CY4-CHOLESTEROL

VI. BILIARY END-PRODUCTS OF CHOLESTEROL METABOLISM*

BY M. D. SIPERSTEIN, FRANKLIN M. HAROLD, I. L. CHAIKOFF, AND

W. G. DAUBEN

(From the Department of Physiology, School of Medicine, and the Department of Chemistry, University of California, Berkeley, California)

(Received for publication, November 23, 1953)

Previous studies have shown that the principal end-products of C14- cholesterol metabolism in bile and feces are bile acids (1, 2). Thus, follow- ing the injection of cholesterol-4-Cl4 into rats, as much as 90 per cent of the Cl4 can be recovered in the bile (3), and, of this, up to 90 per cent is present as bile acids (1). Two of the bile acids have been identified by Bergstrom as taurocholic and taurochenodesoxycholic acids (2, 4).

In the present study, U4-labeled lithocholic acid has been identified as a third bile acid derived from the metabolism of cholesterol-4-U4. The three bile acids in bile of rats that had received cholesterol-4-Cl4 were studied. The time of appearance of isotope in these acids was determined with the aid of filter paper chromatography. A possible scheme of their metabolic interrelations is presented, based on the sequence of appearance of isotopic bile acids. Evidence also presented here shows that no other bile acids are formed in the course of the enterohepatic circulation of bile acids.

EXPERIMENTAL

Radioactive CholesteroZ-Cholesterol-4-Cl4 was prepared by a method de- scribed previously (5). Since C14-labeled cholesterol undergoes decompo- sition on standing (6), it was purified immediately before use by the follow- ing method. The digitonide of the labeled cholesterol was first prepared and then split with pyridine and ether as described by Schoenheimer and Dam (7), and the digitonin was removed by centrifugation. The choles- terol obtained by evaporating the pyridine-ether mixture was dissolved in petroleum ether and applied to an alumina column. The column was washed first with petroleum ether and then with a 9: 1 mixture of petro- leum ether and ethyl ether. The cholesterol wa,s eluted with a 4 : 1 mixture of petroleum ether and ethyl ether, and the chromatography repeated once

* This investigation was supported by a rcsenrch grant from the National Cancer Institute, Sationnl Institutes of Health, United States Public Health Service.

181

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182 C’4-CHOLESTEROL. VI

more. Purity of the isolated cholesterol was established by infra-red anal- ysis and paper chromatography as described by Kritchevsky and Kirk (8).

Treatment of Animals-Male rats of the Long-Evans strain, weighing 250 to 300 gm., were lightly anesthetized with ether, and bile cannulas were inserted into their common bile ducts as previously described (3). 1 to 3 mg. of labeled cholesterol emulsified in saline with Tween 20 were then injected into their tail veins. The rats were placed in restraining cages and given free access to food and water, and bile was collected at intervals up to 6 days.

Chromatographic Analysis of Bile-The bile samples were promptly fro- zen and stored until used. The bile was analyzed for bile acids with the aid of filter paper chromatography. The method used here was a slight modification of that described by Taurog, Tong, and Chaikoff in their thyroid studies (9).

Ascending chromatography was employed, 10 X 37 cm. Whatman No. 1 filter paper being used. A mixture of 35 parts of water and 100 parts of collidine was the developing solvent, and the process was carried out in an ammonia atmosphere. 100 to 200 ~1. of whole bile were applied to the paper in a thin horizontal line approximately 9 cm. long and 6 cm. from the bottom of the paper. Placing the samples at this distance produced the most satisfactory chromatograms. After the solvent was allowed to ascend for 16 to 24 hours, the chromatograms were dried and stapled on x-ray films. After a period of 2 weeks to 3 months, the films (radioauto- graphs) were developed. The bile acids were now visualized by spraying the filter paper chromatograms with a 50 per cent solution of antimony trichloride in glacial acetic acid and drying them at 90-96” for 3 to 5 min- utes to bring out the co1or.l The colors for bile acids thus produced varied widely and were readily outlined in most instances.

Results

Chromatographic Behavior of Pure Bile Acids-This was established by applying approximately 0.5 mg. of the bile acid to a filter paper strip (Table I). In general, the conjugated bile acids moved somewhat faster than the free acids, this effect being more pronounced for the taurine conju- gates than for the glycine conjugates. Thus glycocholic and cholie acids

could not be separated readily, but taurocholic acid moved appreciably faster than did the other two. The number of hydroxyl groups also influenced the rate of movement. The relative rates were monohydroxy > dihydroxy > trihydroxy.

1 Compounds other than steroids yield colors with antimony trichloride. Since antimony trichloride injured the emulsion of the x-ray films, the chromatograms were sprayed after the radioautographs had been prepared.

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SIPERSTEIN, HAROLD, CHAIKOFF, AND DAUBEN 183

Identification of Some Bile Acid End-Products of Cholesterol-.J-C14 Metab- olism-A diagram of a typical chromatogram of rat bile (Fig. 1, A) shows that four bands are usually brought out by spraying with antimony tri-

TABLE I

Characterization oj Pure Bile Acids by Ascending Paper Chromatography in Co&dine-Water

Bile acid

TAithocholic. Taurolithocholic. . Desoxycholic Chenodesoxycholic . Tsurochenodesoxycholic Cholic......................................... Taurocholic Glycocholic 7-Keto-3,12-dihydroxycholanic. Dehydrocholic..

T

COMPOUND

CHOLESTEROL, ETC.

TAUROCHENODESOXYCHOLIC ACID TAUROCHOLIC ACID COMPOUND Y

UNIDENTIFIED

T

[ c

RF

-RON r OS2 073

-

059 - -

039

RIGIN I

YPICAL RAT BILE HROMATOGRAM #PRAYED WITH ib Cl3

-- --

--

A

I N EXPT I

RF

0.83 0.83 0.75 0.71 0.82 0.57 0.73 0.60 0.59 0.95

klor obtained with SbCla

Rose I‘

Purple “ “

Deep rose “ “ ‘I ‘I

Yellow Faint yellow

tADlOAL,TOGRAPHS OF RAT IILE SAMPLES OBTAINED \FTER INJECTION OF :HOLESTEROL-4-C’4

ER INJEC-

I i 15

~~-. ______.. ADIOAUTOGRAPH! F BILE OBTAINED 0 HRS. AFTER YJECTION OF .HOLESTEROL-4- ;I4 IN EXPT. 2

-

- -

FIG. 1. Diagrammatic representation of chromatograms and radioautographs of rat bile. For an explanation, see the text.

chloride. The lowest (i.e. nearest the origin) has a rose color and an RF of about 0.39. At no time in the experiments described below was radioac- tivity found in this band. This, in addition to the band’s slow rate of movement on the filter paper, leads us to believe that it may not represent a bile acid.

The next visible band is located at RF 0.73 and is deep rose in color.

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184 d4-CHOLESTEROL. VI

This position corresponds with that taken by a sample of pure taurocholic acid (Table I). In order to identify this band, the following experiments were carried out.

A rat was injected with cholesterol-4-U4 and a large bile sample was collected at late intervals (see below) ; namely, 48 hours or longer after the injection. Pure taurocholic acid was added to an aliquot of the bile. The mixture was chromatographed, and a radioautograph was prepared from the chromatogram. A single radioactive band was found at RF 0.73, and this corresponded exactly with the single color band obtained by spraying the chromatogram with antimony trichloride.

The bulk of the sample was chromatographed without addition of car- rier. A radioautograph was prepared, and the band corresponding to the radioactive spot at RF 0.73 was cut out and eluted with methanol. The solvent was evaporated, and the residue was dissolved in a small volume of methanol and used for further identification studies as follows: (a) An aliquot of the methanol extract was chromatographed as described above and a radioautograph was prepared. This showed two bands, a major one at RF 0.73 and a minor one at the front. The latter is presumably due to partial oxidation of the compound on the paper. (b) Another aliquot was chromatographed together with authentic taurocholic acid, and a radio- autograph was prepared. After visualization with SbCL, the radioactive band was seen to coincide exactly with the colored band of taurocholic acid at RF 0.73. (c) A third aliquot was hydrolyzed with 2 N NaOH and acidified, and the free bile acids were extracted with ether. The ether extract was chromatographed together with cholic acid, and a radioauto- graph was prepared. Two Cl4 bands were again found, a minor one at the front and a major one coinciding exactly with the color band of cholic acid seen after spraying. (d) A fourth aliquot was hydrolyzed in the presence of carrier cholic acid. The mixture was acidified and the precipitated cholic acid was filtered off and recrystallized to constant specific activity from two solvents, ethyl acetate and aqueous ethanol (Table II).

From the results obtained in (a) to (d), it is clear that the band at RF 0.73 is taurocholic acid.

The third band has a faint purple color and an RF of 0.82. This band was identified as the taurine conjugate of chenodesoxycholic acid, pre- viously reported by BergstrGm and Norman (4) to occur in rat bile. The identification was carried out as described in (a) to (d) above, except that the samples of bile were obtained at t,he early intervals (see below) after the administration of the cholesterol-4-CY4. An aliquot was chromato- graphed together with a sample of authentic taurochenodesoxycholic acid, prepared by the method of Bergstrom and Norman (10). A radioauto- graph was prepared and compared with the colored bands on the chroma-

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SIPERSTEIN, HAROLD, CHAIKOFF, AND DAUBEN 185

togram. The single Cl4 band seen coincided exactly with the band of taurochenodesoxycholic acid at about RF 0.82.

The bulk of the bile sample was used for isolation of taurochenodesoxy- cholic acid, which was carried out exactly as described above for taurocholic acid. The eluate from the chromatogram was used for further identifica- tion as follows. (1) An aliquot was chromatographed as described, and a radioautograph was prepared. Only a single Cl4 band was observed, with an RF of 0.82. Although this suggests that no oxidation took place on the paper, the presence of small amounts of oxidation products is, of course, not ruled out. (2) Another aliquot was chromatographed together with a

TABLE II

Identijkation of Product Obtained by Hydrolysis of Compound at Rp 0.73 As Cholic Acid by Isotope Dilution

I

Sample

30,000 c.p.m. eluted from chromatogram at RF 0.73 and hydrolyzed in pres- ence of 50 mg. carrier cholic acid

Treatment Recovered

w. Recrystallized once from 24.4

ethanol-water and 4 times from ethyl acetate

Recrystallized once more 11.5 from ethyl acetate

“ “ 8.0

C”

Total C.P.lll.

8120 333

4160 362

2720 340

Specific activity,

c.p.m. per mg. cholic

acid

sample of taurochenodesoxycholic acid, and a radioautograph was pre- pared. The single Cl4 band coincided exactly with the colored band corresponding to taurochenodesoxycholic acid. (3) A third aliquot was hydrolyzed as described in (c) above, and chromatographed with chenodes- oxycholic acid. This time, two Cl4 bands were observed on the radioauto- graph, coinciding with two visible bands on the chromatogram. One, at Rp 0.71, corresponds to chenodesoxycholic acid. The other, at RF 0.52, may be due to a breakdown product of chenodesoxycholic acid on treat- ment with alkali, but other explanations are not ruled out. (4) A fourth aliquot was hydrolyzed in alkali as described above, in the presence of car- rier chenodesoxycholic acid. After hydrolysis, the mixture was acidified, and the precipitated chenodesoxycholic acid was recrystallized to con- stant specific activity from aqueous ethanol and petroleum ether-ethyl acetate (Table III).

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186 C14-CHOLESTEROL. VI

From the accumulated evidence it is clear that the band at Rp 0.82 con- sists primarily of taurochenodesoxycholic acid. However, our findings do not exclude the possibility that small amounts of other compounds contrib- uted to this band. Indeed, the observation that taurolithocholic acid ap- pears at Rr 0.83 (Table I) led us to investigate whether lithocholic acid was also a product of cholesterol metabolism. An early bile sample from a rat injected with cholesterol-4-Cl4 was hydrolyzed with 2 N alkali, as already described, after the addition of carrier lithocholic acid. Several recrystal- lizations were then carried out from two solvents, aqueous methanol and

TABLE III

Identification of Product Obtained by Hydrolysis of Compound at RF 0.82 As Chenodesoxycholic Acid by Isotope Dilution

Sample Treatment R ecovered

22,500 c.p.m. eluted from chromatogram at RF 0.82 and hydrolyzed in pres- ence of 35 mg. chenodes- oxycholic acid

Recrystallized once from ethanol-water and 4 times from ethyl ace- tate-petroleum ether

Recrystallized once more from ethyl acetate-pe- troleum ether

“ “

I-

m&z.

7.0

2.5

2.0

C”

Total c.p.m.

3000

1150

900

Specific activity,

c.p.m. per ng. chenc- :soxycholic

acid

430

460

450

aqueous ethanol. The results (Table IV) indicate that lithocholic acid is a minor constituent of rat bile and is derived from cholesterol.

Fourth Band-Just at or behind the solvent front lies a lavender band (Rp 0.98) corresponding to the color and position of cholesterol.

Sequence of Elimination of Products of Metabolism of CL4-Cholesterol in Rat Bile-Two experiments were conducted. In Experiment 1, choles- terol-4-Cl4 was injected into several rats immediately after they were pro- vided with bile cannulas. In these rats the labeled bile acids drain away as soon as they are formed without undergoing enterohepatic circulation (11). In Experiment 2, bile cannulas were inserted into the rats 50 hours after the injection of the labeled cholesterol. Since, in these rats, the labeled bile acids excreted were reabsorbed, Experiment 2 permitted us to study the effect of their recirculation on the appearance of the label in the various bile acids.

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SIPERSTEIN, HAROLD, CHAIKOFF, AND DAUBEN 187

Experiment 1. Injection of Cholesterol-$C14 into Rats Already Provided with Bile Cannulas-Three distinct radioactive bands are visible in the radioautograph of the bile sample obtained during the first 4 hours after the injection of cholesterol-4-Cl4 (Fig. 1, B). The Cl4 band at the front corre- sponds to cholesterol, and the second band to taurochenodesoxycholic acid plus lithocholic (or taurolithocholic) acid. The third band at Rp 0.59 did not correspond to any visible band on the chromatogram; it was not identified, and is designated Compound Y. Occasionally chromatograms were obtained in which Compound Y was absent. It should be noted

TABLE IV

Identification of Lithocholic Acid in Hydrolyzed Rat Bile by Isotope Dilution

Treatment

Recrystallized twice from meth- anol-water

Recrystallized once more from meth- anol-water

I‘ I‘ ‘I “

Experiment 1. C’”

- 1 ml. early bile

sample (32,800 c.p.m.) + 150 mg. carrier litho- cholic acid

Experiment 2. CL4

42 0.9 ml. early bile sample (212,000

11

11 11

c.p.m.) + 150

mg. carrier lithocholic acid

Ei d 6 1 B

:54( .J

32

31 31

that during these 4 hours there was no radioactivity in taurocholic acid. Activity was, however, found in taurochenodesoxycholic acid and at the front as early as 1 hour after the injection of C14-cholesterol.

The radioautograph of the bile collected during the next 3 hours is iden- tical with that shown in Fig. 1, B. The third radioautograph (Fig. 1, C) represents a bile sample collected between 7 and 14 hours. Here for the first time a faint but definite fourth radioactive band is seen, corresponding to taurocholic acid. During the next 9 hours (Fig. 1, D), taurochenodesoxy- cholic acid and Compound Y faded in intensity, leaving most of the radio- activity in taurocholic acid. After 23 hours (Fig. 1, E) no activity was ob- served in Compound Y, and only a very small amount in taurochenodes- oxycholic acid. In fact, it was found that from the 23rd hour until the end of the study, with the exception of small amounts of activity in cholesterol

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188 C’4-CHOLESTEROL. VI

and taurochenodesoxycholic acid, practically all of the Cl4 was present as taurocholic acid (see, for example, 46 to 58 hours, Fig. 1, F).

The actual amounts of Cl4 present in the radioactive bands at various time intervals were determined as follows. Samples of bile were chroma- tographed, and radioautographs were prepared as described above. The

TABLE V

Experiment 1. Distribution of Radioactivity in Sections of Chromatograms of Bile Collected at Various Intervals Following Injection of Cholesterol-Q-C14

Time

hrs.

o-2 2-4 4-7 7-12

22-35 35-45

Per cent total Cl4 on chromatogram recovered in

9.8 11.1 1o.ot 50.6 18.4 5.4 12.6 13.7t 56.4 12.0 4.4 6.3 9.2 60.0 19.8 6.2 8.8 21.5 54.5 9.0 3.5 5.7 72.5 12.4 5.7 3.0 8.4 70.0 10.5 8.3

Compound Y Taurocholic acid TauroChenodeso~ cholic acid*

* Lithocholic (or taurolithocholic) acid is a minor constituent of this band. t At these intervals radioactive bands corresponding to taurocholic acid were not

visible on the radioautographs. The radioactivity found here is attributed to streaking of the Cl4-taurochenodesoxycholic acid.

TABLE VI

Experiment 5’. Filter Paper Chromatography of Whole Rat Bile Obtained by Cannulation 50 Hours after Injection of Cholesterol-.$-C’4

Compound Per cent total activity on paper

Origin................................................. 5.7 Taurocholic acid....... .._.........._.._............ 45.3 Taurochenodesoxycholic acid*. 46.4 Cholesterol and front . _. . 2.4

* See foot-note to Table V.

paper strips were then cut into five sections, as shown by the arrows in Fig. 1, A. Each section was extracted three times with boiling ethyl alcohol, the combined extracts were brought to volume, and an aliquot was taken for Cl4 determination. The results (Table V) fully confirm the impressions gained from the radioautographs. In the early period, radioactivity is found only in taurochenodesoxycholic acid and at the front. In the later samples, taurocholic acid is the dominantly labeled bile acid, whereas the amount of Cl4 has diminished in the other bile acids.

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SIPERSTEIiY, HAROLD, CHAIKOFF, AND DAUBEN 189

Experiment S?. Administration of Cholesterol-.&P 50 Hours before Bile Duct Was Cannulated-In order to see whether any additional bile acids become labeled during the enterohepatic circulation of the bile acids, a rat was injected with cholesterol-4-Cl4 and the bile duct cannulated 50 hours later. Both taurocholic acid and taurochenodesoxycholic acid are strongly labeled, but no new labeled compounds are found (Fig. 1, G). The radio- autographs of samples collected at later intervals (up to 24 hours after can- nulation) closely resemble that shown in Fig. 1, G.

The actual distribution of radioactivity was determined by cutting the paper strip into sections and eluting as described above. The results (Table VI) show that, 50 hours after injection, about half of the radioactiv- ity is still present in taurochenodesoxycholic acid.

DISCUSSION

Three independent procedures were used to identify the biliary end- products of cholesterol metabolism in the rat: (1) direct chromatography of the bile obtained from rats that received cholesterol-4-C14; (2) chroma- tography of the Cl4 bile acids after their hydrolysis; and (3) recrystalliza- tion of the isolated Cl4 bile acids to constant specific activity after the ad- dition of carrier. The evidence confirms previous reports by Bergstrom (2) that taurocholic acid, accompanied by small amounts of taurocheno- desoxycholic acid (4), constitutes the chief biliary excretion product of cholesterol metabolism. In addition, lithocholic acid has been identified as a minor product. It should be noted, however, that, during the first 7 hours after injection of C14-labeled cholesterol into rats already provided with bile cannulas, no radioactivity is found in taurocholic acid. During this early period, the only radioactive bile acids are taurochenodesoxy- cholic acid, lithocholic acid (presumably also conjugated with taurine), and sometimes an unidentified compound which we have designated as Com- pound Y. As the excretory process is followed for longer periods, tauro- chenodesoxycholic acid and Compound Y lose their activity, while tauro- cholic acid becomes the most prominent radioactive bile acid excreted in bile. Because of the very small amounts of lithocholic acid found in rat bile, we were unable to determine whether this bile acid also loses its radio- activity with time.

Previous work on the transformation which bile acids undergo in various animals has led to the suggestion that cholic acid is the primary bile acid formed from cholesterol, and is subsequently reduced to dihydroxy and monohydroxy bile acids (12). Our findings do not lend support to these ideas. On the contrary, we find that both lithocholic and chenodesoxy- cholic acid become labeled long before radioactivity appears in cholic acid. This suggests that both of these bile acids are intermediates in the conver- sion of cholesterol to cholic acid, perhaps by a series of successive hydroxyl-

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190 C’4-CHOLESTEROL. VI

ations leading from lithocholic acid to chenodesoxycholic acid, and eventu- ally to cholic acid. The evidence of Bergstriim et al. (13, 14) indicates, however, that lithocholic and chenodesoxycholic acids give rise directly to an as yet unidentified trihydroxy bile acid not identical with cholic acid.

Experiment 2 was designed to study the effect of the recirculation of the bile acids upon their metabolism. No new labeled bile acids were encoun- tered. This finding suggests that, even though it has been shown that in- testinal microorganisms can oxidize and perhaps also hydroxylate bile acids (15, IS), reactions within the intestine do not modify appreciably the nature of the circulating products of cholesterol metabolism.

The distribution of the Cl4 among the various bile acids in the bile sample taken 50 hours after the injection of cholesterol-4-Cl4 differs considerably from that observed in Experiment 1. In this latter experiment, 50 hours after the injection of the cholesterol-4-C 14, the ratio of radioactivity in taurochenodesoxycholic acid to that in taurocholic is 1:7, whereas in Ex- periment 2 the ratio is 1: 1. The difference in the ratios found in Experi- ments 1 and 2 requires further study.

We are indebted to Dr. N. K. Freeman for the infra-red analyses of the labeled cholesterol. Our thanks are also due to Professor G. A. D. Hasle- wood of Guy’s Hospital Medical School, London, England, for the gift of authentic chenodesoxycholic acid. The assistance of Mr. H. H. Hernan- dez, Dr. S. Hotta, and Dr. M. E. Jayko is gratefully acknowledged.

SUMMARY

1. A method for the paper chromatographic separation of the bile acids is described. The end-products of cholesterol metabolism in the rat were studied with this procedure.

2. Lithocholic acid has been identified as one of the products of cholesterol metabolism.

3. During the first 7 hours following the injection of cholesterol-4-C14, radioactivity is visible only in taurochenodesoxycholic and lithocholic acids and sometimes in an unidentified compound designated here as Com- pound Y.

4. Radioactivity begins to be visible in taurocholic acid about 7 hours after the injection of C14-cholesterol, and this eventually becomes the major radioactive excretion product.

5. The enterohepatic circulation of the bile acids does not affect the na- ture of the metabolites of cholesterol, but does delay the disappearance of radioactivity from taurochenodesoxycholic acid.

6. The possibility that lithocholic and chenodesoxycholic acids are inter- mediates in the conversion of cholesterol to cholic acid is discussed.

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SIPERSTEIN, HAROLD, CHAIKOFF, AND DAUBEN 191

BIBLIOGRAPHY

1. Siperstein, M. D., Jayko, M. E., Chaikoff, I. L., and Dauben, W. G., Proc. Sot. Exp. Biol. and Med., 81, 720 (1952).

2. Bergstrom, S., K. Fysiograf. Sdllskap. Lund, Fiirhandl., 22, No. 16 (1952). 3. Siperstein, M. D., and Chaikoff, I. L., J. Biol. Chem., 198, 93 (1952). 4. Bergstrom, S., and Norman, A., Proc. Sot. Exp. Biol. and Med., 83, 71 (1953). 5. Dauben, W. G., and Eastham, J. F., J. Am. Chem. Sot., 73,326O (1951). 6. Tolbert, B. M., Adams, P. T., Eennett, E. L., Hughes, A. M., Kirk, M. R., Lem-

mon, R. M., Noller, R. M., Ostwald, R., and Calvin, M., J. Am. Chem. Sot., ‘76, 1867 (1953).

7. Schoenheimer, R., and Dam, H., 2. physiol. Chem., 216, 59 (1933). 8. Kritchevsky, D., and Kirk, M. R., J. Am. Chem. Sot., 74, 4484 (1952). 9. Taurog, A., Tong, W., and Chaikoff, I. L., J. Biol. Chem., 184, 83 (1950).

10. BergstrGm, S., and Norman, A., Acta them. Stand., 7, 1126 (1953). 11. Siperstein, M. D., Hernandez, H. H., and Chaikoff, I. L., Am. J. Physiol., 171,

297 (1952). 12. Kataoka, Y., J. Biochem., Japan, 34, 87 (1941). 13. Bergstrom, S., Rottenberg, M., and Sjovall, J., 2. physiol. Chem., 296, 278 (1953). 14. Bergstrom, S., Sjovall, J., and Voltz, J., Acta physiol. Stand., 30, 22 (1953). 15. Baumgartel, T., KZin. Wochschr., 26, 378 (1947). 16. Schmidt, L. H., and Hughes, H. B., J. BioZ. Chem., 143,771 (1942).

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Chaikoff and W. G. DaubenM. D. Siperstein, Franklin M. Harold, I. L.

METABOLISMEND-PRODUCTS OF CHOLESTEROL

-CHOLESTEROL : VI. BILIARY14C

1954, 210:181-191.J. Biol. Chem. 

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