Mikrochim. Acta [Wien] 1991, III, 137-145 Mikrochimica Acta

�9 by Springer-Verlag 1991 Printed in Austria

Estimation of Critical Micelle Concentrations of Bile Acids by Reversed-Phase High Performance Liquid Chromatography I

Roger Shaw 2, William H. Elliott, and B. George Barisas*

Edward A. Doisy Department of Biochemistry, St. Louis University, St. Louis, MO 63104, USA

Abstract. The critical micelle concentration (CMC) for bile salts or other surfac- tants is defined as that solute concentration at which appreciable changes ~in such phenomena as light scattering, surface tension, or solubilization of other organic molecules occur, these changes indicating appearance of surfactant aggregates. The CMC thus reflects hydrophobic interactions of the surfactant with itself. The self-association ofhydrophobic molecules resembles the partition of a solute into the lipophilic phase in reversed-phase high performance liquid chromatography (RPLC): Both processes can be considered as transfers of a molecule from an aqueous to a lipophilic medium. The critical micelle concentra- tion of a particular bile salt, being a measure of its hydrophobic self-association, should therefore be correlated with its chromatographic mobility since they are fundamentally related phenomena. Experimentally, significant correlations be- tween these quantities are obtained, both for bile salts and n-alkyl sulfonates, and only microgram amounts of sample are required for RPLC measurements. Among three homologous series of bile salt surfactants, CMC values predicted from RPLC measurements agree, within a standard error of 7%, with CMC values determined directly. This suggests the applicability of reversed-phase liquid chromatography to the micro-scale determination of critical micelle con- centrations of bile salts, n-alkyl sulfonates, and other homologous series of surfactants.

Key words: bile salts, n-alkyl sulfonates, critical micelle concentrations (CMC), high performance liquid chromatography (HPLC), micelles.

* To whom correspondence should be addressed at the Department of Chemistry, Colorado State University, Fort Collins, CO 80523, USA

1 This work was supported in Part by NIH Grants HL-07878 (W.H.E.) and AI-21873 (B.G.B.) and by a Fulbright Senior Fellowship (B.G.B.). This is paper LXXX in the series "Bile Acids" by W.H.E.

2 Deceased March 29, 1986

138 R. Shaw et al.

Thermodynamic considerations indicate that reversed-phase high performance liq- uid chromatography (RPLC) 3 should be useful in investigating hydrophobic inter- actions between molecules, particularly between surfactants [1]. The R P L C mobili- ties of surfactants and the binding affinities of such molecules to one another in micelles should both reflect the free energies of the transfer of detergent molecules from an aqueous to a hydrophobic phase [ la , 2]. Thus R P L C measurements should consti tute a means of estimating surfactant properties such as critical micelle con- centrations (CMC). Moreover the small sample requirements of R P L C would permit determinat ions on micro-scale amounts of sample. Liquid chromatography has previously been used to study bile salt micelle formation, but chromatographic mobilities have not been successfully compared to corresponding C M C values. A m m o n and Walter [21] used gel exclusion chromatography to determine bile salt m o n o m e r concentrations. Cantafora et al. [20] observed correlations between cho- lesterol-solubilizing capacities of bile salt solutions and their R P L C mobilities. Thus, experimental verification of C M C - R P L C mobility relationships has not yet been presented.

Bile acids 4 are physiologic detergents whose physical and chromatographic properties [3] make them uniquely suited for such studies. They are C24-steroids (Fig. 1) usually containing a cis- or t r a n s - A / B ring junct ion and e- o r / / -o r ien ted nuclear hydroxyl groups. The terminal carboxyl group of the side-chain may be conjugated with the amino acids taurine or glycine. The presence of opposing hydrophil ic (e) and hydrophobic (13) surfaces on the same steroidal skeleton ex- plains the surfactant nature of these compounds [-2]. We report here the use of bile acids as trial surfactants to study the relationship between mobility in R P L C and critical micelle concentrations.

Theory

The mobili ty of a solute in R P L C is best defined by its relative capacity factor rk' [-3]

rk ' - k ' ( x ) k ' (s) ' (1)

where k' is the capacity factor of c o m p o u n d x or the s tandard s. In these studies deoxycholic acid has been chosen as the standard. Since k' is propor t ional to the part i t ion coefficient of the solute from the mobile (aqueous) phase into the stationary (C18-hydrocarbon) phase [4], it must vary with the free energy of part i t ion AGp as follows,

3 Abbreviations used in this paper are: CMC, critical micelle concentration; RPLC, reversed-phase liquid chromatography; 20-MC, 20-methylcholanthrene

4 Bile acids are cholanic acid derivatives whose substituents, trivial names, and abbreviations used in the text are as follows: cholic acid (C), 3a,7c~,12~-trihydroxy-5fl-cholanic acid; chenodeoxycholic acid (CDC), 3~,7~-dihydroxy-5fl-; deoxycholic acid (DC), 3c~,12~-dihydroxy-5fl-, ursodeoxycholic acid (UDC), 3~,7fl-dihydroxy-5fl-; hyocholic acid (HC), 3a,6~,7~-trihydroxy-5fl-; hyodeoxycholic acid (HDC), 3c~,6cc-dihydroxy-5fl-. Only bile acids conjugated with taurine (T) or glycine (G) are included in this study; glucuronides or sulfates are not considered here. Derivatives of 5~-cholanic acid, or allocholanic acid, are designated by (A)

Estimation of Critical Micelle Concentrations of Bile Acids

A H o%~~.,...1,,.,%j,//o H R

H

139

B /5'- side

~ e-side

COzH

Fig. 1. Top view (A) and side view (13) of cholic acid and its conjugates

AGp = --RT(ln k' + to), (2)

where R is the gas constant, T is the absolute temperature, r 0 is a constant. The process of micelle formation can also be treated thermodynamically as the

transfer of detergent molecules from an aqueous to a lipophilic phase, and the free energy change AGM is given [5] by

( A G M = R T l n [ B ] + l n 7 , (3)

where [B] is the concentration of monomers of detergent, 7 is the monomer activity coefficient, [B~] is the concentration of micelles sufficiently large to solubilize an aromatic polynuclear hydrocarbon such as methylcholanthrene, and fiis the average size of such micelles. The detergent concentration [B]c at which solubilization of methylcholanthrene becomes noticeable is the critical micellar concentration. At such concentrations, [B~] << [B]. The activity coefficient can be estimated by a modified Debye-Hiickel equation according to Giintelberg [6] to be 0.010 to 0.042, much smaller than the corresponding ln[B], values. The term ln[B,]/fi should be relatively constant with an assumed value s o for the bile acids under investigation. Incorporation of the above considerations into Eq. (3) yields

A G ~ ~ R T ( l n [ B ] c -- So). (4)

A G e and AGta both represent the transfer of solute molecules from an aqueous to a lipophilic medium and would thus be expected to be related by a linear free energy relationship,

A G M = m A G e - t o R T . (5)

140 R. Shaw et al.

In this view t o represents the difference in the hydrophobicities of the lipophilic environments of the RPLC stationary phase and the detergent micelle and m characterizes the different extents to which a detergent molecule is exposed to these hydrophobic environments. Combining Eqs. (1), (2), (4), and (5) and converting to common logarithms,

log(eMC) = - m log rk' - log k ' ( s ) - ( m r o + s o - t o ) . (6)

Thus, a plot of log(CMC) versus log r k ' for a given homologous series of surfactants is expected to yield a straight line of negative slope.

Experimental

Materials

2-Propanol was Fisher HPLC grade. Water was doubly distilled. Potassium phosphate (analytical grade) was obtained from Mallinckrodt. 6-[14C]-20-Methylcholanthrene (sp. act. 4.61 mCi/mmol) purchased from New England Nuclear Corp. was purified by thin-layer chromatography [7] on plates coated with silica gel H (0.25 mm thick). Elution with benzene/hexane (4 : 6) provided a faintly yellow material which was at least 96~ radiochemically pure. Light and air were excluded as much as possible during work-up. The recovered material was made up to a known concentration in benzene and kept at -20~ under which conditions it was stable for several years.

Cholic acid, hyocholic acid, chenodeoxycholic acid, and deoxycholic acid were taken from stock in this laboratory. Ursodeoxycholic acid was supplied by Sigma Chemical Co. Allocholic acid and allochenodeoxycholic acid were synthesized by allomerization of the corresponding 5fl-bile acids with Raney nickel as described [7]. All of the above compounds were homogeneous by thin-layer chromatog- raphy (chloroform : methanol : acetic acid, 80 : 12 : 3). Sodium salts of taurine- and glycine-conjugated bile acids were prepared by coupling with EEDQ [7]. Sodium tauroursodeoxycholate was produced in 91~o yield. The conjugates were pure by thin-layer chromatography (chloroform/methanol/water, 15 : 10 : 2).

P r o c e d u r e

The capacity factors k' of bile acids were determined [-3] on a #Bondapak/Cla column (30 c m x 0.4 cm I.D.) housed in a Waters Associates ALC 201 instrument containing a U6K loop injector and a R401 differential refractometer. The eluent consisted of 2-propanol/10 mM potassium phosphate (pH 7.0) (160 : 340), flowing at 1 ml/min. Refractometer detection limits for bile acids were approximately 2, 6, and 10 nmols for taurine-conjugated, glycine-conjugated, and unconjugated bile acids, respectively. Typical sample size was 10 #1 containing about 10 #g of bile acid. In each case the sample concentration was well below its measured critical micelle concentration. Thus the primary hydrophobic interaction of the sample was always with the stationary phase rather than with other sample molecules. Relative capacity factors rk' were calculated by Eq. (1) using k' values for deoxycholic acid as the standard.

CMC values were determined similar to the method of Norman [8] with a few variations. Excess 6-[-14C]-20-methylcholanthrene was incubated for 48 h with 1 ml solutions of increasing concentrations of salts of bile acids in vials wrapped in Parafilm and sealed with teflon-lined caps. The amount of solubilized labeled hydrocarbon was measured by assaying the radioactivity in the filtrate after filtra-

Estimation of Critical Micelle Concentrations of Bile Acids 141

tion through glass-wood or a sintered-glass funnel. In most experiments which mimick the conditions of RPLC, the studies were performed at room temperature (23.5-24.5~ in 10 mM potassium phosphate buffer at pH 7 to 8. As a control it was shown that the addition of a steroidal hydrocarbon, 5~-cholestane, to the above bile acid solutions did not alter the pH. Two taurine-conjugated bile acids were studied in water at pH 5-7 at 40~ These particular materials exhibit CMC values independent of temperature over the 25-40~ range and independent of pH at values above pH 5 [2]. Some CMC values were taken from the original 20-MC solubilization measurements of Norman [8] and of Ekwall et al. [9].

Data on solubility of methylcholanthrene in bile acid solutions were smoothed using a variation of the model described by Mazer et al. [10] which takes account of primary and secondary micelle formation. An iterative non-linear least-squares algorithm [11] was implemented in the BASIC language on an IBM-PC/XT com- puter. The apparent CMC 5 was defined operationally as the bile salt concentration at which 20-MC solubilization had risen to twice the baseline value, i.e. where significant 20-MC solubilization occurs. This matter is developed further in the next section.

Results and Discussion

The CMC of a detergent molecule reflects the hydrophobic affinity of the molecule for the micellar environment and numerous methods, both invasive and non- invasive, have been used to measure this quantity [2]. We have chosen to measure CMC values by an invasive method, namely by monitoring the extent of solubiliza- tion of a lipophile by increasing concentrations of the bile acid. There are three reasons for this choice. First, the majority of CMC values reported by others [8, 9, 12] on systems of interest to us have been obtained by this method. Second, the procedure is simple and no sophisticated instrumentation is required. Finally, the CMC values thus obtained are more useful for practical purposes than those obtained from non-invasive experiments since most uses of surfactants involve solubilization of foreign molecules. In contrast to studies involving pure micelles only, the invasive method introduces a second equilibrium between detergent and indicator molecules, which may perturb the hydrophobic interaction between de- tergent molecules. However, it has been argued that if the indicator molecule is highly insoluble in water, the perturbation should be minimal, as has been demon- strated for the detergent sodium lauryl sulfate [12]. Norman's [8] results also show that the molar ratio of solubilized methylcholanthrene to bile acid is very much less than one. Thus, the interference of methylcholanthrene in the bile acid aggregation equilibrium should be minimal.

The graphical method of determining CMC by drawing intersecting lines on solubility curves is not as useful for bile acids as for linear detergents which aggregate

5 It should be emphasized that the term "CMC" as applied to a bile acid is an operational measure of the hydrophobic affinity of a bile acid defined for the purpose of correlation with the rk' of the bile acid in RPLC. It does not imply that the phenomenon of highly-cooperative micelle formation exhibited by such materials as the n-alkyl sulfonates is applicable to the bile acids

142 R. Shaw et al.

~L

i O N.

I I I I

0

/ CMC

10 20 30 4 0

[UDC] (rnM)

Fig. 2. Solubilization curve of 20-methylcholanthrene by sodium ursodeoxycholate ( 0 - 0 ) . Contin- uous curve represents data smoothed by computer fitting to the model of Mazer et al. [10]. The experiment was performed in 10 mM potassium phosphate (pH 7.0), UDC denotes ursodeoxycholate; and 20-MC, 20-methylcholanthrene

much more cooperatively. The data from bile acids do not provide a sharp break in the solubilization curve (Fig. 2), and consequently the uncertainty is high as to the best region to draw the lines. For these reasons, we have chosen to specify the CMC operationally as that concentration of a bile acid at which significant 20-MC solubilization takes place, i.e., where significant deviation takes place from the straight line drawn through the points at low bile acid concentrations. Our results for CMC determinations of bile salts are summarized in Table 1. Where CMC values have been determined from 20-MC solubilization both by ourselves and by others (NaC, NaTC, NaTDC, and NaTCDC), good agreement between these values is observed.

Table 1 also contains values of rk' measured for the various bile acids by RPLC. Plots oflog(CMC) versus log rk' (Fig. 3) demonstrate clearly the correlation between CMC and rk' predicted by Eq. (6). The data resolve themselves into three sets, the unconjugated bile acids and the taurine and glycine conjugates. For the unconju- gated bile salts the slope of the regression line is -0.93, the intercept is 0.72, and the correlation coefficient is 0.98. For the taurine conjugated, bile salts, these quantities are -1.15, 0.52, and 0.98, respectively. For the glycine conjugates, the regression parameters are -1.02, 0.55, and 0.99, respectively. All three lines are essentially parallel with negative slopes. This is precisely the behavior predicted by Eq. (6).

Estimation of Critical Micelle Concentration s of B!le Acids

Table 1. CMC and rk' values for bile acids

143

CMC (mM) log(CMC) Bile acid rk'" log rk 'a (measured) b (measured) b

log(CMC) (predicted) c

C 0.35 -0 .46 13.0 (ref. [9]) 1.11 1.14 HDC 0.36 --0.44 13.0 (this study) 1.10 1.12 UDC 0.34 -0 .47 15.5 (this study) 1.19 1.15 CDC 0.80 -0 .10 7.0 (ref. [8]) 0.85 0.81 DC 1.00 0.00 5.0 (ref. [9]) 0.70 0.72 TC 0.31 -0.51 12.0 (ref. [8]) 1.08 1.11 TAC 0.32 - 0.49 12.6 (this study) 1.10 1.09 TCDC 0.68 -0.17 6.0 (ref. [8]) 0.78 0.72 TACDC 0.69 -0.16 4.7 (this study) 0.67 0.70 TDC 0.83 - 0.08 4.0 (ref. [9]) 0.60 0.61 GC 0.30 -0 .52 12.0 (ref. [8]) 1.08 1.08 GAC 0.29 -0.54 12.3 (this study) 1.09 1.10 GCDC 0.65 - 0.19 6.0 (ref. [8]) 0.78 0.74 GACDC 0.66 - 0.18 5.5 (this study) 0.74 0.73 GDC 0.83 - 0.08 4.0 (ref. [9]) 0.60 0.63

" Experimental rk' and log rk' values from this study b Experimental CMC and log(CMC) values from this study or from other specified sources ~ Iog(CMC) values predicted using Eq. (6) from measurements of rk'

E

0

o

1.2

1.0

. 8

. 6

A U

Hocj[ c

q0c

i I I I I

0 .2 .4

- log rk'

. ' I

/ i'oo I i i i i i

0 .2 .4

- log rk'

, i , i i

C

/ TTGCDC

I I I I I

0 .2 .4

- log rk'

Fig. 3. Relationship between log rk' and log(CMC) for bile salts. Left: free bile salts: intercept, 0.72 slope, -0.93; correlation coefficient, 0.98. Center: taurine conjugates: intercept, 0.52; slope, -1.15 correlation coefficient, 0.98. Right: Glycine conjugates: intercept, 0.55; slope, -1.02; correlatior coefficient, 0.99. Error bars indicate the __+ 15~o estimated standard deviation of individual CM( measurements

I n the p r e c e d i n g p lo t s the l o w e r va lue o b s e r v e d for the i n t e r cep t o f the un.

c o n j u g a t e d bile ac ids re la t ive t o the g lyc ine- a n d t a u r i n e - c o n j u g a t e s is eas i ly under .

144 R. Shaw et al.

stood. It is well known that unconjugated bile acids near neutral pH exhibit lower solubility and higher CMC values than the corresponding conjugates and this is usually attributed to incomplete ionization of the unconjugated acids. On the other hand, in RPLC free bile acids exhibit higher rk' values than the corresponding conjugates. This has been attributed by Shaw et al. [3] to lower solubility of the free salts in the mobile phase causing increased retention times.

Many scientists working with bile acids [2] use the CMC to derive the free energy of micellar aggregation. However, it is questionable if a true CMC actually exists for the bile acids [13]. The lack of a sharp break in the solubilization curve (Fig. 2) strongly argues for the existence of a number of polymeric species in equilibrium in a solution of a bile acid with no specific polymer predominating in the concentration range investigated [14, 15]. This concept is reinforced by results from studies on diffusion [16] and changes in the molal volume [17].

On the other hand, the n-alkyl sulfonates are well known to exhibit highly cooperative micelle formation so that both CMC values and RPLC retention times can readily be determined. A test of the general validity of our method is to examine CMC and rk' values for a homologous series of these materials. Fig. 4 is a plot of log (CMC) versus log rk' for C8-C14 sodium n-alkyl sulfonates in aqueous solutions where data are taken from the literature [18, 19]. The slope of the line is - 1.72 and the correlation coefficient is 0.99. The excellent fit of these data to Eq. (6), which doubtless arises in part from the precision with which CMC values can be obtained for such highly cooperative systems, argues strongly for the general applicability of our method for CMC estimation.

The data thus suggest that CMC values can be accurately estimated from RPLC data, once CMC values for a sufficient number of homologous surfactants have been measured experimentally. The standard deviation of observed log(CMC) values in Table 1 about those predicted by Eq. (6)is 0.03 log units or about 79/0. Thus, within

i J , i

2.0 log CMC vs. log k'

for homologous linear

.EE 1.5 C n - alkylsulfonates

�9 ~ C10 :s o 1.0

0.5

C14 t I I i

-0.6 -0.4 -0.2 0.0 0.2

-log k'

Fig. 4. Relationship between log rk' and log(CMC) for homologous n-alkyl sodium sulfonates. CMC and RPLC data are from Ogino et al. [18] and Boiani [19], respectively. Intercept, 1.61; slope, - 1.72; correlation coefficient, 0.99

Estimation of Critical Micelle Concentrations of Bile Acids 145

a homologous series of surfactants, CMC values predicted from RPLC measure- ments appear to approximate satisfactorily those determined directly.

Acknowledgements. This paper is dedicated to the memory of Dr. Roger Shaw, a talented young biochemist who executed the experimental work of this study while an Assistant Research Professor in the E. A. Doisy Department of Biochemistry, St. Louis University, School of Medicine. Prior to his untimely death on March 29, 1986, he was a member of the scientific staffof the Litton Cancer Research Center, Frederick, MD 21701. We wish to thank Mr. M. Rivetna for his technical assistance and Drs. J. W. Davis and J. C. Lee for useful discussions.

References

[1] C. Horvath, W. Melander, J. Molnar, J. Chromatogr. 1976, 125, 129. [ la] R. Shaw, W. H. Elliott, Fed. Proc. 1980, 39, 1721.

[2] D. M. Small, in: ?he Bile Acids, Chemistry, Physiology and Metabolism, Vol. 1 (P. P. Nair, D. Kritchevsky, eds.), Plenum Press, New York, 1972, pp. 249-356.

[3] R. Shaw, M. Rivetna, W. H. Elliott, J. Chromatogr. 1980, 202, 347. [4] H. Konemann, R. Zelle, F. Busser, W. E. Hammers, J. Chromatogr. 1979, 178, 559. [5] C. Tanford, in: ?he Hydrophobic Effect: Formation of Micelles and Biological Membranes, 2nd

Ed., Wiley, New York, 1980, chapt. 7. [6] R. A. Robinson, R. H. Stokes, in: Electrolyte Solutions, 2nd Ed., Butterworths, London, 1970,

pp. 231,468. [7] R. Shaw, W. H. Elliott, Lipids 1980, 15, 805. [8] A. Norman, Acta Chem. Scand. 1960, 14, 1295. [-9] P. Ekwall, K. Fontell, A. Sten, Proc. 2nd lnt. Congress Surf Act. 1957, Vol. I, p. 357.

[-10] N. A. Mazer, M. C. Carey, R. F. Kwasnick, G. B. Benedek, Biochemistry 1979, 18, 3064. [11] B. G. Barisas, S. J. Gill, Biophysical Chem. 1979, 9, 235. [12] R.J. Williams, J. N. Phillips, K. Mysels, J. Trans. Faraday Soc. 1955, 51,728. [13] W. I. Higuchi, C. C. Su, J. Y. Park, M. H. Alan, J. Phys. Chem. 1981, 85, 127. [14] P. Mukerjee, J. R. Cardinal, J. Pharm. Sci. 1976, 65, 882. [15] A. F. Hofmann, Biochem. J. 1963, 89, 57. [16] B. Lindman, N. Kamenka, H. Fabre, J. Ulmius, T. Weiloch, J. Coll. Interf Sci. 1980, 73, 556. [17] M. Vadnere, R. Natarajan, S. Lindenbaum, J. Phys. Chem. 1980, 84, 1900. [18] K. Ogino, T. Kakihara, M. Abe, Coll. and Polymer Sci. 1987, 84, 604. [19] J. A. Boiana, Anal. Chem. 1987, 59, 2583. [20] A. Cantafora, A. Di Biase, M. Angelico, D. Alvaro, Chromatographia 1987, 24, 277. [21] H. V. Ammon, L. G. Walter, Anal. Chem. 1982, 54, 2079.

Received October 1, 1990. Revision October 4, 1990.