BILE ACIDS

II. METABOLISM OF DEOXYCHOLIC ACID-24-Cl4 AND CHENODEOXYCHOLIC ACID-24-V IN THE RAT*

BY THEODORE A. MAHOWALD,t JOHN T. MATSCHINER,? S. L. HSIA, ROBERT RICHTER, E. A. DOISY, JR., WILLIAM H. ELLIOTT,

AND EDWARD A. DOISY

(From the Departments of Biochemistry and Internal Medicine, St. Louis University School of Medicine, St. Louis, Missouri)

(Received for publication, October 2, 1956)

The development of excellent new chromatographic procedures for the separation of conjugated (1) and free bile acids (2) has facilitated study of the metabolism of U4-labeled deoxycholic acid and chenodeoxycholic acid.l Some of our data are confirmatory of the work conducted in Berg- strom’s laboratory, but additional interesting observations have been made. After intraperitoneal administration of deoxycholic acid-24-Cl4 to rats with bile fistulas, Bergstrom et al. (3) recovered more than half of the radio- activity as labeled cholic acid. In the same type of experiment, cheno- deoxycholic acid-24-Cl4 gave two metabolites which chromatographed similarly to cholic acid, but neither was identical with cholic acid (4). Mahowald et al. (5) and Matschiner et al. (6) have reported the isolation of these metabolites of chenodeoxycholic acid from rat bile.

This report is concerned with a study of the metabolism of deoxycholic acid-24-Cl4 and chenodeoxycholic acid-24-Cl4 in normal rats and in rats with ligated or cannulated bile ducts. The amounts of radioactivity in the excretory products were determined in these animals after intragastric administration, and the bile and urine were fractionated to ascertain the nature of the radioactive metabolites. Absorption studies with the labeled acids were also performed on rats with both cannulated intestinal lym- phatic and bile ducts.

* Preliminary reports of the studies contained in this paper were presented at meetings of the Federation of American Societies for Experimental Biology at Atlan- tic City, April, 1954, and San Francisco, April, 1955.

t The material presented herein is taken, in part, from theses submitted to the Graduate School of St. Louis University by John T. Matschiner and Theodore A. Mahowald in partial fulfilment of the requirements for the degree of Doctor of Phi- losophy in Biochemistry.

1 We are indebted to Dr. Claire E. Graham of The Wilson Laboratories, Chicago, Illinois, for a generous supply of cholic and deoxycholic acids.

781

by guest on June 5, 2018http://w

ww

.jbc.org/D

ownloaded from

782 BILE ACIDS. II

EXPERIMENTAL

Synthesis of Deoxycholic Acid-2.J-C’4 and Chenodeoxycholic Acid-d&C’- The carboxyl-labeled acids used in these studies were prepared from the norbromide through a nitrile synthesis by the method of Bergstrom et al. (7), except that a larger excess of the norbromide was used than was re- ported by these workers. The norbromides were prepared by bromine degradation of the silver salt of the corresponding acid diacetates as re- ported by Brink et al. (8).

A 3-fold molar excess of 23-bromo-3cu, 12a-diacetoxynorcholane (1.6 gm., m.p. 127-129”) was allowed to react with 0.92 mmole of potassium cyanide2 containing 2.0 mc. of CY4. After hydrolysis, 263 mg, of an acidic fraction were obtained (72 per cent). The deoxycholic acid-24-Cl4 was purified by crystallization from acetone and dried in vacua at 147” for 1.5 hours. The labeled acid was characterized by its melting point (176-177.5”), which showed no depression on admixture with authentic deoxycholic acid, and by the preparation of the methyl ester of the diacetate of this acid, m.p. 117.5-118.5” (9). Radioassay of the deoxycholic acid-24-Cl4 was carried out by oxidation of the material to carbon dioxide which was precipitated as BaC03 and counted with a thin window Geiger tube. By comparison with a standard BaC1403 sample, the labeled acid was found to have a specific activity of 3.7 pc. per mg.

Chenodeoxycholic acid-24-Cl4 was prepared by a similar procedure. The reaction mixture contained 770 mg. of 23-bromo-3a, 7or-diacetoxynorcho- lane (m.p. 185-188’), 20.2 mg. of potassium hydroxide, and 32.9 mg. of potassium cyanide2 containing 1 mc. of CY4. After hydrolysis, 156.5 mg. (79.5 per cent) of crude chenodeoxycholic acid-24-Cl4 were obtained. The product was repeatedly crystallized from a mixture of acetone, benzene, and petroleum ether. The purified product, weighing 46 mg. and melting at 144-146” with no depression on admixture with authentic chenodeoxy- cholic acid, was used in the following experiments. Radioassay of the labeled acid by the procedure described for deoxycholic acid-24-Cl4 gave a specific activity of 4.4 fit. per mg.

Distribution Studies-Distribution studies were carried out for each acid on three groups of adult male rats of the St. Louis University colony. One group was prepared for collection of bile by cannulation of the bile duct. A second group was surgically jaundiced by doubly ligating and severing the bile duct. Three normal animals were kept in cages suitable for the collection of the expired air. Under light ether anesthesia, each animal received, by stomach tube, approximately 1 mg. of the labeled acid as the

2 The radioactive potassium cyanide was purchased from the Nuclear Instrument and Chemical Corporation, Chicago, Illinois, on allocation from the Atomic Energy Commission.

by guest on June 5, 2018http://w

ww

.jbc.org/D

ownloaded from

MAHOWALD, MATSCHINER, HSIA, RICHTER, DOISY, 783 ELLIOTT, AND DOISY

sodium salt in 1 ml. of water. The urine and feces and, in appropriate animals, bile or expired air were collected at regular intervals, and aliquots were taken for radioassay. Operative techniques and methods of collec- tion of specimens and Cl4 assay were the same as those previously de- scribed (lo), except that polythene tubing with a beveled tip was used for all cannulations.

After administration of either labeled chenodeoxycholic or deoxycholic acid, the excretion patterns were similar (Fig. 1). The route of excretion was affected by the type of experimental animal. In normal animals,

NC- FECES D (31 CD(J) 3) CD(3

DL- URINE CD(31 D(4)

LL I 4 7

IL- I47

DAYS FIG. 1. Average daily excretion of Cl4 by the principal excretory route. All ani-

mals received approximately 1 mg. of deoxycholic acid-24-Cl4 (D) or chenodeoxy- cholic acid-24-P (CD) intragastrically. The heights of the bars indicate the per- centages of the administered dose recovered. NC represents normal animals, BF, animals with bile fistulas, and DL, animals with ligated bile ducts. The number of animals used is given in parentheses.

essentially all of the radioactivity was excreted in the feces; only 0.2 and 0.5 per cent were found in the urine with deoxycholic acid and chenode- oxycholic acid, respectively, over the 10 day period, and none was found in the expired air. In the animals with bile fistulas, the Cl4 was rapidly excreted in the bile; an average of 1.2 and 1.3 per cent was found in the feces and of only 0.1 and 0.4 per cent in the urine with deoxycholic acid and chenodeoxycholic acid, respectively, over a 5 day period. In animals with ligated bile ducts the urine became the principal excretory route, with an average of 3.7 and 2.9 per cent appearing in the feces over a 9 day period after the administration of labeled deoxycholic acid and chenodeoxycholic acid, respectively.

Fractionation of Biliary Radioactivity-Samples of pooled bile from each animal were deproteinized by the addition of 10 volumes of 95 per cent

by guest on June 5, 2018http://w

ww

.jbc.org/D

ownloaded from

784 BILE ACIDS. II

ethyl alcohol, and the alcoholic filtrates were evaporated to dryness in vucuo. The residues were dissolved in a small volume of water, acidified to pH 1, and diluted with 6 volumes of butyl alcohol. These solutions were washed thoroughly with water, and the washings were reextracted with a small amount of butyl alcohol. The aqueous phases retained from 0 to 0.35 per cent of the biliary radioact.ivity. After evaporation of the butanol extracts to dryness in vacua, the residues were subjected to reverse phase chromatography as described by Bergstriim and Norman (1). In agreement with their results, taurocholic acid, glycocholic acid, and free cholic acid were eluted as described in their report.3 After administration of either labeled deoxycholic or chenodeoxycholic acid, nearly all of the Cl4 in the extracts appeared in the taurine-conjugated fractions. How- ever, 2.2 per cent of the Cl4 in experiments with labeled deoxycholic acid and 1.3 per cent in those with labeled chenodeoxycholic acid appeared to be glycine conjugates. Only 0.4 per cent of the deoxycholic acid radio- activity, but none of the Cl4 of chenodeoxycholic acid, was eluted in free bile acid fractions. Subsequent washing of the column with chloroform removed 3.8 per cent of the chromatographed radioactivity of deoxycholic acid, but none of the Cl4 of chenodeoxycholic acid. The total recovery averaged 97 and 96 per cent, respectively, for these acids.

To examine further the extent of metabolism of the administered acids, the taurine-conjugated fractions from individual columns were combined, the organic solvents were evaporated in vacua, and the residues were dis- solved in approximately 15 ml. of 5 per cent sodium hydroxide and auto- claved at 120” for 3.5 hours. Ether extracts of the basic hydrolysates contained no radioactivity. After acidification and extraction with ether, the aqueous phases retained from 0.2 to 3.1 per cent of the radioactivity.

The ether extracts containing the acids were evaporated, and the residues were chromatographed on partition columns (Celite), with 70 per cent aqueous acetic acid being used as the stationary phase and mixtures of Skellysolve B and benzene as the movable phases (6). The elution pat- tern of the ether-soluble radioactivity from one animal which had been given labeled deoxycholic acid (Rat DBFS) is shown in Fig. 2. The Cl4 in the eluates from Fractions 20-l to 40-4, representing 47 per cent of the biliary radioactivity, was chromatographically identical with deoxycholic acid. The irregular early elution of trace amounts of deoxycholic acid in Fractions 20-l t)o 20-4 has been found to be characteristic of this compound

3 Chromatography of beef bile plus added unconjugated cholic acid in the aqueous methanol and octanol-chloroform system of Bergstrijm and Norman (1) gave the con- firmatory results described in the text. Chromatography of normal rat bile revealed small amounts of glycocholic acid and only traces, if any, of free cholic acid; how- ever, in nutritional experiments, we have observed increased amounts of both glyco- cholic and free cholic acids in rat bile (unpublished experiments).

by guest on June 5, 2018http://w

ww

.jbc.org/D

ownloaded from

MAHOWALD, MATSCHINER, HSIA, RICHTER, DOISY, 785 ELLIOTT, AND DOISY

in the solvent system used. These fractions were mixed with approxi- mately 25 mg. of authentic deoxycholic acid and chromatographed. The radioactivity was cluted coincident with the deoxycholic acid, and three recrystallizations of the chromatographed material, once from a mixture of ethyl acetate and petroleum ether and twice from a mixture of acetone and petroleum ether, did not change the specific activity of the crystals.

The second zone4 of elution of CL4, from Fractions 80-l to 80-4 (Fig. 2), contained 43 per cent of the biliary radioactivity; it included the elution

J II ----T 500 ML. 0 too 200 300 400 10

%C6H6 s 0 1 20 40 * 60 1 80 100 ’ ELUATE

FIG. 2. Chromatographic analysis of the free bile acids obtained from the bile of an animal given deoxycholic acid-24-P. The heights of the bars indicate the per- centages of the chromatographed 0”. The volume of the eluate is given as ml. (Ml.) or liters (L.) and the composition of the eluate (‘$@%,HG) is given as the per cent of benzene in Skellysolve B.

of 50.8 mg. of cholic acid as determined by the furfural-sulfuric acid col- orimetric assay (11). After chromatography of these fractions, the cholic acid was crystallized several times from a mixture of acetone and benzene, and the crystals were dried in vacua at 100” for 10 hours. The cholic acid was identified by its melting point (199-200”), which showed no depres- sion on admixture with authentic cholic acid, and by its specific rotation ([o(]i3 $40’ in met,hanol). Methyl cholate was prepared as a deriva- tive, m.p. 156-157” (12). The specific activity of the cholic acid remained constant during several crystallizations and during the preparation of the derivative, indicating that the radioactivity eluted in the fractions was

* In connection with the discussion of the graphical presentation of data in this and subsequent papers, the term zone is used to designate a peak of radioactivity in a fraction; it may include the eluates preceding and following the peak.

by guest on June 5, 2018http://w

ww

.jbc.org/D

ownloaded from

786 BILE ACIDS. II

present as labeled cholic acid. Upon chromatography of the free bile acids obtained by hydrolysis from each animal, an average of 38.6 per cent (range from 29.3 to 43.5 per cent) of the radioactivity was eluted in the cholic acid fractions and 54 per cent in the deoxycholic acid fractions, with an average over-all recovery of 97 per cent.

The conjugated bile acids obtained from the bile of animals given cheno- deoxycholic acid-24-Cl4 were hydrolyzed, and the free bile acids were ex- tracted by the process used in the experiments with labeled deoxycholic acid. After extraction with ether, the aqueous phases retained from 0.7 to

r

ML. 0 100 200 300 400 500 61

%C6H6 1 0 I 20 I 40 I 60 1 80 I 100 1

ELUATE

FIG 3. Chromatographic analysis of the free bile acids obtained from the bile of an animal given chenodeoxycholic acid-24-W. The heights of the open bars indi- cate the percentages of the chromatographed C14. The heights of the diagonally lined bars from the base line to the top indicate the mg. of cholic acid as determined by calorimetric assay (11). See the legend to Fig. 1 for an explanation of the sym- bols.

3.8 per cent of the radioactivity. The ether extracts were evaporated, and the residues were chromatographed on t,he 70 per cent aqueous acetic acid partition column as described by Matschiner et al. (6). The pattern of elution of cholic acid and the ether-soluble radioactivity from one animal (Rat CDBF1) is shown in Fig. 3. The Cl4 in Fractions 40-l and 40-2 rep- resents 49 per cent of the biliary radioactivity; it was identified as cheno- deoxycholic acid by dilution with authentic chenodeoxycholic acid and crystallization to constant specific activity. The other two zones of Cl4 elution contained 18 and 22 per cent of the chromatographed C14, respec- tively.

Upon chromatography of t,he free acids from each of the other animals, an average of 55 per cent (range from 47 to 64 per cent) of the radioactiv-

by guest on June 5, 2018http://w

ww

.jbc.org/D

ownloaded from

MAHOWALD, MATSCHINER, HSIA, RICHTER, DOISY, 787 ELLIOTT, AND DOISY

ity was eluted in the chenodeoxycholic acid fractions. 15 per cent (range from 12 to 18 per cent) of the chromatographed Cl* was found in the frac- tions immediately preceding cholic acid and 20 per cent (range from 18 to 22 per cent) was found in the fractions following cholic acid, with a total recovery of 97 per cent. The isolation of two new trihydroxycholanic acids from rat bile identical with these labeled metabolites of chenodeoxycholic acid-24-C* is reported in Paper I of this series (6), Acid I being the metab- olite of chenodeoxycholic acid less polar than cholic acid and Acid II the metabolite more polar than cholic acid.

The procedure of protein precipitation, butanol extraction, chromatog- raphy of the butanol extract, and hydrolysis of the taurine-conjugated acids was usually followed. However, in several instances with animals which had been given either labeled deoxycholic acid or chenodeoxycholic acid, the free bile acids were obtained directly by adding sodium hydroxide to the bile to give a concentration of 5 per cent and autoclaving the mix- ture at 120” for 3.5 hours. Ether extracts of the basic hydrolysates con- tained no radioactivity. After acidifying the hydrolysates and extracting with ether, an acid fraction was obtained which contained nearly all of the Cl* present in the original bile. The aqueous phases retained from 0.7 to 1.0 per cent of the radioactivity. Chromatography of the acid fractions gave essentially the same elution pattern of radioactivity as that observed with the free bile acids obtained by the longer procedure described above.

Fractionation of Urinary Radioactivity-The urines from the individual animals with ligated bile ducts which had been given labeled deoxycholic acid or chenodeoxycholic acid were appropriately combined, acidified to pH 1, and extracted with butyl alcohol. The butanol phases were thor- oughly washed with water, and the water was reextracted with a small amount of butyl alcohol. The aqueous phase retained from 0.8 to 7.0 per cent of the urinary radioactivity. Chromatography of the butanol resi- dues on the reverse phase partition column of Bergstrom and Norman (1) gave essentially the same results with each acid. Nearly all the radioac- tivity was eluted in the taurine-conjugated fractions, with an average of 5.6 per cent appearing in later fractions. Washing of the columns with chloroform extracted an average of 2.6 per cent of the chromatographed radioactivity in the experiments with labeled deoxycholic acid and less than 0.5 per cent in those with labeled chenodeoxycholic acid.

After hydrolysis of the conjugated bile acids and extraction of the hydrol- ysate as described for the experiments on bile, the aqueous phases retained from 8.4 to 15.8 per cent and from 5.3 to 21.7 per cent of the radioactivity in the experiments with labeled deoxycholic acid and chenodeoxycholic acid, respectively. These values are significantly higher than those ob-

by guest on June 5, 2018http://w

ww

.jbc.org/D

ownloaded from

788 RILE ACIDS. II

served in the case of the biliary radioactivity (see above). The free urinary bile acids were separated on the acetic acid partition column. Fig. 4 shows the elution pattern of one-tenth of the ether-soluble radioactivity from one animal (Rat DDL,) given labeled deoxycholic acid. Fractions 80-l to SO-3 contained 84.5 per cent of the chromatographed Cl4 and also 1.2 mg. of cholic acid as determined by the furfural-sulfuric acid calorimetric assay (11). Upon chromatography of the free urinary bile acids from each an- imal, an average of 80 per cent of the radioactivity was eluted in the cholic acid fractions. An average of 5.6 per cent was eluted later than cholic acid (more than 500 ml. of eluting solvent), and 6.7 per cent was detected

500 600

%cfjHgI 0 I 20 I 40 I 60 I 80 I 100 1

ELUATE

FIG. 4. Chromatographic analysis of the free bile acids obtained from the urine of a surgically jaundiced animal given deoxycholic acid-24-P. The heights of the bars indicate the percentages of the chromatographed Cl*. See the legend to Fig. 1 for an explanation of the symbols.

in a final washing of the column with acetic acid. Only 2.7 per cent of the chromatographed Cl4 could be detected in the deoxycholic acid fractions. The cholic acid obtained from the total urine of one animal was chromat- ographed and crystallized, and the specific activity was determined. This specific activity remained constant during three crystallizations, once from a mixture of acetone and petroleum ether, once from a mixture of acetone and benzene, and once from a mixture of methanol and water, indicating that the radioactivity in the zone was present as labeled cholic acid.

The free bile acids obtained from the urine of jaundiced animals given labeled chenodeoxycholic acid were chromatographed on acetic acid par- tition columns 5 times as large as the basic size previously described (6). Fig. 5 shows the elution pattern of the ether-soluble radioactivity from one animal (Rat CDDLb). The main zone of elution of Cl4 contains 67 per

by guest on June 5, 2018http://w

ww

.jbc.org/D

ownloaded from

MAHOWALD, MATSCHINER, HSIA, RICHTER, DOISY, 789 ELLIOTT, AND DOISY

cent of the chromatographed C4. Experiments on two additional rats gave confirmatory data, the average of the chromatographed Cl4 in these fractions being 71 per cent. The identification of this metabolite as Acid I and a quantitative study of the amounts of Acid I and cholic acid present in the urine of rats under conditions of surgical jaundice are reported in Paper III of this series (13).

Absorption of Intragastrically and Intraduodenally Administered Deoxy- cholic Acid-2.&C14 and Chenodeoxycholic Acid-b4-@-Absorption studies

! L. 6 0.5 1.0 1.5 2.0 2.5 I .(

%C,H,l 0 I 20 I 40 I 60 I 80 1 100 1

ELUATE

FIG. 5. Chromatographic analysis of free bile acids obtained from the urine of a surgically jaundiced animal given chenodeoxycholic acid-24-W. The heights of the open bars indicate the percentages of the chromatographed CP. The heights of the diagonally lined bars from the base line to the top indicate the mg. of cholic acid in the eluate as determined by calorimetric assay (11). See the legend to Fig. 1 for an explanation of the symbols.

were carried out with each labeled acid on two adult male rats prepared with bile duct and intestinal lymphatic fistulas. Surgical procedures and postoperative treatment were the same as those which have been described previously (14, 15). Deoxycholic acid-24-Cl4 was administered to two an- imals 42 hours after surgery. Under light ether anesthesia, one animal (Rat DLF1) received, by stomach tube, approximately 1 mg. (3.7 PC.) of the labeled acid as the sodium salt in 1 ml. of water. Another animal (Rat DLF$ received the labeled acid intraduodenally, without anesthesia, through an inlying duodenostomy tube inserted through the distal portion of the bile duct. Two similar animals were given approximately 1 mg. of chenodeoxycholic acid-24-Cl4 (4.4 PC.) 48 hours after surgery by stomach tube (Rat CDLFr) and intraduodenally (Rat CDLFZ). The bile, urine,

by guest on June 5, 2018http://w

ww

.jbc.org/D

ownloaded from

790 BILE ACIDS. II

feces, and lymph were collected at definite time intervals, and an aliquot was taken for radioassay as described previously (10).

Less than 0.1 per cent of the radioactivity was recovered in the lymph of these animals in the first 12 hour period, whereas nearly all of the Cl4 was recovered in the bile during the same period, indicating nearly com- plete absorption of the administered bile acids via the portal system. Less than 0.1 per cent of the radioactivity was recovered in the urine and 1.5 per cent in the feces over a 36 hour period.

.5 4 8 12

HOURS

FIG. 6. Cumulative recovery of administered 04 in the bile. The quantities of 04 are expressed as percentages of the Cl4 administered. D, broken line, or CD, solid line, indicates the labeled acid administered, deoxycholic acid-24-Cl4 or cheno- deoxycholic acid-24-04, respectively. LFI, animals which received the bile acid intragastrically under light ether anesthesia 42 or 48 hours (see the text) postoper- atively; LFP, animals which received the acid intraduodenally without anesthesia 42 or 48 hours postoperatively; BF, animals described in Fig. 1, which received the bile acid while under ether anesthesia immediately after surgery.

The rate of recovery of the administered radioactivity in the bile can be seen in Fig. 6. After intraduodenal administration (Rats DLFz and CDLF2), 76 and 91 per cent of the Cl4 were recovered within 1 hour. The slower, irregular biliary recovery after intragastric administration under light ether anesthesia (Rats DLFl and CDLF1) was more similar to the recovery rate observed in the distribution studies, in which the labeled acids were administered intragastrically to rats with cannulated bile ducts immediately after surgery. The average data obtained from these animals (Rats DBF and CDBF) over the first 12 hours are included in Fig. 6 for comparison.

On fractionation of the biliary radioactivity obtained after intraduodenal administration of deoxycholic acid-24-Cl4 (Rat DLF*), 30.1 per cent of the

by guest on June 5, 2018http://w

ww

.jbc.org/D

ownloaded from

MAHOWALD, MATSCHINER, HSIA, RICHTER, DOISY, 791 ELLIOTT, AND DOISY

Cl4 was found in the cholic acid fractions and 65.5 per cent in the deoxy- cholic acid fractions. Fractionation of the biliary radioactivity obtained after intraduodenal administration of chenodeoxycholic acid-24-Cl4 gave 7.0 and 11.6 per cent of the Cl4 in the Acid I and the Acid II fractions, re- spectively, and 71 per cent of the radioactivity in the chenodeoxycholic acid fractions.

DISCUSSION

The excretory pathways of the radioactivity after intragastric adminis- tration of deoxycholic acid-24-Cl4 and chenodeoxycholic acid-24-Cl4 to nor- mal and bile fistula rats shown in Fig. 1 are comparable with the results obtained in Bergstrom’s laboratory with several labeled bile acids admin- istered intraperitoneally (3, 16, 17). The small amounts of Cl4 detected in the feces of the animal which had undergone operation emphasize the efficiency with which the bile acids are absorbed from the gut. The marked difference between the excretory rate in the bile and in the feces is com- patible with the now accepted enterohepatic circulation described for the bile acids (18-20).

The chromatographic behavior of the biliary Cl4 prior to hydrolysis is in accord with the observations of Norman (21) and others that the rat excretes bile acids in the bile mainly as taurine conjugates. The chromat- ographic behavior of the urinary Cl4 from animals with ligated bile ducts suggests that the same conjugation pattern occurs in the urine under these conditions. However, the increased resistance of this material to alkaline hydrolysis as noted in the text may be due to some difference in the chem- ical nature of part of the urinary conjugated radioactivity.

The data shown in Fig. 2 confirm the observations of Bergstrom et al. (3) on the conversion of deoxycholic acid to cholic acid in the rat. The data shown in Fig. 3 indicate that chenodeoxycholic acid is metabolized by the rat to two other bile acids, as was observed earlier by Bergstrom and Sjovall (4). The isolation of these metabolites and their subsequent chemical characterization have shown them to be two new trihydroxycho- lanic acids (5, 6, 22). The preparation of one of these metabolites by par- tial synthesis has been carried out and appears in Paper V (22).

As shown in Fig. 4 and in the text, under conditions of bile duct ligation an average of 80 per cent of the administered deoxycholic acid was con- verted to cholic acid. Less than 3 per cent of the chromatographed Cl4 could be detected in the deoxycholic acid fractions. In similar experi- ments with chenodeoxycholic acid-24-C14, 71 per cent of the chromato- graphed Cl4 appeared in the Acid I fractions and less than 2 per cent could be detected in the chenodeoxycholic acid fractions. A detailed study of

by guest on June 5, 2018http://w

ww

.jbc.org/D

ownloaded from

792 BILE ACIDS. II

the concentrations of Acid I and cholic acid in the urine of animals with ligated bile ducts is reported in Paper III of this series (13).

The small amount of Cl4 observed in the lymph after intragastric or in- traduodenal administration of these acids is in agreement with observa- lions of others on the absorption of C14-labeled cholanic acid (23) and tau- rocholic acid (19) and of taurocholate-S35 (20) in the rat.

SUMMARY

Deoxycholic acid-24-Cl4 and chenodeoxycholic acid-24-Cl4 were prepared by the methods of Bergstrom et al. After intragastric administration to normal animals, the Cl4 was excreted in the feces over a 10 day period. No radioactivity was found in the expired air. In animals with cannulated bile ducts, the radioactivity was rapidly recovered in the bile, while, in an- imals with ligated bile ducts, the urine became the principal excretory route. After intragastric or intraduodenal administration, less than 0.1 per cent of the Cl4 was recovered in the lymph. Animals with cannulated bile ducts converted from 29.3 to 43.5 per cent of the administered deoxycholic acid to cholic acid. The conversion to cholic acid in animals with ligated bile ducts was from 65 to 91 per cent. Chenodeoxycholic acid was metab- olized to the two new bile acids, Acid I and Acid II, in animals with cannu- lated bile ducts. Acid I was formed to the extent of 15 per cent and Acid II to the extent of 20 per cent. In the urine of rats with ligated bile ducts, Acid I represented the main metabolite of chenodeoxycholic acid, with 71 per cent of the chromatographed Cl4 appearing in these fractions.

BIBLIOGRAPHY

1. Berg&cm, S., and Norman, A., Proc. Sot. Exp. Biol. and Med., 83,71 (1953). 2. Mosbach, E. H., Zomzely, C., and Kendall, F. E., Arch. Biochem. and Biophys.,

48, 95 (1954). 3. Bergstriim, S., Rottenberg, M., and Sjijvall, J., 2. physiol. Chem., 295,278 (1953). 4. Bergstrijm, S., and Sjijvall, J., Acta them. &and., 8, 611 (1954). 5. Mahowald, T. A., Elliott, W. H., and Thayer, S. A., Federation Proc., 14, 250

(1955). 6. Matschiner, J. T., Mahowald, T. A., Elliott, W. H., Doisy, E. A., Jr., Hsia, S. L.,

and Doisy, E. A., J. Biol. Chem., 226, 771 (1957). 7. BergstrBm, S., Rottenberg, M., and Voltz, J., Acta them. &and., 7, 481 (1953). 8. Brink, N. G., Clark, D. M., and Wallis, E. S., J. Biol. Chem., 162, 695 (1946). 9. Reichstein, T., and Sorkin, M., Helv. chim. acta, 26, 797 (1942).

10. Bocklage, B. C., Nicholas, H. J., Doisy, E. A., Jr., Elliott, W. H., Thayer, S. A., and Doisy, E. A., J. Biol. Chem., 202,27 (1953).

11. Reinhold, J. G., and Wilson, D. W., J. Biol. Chem., 96, 637 (1932). 12. Fieser, L. F., and Rajagopalan, S., J. Am. Chem. Sot., 72,553O (1950). 13. Mahowald, T. A., Matschiner, J. T., Hsia, S. L., Doisy, E. A., Jr., Elliott, W. H.,

and Doisy, E. A., J. Biol. Chem., 226,795 (1957).

by guest on June 5, 2018http://w

ww

.jbc.org/D

ownloaded from

MAHOWALD, MATSCHINER, HSIA, RICHTER, DOISY, 793 ELLIOTT, AND DOISY

14. Bollman, J. L., Cain, J. C., and Grindlay, J. H., .I. Lab. and Clin. Med., 33,1349 (1948).

15. Hyde, P. M., Doisy, E. A., Jr., Elliott, W. H., and Doisy, E. A., J. Biol. Chem., 209, 257 (1954).

16. BergstrGm, S., Sjavall, J., and Voltz, J., ncta physiol. &and., 30, 22 (1953). 17. Lindstedt, S., and Norman, A., Acta physiol. &and., 34, 1 (1955). 18. Josephson, B., <hysioZ. Rev., 21, 463 (1941). 19. Sjbvall, J., and Akesson, I., Acta physiol. &and., 34, 273 (1955). 20. Portman, 0. W., and Mann, G. V., J. BioZ. Chem., 213,733 (1955). 21. Norman, A., Acta physiol. &and., 32, 1 (1954). 22. Hsia, S. L., Matschiner, J. T., Mahowald, T. A., Elliott, W. H., Doisy, E. A., Jr.,

Thayer, S. A.,Oand Doisy, E. A., J. Biol. Chem., 226, 811 (1957). 23. SjBvall, J., and Akesson, I., Acta physiol. &and., 34, 279 (1955).

by guest on June 5, 2018http://w

ww

.jbc.org/D

ownloaded from

William H. Elliott and Edward A. DoisyS. L. Hsia, Robert Richter, E. A. Doisy, Jr.,

Theodore A. Mahowald, John T. Matschiner,THE RAT

CHENODEOXYCHOLIC ACID-24-C14 INDEOXYCHOLIC ACID-24-C14 AND BILE ACIDS: II. METABOLISM OF

1957, 225:781-794.J. Biol. Chem. 

  http://www.jbc.org/content/225/2/781.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  tml#ref-list-1

http://www.jbc.org/content/225/2/781.citation.full.haccessed free atThis article cites 0 references, 0 of which can be

by guest on June 5, 2018http://w

ww

.jbc.org/D

ownloaded from