THE TRANSFORMATION OF CHOLESTEROL TO COPROSTANOL”

BY R. S. ROSENFELD, DAVID K. FUKUSHIMA, LEON HELLMAN, AND

T. F. GALLAGHER

(From the Sloan-Kettering Institute for Cancer Research, New York, New York)

(Received for publication, June 9, 1954)

The conversion of cholesterol to coprostanol (coprosterol)l has been studied in an attempt to define the mechanism of this transformation. Two principal hypotheses have been advanced to explain this reaction: (1) a direct stereospecific reduction of the double bond, presumably by intestinal microorganisms (1, 2), and (2) a three stage conversion from cholesterol involving the intermediates A4-cholestenone (II) and copros- tanone (III) (3, 4). Fig. 1 illustrates these steps. It has been shown that, after feeding cholesterol-3d,4-Cl4 to humans, the conversion to co- prostanol occurs in large parts by a mechanism in which the hydrogen at C-3 remains intact. Experiments in which the doubly labeled cho- lesterol was incubated with fecal material provided confirmatory evidence of the retention of structure at C-3.

EXPERIMENTAL’

Choleslerol-4-P4-This product was prepared by the method of Belleau and Gallagher (5) from cholestenone-4-Cl4 purchased from the Oak Ridge National Laboratory.

Cholesterol-3d and Epicholesterol-Sd-To a suspension of 2.5 gm. of lith- ium aluminum deuteride3 in 150 ml. of dry ether were slowly added 100 ml. of an ether solution containing 20 gm. of AQholesten-3-one. The mixture was stirred for 3 hours and the excess reagent destroyed with ethyl acetate. The ether solution was then washed with dilute acid and the solvent evap- orated. The residue was dissolved in 250 ml. of ethanol containing 4 ml. of concentrated hydrochloric acid. The solution was refluxed for 30 min- utes and diluted with ether. The ether solution was successively washed

* This investigation was carried out under contract No. AT(30-l)-910 with the United States Atomic Energy Commission and supported by grants from the Anna Fuller Fund, the Lillia Babbitt Hyde Foundation, and the National Cancer Institute, United States Public Health Service (No. C-440).

1 Coprostanol is used throughout this paper to denote coprostan-38-01 instead of the older term “coprosterol.”

2 All melting points are corrected. 3 Obtained from Metal Hydrides Inc., Beverly, Massachusetts, on allocation from

the Atomic Energy Commission.

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302 CHOLESTEROL TO COPROSTANOL

with water, alkali, and water and dried over sodium sulfate. Evaporation of the solvent afforded 19 gm. of crystalline product. Chromatography on silica gel and recrystallization from acetone yielded 13 gm. of cholesterol- 3d; m.p. 149-149.5’, 2.00 atoms per cent excess deuterium (hereafter de- noted by the symbol AD) corresponding to 0.92 gm. atom of deuterium per mole (hereafter denoted by the symbol D).

Epicholesterol-3d (0.75 gm.) was also obtained from the chromatogram and, after recrystallization from acetone, melted at 140-141”.

Proof for Location of Isotope in CholesteroMd-76.5 mg. of cholesterol-3d were diluted with 412.3 mg. of non-isotopic cholesterol (factor 6.39). The diluted cholesterol was analyzed and contained 0.144 D (0.314 AD), which corresponds to 2.01 AD for the undiluted cholesterol. Hydrogenation of 440 mg. of the diluted sterol in 30 ml. of acetic acid and 5 ml. of cyclohexane

I (I)-

FIG. 1. Three stage conversion of cholesterol to coprostanol involving the inter- mediates AQholestenone (II) and coprostanone (III).

with 100 mg. of platinum oxide yielded cholestanol-3d; m.p. 141.5-143’, 0.146 D (0.305 AD). Oxidation of 230 mg. of cholestanoL3d with 2 per cent chromic oxide in acetic acid at room temperature for 2 hours afforded cholestan-3-one; m.p. 129.5-130.5”, 0.003 AD.

This result is in agreement with the findings of Dauben and Eastham (6) who have studied the mechanism of the reduction of ketones with lith- ium aluminum deuteride.

Administration of Cholesterol and Collection of Xamples. Xtudies in Vivo- Cholesterol-4-Cl4 and cholesterol-3d were dissolved in acetone and the material was recrystallized to give doubly labeled material; 435,000 dis- integrations per minute per millimole (d.p.m. per mmole), 0.92 D (2.00 AD). For oral administration, the cholesterol-3d,4-Cl4 in 10 ml. of sesame oil was added to 100 ml. of warm chocolate milk containing 5 gm. of gum acacia and additional chocolate syrup. The mixture was homogenized and fed to the subject 1 hour before the evening meal. Subject A received 0.510 gm. and Subject B was given 0.400 gm. of the labeled cholesterol. Daily stool collections were made and the 2nd and 3rd day collections were examined for fecal sterols.

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ROSENFELD, FUKUSHIMA, HELLMAN, AND GALLAGHER 303

Incubation Xtudies-Two experiments were performed. In the first 0.214 gm. of cholesterol, S,llO,OOO d.p.m. per mmole and 0.92 D (2.00 AD), was used and in the second 0.394 gm. of cholesterol, 3,420,OOO d.p.m. per mmole and 0.92 D (2.00 AD), was incubated. The cholesterol was sus- pended in 10 ml. of 0.9 per cent saline with 1 drop of Tween 80 and the mixture was emulsified by rotation for 2 days in a ridged flask with glass beads. Feces from a normal human were immediately mixed with 50 ml. of 0.9 per cent saline at low speed in a Waring blendor until homogenized. The cholesterol emulsion was added and the mixture was homogenized for an additional 3 minutes. The suspension was transferred to a 2 liter flask with 50 ml. of saline. The contents were incubated at 37” for 50 hours with slow stirring through a mercury seal. Air was initially present in the flask and no deliberate change in this atmosphere was made for the period of incubation.

Isolation of Fecal Xterols-With Subject A, the 24 hour collections were homogenized and extracted three times with acetone. The combined acetone extract (1 liter) was filtered and the solvent was removed. The residue was refluxed for 3 hours with 200 ml. of 5 per cent potassium hy- droxide in 90 per cent ethanol. The cooled alkaline solution was diluted with an equal volume of water. In Subject B, the homogenized feces were mixed with 50 gm. of potassium hydroxide and 150 ml. of ethanol and re- fluxed for 4 hours. The incubation experiments were treated in the same manner as the material from Subject B. Each alkaline mixture was ex- tracted three times with a total of approximately 2 liters of petroleum ether (b.p. 60”). The petroleum ether solution was washed with three 200 ml. portions of 50 per cent aqueous ethanol which were discarded. After dry- ing over sodium sulfate and removal of the petroleum ether, the residual orange oil crystallized on standing. Each non-saponifiable fraction was chromatographed on 75 times its weight of Merck acid-washed alumina and material was eluted from the column with petroleum ether-benzene mix- tures. In each case, three crystalline fractions were obtained which, in order of elution, were coprostanone, coprostanol, and cholesterol.

Purijication of Steroids Isolated from Non-Saponifiable Fraction of Feces- A general description of the purification procedure is presented for each compound. Variations in the procedures are noted for specific samples.

Coprostanone-The crude material was sublimed in a good vacuum and the sublimate was chromatographed on silica gel (adsorbent to substance = 150: 1). Coprostanone, eluted with petroleum ether-10 per cent ether, was recrystallized twice from methanol. This substance was obtained from each of the six non-saponifiable fractions examined. Coprostanone from the studies in vivo melted from 66-74”; the incubation experiments afforded material melting from 68-71”. The infra-red spectra were iden- tical with that of an authentic sample of coprostanone.

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304 CHOLESTEROL TO COPROSTANOL

Coprostanol-This substance comprised the largest amount of the steroid fraction of the feces and the infra-red spectrum of the crystalline material was identical with that of pure coprostanol. The coprostanol was re- crystallized twice from methanol and acetylated with acetic anhydride and pyridine. The product was treated with perbenzoic acid in benzene solu- tion for 2 days in the refrigerator. Chromatography on alumina afforded in the petroleum ether eluates coprostanyl acetate which was recrystallized twice from methanol.

Subject A-Day 2, coprostanol; m.p. 105-106”. Coprostanyl acetate; m.p. 89%89.5’, 6000 d.p.m. per mmole, 0.013 D (0.026 AD). Day 3, co- prostanol; m.p. 99-101’. Coprostanyl acetate; m.p. 94-95”, 25,800 d.p.m. per mmole, 0.050 D (0.099 AD).

Subject B-Day 2, coprostanol; m.p. 98-99”. Coprostanyl acetate; m.p. E&90”, 23,200 d.p.m. per mmole, 0.026 D (0.054 AD). Day 3, co- prostanol; m.p. 97-99”. Coprostanyl acetate; m.p. 87-89”, 3000 d.p.m. per mmole, 0.009 D (0.017 AD).

Incubation l-Coprostanol acetate was treated with perbenzoic acid for 2 hours at room temperature instead of the longer reaction time described above. Coprostanol; m.p. 103-105”. Coprostanyl acetate; m.p. 87.5- 88.5“, 945,000 d.p.m. per mmole, 0.151 D (0.302 AD).

Incubation W-Coprostanol; m.p. 99-101’. Coprostanyl acetate; m.p. 89-90”, 328,000 d.p.m. per mmole, 0.110 D (0.220 AD).

Cholesterol-The fraction eluted from the column with petroleum ether- benzene (1:1) consisted of cholesterol contaminated with other material, as judged by infra-red spectrometry. The contaminant was shown to be cholestanol. The sterol mixture was recrystallized three times from ace- tone and then brominated in glacial acetic acid. The 5,6-dibromocho- lesterol was debrominated with zinc in glacial acetic acid and the recovered cholesterol was recrystallized from acetone.

Subject A-Day 3, cholesterol; m.p. 142-146’, 14,200 d.p.m. per mmole, 0.019 D (0.014 AD).

Subject B-Day 2, cholesterol; m.p. 146-148”, 11,600 d.p.m. per mmole, 0.030 D (0.022 AD). Day 3, cholesterol; m.p. 146-147”. No radioactivity was detected and therefore the compound was not analyzed for deuterium.

Incubation I-The cholesterol obtained by debromination of 5, Gdi- bromocholesterol with zinc and acetic acid melted from 144-146”. Since this treatment causes some acetylation of the hydroxyl group (7), the prod- uct was refluxed with 2 N potassium hydroxide in 90 per cent methanol. The recovered cholesterol was recrystallized twice from acetone; m.p. 14%148.5”, 4,130,OOO d.p.m. per mmole, 0.455 D (0.330 AD). The radio- activity and deuterium content of the cholesterol were unchanged by sa- ponification.

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ROSENFELD, FUKUSHIMA, HELLMAN, AND GALLAGHER 305

Incubation .&-Bromination of the crystalline sterol was carried out as above. The 5,6-dibromocholesterol was debrominated by refluxing with sodium iodide in ethanol-benzene solution and the recovered cholesterol was recrystallized from acetone; m.p. 147-148’. This material was acety- lated with acetic anhydride and pyridine and the product was chromato- graphed on alumina. The crystalline eluate melted at 114.5-116” after one recrystallization from methanol. The cholesteryl acetate was saponified with ethanolic potassium hydroxide and the product was again chromato- graphed on alumina. Cholesterol so obtained was recrystallized once from acetone; m.p. 147-148.5”, 1,590,OOO d.p.m. per mmole, 0.404 D (0.239 AD). Radioactivity and deuterium measurements on cholesterol obtained during stages of the purification showed no appreciable change in isotopic com- position.

Oxidation of Coprostanol-Coprostanyl acetate, obtained as the ultimate product in the purification of coprostanol, was saponified and oxidized at room temperature with 2 per cent chromic oxide in acetic acid. The iso- lated coprostanone was chromatographed on alumina and the crystalline material, eluted from the column with petroleum ether-benzene (9: I), was recrystallized from ethanol; m.p. 63-64”. The compound was dissolved in methanol and refluxed with 5 per cent ethanolic potassium hydroxide for 3 hours. Coprostanone obtained from the alkali equilibration was chromatographed on alumina and recrystallized three times from methanol. The infra-red spectrum was identical with that of an authentic sample of coprostanone.

Incubation I-Coprostanone; m.p. 62.5-64”, 898,000 d.p.m. per mmole, 0.058 D (0.127 AD). The Cl4 and deuterium content of the coprostanone was the same before and after treatment with alkali.

Incubation .%-Coprostanone obtained by oxidation of the saponified coprostanyl acetate was isolated and characterized after equilibration with alkali; m.p. 63.5-65.5”, 228,000 d.p.m. per mmole, 0.016 D (0.034 AD).

Isolation of Cholestanol-The crude cholesterol obtained by chromatogra- phy of the non-saponifiable fraction of Subject B (Day 2) was brominated in acetic acid. The suspension was filtered and an ether solution of the filtrate was washed free of bromine with sodium sulfite solution. The ether-soluble material was treated with powdered zinc in glacial acetic acid and then acetylated. The acetylated material was treated with perbenzoic acid to epoxidize any cholesteryl acetate that remained and the reaction product was chromatographed on alumina. Crystalline material was ob- tained from the petroleum ether-benzene eluates (19: 1) and was identified as cholestanyl acetate by infra-red spectrometry. The substance was not examined further.

Isotopic Anal@s-Deuterium analyses were performed according to the

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306 CHOLESTEROL TO COPROSTANOL

procedure described by Fukushima and Gallagher (8). Cl4 measurements were made on stainless steel planchets with a 1.63 cm.-2 sample area by using windowless gas flow counters (Tracerlab, Inc.); the results were cal- culated to infinite thickness. Coprostanol, coprostanyl acetate, and co- prostanone were melted on the planchets prior to counting, while cholesterol was precipitated as the digitonide and was plated from an acetone-alcohol suspension (9). Cl4 and deuterium measurements were made on cholesterol digitonide.

Studies in Viva-The radioactivity and deuterium content of the sterols isolated from the fecal extracts of the two subjects fed cholesterol-3d ,4- Cl4 is shown in Table I. The measurements were made on the most highly purified samples of coprostanyl acetate and cholesterol, as described in the

TABLE I

Isotope Analysis of Fecal Sterols Derived from Fed Cholesterol-Sd,4-C’4

Chol$erol Subject A Subject B _“-

stero1s from feces Sterols from feces

I I IDilution of I 1 Dilution of

Day Cholesterol Coprostanol c,~c~l~;l Cholesterol Coprostanol coprostanol

D.p.m. calculated from

D

+% D if D lgg $;I C1’ ,, 12; ,, iggf$

~I-;,-,,~,,,l,,,n~~~fiI,~~),,,,l,~,, 0.011 6.7 84 65 0.030 11.6 0.027 23.4 34 18

experimental section. Cl4 and deuterium analyses were also made on cer- tain samples of coprostanol and cholesterol during the purification stages so that isotopic homogeneity could be established. The low content of both Cl4 and deuterium in coprostanol and cholesterol is probably the result of absorption of cholesterol by the subjects, as well as dilution of the labeled material by both cholesterol and coprostanol in the gastrointestinal tract.

The dilution of both isotopes in coprostanol is also shown in Table I. These dilution factors were calculated by dividing the original radioactivity or deuterium content of the fed cholesterol by the appropriat,e value in the recovered coprostanol. The dilution, based on either Cl4 or deuterium, was of the same order of ma.gnitude for each day. If the over-all con- version of cholesterol to coprostanol merely involved a saturation of the 5,6 double bond of the former, the dilution, calculated from either deu- terium or C14, must of necessity be identical. On the other hand, if the conversion of cholesterol to coprostanol involved oxidation at C-3 with

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ROSENFELD, FUKUSHIMA, HELLMAN, AND GALLAGHER 307

subsequent reduction of the resultant ketone (Fig. l), there could be no deuterium present at C-3 and, therefore, the dilution of deuterium would approach infinity. Since it can be seen in Table I that this did not occur, it is reasonable to assume that the cholesterol that was converted to copros- tanol was reduced without prior or subsequent oxidation at C-3.

Incubation &%,&es-The Cl4 content of cholesterol and coprostanol re- covered from the incubation of feces with cholesterol-3d,4-Cl4 is shown in Table II. It may be noted that in the first incubation experiment 36 per cent of the radioactivity present in the cholesterol that was added to the fecal suspension was recovered as coprostanol; in the second incubation 27 per cent of the radioactivity was found in coprostanol. That these figures represent a good approximation of the actual conversion of cholesterol to coprostanol and do not result from contamination of coprostanol by un-

Incubation

1 2

TABLE II

Conversion of Cholesterol to Coprostanol by Incubation System

Sterols recovered

Cholesta&d,4-C!‘d Cholesterol I Coprostanol

Total radioactivity Total radioactivity

WT. a.p.nt. x 10-s mg. d.p.m. x 10-6 per cent mg. d.p.m. x 10-s per cent

214 4.47 222 1.79 40 905 1.60 36 394 3.42 443 1.46 43 1204 0.92 27

changed cholesterol is apparent for two reasons: (1) Chromatography of the non-saponifiable portion of the feces efficiently separated coprostanol from cholesterol in well spaced fractions with no overlap and infra-red examination of the appropriate fractions confirmed the identity of the compounds. (2) On purification of each sterol as described in the experi- mental section, the specific activity increased about 20 to 30 per cent and reached a constant value. This shows that the impurities present in the compounds obtained in the chromatographic separation were either non- radioactive, had the same specific activity, or were of lower radioactivity. Therefore, the transformation of cholesterol to coprostanol by incubation with feces in vitro has been unequivocally demonstrated.

DISCUSSION4

Both the feeding experiments and the incubation studies show that the dilutions of deuterium and Cl4 are essentially of the same order and in the

4 In discussing the data, the assumption has been made that deuterium at C-3 enters into reaction identically with protium.

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308 CHOLESTEROL TO COPROSTANOL

incubation system, where less complicated conditions exist, the retention of the isotopic label at C-3 is beyond question. It is, therefore, unlikely that coprostanol arises from cholesterol principally by the sequence shown in Fig. 1. For this to occur, the deuterium atom at C-3 would be completely lost and the coprostanol would have contained Cl4 but no deuterium.

It might be argued that a portion of the cholesterol-3d,4-Cl4 lost deu- terium by oxidation to cholestenone and the unsaturated ketone was in turn saturated without loss of deuterium by the same reduced coenzyme that had acted as a hydrogen acceptor in the oxidation step. This would be equivalent to a reshuffling of deuterium with no net loss of isotope so that Cl4 and deuterium would be in essentially constant ratio in both cho- lesterol and coprostanol. While this might be a permissible explanation for the results obtained when cholesterol was fed, it is not applicable to the incubation experiments. This is apparent from the isotopic ratio cal- culated as follows:

D gm. atoms deuterium per mole compound -= C’4 (d.p.m. per mmole compound)/107

The ratio, while arbitrary, affords a convenient comparison of the dilution of both isotopes relative to each other. The ratio of the isotopes, as well as the Cl4 and deuterium content of the cholesterol and coprostanol in the fecal incubations, is shown in Table III. In the first incubation, the iso- topic ratio of the cholesterol added to the feces was 1.13 and that of th, cholesterol isolated after incubation was 1.10. It is immediately eviden from the unchanged isotopic ratio that the cholesterol not converted tc other substances was merely diluted by non-isotopic cholesterol in the feces. The corresponding isotopic ratio for coprostanol, however, leads to a different conclusion in that the D : Cl4 ratio of 1.60 was greater than that for cholesterol. Thus, in coprostanol, the Cl4 was diluted to a greater extent than was deuterium. Therefore, the transformation of cholesterol to coprostanol cannot be only a simple hydrogenation of the 5,6 double bond, since this would result in coprostanol with the same D : Cl4 ratio as in the starting material. In the second incubation, essentially the same results were obtained. The D : Cl4 ratio was greater in coprostanol (3.33) than in the cholesterol (2.69) added to the fecal material.

The presence of deuterium elsewhere than at C-3 in coprostanol was established by oxidation to the corresponding ketone. The coprostanone obtained had an appreciable amount of deuterium in the molecule (Table IV) and the isotope was not lost after equilibration with base. The deu- terium lost upon oxidation to coprostanone is equivalent to the carbon- bound deuterium at C-3. Therefore, in the first incubation, 0.093 gm. atom of deuterium was present at C-3 in coprostanol, while, in the second

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ROSENFELD, FUKUSHIMA, HELLMAN, AND GALLAGHER 309

experiment, the corresponding value was 0.094 gm. atom of deuterium, as shown in Table IV. It is now possible to compare the D : Cl4 ratio in the original cholesterol with the isotopic ratio in coprostanol for which the deuterium content has been corrected for isotope elsewhere than at C-3. When thus corrected, in each experiment the ratio is virtually the same in the coprostanol and in the original cholesterol.

A plausible explanation can be advanced for these results. It is clear that coprostanol contained deuterium elsewhere than at C-3 and the most reasonable site for this additional isotope is at C-5 and C-6. Since the

TABLE III

Specijk Activity and Deuterium Content of Sterol ,from Incubation Experiments

CholesterolL3d,?-C’a added to medium

Sterols recovered

Cholesterol I Coprostanol Incuba- tion

/ D --,-- 1 0.92 2 0.92

D.p.m. per mmole g4

D.p.m. D per mmole

x 10-r x 10-7

0.811 1.13 0.455 0.413 0.342 2.69 i 0.404 0.159

TABLE IV

--

1.10 0.151 0.095 1.60 2.54 0.110 0.033 3.33

D.p.m. per mmole

x 10-r .___

Deuterium to Cl4 Ratios in Cholesterol and Coprostanol

D 0

Coprostanol

Incubation D

Cllolesterol, c4 Coprostanone, D

D* D

G __-~-

1 1.13 0.058 0.093 0.98 2 2.69 0.016 0.094 2.86

* Gm. atoms of deuterium per mole at C-3 only.

only source of deuterium was C-3 in cholesterol, it follows that some of the cholesterol must have been used as a deuterium donor for other cholesterol molecules that were ultimately converted to coprostanol.

Thus some cholesterol must have been converted to substances other than coprostanol with a net loss of Cl4 from the system cholesterol-copros- tanol and a transfer or addition of deuterium to other cholesterol molecules that were reduced to coprostanol must have occurred. Support for this explanation would be provided by the actual location of the deuterium atoms elsewhere in coprostanol than at C-3 and investigations toward this end are now in progress. It is pertinent in this connection to note that Stadtman, Cherkes, and Anfinsen (10) have shown that cholestenone can be further oxidized by certain bacteria to substances such as A4-cholestene-

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310 CHOLESTEROL TO COPROSTANOL

3,6-dione, 5-keto-3,5-seco-4-norcholestan-3-oic acid (Windaus’ keto acid), and other products as yet uncharacterized. Also Wainfan et al. (11) have reported that cholesterol-4-C4 can be partially destroyed by incubation with rat feces and these workers have isolated cholesterol-oxidizing bacteria from the intestinal contents of cholesterol-fed rats.

The fact of incorporation of deuterium that could only have been de- rived from C-3 elsewhere in the coprostanol molecule can be considered strong evidence for the participation of an organized cellular structure in the reaction. This, then, could mean only that the cholesterol added to the fecal suspension must either have been absorbed into bacterial cells or transformed on the surface of the organisms. In all events, it is evident that the enzyme systems responsible for the reduction were not excreted into the medium by the bacterial cells that produced them. These facts probably explain the relatively slow transformation of cholesterol to cop- rostanol.

SUMMARY

1. The transformation of cholesterol to coprostanol has been studied in human subjects and in incubation experiments with the aid of cholesterol- 3d, 4-04.

2. Cholesterol-3d has been prepared from A5-cholesten-3-one by reduc- tion with lithium aluminum deuteride.

3. The separation of coprostanone, coprostanol, and cholesterol by chro- matography of the fecal non-saponifiable fraction on alumina has been described.

4. Coprostanol isolated in both in vivo and incubation studies contains deuterium and Cl4 and the unequivocal conversion of cholesterol to cop- rostanol by an incubation system has been demonstrated.

5. The data suggest that coprostanol is produced from cholesterol prin- cipally by a process not involving the hydroxyl group at C-3, but by direct saturation of the 5,6 double bond of cholesterol. In the incubation studies, appreciable amounts of deuterium were introduced into coprostanol at positions other than at C-2, C-3, and C-4; this isotope is presumably at C-5 or C-6.

The authors gratefully acknowledge the technical assist,ance of Mrs. Evelyn Meyer who isolated and purified the compounds and Miss Josephine Leong and Mr. Milton Heffler who performed the deuterium analyses.

BIBLIOGRAPHY

1. Bondzynski, S., and Humnicki, V., Z. physiol. Chenz., 22, 396 (1896). 2. Schoenheimer, R., Behring, H., Hummel, R., and Schindel, L., Z. physiol. Chwn.,

192, 73 (1930).

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ROSENFELD, FUKUSHIMA, HELLMAN, AND GALLAGHER 311

3. Schoenheimer, R., Rittenberg, D., and Graff, M., J. Biol. Chem., 111, 183 (1935). 4. Rosenheim, O., and Webster, T. A., Biochem. J., 37, 513 (1943). 5. Belleau, B., and Gallagher, T. F., J. Am. Chem. Sot., 73, 4458 (1951). 6. Dauben, W. G., and Eastham, J. F., J. Am. Chem. Sot., 73, 3260 (1951). 7. Fieser, L. F., J. Am. Chem. Sot., 76,542l (1953). 8. Fukushima, D. K., and Gallagher, T. F., J. Biol. Chem., 198, 861 (1952). 9. Rosenfeld, R. S., Hellman, L., Considine, W. J., and Gallagher, T. F., J. Biol.

Chem., 208, 73 (1954). 10. Stadtman, T. C., Cherkes, A., and Anfinsen, C. B., J. Biol. Chem., 206,511 (1954). 11. Wainfan, E., Henkin, G., Rittenberg, S. C., and Marx, W., J. BioZ. Chem., 207,

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Hellman and T. F. GallagherR. S. Rosenfeld, David K. Fukushima, LeonCHOLESTEROL TO COPROSTANOL

THE TRANSFORMATION OF

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