On the Transformation and Enterohepatic Circulation

of Cholic Acid in the Rat

BILE ACIDS AND STEROIDS 68

ARNE NORMAN AND JAN SJ~VALL

From the Department of Physiological Chemistry, University of Lund, Lund, Sweden

(Received for publication, April 14, 1958)

When labeled cholic acid is given to rats, the radioactivity can be quantitatively recovered in feces mainly as various un- identified transformation products (I, 2). These products are formed through the action of intestinal microorganisms since they are not found in germ-free rats or rats treated with chemo- therapeutics (3,4).

In a series of papers, Schmidt et al. (5-7) studied the metab- olism of cholic acid in the guinea pig. They found that cholic acid administered per OS was quantitatively recovered in feces as keto acids. It has been shown that in the rabbit, and in man, deoxycholic acid is formed from cholic acid during the entero- hepatic circulation, probably through the action of intestinal microorganisms (8-10).

Several papers on the action of different microorganisms on cholic acid have been published. Alcaligenes faecalis and several strains of Escherichia coli can oxidize the hydroxyl groups and give various keto acids (6, 11). The conversion to 3-keto- A4-cholenic acid derivatives in Nocardia and Xtreptomyces cul- tures has been demonstrated (12, 13), and some fungi can oxidize the side chain (14, 15). Reduction of keto groups has also been reported (13, 16).

In bile, cholic acid occurs conjugated with taurine and glycine and these conjugates can be split by different strains of Clostridia and enterococci (17).

From the above it is clear that cholic acid can be transformed in various ways during the passage through the intestine. The aim of the present investigation was to find out where these transformations take place, and to ascertain the nature of the products formed and whether these products are reabsorbed so as to take part in the enterohepatic circulation.

EXPERIMENTAL

Bile Acid Samples-Cholic acid-24-Cl4 and deoxycholic acid-24-Cl4 were prepared according to the method of Bergstrom et al. (18).

7-Ketodeoxycholic acid-24-Cl4 (3a, 12a-dihydroxy-7-ketocho- lanic acid) was prepared by oxidation of 20 mg. of labeled cholic acid with N-bromosuccinimide (19). It was purified by reversed phase chromatography with Phase System C2 (see below).

3cr, 7/l, 12ol-trihydroxycholanic acid was synthesized according to the method of Samuelsson (21) and was kindly supplied by him.

12-Ketolithocholic acid (3cr-hydroxy-12.ketocholanic acid) was synthesized according to Bergstrom and Haslewood (22) at a 160-161” m.p.

7-Ketodeoxycholic acid was prepared as the labeled acid and crystallized as the methyl ester from ethyl acetate-light pe- troleum at a 153-155” m.p.

Administration of Labeled Compounds-White rats of the Institute stock weighing 200 to 300 gm. were used.

The animals were kept in metabolic cages during the experi- ments. Bile duct-cannulated animals were kept in restraining cages as described previously (20). During the experiments the rats were given white bread and water ad libitum.

The labeled bile acids were given as sodium salts dissolved in saline either intraperitoneally or into the cecum. In the latter case the cecal wall was obliquely perforated with a fine needle, and 0.2 ml. of the bile salt solution was injected into the cecal content. By this method no leakage through the injection hole oould be observed.

Extraction of Labeled Material-In the experiments bearing on the distribution of labeled material in the animals, the gastro- intestinal tract was removed and the small intestine was cut at the gastric and celiac junctions. It was then divided into three equal parts. The contents of these parts and of the large intestine were washed out with approximately 50 ml. of saline. These washings were evaporated to dryness and extracted as described below.

The intestinal wall and the other organs studied were homog- enized in 80 per cent ethanol in a Waring Blendor and extracted twice by refluxing for 2 hours with 80 per cent ethanol on a boiling water bath. The intestinal washings and the feces were extracted in the same way, without homogenization. The extracts were evaporated and the radioactivity was determined on a suitable aliquot.

Before being subjected to chromatography the labeled com- pounds were extracted with butanol as described earlier (23).

Conjugated bile acids were hydrolyzed in N KOH for 6 hours at 120” in a sealed tube. After acidification the free bile acids were extracted with ether.

Chromatographic Methods-Reversed phase partition chroma- tography was performed as described previously (24-26).

The following phase systems were used:

Phase system

Moving phase (ml.) Stationary phase (ml.)

Cl Methanol-water 150: 150 Chloroform-isooctanol 15: 15 c2 Methanol-water 144: 156 Chloroform-isooctanol 15: 15 c3 Methanol-water 140: 160 Chloroform-isooctanol 15: 15 Fl Methanol-water 165: 135 Chloroform-heptane 45:5 F2 Methanol-water 180: 120 Chloroform-heptane 40: 10

872

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October 1958 A. Norman and J. &&all 873

System Cl was used for the separation of both free bile acids hydrophobic Super Cel. A column of this size was used when from conjugated ones and of cholic acid from its monoketo the amount of material to be analyzed was less than 50 mg. For derivatives. A better separation of the latter acids could be larger amounts, the columns were correspondingly increased. achieved in systems C2 and C3. Di- and triketo derivatives of For the sake of comparison the effluent volumes of all figures cholic acid, acids less hydroxylated than cholic acid and their have been corrected to correspond to a 4.5 gm. column. keto derivatives, were separated with Fl and F2; F2 is used for Paper chromatography (27) was used for the separation of the more hydrophobic acids. chenodeoxycholic and deoxycholic acids.

4 ml. of the stationary phase was supported on 4.5 gm. of IdentQication-The separated peaks of radioactivity were

TABLE I

Percentage of radioactivity recovered from different organs and feces of rats given cholic acid-24-P intraperitoneally

GRB GRC GRD GRE GRF GRK GRL Rat GRA - . . -

Amount given milligrams. c.p.m. X lo+..

Feeding conditions*. Killed after (no. of

hours).

Liver..................

0.65 0.65 0.65 0.65 45 45 27 27

Fasted Fasted Fasted Fasted

24 24 48 48

4 4 3 3

Small intestine Contents

Upper

Middle. Lower. Total..

Wall.

Large intestine

Contents + wall.. Feces................ Total. .

Total recovery..

--

-

84t 13

101

W 48 33 11 8 10

18 26 __ 101 77 72

GRG GRH

1.98 1.98 1.75 1.70 0.25 0.25 456 456 373 369 515 515

Fed Fed Fasted Fasted Fed Fed

24 24 24 24 70

2 3 2 1

5 6

15 16 10 8 30 30

4 3

7

20 38 65

6

30 39 8 5

38 44

2

3

29 18

50 12

9 2

11

7 3

10

70

1

4

9 20

33 7

10 35 45

2 5

9 16 3

17 44

61

74 80 75 83 86 81

* The animals were either fasted or fed ad libitum during the experimental period. t Including large intestine and feces.

TABLE II

Percentage of distribution of radioactivity remaining in different parts of enterohepatic circulation of rats given cholic acid-24-W

GRE GRF GRK GRL GRM GRN GRH Rat

Amount given milligrams c.p.m. X lo+.. _.

Feeding conditions*.

Killed after (no. of hours).

1.98 1.98 0.25 0.25 456 456 515 515

Fed Fed Fed Fed

24 24 70 70

Liver....................... 2 3

Small intestine

Contents Upper.................. 8 8 Middle 23 22

Lower. 16 11 Wall. 6 4

Total. 53 45

Large intestine Contents + wall.. 45 52

2

7

17 40 14

78

20

1.75 1.70 373 369 Fast. Fast. 24 24

4

0.5 0.5 1000 1000

Fed Fed 70 70

3 3 2 3

7 13

24 7

51 70 73

4 10 40 25 25 47 17 7 86 89

45 27 24 12 8 -

* The animals were either fasted or fed ad Zibitum during the experimental period.

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874 Transformation of Cholic Acid Vol. 233, No. 4

rerun together with unlabeled reference substances so that the RESULTS

rates of elution could be compared. Final identification was done by determining the specific activity after recrystallization A. Distribution of Isotope in Rats at Different Times after

with unlabeled reference compounds. For identification pur- Intraperitoneal Administration of Cholic Add-Z&P

poses sulfuric acid spectra were recorded with a self-recording Table I gives the percentage of isotope in liver, intestinal spectrophotometer (Perkin-Elmer Spectracord 4000) after the tract, and feces at different times after the intraperitoneal purification of the bile acids by means of paper chromatography injection of labeled cholic acid. In some rats, the stomach,

(28). spleen, kidneys, urine, and the expired COz were also analyzed Method of Radioassay-The radioactivity of the bile acids, as for isotope, but the radioactivity, if detected at all, never ac-

well as that of biologic extracts, was determined as described counted for more than 1 per cent of the dose given. The some- earlier (20) by counting of an infinitely thin layer on aluminium what low total recoveries can be accounted for by losses in the planchets under a Tracerlab TGC-2 GM tube or in a Tracerlab collection of feces, in the intestinal washings, and in the extrac- gas flow counter.

c.p.m. a -3

110 r

tion procedures.

Bile

-3 c.am.xlO ,

Ii Small intestine I

Phase system Fl Phase system Cl

ml effluent

FIG. 1. Separation of hydrolyzed labeled material found in bile and small intestinal contents 3 days after the administration of labeled cholic acid. The front bands obtained with Phase System Fl were rerun with Phase System Cl.

:.p.m. x 16’

!5

IO

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October 1958 A. Norman and J. Sjiivall 875

Since the bile acids are fairly rapidly excreted in the feces, better information on the distribution of the isotope in the body would be gained by correcting for the isotope excreted. Table II shows that the distribution of the isotope still remaining in the body is essentially the same 24 and 70 hours after the in- jection of the labeled cholic acid. Only 2 to 4 per cent of the label is present in the liver, practically all of it being confined to the intestinal tract. The small intestinal wall contains ap- proximately twice the amount found in the liver, the figures being fairly variable because of the difficulty in developing a standardized technique for the washing out of the intestinal contents. The large intestinal wall was analyzed in some cases but contained less than 1 per cent of the total radioactivity. Most of the isotope is present in the contents of the lower two-

c.p.m. x lc3

10 0

F

thirds of the small intestine and in the contents of the large intestine, mainly in the cecum.

When the rats were fasting during the experiments the results were essentially the same, as can be seen in Table I and II. However, a somewhat smaller proportion of the isotope was recovered from the large intestine and feces as compared with the normal animals.

B. Separation of Labeled Material in Bile, Intestinal Contents, and Feces 3 Days after Intraperitoneal

Injection of Cholic Acid-RQ-04

It is known that all the bile acids excreted in the bile of bile fistula rats are conjugated, whereas most of the fecal bile acids are unconjugated (1). In order to ascertain where the splitting

Large intestine

ct2.m.x G3,

Feces

Phase system Fl Phase system Cl

C.

-8

-6

-4

-2

5 1500

-I(

i

2

.p.m. x 1ti3

:.p.m.x 1G3

!5

ml effluent

FIG. 2. Separation of unhydrolyzed labeled material from feces and contents of the large intestine 3 days after the administration of labeled cholic acid. Chromatograms of hydrolyzed small intestinal contents from the same rat are shown in Fig. 1. For explanations see Fig. 1.

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876 Transformation of Cholic Acid

3

4

1

hl

‘,o

2 ci

6-

!4-

12-

0 n-

18 r A ’

18-

6-

Vol. 233, No. 4

B

0 40 80 120 60 110 160 60 ii0 160 ml effluent

Phase system F2 Phase system Fl

FIG. 3. Left curve. Fecal bile acids retained in the column after the chromatography shown in the left lower curve of Fig. 2. Deoxy- cholic acid (18 to 25 ml. effluent) and lithocholic acid (60 to 80 ml.) were added as reference substances. Right curves. Rechromatog- raphy of the radioactive bands A and B of left curve together with unlabeled 12-ketolithocholic acid. -, radioactivity; -----, titration.

of the conjugates takes place, the butanol extracts of the ma- terial from different levels of the intestinal tract were chroma- tographed with Phase System Cl. In this way taurine and glycine conjugates were separated from the free bile acids.

It was found that with the possible exception of 1 per cent, all the labeled bile acids present in the small intestine were conju- gated. Similarly all the bile acids were conjugated in the bile of rats, where the labeled cholic acid had circulated for 3 days before a bile fistula was made. In the cecum, however, the peptide bonds had been split and the conjugated bile acids usually comprised less than 20 per cent in the rats examined. In feces, even smaller amounts of conjugated bile acids were present.

Figs. 1 and 2 show the separation of labeled products from hydrolyzed small intestinal contents, unhydrolyzed large in- testinal contents, and feces of a rat in which radioactive cholic

10

20

i d. ri 0

0 10 20 30 10 cm

2 I start

I , , e3.m ezLa -

CD D Solvent direction

FIG. 4. Descending paper chromatography of the fecal di- hydroxycholanic acid band shown in the left lower curve of Fig. 2. The chromatogram with spots of the reference substances, chenodeoxycholic (CD) and deoxycholic (D) acids, is reproduced below the diagram showing the distribution of radioactivity in the paper.

acid had taken part in the enterohepatic circulation for 3 days. Fig. 1 also shows the chromatograms of the hydrolyzed bile of a rat where the cholic acid had circulated under similar conditions. Instead of the rat being killed, however, a bile fistula was made after 3 days. It is seen that some of the cholic acid has been transformed to metabolites which show the same elution rates in the chromatograms of the different materials analyzed. In the bile and the small intestinal contents, the amount of metab- olites is not as large as in the large intestinal contents and feces. The degree of transformation varied greatly in different animals. In addition to the labeled compounds shown in Figs. 1 and 2, small amounts of less polar radioactive substances remained in the stationary phase of Phase System Fl. They were separated with Phase System F2, and Fig. 3 shows the chromatogram of material from the feces.

C. Nature of Different Labeled Compounds Isolated from Bile, Intestinal Contents and Feces 3 Days after

Injection of Chotic Acid-24-C14

Metabolites Isolated with Phase Systems F1 and FZ-In Phase System Fl the peaks of the deoxycholic and chenodeoxycholic acids appear at approximately 50 ml. effluent. Cholic acid and its monoketo derivatives are eluted with the front. Diketo derivatives of cholic acid appear between the front and the deoxycholic acid band. When chromatographies with this system are stopped just after the appearance of deoxycholic acid, the stationary phase retains dehydrocholic acid, keto derivatives of dihydroxycholanic acids and monohydroxycholanic acids. Figs. 1 and 2 show two peaks of radioactivity in Phase System Fl, one appearing with the front and the other at the place of deoxycholic-chenodeoxycholic acids. In order to determine the nature of the latter peak, descending paper chromatographies were run, a phase system being used which was suitable for the separation of deoxycholic and chenodeoxy- cholic acids. The main part of the activity appeared at the

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October 1958 A. Norman and J. Xj6vall 877

same place as deoxycholic acid (Fig. 4). No change in the specific activity was observed after several recrystallizations with unlabeled deoxycholic acid in different solvents (Table III).

When the deoxycholic acid had been eluted in Phase System

TABLE III

Recrystallizations of different radioactive bands chromatographically isolated from intestinal contents and feces of

rats given cholic acid-24-P

Sample

Dihydroxycholanic acid band from fe- ces and large in- testine (Fig. 2)

Cholic acid band from feces (Fig. 2)

Methyl ester of 7- Methyl es- ketodeoxycholic ter of 7- acid band from in- ketode- testinal contents oxycholic and feces (Fig. 5B) acid

12-ketolithocholic 12-keto- acid band from fe- lithocho- ces (Fig. 3A) lit acid

nactive bile a& added,

Deoxycho- lit acid

Cholic acid

Bile Intestinal contents and feces

T-

EtOAc EtOAc HAc-water HAc-water EtOH-water

EtOAc EtOAc HAc-water HAc-water EtOH-water

EtOAc-light petroleum

Acetone-water Acetone-water EtOAc-light

petroleum

EtOAc Acetone-water EtOAc-light

petroleum Acetone-water

‘c

_

Yeighl

5770

5550 5900 5250 5550

106 5390 82 4700 59 $950 48 5150 36 5156 25 5300

75 1000 50 830

42 800 32 870 27 835

75 850 65 830 38 885 19 830

12 760

Fl, the isotope remaining in the stationary phase was rerun with Phase system F2, together with unlabeled deoxycholic and lithocholic acids. Fig. 3 shows a chromatogram of fecal ma- terial. Three radioactive bands appeared between the deoxy- cholic and the lithocholic acids, and two other bands were found after the lithocholic acid. The fractions containing the first two bands (Fig. 3A) and the third band (Fig. 3B) were rerun with Phase System Fl together with unlabeled 12-ketolitho- cholic acid. One of the bands was eluted at the place of 1% ketolithocholic acid, the other two being, respectively, more and less polar than this acid. The fractions containing 12-keto- lithocholic acid were recrystallized with unlabeled 12-keto- lithocholic acid, and the specific activity remained unchanged (Table III). The nature of the other labeled compounds ap- pearing in Phase System F2 is still unknown, but one of them appears at the place of 3,12-diketocholanic acid (65 to 85 ml. effluent). Together with 12-ketolithocholic acid these com- pounds comprised about 10 per cent of the total labeled products excreted in the feces of the rats examined1 Similar chroma- tographies were run with material from the large and small intestine and bile, and labeled bands appeared at approximately the same place as in the chromatogram shown in Fig. 3. The degree of transformation into these metabolites in the large intestine was about the same as in feces, whereas in bile and small intestine these metabolites comprised less than 2 per cent of the total radioactivity.

Metabolites Isolated with Phase Systems C1 and CL%--The front peak in Phase System Fl was rerun with Phase System Cl. The chromatograms are seen in Figs. 1 and 2 which show the presence of two metabolites eluted before the main peak that appears at the place of cholic acid. The front peaks in Fig. 2 consist of the remaining conjugated bile acids and are therefore not present in the hydrolyzed samples of Fig. 1. The cholic acid band (80 to 110 ml. effluent) was collected and recrystallized with unlabeled cholic acid without any change in the specific activity (Table III).

The fractions that contain the double peak appearing before _ cholic acid were combined. These fractions from the chroma

20 80 140 20 80 140 20 SO 140 ml effluent ml effluent

FIG. 5. Rechromatography with Phase System C2 of the radioactive bands appearing between 40 and 70 ml. effluent in the chromato- FIG. 5. Rechromatography with Phase System C2 of the radioactive bands appearing between 40 and 70 ml. effluent in the chromato- grams shown in Figs. 1 and 2 (right curves). grams shown in Figs. 1 and 2 (right curves). For further explanations see the text. For further explanations see the text. Reference substance: 7-ketodeoxycholic acid (80 to Reference substance: 7-ketodeoxycholic acid (80 to 110 110 ml. effluent). --, ml. effluent). --, radioactivity; -----, titration values. radioactivity; -----, titration values.

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878 Transformation of Cholic Acid Vol. 233, hTo. 4

tograms of the intestinal contents and feces (Figs. 1 and 2) were rerun with Phase System C2. In this way a partial separation of the two peaks was obtained. Each peak was then rerun together with unlabeled 7-ketodeoxycholic acid in the same solvent system. These chromatograms are shown in Fig. 5, where the corresponding chromatogram of the double peak in bile is also seen. Both bile and the combined intestinal contents and feces yield a radioactive band which is eluted with the same rate as 7-ketodeoxycholic acid. The band from the intestinal contents and feces (Fig. 5B) was isolated and 7-ketodeoxycholic acid was added. After treatment with diazomethane, the methyl ester was recrystallized several times without change in the specific activity (Table III).

The first part of the double peak was eluted just before 7-ketodeoxycholic acid (Fig. 5A). Samuelsson (21) has shown

that synthetic 3or, 7p, 12cu-trihydroxycholanic acid appears at this place. The titration curve of the chromatogram of bile, as well as that of the chromatogram of the intestinal contenm and feces, revealed the presence of an acidic compound being eluted at exactly the same place as the radioactive band. This band (Fig. 5A) was collected, and it weighed about 2 mg. An aliquot was used for ascending paper chromatography with ethylene chloride-heptane 50:50 as moving phase and 70 per cent acetic acid as stationary phase. After the paper was sprayed with phosphomolybdic acid solution, heating revealed a spot at the place of 3a!, 70,12cu-trihydroxycholanic acid. The radioactivity coincided with this spot. The sulfuric acid spectrum of this metabolite was identical with that of synthetic 301,7@, 12a-trihydroxycholanic acid, as seen in Fig. 6. The 3a, 7@, 12a-trihydroxycholanic acid bands from bile and in-

FIG. 6. Sulphuric acid spectra after ascen.ding paper chromatography of 3a,7& 12a-trihydroxycholanic acid(-----) and Peak A in

Fig. 5. Samples heated simultaneously for 15 minutes at 60” in 65 per cent sulfuric acid.

8

Bile Intestinal contents and feces

ml effluent

FIG. 7. Rechromatography with Phase System C3 of labeled material appearing before 7-ketodeoxycholic acid in the chromatograms

shown in Fig. 5. Reference substance: 3a:,7@,12cu-trihydroxycholanic acid. -- radioactivity; -----, titration values.

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*October 1958 A. Norman and J. Sjiivall 879

testinal contents and feces were rerun together with unlabeled isotope excreted in the bile was determined, as was the radio- ICY, 7@, 12a-trihydroxycholanic acid. As seen in Fig. 7, the activity of the combined extracts of feces and large intestine. elution rate of the radioactive compound was identical with that The results are summarized in Table IV. Cholic, 7-ketodeoxy- of the carrier added. The bands were treated with chromic cholic, and deoxycholic acids were absorbed to approximately acid under conditions known to oxidize cholic acid to dehydro- the same extent. In some animals, the rate of biliary excretion cholic acid (29) and then chromatographed with Phase System of isotope was followed. A representative excretion curve is Cl. The radioactivity non- moved with dehydrocholic acid shown in Fig. 8 together with a curve for the excretion of orally added a,s carrier. administered cholic acid. The absorntion from the cecum is

D. Absorption and Transformation of %$-C14-labeled Cholic Acid much slower and less complete than that from the small intestine.

and Some of Its Principle Metabolites When In order to produce the smallest possible surgical trauma, 4

Injected into Cecum of Bile Fistula Rats control animals were injected with cholic acid 24 hours before

From the previous sections it is evident that the microbial % transformations of cholic acid in the rat mainly take place after

IOOr

the entry of the bile acid into the cecum, where several metab- olites of cholic acid are found. In addition to unchanged cholic acid, deoxycholic, 7-ketodeoxycholic, 12-ketolithocholic and, *o- very probably, 3a:, 7p, 12ol-trihydroxycholanic acids have been identified. In bile and intestinal contents only a small per cent of these metabolites are found. The question arises whether so- an absorption of bile acids from the cecum is possible, and whether the metabolites found are not only bacterial products, but have been formed through the combined action of microbial and liver enzymes during an enterohepatic circulation of the ” bile acids. Therefore cholic, 7-ketodeoxycholic and deoxycholic i acids were injected into the cecum of bile fistula rats. Under ‘0 these conditions, when the enterohepatic circulation is inter- f 20.. rupted, it is possible to study the microbial metabolites formed E nJ in the cecum and their absorption and eventual transformation 2 in the liver before being excreted into the bile. s 0 I I I 1 1

Amount Absorbed from Cecum-Less than 1 mg. of the labeled 0 10 20 30 40 h0”::

bile acid as the sodium salt in 0.2 ml. of saline was injected into the cecal content of bile fistula rats. 48 hours after the injection

FIG. 8. Isotope excreted in the bile following the administra- tion of cholic acid-24-W per OS (A--A) and into the cecum

the rats were killed and the large intestine removed. The (O--O) of bile fistula rats.

TABLE IV

Absorption of di$erent P-labeled bile acids injected into cecum of bile duct-cannulated rats and rats on which bile jistula was performed 2.J hours after injection _

Rat Bile acid administered

GR-I

GR-Cl

,GR-C2

GR-Dl

GR-D2

GR-71

GR-72

GR-V GR-X GR-Y GR-Z

Amount of bile acid given

- I

WT.

Clholic acid

Cholic acid

Cholic acid

Deoxycholic acid

Deoxycholic acid

‘I-ketodeoxycholic

acid

7-ketodeoxycholic

acid Cholic acid Cholic acid Cholic acid

Cholic acid

0.20 46

0.25 500

0.25 500

0.90 450

0.65 330

0.45 120

0.40

0.25 0.25 0.25

0.25

110

120 120 120

120

-

c .p.m. x 10-a

Time for administration

i hours after cannulation

[mmediately after cannula- tion

Immediately after cannula- tion

Immediately after cannula-

tion Immediately after cannula-

tion Immediately after cannula-

tion

Immediately after cannula-

tion 24 hours before cannulation 24 hours before cannulation

24 hours before cannulation 24 hours before cannulation

Recovery, ;b of given amount

- I % of recovered amount

found

Bile (

-

:ecum and feces

80 51 49

84 42 58

86 64 36

74 34 66

96 72 28

70 73 27

81 79 21

84 65 35 86 52 48 85 59 41

80 53 47

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880 Transformation of Cholic Acid Vol. 233, No. 4

8

t

Cholic acid meta$

A

.ites 100 8

C D B

7- ketodeoxycholic acid metabolites

0 60 120 180 0 30 60 60 120 180

1 Y

s

;

20 0 60 120 180 0 30 60 60 120 180

ml effluent Phase system C2 Phase system Fl

Deoxycholic acid metabolites 60- D

2- 6

CA 30- I B

FIG. 9. Chromatographic separations of labeled products in cecum and feces after the injection of labeled cholic, 7-ketodeoxycholic and deoxycholic acids into the cecum of bile fistula rats. Front bands obtained with Phase System Fl were rerun with Phase System C2. Reference substances: ‘I-ketodeoxycholic (A), 12.ketolithocholic (B), cholic (6) and deoxycholic (D) acids. --, radioactivity; -----, titration.

the bile duct cannulation. Since a small amount of the bile acid absorbed before cannulation has probably been lost by fecal excretion, the biliary excretion of isotope represents a minimum value for the cecal absorption in these animals.

Microbiological Transformation of Bile Acids in Large In- testinal Contents and Feces-The combined ethanolic extracts of feces and large intestinal contents of rats, cannulated before the intracecal injection, were evaporated and extracted with ether from an acidified water solution. These extracts were subjected to chromatography with Phase System Fl. The front peaks of these chromatograms were extracted with ether and rerun with Phase System C2. The results obtained after the injection of the different acids into the cecum are shown in Fig. 9.

The major part of the cholic acid-24-Cl4 injected has been transformed into various labeled compounds which are eluted at approximately the same rate as the cholic acid metabolites

isolated from feces of normal rats. In addition to the front band, the chromatogram with Phase System Fl shows two main peaks at the place of deoxycholic (D) and 12-ketolithocholic (B) acids. Minor radioactive peaks are seen after the 12-keto- lithocholic acid band. With the aid of Phase System C2 the front band was shown to consist of several compounds. In addition to unchanged cholic acid (C) some more hydrophilic metabolites were eluted, one of these at the place of 7-keto- deoxycholic acid (A). 7-Ketodeoxycholic, deoxycholic, and 12-ketolithocholic acids were further identified by isotope dilution as described in the foregoing section. When the labeled bands appearing before 7-ketodeoxycholic acid were rechroma- tographed together with unlabeled 3ar, 7p, 12or-trihydroxy- cholanic acid, part of the isotope appeared at the place of this acid. The small amount of radioactivity did not admit of further identification.

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October 1958 A. Norman and J. Sjiivall 881

The experiments with 7-ketodeoxycholic acid showed an acids are completely conjugated in the liver before their excre- almost complete conversion of this acid. The main metabolite tion in the bile. Therefore, the bile samples had to be hy- (D) was identified as deoxycholic acid by isotope dilution. drolyzed before being subjected to chromatography with Phase Minor radioactive bands were found, partly as a double peak at System Fl. After ether extraction the front peak of this the place of 12-ketolithocholic acid, partly before cholic acid. chromatogram was rerun with Phase System C2. Fig. 10 Most of the radioactivity, more hydrophilic than cholic acid, shows the chromatograms of the bile samples from the same appeared at the place of 3cr, 7p, 12Lu-trihydroxycholanic acid. rats as those used in the experiments shown in Fig. 9. Owing to the small amounts of radioactivity in these bands, After injection of labeled cholic acid into the cecum of bile further identifications could not be carried out. No isotope fistula rats a large number of metabolites appear in the bile. was found at the place of cholic acid. Thus, radioactive bands are found at the place of deoxycholic,

In contrast to the extensive transformation of cholic and cholic, i-ketodeoxycholic, and 301,7p, 12cr-trihydroxycholanic 7-ketodeoxycholic acids, the main part of the deoxycholic acid is acids. About 1 per cent of the isotope is found in the form of left unchanged (D). Minor radioactive bands are eluted after compounds more hydrophobic than deoxycholic acid. If this deoxycholic acid, the largest ones in the shape of a double peak picture is compared with the corresponding chromatograms of at and after 12-ketolithocholic acid. Furthermore, there is a the fecal acids of the same rat (Fig. 9), it is seen that the ratio minor transformation into more hydrophilic compounds, which of cholic acid to deoxycholic acid is higher in the bile. This can are eluted before and after cholic acid. Their nature is un- partly be explained by the conversion of deoxycholic acid to known. cholic acid, which is known to take place in the rat liver (30).

Nature of Bile Acids Absorbed and Excreted in Bile-It is well The proportion of compounds which are more hydrophobic than known from a large number of experiments that various bile deoxycholic acid is much lower in bile than in feces.

Cholic acid metabolites.

6r

0 50 100 150 200 0 LO 80 120 160

Deoxycholic acid metabolites.

160 D

30

0 50 100 150 200 0 40 80 120 __. '

Phase system C2 ml effluent

Phase system Fl

FIG. 10. Chromatographic separations of the labeled products in the bile of the same rats as those used for the experiments shownin Fig. 9. For explanations see Fig. 9.

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882 Transformation of Cholic Acid Vol. 233, No. 4

In our experiments, 7-ketodeoxycholic acid is the main labeled product in bile after the injection of this acid into the cecum. It has been identified by means of isotope dilution. The peak appearing before 7-ketodeoxycholic acid in the chroma- togram was rerun with phase system C3 and the labeled com- pound wal.s eluted at the sa.me place R.S 3or,7p, 12cr-trihydroxy- cholanic acid added as carrier. The other metabolites that were found appeared at the places of cholic and deoxycholic acids. Some very small bands were eluted after the latter acid. As pointed out above, no cholic acid was formed in the cecum from 7-ketodeoxycholic acid. The cholic acid band found in the bile is probably cholic acid formed in the liver from the deoxycholic acid absorbed from the cecum. A reduc- tion of 7-ketodeoxycholic acid by liver enzymes, however, cannot be excluded. Further investigations on this problem are de- scribed below, in ‘Section E.”

The chromatographic analyses of the metabolites in bile after the injection of deoxycholic acid into the cecum are seen in the lower curves of Fig. 10. Since most of the deoxycholic acid is left unchanged in the cecum, the labeled products in bile will appear mainly as deoxycholic acid and its liver metabolite, i.e. cholic acid. The small amounts of microbial metabolites are also absorbed and give rise to the bile metabolites separated

from the cholic and deoxycholic acids. The main part of these metabolites is eluted before cholic acid.

E. Metabolism of 7-Ketodeoxycholic Acid

In order to get a better idea of the enterohepatic circulation of the bile acids, it is necessary to know the liver metabolism of the individual microbial products.

Deoxycholic acid is known to be 7a-hydroxylated in rat liver and the cholic acid thus formed is not further transformed (30). It is, however, unknown what becomes of 3a, 7/3,12&rihydroxy- cholanic and 7-ketodeoxycholic acids, and the metabolism of the latter acid has now been investigated.

When labeled 7-ketodeoxycholic acid was given intraper- itoneally to bile fistula rats, only one radioactive band which was eluted before cholic acid could be found in the chroma- tograms of hydrolyzed bile (Fig. 11, left curve). An aliquot of this band was rerun with Phase System C2, and the radio- activity appeared at the place of 7-ketodeoxycholic acid added as carrier (Fig. 11, middle curve). Another aliquot was reduced with sodium borohydride and the radioactivity was now eluted together with the unlabeled cholic acid that had been added (Fig. 11, right curve). The results thus indicate that the 7- ketodeoxycholic acid had been excreted unchanged in the bile.

ml effluent Phase system Cl Phase system C2 Phase system Cl

FIR. 11. Chromatography of hydrolyzed labeled material excreted in the bile of a bile Gslula rat given 7-ketodeoxycholic acid-240 intraperitoneally. Left curve: with unlabeled cholic acid (C). Middle curve: with unlabeled ‘I-ketodeoxycholic acid (A). Right curve: with unlabeled cholic acid (C) after reduction with sodium borohydride. --, radioactivity; -----, titration.

C 8

0 60 120 180 0 35 70 70 110 150 ml effluent

Phase system C 1 Phase system Fl

FIG. 12. Chromatography of fecal acids excreted on the second and third day after administration of 7-ketodeoxycholic acid-24.C14. The front bands obtained with Phase System Fl were rerun with Phase System Cl. Reference substances: cholic (C), deoxycholic (D) and 12.ketolithocholic (B) acids. --, radioactivity; -----, titration.

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October 1958 A. Norman and J. &hall 883

Chromatographic analysis of the labeled products excreted in feces of normal rats after the administration of labeled 7-keto- deoxycholic acid revealed only a small amount of the unchanged acid (Fig. 12). The main metabolites isolated were identified by means of isotope dilution and proved to be deoxycholic and 12-ketolithocholic acids. A small amount of isotope appeared in the cholic acid band and probably represented cholic acid secondarily formed by the liver from the deoxycholic acid. The peaks of radioactivity eluted before the 7-ketodeoxycholic acid have not been identified.

DISCUSSION

Distribution of Bile Acids in Rats-By far the largest amount of the bile acids in the rat (which has no gallbladder) is present in the contents of the small intestine and the cecum. The liver and small intestinal wall contain only about 3 and 9 per cent, respectively. The bile acids are readily absorbed from the small intestine, and they can also be absorbed from the cecum although to a lesser extent and not so rapidly. The bile acids of the rat might, therefore, be regarded as being distributed between at least two pools with different times of enterohepatic circulation. In fasting animals the proportion of bile acids present in the cecum tended to be lower than in animals fed ad l&turn. Different factors, e.g. the diet, may cause a greater loss of bile acids from the small intestine into the cecum, and the incomplete reabsorption here results in an increased fecal excretion of bile acids. Thus, factors influencing the distribu- tion of bile acids in the intestinal tract can affect the half-life of these acids. The important effect of the intestinal flora on the half-life will be discussed later.

Microbiological Transformations of Bile Acids-The extensive transformation of the bile acids before they are excreted in the feces has been demonstrated by several investigators. Analysis of the radioactive bile acids in different parts of the intestinal tract, 3 days after the administration of labeled cholic acid to rats, revealed that the action of microorganisms did not start until the bile acids had entered into the cecum. In the small intestine the bile acids were found completely conjugated mainly with taurine, whereas in the cecum the main part was unconju- gated. The splitting of the peptide bond of conjugated bile acids is due to the action of microorganisms, e.g. different strains of Clostridia and enterococci, whereas the digestive enzymes have no effect on these acids (3, 17).

In addition to labeled free cholic acid, a large number of other radioactive compounds were found in the cecum. Several of these compounds could also be found in bile and small in- testinal cont.ents, where they were present as conjugates. The ratio of metabolites to unchanged cholic acid was about the same in bile and small intestine, increased markedly in the recum, and was still higher in feces. As is shown in Fig. 13, the main part of the metabolites thus formed during the entero- hepatic circulation of cholic acid (Z) was identified as deoxy- cholic acid (IV), 7-ketodeoxycholic acid (ZZ), 30(, 7p, 12&ri- hydroxycholanic acid (ZZZ) and 12-ketolithocholic acid (V). Small amounts of isotope were recovered as four unidentified bands more hydrophobic than deoxycholic acid.

By isolating the transformation products excreted in feces of bile fistula rats, into whose cecum labeled cholic acid had been injected, the liver metabolism of the bile acids could be excluded. Among the purely microbiological metabolites thus formed, SCornpounds ZZ, IV, and 1’ could be separated and identified

and a radioactive band was found at the place of Compound III. By the same technique it was shown that neither II nor IV were end products in the microbial conversion of bile acids. These experiments give an idea of the microbiological trans- formation products that can be formed in the large intestine of the rat. It is reasonable to assume that the formation of these products can occur in various strains of bacteria. The pre- cursor, intermediates, and end products need not be the same in the different strains. It is thus obvious that the compounds isolated cannot be fitted in a general reaction scheme. The main metabolites formed from the acids I, II, and IV are sum- marized in Fig. 13. Dotted arrows indicate the formation of compounds identified by their chromatographic behavior only.

Experiments in vitro have shown that different bacteria can oxidize the hydroxyl groups of cholic acid. Alcaligenes jaecalis dehydrogenates cholic acid in stages at C-7, C-12, and C-3, finally producing a good yield of dehydrocholic acid (6). Various species of Clostridia and Escherichia coli rapidly convert cholic acid into 7-ketodeoxycholic acid; C-3 and C-12 are, however, not oxidized (31). In the present investigation it has been shown that bacteria present in the large intestine of the rat oxidize hydroxyl groups at C-7 and C-12. Thus 7-ketodeoxy- cholic and 12-ketolithocholic acids have been identified as products of cholic acid. The larger proportion of the former product indicates an easier oxidation of C-7 than C-12. No 3oc-hydroxy-7,12-diketocholanic acid (VI) could be found. Its formation and subsequent rapid conversion into other products, e.g. into 12-ketolithocholic acid cannot, of course, be excluded. The oxidation of C-3 does not seem to be an im- portant reaction in the rat. No labeled dehydrocholic acid (VII) could be found and only trace amounts of isotope were found at the place of dehydrodeoxycholic acid (V) in the chroma- tograms of feces. Several investigators have shown an oxidation of cholic acid to 3-keto-A4-cholanic acid derivatives (12, 15), and some of the unidentified labeled products might represent such compounds. The amount of unidentified metabolites is, how- ever, very small as compared with that of the identified ones.

Experiments in vitro have shown that various intestinal bacteria can effect the reduction of keto groups in the cholanic acid nucleus (32). In our experiments on the bacterial trans- formation of 7-ketodeoxycholic acid it was shown that this acid was almost completely transformed into deoxycholic acid. No intermediate formation of cholic acid could be demonstrated. A small amount of isotope was found at the place of 3ar, 7p, 12~ trihydroxycholanic acid, as was also the case in the experiments with cholic acid. It seems probable that the former compound, normally found in the rat, is of bacterial origin. It might possibly be formed by a stereospecific reduction of the 7-keto- deoxycholic acid formed from cholic acid.

The formation of deoxycholic acid mentioned above can take place either from cholic acid or 7-ketodeoxycholic acid. Micro- organisms capable of carrying out these reactions are not known. Common intestinal microorganisms, e.g. E. coli and Clostridia, form 7-ketodeoxycholic acid as an end product from cholic acid. It is, therefore, reasonable to assume that at least some of the deoxycholic acid has been formed indirectly from cholic acid via 7-ketodeoxycholic acid. Whether the latter acid is a neces- sary intermediate in the formation of deoxycholic acid cannot be decided in our experiments. Studies on the reaction mech- anism with the aid of 7&T-cholic acid are being made at this Institute.

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884 Transformation of Cholic Acid Vol. 233, No. 4

O:H UP

R

HO”’ OH

Ill

II aP

R

.,.&R ---&&b” ;gR

P m mI

FIG. 13

No evidence has been obtained for a removal of a hydroxyl or keto group at C-12 analogous to the reaction at C-7.

Enterohepatic Circulation of Cholic Acid-From the foregoing it is evident that the conjugated bile acids are extensively hydrolyzed by the large intestinal microorganisms, which also produce several oxidation and reduction products of cholic acid. The degree of conversion and the relative proportions of the metabolites formed vary in different animals. A slow and incomplete bile acid absorption occurs in the cecum, and the microbial metabolites appear in the bile. Our experiments have shown that deoxycholic and 7-ketodeoxycholic acids formed from cholic acid in the cecum are absorbed and can be found as taurine conjugates in the bile. These results are in accordance with the finding that the deoxycholic acid present in human and rabbit bile is formed through a bacterial action on cholic acid during the enterohepatic circulation (8-10).

The extent of bile acid absorption from the cecum during the normal enterohepatic circulation cannot be assessed in the present investigation. The ratio in the bile of cholic acid to its bacterial metabolites is of no value for this estimation since two of the main metabolites are partly reconverted to cholic acid during the enterohepatic circulation. 7-Ketodeoxycholic acid is not metabolized by the rat liver but can be transformed in the cecum to deoxycholic acid, which by rat liver enzymes is hy- droxylated to cholic acid.

As mentioned before, the intestinal flora has a dominating

influence on the rate of fecal excretion of bile acids. Thus the half-life of cholic acid is about 5 times longer in germ-free than in normal rats (3). It might be possible to explain this finding by assuming the formation of microbial transformation products practically unabsorbable from the cecum. Our experiments, however, do not support the existence of such a mechanism.

SUMMARY

Cholic acid-24-U* was given intraperitoneally to rats and the animals were killed after different periods of time. The distribu- tion of isotope in the body and the nature of the labeled products in bile, intestinal contents, and feces were studied. The follow- ing results were obtained:

1. By far the greatest part of the isotope not excreted was present in the contents of the small and large intestine, mainly in the former. Only about 9 and 3 per cent were found in the small intestinal wall and liver, respectively.

2. In bile and small intestinal contents the bile acids occur in a conjugated form, whereas in the large intestine free bile acids are mainly found.

3. During the enterohepatic circulation the cholic acid is partly transformed. The amount of metabolites thus present is much higher in the large intestine than in the small intestine and bile.

4. The main metabolites were found to be 301,7p, 12cr-tri- hydroxycholanic acid, 3or, 12a-dihydroxy-7-ketocholanic acid,

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October 1958 A. Norman and J. Sjiivall 885

deoxycholic acid and 3cr-hydroxy-12-ketocholanic acid. Small amounts of at least four unidentified labeled compounds, one of them possibly 3,7-diketocholanic acid, were also found.

In order to investigate the formation of cholic acid metab- olites, cholic acid-24-C14, 3a, 12a-dihydroxy-7-ketocholanic acid- 24-Cl4 and deoxycholic acid-24-Cl4 were injected into the cecum of bile fistula rats. The nature of the labeled products in bile and feces was studied and the following results were obtained:

1. Among the purely microbial metabolites of cholic acid, SLY, 12a-dihydroxy-7-ketocholanic, deoxycholic and Ba-hydroxy- 12-ketocholanic acids were identified. Several unidentified products were found, one of them was probably 3c~,7p, 12a- trihydroxycholanic acid. 3a, 12a-dihydroxy-7-ketocholanic acid was mainly transformed into deoxycholic acid. Deoxycholic acid was only slowly metabolized by the microorganisms to unidentified compounds.

2. More than 50 per cent of the isotope injected into the

cecum was recovered in the bile. Chromatographic separation of the labeled products after hydrolysis showed the presence of radioactive bands at the point corresponding to the unchanged acid injected, as well as at the points corresponding to its prin- cipal microbial metabolites. The main part of the deoxycholic acid formed was converted to cholic acid during the liver pas- sage. No transformation of 3cu, 12c+dihydroxy-7-ketocholanic acid in the liver was observed.

The implications of these findings have been discussed.

Acknowledgments-The technical assistance of Mrs. Ann-Mari Andersson, Miss Margret M%nsson and Miss Margaretha Persson is gratefully acknowledged.

This work is part of investigations supported by Statens Medicinska Forskningsr%d and Knut och Alice Wallenbergs Stiftelse and the National Institutes of Health (H 2842), United States Public Health Service, Bethesda, Maryland.

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Arne Norman and Jan SjövallRat: BILE ACIDS AND STEROIDS 68

On the Transformation and Enterohepatic Circulation of Cholic Acid in the

1958, 233:872-885.J. Biol. Chem. 

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