; Mayer, L. D.; Cullis, P. R. In Liposome Preparation and Related Techniques; Gregoriadis, G., Ed.; CRC Press: London, 1993. (36) Mayer, L. D.; Hope, M. J.; Cullis, P. R. Biochim. Biophys. Acta 1986, 858, 161. (37) Bartlett. J. Biol. Chem. 1959, 234, 234.

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Table 1. Steady-State Anisotropy of DPH and TMA-DPH in EPC, HEPC, and ESM Vesiclesa lipid composition EPC HEPC ESM

DPH r

TMA-DPH r

0.073 ( 0.002 0.314 ( 0.001 0.223 ( 0.002

0.199 ( 0.001 0.330 ( 0.001 0.226 ( 0.004

a Large vesicles were prepared at 55 °C by extrusion through 400 nm polycarbonate filters from EPC, HEPC, or ESM. Anisotropy measurements were carried out at 25 °C with DPH or TMA-DPH as a fluorescent probe. Values are given as the average value ( range from two different batches of vesicles.

phospholipid was dried under argon. Vesicles were then prepared as described above. DPH (0.5 mol %) and TMA-DPH (0.5 mol %) were added to the vesicle solutions after extrusion. DPH was added to preformed vesicles from an acetonitrile stock solution, TMA-DPH was added from an ethanol stock solution. The final acetonitrile or ethanol concentration in the assay solution did not exceed 0.5 vol %. Excitation was carried out at 360 nm, and emission was recorded at 430 nm, at a temperature of 25 °C, using a Quantamaster 1 spectrofluorimeter. The steady-state anisotropy, r, is defined as

r ) (I|| - GI⊥)/(I|| + GI⊥) where I|| and I⊥ are the fluorescence intensities with the analyzer parallel and perpendicular to the vertical polarizer, respectively.38 G represents the ratio of the sensitivities of the detection system for vertically and horizontally polarized light.38 Determination of the cmc for the Bile Salts. The cmc’s for the bile salts were determined because we wanted to use bile salt concentrations below the cmc in experiments. If the bile salt concentrations are too close to the cmc, micelle/membrane interactions may complicate the observed processes, and membrane solubilization becomes a greater risk as well.31 8-Anilinonaphthalene-1-sulfonate (ANS) was used as fluorescence probe to determine the cmc for cholate, deoxycholate, glycocholate, and taurocholate. The cmc of the surfactant was determined by subsequently adding 2 µL of bile salt from a stock solution into a 10 µM solution of ANS. Upon approaching the cmc of the surfactant, one could observe a gradual shift to a higher fluorescence intensity due to the partitioning of ANS into the hydrophobic core of the micelles. Excitation was carried out at 370 nm, and emission was recorded at 430 nm, at room temperature, using a Hitachi F-2000 spectrofluorimeter (Hitachi Ltd., Tokyo, Japan). The acquired cmc values were as follows: cholate, 9.0 ( 1 mM; deoxycholate, 1.8 ( 0.2 mM; glycocholate, 7.5 ( 0 mM; taurocholate, 7.0 ( 0.5 mM. These results are in agreement with earlier findings.39

Results Steady-State Anisotropy for Phosphatidylcholine and Sphingomyelin in Unilamellar Vesicles. Lipid packing in the phospholipid membranes was studied due to its possible effect on the partitioning of bile salts into phospholipid membranes. Steady-state anisotropy measurements of DPH and TMA-DPH40 were employed to study the membrane fluidity of EPC, HEPC, and ESM vesicles (Table 1). With DPH as a probe, the anisotropy value was significantly lower for EPC compared to the other phospholipids. ESM has mainly saturated chains with the 16:0-SM species predominating, whereas HEPC (38) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Publishers: New York, 1999. (39) Jones, M. N.; Chapman, D. In Micelles, Monolayers and Biomembranes; Wiley-Liss: New York, 1995. (40) Prendergast, F. G.; Haugland, R. P.; Callahan, P. J. Biochemistry 1981, 20, 7333. (41) Ramstedt, B.; Leppimaki, P.; Axberg, M.; Slotte, J. P. Eur. J. Biochem. 1999, 266, 997. (42) Avanti Polar Lipids, I. Avanti Polar Lipids, Inc catalog; 1995; p 14.

Figure 1. Titration of cholate and deoxycholate (1 mM) with large unilamellar vesicles composed of EPC and HEPC (30 mM) in buffer. The experiment was performed by titrating 10-µL aliquots of vesicle solution into the bile salt suspension (V ) 1.335 mL) in the sample cell.

consists mainly of 16:0- and 18:0-PC:s, while over 40% of EPC is unsaturated (18:1- and 18:2-PC:s). When TMADPH was used as a probe, the steady-state anisotropy measurements showed that the polar regions of HEPC vesicles were more ordered and condensed than the corresponding regions of the two other phospholipids. Vesicle Stability in the Presence of Bile Salts. To study how bile salts affected vesicle stability, we measured light scattering from the vesicles in a fluorescence spectrophotometer. Experiments were performed by titrating 10-µL aliquots of EPC or ESM (10 and 30 mM) vesicles into the cuvette containing cholate, deoxycholate, glycocholate, or taurocholate at different concentrations (0.3, 0.6, and 1 mM). Titration of vesicles into buffer was used as a reference. The volume in the cuvette in the scattering experiments was equal to the volume in the ITC sample cell (1335 µL). The experiments were performed at 25 °C. We found that cholate, glycocholate, and taurocholate (up to 1 mM) did not solubilize EPC or ESM vesicles, whereas deoxycholate above 0.6 mM solubilized ESM but not EPC vesicles (concentrations higher than 1 mM were not tested). At 0.3 mM deoxycholate did not solubilize ESM vesicles (data not shown). Partition studies were consequently performed with bile salt concentrations that did not cause vesicle solubilization. Isothermal Titration Calorimetry. The thermodynamics of cholate, deoxycholate, glycocholate, and taurocholate partitioning into sphingomyelin and phosphatidylcholine membranes was studied at 25 °C with isothermal titration calorimetry. A typical titration pattern of a partitioning experiment, where deoxycholate partitioned into EPC vesicles, is shown as the second trace in Figure 1. Titration calorimetry experiments were performed by adding aliquots of the suspension of phospholipid vesicles from the syringe into the sample cell containing the bile salt solution. Each titration of vesicles into the sample cell gave rise to a heat of reaction, caused by the partitioning of bile salts into the bilayers. The heats of reactions decrease because after each injection of vesicles

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Table 2. Partitioning of Cholate and Deoxycholate into EPC and HEPC Membranesa EPC Membranes bile salt species

K (M-1)

∆H (kJ mol-1)

∆G (kJ mol-1)

∆S (J K-1 mol-1)

cholate deoxycholate

123 ( 1 410 ( 10

11 ( 0.5 9 ( 0.5

-11.9 -14.9

77 80

HEPC Membranes bile salt species

K (M-1)

∆H (kJ mol-1)

∆G (kJ mol-1)

∆S (J K-1 mol-1)

cholate deoxycholate

275 ( 5

7 ( 0.5

-13.9

70

a

Aliquots (10 µL) of phospholipid vesicles (30 mM) in buffer were injected into the sample cell containing the bile salt solution (1 mM) at 25 °C. The partition coefficients are calculated under the assumption that all phospholipid is available for partitioning. Values are given as the average value ( range from two different experiments.

fewer bile salt molecules were available to partition. The partitioning experiment served to measure the K value of the surfactant between the bilayer and the water phase and the change in enthalpy (∆H) derived from the transfer of surfactant from the water phase into the phospholipid bilayer. The K value as used in our work refers to the KL value as described in a recent review by Lasch.43 The K value describes the relationship between the amount of detergent bound to the membrane and free detergent in the solution. Wenk and co-workers have successfully established a two-parameter partition model to acquire the K value from detergent/membrane partitioning experiments, and we have used their model as a tool to analyze our data.28 Knowledge of the K value and ∆H enabled calculation of the free energy of binding (∆G) and the change in entropy (∆S), according to

∆G ) -RT ln K ) ∆H - T∆S where R is the gas constant and T is the absolute temperature.44 Partitioning of Cholate and Deoxycholate into Bilayers Composed of EPC or HEPC. The partitioning of cholate and deoxycholate into large unilamellar vesicles composed of EPC or HEPC was conducted to establish a picture of the effect of acyl chain composition on partitioning. A common theme was observed. A substantially higher degree of partitioning was detected for EPC compared to HEPC (Table 2 and Figure 1). The heat of reaction decreased much faster after each injection for deoxycholate compared to cholate when they partitioned into EPC bilayers. This suggests that deoxycholate was transferred into the bulk hydrocarbon moiety of the membrane or bound to the membrane interface to a higher degree than cholate, hence giving a higher K value. Detergents with a low hydrophilic/hydrophobic balance can be expected to have a low cmc but high K value.15 A high K value characterizes a high degree of partitioning.28,45 The transbilayer movement (flip-flop) of fully ionized cholate and deoxycholate has been shown to be fast enough to assume an equal distribution between the membrane hemileaflets.46 Although the bile salts in the present study (at pH 7.4) are mostly ionized, we still consider it likely that under the time scale of the (43) Lasch, J. Biochim. Biophys. Acta 1995, 1241, 269. (44) Atkins, P. W. In Physical Chemistry, 6th ed.; Oxford University Press: Oxford, 1998. (45) Kamp, F.; Hamilton, J. A.; Westerhoff, H. V. Biochemistry 1993, 32, 11074. (46) Donovan, J. M.; Jackson, A. A. Biochemistry 1997, 36, 11444.

Figure 2. Titration of cholate, deoxycholate, glycocholate, and taurocholate (0.3 mM) with large unilamellar vesicles composed of EPC (10 mM) in buffer. The experiment was performed by titrating 10-µL aliquots of vesicle solution into the bile salt suspension (V ) 1.335 mL) in the sample cell.

experiments, the bile salts distribute among both monolayers of the vesicle membranes. It should be noted that because the bile salts are ionized, their accumulation in the membranes eventually would affect the partitioning process because of increased electrostatic repulsion. This likely effect has not been corrected for in this study. Deoxycholate partitioned into EPC membranes with a K value of 410 ( 10 M-1 at 25 °C, whereas cholate gave a much lower K value (123 ( 1 M-1, Table 2). The higher K value for deoxycholate is likely to be explained the absence of its C-7 hydroxyl group which is present in cholate, i.e., making deoxycholate more hydrophobic than cholate (Chart 1).47 Dihydroxy bile salts are known to partition into lipid membranes to a higher degree than trihydroxy bile salts.47 Cholate was unable to partition into HEPC membranes, while deoxycholate partitioned into HEPC membrane giving a K value of 275 ( 5 M-1. The transfer of deoxycholate into EPC and HEPC bilayers was accompanied by a ∆H of 9 ( 0.5 and 7 ( 0.5 kJ mol-1, respectively, while cholate partitioned into EPC with a ∆H of 11 ( 0.5 kJ mol-1. Interactions of Bile Salts with EPC and ESM Bilayers. Due to solubilization of ESM membranes at a deoxycholate concentration of 1 mM, the bile salt concentrations were lowered to 0.3 mM to allow for pure partitioning. Cholate partitioned into EPC and ESM membranes at these concentrations to such a low extent that ∆H and K values could not be calculated (the heat of reaction was about 50 ( 2 µJ/injection; see Figures 2 and 3). The lack of signal was not due to ∆H being close to zero (which is possible for some membrane partitioning processes29) but rather that the δhi signal was too low at this detergent concentration to allow for accurate measurements with the ITC system used. Deoxycholate partitioned into ESM bilayers with a K value of 2200 ( 100 M-1, which is markedly higher compared to the K value obtained for EPC (550 ( 30 M-1, Table 3). The ∆H was larger for deoxycholate partitioning into ESM (13.2 ( 2.8 kJ mol-1) compared to the value measured for EPC (47) Schubert, R.; Jaroni, H.; Schoelmerich, J.; Schmidt, K. H. Digestion 1983, 28, 181.

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Figure 3. Titration of cholate, deoxycholate, glycocholate, and taurocholate (0.3 mM) with large unilamellar vesicles composed of ESM (10 mM) in buffer. The experiment was performed by titrating 10-µL aliquots of vesicle solution into the bile salt suspension (V ) 1.335 mL) in the sample cell. Table 3. Partitioning of Deoxycholate into EPC and ESM Membranesa membrane type

K (M-1)

∆H (kJ mol-1)

∆G (kJ mol-1)

∆S (J K-1 mol-1)

EPC ESM

520 ( 30 2200 ( 100

10.8 ( 1.3 13.2 ( 2.8

-15.5 -19.1

86.5 109

a Aliquots (10 µL) of phospholipid vesicles (10 mM) in buffer were injected into the sample cell of the containing bile salt solution (0.3 mM) at 25 °C. The partition coefficients are calculated under the assumption that all phospholipid is available for partitioning. Values are given as the average value ( range from five different experiments.

membranes (10.8 ( 1.3 kJ mol-1). The ∆S was also higher for deoxycholate partitioning into ESM (109 J K-1 mol-1) than for the partitioning into EPC (86.5 J K-1 mol-1). Both glycocholate and taurocholate failed to give partition heats at 0.3 mM (or even 1 mM, data not shown) when exposed to either EPC or ESM membranes (Figures 2 and 3). However, at 30 or 37 °C both glycocholate and taurocholate increasingly caused solubilization of ESM vesicles (data not shown). Heat Capacity. Partitioning of amphiphiles into lipid membranes is an endothermic heat dependent process where ∆HoD decreases linearly with increasing temperature.29,48 The temperature dependency of deoxycholate (1 mM) partitioning into EPC vesicles (30 mM) was determined, giving a molar heat capacity change (∆Cp) of -103.5 cal mol-1 K-1 (data not shown). Such large negative ∆Cp values are typical for hydrophobic interactions.49 Other groups have presented similar results for the partitioning of amphiphilic compounds into lipid bilayers.28,29,50 Discussion The most important advantage of this study, compared to other studies in which partitioning of bile salts into (48) Seelig, J.; Ganz, P. Biochemistry 1991, 30, 9354. (49) Baldwin, R. L. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 8069. (50) Beschiaschvili, G.; Seelig, J. Biochemistry 1992, 31, 10044.

lipid membranes has been examined, is the thermodynamical description of the partitioning process which can be acquired by using isothermal titration calorimetry. In this study we have examined the partitioning of different bile salts into large unilamellar vesicles composed of EPC, HEPC, or ESM. The Partitioning Process. Lipid vesicles are titrated into a bile salt solution, causing the bile salt molecules to partition into the vesicle membrane. If the detergent is applied at low enough concentration, cD < cmc, membrane partitioning is the only process to be considered.28 The partitioning of amphipathic molecules between aqueous solution and bulk hydrocarbon is an equilibration process that is governed by thermodynamic parameters. The net enthalpy change caused by the partitioning is a result of dehydration of the nonpolar moieties of the amphipathic molecule, induced disordering, and disorientation of the membrane lipids, affecting the interactions of the polar headgroups with water in the interfacial region of the lipid bilayer. The electrostatic repulsive forces between the polar interface of the membrane and the hydrophobic moiety of the amphipathic molecule also have a marked impact on the net enthalpy change.29 The molecular interpretation of the partitioning process focuses on the hydrophobic effect, where the reduction of the intermolecular hydrogen bonding capacity of water is caused by the hydrophobic moieties of the bile salts.51 Hence, it is the removal of the hydrophobic moieties from the polar phase to the bulk hydrocarbon in the lipid membrane that increases the overall entropy of the system and makes partitioning possible.28,29 It is unknown whether bile salts partition into membranes as monomers or dimers and localize in the nonpolar bulk hydrocarbon or bind to the membrane interface as monomers with their nonpolar moieties oriented toward the nonpolar acyl chains and their polar moieties oriented toward the water phase. Surface chemistry techniques have established that undissociated bile acids orient with their sterol nuclei parallel to the aqueous interface, in a manner that allows their hydrophilic groups to interact with water.10,52-54 These results are in agreement with NMR and monolayer studies which suggest that bile salt molecules are oriented with their steroid nuclei parallel to the membrane interface rather than to be incorporated into the nonpolar interior of the membrane.13,55 However, it has also been shown that bile salts can be incorporated perpendicular with respect to the membrane interface.56 It has been suggested that some bile salts have the ability to partition into membranes as dimers, avoiding contact between the polar groups of the bile salts and the hydrophobic acyl chains.10,57,58 Our results do not allow us to determine which of the described events are more likely, or if both may be operable under our experimental conditions. (51) Tanford, C. In The Hydrophobic Effect: Formation of Micelles and Biological Membranes, 2nd ed.; Wiley-Interscience: New York, 1980. (52) Ekwall, P.; Ekholm, R.; Norman, A. Acta Chem. Scand 1957, 11, 693. (53) Miyoshi, H.; Nagadome, S.; Sugihara, G.; Kagimoto, H.; Ikawa, Y.; Igimi, H.; Shibata, O. J. Colloid Interface Sci. 1991, 149, 216. (54) Shibata, O.; Miyoshi, H.; Nagadome, S.; Sugihara, G.; Igimi, H. J. Colloid Interface Sci. 1991, 146, 594. (55) Fahey, D. A.; Carey, M. C.; Donovan, J. M. Biochemistry 1995, 34, 10886. (56) Saito, H.; Sugimoto, Y.; Tabeta, R.; Suzuki, S.; Izumi, G.; Kodama, M.; Toyoshima, S.; Nagata, C. J. Biochem. (Tokyo) 1983, 94, 1877. (57) Carey, M. C. Bile Acids in Gastroenterology; MTP Press: Lancaster, 1982 (58) Schubert, R.; Beyer, K.; Wolburg, H.; Schmidt, K. H. Biochemistry 1986, 25, 5263.

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The hydrophilic-hydrophobic balance of the bile salts was found to have a significant impact on the partitioning coefficient. The other thermodynamical parameters were found to be less dependent on the hydrophilic-hydrophobic balance of the bile salts. It has been shown that more hydrophobic molecules generally have a higher K value than less hydrophobic molecules.28 Lichtenberg has presented rather interesting results in which he demonstrated the correlation of membrane/water partitioning coefficients of detergents with the cmc.15 Heerklotz and Seelig showed recently that detergents can be grouped into two classes so that the product K‚cmc is either smaller or larger than 1.30 They denoted detergents with K‚cmc < 1 as “strong” detergents and K‚cmc > 1 as “weak” detergents. According to this classification, cholate would be a “weak” detergent (K‚cmc ) 1.107) and deoxycholate a “strong” detergent (K‚cmc ) 0.738). Kamp and Hamilton have presented membrane/water K values for the partitioning of cholic acid and deoxycholic acid into EPC membranes.45 In their study deoxycholic acid partitioned into EPC membranes with a molar K value of 667 M-1, whereas cholic acid gave a K value of 187 M-1. These results are not very different from our results with the salt form of the same bile acids (410 ( 10 and 123 ( 1 M-1, respectively, Table 2). In our calculations we have assumed that the outer and inner leaflets of the vesicle bilayers contain equal amount of lipids.46 Kamp and Hamilton acquired the K values with a fluorometric method and further assumed a 2:1 distribution of un-ionized cholic acid molecules between the outer and inner leaflets in their calculations.45 Partitioning Thermodynamics and Membrane Solubilization. We observed significant differences in the degree of deoxycholate partitioning into phosphatidylcholine (K ) 520 ( 30 M-1) compared to sphingomyelin vesicles (K ) 2200 ( 100 M-1). Both phospholipids have the same polar headgroup (phosphoryl choline), but a closer look at the rest of the structures of these two molecules elucidates their differences. Sphingomyelin has sphingosine as the hydrophobic backbone, together with an amid-linked acyl chain while phosphatidylcholine has a glycerol backbone with two acyl chains linked to it with carbonyl ester linkages.8,59 These structural differences result in sphingomyelins additional ability, compared to phosphatidylcholines, to participate in intermolecular hydrogen bonding.8,60,61 Natural sphingomyelins are also known to have stronger van der Waals intermolecular (59) Silvius, J. R. In Phospholipids Handbook; Cevc, G., Ed.; Marcel Dekker: New York, 1993; p 1.

Ollila and Slotte

forces than natural glycerophospholipids, because of the difference in acyl chain unsaturation.8 The extra hydrogen bonding ability and the strong van der Waals intermolecular forces that natural sphingomyelins possess are expected to increase the cohesive interactions in the membranes. Since ESM and EPC differ markedly in their hydrogen-bonding properties, it is possible that the increased binding of deoxycholate to ESM membranes was stabilized by hydrogen bonding in a way not possible in the EPC membrane. This explanation is also substantiated by the larger enthalpy seen for deoxycholate partitioning into ESM membranes as compared to EPC membranes (13.2 and 10.8 kJ mol-1, respectively). The observation that sphingomyelin membranes are more easily solubilized by detergents than natural phosphatidylcholine membranes have been explained by the different capacity of sphingomyelin and phosphatidylcholine membranes to accommodate detergents before the membranes are saturated and solubilization occurs.14 Cytotoxic Effect of Bile Salts. Several recent studies show that sphingomyelin, together with cholesterol, exhibits a protective role in cell membranes toward the cytotoxic effect of bile salts.20,21 Earlier studies have also shown that bile salts are capable of partitioning into or binding to lipid membranes and that the partitioning reflects their ability to induce cell damage.12,58,62,63 Deoxycholate partitioned both into EPC and HEPC membranes to a higher degree than cholate. This clearly shows the differences in partitioning strength of dihydroxy bile salts compared to trihydroxy bile salts. These results are in agreement with earlier studies.64 The two conjugated bile salts glycocholate and taurocholate were not able to partition into EPC or ESM membranes at concentrations of 0.3 or 1 mM at T ) 25 °C. In conclusion, this study has demonstrated that bile salts partition very differently into sphingomyelin membranes as compared to phosphatidylcholine bilayers. The difference in hydrogen bonding properties between sphingomyelin and phosphatidylcholine membranes probably explains some of the difference. LA0014196 (60) Pascher, I. Biochim. Biophys. Acta 1976, 455, 433. (61) Boggs, J. M. Biochim. Biophys. Acta 1987, 906, 353. (62) Scholmerich, J.; Becher, M. S.; Schmidt, K.; Schubert, R.; Kremer, B.; Feldhaus, S.; Gerok, W. Hepatology 1984, 4, 661. (63) Schmucker, D. L.; Ohta, M.; Kanai, S.; Sato, Y.; Kitani, K. Hepatology 1990, 12, 1216. (64) Schubert, R.; Schmidt, K. H. Biochemistry 1988, 27, 8787.