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Removal of sodium ion from bile acid solution using diffusional dialysis with cation exchange membrane
Separation and Purification Technology 33 (2003) 189 /197 www.elsevier.com/locate/seppur
Removal of sodium ion from bile acid solution using diffusional dialysis with cation exchange membrane Hiroyoshi Inoue * Radioisotope Institute for Basic and Clinical Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan Received 29 October 2002; received in revised form 5 January 2003; accepted 11 January 2003
Abstract The behavior of sodium ions passing through a CM1 cation exchange membrane was studied with electroconductive and diffusional membrane permeabilities based on nonequilibrium thermodynamics to remove sodium ion from bile acid solution. These membrane permeabilities of sodium ion in order of amplitude showed sodium chloride / sodiumtaurocholate /sodium cholate systems in all cases. In the sodium cholate and sodium taurocholate systems, the curves relating the electroconductive membrane permeability with concentration showed distinct changes in gradient at around the concentration of primary micelle formation of each bile acid solution. This was attributable to an increase in membrane/solution distribution of bile acid anion on basis of the micelle formation. Using diffusional dialysis, 7.3% of sodium ion was removed from rat serum. Sodium ion was significantly reduced with increasing total bile acid concentration in rat serum (P B/0.05; correlation coefficient / /0.606). # 2003 Elsevier B.V. All rights reserved. Keywords: Cation exchange membrane; Sodium; Cholate; Taurocholate; Rat serum; Membrane permeability
1. Introduction Bile salts undergo enterohepatic circulation, but bile acids possess a degree of hepatotoxicity, and hence have to be excreted rapidly into bile ducts. Most chronic cholestatic liver diseases, especially primary biliary cirrhosis (PBC) and primary sclerosing cholargitis (PSC), result from progressive destruction or loss of intrahepatic or extrahepatic bile ducts. For treating these diseases, the hydrophilic ursodeoxycholic acid (UDCA) is clini-
* Tel.: /81-942-31-7584; fax: /81-942-31-7702. E-mail address: [email protected] (H. Inoue).
cally applied on basis of stimulating the excretion of bile acids and other potentially hepatotoxic compounds [1]. It may also protect against injury of the bile ducts by bile acids by interfering with the uptake of bile acids into the hepatocytes. The uptake of bile acids into hepatocytes is mediated by a relatively specific Na -dependent transporter and also by a Na -independent transporter with a broader substrate specificity that may be involved in the uptake of non-bile acid organic anions [2,3]. The Na -dependent pathway accounts for more than 80% of taurocholate uptake and for less than 50% of cholate uptake [4,5]. Thus it is predicted that by abolishing the Na gradient in the basolateral hepatocyte membrane the blood bile
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acids are not taken up into the hepatocytes and circulate in body. Attempts have been made to improve the hemodialysis process by removing various ions from blood with a membrane or a column [6,7]. In this process, the ion permeation across the membrane, regarded as a multicomponent ionic system, has been too complicated to describe in an analytical form, and ionic transport too hard to control. The addition of charged sites into the membrane provides ionic permselectivity [8]. The ionic permselectivity in the charged membrane can be achieved by controlling the membrane/solution ionic distribution and the ionic diffusion within the membrane. The design of the removal processes is important for studying the behavior of sodium ion and bile acid anion within the membrane phase and in the boundary between the membrane and solution phases. Phenomenological analyses based on nonequilibrium thermodynamics are suitable for the various transport phenomena arising across a membrane. In the present study, the transmembrane potential, the membrane conductance, the fluxes of bile acid salt, as well as the concentrations of bile acid anion within a membrane were measured to quantitatively estimate the Na selective transport performance in the sodium chloride (NaCl), the sodium cholate (NaCA), or the sodium taurocholate (NaTCA) single salt system with a cation exchange membrane. We finally tried to remove the Na from the rat serum using diffusional dialysis with the cation exchange membrane.
2. Experimental procedures 2.1. Bile acid solution preparation NaCl aqueous solution was prepared using conductive water (]/17 MV cm) and guaranteed reagent grade NaCl (Nacalai Tesque, Japan) without further purification. NaCA and NaTCA aqueous solutions were prepared from conductive water and guaranteed reagent grade bile salts (Nacalai Tesque, Japan) which were purified by foam fractionation [9,10].
The apparent critical micellar concentration (CMC) of each bile salt was measured fluorimetrically using the polar probe diphenylhexatriene [11]. A 1-ml aliquot of 10 mmol dm 3 diphenylhexatriene in tetrahydrofuran was added to different concentrations of bile salts dissolved in 2.0 cm3 of Tris /saline buffer, pH 7.2, I /0.1. The tubes were incubated in the dark at 37 8C for 30 min and the resulting fluorescence was measured at an excitation wavelength of 355 nm and an emission wavelength of 430 nm (RF-5000; Shimadzu Corp., Tokyo). Sodium activity coefficients in bile acid solutions were calculated from the equation E /E0/ S log aNa, where E is the potential between an Ag/ AgCl wire in a saturated KCl bridge and the sodium sensitive electrode, both immersed in the unknown solution. E0 and S were determined by including samples of 10 and 100 mmol dm 3 NaCl with each series of unknowns. The sodium activity coefficients, gNa, in 10 and 100 mmol dm 3 NaCl were taken as 0.909 and 0.787, respectively [12]. 2.2. Membrane transport measurement A highly selective cation-exchange membrane, Neocepta CM1 (Tokuyama Corp., Japan), was used throughout the present study after immersion in 2 /10 3 mol dm 3 bile acid for a few days to replace all the exchangeable ions with Na . The CM1 membrane is composed of a polyvinyl chloride with an ion exchange group of sulfonic acid type and its basic physicochemical properties are as follows: exchange capacity, 2.4 mequiv gdry per resin; water content, 38%; thickness, 0.14 mm. Transmembrane potentials, conductances and ion fluxes were measured with the experimental setup shown in Fig. 1. The system consisted of solution phase I, where the bile acid concentration was varied from 2 /10 4 to 2 /10 2 mol dm 3, and solution phase II, where the bile acid concentration was fixed at 2 /10 3 mol dm 3, separated by the CM1 membrane. The membrane potential measurements were performed with calomel electrodes connected to a high-input impedance voltmeter (Takeda Riken; TR6856); conductance measurements were performed by
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Fig. 1. Schematic representation of experimental assemblies for the electrochemical measurements. Both solution phases were stirred at 90 rpm.
means of filling the chamber with mercury as a conductivity electrode connected to an impedance bridge (Yokogawa /Hewlett /Packard; 4255A) and inter-compartmental ion fluxes were estimated by means of tracing the time changes in solution conductivities of both solution phases. The concentrations of bile acid within the membrane phase were measured by the following procedure. A piece of the CM1 membrane, preweighed at the reference conditions in which all the membranes were equilibrated sufficiently with 2 /10 3 mol dm 3 bile acid solution, was immersed in the test solution containing [2,4-3H(N)]-cholic acid (American Radiolabeled Chemicals Inc.) or [24-14C]taurocholic acid (American Radiolabeled Chemicals Inc.) for 96 h. The test membrane, after rinsing with conductive water and wiping off the adhering surface solution, was measured with a liquid scintillation counter (Beckman; LS6500). All measurements were carried out at 369/1 8C.
2.3. Diffusional dialysis for serum Blood collecting procedures used in this study were approved by the internal animal use committee of Kurume University School of Medicine. Male Sprague /Dawley rats (Seac Yoshitomi; Japan), weighing 200 /250 g, were housed individually in temperature-controlled rooms (239/ 2 8C, humidity 559/10%). Blood samples (1.5 cm3) were collected to take the serum from the femoral vein every 2 days. The sera were then stored at /80 8C until the bile acid transport experiments were performed. The total bile acid concentration of serum samples was measured using a commercial kit (Wako Chemical Ltd., Osaka). Removal of Na from the serum using diffusional dialysis was performed for 60 min with the experimental set-up shown in Fig. 2. The volume of all sample phases including rat serum was 20
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3. Results
Fig. 2. Schematic representation of experimental assemblies for the diffusional dialysis treatment of rat serum. Both solution phases were stirred at 120 rpm.
cm3, while the volume of washing phases was 40 cm3 and was flushed with distilled water at 12 cm3 min 1. The experimental assemblies were kept at 369/1 8C. All cation concentrations were carefully measured with a polarized Zeeman atomic absorption spectrophotometer (180-80; Hitachi, Tokyo). The chloride ion concentration of rat serum was measured using a commercial kit (Wako Chemical Ltd.).
Sodium activity coefficients, gNa , for NaCl, NaCA, and NaTCA solutions are shown in Fig. 3. It can be seen that for any given concentration level, gNa in the NaCA and NaTCA solutions was less than the value for gNa when chloride was the accompanying anion in the solution. Taurine conjugation appreciably affected the sodium activity coefficient gNa at concentrations greater than 10 2 mol dm 3. Fig. 4 summarizes the electrochemical results obtained in the concentration-cell system. Transmembrane potential data in Fig. 4(a) indicate that the CM1 membrane reveals Nernstian responses except at concentrations of phase I below 10 3 mol dm 3. Of the three systems, the NaCA system showed the largest deviation from reasonable cation transport. Membrane conductance shown in Fig. 4(b) has been shown to be almost constant on the used electrolyte concentration range. The membrane conductance was NaCl, NaTCA and NaCA in order of amplitude in all concentration ranges. The inter-compartmental ion flux data for NaCl, NaCA and NaTCA are given in Fig. 4(c). The flux of Na across the membrane was NaCl / NaTCA /NaCA systems in order of amplitude in all cases. The membrane permeability matrix theory has shown to be useful for quantitatively analyzing the permselective ion transport across a charged membrane [13 /18]. Membrane transport parameters, such as the number of Na and bile acid anions X transported , tNa and tX, and conductive membrane permeability to Na , PNa, can be estimated by the following relations: V0 tNa VNa tX VX
gNa Gm
RT F
PNa Fig. 3. Activity coefficient of NaCl (k), NaCA ('), and NaTCA (j). See the text for detail.
VNa
ln
gX Gm
VX
I PNa aII Na PX aX
PNa aINa PX aII X
RTtNa Gm 1=2 2 F (aINa aII Na )
F (V VNa )=2RT sinh[F (V VNa )=2RT]
(1) (2)
where V0 is the transmembrane potential in the
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absence of electric membrane current, Gm the membrane conductance, a the activity, g the ionic membrane conductance, F the Faraday constant, R the gas constant, and T is the absolute temperature. The superscripts I and II identify two solution phases separated by the CM1 membrane; the subscripts Na and X refer to Na and bile acid anion X ; VNa and VX are the Nernst equilibrium membrane potentials for Na and X , respectively. When, however, the membrane current is absent, the diffusional membrane permeability to Na , P0Na, is defined as: P0Na
JNa aII Na
exp(FV0 =2RT) aINa exp( FV0 =2RT) (3)
where J is the ionic flux across the membrane. Fig. 5 summarizes the electroconductive and diffusional membrane permeabilities to Na for the NaCl, NaCA and NaTCA systems. The electroconductive membrane permeabilities of Na , PNa, representing the solution/membrane ion distribution due to electrostatic effects, were calculated using Eq. (2), the transmembrane potential data, and the membrane conductance data (Fig. 5(a)). PNa decreased monotonously with increasing hypertonic bile acid solution. In the NaCA and NaTCA systems, the curve relating the electroconductive membrane permeability with the concentration showed a distinct change in gradient of the phase I solution activity at around 3.9 and 6.3 mmol dm 3, respectively. The diffusional membrane permeabilities of Na , P0Na, representing the migration speed of Na within the membrane phase, were calculated using Eq. (3) and the transmembrane potential data, the ion flux data (Fig. 5(b)). P0Nas in all systems exhibited little dependence on the solution activity of phase I which differed from that for PNa. Over the
Fig. 4
Fig. 4. Transmembrane potential (a), membrane conductance (b) and inter-compartmental ion flux (c) as a function of the solution activity of phase I for NaCl (k), NaCA ('), and NaTCA (j) concentration-cell systems with the CM1 membrane. The broken line in (a) refers to the data derived from the Nernst equation.
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Fig. 5. Electroconductive (a) and diffusional (b) membrane permeabilities of sodium ion as a function of the solution activity of phase I for NaCl (k), NaCA ('), and NaTCA (j) concentration-cell systems with the CM1 membrane. The arrows in (a) refer to the inflexion point of slopes.
concentration range studied both P0Na and PNa showed the order of amplitude for the studied ions systems of NaCl /NaTCA /NaCA. P0Na in NaCl system was nearly six times that of the NaCA system and nearly three times that of the NaTCA system. Fig. 6 illustrates the bile acid concentration within the membrane as a function of the external solution concentration. The bile acid that was co-
Fig. 6. Cholic acid (') and taurocholic acid (j) concentrations (mol g-wet membrane weight) within the membrane phase as a function of the external concentrations. Each value is mean9/ S.D. represented by vertical bar, for six to eight samples per point.
ion with fixed charged sites within the membrane increased with the external solution concentration. The amounts of TCA anion taken up were always larger than those of the CA anion. The difference in concentration between CA anion and TCA anion within the membrane decreased gradually as osmosis progressed. The ratios of TCA anion to CA anion were approximately 8.3 and 1.3 at 2 / 10 4 and 2 /10 2 mol dm 3, respectively. The curve relating the bile acid concentration within
Fig. 7. Removal of sodium ion from rat serum with diffusional dialysis treatment as a function of the total bile acid concentration of the serum.
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the membrane with the external concentration showed a change of slope around 2 /10 3 mol dm 3. Using diffusional dialysis treatment for rat serum, the concentration of sodium ion was reduced as shown in Fig. 7. The values in Fig. 7 were corrected with an increase in solution volume of sample phase according to hydrostatic pressure. Na decreased significantly with increasing total bile acid concentration in rat serum (P B/0.05; correlation coefficient / /0.606). Diffusional dialysis treatment also removed other cations from rat serum. For example, potassium, calcium, and magnesium ions were reduced by 6.6, 24.2, and 11.2%, respectively (Fig. 8). However, the decrease in these cations did not have significant correlation with the total bile acid concentration. Correlation coefficients in the serum were: K, /0.289; Ca, 0.390; Mg, /0.427.
4. Discussion In this study, the selective removal of Na from the bile acid solution was attempted with a cation exchange membrane. It is important for ion transport to verifiably evaluate the effective concentration of Na in bile acid solution as the ‘activity coefficient’. The behavior of Na in solution phase has to be precisely understood in order to estimate Na transport across the membrane. For bile acid solution, as noted in Fig. 3, activity coefficients for Na in NaCA and NaTCA solutions were considerably less than the
Fig. 8. Removal of cation from rat serum with diffusional dialysis treatment. Each value is mean9/S.D. represented by the vertical bar, for 16 samples per point.
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corresponding values expected with NaCl solution. This suppression of activity coefficients might result from a lowering due to interaction between Na and bile acid anion in solution, or that some Na may be bound into the micelle structure in the presence of negatively charged micelles. These effects would exactly mirror the removal of Na from the solution. Therefore, the activities derived from the activity coefficients measured in Fig. 3 were used in the electrochemical and ion flux measurements and the change in membrane permeabilities as shown in Figs. 4 and 5. Generally, in treating the transport of equivalent ions across the ion exchange membrane, the smaller the radius of the counter-ion having the same charge as the site within the membrane, the more the counter-ion enters into the membrane via a molecular sieve effect. Thus the transport of the ion is accelerated. In short, the uptake of the counter-ion into the membrane increases the apparent ion exchange capacity of the membrane. However, the present study showed the reverse in that the CA anion, although smaller, was incorporated into the membrane less than the larger TCA anion. Further, PNa and P0Na for the NaTCA system were always larger than those for the NaCA system (Fig. 5). These results might be attributable to the physicochemical properties of bile acid solutions. Bile acid anions consist of a hydrophilic moiety and a hydrophobic moiety. Taurine conjugation enhances the hydrophobic properties of bile acid anions, whereas the polymer membrane possesses a large hydrophobic region in the membrane support matrix. Therefore, more TCA anion might enter into the membrane than CA anion (Fig. 6) although the greater size of the TCA anion may confer a disadvantage by making it more difficult to penetrate the membrane. In addition, the greater uptake of TCA anion may be due, at least in part, to the greater size of the TCA anion creating a lower anionic charge density through charge delocalization. This decreases the electrostatic repulsion between the bile acid anion and the negatively charged sites within the membrane and allows the bile acid anion to permeate readily into the membrane. The formation of micelles also influences Na transport. Changes in slope in the concentration
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dependency of PNa were observed in NaCA and NaTCA systems, but not in the NaCl system (Fig. 5). The bile acid anions also tended to display stronger binding affinity to the membrane at concentrations above 2 /4 mol dm 3 for both the NaCA and NaTCA systems (Fig. 6). The CMCs in NaCA and NaTCA solutions were fluorimetrically measured at 37 8C and were 9.6 and 7.8 mmol dm 3, respectively. On the other hand, the curve change points of phase I concentration in PNa as shown in Fig. 5 are rigorously corrected by dividing by the activity coefficients, for NaCA, 4.2 mmol dm 3, and for NaTCA, 6.7 mmol dm 3. These values are smaller than the above CMCs measured fluorimetrically. CMC in bile acid solution appears not as a point but over a certain range since bile salts have lower aggregation number compared with conventional aliphatic surfactants. For example, for NACA solution, lower values of 4 [19] and higher values of 16 [20] have been reported from different measurements. These broad values could be explained by the primary /secondary micelle model suggested by Small [21]. The primary micelles are formed in such a way that the hydrocarbon backs of the steroid nucleus associate. The secondary micelles are then formed by the aggregation of these primary micelles. This model invokes a stepwise aggregation mechanism. Therefore, in the present study, the affinity of bile acid anions for the membrane might have begun to increase from primary micelle formation in the bile acid solution and PNa increased with increasing affinity of the bile acid anion. It was more profitable to remove the divalent cations, Ca (24.2%) and Mg (11.2%) from rat serum than the univalent cations, Na (7.3%) and K (6.6%), as shown in Fig. 8. Increasing phase I solute concentration also influenced the removal efficacy by decreasing Na permeability, as shown in Fig. 5(a). In the system below [22]: Solution phase I NaCl, 10 3 / 10 1 mol dm 3
Membrane phase Cation exchange membrane
Solution phase II CaCl2, 10 3 / 10 1 mol dm 3
Cation transport across a cation exchange membrane involves competition between the cationic distribution process between the solution and the membrane due to the electrostatic effects, and the cation migration speed in the diffusion process within the membrane. In the above system, the concentration within the membrane of Ca2, m m Cm Ca, was nearly as large as that of Na , CNa. CNa/ m CCa first exceeded unity in the solution phase concentration range above the external solution concentration ratio, CNaCl/CCaCl2 /100. The ratio of Na to Ca2 for the diffusion coefficient within the membrane exceeded unity above CNaCl/CCaCl2 /1/50. For rat serum in the present study, since the concentration of Na , 1399/4.6 mmol dm 3, was on average 597 and 1578 times the concentrations of Ca2, 0.2339/0.033 mmol dm 3, and Mg2, 0.08819/0.010 mmol dm 3, respectively, the Na flux across the membrane was approximately 179 times that of the Ca2 flux and 1029 times that of the Mg2 flux. This indicates that, in the CM1 membrane, although divalent ions are somewhat advantageous for membrane transport, most transport of cations was promoted along with the electrochemical potential gradients. The relationship between the cation removal and the bile acid concentration in the rat serum is significant for only Na not for K , Ca2 or Mg2 (Fig. 7). In the present study, the anions in the rat serum consisted of a majority of chloride ion, carbonate anion, protein, and a minority of bile acid anions at approximately one-five thousandth the concentration of the chloride ion, 1039/ 5.1 mmol dm 3,. Since Na transport across the membrane has no charge effect unlike for Ca2 and Mg2, and is very much larger than for other univalent cations, the influence of bile acid anions that cannot penetrate the membrane might be revealed only in Na transport. At this point, we cannot determine further detail. In the near future, we will attempt to study Na removal with a divalent ion-exclusion type cation exchange membrane to increase Na selectivity and with electrical dialysis to increase removal efficacy.
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Acknowledgements This work was partly supported by a Grant-inAid for Scientific Research (No. 14580543) from the Japan Society for the Promotion of Science, and by the Salt Science Research Foundation (No. 0102). The author wishes to thank Tokuyama Corp, Japan for supplying the Neocepta CM1 membrane and Mayumi Kagoshima for her excellent technical assistance.
References [1] U. Beuers, J.L. Boyer, G. Paumgaetner, Ursodeoxycholic acid in cholestasis: potential mechanisms of action and therapeutic applications, Hepatology 28 (1998) 1449. [2] B. Hagenbuch, B. Stieger, M. Foguet, H. Lubbert, P.J. Meier, Functional expression cloning and characterization of the hepatocyte Na /bile acid cotransport system, Proc. Natl. Acad. Sci. USA 88 (1991) 10629. [3] R.P.J. Oude Elferink, D.K.F. Meijer, F. Kuiper, P.L.M. Jansen, A.K. Groen, G.M.N. Groothuis, Hepatobiliary secretion of compounds; molecular mechanisms of membrane transport, Biochim. Biophys. Acta 1241 (1995) 215. [4] H. Kouzuki, H. Suzuki, K. Ito, R. Ohashi, Y. Sugiyama, Contribution of sodium taurocholate co-transporting polypeptide to the uptake of its possible substrates into rat hepatocytes, J. Pharmacol. Exp. Ther. 286 (1998) 2043. [5] B.F. Scharschmidt, J.E. Stephens, Transport of sodium, chloride and taurocholate by cultured rat hepatocytes, Proc. Natl. Acad. Sci. USA 78 (1981) 986. [6] M. Higa, A. Kira, A. Tanioka, K. Miyasaka, New hemodialysis method using positively charged membrane dialyzer and/or polycation dialysate, Ind. Eng. Chem. Res. 32 (1993) 917. [7] J. Sone, T. Saibara, H. Himeno, K. Yamasaki, K. Miyamoto, T. Maeda, S. Onishi, Y. Yamamoto, K. Park, T. Okumiya, M. Sasaki, Assessment of bilirubin clearance capacity of a newly developed ion-exchange adsorption column and its possible use as a supportive therapy in hepatorenal syndrome, J. Clin. Apheresis 5 (1990) 123. [8] H. Inoue, K. Kaibara, N. Ohta, S. Nakabo, A. Fujita, H. Kimizuka, Ion transport across charged ultrafiltration membrane, aqueous metal chloride solution /sulfonate polysulfone membrane system, Mem. Fac. Sci. Kyushu. Univ. 16 (1988) 137.
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[9] Y. Moroi, M. Kitagawa, H. Itoh, Aqueous solubility and acidity constants of cholic, deoxycholic, chenodeoxycholic, and ursodeoxycholic acids, J. Lipid Res. 33 (1992) 49. [10] P. Mukerjee, Y. Moroi, M. Murata, Y.S. Yang, The physical chemistry of bile in health and disease, Hepatology 4 (1984) 61S. [11] A. Chattopadhyay, E. London, Fluorimetric determination of critical micelle concentration avoiding interference from detergent charge, Anal. Biochem. 139 (1984) 408. [12] E.W. Moore, J.M. Dietschy, Na and K activity coefficients in bile and bile salt determined by glass electrodes, Am. J. Physiol. 206 (1964) 1111. [13] K. Kaibara, H. Inoue, H. Kimizuka, Multi-ionic potential and membrane permeability matrix. II. Na /Ca2 biionic potential and membrane and effects of Cl on cation transport, Bull. Chem. Soc. Jpn. 60 (1987) 3175. [14] K. Kaibara, H. Inoue, S. Tsuruyama, H. Kimizuka, Study of ion transport across amphoteric ion-exchange membrane. V. Nonequilibrium thermodynamic analyses of ion selectivity, Bull. Chem. Soc. Jpn. 61 (1988) 1517. [15] H. Kimizuka, Y. Nagata, K. Kaibara, Nonequilibrium thermodynamics of the ion and solvent transports through ion-exchange membrane, Bull. Chem. Soc. Jpn. 56 (1983) 2371. [16] H. Inoue, M. Kagoshima, Removal of 125I from radioactive experimental waste with an anion exchange paper membrane, Appl. Radiat. Isot. 52 (2000) 1407. [17] H. Inoue, Influence of glucose and urea on 125I transport across an anion exchange paper membrane, Appl. Radiat. Isot. 54 (2001) 595. [18] H. Inoue, Transport of 125I and 36Cl across an anionexchange paper membrane, Appl. Radiat. Isot. 56 (2002) 659. [19] B. Lindman, N. Kamenka, H. Fabre, J. Ulmius, T. Eieloch, Aggregation, aggregate composition, and dynamics in aqueous sodium cholate solution, J. Coll. Interf. Sci. 73 (1980) 556. [20] R. Zana, D. Guveli, Fluorescence probing study of the association of bile salts in aqueous solutions, J. Phys. Chem. 89 (1985) 1687. [21] P.P. Nair, D. Kritchevsky, The Bile Acids; Chemistry, Physiology, and Metabolism, Plenum Press, New York, 1971. [22] K. Kaibara, H. Inoue, T. Aritomi, Multi-ionic potential and membrane permeability matrix. III. Diffusion and concentration of ions within membrane phase as a controlling factor to ion permselectivity, Bull. Chem. Soc. Jpn. 62 (1989) 2362.
Removal of sodium ion from bile acid solution using diffusional dialysis with cation exchange membrane Hiroyoshi Inoue * Radioisotope Institute for Basic and Clinical Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan Received 29 October 2002; received in revised form 5 January 2003; accepted 11 January 2003
Abstract The behavior of sodium ions passing through a CM1 cation exchange membrane was studied with electroconductive and diffusional membrane permeabilities based on nonequilibrium thermodynamics to remove sodium ion from bile acid solution. These membrane permeabilities of sodium ion in order of amplitude showed sodium chloride / sodiumtaurocholate /sodium cholate systems in all cases. In the sodium cholate and sodium taurocholate systems, the curves relating the electroconductive membrane permeability with concentration showed distinct changes in gradient at around the concentration of primary micelle formation of each bile acid solution. This was attributable to an increase in membrane/solution distribution of bile acid anion on basis of the micelle formation. Using diffusional dialysis, 7.3% of sodium ion was removed from rat serum. Sodium ion was significantly reduced with increasing total bile acid concentration in rat serum (P B/0.05; correlation coefficient / /0.606). # 2003 Elsevier B.V. All rights reserved. Keywords: Cation exchange membrane; Sodium; Cholate; Taurocholate; Rat serum; Membrane permeability
1. Introduction Bile salts undergo enterohepatic circulation, but bile acids possess a degree of hepatotoxicity, and hence have to be excreted rapidly into bile ducts. Most chronic cholestatic liver diseases, especially primary biliary cirrhosis (PBC) and primary sclerosing cholargitis (PSC), result from progressive destruction or loss of intrahepatic or extrahepatic bile ducts. For treating these diseases, the hydrophilic ursodeoxycholic acid (UDCA) is clini-
* Tel.: /81-942-31-7584; fax: /81-942-31-7702. E-mail address: [email protected] (H. Inoue).
cally applied on basis of stimulating the excretion of bile acids and other potentially hepatotoxic compounds [1]. It may also protect against injury of the bile ducts by bile acids by interfering with the uptake of bile acids into the hepatocytes. The uptake of bile acids into hepatocytes is mediated by a relatively specific Na -dependent transporter and also by a Na -independent transporter with a broader substrate specificity that may be involved in the uptake of non-bile acid organic anions [2,3]. The Na -dependent pathway accounts for more than 80% of taurocholate uptake and for less than 50% of cholate uptake [4,5]. Thus it is predicted that by abolishing the Na gradient in the basolateral hepatocyte membrane the blood bile
1383-5866/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1383-5866(03)00007-8
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acids are not taken up into the hepatocytes and circulate in body. Attempts have been made to improve the hemodialysis process by removing various ions from blood with a membrane or a column [6,7]. In this process, the ion permeation across the membrane, regarded as a multicomponent ionic system, has been too complicated to describe in an analytical form, and ionic transport too hard to control. The addition of charged sites into the membrane provides ionic permselectivity [8]. The ionic permselectivity in the charged membrane can be achieved by controlling the membrane/solution ionic distribution and the ionic diffusion within the membrane. The design of the removal processes is important for studying the behavior of sodium ion and bile acid anion within the membrane phase and in the boundary between the membrane and solution phases. Phenomenological analyses based on nonequilibrium thermodynamics are suitable for the various transport phenomena arising across a membrane. In the present study, the transmembrane potential, the membrane conductance, the fluxes of bile acid salt, as well as the concentrations of bile acid anion within a membrane were measured to quantitatively estimate the Na selective transport performance in the sodium chloride (NaCl), the sodium cholate (NaCA), or the sodium taurocholate (NaTCA) single salt system with a cation exchange membrane. We finally tried to remove the Na from the rat serum using diffusional dialysis with the cation exchange membrane.
2. Experimental procedures 2.1. Bile acid solution preparation NaCl aqueous solution was prepared using conductive water (]/17 MV cm) and guaranteed reagent grade NaCl (Nacalai Tesque, Japan) without further purification. NaCA and NaTCA aqueous solutions were prepared from conductive water and guaranteed reagent grade bile salts (Nacalai Tesque, Japan) which were purified by foam fractionation [9,10].
The apparent critical micellar concentration (CMC) of each bile salt was measured fluorimetrically using the polar probe diphenylhexatriene [11]. A 1-ml aliquot of 10 mmol dm 3 diphenylhexatriene in tetrahydrofuran was added to different concentrations of bile salts dissolved in 2.0 cm3 of Tris /saline buffer, pH 7.2, I /0.1. The tubes were incubated in the dark at 37 8C for 30 min and the resulting fluorescence was measured at an excitation wavelength of 355 nm and an emission wavelength of 430 nm (RF-5000; Shimadzu Corp., Tokyo). Sodium activity coefficients in bile acid solutions were calculated from the equation E /E0/ S log aNa, where E is the potential between an Ag/ AgCl wire in a saturated KCl bridge and the sodium sensitive electrode, both immersed in the unknown solution. E0 and S were determined by including samples of 10 and 100 mmol dm 3 NaCl with each series of unknowns. The sodium activity coefficients, gNa, in 10 and 100 mmol dm 3 NaCl were taken as 0.909 and 0.787, respectively [12]. 2.2. Membrane transport measurement A highly selective cation-exchange membrane, Neocepta CM1 (Tokuyama Corp., Japan), was used throughout the present study after immersion in 2 /10 3 mol dm 3 bile acid for a few days to replace all the exchangeable ions with Na . The CM1 membrane is composed of a polyvinyl chloride with an ion exchange group of sulfonic acid type and its basic physicochemical properties are as follows: exchange capacity, 2.4 mequiv gdry per resin; water content, 38%; thickness, 0.14 mm. Transmembrane potentials, conductances and ion fluxes were measured with the experimental setup shown in Fig. 1. The system consisted of solution phase I, where the bile acid concentration was varied from 2 /10 4 to 2 /10 2 mol dm 3, and solution phase II, where the bile acid concentration was fixed at 2 /10 3 mol dm 3, separated by the CM1 membrane. The membrane potential measurements were performed with calomel electrodes connected to a high-input impedance voltmeter (Takeda Riken; TR6856); conductance measurements were performed by
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Fig. 1. Schematic representation of experimental assemblies for the electrochemical measurements. Both solution phases were stirred at 90 rpm.
means of filling the chamber with mercury as a conductivity electrode connected to an impedance bridge (Yokogawa /Hewlett /Packard; 4255A) and inter-compartmental ion fluxes were estimated by means of tracing the time changes in solution conductivities of both solution phases. The concentrations of bile acid within the membrane phase were measured by the following procedure. A piece of the CM1 membrane, preweighed at the reference conditions in which all the membranes were equilibrated sufficiently with 2 /10 3 mol dm 3 bile acid solution, was immersed in the test solution containing [2,4-3H(N)]-cholic acid (American Radiolabeled Chemicals Inc.) or [24-14C]taurocholic acid (American Radiolabeled Chemicals Inc.) for 96 h. The test membrane, after rinsing with conductive water and wiping off the adhering surface solution, was measured with a liquid scintillation counter (Beckman; LS6500). All measurements were carried out at 369/1 8C.
2.3. Diffusional dialysis for serum Blood collecting procedures used in this study were approved by the internal animal use committee of Kurume University School of Medicine. Male Sprague /Dawley rats (Seac Yoshitomi; Japan), weighing 200 /250 g, were housed individually in temperature-controlled rooms (239/ 2 8C, humidity 559/10%). Blood samples (1.5 cm3) were collected to take the serum from the femoral vein every 2 days. The sera were then stored at /80 8C until the bile acid transport experiments were performed. The total bile acid concentration of serum samples was measured using a commercial kit (Wako Chemical Ltd., Osaka). Removal of Na from the serum using diffusional dialysis was performed for 60 min with the experimental set-up shown in Fig. 2. The volume of all sample phases including rat serum was 20
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3. Results
Fig. 2. Schematic representation of experimental assemblies for the diffusional dialysis treatment of rat serum. Both solution phases were stirred at 120 rpm.
cm3, while the volume of washing phases was 40 cm3 and was flushed with distilled water at 12 cm3 min 1. The experimental assemblies were kept at 369/1 8C. All cation concentrations were carefully measured with a polarized Zeeman atomic absorption spectrophotometer (180-80; Hitachi, Tokyo). The chloride ion concentration of rat serum was measured using a commercial kit (Wako Chemical Ltd.).
Sodium activity coefficients, gNa , for NaCl, NaCA, and NaTCA solutions are shown in Fig. 3. It can be seen that for any given concentration level, gNa in the NaCA and NaTCA solutions was less than the value for gNa when chloride was the accompanying anion in the solution. Taurine conjugation appreciably affected the sodium activity coefficient gNa at concentrations greater than 10 2 mol dm 3. Fig. 4 summarizes the electrochemical results obtained in the concentration-cell system. Transmembrane potential data in Fig. 4(a) indicate that the CM1 membrane reveals Nernstian responses except at concentrations of phase I below 10 3 mol dm 3. Of the three systems, the NaCA system showed the largest deviation from reasonable cation transport. Membrane conductance shown in Fig. 4(b) has been shown to be almost constant on the used electrolyte concentration range. The membrane conductance was NaCl, NaTCA and NaCA in order of amplitude in all concentration ranges. The inter-compartmental ion flux data for NaCl, NaCA and NaTCA are given in Fig. 4(c). The flux of Na across the membrane was NaCl / NaTCA /NaCA systems in order of amplitude in all cases. The membrane permeability matrix theory has shown to be useful for quantitatively analyzing the permselective ion transport across a charged membrane [13 /18]. Membrane transport parameters, such as the number of Na and bile acid anions X transported , tNa and tX, and conductive membrane permeability to Na , PNa, can be estimated by the following relations: V0 tNa VNa tX VX
gNa Gm
RT F
PNa Fig. 3. Activity coefficient of NaCl (k), NaCA ('), and NaTCA (j). See the text for detail.
VNa
ln
gX Gm
VX
I PNa aII Na PX aX
PNa aINa PX aII X
RTtNa Gm 1=2 2 F (aINa aII Na )
F (V VNa )=2RT sinh[F (V VNa )=2RT]
(1) (2)
where V0 is the transmembrane potential in the
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absence of electric membrane current, Gm the membrane conductance, a the activity, g the ionic membrane conductance, F the Faraday constant, R the gas constant, and T is the absolute temperature. The superscripts I and II identify two solution phases separated by the CM1 membrane; the subscripts Na and X refer to Na and bile acid anion X ; VNa and VX are the Nernst equilibrium membrane potentials for Na and X , respectively. When, however, the membrane current is absent, the diffusional membrane permeability to Na , P0Na, is defined as: P0Na
JNa aII Na
exp(FV0 =2RT) aINa exp( FV0 =2RT) (3)
where J is the ionic flux across the membrane. Fig. 5 summarizes the electroconductive and diffusional membrane permeabilities to Na for the NaCl, NaCA and NaTCA systems. The electroconductive membrane permeabilities of Na , PNa, representing the solution/membrane ion distribution due to electrostatic effects, were calculated using Eq. (2), the transmembrane potential data, and the membrane conductance data (Fig. 5(a)). PNa decreased monotonously with increasing hypertonic bile acid solution. In the NaCA and NaTCA systems, the curve relating the electroconductive membrane permeability with the concentration showed a distinct change in gradient of the phase I solution activity at around 3.9 and 6.3 mmol dm 3, respectively. The diffusional membrane permeabilities of Na , P0Na, representing the migration speed of Na within the membrane phase, were calculated using Eq. (3) and the transmembrane potential data, the ion flux data (Fig. 5(b)). P0Nas in all systems exhibited little dependence on the solution activity of phase I which differed from that for PNa. Over the
Fig. 4
Fig. 4. Transmembrane potential (a), membrane conductance (b) and inter-compartmental ion flux (c) as a function of the solution activity of phase I for NaCl (k), NaCA ('), and NaTCA (j) concentration-cell systems with the CM1 membrane. The broken line in (a) refers to the data derived from the Nernst equation.
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Fig. 5. Electroconductive (a) and diffusional (b) membrane permeabilities of sodium ion as a function of the solution activity of phase I for NaCl (k), NaCA ('), and NaTCA (j) concentration-cell systems with the CM1 membrane. The arrows in (a) refer to the inflexion point of slopes.
concentration range studied both P0Na and PNa showed the order of amplitude for the studied ions systems of NaCl /NaTCA /NaCA. P0Na in NaCl system was nearly six times that of the NaCA system and nearly three times that of the NaTCA system. Fig. 6 illustrates the bile acid concentration within the membrane as a function of the external solution concentration. The bile acid that was co-
Fig. 6. Cholic acid (') and taurocholic acid (j) concentrations (mol g-wet membrane weight) within the membrane phase as a function of the external concentrations. Each value is mean9/ S.D. represented by vertical bar, for six to eight samples per point.
ion with fixed charged sites within the membrane increased with the external solution concentration. The amounts of TCA anion taken up were always larger than those of the CA anion. The difference in concentration between CA anion and TCA anion within the membrane decreased gradually as osmosis progressed. The ratios of TCA anion to CA anion were approximately 8.3 and 1.3 at 2 / 10 4 and 2 /10 2 mol dm 3, respectively. The curve relating the bile acid concentration within
Fig. 7. Removal of sodium ion from rat serum with diffusional dialysis treatment as a function of the total bile acid concentration of the serum.
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the membrane with the external concentration showed a change of slope around 2 /10 3 mol dm 3. Using diffusional dialysis treatment for rat serum, the concentration of sodium ion was reduced as shown in Fig. 7. The values in Fig. 7 were corrected with an increase in solution volume of sample phase according to hydrostatic pressure. Na decreased significantly with increasing total bile acid concentration in rat serum (P B/0.05; correlation coefficient / /0.606). Diffusional dialysis treatment also removed other cations from rat serum. For example, potassium, calcium, and magnesium ions were reduced by 6.6, 24.2, and 11.2%, respectively (Fig. 8). However, the decrease in these cations did not have significant correlation with the total bile acid concentration. Correlation coefficients in the serum were: K, /0.289; Ca, 0.390; Mg, /0.427.
4. Discussion In this study, the selective removal of Na from the bile acid solution was attempted with a cation exchange membrane. It is important for ion transport to verifiably evaluate the effective concentration of Na in bile acid solution as the ‘activity coefficient’. The behavior of Na in solution phase has to be precisely understood in order to estimate Na transport across the membrane. For bile acid solution, as noted in Fig. 3, activity coefficients for Na in NaCA and NaTCA solutions were considerably less than the
Fig. 8. Removal of cation from rat serum with diffusional dialysis treatment. Each value is mean9/S.D. represented by the vertical bar, for 16 samples per point.
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corresponding values expected with NaCl solution. This suppression of activity coefficients might result from a lowering due to interaction between Na and bile acid anion in solution, or that some Na may be bound into the micelle structure in the presence of negatively charged micelles. These effects would exactly mirror the removal of Na from the solution. Therefore, the activities derived from the activity coefficients measured in Fig. 3 were used in the electrochemical and ion flux measurements and the change in membrane permeabilities as shown in Figs. 4 and 5. Generally, in treating the transport of equivalent ions across the ion exchange membrane, the smaller the radius of the counter-ion having the same charge as the site within the membrane, the more the counter-ion enters into the membrane via a molecular sieve effect. Thus the transport of the ion is accelerated. In short, the uptake of the counter-ion into the membrane increases the apparent ion exchange capacity of the membrane. However, the present study showed the reverse in that the CA anion, although smaller, was incorporated into the membrane less than the larger TCA anion. Further, PNa and P0Na for the NaTCA system were always larger than those for the NaCA system (Fig. 5). These results might be attributable to the physicochemical properties of bile acid solutions. Bile acid anions consist of a hydrophilic moiety and a hydrophobic moiety. Taurine conjugation enhances the hydrophobic properties of bile acid anions, whereas the polymer membrane possesses a large hydrophobic region in the membrane support matrix. Therefore, more TCA anion might enter into the membrane than CA anion (Fig. 6) although the greater size of the TCA anion may confer a disadvantage by making it more difficult to penetrate the membrane. In addition, the greater uptake of TCA anion may be due, at least in part, to the greater size of the TCA anion creating a lower anionic charge density through charge delocalization. This decreases the electrostatic repulsion between the bile acid anion and the negatively charged sites within the membrane and allows the bile acid anion to permeate readily into the membrane. The formation of micelles also influences Na transport. Changes in slope in the concentration
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dependency of PNa were observed in NaCA and NaTCA systems, but not in the NaCl system (Fig. 5). The bile acid anions also tended to display stronger binding affinity to the membrane at concentrations above 2 /4 mol dm 3 for both the NaCA and NaTCA systems (Fig. 6). The CMCs in NaCA and NaTCA solutions were fluorimetrically measured at 37 8C and were 9.6 and 7.8 mmol dm 3, respectively. On the other hand, the curve change points of phase I concentration in PNa as shown in Fig. 5 are rigorously corrected by dividing by the activity coefficients, for NaCA, 4.2 mmol dm 3, and for NaTCA, 6.7 mmol dm 3. These values are smaller than the above CMCs measured fluorimetrically. CMC in bile acid solution appears not as a point but over a certain range since bile salts have lower aggregation number compared with conventional aliphatic surfactants. For example, for NACA solution, lower values of 4 [19] and higher values of 16 [20] have been reported from different measurements. These broad values could be explained by the primary /secondary micelle model suggested by Small [21]. The primary micelles are formed in such a way that the hydrocarbon backs of the steroid nucleus associate. The secondary micelles are then formed by the aggregation of these primary micelles. This model invokes a stepwise aggregation mechanism. Therefore, in the present study, the affinity of bile acid anions for the membrane might have begun to increase from primary micelle formation in the bile acid solution and PNa increased with increasing affinity of the bile acid anion. It was more profitable to remove the divalent cations, Ca (24.2%) and Mg (11.2%) from rat serum than the univalent cations, Na (7.3%) and K (6.6%), as shown in Fig. 8. Increasing phase I solute concentration also influenced the removal efficacy by decreasing Na permeability, as shown in Fig. 5(a). In the system below [22]: Solution phase I NaCl, 10 3 / 10 1 mol dm 3
Membrane phase Cation exchange membrane
Solution phase II CaCl2, 10 3 / 10 1 mol dm 3
Cation transport across a cation exchange membrane involves competition between the cationic distribution process between the solution and the membrane due to the electrostatic effects, and the cation migration speed in the diffusion process within the membrane. In the above system, the concentration within the membrane of Ca2, m m Cm Ca, was nearly as large as that of Na , CNa. CNa/ m CCa first exceeded unity in the solution phase concentration range above the external solution concentration ratio, CNaCl/CCaCl2 /100. The ratio of Na to Ca2 for the diffusion coefficient within the membrane exceeded unity above CNaCl/CCaCl2 /1/50. For rat serum in the present study, since the concentration of Na , 1399/4.6 mmol dm 3, was on average 597 and 1578 times the concentrations of Ca2, 0.2339/0.033 mmol dm 3, and Mg2, 0.08819/0.010 mmol dm 3, respectively, the Na flux across the membrane was approximately 179 times that of the Ca2 flux and 1029 times that of the Mg2 flux. This indicates that, in the CM1 membrane, although divalent ions are somewhat advantageous for membrane transport, most transport of cations was promoted along with the electrochemical potential gradients. The relationship between the cation removal and the bile acid concentration in the rat serum is significant for only Na not for K , Ca2 or Mg2 (Fig. 7). In the present study, the anions in the rat serum consisted of a majority of chloride ion, carbonate anion, protein, and a minority of bile acid anions at approximately one-five thousandth the concentration of the chloride ion, 1039/ 5.1 mmol dm 3,. Since Na transport across the membrane has no charge effect unlike for Ca2 and Mg2, and is very much larger than for other univalent cations, the influence of bile acid anions that cannot penetrate the membrane might be revealed only in Na transport. At this point, we cannot determine further detail. In the near future, we will attempt to study Na removal with a divalent ion-exclusion type cation exchange membrane to increase Na selectivity and with electrical dialysis to increase removal efficacy.
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Acknowledgements This work was partly supported by a Grant-inAid for Scientific Research (No. 14580543) from the Japan Society for the Promotion of Science, and by the Salt Science Research Foundation (No. 0102). The author wishes to thank Tokuyama Corp, Japan for supplying the Neocepta CM1 membrane and Mayumi Kagoshima for her excellent technical assistance.
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