Biotechnol. Prog. 1990, 6, 62-66
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Two-Barrel Bile-Acids-Sensitive Microelectrodes Based on Liquid Ion Exchanger Yu Bi' and Hong Wen-bin Laboratory of Cell Physiology, Department of Scientific Instruments, Zhejiang University, Zhejiang Province, China
Several liquid membrane microelectrodes sensitive to bile acids (two barrel, tip diameter about 0.5 pm) are described. The results of different liquid ion exchangers such as Aliquat 336/decanol, trioctylmethylammonium/decanol, hexadecyltrimethylammonium/decanol, benzyldimethylhexadecylammonium/decanol, hexadecyltributylammonium/5% hexachlorobenzene + 0.5% bromoacetanilide in o-dichlorobenzene are compared with each other, and the better one among them is the mixture of benzyldimethylhexadecylammonium cholate/decanol with hexadecyltributylammonium taurocholate/5% hexachlorobenzene + 0.5% bromoacetanilide in o-dichlorobenzene because of its quicker response time and low drift. The calibration curves, slopes, test limits, selective coefficients, drifts, and response times of the various bile-acids-sensitive microelectrodes in different calibration solutions were demonstrated and compared with each other.
Introduction The liver, an organ essential to life, is the largest digestive gland in our body and has a multitude of functions within the environment of the organism. Bile acid is one of the most important components in bile excreted by hepatocytes and plays a critical role in promoting bile flow and absorption of lipophilic compounds in the intestine by micellization. In addition, bile acids represent the major route for the elimination of cholesterol via bile acid formation ( I ) . However, very little is known about the respective roles of hepatocyte organelles in the formation and secretion of bile (2). There are many methods by which bile acids can be measured, for example, calorimetric ( 3 ) , radiochemical ( 4 ) , spectrophotometric ( 5 ) ,and enzymatic (6) methods and gas chromatography (7), thin-layer chromatography (8), and high-performance liquid chromatography (9).The disadvantage of all the methods mentioned above is that the space resolution is too low to be used at cellular and subcellular level, especially in living cells. The advantage of the ion-sensitive microelectrode is its very high space resolution, by which some interesting ion activities inside and outside the living cells can be monitored continuously for a relatively long time without great damage to the living cell. This characteristic forced us to develop some new ion-sensitive microelectrodes especially for some very important organic ions, such as acetylcholine (10-12), serotonin (13), and histamine (14). In the present study, the preparation of an organic liquid ion exchanger sensitive to bile acids, the construction of two-barrel bile-acids-sensitivemicroelectrodes, their calibration curves in different inorganic and organic ion solutions, detection limits, slope, selective coefficients, and drifts are described.
Materials and Reagents Two-barrel glass rodlets (Thetaglass) were obtained from Neumann, Munich, FRG, or Fa. H. Kugelstatter, Garching, FRG. N,N-Dimethyltrimethylsilylamine,tributylamine, l-bromohexadecane, ethanol, taurocholic acid sodium salt, 4-bromoacetanilide, 1,2-dichlorobenzene,and
Aliquat 336 (MW 404.17) were bought from Fluka Chemie, AG Buchs, FRG. Hexachlorobenzene and deoxycholic acid sodium salt were obtained from Aldrich Chemie, Steinheim, FRG. Cholic acid sodium salt, deoxycholic acid free acid, chenodeoxycholic acid, ursodeoxycholic acid, bile bovine, l-decanol, benzyldimethylhexadecylammonium chloride, hexadecyltrimethylammonium bromide, and L-(+)-lacticacid free acid were purchased from Sigma, St. Louis, MO. Chloroform, ammonium acetate, potassium nitrate, sodium chloride, and disodium phosphate were obtained from E. Merck, AG Darmstadt, FRG, and trioctylmethylammonium chloride (MW 442) was bought from Merck, Schuchardt, FRG. Diethyl ether was obtained from Carl Roth K G, Karlsruhe, FRG. Medium DMEM (Minimum Essential Medium after Dulbecco) was purchased from Boehringer, Mannheim, FRG.
Apparatus and Measurements The pipette puller, Sacks flaming micropipette puller, Model PC-84, was used for the making of the glass microelectrode. The microelectrode amplifier (Max-Planck-Institut fur Systemphysiologie, Dortmund, FRG) and a six-channel recorder BBC Goerz Metrawatt type SE 461 were used for the measurements and recording of the ion-sensitive microelectrode potentials. A saturated calomel electrode was employed as the reference electrode. The measuring cell is Ag-AgC1/0.5 M KCl/liquid membrane/sample/0.9 70NaCl/calomel electrode. All the measurements were performed at room temperature (22 "C) and under the Faraday cage. All data points in the figures are expressed as the mean f the standard deviation. The slope, test limit, selective coefficient, drift, and response time were determined by the methods described by the International Union of Pure and Applied Chemistry (IUPAC) (15). Potentiometric selectivity coefficients of the bile-acidssensitive microelectrode were measured by the mixedsolution method recommended by IUPAC.
8756-7938/90/3006-0062$02.50/0 0 1990 American Chemical Society and American Institute of Chemical Engineers
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Preparation of the Liquid Ion Exchangers Sensitive to Bile Acids 1. Benzyldimethylhexadecylammonium Cholate. A 50-mL amount of 0.1 M cholic acid sodium salt aqueous solution was mixed with 50 mL of 0.1 M benzyldimethylhexadecylammonium chloride/chloroform solution for 10 min and then kept at room temperature for 1 h. The chloroform layer was separated from the aqueous solution. Added dropwise into the chloroform solution was 200 mL of diethyl ether with stirring over a period of 4 h until no more white precipitate formed. The precipitate was filtered and dried at room temperature to give a solid material complex of cholic acid with benzyldimethylhexadecylammonium. The liquid ion exchanger sensitive to cholic acid was prepared by making a solution of 2% (w/w) benzyldimethylhexadecylammonium cholate in decanol. 2. TrioctylmethylammoniumCholate (MW 814.6). A 10-mL amount of lo-, M cholic acid sodium salt was mixed with 10 mL of 10% trioctylmethylammonium chloride/decanol solution for 10 min, and then the mixture was kept at room temperature for 24 h. The upper layer of the decanol solution was separated as the liquid ion exchanger. 3. Aliquat 336 Cholate (MW 776.77). A 10-mL amount of lo-, M cholic acid sodium salt was mixed with 10 mL of 10% Aliquat 336/decanol solution for 10 min, and then the mixture was kept at room temperature (22 "C) for 24 h. The upper layer of the decanol solution was separated from the aqueous solution as the liquid ion exchanger. 4. Hexadecyltrimethylammonium Cholate. A 10mL amount of lo-, M cholic acid sodium salt was mixed with 10 mL of 10% hexadecyltrimethylammonium bromide in decanol thoroughly for 10 min. The mixture was kept at room temperature (22 "C) for 24 h. The upper layer of the decanol solution was separated from the aqueous solution as the liquid ion exchanger. 5 . Hexadecyltributylammonium Taurocholate. Hexadecyltributylammonium ion was prepared by refluxing 0.1 M tributylammonium with 0.1 M bromohexadecane in 100 mL of ethanol for 24 h, and then the solution was heated to 78 "C in an oil bath to evaporate the ethanol and to obtain hexadecyltributylammonium bromide. The hexadecyltributylammonium bromide was mixed with 0.1 M taurocholic acid sodium salt in a 100mL aqueous solution. A 100-mL membrane solution (5% hexachlorobenzene/0.5% bromoacetanilide in o-dichlorobenzene) was mixed with the aqueous solution mentioned above for 30 min. The hexadecyltributylammonium taurocholate complex was extracted into the membrane solution to form a liquid ion exchanger with a concentration of 0.1 M hexadecyltributylammonium taurocholate in membrane solution. 6. Combination of Liquid Ion Exchanger 1 with 5. Two parts of 2% (w/w) benzyldimethylhexadecylammonium cholate in decanol was mixed with one part of 0.1 M hexadecyltributylammonium taurocholate in 5% hexachlorobenzene/0.5% bromoacetanilide/o-dichlorobenzene solution to form a new liquid ion exchanger.
Preparation of the Two-Channel Microelectrode Sensitive to Bile Acid The two-barrel glass rodlets were pulled by the microelectrode puller to form glass microelectrodes with a tip diameter of about 0.5 pm. The tip of the glass microelectrode was set in the vapor of N,N-dimethyltrimethylsilylamine for 1s. Then, the electrolyte of low2M KC1
i
100
------
,
2
_-_.............. -3 4
Figure 1. Calibration curves of the bile-acids-sensitive microelectrode calibrated in (1) 10-2-10-6 M cholate Na in medium DMEM + 10% calf serum; ( 2 ) 10-2-10-s M cholate Na in tyrode solution + 10 mM HEPES (pH = 7.4); (3) 10-2-10* M cholate Na in 0.9% NaCl; (4) 240002% bile bovine in 0.9% NaCl. The liquid ion exchanger of the microelectrode is 2% (w/w) benzyldimethylhexadecylammonium cholate in decanol.
was injected into the reference channel from the backend of the dc channel of the glass microelectrode. The silanization of the bile-acids-sensitive channel was performed by injecting a small drop of 100% N,N-dimethyltrimethylsilylamine into the bile acids channel from the back-end of the bile-acids-sensitive channel of the glass microelectrode. The N,N-dimethyltrimethylsilylamine solution in the bile-acids-sensitivechannel evaporated thoroughly in about 1 h. After silanization, the microelectrode was kept at room temperature for 10 h, and then a small drop of liquid ion exchanger sensitive to bile acids was injected into the tip of the glass microelectrode from the back-end of the bile-acids-sensitive channel. After that, the electrolyte of M KC1 was injected into the bile-acids-sensitivechannel from the back-end of the glass microelectrode. A silver-silver chloride wire with a diameter of 0.1 mm was inserted into the dc and bile-acidssensitive channel separately from the back-ends of the microelectrode.
Results and Discussion The calibration curves of the bile-acids-sensitive microelectrode with the liquid ion exchanger of 2 % benzyldimethylhexadecylammonium cholate in decanol in different calibration solutions are shown in Figures 1 and 2. It is shown in Figure 1 that calibration curve 2 of the bile-acids-sensitive microelectrode in tyrode calibration solution is very similar to curve 1 in medium DMEM. We can easily use tyrode solution as a calibration solution from which a result can be obtained similar to that in medium DMEM. In Figure 1,the relationship between calibration curves 3 and 4 in cholate Na and bile bovine is shown. The tendency of the two calibration curves is similar. The result is that the different activities of bile acids in bile bovine can be monitored by a bile-acids-sensitive microelectrode. In Figure 2, calibration curves 1-3 in three other bile acids are shown. The microelectrode is also sensitive to these three kinds of bile acids, but the sensitivities t o different bile acids are not similar. In Figure 2, calibration curves 4-6 in 10-2-10-s M cholate Na with different inorganic solutions are shown. The anions CH,COO-, NO,-, and HP0,'- do not interfere with the measurements of the bile acids obviously. In Figure 3, calibration curves 1 and 2 of the bile-acids-sensitive microelectrode with the liquid ion exchanger of 10% trioctylmethylammonium cholate in decanol are shown. The inorganic anion NO,- does not interfere with the measurement of the bile acid obviously.
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-mV
1
-m V
(4)
100
150
100 1001
50
Figure 2. Calibration curves of the bile-acids-sensitive microelectrode calibrated in (1) 10-2-104 M deoxycholic acid in 0.9% NaCl; (2) 10-2-104 M chenodeoxycholic acid in 0.9% NaCl; (3) 10-2-10" M ursodeoxycholic acid in 0.9% NaCl; (4) 10-2-10* M cholate Na in M Na2HP04; (5 10 2-10" M cholate Na in M M CH,COOHN,; (6) 10-2-10 M cholate Na in KNO,. The liquid ion exchanger of microelectrode is 2% (w/ w) benzyldimethylhexadecylammonium cholate in decanol.
-d-
Figure 3. Calibration curves of the bile-acids-sensitive microelectrodes calibrated in (1) 10-'-10" M cholate Na in M KNO,; (2) 10-2-10" M cholate Na in 0.9% NaCl. The liquid ion exchanger for the microelectrode is 10% trioctylmethylammonium cholate (MW 814.6) in decanol. Calibration curves in 10-2-10* M cholate Na + 0.9% NaCl by two kinds of microelectrodes with liquid ion exchanger. (3) 10% (w/w) trioctylmethylammonium cholate (MW 814.6) in decanol; (4) 2% (w/ w) benzyldimethylhexadecylammonium cholate in decanol.
Calibration curves 3 and 4 between the bile-acids-sensitive microelectrodes with 10% trioctylmethylammonium cholate in decanol and with 2% benzyldimethylhexadecylammonium cholate in decanol are compared with
F i g u r e 4. Calibration curves of the bile-acids-sensitive microelectrodes calibrated in (1) 10-2-10* M deoxycholic acid Na in 0.9% NaC1; (2) 10-2-10* M taurocholate Na in 0.9% NaC1; (3) 10-2-10* M cholate Na in 0.9% NaCl. The liquid ion exchanger for the microelectrode is 2 parts of 2% (w/w) benzyldimethylhexadecylammonium cholate in decanol mixed with 1 part of 0.1 M hexadecyltributylammonium taurocholate in 5% hexachlorobenzene/0.5% bromoacetanilide/o-dichlorobenzene.
each other in Figure 3. The sensitivity to bile acid of these two kinds of bile-acids-sensitive microelectrodes is very similar. The calibration curves of the microelectrode with combined liquid ion exchanger 4 and 5 (2 parts of 2 % benzyldimethylhexadecylammonium cholate in decanol mixed with 1part of 0.1 M hexadecyltributylammonium taurocholate in dichlorobenzene) in different calibration solutions are shown in Figure 4. The slopes, test limits, selective coefficients, drifts, and response times of the bile-acids-sensitive microelectrode with six kinds of liquid ion exchangers in different calibration solutions are shown in Table I. The sensitivity of the bile-acids-sensitive microelectrode with liquid ion exchanger 1 is better than the others, but the drift is higher and the response time is longer than the others as seen in the table. The response time of the microelectrode with liquid ion exchanger 5 is quicker than the others, but its drift is higher. Although the slope of the bile-acids-sensitive microelectrode with liquid ion exchanger 6 is smaller, its response time is quicker and the drift is lower than the others. Liquid ion exchanger 6 is a better one for making microelectrodes sensitive to bile acids. In the past years, some ion-selective electrodes based on liquid membrane ion exchangers have been made for the measurements of some inorganic and organic anions
Table I. Slope, Test Limits, Selective Coefficients (K$&eio,,) Drifts, and Response Times ( T9,,) of the Bile-Acids Sensitive Microelectrode with Various Liquid Ion Exchangers in Different Calibration Solutions drift in 10-3 M liquid ion exchanger slope 10-4test cholate, T,(10-3-104 M), electrolyte concn, M (10-3-10-4 M) limit a h b i , mV/h min 1 2% benzyldimethylhexadecylammonium NaCl 0.16 24 1 6.3 x 10-4 3 cholate in decanol 0.01 10-3 KNOz 50 -3 3 0.001 0.01 10-3 Na,HPO, 0.001 47 10 4 medium DMEM 24 1 3 -5 24 1 4 tyrode -5 KNO, 2 10% trioctylmethylammonium cholate 0.001 54 0.1 10-2 -1 2 (MW 814.6) 0.16 NaCl 16 0.1 6.3 x 10-5 1.5 2 0.16 NaCl 1 6.3 x 10-4 -2 2 6 3 10% Aliquat 336-cholate (MW 776.77) medium DMEM 12 1 -1 2 4 10% hexadecyltrimethylammonium tyrode 1 19 2 20 cholate in decanol 1 2 DMEM 15 10 5 0.1 M hexadecyltributylammonium NaCl 0.16 10 1 6.3 x 10-4 -5 1 taurocholate in o-dichlorobenzene 6 mixture of ion exchanger 0.16 1 6.3 x 10-4 -1 NaCl 9 1 1 and 5 -1 lactic acid 0.001 1 38 0.1 10-2
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such as bile salts (I6-I8), benzoate ( I 9 ) ,perchlorate, chloride (20,21),nitrate (22-24), and nicotinate (25). Because these anion-sensitive electrodes mentioned above are not microelectrodes, they have a relatively big sensor area and quick response time. The anion exchangers used by authors mentioned above cannot be used directly to manufacture the anion-sensitive microelectrode (tip diameter about 0.5 pm, two channel), because these liquid ion exchangers have relatively high electric resistance, and the microelectrodes made by these exchangers have too long a response time to be used. In the present experiment, this difficulty was overcome by combining two different liquid ion exchangers to form a mixture with low electric resistance (about 10" Q compared with lo1' Q of the other liquid ion exchangers) and quick response time (see Table I). In this study, a special silanization method for the making of a two-channel microelectrode was developed. The result of silanization of the bile acid channel of the microelectrode was excellent, and, at the same time, the dc channel of the microelectrode retained its hydrophilic characteristic thoroughly. Liver cell culture is a model in vitro system for the study of the regulation of bile acid metabolism (26). Although much work has recently been focused on elucidating the mechanism of bile acid uptake at the sinusoidal membranes and excretion at the canalicular membranes, little is known about the process by which bile acids transverse the hepatocyte from the sinusoidal to the canalicular pole ( I ) . Direct studies of primary canalicular bile formation have been hampered by the relative inaccessibility of tiny (1-2-pm diameter) canalicular spaces, which has prevented direct sampling or quantitative measurements of the secretion or the application of classical electrophysiological techniques to study the mechanisms of canalicular bile formation (27). Exact knowledge about the composition of the primary bile is, however, a prerequisite for an understanding of the biophysical and biochemical processes involved in bile secretion (28).
The subcellular distribution of the major bile acids in rat liver has been studied by the application of gas-liquid partition chromatographic methods (29). The authors reported that the cholic acid in rat liver homogenate is M, and all bile acids together are obvihigher than ously higher than M. It is possible to guess that the concentration of the bile acids at the canalicular pole would be much higher than M, which is detectable by the bile-acids-sensitive microelectrode described by us. In the present study, the development of a two-barrel microelectrode sensitive to bile acids is one of the possible ways for dynamic determination of bile acid activity in the study of the living cellular or subcellular level of the liver because the potential difference between the output of dc and the bile acid channel of the microelectrode amplifier is taken to present the signals for bile acid ion activity that is free from bioelectrical signals.
Conclusion Several organic compounds were tested and employed in the preparation of a microelectrode sensitive to cholic acid. The better one among them is the mixture of benzyldimethylhexadecylammonium cholate/decanol with hexadecyltributylammonium taurocholate/5% hexachlorobenzene + 0.5 % bromoacetanilide in o-dichlorobenzene. The specifications of the microelectrode with this kind of liquid ion exchanger are (a) two channels, one chan-
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ne1 for dc, the other one for bile acid (tip diameter about 0.5 pm); (b) detection limit, M cholate (tested in cholate + tyrode solution); (c) no serious interference from ions such as NO3-, CH,COO-, C1-, and HP0,2-; (d) sensitive to several bile acids such as deoxycholic acid, chenodeoxycholic acid, and ursodeoxycholic acid; and (e) drift of the bile acid channel, about 1 mV/h.
Acknowledgment We are most grateful to Stifung Volkswagenwerk,FRG; the Sciences Foundation of Educational Committee of China; the National Natural Sciences Foundation of China; the Max-Planck-Institut fur Systemphysiologie, FRG; and the Wuangkuanchen Foundation for their financial support.
Literature Cited (1) Stolz, A.; Takikawa, H.; Sugiyama, Y.; Kaplowitz, N. In Bile Acids and the Liver; Paumgartner, G., Stiehl, A., Gerok, W., Eds.; M T P Press: Lancaster, Boston, T h e Hague, Dordrecht, 1987; p 125. (2) Jones, A. L.; Schmucker, D. L.; Renston, R. H.; Murakami, T. Dig. Dis. Sci. 1983, 25 (8), 609 (August). (3) Biader Ceipidor, U.; Curini, R.; D'Ascenzo, G.; Tomassetti, M. Thermochim. Acta 1981,46, 269. (4) Minder, E.; Karlaganis, G.; Schmied, U.; Vitins, P.; Gustav, P. Chin. Chim. Acta 1979, 92, 177. (5) Biader Ceipidor, U.; Curini, R.; D'Ascezo, G.; Tomassetti, M. Thermochim. Acta 1981,46, 279. (6) Talalay, P. Methods Biochem. Anal. 1960, 8, 119. (7) Setchell, K. D. R.; Matsui, A. Clin. Chim. Acta 1983,127, 1. (8) Brusgaard, A. Clin. Chim. Acta 1970,28, 495. (9) Sian, M. S.; Harding, A. J. Clin. Chim. Acta 1978, 98, 243. (10) Yu, B.; Weigelt, H. Znt. J. Microcirc. 1986, 3, 505. (11)Yu, B.; Zeng, X. Y.; Weigelt, H. Znt. J. Microcirc. 1986, 5, 107. (12) Yu, B. Chinese Patent Application No. 87014761, 1987. (13) Yu, B. Chin. J. Physiol. Sci. 1989, 5 (l), 10. (14) Yu, B. Biosensors 1989, 4, 373. (15) International Union of Pure and Applied Chemistry. Analytical Chemistry Division Commission on Analytical Nomenclature. Pure Appl. Chem. 1976,48, 127. (16) Gilligan, T. J.; Cussler, E. L.; Evans, D. F. Biochim. Biophys. Acta 1977,627, 627. (17) Campanella, L.; Sorrentino, L.; Tomassetti, M. Analyst 1983,108, 1490. (18) Campanella, L.; Sorrentino, L.; Tomassetti, M. Anal. Lett. 1982,15 (B18), 1515. (19) Amorso, D.; Campanella, L.; De Angelia, G.; Ferri, T.; Morabite, R. Membr. Sci. 1983, 16, 259. (20) Coetzee, C. J.; Freiser, H. Anal. Chem. 1968, 40, 2071. (21) Coetzze, C. J.; Freiser, H. Anal. Chem. 1969, 41, 1128. (22) Dobbelstein, T. N.; Diehl, H. Talanta 1969, 16, 1341. (23) Davies, J. E. W.; Moody, G. J.; Thomas, J. D. R. Analyst 1972, 97, 87. (24) Qureshi, G. A.; Lindquist, J. Anal. Chim. Acta 1973, 67, 243. (25) Campanella, L.; De Angelis, G.; Ferri, T.; Gozzi, D. Analyst 1977, 102, 723. (26) Everson, G. T. In Bile Acids and the Liver with an Update on Gallstone Disease; Paumgartner, G., Stiehl, A., Gerok, W., Eds.; M T P Press: Lancaster, Boston, The Hague, Dordrecht, 1987; p 61. (27) Gautam, A.; Scaramuzza, D. M.; Boyer, J. L. In Bile Acids and the Liver with an Update on Gallstone Disease; Paumgartner, G., Stiehl, A., Gerok, W., Eds.; M T P Press: Lancaster, Boston, The Hague, Dordrecht, 1987; p 163. (28) Petzinger, E.; Foellmann, W.; Acker, H.; Hentschel, J.;Zierold, K.; Kinne, R. Zn Vitro Cellular Dev. Biol. 1988, 24 (6),491. (29) Okishio, T.; Nair, P. P. Biochemistry 1966, 5, 3662. Accepted November 29, 1989.
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Registry No. Cholic acid sodium salt, 361-09-1; benzyldimethylhexadecylammonium chloride, 122-18-9; benzyldimethylhexadecylammonium cholate, 88428-97-1; trioctylmethylammonium chloride, 5137-55-3; trioctylmethylammonium cholate, 124536-24-9; hexadecyltrimethylammonium bromide, 57-09-0;
Biotechnol. Prog., 1990, Vol. 6, No. 1
hexadecyltrimethylammonium cholate, 56617-36-8;bromohexadecane, 112-82-3; hexadecyltributylammonium bromide, 643967-4; taurocholic acid sodium salt, 145-42-6; hexadecyltributylammonium taurocholate, 124536-25-0.
