Interaction of Bile Salts with Model Membranes Mimicking the

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Interaction of bile salts with model membranes mimicking the gastrointestinal epithelium: a study by isothermal titration calorimetry AUTHOR NAMES Filipe M. Coreta-Gomesa), b), Patrícia A. T. Martins a), b), Adrián Velazquez-Campoy c), d), Winchil L. C. Vaz a), b), Carlos F. G. Geraldesb),e), Maria João Moreno a), b)*

AUTHOR ADDRESS a) b) c)

Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal

Coimbra Chemistry Center, University of Coimbra, 3004-535 Coimbra, Portugal

Institute of Biocomputation and Physics of Complex Systems (BIFI), Joint Unit IQFR-CSIC-

BIFI, Universidad de Zaragoza, Zaragoza, Spain; Department of Biochemistry and Molecular and Cell Biology, University of Zaragoza, Zaragoza, Spain d) e)

Fundación ARAID, Diputación General de Aragón, Spain

Department of Life Sciences, Faculty of Science and Technology, University of Coimbra,

CORRESPONDING AUTHOR: Maria João Moreno, Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal; [email protected]

KEYWORDS Bile Salts/ Membranes/ Isothermal Titration Calorimetry/ Partition/ Translocation

ACS Paragon Plus Environment

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ABSTRACT Bile salts (BS) are bio-surfactants synthesized in the liver and secreted into the intestinal lumen where they solubilize cholesterol and other hydrophobic compounds facilitating their gastrointestinal absorption. Partition of BS towards biomembranes is an important step in both processes. Depending on the loading of the secreted BS micelles with endogeneous cholesterol and on the amount of cholesterol from diet this may lead to the excretion or absorption of cholesterol, from cholesterol saturated membranes in the liver or to gastrointestinal membranes, respectively. The partition of BS towards the gastrointestinal membranes may also affect the barrier properties of those membranes affecting the permeability for hydrophobic and amphiphilic compounds. Two important parameters in the interaction of the distinct BS with biomembranes are their partition coefficient and the rate of diffusion through the membrane. Altogether, they allow the calculation of BS local concentrations in the membrane as well as their asymmetry in both membrane leaflets. The local concentration and, most importantly, its asymmetric distribution in the bilayer are a measure of induced membrane perturbation which is expected to affect significantly its properties as a cholesterol donor and hydrophobic barrier. In this work we have characterized the partition of several BS, non-conjugated and conjugated with glycine, to large unilamellar vesicles (LUV) in the liquid-disordered phase and with liquidordered/liquid-disordered phase coexistence, using isothermal titration calorimetry (ITC). The partition into the liquid-disordered bilayer was characterized by large partition coefficients and favored by enthalpy while association with the more ordered membrane was weak and driven only by the hydrophobic effect. The tri-hydroxy BS partitions less efficiently towards the


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membranes but shows faster translocation rates, in agreement with a membrane protective effect of those BS. The rate of translocation through the more ordered membrane was faster indicating accumulation of BS at specific locations in this membrane.

INTRODUCTION The function of cell membranes is ambivalent: on one hand they form a structured interface ensuring integrity between two separated aqueous media; on the other hand they must be permeable enough to guarantee cell homeostasis. A good example of this dual function may be found in the gastrointestinal membrane (GIM). Prior to absorption at the intestine, non-polar molecules (e.g. Cholesterol) need to get into close proximity of the GIM.1-4 Dietary micelles in the intestinal lumen - rich in bile salts (BS), phospholipids (PL) and fatty acids (FA) - are the common vectors for solubilization of hydrophobic compounds and once in the vicinity of GIM they permit the absorption of the nonpolar molecules by either passive diffusion and/or protein mediated processes.5,6 Glycine conjugates of BS are the most abundant, and among them glycodeoxycholic acid (GDCA), glycochenodeoxycholic acid (GCDCA) and glycocholic acid (GCA).2 The bile salts are produced in the liver, stored in the gallbladder and secreted into the intestinal lumen after eating. Depending on the cholesterol concentration in the liver and bile canaliculi membranes they may present distinct levels of saturation with cholesterol. Once discharged into the intestinal lumen BS micelles mix with the dietary compounds to form dietary micelles and/or larger aggregates. Depending on the total concentration of cholesterol and other compounds (namely phytosterols, fatty acids and fibers), the amount of cholesterol in the dietary micelles may be larger or smaller


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than in the BS micelles secreted and this entero-hepatic cycle may lead to absorption or excretion of cholesterol, respectively.7 In addition to this function as vehicles for the transport of hydrophobic molecules, BS also interact with the lipid membranes and may profoundly alter their properties with impact on the barrier functions.6,8-10 At low concentrations, BS interact mostly with the surface of lipid bilayers inserting only the non-polar face of their rigid structure.11 This leads to an increase in the membrane fluidity,8,12 in contrast to the condensing effect of the structurally related cholesterol.13 As the BS concentration increases, they tend to aggregate in the membrane forming polar pores through which solutes may permeate and eventually lead to membrane solubilization.8 The outcome of BS interaction with biomembranes (increased fluidity, pore formation or solubilization) depends on the local concentration in the lipid bilayer. This is a function of the partition coefficient between the aqueous phase and the membrane, which depends on BS structure. Some results may be found in the literature regarding the partition coefficient of BS into membranes of physiological relevance.8,14-18 The major purpose of those studies was to characterize gallstone solubilization by BS and, consequently, the ratio of bound BS to total lipid was quite high (larger than 0.1). An exception is noted for the work of Heuman and co-workers where the partition coefficient was measured at BS/Lipid ratios from 0.01 up to nearly 1 and showed significant variations on the affinity of BS to the membrane.15 This is due both to the negative charge imposed on the lipid bilayer by the BS (that hinders the binding of further BS molecules) and to changes in the membrane structure and properties upon partition of the BS.8,15 Therefore, the partition coefficient measured at high local BS concentrations is not the intrinsic one and may not be used to rationalize quantitatively the relative affinities of different BS to unperturbed membranes. Additionally, its validity to predict the local concentration of BS in


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conditions different from those used in the specific experiment is limited. To obtain the intrinsic partition coefficients, the lipid to bound ligand ratio should be small. The effect of the local ligand concentration has been characterized by us for two structurally unrelated small molecules bearing a single charge (chlorpromazine19 and sodium dodecyl sulfate20). We found that below a lipid to bound ligand ratio of 25 (local concentration of ligand in the membrane higher than 4 mol%), significant effects are observed on the partition coefficient obtained and interaction enthalpy variation.19,20 In this work we have characterized the partition of several BS (structures in Figure 1) to lipid bilayers prepared from pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and a lipid mixture of POPC, Egg Sphingomyelin (SpM) and Chol at the molar ratio of 1:1:1. The latter is a good model for gastrointestinal epithelial membranes21 and pure POPC was used because it is the most abundant phospholipid in eukaryotic membranes and generally leads to large interaction enthalpies allowing the use of small BS concentrations.19,20,22,23 Another important parameter in the interaction of BS with membranes is its rate of translocation between leaflets (flip-flop). This rate is expected to depend significantly on the local concentration of BS in the membrane due to the formation of BS aggregates at high concentrations that generate polar pathways through the lipid bilayer. For BS in the monomeric form, the rate of translocation has been reported to be fast for BS in the neutral form and very slow for ionized BS (negligible in 24 h)24 although a moderately fast translocation has been obtained by other authors at neutral pH values.25 We try to solve this controversy by evaluating the rate of translocation of BS in the distinct membranes using the uptake and release protocol for ITC.19,26


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Figure 1. Chemical structure of the bile salts used in this work. The structure of the glycine conjugate is shown; un-conjugated bile salts have an OH group instead of the glycine (inside the square) and lack the G in their abbreviated name.

The enthalpy variation upon partition to the lipid bilayer may give important information regarding the forces involved in the interaction and the effects of BS on the properties of the bilayers. Interaction with lipid bilayers in the liquid-disordered phase is usually accompanied by a negative enthalpy variation reflecting the good solvation properties of the membrane.20,27 In contrast, partition into membranes in the liquid-ordered (or gel) phase is due mainly to the hydrophobic effect and leads to positive enthalpy variations.16,28-31 The phase behavior of the ternary mixture of POPC:SpM:Chol used in this work has been studied by several authors and at 37 oC some found evidences for the coexistence of liquid-ordered/liquid-disordered phases,32 while others for a pure liquid-disordered phase.33,34 This controversy may be due to the different techniques used which are sensitive to distinct length and time scales of the phase heterogeneity. The enthalpy variation and the partition coefficient for the interaction of solutes with POPC:SpM:Chol bilayers may add important information regarding the phase properties of those membranes. When using ITC, the enthalpy variation obtained directly in a partition experiment results from several contributions, namely the intrinsic partition enthalpy, the heat of dilution and changes in


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the ionization of the solute (which depend on the ionization enthalpy of the pH buffer).35,36 The intrinsic enthalpy variation of partition and the number of protons released/captured by the solute upon partition may be obtained through the measurement of the overall enthalpy variation in pH buffers with distinct ionization enthalpies.37,38 Due to this contribution, buffers with large ionization enthalpies are erroneously considered less suitable for ITC measurements. This may however be of great practical utility when the intrinsic association enthalpy is very low and is commonly used for protein-ligand assays.39,40 In this work we have used this approach to characterize the interaction of both un-conjugated and glycine-conjugated bile salts with POPC membranes, obtaining the intrinsic partition enthalpy variation and the change in the ionization state of the BS upon association with the membrane. The global charge of the BS when associated with the lipid bilayer (and the corresponding apparent pKa values) may in this way be obtained facilitating the interpretation and prediction of their rates of translocation through the bilayer.

EXPERIMENTAL Sodium salts of GCDCA, GCA, GDCA, and acid form of DCA, CDCA and CA were purchased from Sigma. Stock solutions of glycine conjugated BS at high concentration were prepared directly in the required buffer and whenever necessary the pH was adjusted to 7.4, smaller concentrations were prepared by dilution. High concentrations of the unconjugated BS in aqueous solvents could not be prepared directly due to the formation of gels. In this case, stock solutions were prepared in methanol and the aqueous solutions were prepared by measuring the required volume of the stock solution followed by evaporation of the methanol and solubilization


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of the BS film in the aqueous solvent. The final value of pH was verified and adjusted to 7.4 with HCl or NaOH when necessary. The stock solutions were kept at 4 oC and used within two weeks. The lipids POPC, Chol and SpM were purchased from Avanti Polar Lipids (Alabaster, AL, USA). The required aqueous suspensions of lipids were prepared by evaporating a solution of the necessary lipid mixture in an azeotropic solution of chloroform:methanol (87:13, v/v), blowing dry nitrogen over the heated solution and leaving the residuum in a vacuum desiccator for at least 8 h at 23ºC in the dark. The solvent free residue was then hydrated with one of the pH buffers used, namely: 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES); Phosphate; or Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer (all at 10 mM, pH 7.4), each containing 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), and 0.02% w/v of sodium azide (NaN 3 ). The hydrated lipid was subject to several cycles of vortex/incubation at a temperature above the lipid main transition temperature (room temperature for POPC and 60ºC for POPC:Chol:SpM) for at least 1 hour to produce a suspension of multilamellar vesicles. These vesicles were then extruded at the same incubation temperature, using a minimum of 10 passages, through two stacked polycarbonate filters (Nucleopore) with a pore diameter of 0.1 μm to obtain large unilamellar vesicles (LUV). For the preparation of the lipid vesicles containing BS (release experiments), a concentrated solution of BS (a film for the case of the unconjugated BS) was equilibrated with the preformed lipid vesicles for 24 h at 37 ºC with gentle stirring. The final phospholipid (POPC and SpM) concentration was determined using a modified version of the Bartlett phosphate assay and the final Chol concentration was determined by the Lieberman−Burchard method, as previously described.19


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Titrations were performed on a VP-ITC instrument from MicroCal (Northampton, MA) at 37 °C. The stirring speed was 296 rpm and reference power was 10 μcal s−1. As recommended by the manufacturer, a first injection of 4 μL was performed and the titration proceeded with additions of 10 μL LUVs suspension per injection. Two avoid reduction in the available amount of ligand due to adsorption to some equipment parts, the cell was pre-rinsed with a solution having the same composition of the one used in the experiment. All solutions were degassed for 15 min. Two protocols for the ITC experiments were used in this work: (I) uptake, in which liposomes were injected into a BS solution in the cell, and (II) release, in which a liposome solution containing BS was injected into the cell containing buffer. Altogether, those protocols allow obtaining the thermodynamic parameters for the interaction as well as a qualitative evaluation of the translocation rate constant.19,26 The thermograms were integrated using the data analysis software Origin 7.0 as modified by MicroCal and the resulting differential titration curves were fitted with the appropriate equations using Microsoft Excel and the Add-In Solver.20 The predicted heat evolved in each titration step and the best fit of the model to the experimental values were obtained by least square minimization considering a simple partition (partition coefficient defined as the ratio of the local ligand concentration in both phases) as previously described.19 We have considered both a fixed amount of available lipid (total or half depending on the relative rates of translocation) and a variable fraction of accessible lipid, γ, obtained from the best global fit of uptake and release protocols, as explained elsewhere. 19 The molar volumes, VL considered for the lipids used in this work were 0.76 dm3 mol−1 for POPC and SpM, and 0.39 dm3 mol−1 for cholesterol.41 For the mixture POPC:Chol:SpM 1:1:1 a weighted average value was used leading to 0.62 dm3 mol−1.


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RESULTS AND DISCUSSION Partition of BS into bilayers in the liquid disordered phase (POPC) using the uptake protocol. The concentration of solute (designed as ligand in this work to highlight their ability to associate with several biological binding agents including membranes and proteins) used in partition experiments influences significantly the parameters obtained, both the partition coefficient and the interaction enthalpy. We have previously characterize this effect for the association of the small molecules CPZ (univalent cation) and SDS (univalent anion) with POPC membranes and found that at the high values of ionic strength used (150 mM), no significant effects are observed above a lipid to bound ligand ratio of 25 (local concentration of ligand in the membrane below 4 mol%).19,20 Given the similar molecular weight of the BS and their single charge at neutral pH values, similar effects of ligand concentration are expected. We have nevertheless performed some experiments with DCA where the total concentration was changed from 10 to 100 µM leading to a minimum POPC to bound ligand ratio (at the first lipid addition) from near 100 to 10 respectively. The results obtained were in agreement with a non-perturbing effect for local concentrations of ligand below 4 mol% (results not shown). The large enthalpy variation associated with the partition of most BS to the membranes used in this study allowed us to actually use even smaller local ligand concentrations (typically below 2 mol% POPC and below 0.5 mol% for POPC:SpM:Chol membranes). At those small local concentrations the partition parameters are the intrinsic ones and therefore, the standard state considered in this work corresponds to infinite dilution. In a representative experiment for the titration of DCA with POPC LUVs, 10 mM of lipid was loaded in the syringe to titrate a solution of DCA in the cell with a concentration of 10 μM, Figure 2 Plot A. The final lipid concentration in all independent LUV preparations was


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quantified and small variations are obtained, the concentrations given in the figures correspond to the values observed in the particular experiment shown. The lipid concentration accessible to the ligand, and not the total lipid concentration, is the relevant parameter to obtain the partition coefficient from the analysis of the experimental results. At this stage we have assumed slow translocation (a fraction of accessible lipid, γ, equal to 0.5) as this is generally assumed for the ionized form of BS at low local concentrations.4,24 This question will be addressed later.

-4

-12 0.0

0.0

dQ/dt (µJ/s)

dQ/dt (µJ/s)

-8

-5

-0.2

-0.4

-10 -0.2

-0.4 30

0.5

0.0

60

90

120 150

30

Time (min)

1.0

[POPC] (mM)

1.5

0

2

4

60

90

120 150

6

8

Time (min)

Heat/peak (µJ)

B

A Heat/peak (µJ)

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-15

[POPC] (mM)

Figure 2. Typical results for the titration of unconjugated BS at 37 ºC in HEPES buffer: DCA 10 µM in the cell with 9.3 mM POPC in the syringe (plot A); and CA 30 µM in the cell with 50 mM POPC in the syringe (plot B). The line is the best fit with a simple partition model for K Pobs =2.8×103, ∆H 0 = -13 kJ mol-1 and

K Pobs =3.2×102, ∆H 0 = -4.6 kJ mol-1, respectively,

considering that only the lipid in the outer leaflet is accessible to the BS (γ=0.5). The insets show the respective thermograms.

As observed in Figure 2, the partition of DCA to the POPC membrane is an exothermic process. The ratio of lipid to bound BS was equal to 50 in the first 10 µL injection and increased to 100 when 50% DCA is associated with the liposomes. At this small local concentration of ligand no membrane perturbation is expected and therefore, the partition coefficient obtained is the intrinsic one without the need to correct for electrostatic effects20. To determine the effect of BS


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hydrophobicity in their partition to membranes, we have also performed the titration of CDCA and CA. Typical results obtained for the less hydrophobic BS with three hydroxyl groups (CA) are shown in Figure 2B. The effects of conjugation with glycine were also evaluated; the results obtained are collected in Table 1. A first observation that may be taken is that the observed partition coefficient depends on the number and position of the hydroxyl groups in the BS in the order CDCA>DCA>>CA. The same trend was observed earlier for taurine conjugates,18 although with somewhat distinct partition coefficients and relative values. For the glycine conjugates the partition coefficients obtained for GDCA and GCDCA are essentially the same, both being much larger than that of GCA in agreement with previous work reported in literature for conjugated and non-conjugated BS.14,15,17,18,42 The partition coefficient obtained for the glycine conjugates is only slightly smaller than that of the unconjugated BS. In fact, for small local concentrations in the membrane, BS conjugation does not seem to influence strongly the partition coefficient obtained. The value obtained in this work for CA and GCA is not very different from the values encountered in the literature for CA (4.2×102),8 and TCA (3.1×102).15 Similarly, the partition coefficient reported for UDCA (3.7×102)8 is only twice that of TUDCA (1.5×102) or GUDCA (1.3×102)14, obtained at lipid to BS ratios of 100, 17 and 50 respectively. The enthalpy variation upon association with the POPC membranes is also shown in Table 1 and is always negative in spite of significant variations for distinct BS. The enthalpy variation is more exothermic for DCA and CDCA than for CA (in qualitative agreement with previous results)16 suggesting a better solvation of the di-hydroxy BS by the POPC bilayer. This result is compatible with a more superficial location of CA in the bilayer as is generally considered.42 Conjugation with glycine does not significantly affect the enthalpy variation upon partition to the POPC bilayers except for a smaller differentiation between di and tri-hydroxy BS. The enthalpy


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variation reported in Table 1 is the one observed directly in a titration experiment ( ∆H o,obs ) and is the result from several processes; namely changes in the ionization state of the solute, corresponding variations in the buffer ionization, and interaction with the lipid bilayer. To allow the interpretation of the enthalpy variation obtained for the different BS in terms of their interaction with the POPC bilayer it is therefore necessary to characterize the contributions from changes in their ionization state. This may be done through the dependence of the observed o,ioniz enthalpy variation ( ∆H o,obs ) on the ionization enthalpy of the buffer used ( ∆H buffer ), which will

be discussed in the next section. Table 1. Partition coefficient ( K Pobs ) and enthalpy variation ( ∆H o,obs ) for the interaction of BS with POPC bilayers. The left columns consider an equilibration factor equal to 0.5 (slow translocation) while the columns to the right show the results obtained by the best global fit of uptake and release experiments. The values show correspond to the average and standard deviation of at least 3 (typically 5) independent experiments. The temperature was 37 ºC and the aqueous buffer was HEPES at pH=7.4. Uptake Protocol

Uptake & Release Protocol

(assuming γ=0.5)

(γ from the global fit )

BS 3 K Pobs (×10 )

∆H o,obs

3 K Pobs (×10 )

-1

∆H o,obs

γ

-1

(kJ mol )

(kJ mol )

DCA

2.5 ± 0.3

-13 ± 2

1.9 ± 0.3

-14 ± 2

0.7± 0.2

CDCA

3.3 ± 0.6

-14 ± 3

1.9 ± 0.4

-11 ± 1

0.9 ± 0.2

CA

0.7 ± 0.2

-8 ± 3

0.3 ± 0.1

-5 ± 1

1.0 ± 0.2


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GDCA

2.0 ± 0.8

-15 ± 4

GCDCA

2.1 ± 0.9

-16 ± 2

0.33 ± 0.10

-13 ± 4

GCA

Contribution of changes in ionization to the overall enthalpy variation observed. The interaction of the distinct BS with POPC bilayers was characterized in three pH buffers with distinct ionization enthalpies, namely phosphate, HEPES and Tris.35 The dependence of the observed partition enthalpy variation on the ionization enthalpy of the different buffers is shown in Figure 3. 20

∆H o, obs (kJ mol-1)

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10 0 -10 -20 -30 0

a

20

40

ioniz (kJ mol-1) ∆H o,buffer

Figure 3. Dependence of the observed enthalpy variation (obtained from the best fit using the value of γ obtained in this work) due to partition into POPC membranes ( ∆H o,obs ) on the buffer’s o,ioniz ionization enthalpy ( ∆H buffer ), for DCA (), CDCA (), CA () and GDCA (■). The enthalpy

of ionization for the different buffers was obtained from literature

35,36

taking into account the

different temperature. The lines are the best linear fit from which the number of protons released by the buffer (captured by the BS, ∆nH+ ), and the intrinsic enthalpy variation due to partition ( ∆H Po ) are obtained, slope and intercept respectively (Tables 2 and 3).


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There is a strong dependence of the observed enthalpy variation with the buffer’s ionization enthalpy for the case of unconjugated BS indicating a significant variation in their ionization state when associating with the bilayer. In contrast, the observed enthalpy variation was not significantly affected by the buffer for the case of the glycine conjugate GDCA. The number of protons captured by the BS upon partition to the lipid bilayer may be obtained from the slope and is given in Table 2 together with the charge and apparent ionization constant ( pK aL ) of the BS when associated with the bilayer. The intercept of the dependence of ∆H o,obs on the ionization enthalpy of the buffer gives the intrinsic enthalpy variation associated with partition ( ∆H Po ) and is shown in Table 3. Table 2. Number of protons released by the buffer (captured by the BS), ∆nH+ , due to the partition of the distinct BS into POPC bilayers. The pK a values of BS in the aqueous media ( pK aW ) were taken from the literature43 and are also shown, as well as the calculated global W L charge of the BS in water ( zBS ) and in the bilayer ( zBS ) at pH=7.4 in the bulk aqueous solution.

The apparent ionization constant of the BS when associated with the lipid bilayer ( pK aL ) was calculated from the fraction of ionized form, assuming the same pH as in the bulk aqueous phase, and is also given. BS

∆nH+

pK aW a

DCA

0.57 ± 0.05

5.02

CDCA

0.52 ± 0.03

4.98

W zBS

L zBS

pK aL

∆pK a

-0.43

7.5

2.5

-0.48

7.4

2.5

-1.0

CA

0.51 ± 0.07

5.00

-0.49

7.4

2.4

GDCA

-0.07 ± 0.09

3.86

-1.0

3.9

0.0


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a

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values of pK aW taken from reference 43

The neutral form of the unconjugated BS is significantly stabilized when associated with the POPC bilayer leading to a change of nearly 0.5 charge units for all BS, corresponding to a increase of approximately 2.5 units in their apparent pK aL (when calculated relative to the pH value of the bulk aqueous solution). In contrast, the interaction of the glycine conjugated BS (GDCA) does not lead to variations in its ionization state. The increased stability of the ionized form for glycine conjugated BS (lower pK aW ) leads to a larger energetic cost for neutralization upon association with the lipid membrane and partially explains this result. However, if this was the only variable one would expect ∆nH+ equal to 0.1. The stabilization of the neutral form of unconjugated BS, together with the large and favorable enthalpy variation associated with partition to the POPC bilayer, suggest the formation of a hydrogen bond between the BS and POPC. The establishment of the hydrogen bond requires protonation for the case of unconjugated BS but not for the glycine conjugates. In addition, the increased distance from the carboxylic group and the hydrophobic fused rings in the case of glycine conjugated BS permits a close interaction with the lipid bilayer while maintaining the ionized group in the aqueous phase. The stabilization of the neutral form of the un-conjugated BS associated with the POPC bilayer is significantly larger than that obtained in BS micelles44. This further supports the stabilization of the neutral form due to the establishment of a hydrogen bond with POPC. When the intrinsic thermodynamic parameters for partition of the distinct BS are compared (Table 3), it is observed that the partition of the glycine conjugates is accompanied by a small favorable entropy increase while the entropy of the system actually decreases for the case of the un-conjugated BS, an exception being noticed for CA where no entropy variation is observed.


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Large positive variations in the system entropy are usually considered a fingerprint for the removal of non-polar surfaces from water due to the hydrophobic effect.45,46 The results obtained may therefore be interpreted as the hydrophobic effect not being the main driving force for the interaction. In fact, the enthalpy variation observed upon transfer of a non-polar molecule from water to a non-polar media depends on the interactions established between the molecule being transferred and the non-polar media and is partially compensated by a variation in the system entropy.45,47 Lipid bilayers have a high degree of organization with transverse polarity gradients, and the alignment of the dipole moments of the amphiphilic lipid molecules generates electric fields of very high magnitude.48 Strong interactions, stabilized by enthalpy, may therefore be established between amphiphilic molecules and lipid bilayers. We are currently exploring this question using Molecular Dynamics simulations. Table 3. Intrinsic thermodynamic parameters of the partition of unconjugated and glycine conjugated BS to POPC lipid membranes at 37 ºC. The enthalpy variation for the unconjugated BS was obtained from the intercept of the dependence with the buffer ionization enthalpy while that of the glycine conjugates is the same as in Table 1. ∆H Po

∆G o

T ∆S o

(kJ mol-1)

(kJ mol-1)

(kJ mol-1)

DCA

-25 ± 2

-19.4 ± 0.3

-5 ± 2

CDCA

-22 ± 1

-19.5 ± 0.5

-3 ± 1

CA

-14 ± 2

-14.9 ± 0.7

1±2

GDCA

-15 ± 4

-19.6 ± 1.0

5±4

GCDCA

-16 ± 2

-19.7 ± 1.1

4±3

BS


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GCA

-13 ± 4

-14.9 ± 0.8

Page 18 of 31

2±4

Qualitative evaluation of the rate of translocation – the uptake and release protocol. In the previous sections it was assumed that throughout the titration only the lipids in the outer monolayer of the liposomes were accessible to the BS (γ = 0.5). This was justified by the usual assumption that the rate of translocation of BS in the ionized form is very small8,24 and by the fact that only 0.4 % of un-conjugated and 0.03 % of glycine conjugated BS are in the neutral form in the aqueous phase at pH=7.4. The observation that significant changes in the ionization state of un-conjugated BS take place upon association with the POPC membranes justifies a closer evaluation of the rate of translocation through the membrane. We have performed release experiments to qualitatively evaluate the rate of BS translocation following the uptake and release protocol.26 The ratio of lipid to BS used in the release experiments was carefully chosen to match the conditions observed at half titration in the respective uptake experiment. Figure 4 shows typical results obtained for the uptake and release protocols for the association of CDCA and CA with POPC membranes. From these experiments it is possible to determine the partition coefficient, the enthalpy variation associated with partition ( ∆H o,obs ) and the equilibration factor (γ ) which includes information regarding the relative characteristic time of BS translocation and the duration of the titration experiment. The parameters obtained for all the unconjugated BS are given in Table 1, the equilibration factor obtained for the glycine conjugates was close to 0.5 for all BS studied.


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8

0

-8

1.4 1.2

1.4

χ2

χ2

0 1.8

0.5 0.7 0.9

0.5 0.7 0.9

γ

0.0

0.5

[L] (mM)

-15

1.0

1.0

Heat/peak (µJ)

15

Heat/peak (µJ)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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γ

1.0 0

3

6

[L] (mM)

Figure 4. Representative titration curves obtained at 37 ºC following the uptake (○) and release (□) protocols for association of BS with POPC membranes in HEPES buffer at pH=7.4. The lines are the global best fits19 and the insert shows the dependence of the square deviation of the global best fit (χ2) as a function of γ (■) and the confidence interval at 95% (─).49 Plot A:

[CDCA ]cell

uptake

= 10 µM, [ POPC]syr = 9 mM, L:BSrelease =90, K Pobs = 2.3×103, ∆H o,obs = -11 kJ mol-1

and γ = 0.73. Plot B: [ CA ]cell = 30 µM, [ POPC]syr = 50 mM, L:BSrelease =110, K Pobs = 2.8×102, uptake

∆H o,obs = -5.1 kJ mol-1 and γ = 1. The results obtained clearly show that γ and therefore the translocation rate, is dependent on the molecular structure of the BS when unconjugated. A faster translocation is obtained for the trihydroxy BS (CA, Table1) leading to equilibration with the whole lipid pool during the titration experiment (γ = 1). Intermediate values are obtained for the di-hydroxy BS with DCA being the one with the slower translocation (γ = 0.7 ± 0.2). The equilibration factor for the glycine conjugates was always close to 0.5 indicating that translocation is slower than the time required to perform the titration (a few minutes for each titration step). The unexpected faster translocation obtained for the most hydrophilic CA may be related with local defects induced in the membrane, in agreement with the decrease in the membrane order observed by NMR50 and fluorescence anisotropy.8


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Page 20 of 31

A quantitative evaluation of the rate of translocation is not possible with this approach but intermediate values of γ indicate that the characteristic time for translocation is between 5 and 30 min.19 The results obtained are compatible with the work of Donovan and co-workers that found 15 minutes as the upper limit for the characteristic time of GDCA translocation through EggPC membranes at a lipid:BS ratio of 25.14 Partition of BS into membranes of POPC:SpM:Chol (1:1:1). The lipid composition of gastrointestinal membranes is enriched in sphingomyelin and cholesterol21 and the partition of BS into those membranes is of high relevance in the recirculation of BS as well as on their effect on membrane permeability. It has already been shown that the presence of high cholesterol levels decreases the partition coefficient of solutes in general19,29-31 and of BS in particular8,12,14,15. Partition into sphingomyelin containing membranes (pure and cholesterol mixtures) is also less extensive

16,19,29-31

although one study showed that 30 mol% sphingomyelin did not affect the

partition of CA into EggPC bilayers.8 We have characterized the association of the BS with lipid bilayers composed of POPC:SpM:Chol (1:1:1) as a model for the outer leaflet of the gastrointestinal membranes. Some representative results are shown in Figure 5, they are all collected in Table 4 except for the most hydrophilic BS (GCA) whose affinity for those membranes was too small to be measured accurately.


15

0

0 -15

-40

1.4 1.2

χ2

-80

1.0

1.2 1.1 1.0 0.3 0.5 0.7 0.9

0.3 0.5 0.7 0.9

-120 0

γ

γ

2

4

6

-30

Heat/peak (µJ)

40

χ2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Heat/peak (µJ)

Page 21 of 31

0

[L] (mM)

2

4

6

-45 8

[L] (mM)

Figure 5. Representative titration curves obtained at 37 ºC following the uptake (○) and release (□) protocols for association of BS with POPC:SpM:Chol 1:1:1 membranes in HEPES buffer at pH=7.4. The lines are the global best fits19 and the inset shows the dependence of the square deviation of the global best fit (χ2) as a function of γ (■) as well as the confidence interval at 95% (─) or 67% (---).49 Plot A: [ DCA ]cell = 20 µM, [ POPC]syr = 60 mM, L:BSrelease =220, uptake

K Pobs = 1.9×102, ∆H o,obs = 50 kJ mol-1 and γ = 1. Plot B: [ CA ]cell = 30 µM, [ POPC]syr uptake

mM, [ POPC]syr

release

uptake

= 60

= 52 mM, L:BSrelease =250, K Pobs = 9.2×101, ∆H o,obs = 16 kJ mol-1 and γ = 0.93.

The values obtained for the partition coefficient are nearly an order of magnitude smaller than those for association with POPC bilayers indicating a poor solvation of the bile salts by the POPC:SpM:Chol 1:1:1 membrane. In fact, the interaction enthalpy variation is always positive, suggesting that partition is mainly driven by the hydrophobic effect and that the accommodation of the ligand in the membrane requires the disruption of lipid-lipid interactions. This is in agreement with previous studies on partition into cholesterol enriched bilayers and reflects the increased order of the membranes.13 As observed for partition into POPC membranes, conjugation with glycine leads to a small decrease in the partition coefficient. The large and positive enthalpy variation obtained for the interaction of all BS with this ternary lipid bilayer may also reflect membrane perturbation, see below.


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Page 22 of 31

Table 4. Partition coefficient and enthalpy variation for the interaction of BS with lipid membranes composed of POPC:SpM:Chol 1:1:1, at 37 oC in HEPES buffer at pH=7.4. The equilibration factor was considered equal to 1 (fast translocation) for all BS. The values show correspond to the average and standard deviation of at least 3 (typically 5) independent experiments.

BS

2

∆H o,obs

∆G o

T ∆S o

(kJ mol-1)

(kJ mol-1)

(kJ mol-1)

K Pobs (×10 )

DCA

2.1 ± 1.0

53 ± 17

-13.8 ± 1.2

67 ± 17

CDCA

1.6 ± 0.5

36 ± 12

-13.0 ± 0.8

49 ± 12

CA

0.7 ± 0.3

37 ± 25

-11.1 ± 1.1

48 ± 23

GDCA

1.3 ± 0.3

29 ± 6

-12.6 ± 0.6

42 ± 6

GCDCA

1.2 ± 0.7

21 ± 9

-12.3 ± 1.5

33 ± 9

Surprisingly, the best fit of the uptake and release protocols performed for the unconjugated BS indicates that translocation occurs faster than the time required to perform the titration (γ = 1). This is contrary to the results obtained with binary mixtures of PC or SpM with cholesterol.14,51 The fast rates of translocation observed may result from phase separation in this ternary lipid mixture. The phase behavior of POPC:SpM:Chol mixtures have been studied by several authors and there is evidence for coexistence of liquid-ordered and liquid-disordered phases at 37 oC.32 If the partition of the BS into one of the distinct phases (presumably that enriched in POPC) or to the phase boundaries is preferential, its local concentration would be higher and could lead to aggregation and facilitate its translocation.8 In agreement with this interpretation, we observed a strong dependence of the parameters obtained with the lipidic sample preparation, which is


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illustrated by the large standard deviation. We have also observed some dependence on the concentration of BS pointing towards a decrease in the partition coefficient and a concomitant increase in the enthalpy variation as the ligand concentration is increased. This effect has not been systematically studied but suggests a significant perturbation of the lipid bilayer even at the small concentrations used in this study. The results shown in Table 4 are the average values obtained for a total BS concentration from 20 to 50 µM which lead to a ratio of lipid to bound BS larger than 200 throughout the titration.

CONCLUSIONS The results of this work quantitatively show that the partition of BS to lipid bilayers is higher for the most hydrophobic di-hydroxy BS, CDCA and DCA, and lowest for the tri-hydroxy CA, both for unconjugated and glycine conjugated forms. Partition of BS to POPC membranes is enthalpically driven, being more exothermic for DCA and less exothermic for GCA, while the association with POPC:SpM:Chol 1:1:1 membranes is always endothermic with larger enthalpy variations being obtained for the unconjugated BS. Those results cannot be quantitatively compared with data available in literature due to the very high local concentration of BS used in those studies which affects the observed partition and interaction enthalpy variation. Significant changes in the ionization state of unconjugated BS were observed upon partition to POPC bilayers, with the stabilization of the neutral form and a shift of 2.5 units in their apparent pK a , while no variation was observed for the glycine conjugates. Together with the large and favorable enthalpy variation, this result supports the formation of a hydrogen bond between POPC and the neutral form of the unconjugated BS as well as with the ionized form of glycine conjugates (possibly with the amide group).


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Page 24 of 31

Qualitative information regarding the rate of translocation through the lipid bilayer was also obtained showing that the unconjugated BS translocate faster than their glycine conjugates and that the translocation of the tri-hydroxy BS (CA) is faster than that of the di-hydroxy BS (DCA and CDCA). Translocation through the ternary lipid bilayer of POPC:SpM:Chol 1:1:1 was faster than through the POPC membrane, suggesting the preferential interaction of the BS with particular regions within a laterally heterogeneous membrane32-34 (fluid domains and/or interfaces) leading to a corresponding increase in the local BS concentration. The less extensive partition of the BS for the cholesterol enriched bilayers supports an improved resistance of those membranes for BS solubilization. On the other hand, the faster translocation observed facilitates the passive diffusion of BS across gastrointestinal and bile canaliculi membranes with relevance for the re-absorption of BS at the intestine and excretion into the gallbladder respectively.

ACKNOWLEDGMENTS This work was supported by research Grants from the Portuguese Ministry for Science and Higher Education through Fundação para a Ciência e a Tecnologia (FCT) via the PTDC program cofinanced by the European Union (project IN0479-AI E-07/12, and UID/QUI/00313/2013 UID/QUI/00313/2013). Filipe Coreta-Gomes acknowledges FCT for the Ph.D. fellowship (SFRH/BD/40778/2007) and the short term fellowship ASTF 147-2013 from European Molecular Biology Organization. Patrícia A. T. Martins acknowledges FCT for the Ph.D. fellowship SFRH/BD/38951/2007.


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TOC


Interaction of Bile Salts with Model Membranes Mimicking the

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