THE EXTRACTION AND SEPARATION OF BILE ACIDS

BY EDWARD H. AHRENS, JR.,* AND LYMAN C. CRAIG

(From the Laboratories of The Rockefeller Institute for Medical Research, New York, New York)

(Received for publication, November 14, 1951)

As experience is gained in the attempt to apply counter-current dis- tribution to the separation of complicated biological substances from the mixtures in which they naturally occur, it becomes abundantly clear that the solubility and the hydrophobic or hydrophilic properties of known pure components are markedly altered in such mixtures. Thus, one solute may carry with it into solution a second solute which by itself has poor solubility, or it may partition a hydrophilic solute predominantly into an organic phase.

Although the delicate balances required in living systems probably de- pend to a large extent on such interactions, separation procedures in the laboratory are rendered thereby much more uncertain and difficult. A familiar example is to be found in bile. The components of bile have long been known to have unusual solubilizing capabilities which have been thought to play a role in the absorption from the intestine into the blood stream of various lipide substances. Also it is well known that any at- tempt to isolate the components of bile without first modifying their prop- erties by hydrolysis will almost certainly meet with considerable difficulties.

Since strong association tendencies are characteristic of such substances, it would appear that a profitable approach to their separation and charac- terization might come through the compounding of systems suitable for counter-current distribution which would take advantage of these associa- tion tendencies.

This line of thought has resulted in a study of the separation by counter- current distribution of the known components of bile, both hydrolyzed and unhydrolyzed. Our present objective has not been to isolate and finally to characterize the known and unknown components of bile, but rather to make a preliminary survey of systems and the components of systems which might be specifically useful. It is obvious that, with solutes which deviate strongly from the ideal, the components of a system can be as- sessed properly only in the light of their effect on the partition ratios of those solutes through the concentration gradients which are necessarily a part of the distribution scheme. Actual distributions with many transfers are therefore required for critical evaluation.

* Senior Fellow of The National Foundation for Infantile Paralysis, Inc.

763

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764 SEPARATION OF BILE ACIDS

It is shown in another report (1) that solvent systems containing acetic acid permit almost ideal behavior in the separation of the higher fatty acids by counter-current distribution. The effect of this solvent appears to be due to a competitive association in which the overpowering numbers of acetic acid molecules insure that the vast majority of associated molec- ular complexes consist of single molecules of fatty acid with acetic acid. As a result distributions proceed largely as if only monomeric species of the solute of interest were involved.

Advantage was taken of this observation when the distribution of the free bile acids was approached. In systems containing high concentrations of acetic acid, cholic, desoxycholic, and lithocholic acids and their dehydro derivatives behaved ideally and permitted their separation. This state of affairs held also for the glycine-conjugated bile acids, but with the taurine conjugates such systems gave partition ratios too low for practical use.

From a physiological point of view, the greatest interest centers upon the separation and characterization of the unhydrolyzed components. A distinct advantage would be gained if the usual hydrolysis of bile extracts could be avoided, since conditions which guarantee complete hydrolysis without inducing possible changes in chemical structure have not been defined as yet. Also the lack of quantitative data on the interrelationships of the various conjugated bile acids in normal and abnormal biles added considerably to the desirability of studying the intact substances as they naturally occur.

Materials and Methods

The known samples of bile acids used in setting up suitable solvent sys- tems and in preliminary distributions were obtained from various sources. The majority had been synthesized by George A. Breon and Company and were furnished to the authors by Dr. Herbert Jaffe of the Rockefeller Institute, to whom thanks are due. Others synthesized or isolated from natural sources were supplied by G. D. Searle and Company through the courtesy of Dr. A. L. Raymond. The various acids studied are listed with source references in Table I.

Fresh ox bile was obtained by gallbladder puncture from a local slaugh- terhouse. 200 ml. lots were frozen in dry ice and acetone within 4 hours of collection, and were either lyophilized or stored at -20”.

Partition ratios mere determined for the most part at concentrations of 1 per cent, and isotherms were plotted over a concentration range of 0.5 to 3 per cent. Concentrations in the two phases were measured by a rapid microgravimetric procedure, accurate to ho.02 mg., described by Craig et al. (2). To volatilize all traces of water from these samples,

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E. H. AHRENS, JR., AND L. C. CRAIG 765

preliminary dryings on a steam bath were followed by heating at 100” for 20 minutes under a high vacuum.

Distributions of more than 100 transfers were made in the all-glass, fully

TABLE I

Partition Ratios* of Free and Conjugated Bile Acids in Various Solvent Systems

Bile acids, source E iyster A

System B

-

B L

14

7

SyFm iystem “‘S&an C

0.01:

0.06 2.25 0.25 0.16 0.18

Conjugated

Free

Sodium taurocholate, Searle Glycocholic acid, Breon, Searle Glycodesoxycholic acid, Searle Dehydrocholic acid, Searle 3,12-Dihydroxy-7-ketocholanic

acid, Breon 3,7-Dihydroxy-12-ketocholanic

acid, Breon 3-Hydroxy-7,12-diketocholanic

acid, Breon Cholic acid, Breon Hydrodesoxycholic acid (3,6-di-

hydroxycholic), Searle Dehydrodesoxycholic acid, Searle wDehydrohydrodesoxycholic

acid, Searle P-Dehydrohydrodesoxycholic

acid, Searle 3-Hydroxy-12-ketocholanic acid,

Breon Desoxycholic acid, Searle, Breon Dehydrolithocholic acid, Searle Lithocholic acid, Searle

-

0.00

0.01

0.06

0.24

,

0.024

0.07:

0.14

0.49

1.14 7.4 7.7

0.20

0.20

0.31 0.88

1.05 1.05

1.06

1.24

2.14

12.4

0.94

6.2 12.8

* 100 mg. of solute per 10 ml. of each previously equilibrated phase. System A, 2,2,4-trimethylpentane or n-heptane-HOAc (97.5 per cent); System

B, isopropyl ether (75 per cent), n-heptane (25 per cent)-HOAc (60 per cent); System C, isopropyl ether (85 per cent), n-heptane (15 per cent)-HOAc (60 per cent); Sys- tem D, n-butanol (30 per cent), n-heptane (70 per cent)-HOAc (10 per cent); System E, set-butanol-HOAc (3 per cent).

automatic machine described by Craig et al. (3). Shorter runs were made in the hand-operated glass apparatus reported by Craig and Post (4).

The evaporation of solvents was carried out at temperatures below 30” under a vacuum in the rotary distillation apparatus described by Craig el al. (5). Foaming was controlled easily by addition of n-butanol to the distilland.

Extractions of bile acids from bile were checked at various stages by the

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766 SEPARATION OF BILE ACIDS

Pettenkofer reaction as follows. Dried residues were taken up in about 0.5 ml. of ethanol, to which a drop of saturated sucrose solution was then added. About 2 ml. of concentrated sulfuric acid were dropped into the mixture with development of a deep Burgundy red color in the presence of 1 mg. or more of bile acid.

Amino acids conjugated to the bile acids were identified by paper chro- matography in two solvent systems.’ In a system consisting of 75 ml. of set-butanol, 15 ml. of 88 per cent formic acid, and 10 ml. of water, the Rp values of glycine and taurine were 0.25 and 0.09, respectively. In an alkaline system of 100 ml. of set-butanol and 40 ml. of 3 per cent aqueous ammonia, Rp values were 0.19 and 0.24 for glycine and taurine. Under these conditions no other ninhydrin spots were found in hydrolysates of conjugated bile acids.

Distribution Studies with Known Bile Acids

Distribution of Free Bile Acids and Glycine Conjugates-In a system con- sisting of n-heptane or isooctane and 97.5 per cent acetic acid (2.5 per cent H&), it was found that the higher fatty acids could be separated readily from the bile acids. The partition ratios (K values) of the fatty acids (C,, and longer) were all higher than 1.9, while the K values of the bile acids ranged from 0.24 (lithocholic acid) to 0.004 (glycocholic acid). The spread of K values of the bile acids in this system (System A, Table I) gave promise that more advantageous systems could be devised either by lowering the concentration of acetic acid or by adding an upper phase with more attracting power. Thus, in isopropyl ether-acetic acid (50 per cent), glycocholic acid had a K of 0.23, but desoxycholic acid was found com- pletely in the upper phase. In isopropyl ether (75 per cent), n-heptane (25 per cent)-acetic acid (60 per cent) the range of K was 7.7 (lithocholic acid) to 0.02 (glycocholic acid). Several distributions of known bile acid mixtures were carried out in this system (System B, Table I) for 100 or more transfers. Since partition isotherms were essentially linear, there was close correspondence between experimental and theoretical curves. It was demonstrated that mixtures of cholic, desoxycholic, and lithocholic acids could be completely separated in 55 transfers, and mixtures of the three corresponding dehydro (keto) acids in 70 transfers. Synthetic mix- tures of all six acids were separated into five components in 400 transfers. Lithocholic and dehydrolithocholic acids, with K values during individual distributions of 5.8 and 5.1, ran together with a K of 5.15. Another ap- proach to complete resolution of mixtures of hydroxyl and ketonic acids probably could be made by a preliminary extraction of the ketonic acids with Girard’s Reagent T (6), followed by regeneration and distribution

1 Gregory, J. D., unpublished data.

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E. H. AHRENS, JR., AND L. C. CRAIG 767

of the two groups, after which 70 transfers for each group should be suf- ficient. This, however, has not actually been done in this laboratory.

Variation of the isopropyl ether concentration in System B considerably affects the partition ratios of the various bile acids. Thus, raising the isopropyl ether-heptane ratio from 50:50 to 75:25 to 85: 15 raised the K of cholic acid from 0.02 to 0.14 to 0.31. The latter system (System C, Table I) was finally adopted for use in subsequent distributions of the free and glycine-conjugated bile acids.

Separation of Taurine-Conjugated Acids from Other Bile Acids-It was found desirable to separate as a group those bile acids conjugated to taurine on the one hand from the free bile acids and those conjugated to glycine on the other. Since the taurine conjugates are more hydrophilic than the glyco or free acids, it was necessary to devise systems in which the attrac- tion of the aqueous lower phase was greatly diminished, relative to the systems already discussed. This was accomplished by decreasing the ace- tic acid concentration of the lower phase. Thus the K of glycocholic acid in n-butanol (30 per cent), n-heptane (70 per cent)-acetic acid (40 per cent) was 0.006, and with successively diminishing concentrations of acetic acid the K rose to 0.034 (30 per cent), 0.46 (20 per cent), 2.26 (10 per cent), and 4.4 (5 per cent). The 10 per cent acetic acid system was used in several distributions of about thirty transfers to separate the tauro acids from the glyco acids and free acids (System D, Table I). In retrospect, fewer transfers might be required if the acetic acid concentra- tion were lowered to 5 per cent (sodium taurocholate K = 0.04, glycocholic acid K = 4.4). Deviations from linear isotherms in weak acetic acid systems are of no great importance in these group separations since there is no advantage in subjecting these distributions to mathematical in- terpretation.

Distribution of Taurine Conjugates-The separation of taurine-conju- gated acids from each other necessitated the use of aqueous sytems such as those applied to the separation of amino acid mixtures (7), because of the strong hydrophilic properties of these compounds. When it was found that the K of sodium taurocholate in set-butanol-acetic acid (3 per cent) (System E) was about 1, a distribution of 660 transfers was carried out on a 0.5 gm. sample of this salt (Fig. 1). Although the peak of the bile acid curve traveled at a calculated K of 1.03, there was considerable phase dis- tortion and marked skewing of the curve to the right. That this was due to non-linearity of the partition isotherm was demonstrated when K values determined at various points along t.he two limbs were found to fall from 2.1 at 0.025 per cent concentration (Tube 333) to 1.5 at 0.05 per cent concentration at the peak (Tube 340). However, K values at similar concentration levels on both sides of the curve were almost identical,

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768 SEPARATION OF BILE ACIDS

indicating homogeneity of the solute band. The reason for the irregularity of the right limb of the curve was not apparent.

To combat the tendency toward skewing, numerous attempts have been made to find a system to which more acetic acid can be added, while at the same time opposing this agent with a sufficiently attractive organic sol-

-butanol /HOAC (3%)

Tube No. FIN. 1. Distribution pattern of sodium taurocholate isolated from natural sources

(Searle). At 225 transfers, the salt band (K = 0.218) was removed before recycling.

vent to bring the taurine conjugates into the upper phase. Larger con- centrations of acetic acid cannot be used in System E because mutual solubility is reached at about 6 per cent acetic acid concentration. If some other organic solvent is added in order to permit a higher concen- tration of acetic acid, the effect of both steps is to force the solute almost entirely into the aqueous phase. Also, the addition of n-butanol, iso- propyl ether, diethyl ether, or heptane produces troublesome emulsifica- tion. It will be noted below that, although distributions were carried out in System E with good separation of taurocholic from taurodesoxycholic

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E. H. AHRENS, JR., AND L. C. CRAIG 769

acid, marked skewing made quantitative evaluation of the distribution difficult.

It is conceivable that a carrier like lauryl amine (8) might be useful to attract these compounds into an organic solvent. However, experiments with non-volatile carriers have not been made by us, primarily because of interference with direct gravimetric analysis.

Fractionation of Bile

Extraction of bile with ethanol is the procedure generally used to sepa- rate the bile acids from the precipitated mucoproteins. Thus, for small scale extractions bile is added to about 25 volumes of ethanol. In the present experiments considerable economy in solvent volume was made by repeated extractions of lyophilized bile powder with small volumes of solvent. It was convenient to lyophilize 200 ml. quantities of bile to which equal volumes of water had first been added with thorough stirring. Thus, 200 ml. of bile, sp. gr. 1.022, yielded about 16 gm. of powder which ap- peared dry but was extremely hygroscopic. This powder was immediately mixed with 400 ml. of absolute ethanol with a footed rod to break up all the lumps. The centrifuged mixture could be cleanly decanted. Three such extractions were carried out. The third extract was found to be Pettenkofer-negative. Typically, the residue was white and had a dry weight of about 0.7 gm. The extract containing bile acids, lipides, and inorganic salts was evaporated quickly to near dryness.

The separation of the bile acids from the other components in the ex- tract proved to be difficult, and several procedures were explored. Clas- sical extraction in repeated single stages with ether and with heptane was compared with preparative counter-current distribution of the entire ex- tract.

Fractionation of Bile Extract by Classical Single Stage Exlractions-The ethanol extract of 200 ml. of bile was taken up in 100 ml. of water and brought to pH 9 with sodium carbonate. The mixture was extracted three times with equal volumes of ether in order to remove cholesterol, cholesterol esters, and triglycerides. The phases were separated by cen- trifugation. The ether extracts were back-washed with water which was returned to the aqueous fraction. The aqueous fraction was taken to pH 2 with H&304 and extracted once with heptane to remove free fatty acids. Bile acids in the neutralized aqueous fraction were freed of inorganic salts by extraction of the dried residue with absolute ethanol. The ethanol extract was taken to dryness, leaving a greenish white amorphous powder (final fraction) which was moderately hygroscopic. The ether and heptane extracts were taken to dryness also, leaving in the first case a yellow oily residue with some crystals and a brownish solid in the second.

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770 SEPARATION OF BILE ACIDS

The ether, heptane, and final fractions weighed 425 mg., 190 mg., and 13.8 gm., respectively. Subsequent fractionation by counter-current dis- tribution demonstrated that all three fractions contained bile acids, 250 mg., 100 mg., and 12.3 gm., respectively. Cholesterol and cholesterol esters (qualitative Liebermann-Burchard reaction) were present only in the ether fraction and totaled 120 mg. There were about 80 mg. of free fatty acids in the heptane fraction. Lipide phosphorus was found in all fractions: ether 2 mg., heptane 0.6 mg., and final fraction 34 mg. Thus 97 per cent of the bile acids and 93 per cent of the phospholipides of bile were found in the final fraction. This fraction also contained 9 per cent ash, much of which could be accounted for as sodium salts; nitrogen analyses of 3.7 per cent indicated the presence of a small amount of non- protein nitrogen compounds other than those conjugated to known bile acids (glycolithocholic acid N = 3.23 per cent).

Fractionation of Bile Extracts by Preparative Counter-Current Distribu- tion-The entire residue from an alcohol extract of 200 ml. of bile was distributed between heptane and 97.5 per cent acetic acid (System A). Emulsions did not form during this distribution. Twenty transfers com- pletely separated the inorganic salts, bile acids, and phospholipides on the one hand, from the remaining lipide components on the other. In a representative distribution 15.1 gm. of solutes, including the bile acids, were found in the aqueous phase in five tubes at one end of the train and were separated by three tubes from 210 mg. of lipides which favored the heptane phase in ten tubes at the other end of the train. The complete- ness of this separation was verified by lipide phosphorus analyses and by Liebermann-Burchard and Pettenkofer reactions. This procedure pro- duced the desired quantitative separation of the bile acids from the other lipides of bile, while the classical extraction described above led to in- complete separation of bile acids despite a considerably larger expendi- ture of time and effort. Since further characterization of the components in the small heptane fraction was not the primary interest of this study, this fraction was set aside. Systems are described in other reports from this laboratory (1)2 in which the fatty acids, cholesterol, and cholesterol esters of this fraction can be separated for identification.

Fractionation of Bile Acid Groups into Individual Components-The mix- ture of solutes which favored the aqueous phase in the above distribution consisted of inorganic salts, taurine-conjugated bile acids, glycine conju- gates, and free bile acids. The phospholipides of bile also remained in this fraction. It was found useful to make a second preparative dis- tribution of this mixture in order to isolate the salts and taurine conju- gates. This was accomplished in System D in thirty-six transfers in a

2 Ahrens, E. H., Jr., and Craig, L. C., J. Biol. Chem., in press.

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E. H. AHRENS, JR., AND L. C. CRAIG 771

twelve tube train. When the entire mixture (15.1 gm.) was thus dis- tributed, almost complete separation was achieved between 11.1 gm. of taurine conjugates (plus inorganic salts) and 4.0 gm. of a mixture of glycine conjugates, free bile acids, and phospholipides. The former group pre- ferred the aqueous phase, while the latter group favored the organic phase. The separated groups were recovered and taken to dryness.

Glycine Conjugates-The smaller fraction from the second preparative distribution was then run in System C. The first of three successive anal- yses is shown at the top of Fig. 2. At 216 transfers the phospholipides occupied a sharp band in Tubes 160 to 215, and three bile acid peaks at Tubes 10, 34, and 78 were found. At this stage the phospholipide band (1.7 gm.) was removed from the train and replaced with fresh solvent phases. The upper phases were recycled until 412 transfers had been accomplished. Analysis showed that the band which at 216 transfers had its peak at Tube 78 had now moved along to occupy Tubes 120 to 170 with its peak at 145; the experimental and theoretical curves coin- cided. This fraction (K = 0.531) was removed and replaced with fresh solvents. The two other bands, with peaks now at Tubes 25 and 67, were left in the machine; recycling was carried to 902 transfers. The final analysis at the bottom of Fig. 2 showed the appearance now of three distinct bands.

Comparison of calculated and experimental curves indicated that the fraction with K of 0:072 was contaminated with a small amount of material with a lower K, denoted by the lack of fit of the extreme left limbs of the curves. This could be explained by a carry over of a very small amount of taurine-conjugated acids from the previous preparative distribution. The contents of Tubes 50 to 75 were pooled for further analysis. The curves of the small intermediate band (K = 0.132) showed some over- lapping by each of the neighboring bands. The broad right limb of the major peak (K = 0.202) suggested the presence of a minor component with slightly higher K. The contents of Tubes 90 to 115 and 140 to 165 were separately recovered and taken to dryness.

The residue from the band with K of 0.202 crystallized readily from acetone to give a substance melting at 113-115”. This apparently is the acetone complex of glycodesoxycholic acid in a 1: 1 molar ratio. The material did not lose the molecule of acetone on drying in a high vacuum at 110”.

C29H4B06N. Calculated. C 68.7, H 9.74, N 2.76 Found. “ 68.74, “ 9.50, “ 3.06

The residue from the band of K 0.072 did not crystallize readily from solvents. However, when taken up in glacial acetic acid and lyophilized, a residue which appeared crystalline was obtained. It apparently was

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772 SEPARATION OF BILE ACIDS

FIG. ox bile.

Glycine - Conjugated Bile Acid Group

10 - Lower phase - Upper ‘1

- C---O Theoretical

I Al ‘36

5

4 x=0.072 K=O.ZOZ

t

3 @d

20 40 60 80 100 120 140 160 220 Tube No.

2. Distribution pattern of glycine-conjugated bile acid group isolated 1 irom

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E. 1~. AHRENS, JR., AND L. C. CRAIG 773

also a choleic acid type of complex cont.aining 1 M of glycocholic acid and 1 M of acetic acid. It did not lose the molecule of acetic acid on drying to constant weight at 80” under a high vacuum.

C26H4306N. Calculated. C 65.1, H 9.20, N 2.72 Found. “ 65.28, “ 9.08, “ 2.94

This distribution demonstrated the presence of at least four bile acid components in addition to a single band of phospholipides. In order of increasing K values, the proportions of these four components were ap- proximately 31, 7.7, 55, and 6.3 per cent. Elementary analyses and paper chromatography after hydrolysis indicated that all were glycine con- jugates. Although the substituents on the cholanic acid nucleus have not been established with complete certainty in each case, the elementary data and partition ratios suggest that the K = 0.072 band was glycocholic acid, and the K = 0.202 band glycodesoxycholic acid (see Table I). The bands with K values of 0.132 and 0.542 do not correspond to available known conjugated bile acids and presumably are new compounds. They did not crystallize. It is of interest that two different lots of fresh ox bile analyzed in this manner failed to show evidence of free bile acids. How- ever, the second sample of bile contained another major bile acid band with K = 1.23, which failed to appear in the distribution just detailed. This component could be glycolithocholic acid.

Tuurine Conjugates-The taurine conjugates (plus inorganic salts) from a preparative distribution in n-butanol (30 per cent), n-heptane (70 per cent)-acetic acid (20 per cent) were run in System E. Since mutual solubility of the two phases was produced by amounts of mixed solutes in excess of 150 mg. per 10 ml. of each phase, it was necessary to limit the quantity distributed to 1 gm. out of the total fraction of 11.1 gm. The three analyses after successive numbers of transfers are shown in Fig. 3. After 210 transfers a clearly separated salt band in Tubes 10 to 80 was removed and replaced with fresh solvent. Two bile acid peaks began to appear at this stage, and at 398 transfers these components were more sharply separated. Marked skewing of both bands was apparent. On the right there was a tailing out of glycine conjugates and of phospho- lipides incompletely removed by the previous preparative distribution in the 20 per cent acetic acid system. (It should be noted that subsequent preparative distributions carried out in 10 per cent acetic acid have pro- duced clean cut separations of the glycine conjugates.) This tail in Tubes 280 to 380 was removed, solvents were replaced, and recycling continued to 803 transfers. The final analysis at the bottom of Fig. 3 showed fairly complete separation of the two major components. There was very marked skewing due to a partition isotherm in which rising concentrations

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774 SEPARATION OF BILE ACIDS

FIG.

- Lower phase - Upper ‘1 o---o Theoretical

g 0.6 I I Removed

:; ’

:: !! :

1.0

0.4 ii 1% II

I I T 180 200 220 240 260 280 300 320 340 360 380

0.8

0.6

$0.4

L$ 0.2

420 440 460 480 500 520 540 560 580 600 620 Tube No.

3. Distribution pattern of taurine-conjugated bile acid group from ox bile

produced decreasing partition ratios. The same irregularity of the right limb of the curve and skewing was observed also in the distribution of pure sodium taurocholate discussed above (Fig. 1). It is of interest that the calculated K at the peak of the curve of the known bile acid was 1.03 in

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E. H. AHRENS, JR., .4ND L. C. CRAIG 775

Fig. 1, comparing well with the calculated K of 1.14 for the corresponding component in the present distribution. Present data suggest that the band on the right (calculated K at the peak = 1.70) was taurodesoxycholic acid.

Sodium analyses of the tubes in the salt band showed concentrations ranging from 0.10 m.eq. per liter in Tube 20 to 2.70 m.eq. per liter in Tube 50. Potassium concentrations ranged up to 0.12 m.eq. per liter in Tube 50. Sodium acetate was obtained from the band by crystallization from absolute ethanol. By contrast, sodium analyses of the band on the left (Fig. 3, bottom) showed that at least 95 per cent of this band was free acid. There appears to be no ready explanation for the presence of the small percentage of sodium in this band.

In view of the present technical difficulties encountered in distributing the taurine conjugates, it may be advantageous, when quantitative deter- mination of the component acids is desired, to hydrolyze the mixture and then to distribute the free acids in System C. Since any unhydrolyzed taurine-conjugated acids will have an exceedingly low K in System C, they will be separated readily from cholic acid, the free acid with the lom- est K. Such an operation will give a quantitative picture of the complete- ness of hydrolysis at the same time that the component acids are being evaluated.

This approach was carried out on an aliquot of the taurine conjugates isolat.ed in a preparative distribution in System D. Hydrolysis was car- ried out with saturated barium hydroxide by refluxing in a stream of ni- trogen for 5 hours. The bile acids were recovered from the hydrolysis mixture by extraction with see-butanol and were distributed for 67 trans- fers in System C. Three peaks appeared: a large peak of unhydrolyzed acids with K of 0.001, and two smaller peaks with K values of 0.254 and 1.79. All bands gave a positive Pettenkofer test. Since it was estimated that hydrolysis was only 30 per cent complete under these conditions, further characterization of the isolated components was not carried out. However, the two free acid peaks were indicated to be cholic and des- oxycholic acids by comparison with the K of the known compounds. If hydrolysis of the mixed conjugates is assumed to have occurred on a ran- dom basis, the proportions of the two acids can be calculated to have been 54 and 46 per cent, respectively, in the original mixture of taurine conjugates. These figures agree well with the relative amounts of the two acids calculated from the areas under the curves in Fig. 3.

DISCUSSION

The difficulties in working with bile are well known. The strong solu- bilizing and surface-active properties of its many components cause nearly every undesirable behavior an investigator ordinarily seeks to avoid when

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776 SEPARATION OF BILE ACIDS

attempting separations. This was clearly brought out by the incomplete separations obtained with single stage extractions.

On the other hand, counter-current distribution is capable of achieving the desired result. An association complex of the type existing in bile can be considered in terms of a reversible equilibrium which in its simplest form might be expressed by Equation 1, where X and Y are single solutes and n and n’ are

X,Y,’ = nX + n’Y (1)

the number of molecules of each in the complex or micelle. At each stage of the distribution different proportions of X and Y mill be carried forward if the partition ratios of the monomers, X and Y, are different. How- ever, the rate at which the X and Y bands separate from each other will not be as rapid as would be expected from their respective K values, since the equilibrium of Equation 1 always must be satisfied. Therefore, more transfers are required to give clear cut separation, but the ultimate ob- jective is nevertheless attainable. This conclusion is supported by the experimental results reported here in which at least seven conjugated bile acids have been separated from bile.

In general, solutes which tend to associate in mixtures to form mixed micelles, as in Equation 1, also tend to form simple micelles with their own species. This leads to skewed partition isotherms and to serious difficulty in separations when the partition ratios of the components of a mixture are not far removed from each other. Fortunately, this state of affairs has been satisfactorily overcome with the systems reported here, except in the case of the tauro conjugates.

In regard to the relative positions of the various bile acid bands in distributions, the successive addition of hydroxyl or ketonic groups to the cholanic acid nucleus at positions 3, 7, and 12 produced successively greater attraction for the aqueous phase. Strangely enough, when oxygen was introduced as a ketonic group, a greater hydrophilic effect was pro- duced than when it was introduced as a hydroxyl. The reason for this behavior is not apparent at the present time. The conjugated acids were uniformly more hydrophilic than the free acids. Taurine added a greater hydrophilic attraction than did glycine.

The systems presented in this report permitted the demonstration in ox bile of two taurine conjugates and five glycine conjugates. The former outweighed the latter 5 to 1. xo free bile acids were found. The steps by which these components were demonstrated are diagrammed in Fig. 4. Group separations were followed by distributions which separated each group into individual components. At each step an entire fraction or appropriate aliquot from a preceding distribution was further subdivided

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E. H. AHRENS, JR., AND L. C. CRAIG 777

so that there were no discards or residues. In addition to showing the presence of known compounds, counter-current distribution permitted the demonstration of two components apparently unreported heretofore. When partition isotherms were linear, good correspondence between math- ematical and experimental curves gave strong evidence of homogeneity.

Ox Bile I

Ethanol &traction

I Distribution #l

9’7% HOAc : n-Heptane (n = 20)

Bile acids Phospholipides

I DistribAtion #2

Fatty acids Triglycerides Cholesterol and esters

10% HOAc : n-Butanol, n-Heptane (n = 40)

/ Tauro - bile acids

Distribution #3 Distribution #4 3% HOAc : set-Butanol 60% HOAc : Isopropyl ether, n-Heptane

(n = 800) (n = 900)

\ Glyco - bile acids Free bile acids Phospholipides

L..n FIG. 4. Flow sheet for extractions and distributions used in preliminary separa-

tion of the components of ox bile. 72, the number of transfers used.

In these cases the quantity of solute in each band could be calculated and the quantitative interrelationships of the various fractions defined. It is well to note that such calculations are not complicated by the uncertain- ties created by employing hydrolytic techniques, by the addition of com- plexing agents, salts, etc. When partition isotherms were non-linear and the curves skewed, the amount of solute in a band could be derived by weighing the recovered residue. In all cases, proof of separation of in- dividual components was followed by recovery of fractions for further investigation.

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778 SEPARATION OF BILE ACIDS

It is obvious that the picture defined in this series of operations differs in important respects from that which results when other published frac- tionation approaches are applied. Since attention is paid simultaneously to all fractions of the whole and since losses are negligible, this approach may be thought of as “all-inclusive analysis,” or analysis of the whole. The combined analytical and preparative features of counter-current dis- tribution make all-inclusive analysis possible in this field for the first time.

SUMMARY

1. The application of counter-current distribution to the extraction of bile acids from the other components of bile and to the subsequent separa- tion of the various conjugated and free bile acids into individual compo- nents is described.

2. The strong association forces between the various lipide and bile acid constituents of bile were found to create considerable difficulty in the extraction of bile acids by classical techniques. These forces are easily overcome by counter-current distribution however, with clean cut separation of the bile acids from the other components.

3. Solvent systems are presented for (1) separation of bile acids from other bile components, (2) separation of taurine-conjugated bile acids from free bile acids and glycine conjugates, (3) separation of taurine con- jugates into individual components, and (4) separation of glycine conju- gates and free acids into individual components.

4. Studies on fresh ox bile by the above approach demonstrated two taurine conjugates and five glycine conjugates. The former outweighed the latter 5 to 1. No free bile acids were found.

5. Counter-current distribution makes possible for the first time in this field “allinclusive analysis” of the component’s of an extract.

The authors are indebted to Dr. W. J. Eisenmenger of the Hospital of the Rockefeller Institute for sodium and potassium analyses. The tech- nical assistance of Miss Dorris McNamara is gratefully acknowledged.

BIBLIOGRAPHY

1. Ahrens, E. H., Jr., and Craig, L. C., J. Biol. Chem., 196, 299 (1952). 2. Craig, L. C., Hausmann, W., Ahrens, E. H., Jr., and Harfenist, E. J., Anal. Chem.,

23, 1326 (1951). 3. Craig, L. C., Hausmann, W., Ahrens, E. H., Jr., and Harfenist, E. J., Anal. Chem.,

23, 1236 (1951). 4. Craig, L. C., and Post, 0. H., Anal. Chem., 21, 500 (1949). 5. Craig, L. C., Gregory, J. D., and Hausmann, W., Anal. Chem., 22, 1462 (1950). 6. Hughes, H. B., J. BioZ. Chem., 143, 11 (1942). 7. Craig, L. C., Anal. Chem., 22, 1346 (1950). 8. O’Keeffe, A. E., Dolliver, M. A., and Stiller, E. T., J. Am. Chem. Sot., 71, 2452

(1949).

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Edward H. Ahrens, Jr. and Lyman C. CraigOF BILE ACIDS

THE EXTRACTION AND SEPARATION

1952, 195:763-778.J. Biol. Chem. 

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