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Further studies of the intestinal degradation products of cholic acid-24-C14 in rats: Formation of deoxycholic acid
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
78,
125-137
(1958)
Further Studies of the Intestinal Degradation Products of Cholic Acid-24-Cl4 in Rats: Formation of Deoxycholic Acid’ Oscar W. Portman From
the Department Public Health, Received
of Nutrition, Harvard Boston, Massachusetts March
School
of
18, 1958
It was shown in previous studies (l-3) that diet influences the biliary and fecal excretion of bile acids by the rat. The turnover of bile acids is also greatly prolonged when the bacterial flora is reduced or absent (4, 5). Taurocholic acid, the principal bile acid in rat bile (6), is hydrolyzed, and its steroid portion is modified to one or more less polar compounds in its passage from the duodenum to the feces. The inhibition of the bacterial flora with antibiotics prevents these changes (7). It is a reasonable hypothesis that the bacterial modification of bile acids in the gut inhibits their absorption and hastens their disappearance from the body. It was observed in a previous study involving the administration of cholate-24-Cl4 (3) to rats that in all dietary groups the feces contained one peak of radioactivity in the dihydroxycholanic acid region. Abel1 et al. (8) had already observed that the feces of dogs contain large quantities of deoxycholic acid. Carey and Watson (32) and Lindstedt (9) had identified deoxycholic acid in human feces, and Lindstedt and Sjijvall (10) had shown that cholic acid is converted to deoxycholic acid in the rabbit intestine and is subsequently reabsorbed and excreted in the bile. The rat usually excretes little or no deoxycholic acid into the bile (6). The rat liver, however, has the ability to convert deoxycholic acid to cholic acid (11). Thus at least one of three conditions appears to exist in the rat: (a) The intestinal conversion of cholic to 1 This research was supported in part by grants-in-aid from the Life Insurance Medical Research Fund, New York; National Heart Institute (Gr. No. H-136), U. S. Public Health Service, National Institutes of Health, Bethesda, Md.; John A. Hartford Memorial Fund, and the Fund for Research and Teaching, Department of Nutrition. 125
126
PORTMAN
deoxycholic acid does not occur, (b) the conversion does not occur in the area of intestinal reabsorption of bile salts, or (c) the liver is extremely efficient in reconverting absorbed deoxycholic acid to cholic acid. The dehydroxylation of steroid compounds by endogenous or bacterial enzymes of the gut has not been a well-studied transformation [see t’he review of Talalay (la)]. Clearly the ability of bacteria to adapt their metabolism to use various groupings of steroid compounds is enormous. The present study deals with the identification of the intestinal degradation products of radiocholate in the rat and with the metabolism of t’hese products and the anatomical sites of their formation. MATERIALS
,4ND
&hTHODS
Cholic acid-24-C14, synthesized by the method of Bergstriim et al. (13), was the same material described in a previous study (3). The reference bile acids and their sources were also previously described (3). Bile acids were isolated from feces and from samples at various sites in the glut, by repeatedly extracting the crushed solids with hot ethanol. The ethanolic ext,racts were then cleared of lipides by the method of Josephson (14). The bile acids were chromatographed on paper by the method of Sjijvall (15), or on the columns described by Mosbach et al. (16). Hydrolysis of conjugated bile acids, when performed, was accomplished in 4 N KOH in sealed tubes heated to 125’ for 3 hr. Radioassay of increments of the paper chromatographs or of aliquots from the column chromatographs were performed as previously described (3). Spectrophotometric measurement.s were performed on samples of the separated bile acids using the reactions of Minibeck (17) and of Szalkowski and Mader (18). The relative specificity of these reactions has been demonstrated (19). The infrared spectrophotometry was performed on methyl esters of bile acids in CS, using a Perkin-Elmer continuous reading machine with a 0.05.ml. microcell. Ketosteroids were isolated from extracts containing bile acids by t,he Hughes modification (20) of the Girard T reaction. Carrier dehydrocholic acid was added t.o labeled material, and the mercuric iodide hydrazone salt, after washing, was plated directly for radioassay. The control for completeness of removal of nor!ketonic labeled material was a similar processing of unlabeled dehydrocholic acid plus cholic acid-24-04. RESULTS
Excretion of Labeled Ketosteroids in Feces of Rats Treated with Cholate-f?/,-F4 Samples of alcoholic extracts of feces from rats injected intraperitoneally with 1 mg. (2.2 PC.) cholate-24-U4 were taken for the isolation of labeled ketosteroids. The percentages of total activity in the samples that were in compounds having one or more carbonyl groupings are
FORMATION
OF
DEOXYCHOLIC
TABLE Ezcretion
of Labeled with
127
ACID
I
Ketosteroids (20) in the Feces of Rats Injected Intraperitoneally Cholic Acid-24-C14 (pH 7.4 Phosphate Buffer)
Diet
Time
after
injection
Percentage of total radioactivity in sample as ketosteroids
days
Purina
chow
Synthetic
(starch)
Synthetic
(sucrose)
O-l 24 o-l 24 24 O-l 2-4
6.3” 18.8 7.4 5.9 9.1 4.1 7.9
Q When a small amount of labeled cholic acid was added to dehydrocholic acid and the above ketosteroid isolation was carried out, an average of 2.6yo of the total radioactivity remained with the isolated ketosteroids.
shown in Table I. The means of three determinations on animals from three diet groups are presented. Fecal collections taken during the first 24 hr. and between the 48th and 96th hr. after administration of radiocholate were used. A range of between 4 and 19 % of the total activity was present in the ketosteroid fraction. It should be noted that 2% of a trace amount of radiocholate added to dehydrocholic acid remained in the ketosteroids isolated by the Hughes procedure. Isolation of the Principal Labeled Component in Fecesof Rats Injected with Cholate-Z?4-C14 Since the labeled components of the feces of rats with intact intestinal flora and which have been injected with radiocholate are largely unconjugated, the system of column chromatography described by Mosbach et aE. (16) was particularly well suited for separations. The feces from two rats fed a synthetic diet free of cellulose, each injected with 1 mg. radiocholate (2.2 PC.), were collected between the 24th and 48th hr. after injection and processed as described above. The crude bile acids when subjected to chromatography gave a radioactivity distribution as indicated in Fig. 1. The notations IPE 40 and IPE 60 are the same as those used by Mosbach et al. (16) in an identical system; i.e., IPE 40 indicates isopropyl ether-ligroine 40:60 (U/V) and IPE 60 indicates isopropyl ether-ligroine 60:40 (U/V). This large peak in the
128
PORTMAN
dihydroxy~~olanic region was invariable in about 20 samples. Lesser and more variable amounts (depending on diet) of fecal ~~di~~ctivity were present in the more polar IPE 60 region. The material in the WE: 40 peak was tentatively designated bile acid “D” (or dihydroxycholnniclike).
The spectra of cholic, deoxycholic, and chenodeoxycholic acids and bile :kcid D were determined in the reagent, of Minibeck (17). ‘l’hc material used as bile acid D in this irt&mce was tube Ko. 10 of t’hr &romatograph of feces shown in Fig. 1. The spectra of chenodeosychdie, deoxycholic, and bile acid I> were essentially ident,icnl in t,hr region between 300 and 400 rnk. The spectra between 600 and 720 rnp of 100 pg. of each of these four bile acids in the salicaldehyd~~~SO* reaction of S~alko~vs~ and Mader (18) were determined. The cholic acid and ohenodeoxycholic :zid gave essentially no reading in this range. The spectra of deoxycholic acid and bile acid D in this highly specific reaction appeared very near1.v identical. 6000
i-‘IPE
40
WIPE
60
TUBE
NUMBER
40 FIG. 1. Separation of the radioactivity in :t crude: est,ritet of feces collec:t.eti from two rats between the 24th and 48th hr. after the intraperitoneal administration of cholic acid-24-W (1 mg., 2.2 pt./rat). The chromat’ographic system and the IPE symbols are those of Mosbsch et aE. (16) (see test). The IPI’, 40 region (‘orresponds t80 the dihydroxyc~~olal~ie acids and the IPE 60 region to the trihyctrctsycholanic acids. Individual fractions were approximately 10 ml.
FORMATION
OF
DEOXYCHOLIC
129
ACID
Bile acid D and certain standard bile acids (cholic, deoxycholic, and chenodeoxycholic) were methylated and analyzed in the 900-1200 cm.-1 region of the infrared (21). Absorption maxima were located at 1160, 1120, 1065, 1040, and 960 cm.-l with lesser peaks at 975 and 920 cm.-‘. This spectrum was nearly identical with that of methyl deoxycholate and with that of Wootton (21) for methyl deoxycholate. There was no indication of the presence of any methyl chenodeoxycholate or methyl cholate. Repeated Chromatography
of Bile Acid D Plus Carrier
Deoxycholic
Acid
Several different. samples containing bile acid D-Cl* were chromatographed with carrier deoxycholic acid on the columns described by Mosbach et al. (16). Figure 2 shows the correlation of radioactivity and the optical density at 680 rnp in the Szalkowski-Mader reaction using aliquots from a column to which 1 mg. of bile acid D and 20 mg. of authentic deoxycholic acid were added. Bile acid D was inseparable from authentic deoxycholic acid. COUNTS
13 --WIPE
40
O.D.
680
WIPE
PER
MINUTE
my 60
w 2000 l2 5 1500 5 P 2
1000
I
5 0 0
500
I l-l
5 TUBE
FIG. 2. The distribution of the Saalkowski-Mader reagent when approximately 1 mg. of of authentic deoxycholic acid of Mosbach et al. (16). See text
NUMBER
radioactivity and of optical density at 680 rnp in (18) (which is highly specific for deoxycholic acid) labeled bile acid D isolated from feces and 20 mg. were chromatographed together using the system and Fig. 1.
130
PORTMAN
Recrystallization
of Labeled Bile Acid D and Carrier
Deoxycholic
Acid
A sample of bile acid D-P representing the entire dihydroxy peak of radioactivity in one chromatogram was added to 30 mg. deoxycholic acid. Material with a specific activity of 103.3 counts/min./mg. deoxycholic acid resulted. The material was recrystallized two times from alcohol-water, then three times from ethyl acetate-ligroine. Specific activities of 90.2, 89.4, 100.0, 98.8, and 98.0 counts/min./mg. deoxycholic acid were found after the successive recrystallizations. Absorption of Bile Acid D Administered Intraduodenally and Its Conversion to Trihydroxycholanic Acid Since Bergstrom et al. (11) have demonstrated the hepatic conversion of deoxycholic to cholic acid and of chenodeoxycholic acid to other trihydroxycholanic acids [identified by Hsia et al. (22, 23) as trihydroxycholanic acids isomeric with hyocholic acid] by the rat, it was decided to evaluate the absorption and metabolism of bile acid D. Approximately 2 mg. bile acid D or 2 mg. cholic acid (each weakly labeled) was the test dose. The material was administered via indwelling duodenal tubes to rats with bilinry fistulae. Figure 3 represent’s t,he cumuIOO-
4
0
12
FIG. 3. The cumulative i&red either 2 mg. of bile intraduodenally. Bile acid viously treated with cholic
-
CHOLIC
*----a
BILE
16
20 TIME
ACID-C”I ACID
O-C’4
26 24 (HOURS)
32
36
40
44
40
biliary excretion of radioactivity from rats admin. acid D or 2 mg. of cholic acid (both weakly labeled) D was isolated chromat,ographically from r:tt,s preacid-24-C” (see text).
FORMATION
OF
DEOXYCHOLIC
0 COUNTS PER MINUTE ----0.0. SZALKOWSKI-MADER -0.R MINIBECK 39Omy FIPE 60
I-+IPE40
131
ACID
66Omp
n
c’ z 0.400
-I
= 0
2
TU6E
NUMBER
4. The chromatographic distribution of radioactivity and optical densities of biliary bile acids (after alkaline hydrolysis) from a rat administered 2 mg. of bile acid D-Cl4 intraduodenally. The chromatographic system is that of Mosbach et al. (16) (see text). Ten milligrams of carrier deoxycholic acid was added to the material before chromatography; 5-ml. fractions were collected in the IPE 40 region and lo-ml. fractions in the IPE 60 region. FIG.
lative biliary excretion of radioactivity by rats administered either bile acid D or cholic acid. For the rats administered bile acid D there was a lower recovery of administered radioactivity and a much more rapid excretion of that radioactivity which was excreted. When hydrolyzed bihary bile acids from one of the bile acid D rats were combined with 10 mg. of carrier deoxycholic acid and subjected to Mosbach chromatography, the distribution of bile acids and of radioactivity seen in Fig. 4 was observed. Approximately one-third of the radioactivity recovered had been transferred in a single enterohepatic circulation to material moving as trihydroxycholanic acid. Aliquots of the chromatographic collections were subjected to the SzalkowskiMader reaction and read at 680 rnp (specific for deoxycholic acid), and other aliquots were read in the Minibeck reaction at 390 mp (fairly specific for cholic acid). It was apparent that the peaks of greatest optical densities corresponded to the radioactivity peaks. There was a small peak of radioactivity in an area of slightly greater polarity than the cholic acid peak. This, perhaps, corresponds to the new deoxycholic acid metabolite described by Ratliff et al. (24).
132
PORTMAN
Anatomical Sites of the Conversion of Cholic Acid-@04 to Deoxycholic Acid A male rat weighing 300 g. which had been fed Purina chow was injected intraperitoneally with 2 mg. (4.4 PC.) cholic acid-24-P (in pH 7.4 phosphate buffer). Twenty-four hours later, the peritoneal cavity was opened, a bile cannula was inserted, and the duodenum, ileumjejunum, and cecum-colon were quickly isolated with ligatures. Samples of the contents of each intestinal segment, 0.1 ml. bile, 0.5 ml. portal sera, and the 24-hr. collection of feces were transferred to ethallol. After initial isolation of the fractions containing bile acids, part, of each fraction was hydrolyzed and subjected to Mosbach chromatography, and part was run directly on ascending paper chronyntogrums by the method of Sjiivall (15). Figure 5 shows the chromatography of free (hydrolyzed) bile acids from the various fractions. The only lahrlcci I--IPE
40
kIPE
60
PORTAL
200 100
COLON
400
FECES
SERA
200 5
IO
15 TUBE
20 25 NUMBER
30
35
40
FIG. 5. The chromatographic distribution of radioactivity in hydrolyzed amples of bile, portal sera, feces, and contents of various segments of the gastrointestinal tract of a rat on a diet of Purina chow that had been injected one da> previously with 2 mg. (4.4 PC.) of cholic acid-24-W (pEI 7.4 phosphate buffer). The IPE 40 region is the dihydroxycholanic acid region, and the IPE 60 region is the trihydroxycholanic acid region. The paper chromatographic system of Sjijvall (15) indicated that the radioactivity in the cecum-colon and in the feces was associated with free bile acids and that the radioact,ivity in the other fo,ll samples was associated with taurine conjugates.
FORMATION
OF
DEOXYCHOLIC
ACID
133
material in bile, portal sera, and duodenal and ileo-jejunal contents migrated like cholic acid. The principal peak in the chromatograph of material from the cecum-colon was a peak in the dihydroxycholanic acid region (identified as deoxycholic acid). However, the extract of feces when chromatographed showed about 60% of the radioactivity in the IPE 40 (dihydrocholanic acid region) and 40% in the IPE 60 region. This type of pattern in the feces was almost invariably seen in the rats fed Purina chow regardless of how long after the radiocholate administration the fecal collection was taken. Preliminary evaluation of the material associated with the radioactivity in the IPE 60 region for feces indicated that little or none of it was cholic acid. Several samples of portal sera have been taken at different periods after the earlier intraperitoneal administration of radiocholate, and no labeled material other than taurocholate has ever been observed. The separation of conjugated from nonconjugated bile acids by paper chromatography (15) in the various fractions indicated that hydrolysis of steroid-peptide bonds began and was largely completed in the cecum-colon. Absorption
of Cholate-24-C14 and Biosynthetic from the Cecum-Colon
Deoxycholate-24-C14
Two male rats were prepared with biliary catheters and with catheters secured in the lumina of the ceca with purse-string sutures. The ileo-cecal valves were closed with circumferential ligatures. One received a small labeled dose of radiocholate (about 0.5 mg.) via the indwelling intracecal catheter, and the other received a similar dose of deoxycholate-C14 (in pH 7.4 phosphate buffer). A considerable quantity of the intubated radioactivity was rapidly excreted into the bile of both animals. Of the radioactivity from the rat intubated with bile acid D, 29.8 % was recovered in 18 hr., and 69.5 % of the radioactivity from the cholate rat was similarly recovered. The animals died 25 and 23 hr., respectively, after the time of intubation, presumably a result of the gastrointestinal obstruction, DISCUSSION
The apparently complex mixtures of bile acids found in the feces of experimental animals and of man has been a distinct impediment to the study of sterol balance as well as to attempts to understand the metabolism of bile acids. The results of a previous study (3), however, had indicated that rats fed purified rations without added cellulose
134
PORTMAN
excreted all of a trace dose of labeled cholic acid as a single peak in the dihydroxycholanic acid region. Column chromatography of the type described by Mosbach et al. (16) was used to isolate larger samples of this radioactive metabolite of radiocholate. The characterization of this compound was clearly consistent with a dihydroxycholanic acid. Furthermore, detailed chemical and biological characterization indicated that this bile acid was deoxycholic acid. This finding is consistent with the previous observations of Abel1 et al. (8) in dogs and of Lindstedt (9, 10) in humans and rabbits. It is not clear how the conversion of taurocholate to unconjugated deoxycholate in the cecum is related to the observations of Lindstedt and Norman (4) and of Gustafsson et al. (5) that rats with sterile gustrointestinal tracts (as a result of antibiotic therapy or germ-free raising techniques) had much larger (about fivefold greater) biological half lives of administered radiocholate than did controls with intact intestinal flora. It would be attractive to postulate that the absorption of bile salts from the cecum and colon is possible and that cholate is more readily absorbed than deoxycholate. Sjovall (25) has shown that, chol:tt,e is absorbed from the cecum, and we have confirmed this. The above observations indicated that deoxycholate is somewhat lesswell absorbed from the duodenum or cecum than cholate; however, the results are too few and the procedures too artificial to allow clear conclusions to be drawn. Deoxycholic acid has the unique property of forming complexes with fatty acids, so-called choleic acids, that do not tend to strongly dissociate even at alkaline pH. This suggests that in vivo deoxycholic acid may be in an unusual physical form with biological properties different from pure deoxycholic acid. The failure to find any labeled metabolite other than taurocholate in portal sera of rats previously treated with radiocholate strongly suggests that, as earlier reported (l), this is quantitatively the most important bile acid absorbed from the gut of the rat. If the above experiments are correctly interpreted and deoxycholic acid is the principal bile acid in the feces of the rat, it would clearly indicate that the enzymes in the cecum and colon, whether of endogenous or bacterial origin, favor reduction. This would not have been t,he supposition from the work of Schmidt and Hughes (26) in the guinea pig or from the more recent studies using bacteria or bacterial enzyme preparations [e.g. (27-29)], all of which have demonstrated the keto oxidation of hydroxyl groups of bile acids. The importance of this de-
FOR&fATION
OF DEOXYCHOLIC
ACID
135
hydroxylation to the body steroid economy and the manner in which nutritional factors affect this economy in various species remain to be established. Furthermore, the identity and biological properties of the more polar degradation products of cholic acid which are found in the feces of rats fed Purina chow or synthetic diets with added roughage are still unknown. Since completion of this study Dr. James Hamilton of Tulane University has kindly evaluated the deoxycholic acid isolated from feces in these studies using his newer techniques (30), and he has observed minor contamination with a monohydroxy, monoketo acid. A recent preliminary report of Norman and Sjovall (31) describes the conversion of radiocholic acid to 3cu-hydroxy, 12-keto cholanic acid in the colon of the rat. ACKNOWLEDGMENTS The author wishes to thank Dr. Claire Zomzely for her advice on the column chromatographic separation of bile acids, Dr. Erwin Mosbach of Columbia University for characterizing the cholic acid-24-W and for the gift of our chenodesoxycholic acid, Dr. Frederick Uhle of the Department of Pharmacology for preparation of the diazomethane used in methylation of bile acids, and Mr. Jerry Dudek of the Department of Physical Chemistry, Harvard University, for guidance and equipment in determination of the infrared spectra. SUMMARY
Cholic acid-24-Cl4 was injected intraperitoneally into rats, and the intestinal degradation products of cholate were studied in the intestinal tract and feces. A relatively small amount of the radioactivity (4-19 %) was present in compounds with carbonyl groupings. When extracts of feces were chromatographed on the columns described by Mosbach et al. (16), a consistent sharp peak of radioactivity was observed in the dihydroxycholanic acid region. This material, tentatively described as bile acid D, was characterized by its absorption spectrum in the visible and near ultraviolet regions after treatment with the reagents of Minibeck (17) and of Szalkowski and Mader (18), and the methyl ester was characterized by its infrared absorption spectrum. These reactions indicated that bile acid D was essentially pure deoxycholic acid. When bile acid D was added to large quantities of deoxycholic acid and chromatographed, the peak of radioactivity corresponded to the deoxycholic acid peak. Recrystallization of bile acid D and carrier deoxycholic acid did not change the specific activity. Biosynthetic deoxycholate ad-
136
PORTMAN
ministered into the duodenum was one-third converted in a single hepatic circulation to a labeled trihydroxycholanic acid. The conversion of labeled taurocholate to free deoxycholate was shown to occur in the cecum. Biosynthetic deoxycholic acid was partially absorbed when administered into the duodenum or cecum, but in neither case was it absorbed to the extent of labeled cholic acid. No labeled compound PXcept taurocholate has yet been identified in portal sera. The significance of these transformations in the body steroid economy was discussed. REFERENCES
1. PORTMAN, 0. W., AND MANN, G. V., J. Biol. Chem. 213, 733 (1955). 2. PORTMAN, 0. W., MANN, G. V., AND WY~~~ICI, A. I’., Brch. Biochem. Nioph!js. 69, 224 (1955).
3. PORTMAN, 0. W.,
AND MURPHY, I?., Arch. Biochem Riophys.
76, 367 (;\r~g.
1958).
LINUSTEDT, S., AND NORMAN, A., Acta Physiol. Stand. 38, 129 (1956). GUSTAFSSON, B. E., BERGSTROM, S., LINDSTEDT, S., AKD NORMAX, :I.: I',oc. Sot. Exptl. Biol. Med. 94, 467 (1957). 6. BERGSTROM, S., AND NORMAN, A., Proc. Sot. h’xptl. Biol. Med. 33, 71 (I%%I. 7. NORMAN, A., Acta Physiol. &and. 33, 99 (1955). 8. ABELL, L. L., MOSBACH, E. I-I., AND KENDALL, F. E., J. BioZ. Chem. 220, 5’27 4. 5.
(1956).
9. LINDSTEDT, S., Arkiu Kemi 11, 145 (1957). 10. LINDSTEDT, S., AND SJ~VALL, J., Acta Chem. &and. 11, 421 (1957). 11. BERGSTROM, S., ROTTENBERG, M., AND S.riiv.4Lrd, J., Z. physiol. C‘hem. 296, 278 (1953). 12. 13.
TALALAY, P., Physiol. Revs. 37, 362 (1957). BERGSTROM, S., ROTTENBERG, M., AND VOLTZ, J., Acta Chew &and.
38, 481
(1953). 14. 15. 16.
JOSEPHSON, B., Biochem. J. 292, 1918 (1935). SJ~VALL, J., Acta Chem. b’cand. 9, 1034 (1955). MOSBACH, E. H., ZOMZELY, C., AND KENDALL, F. E., ilrch.
Biodum.
Bioph!/s.
48, 95 (1954). 17. MINIBECH, H., Biochem. 2. 237, 29 (1938). 18. SZALKOWSKI, C. R., AND MADER, W. J., Anal. Chem. 24, 1602 (19,52). 19. WYSOCKI, A. P., YORTMAN, 0. W., AND MANN, G. V., Arch. Biochem. Hioph!ja. 69, 213 (1955). 20. HUGHES, H. B., J. Biol. Chem. 140.21 (1941). 21. WOOTTON, I. D. P., Biochem. J. 63, 85 (1953). 22. HsIA,S. L.,MATSCHINER, J.T., MAHOWALII, T. A., ELLIOTT,~. H., DOISY, E. A., JR., THAYER, S. A., AND DOISY, E. A., J. Biol. Chem. 226, 811 (1957). 23. HSIA, S. L.,MATSCHINER, J. T., MAHOWALD, T. A., ELLIOTT, W. H., DOISY, E. A., JR., THAYER, S. A., AND DOISY, E. A., J. BioZ. Chem. 226,667 (1957). 24. RATLIFF, R. L., RAY,P. D., MATSCHINER, J. T., DOTY, W. H., ASU NOISY, E. A., Federation Proc. 17, 294 (1958).
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OF
DEOXYCHOLIC
ACID
137
2.5. SJ~~VALL, J., to be published; quoted by Lindstedt, S., and Norman, A., Acta Physiol. Stand. 38, 121 (1956). 26. SCHMIDT, L. H., AND HUGHES, H. B., J. Biol. Chem. 143, 771 (1942). 27. HALPERIN, A. H., QUASTEL, J. H., AND SCHOLEFIELD, P. G., Arch. Biochem. Biophys. 62, 5 (1954). 28. MARCUS, P. I., AND TALALAY, P., J. Biol. Chem. 218, 661 (1956). 29. HAYAKAWA, S., FUJII, T., SABURI, Y., AND EGUCHI, T., Nature 179,537 (1957). 30. HAMILTON, J. G., AND DIECRERT, J. W., Federation Proc. 17,478 (1958). 31. NORMAN, A., AND SJ~VALL, J., Biochim. et Biophys. Acta 29,467 (1958). 32. CAREY, C. B., JR., AND WATSON, C. J., J. Biol. Chem. 216, 847 (1955).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
78,
125-137
(1958)
Further Studies of the Intestinal Degradation Products of Cholic Acid-24-Cl4 in Rats: Formation of Deoxycholic Acid’ Oscar W. Portman From
the Department Public Health, Received
of Nutrition, Harvard Boston, Massachusetts March
School
of
18, 1958
It was shown in previous studies (l-3) that diet influences the biliary and fecal excretion of bile acids by the rat. The turnover of bile acids is also greatly prolonged when the bacterial flora is reduced or absent (4, 5). Taurocholic acid, the principal bile acid in rat bile (6), is hydrolyzed, and its steroid portion is modified to one or more less polar compounds in its passage from the duodenum to the feces. The inhibition of the bacterial flora with antibiotics prevents these changes (7). It is a reasonable hypothesis that the bacterial modification of bile acids in the gut inhibits their absorption and hastens their disappearance from the body. It was observed in a previous study involving the administration of cholate-24-Cl4 (3) to rats that in all dietary groups the feces contained one peak of radioactivity in the dihydroxycholanic acid region. Abel1 et al. (8) had already observed that the feces of dogs contain large quantities of deoxycholic acid. Carey and Watson (32) and Lindstedt (9) had identified deoxycholic acid in human feces, and Lindstedt and Sjijvall (10) had shown that cholic acid is converted to deoxycholic acid in the rabbit intestine and is subsequently reabsorbed and excreted in the bile. The rat usually excretes little or no deoxycholic acid into the bile (6). The rat liver, however, has the ability to convert deoxycholic acid to cholic acid (11). Thus at least one of three conditions appears to exist in the rat: (a) The intestinal conversion of cholic to 1 This research was supported in part by grants-in-aid from the Life Insurance Medical Research Fund, New York; National Heart Institute (Gr. No. H-136), U. S. Public Health Service, National Institutes of Health, Bethesda, Md.; John A. Hartford Memorial Fund, and the Fund for Research and Teaching, Department of Nutrition. 125
126
PORTMAN
deoxycholic acid does not occur, (b) the conversion does not occur in the area of intestinal reabsorption of bile salts, or (c) the liver is extremely efficient in reconverting absorbed deoxycholic acid to cholic acid. The dehydroxylation of steroid compounds by endogenous or bacterial enzymes of the gut has not been a well-studied transformation [see t’he review of Talalay (la)]. Clearly the ability of bacteria to adapt their metabolism to use various groupings of steroid compounds is enormous. The present study deals with the identification of the intestinal degradation products of radiocholate in the rat and with the metabolism of t’hese products and the anatomical sites of their formation. MATERIALS
,4ND
&hTHODS
Cholic acid-24-C14, synthesized by the method of Bergstriim et al. (13), was the same material described in a previous study (3). The reference bile acids and their sources were also previously described (3). Bile acids were isolated from feces and from samples at various sites in the glut, by repeatedly extracting the crushed solids with hot ethanol. The ethanolic ext,racts were then cleared of lipides by the method of Josephson (14). The bile acids were chromatographed on paper by the method of Sjijvall (15), or on the columns described by Mosbach et al. (16). Hydrolysis of conjugated bile acids, when performed, was accomplished in 4 N KOH in sealed tubes heated to 125’ for 3 hr. Radioassay of increments of the paper chromatographs or of aliquots from the column chromatographs were performed as previously described (3). Spectrophotometric measurement.s were performed on samples of the separated bile acids using the reactions of Minibeck (17) and of Szalkowski and Mader (18). The relative specificity of these reactions has been demonstrated (19). The infrared spectrophotometry was performed on methyl esters of bile acids in CS, using a Perkin-Elmer continuous reading machine with a 0.05.ml. microcell. Ketosteroids were isolated from extracts containing bile acids by t,he Hughes modification (20) of the Girard T reaction. Carrier dehydrocholic acid was added t.o labeled material, and the mercuric iodide hydrazone salt, after washing, was plated directly for radioassay. The control for completeness of removal of nor!ketonic labeled material was a similar processing of unlabeled dehydrocholic acid plus cholic acid-24-04. RESULTS
Excretion of Labeled Ketosteroids in Feces of Rats Treated with Cholate-f?/,-F4 Samples of alcoholic extracts of feces from rats injected intraperitoneally with 1 mg. (2.2 PC.) cholate-24-U4 were taken for the isolation of labeled ketosteroids. The percentages of total activity in the samples that were in compounds having one or more carbonyl groupings are
FORMATION
OF
DEOXYCHOLIC
TABLE Ezcretion
of Labeled with
127
ACID
I
Ketosteroids (20) in the Feces of Rats Injected Intraperitoneally Cholic Acid-24-C14 (pH 7.4 Phosphate Buffer)
Diet
Time
after
injection
Percentage of total radioactivity in sample as ketosteroids
days
Purina
chow
Synthetic
(starch)
Synthetic
(sucrose)
O-l 24 o-l 24 24 O-l 2-4
6.3” 18.8 7.4 5.9 9.1 4.1 7.9
Q When a small amount of labeled cholic acid was added to dehydrocholic acid and the above ketosteroid isolation was carried out, an average of 2.6yo of the total radioactivity remained with the isolated ketosteroids.
shown in Table I. The means of three determinations on animals from three diet groups are presented. Fecal collections taken during the first 24 hr. and between the 48th and 96th hr. after administration of radiocholate were used. A range of between 4 and 19 % of the total activity was present in the ketosteroid fraction. It should be noted that 2% of a trace amount of radiocholate added to dehydrocholic acid remained in the ketosteroids isolated by the Hughes procedure. Isolation of the Principal Labeled Component in Fecesof Rats Injected with Cholate-Z?4-C14 Since the labeled components of the feces of rats with intact intestinal flora and which have been injected with radiocholate are largely unconjugated, the system of column chromatography described by Mosbach et aE. (16) was particularly well suited for separations. The feces from two rats fed a synthetic diet free of cellulose, each injected with 1 mg. radiocholate (2.2 PC.), were collected between the 24th and 48th hr. after injection and processed as described above. The crude bile acids when subjected to chromatography gave a radioactivity distribution as indicated in Fig. 1. The notations IPE 40 and IPE 60 are the same as those used by Mosbach et al. (16) in an identical system; i.e., IPE 40 indicates isopropyl ether-ligroine 40:60 (U/V) and IPE 60 indicates isopropyl ether-ligroine 60:40 (U/V). This large peak in the
128
PORTMAN
dihydroxy~~olanic region was invariable in about 20 samples. Lesser and more variable amounts (depending on diet) of fecal ~~di~~ctivity were present in the more polar IPE 60 region. The material in the WE: 40 peak was tentatively designated bile acid “D” (or dihydroxycholnniclike).
The spectra of cholic, deoxycholic, and chenodeoxycholic acids and bile :kcid D were determined in the reagent, of Minibeck (17). ‘l’hc material used as bile acid D in this irt&mce was tube Ko. 10 of t’hr &romatograph of feces shown in Fig. 1. The spectra of chenodeosychdie, deoxycholic, and bile acid I> were essentially ident,icnl in t,hr region between 300 and 400 rnk. The spectra between 600 and 720 rnp of 100 pg. of each of these four bile acids in the salicaldehyd~~~SO* reaction of S~alko~vs~ and Mader (18) were determined. The cholic acid and ohenodeoxycholic :zid gave essentially no reading in this range. The spectra of deoxycholic acid and bile acid D in this highly specific reaction appeared very near1.v identical. 6000
i-‘IPE
40
WIPE
60
TUBE
NUMBER
40 FIG. 1. Separation of the radioactivity in :t crude: est,ritet of feces collec:t.eti from two rats between the 24th and 48th hr. after the intraperitoneal administration of cholic acid-24-W (1 mg., 2.2 pt./rat). The chromat’ographic system and the IPE symbols are those of Mosbsch et aE. (16) (see test). The IPI’, 40 region (‘orresponds t80 the dihydroxyc~~olal~ie acids and the IPE 60 region to the trihyctrctsycholanic acids. Individual fractions were approximately 10 ml.
FORMATION
OF
DEOXYCHOLIC
129
ACID
Bile acid D and certain standard bile acids (cholic, deoxycholic, and chenodeoxycholic) were methylated and analyzed in the 900-1200 cm.-1 region of the infrared (21). Absorption maxima were located at 1160, 1120, 1065, 1040, and 960 cm.-l with lesser peaks at 975 and 920 cm.-‘. This spectrum was nearly identical with that of methyl deoxycholate and with that of Wootton (21) for methyl deoxycholate. There was no indication of the presence of any methyl chenodeoxycholate or methyl cholate. Repeated Chromatography
of Bile Acid D Plus Carrier
Deoxycholic
Acid
Several different. samples containing bile acid D-Cl* were chromatographed with carrier deoxycholic acid on the columns described by Mosbach et al. (16). Figure 2 shows the correlation of radioactivity and the optical density at 680 rnp in the Szalkowski-Mader reaction using aliquots from a column to which 1 mg. of bile acid D and 20 mg. of authentic deoxycholic acid were added. Bile acid D was inseparable from authentic deoxycholic acid. COUNTS
13 --WIPE
40
O.D.
680
WIPE
PER
MINUTE
my 60
w 2000 l2 5 1500 5 P 2
1000
I
5 0 0
500
I l-l
5 TUBE
FIG. 2. The distribution of the Saalkowski-Mader reagent when approximately 1 mg. of of authentic deoxycholic acid of Mosbach et al. (16). See text
NUMBER
radioactivity and of optical density at 680 rnp in (18) (which is highly specific for deoxycholic acid) labeled bile acid D isolated from feces and 20 mg. were chromatographed together using the system and Fig. 1.
130
PORTMAN
Recrystallization
of Labeled Bile Acid D and Carrier
Deoxycholic
Acid
A sample of bile acid D-P representing the entire dihydroxy peak of radioactivity in one chromatogram was added to 30 mg. deoxycholic acid. Material with a specific activity of 103.3 counts/min./mg. deoxycholic acid resulted. The material was recrystallized two times from alcohol-water, then three times from ethyl acetate-ligroine. Specific activities of 90.2, 89.4, 100.0, 98.8, and 98.0 counts/min./mg. deoxycholic acid were found after the successive recrystallizations. Absorption of Bile Acid D Administered Intraduodenally and Its Conversion to Trihydroxycholanic Acid Since Bergstrom et al. (11) have demonstrated the hepatic conversion of deoxycholic to cholic acid and of chenodeoxycholic acid to other trihydroxycholanic acids [identified by Hsia et al. (22, 23) as trihydroxycholanic acids isomeric with hyocholic acid] by the rat, it was decided to evaluate the absorption and metabolism of bile acid D. Approximately 2 mg. bile acid D or 2 mg. cholic acid (each weakly labeled) was the test dose. The material was administered via indwelling duodenal tubes to rats with bilinry fistulae. Figure 3 represent’s t,he cumuIOO-
4
0
12
FIG. 3. The cumulative i&red either 2 mg. of bile intraduodenally. Bile acid viously treated with cholic
-
CHOLIC
*----a
BILE
16
20 TIME
ACID-C”I ACID
O-C’4
26 24 (HOURS)
32
36
40
44
40
biliary excretion of radioactivity from rats admin. acid D or 2 mg. of cholic acid (both weakly labeled) D was isolated chromat,ographically from r:tt,s preacid-24-C” (see text).
FORMATION
OF
DEOXYCHOLIC
0 COUNTS PER MINUTE ----0.0. SZALKOWSKI-MADER -0.R MINIBECK 39Omy FIPE 60
I-+IPE40
131
ACID
66Omp
n
c’ z 0.400
-I
= 0
2
TU6E
NUMBER
4. The chromatographic distribution of radioactivity and optical densities of biliary bile acids (after alkaline hydrolysis) from a rat administered 2 mg. of bile acid D-Cl4 intraduodenally. The chromatographic system is that of Mosbach et al. (16) (see text). Ten milligrams of carrier deoxycholic acid was added to the material before chromatography; 5-ml. fractions were collected in the IPE 40 region and lo-ml. fractions in the IPE 60 region. FIG.
lative biliary excretion of radioactivity by rats administered either bile acid D or cholic acid. For the rats administered bile acid D there was a lower recovery of administered radioactivity and a much more rapid excretion of that radioactivity which was excreted. When hydrolyzed bihary bile acids from one of the bile acid D rats were combined with 10 mg. of carrier deoxycholic acid and subjected to Mosbach chromatography, the distribution of bile acids and of radioactivity seen in Fig. 4 was observed. Approximately one-third of the radioactivity recovered had been transferred in a single enterohepatic circulation to material moving as trihydroxycholanic acid. Aliquots of the chromatographic collections were subjected to the SzalkowskiMader reaction and read at 680 rnp (specific for deoxycholic acid), and other aliquots were read in the Minibeck reaction at 390 mp (fairly specific for cholic acid). It was apparent that the peaks of greatest optical densities corresponded to the radioactivity peaks. There was a small peak of radioactivity in an area of slightly greater polarity than the cholic acid peak. This, perhaps, corresponds to the new deoxycholic acid metabolite described by Ratliff et al. (24).
132
PORTMAN
Anatomical Sites of the Conversion of Cholic Acid-@04 to Deoxycholic Acid A male rat weighing 300 g. which had been fed Purina chow was injected intraperitoneally with 2 mg. (4.4 PC.) cholic acid-24-P (in pH 7.4 phosphate buffer). Twenty-four hours later, the peritoneal cavity was opened, a bile cannula was inserted, and the duodenum, ileumjejunum, and cecum-colon were quickly isolated with ligatures. Samples of the contents of each intestinal segment, 0.1 ml. bile, 0.5 ml. portal sera, and the 24-hr. collection of feces were transferred to ethallol. After initial isolation of the fractions containing bile acids, part, of each fraction was hydrolyzed and subjected to Mosbach chromatography, and part was run directly on ascending paper chronyntogrums by the method of Sjiivall (15). Figure 5 shows the chromatography of free (hydrolyzed) bile acids from the various fractions. The only lahrlcci I--IPE
40
kIPE
60
PORTAL
200 100
COLON
400
FECES
SERA
200 5
IO
15 TUBE
20 25 NUMBER
30
35
40
FIG. 5. The chromatographic distribution of radioactivity in hydrolyzed amples of bile, portal sera, feces, and contents of various segments of the gastrointestinal tract of a rat on a diet of Purina chow that had been injected one da> previously with 2 mg. (4.4 PC.) of cholic acid-24-W (pEI 7.4 phosphate buffer). The IPE 40 region is the dihydroxycholanic acid region, and the IPE 60 region is the trihydroxycholanic acid region. The paper chromatographic system of Sjijvall (15) indicated that the radioactivity in the cecum-colon and in the feces was associated with free bile acids and that the radioact,ivity in the other fo,ll samples was associated with taurine conjugates.
FORMATION
OF
DEOXYCHOLIC
ACID
133
material in bile, portal sera, and duodenal and ileo-jejunal contents migrated like cholic acid. The principal peak in the chromatograph of material from the cecum-colon was a peak in the dihydroxycholanic acid region (identified as deoxycholic acid). However, the extract of feces when chromatographed showed about 60% of the radioactivity in the IPE 40 (dihydrocholanic acid region) and 40% in the IPE 60 region. This type of pattern in the feces was almost invariably seen in the rats fed Purina chow regardless of how long after the radiocholate administration the fecal collection was taken. Preliminary evaluation of the material associated with the radioactivity in the IPE 60 region for feces indicated that little or none of it was cholic acid. Several samples of portal sera have been taken at different periods after the earlier intraperitoneal administration of radiocholate, and no labeled material other than taurocholate has ever been observed. The separation of conjugated from nonconjugated bile acids by paper chromatography (15) in the various fractions indicated that hydrolysis of steroid-peptide bonds began and was largely completed in the cecum-colon. Absorption
of Cholate-24-C14 and Biosynthetic from the Cecum-Colon
Deoxycholate-24-C14
Two male rats were prepared with biliary catheters and with catheters secured in the lumina of the ceca with purse-string sutures. The ileo-cecal valves were closed with circumferential ligatures. One received a small labeled dose of radiocholate (about 0.5 mg.) via the indwelling intracecal catheter, and the other received a similar dose of deoxycholate-C14 (in pH 7.4 phosphate buffer). A considerable quantity of the intubated radioactivity was rapidly excreted into the bile of both animals. Of the radioactivity from the rat intubated with bile acid D, 29.8 % was recovered in 18 hr., and 69.5 % of the radioactivity from the cholate rat was similarly recovered. The animals died 25 and 23 hr., respectively, after the time of intubation, presumably a result of the gastrointestinal obstruction, DISCUSSION
The apparently complex mixtures of bile acids found in the feces of experimental animals and of man has been a distinct impediment to the study of sterol balance as well as to attempts to understand the metabolism of bile acids. The results of a previous study (3), however, had indicated that rats fed purified rations without added cellulose
134
PORTMAN
excreted all of a trace dose of labeled cholic acid as a single peak in the dihydroxycholanic acid region. Column chromatography of the type described by Mosbach et al. (16) was used to isolate larger samples of this radioactive metabolite of radiocholate. The characterization of this compound was clearly consistent with a dihydroxycholanic acid. Furthermore, detailed chemical and biological characterization indicated that this bile acid was deoxycholic acid. This finding is consistent with the previous observations of Abel1 et al. (8) in dogs and of Lindstedt (9, 10) in humans and rabbits. It is not clear how the conversion of taurocholate to unconjugated deoxycholate in the cecum is related to the observations of Lindstedt and Norman (4) and of Gustafsson et al. (5) that rats with sterile gustrointestinal tracts (as a result of antibiotic therapy or germ-free raising techniques) had much larger (about fivefold greater) biological half lives of administered radiocholate than did controls with intact intestinal flora. It would be attractive to postulate that the absorption of bile salts from the cecum and colon is possible and that cholate is more readily absorbed than deoxycholate. Sjovall (25) has shown that, chol:tt,e is absorbed from the cecum, and we have confirmed this. The above observations indicated that deoxycholate is somewhat lesswell absorbed from the duodenum or cecum than cholate; however, the results are too few and the procedures too artificial to allow clear conclusions to be drawn. Deoxycholic acid has the unique property of forming complexes with fatty acids, so-called choleic acids, that do not tend to strongly dissociate even at alkaline pH. This suggests that in vivo deoxycholic acid may be in an unusual physical form with biological properties different from pure deoxycholic acid. The failure to find any labeled metabolite other than taurocholate in portal sera of rats previously treated with radiocholate strongly suggests that, as earlier reported (l), this is quantitatively the most important bile acid absorbed from the gut of the rat. If the above experiments are correctly interpreted and deoxycholic acid is the principal bile acid in the feces of the rat, it would clearly indicate that the enzymes in the cecum and colon, whether of endogenous or bacterial origin, favor reduction. This would not have been t,he supposition from the work of Schmidt and Hughes (26) in the guinea pig or from the more recent studies using bacteria or bacterial enzyme preparations [e.g. (27-29)], all of which have demonstrated the keto oxidation of hydroxyl groups of bile acids. The importance of this de-
FOR&fATION
OF DEOXYCHOLIC
ACID
135
hydroxylation to the body steroid economy and the manner in which nutritional factors affect this economy in various species remain to be established. Furthermore, the identity and biological properties of the more polar degradation products of cholic acid which are found in the feces of rats fed Purina chow or synthetic diets with added roughage are still unknown. Since completion of this study Dr. James Hamilton of Tulane University has kindly evaluated the deoxycholic acid isolated from feces in these studies using his newer techniques (30), and he has observed minor contamination with a monohydroxy, monoketo acid. A recent preliminary report of Norman and Sjovall (31) describes the conversion of radiocholic acid to 3cu-hydroxy, 12-keto cholanic acid in the colon of the rat. ACKNOWLEDGMENTS The author wishes to thank Dr. Claire Zomzely for her advice on the column chromatographic separation of bile acids, Dr. Erwin Mosbach of Columbia University for characterizing the cholic acid-24-W and for the gift of our chenodesoxycholic acid, Dr. Frederick Uhle of the Department of Pharmacology for preparation of the diazomethane used in methylation of bile acids, and Mr. Jerry Dudek of the Department of Physical Chemistry, Harvard University, for guidance and equipment in determination of the infrared spectra. SUMMARY
Cholic acid-24-Cl4 was injected intraperitoneally into rats, and the intestinal degradation products of cholate were studied in the intestinal tract and feces. A relatively small amount of the radioactivity (4-19 %) was present in compounds with carbonyl groupings. When extracts of feces were chromatographed on the columns described by Mosbach et al. (16), a consistent sharp peak of radioactivity was observed in the dihydroxycholanic acid region. This material, tentatively described as bile acid D, was characterized by its absorption spectrum in the visible and near ultraviolet regions after treatment with the reagents of Minibeck (17) and of Szalkowski and Mader (18), and the methyl ester was characterized by its infrared absorption spectrum. These reactions indicated that bile acid D was essentially pure deoxycholic acid. When bile acid D was added to large quantities of deoxycholic acid and chromatographed, the peak of radioactivity corresponded to the deoxycholic acid peak. Recrystallization of bile acid D and carrier deoxycholic acid did not change the specific activity. Biosynthetic deoxycholate ad-
136
PORTMAN
ministered into the duodenum was one-third converted in a single hepatic circulation to a labeled trihydroxycholanic acid. The conversion of labeled taurocholate to free deoxycholate was shown to occur in the cecum. Biosynthetic deoxycholic acid was partially absorbed when administered into the duodenum or cecum, but in neither case was it absorbed to the extent of labeled cholic acid. No labeled compound PXcept taurocholate has yet been identified in portal sera. The significance of these transformations in the body steroid economy was discussed. REFERENCES
1. PORTMAN, 0. W., AND MANN, G. V., J. Biol. Chem. 213, 733 (1955). 2. PORTMAN, 0. W., MANN, G. V., AND WY~~~ICI, A. I’., Brch. Biochem. Nioph!js. 69, 224 (1955).
3. PORTMAN, 0. W.,
AND MURPHY, I?., Arch. Biochem Riophys.
76, 367 (;\r~g.
1958).
LINUSTEDT, S., AND NORMAN, A., Acta Physiol. Stand. 38, 129 (1956). GUSTAFSSON, B. E., BERGSTROM, S., LINDSTEDT, S., AKD NORMAX, :I.: I',oc. Sot. Exptl. Biol. Med. 94, 467 (1957). 6. BERGSTROM, S., AND NORMAN, A., Proc. Sot. h’xptl. Biol. Med. 33, 71 (I%%I. 7. NORMAN, A., Acta Physiol. &and. 33, 99 (1955). 8. ABELL, L. L., MOSBACH, E. I-I., AND KENDALL, F. E., J. BioZ. Chem. 220, 5’27 4. 5.
(1956).
9. LINDSTEDT, S., Arkiu Kemi 11, 145 (1957). 10. LINDSTEDT, S., AND SJ~VALL, J., Acta Chem. &and. 11, 421 (1957). 11. BERGSTROM, S., ROTTENBERG, M., AND S.riiv.4Lrd, J., Z. physiol. C‘hem. 296, 278 (1953). 12. 13.
TALALAY, P., Physiol. Revs. 37, 362 (1957). BERGSTROM, S., ROTTENBERG, M., AND VOLTZ, J., Acta Chew &and.
38, 481
(1953). 14. 15. 16.
JOSEPHSON, B., Biochem. J. 292, 1918 (1935). SJ~VALL, J., Acta Chem. b’cand. 9, 1034 (1955). MOSBACH, E. H., ZOMZELY, C., AND KENDALL, F. E., ilrch.
Biodum.
Bioph!/s.
48, 95 (1954). 17. MINIBECH, H., Biochem. 2. 237, 29 (1938). 18. SZALKOWSKI, C. R., AND MADER, W. J., Anal. Chem. 24, 1602 (19,52). 19. WYSOCKI, A. P., YORTMAN, 0. W., AND MANN, G. V., Arch. Biochem. Hioph!ja. 69, 213 (1955). 20. HUGHES, H. B., J. Biol. Chem. 140.21 (1941). 21. WOOTTON, I. D. P., Biochem. J. 63, 85 (1953). 22. HsIA,S. L.,MATSCHINER, J.T., MAHOWALII, T. A., ELLIOTT,~. H., DOISY, E. A., JR., THAYER, S. A., AND DOISY, E. A., J. Biol. Chem. 226, 811 (1957). 23. HSIA, S. L.,MATSCHINER, J. T., MAHOWALD, T. A., ELLIOTT, W. H., DOISY, E. A., JR., THAYER, S. A., AND DOISY, E. A., J. BioZ. Chem. 226,667 (1957). 24. RATLIFF, R. L., RAY,P. D., MATSCHINER, J. T., DOTY, W. H., ASU NOISY, E. A., Federation Proc. 17, 294 (1958).
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OF
DEOXYCHOLIC
ACID
137
2.5. SJ~~VALL, J., to be published; quoted by Lindstedt, S., and Norman, A., Acta Physiol. Stand. 38, 121 (1956). 26. SCHMIDT, L. H., AND HUGHES, H. B., J. Biol. Chem. 143, 771 (1942). 27. HALPERIN, A. H., QUASTEL, J. H., AND SCHOLEFIELD, P. G., Arch. Biochem. Biophys. 62, 5 (1954). 28. MARCUS, P. I., AND TALALAY, P., J. Biol. Chem. 218, 661 (1956). 29. HAYAKAWA, S., FUJII, T., SABURI, Y., AND EGUCHI, T., Nature 179,537 (1957). 30. HAMILTON, J. G., AND DIECRERT, J. W., Federation Proc. 17,478 (1958). 31. NORMAN, A., AND SJ~VALL, J., Biochim. et Biophys. Acta 29,467 (1958). 32. CAREY, C. B., JR., AND WATSON, C. J., J. Biol. Chem. 216, 847 (1955).