TH*: JOURNAL OP BIOLOGICAL CHEMISTRY Vol. 24R, No. 22, Issue of November 25, pp. G737-6744, 1971

Printed in U.S.A.

Studies of the Metabolism of 5&holesta-8,14-dien-3@ol and

5~-Cholesta-7,14-dien-3~-ol in Rat Liver

Homogenate Preparations*

(Received for publication, March 8, 1971)

BARRY N. LUTSKY,$ J. A. MARTIN, AND G. J. SCHROEPFER, JR.

From the Department of Biochemistry, School of Chemical Sciences, University of Illinois, Urbana, Illinois 61801

SUMMARY

[3a-3H]Cholesta-7, 14-dien-3/I-01 has been prepared by chemical synthesis and incubated with rat liver homogenate preparations. Under aerobic conditions, the incorporation of label into cholesterol, cholest-7-en-3P-01, and cholest-8(14)- en-36-01 was shown. Under anaerobic conditions, labeled cholest-8(14)-en-3/3-ol and cholest-7-en-3P-ol were formed. A method for the separation of the acetate derivatives of cholesta-8,14-dien-3P-01, cholesta-7,14-dien-3fi-01, and 7- dehydrocholesterol has been described. Employing this method, the convertibility of labeled cholesta-8,14-dien-3fl- 01 to cholesta-7,14-dien-30-01 upon incubation with washed rat liver microsomes has been investigated. Significant con- version of the A8V14-sterol to the A 7P14-sterol was not observed.

The results of recent investigations have provided evidence suggest.ing a possible intermediary role for sterols with a AsJ4-

diene system in the biosynthesis of cholesterol (l-6). We have previously reported the incorporation of the label of [301-3H]cho- lesta-8,14-dien-30.011 into cholesterol and cholest-7-ell-3P-ol upon incubation of this substrate with rat liver homogenate preparations (1, 2). Gnder anaerobic conditions, labeled cho- lest-8.en-3fi-01, cholest-8(14)-en-3P-01, and cholest-7-en-3P-ol were formed (2).

In the present study, [3a-3H]cholesta-7, 14-dien-3/3-ol has been prepared by chemical synthesis and incubated with rat liver homogenate preparations. Under aerobic conditions, the incorporat.ion of label into cholest-8(14)-en-3/?-01, cholest-7-en- 3fi-01, and cholesterol was observed. Under anaerobic condi- tions, labeled cholest-8(14)-en-30-01 and cholest-7-ea-3@-ol were formed.

The isomerization of a number of A*-sterols to AT-sterols can

* This research was supported by Grant HE 09501 from the National Heart, Institute, National Institutes of Health.

$ Supported by Training Grant 2G-321 from the National Institutes of Health.

1 The configuration of the hydrogen at carbon atom 5 in the various sterols mentioned in this paper is 01. The designation of the configuration as 501 is omitted throughout the text to conserve space.

readily be shown upon incubation with isolated rat liver micro somes under anaerobic conditions (7-9). As an extension o our previous work on the metabolism of cholesta-8,14-dien-3P-o and aided by the development of a chromatographic method permitting the separation of the A 8~ and A7s14-steryl acetates, we have investigated the convertibility of cholesta-8,14-dien- 3fl-01 to cholesta-7,14-dien-3fi-ol under the conditions described above. We have been unable to demonstrate significant COII- version of the Aas14-sterol to the A 7v14-sterol under these conditions.

EXPERIMENTAL PROCEDURE AP\‘D RESULTS

General Procedure-Procedures used for the measurement of melting points, calorimetric assay of sterols and steryl acetat,es, gas-liquid chromatographic separation of the various sterols and steryl acetates on columns of 3% &F-l on Gas-chrom Q,, gas- liquid radiochromatographic analyses, thin layer radiochroma- tographic assays, measurement of radioactivity, separation of sterols and steryl acetates on columns of silicic acid-Super Cel and neutral alumina-Super Cel-silver nitrate, thin layer chroma- tographic analyses on plates of alumina-silver nitrate and Silica Gel G, purification of cholesterol by way of the dibromide, preparation of the 10,000 x g supernatant fraction of homoge- nates of rat liver, incubation of sterols with homogenate prepara- tions under aerobic and anaerobic conditions, preparation of steryl acetates, elemental analyses, aud the recording of mass spectra and nuclear magnetic resonance spectra have been described previously (2, 8, 10, 11).

The preparation of 3P-acctosy-cholest-7-ene, 3P-acetoxy- cholest%ene, 3/3-acetoxy-cholest-8(14)-ene, 3@-acetoxy-choles- tane, [3a-3H]cholesta-8,14-diell-3fl-ol, cholesta-8,14-dien-30-01, [3&H]cholest&en-3P-01, and 3P-acetoxy-cholesta-8,14-diene have been described previously (2, 8, 11). 30.[V4C]Acetoxg- cholesta-5,7-diene was prepared from 7-dehydrocholesterol by treatment with [I-14C]acetic anhydride and pyridine as described previously . (8). Tritium-labeled sodium borohydride was pur- chased from New England Nuclear.

Preparation of @,7oc- Diacetoxy-cholest-8(14) -ene-3@,7a- Diacetoxy-cholest-8(14)-ene (3.6 g) was prepared from 3fl-ace- toxy-cholest-7-ene (12 g) by treatment with selenous acid nccord- ing to the method of Fieser and Ourisson (12). The product melted at 137.0-138.5” (literature: 138.5-139.5” (12)) and showed a single component on thin layer chromatographic analysis on a Silica Gel G plate (solvent system: benzene-ethyl acetate, 3 : 1)

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and on Silica Gel G-silver nitrate plates (solvent systems: ben- zene and chloroform-acetone, 98 :2). Mass spectral analysis showed a molecular ion of low intensity (-0.1%) at m/e 486 and prominent ions at m/e 426 (M-CH&OOH), m/e 411 (M- CH,-COOH), m/e 366 (M-CH&OOH-CH&OOH), m/e 311 (M-CH,COOH-R, where R equals the alkyl side chain, C8Hi7), and m/e 253 (M-CH3COOH-CH&OOH-R). The nuclear magnetic resonance spectrum (60 mHz) showed single protons at 4.6 7 and 5.5 7 (broad), attributable to the 7p- and Sa-protons, respectively. No absorption due to olefinic protons was ob- served. Two sharp absorption peaks at 8.0 7 to 8.1 7, corre- sponding to the 6 protons of the acetate moieties, were present. The infrared spectrum was consistent with the assigned structure. No specific absorption in the ultraviolet spectrum (340 to 220 nm) was observed.

Preparation of Cholest-8(1&en-S/3, Yoc-dial and Sp, 7a-Di- beruoyloxy-cholest-8(14)- me-3/3,7a!-Diacetoxy-cholest -8(14)-ene (0.9 g) in ether (50 ml) was added to a slurry of lithium aluminum hydride (0.4 g) in ether (10 ml), and the resulting mixture was heated under reflux for 1 hour. The excess reagent was cau- tiously decomposed by the addition of ethyl acetate and water (50 ml). The resulting mixture was extracted three times with ether (50-ml portions), and the combined extracts were washed with water and dried over anhydrous sodium sulfate. Evapora- tion of the solvent yielded a clear glass which was crystallized from methanol to give cholest-8(14)-en-3/3,7a-diol in the form of compact needles melting at 157-158” (literature: 157-158” (12)).

3/3,7ol-Dibenzoyloxy-cholest-8(14)-ene was prepared from the diol in 84% yield by treatment with benzoyl chloride in pyridine. The product crystallized from chloroform-methanol in the form of small white needles which melted at 154-155” (literature: 152.5-153.5” (12)).

Preparation of Sfl-acetoxy-cholesta-7,1&2iene by Pyrolysis of @,7or- Diacetoxy-cholest-8(14)-ene-3P,70(-Diacetoxy-cholest-8- (14)ene (1 to 2 g) was heated at 200” at reduced pressure (0.01 mm of Hg) for 1 hour. Isolation of pure 3&acetoxycholesta- 7,14-diene from the resulting yellow oil was effected by (a) chromatography on silicic acid columns (eluting solvent, ben- zene) followed by recrystallization from acetone-water and methanol-ether or (b) chromatography on Silica Gel G-silver nitrate columns (eluting solvent: n-hexane-benzene, 60:40) followed by recrystallization from acetone-water and methanol- ether. Yields of the pure diene varied from 65 to 70%. In a typical experiment, the oil obtained by pyrolysis (as described above) of 1.4 g of the 3P,7ac-diacetoxy-cholest-8(14)-ene was applied to a silicic acid column (50 x 1.8 cm; 60 g). With the use of benzene as the eluting solvent, fractions 20 ml in volume (5 min per fraction) were collected. The contents of Fractions 8 through 15, a clear oil which solidified on standing, were pooled and crystallized from acetone-water and from methanol-ether, yielding 3/3-acetoxy-cholesta-7,14-diene (0.8 g). The product showed a single component upon thin layer chromatographic analysis on a Silica Gel G plate (solvent, benzene) and on a Silica Gel G-silver nitrate plate (solvent, benzene). A single component was also noted on gas-liquid chromatographic anal- ysis on a 30/, &F-l column. Melting points of three samples of the product were 84-85”, 84.0-85.5”, and 86-87” (literature: 86-87” (13)). The ultraviolet spectrum (ethanol) showed a x max at 242 nm (e 9700). The infrared and nuclear magnetic resonance (100 mHz) spectra were compatible with the assigned

structure. The nuclear magnetic resonance spectrum indicated the presence of 2 olefinic protons (4.25 7 and 4.53 T) and disap- pearance of one of the two sharp absorption peaks in the region 8.0 to 8.1 7, which were seen in the spectrum of the starting material and ascribable to the protons of the acetate moieties.

3P-Acetoxy-cholesta-7,14--diene (m.p. 85-86”; X~~~nol 242 nm (E 9100)) was also prepared in high yield (78%) by treatment of 30,7or- diacetoxy - cholest - 8( 14) - ene with N , N - dimethyl- aniline by a minor modification of the method of Fieser and Ourisson (12).

Preparation of So-Benzoyloxy-cholesta-7, I.&diem by Pyrolysis of ?I/?, 7oc-Dibenzoyloxy-cholest-8(14)-ene-Pyrolysis of the 3p, 7a- dibenzoate also led to the formation of the corresponding A-1J4- steryl benzoate in high yield. 3/3,7a - Dibenzoyloxy - cholest- 8(14)-ene (0.5g) was heated at 200” at reduced pressure (0.01 mm Hg) for 1 hour. The product was dissolved in chloroform (10 ml) and washed with 1 N NaOH (5 ml) and water (10 ml). The organic phase was dried over anhydrous sodium sulfate and, after evaporation of the solvent, the residue was crystallized from chloroform-methanol to yield 3fl-benzoyloxy-cholesta-7,14- diene (0.31 g) in the form of needles melting at 152” (literature: 151-152” (12)). The nuclear magnetic resonance spectrum (60 mHz) showed absorption due to olefinic protons at 4.25 7 (multiplet - 1 H) and 4.49 7 (multiplet - 1 H). S&Benzoyl- oxy-cholesta-7,14-diene (m.p. 152”) was also prepared in 70% yield by a modification of the method of Fieser and Ourisson (12) which employs treatment of the 3p, 7or-dibenzoyloxy-A*(‘*)- sterol with N,N-dimethylaniline. The melting points of the two samples of the A7J4-steryl benzoate were not depressed upon admixture.

Preparation of Cholesta-7,14-dien-Sfl-ol-3P-Acetoxy-cholesta- 7,14-dienc (1.85 g) was heated under reflux with 10% ethanolic KOIT (200 ml) for 1 hour. Water (200 ml) was added and the resulting mixture was extracted three times with petroleum ether (400.ml portions). The pooled extracts were washed with water and dried over anhydrous sodium sulfate. The residue obtained upon evaporation of the solvent was dissolved in 8 ml of chloroform-acetone (98:2), and 2-ml aliquots were applied to each of four alumina-Super Cel-silver nitrate columns (55 x

1.8 cm). With the use of the same mixture as the eluting solvent, fractions 7.8 to 9.3 ml in volume (10 min per fraction) were collected. The contents of Fractions 20 through 100 were pooled and recrystallized from acetone-water and methanol- ether to yield cholesta-7,14-dien-3/Sol (1.4 g) which melt.ed at 104-105”. Thin layer chromatographic analysis on plates of Silica Gel G and Silica Gel G-silver nitrate (solvent systems: benzene-ethyl acetate, 3 : 1, and chloroform-acetone, 98 :2) indicated a single component. A single component was also noted upon gas-liquid chromatographic analysis on a 3% &F-l column. The ultraviolet spectrum showed a maximum at 242 nm (E 9440, ethanol). The mass spectrum showed a molecular ion at m/e 384. The infrared and nuclear magnetic resonance spectra were compatible with the assigned structure, the infrared spectrum showing loss of the ester carbonyl absorption (1730 cm-) of the starting material and the appearance in the product of a broad absorption (O-H stretch) at 3600 cm+ in the product. The nuclear magnetic resonance spectrum (100 mHz) showed absorption due to 2 olefinic protons at 4.24 7 and 4.51 7, a single proton at 6.5 T (broad), corresponding to the 3ol-proton, and disappearance of the acetoxy protons (at 8 T) seen in the starting material.

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Preparation of Cholesta-7,1&lien-S-one-Cholesta-7,14-dien- 3-one was prepared from cholesta-7,14-dien-3/3-ol by treatment with chromium trioxide in pyridine, and the crude ketone was purified as described previously for the preparation of cholesta- 8,14-dien-3-one (2). The purified cholesta-7,14-dien-3-one, after crystallization from methanol, melted at 129.0-129.5” (literature: 129.0-129.4” (12)). A single component was noted on thin layer chromatographic analysis on plates of Silica Gel G (solvent systems: chloroform and benzene-ethyl acetate, 3:l) and Silica Gel G-silver nitrate (solvent systems: chloroform and benzene-ethyl acetate, 3 : 1) and on gas-liquid chromatographic analysis on a 3% &F-l column. The mass spectrum (molecular ion at m/e 382), the infrared spectrum (carbonyl stretch at 1715 cm-l), and nuclear magnet’ic resonance spectrum (2 olefinic protons at 4.22 r and 4.48 r and disappearance of the absorption due to the 3oL-proton of the starting material) were compatible with the assigned structure. The ultraviolet spectrum showed a maximum at 242 nm (E 9870, ethanol) (literature: X,,, 242 nm (c 9700) (12)).

Preparation of [Sol-311]Cholesta-7, I,+dien-So-ol-To cholesta- 7,14-diei1-3-one (100 mg) in ethanol (25 ml) was added sodium borotrit,ide (20 mCi, 3.0 mg). After stirring for 1 hour at room temperature, water (25 ml) was added, and the resulting mixture was extracted four times with ether (loo-ml portions). The combined ether solutions were washed with water and dried over anhydrous sodium sulfate. Thin layer radiochromato- graphic analysis of the reduction products on a Silica Gel G plate indicated a ratio of 3&hydroxysterol to Sa-hydroxysterol of 77 :23. The labeled cholesta-7,14-dien-3P-ol was purified by way of the digitonide (lo), and the regenerated free sterol was acetylated by treatment with acetic anhydride in pyridine. The labeled steryl acetate (1.11 x lOlo cpm) was further purified by chromatography on two columns (50 x 1 cm) of Silica Gel G-silver nitrate. With a mixture of n-hexane and benzene (60:40) as the eluting solvent, fractions 4.8 ml in volume (30 min per fraction) were collected. The contents of Fractions 18 through 37 were pooled and saponified in the usual way with 10% ethanolic KOH. The recovered free sterol (9.7 x lo9 cpm) was further purified by chromatography on two silicic acid- Super Cel columns (50 x 1 cm). With benzene as the eluting solvent, Fractions 3.5 ml in volume (20 min per fraction) were collected. The contents of Fractions 34 through 50 were pooled. The specific activity of the product was 1.36 x lo8 cpm per mg. Two recrystallizations from methanol yielded short needles melting at 105.~106.0” (specific activity, 1.39 x lOa cpm per mg). A4 recrystallization from acetone-water resulted in no change in melting point or significant change in specific activity (1.37 x lo* cpm per mg). The ultraviolet spectrum indicated an absorption maximum at 242 nm (E 9500, ethanol). The radiopurity was judged to be in excess of 98oj, on the basis of (a) gas-liquid radiochromatographic analysis on a 3% &F-l column, (b) thin layer radiochromatographic analysis on Silica Gel G plates (solvent &ems: chloroform-acetone, 98:2, and benzene-ethyl acetate, 3 : l), (c) thin layer radiochromatographic analysis on Silica Gel G-silver nitrate plates (solvent systems: same as indicated above), (d) chromatographic analysis of the acetate derivative on an alumina-Super Cel-silver nitrate column (50 x 1 cm) with the use of a mixture of chloroform and acetone (98:2) as the eluting solvent, and (e) chromatographic analysis of the acetate derivative on a Silica Gel G-Super Cel-silver nitrate column (50 x 1 cm) (see below for description of prepara-

tion of column) with a mixture of n-hexane and benzene (60 :40) as the eluting solvent. Possible contamination of the A7*14- [3H]sterol with cholesterol, cholest&en-3P-01, cholest-8(14)- en-3@-ol, and cholest-7-en-30-01 was less than 0.02%.

Chromatographic Separation of SP-acetoxy-cholesta-8, IQliene, SP-Acetoxy-cholesta-7,l&?iene, and Sfl-Acetoxy-cholesta-5,7-diene -For studies of the intermediary metabolism of cholesta-8,14- diem3fl-01, cholesta-7,14-dien-30-01, and cholesta-5,7-dien-3P- 01, a method was required for the separation of these isomeric conjugated dienes. Gas-liquid chromatography for the acetate derivatives on a 3% &F-l on Gas-chrom Q column under condi- tions described previously (11) permits the separation of the ASS’-isomer from the As+14- and A7s14-isomers. However, 3& acetoxy-cholesta-8,14-diene and 3fl-acetoxy-cholesta-7,14-diene have the same retention time on this column under the conditions studied. Similarly, column chromatography on alumina-Super Cel-silver nitrate columns (2, 8, 10, 11, 14), either in the form of the free sterols or the acetate derivatives, results in a separa- tion of the AZz7-isomer from the A8,14- and A7*14-isomers. How- ever, although a partial resolution of the A8,14- and A7v%somers has been observed on these columns, the separations have fre- quently been poor, and the recoveries of these sterols from the columns has been highly variable and frequently quite low. The chromatographic procedure described below, employing Silica Gel G-Super Cel-silver nitrate columns and representing a modification of a method described by Galli and Grossi Paoletti (15), has yielded more satisfactory separations of the sterols in question. Although the recoveries of these sterols from the column have generally been much higher, some variation in recoveries has been encountered with different preparations of the adsorbent. However, the method has proved very useful in the purification and isolation of the sterols under considera- tion. The present report constitutes, to our knowledge, the first description of a chromatographic separation of a A*~%terol from a A7*%terol.

Silica Gel G (20 g, Merck (U. S.) and HyAo Super Cel (20 g, Johns-Manville), were thoroughly mixed in a l-liter, round bottomed flask. Silver nitrate (8 g) in water’ (120 ml) was added, and the contents of the flask were thoroughly mixed. The mixture was frozen in an acetone-Dry Ice bath and lyo- philized for 24 hours. The resulting buff-colored powder was stored overnight in a vacuum desiccator over Drierite (W. A. Hammond Drierite Company, Xeria, Ohio) prior to use. The solvent (n-hexane-benzene (60:40) or n-hexane-benzene (70 :30)) was added to the dry powder, the resulting slurry was thoroughly mixed and poured into glass columns (100 x 1 cm or 50 x 1 cm), and the columns were packed under a pressure of approxi- mately 5 p.s.i. of nitrogen. The steryl acetates were applied to the columns in a small volume (~1 ml) of the solvent, and the columns were eluted with the same solvent.

Fig. 1 illustrates the chromatogram obtained upon analysis of a mixture ?f cholesteryl acetate (3.8 mg), 3P-acetoxy-[3a-3H]- cholesta-7,14-diene (1.3 pg, 1.8 x lo5 cpm), 3@acetoxycholesta- 8,14-diene (3.3 mg), and 3fi-[l-14C]acetoxy-cholesta- 5,7-diene (60 pg, 9 x lo* cpm) on a column (100 x 1 cm) using a mixture of n-hexane and benzene (60:40) as the solvent. Fract.ions 2.7 ml in volume (30 min per fraction) were collected.

Fig. 2 shows the chromatogram obtained upon analysis of a mixture of 3/?-acetoxy-[3Lu-3H]cholesta-7, 14.diene (48 pg, 6.7 x lo6 cpm) and 3/3-acetoxy-cholesta-8,14-diene (7.8 mg) on a

column (50 x 1 cm) using a mixture of n-hexane and benzene

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FRACTION NUMBER

FIG. 1. Chromatographic separation of acetates of cholesterol, cholesta-7,14-dien-3&ol, cholesta-8,14-dien-36-01, and 7-dehy- drocholesterol on a column (100 X 1 cm) of Silica Gel G-Super Cel- silver nitrate. A-A., steryl acetate determined colorimetri- tally. The Jirst peak is due to cholesteryl acetate and the second peak is due to 3p-acetoxy-cholesta-8,14-diene. The steryl acetates were assayed calorimetrically at 620 nm, 30 and 7 min after the addition of the Liebermann-Burchard reagent, respectively. O-O, 3H radioactivity due to 3p-acetoxy-[3&H]cholesta-7,14- diene. O---O, l4C radioactivity due to 38.[PC]acetoxy- cholestad,7-diene. The solvent was a mixture of hexane and benzene (60:40). Fractions 2.7 ml in volume were collected. The flow rate was 0.09 ml per min.

FRACTION NUMBER

FIG. 2. Chromatographic separation of acetates of cholesta- 7,14-dien-3p-ol and cholesta-8,14-diene-30-01 on a column (50 X 1 cm) of Silica Gel G-Super Cel-silver nitrate. O--O, 3H radioactivity due to 3p-acetoxy-[3&H]cholesta-7,14-diene; A-A, 36.acetoxy-cholesta-8,14-diene measured colorimetri- tally. The solvent was a mixture of hexane and benzene (70:30). Fractions 3.8 ml in volume were collected. The flow rate was 0.19 ml per min.

(70:30) as the solvent. Fractions 3.8 ml in volume (20 min per fraction) were collected. The recoveries of the 3P-acetoxy- [3cu-3H]cholesta-7, 14.diene and the 3P-acetoxy-cholesta-8,14- diene from the column were 66 and 74%, respectively.

Incubations of [Sa-3H]cholesta-7, l&Sien-S&o1 with Rat Liver Homogenate Preparations Under Aerobic Conditions--[3ar-3H]Cho- lesta-7,14-dien-3P-ol (50 pg, 6.86 x lo6 cpm) in propylene glycol (0.1 ml) was incubated for 3 hours at 37” under aerobic conditions with 15 ml of a 10,000 X g supernatant fraction of a rat liver homogenate preparation to which 10 ml of potassium phosphate buffer (0.1 M, pH 7.4) was added. The sterols (95% recovery of the incubated radioactivity) were isolated from the saponified incubation mixture as described previously and acetylated with acetic anhydride and pyridine. The labeled acetates were ap- plied to a Silica Gel G-Super Gel-silver nitrate column (50 x 1 cm) along with unlabeled 3P-acetoxy-cholesta-8,14-diene (5.2 mg). With a mixture of hexane and benzene (70 :30) as the eluting solvent, fractions 3.5 ml in volume (15 min per fraction)

20 40 60

FRACTION NUMBER

FIG. 3. Alumina-Super Cel-silver nitrate column chromato- graphic analysis of acetate derivatives of monounsaturated sterols recovered after incubation of [3c@H]cholesta-7,14-dien-3p-ol with a rat liver homogenate preparation under aerobic conditions. O--O, radioactivity. The radioactivity in Fractions 76 through 96 has been reduced by a factor of 10 to facilitate presentation. A--A, steryl acetate measured calorimetrically. The jfirst peak is due to 3p-acetoxy-cholest-8(14)-ene, the second peak is due to 3P-acetoxy-cholest-7-ene, and the third peak is due to cholesteryl acetate.

were collected. Approximately 567, of the radioactivity was recovered in Fractions 15 through 26, corresponding to the mo- bility of saturated and monounsaturated (A*, A*(14), A7, and A3) C27 steryl acetates in this system. The contents of these frac- tions were pooled and applied to an alumina-Super Gel-silver nitrate column (100 x 1 cm) along with unlabeled 3fl-acetoxy- cholest-8(14)-ene (10.8 mg) and 3fi-acetoxy-cholest-7-ene (3.8 mg). With hexane-benzene (9O:lO) as the eluting solvent, frac- tions 2.5 ml in volume (30 min per fraction) were collected. The resulting chromatogram (Fig. 3) shows that most (86%) of the recovered radioactivity corresponds in mobility to that of cho- lesteryl acetate (center of peak at Fraction 80). Approximately 12.3 and 1.570 of the recovered radioactivity corresponded in mobility to that of authentic 3fi-acetoxy-cholest-7-ene and 3/3- acetoxy-cholest-8(14)-ene, respectively. No indication of the presence of radioactivity with the mobility of Sfi-acetoxy-cholest- 8-ene was noted. Additional incubations were carried out on a large scale to confirm these findings and to permit detailed char- acterization of the labeled products.

[3cr-3H]Cholesta-7,14-dien-3P-ol (200 pg, 2.74 X lo7 cpm) in propylene glycol (0.4 ml) was incubated in duplicate with loo-ml portions of a 10,000 x g supernatant fraction of a rat liver ho- mogenate preparation for 3 hours at 37” under aerobic conditions. A third incubation was carried out as described above with the exception that the enzyme preparation was heated at 100” for 30 min prior to the addition of substrate. The sterols ( >95% recovery of the incubated radioactivity in each case) were iso- lated from the saponified incubation mixtures as described pre- viously.

The sterofs recovered from the first aerobic incubation were treated with acetic anhydride and pyridine as described previ- ously, and the labeled acetates were applied to a Silica Gel G- Super Cel-silver nitrate column (50 x 1 cm) along with unlabeled 3P-acetoxy-cholesta-7,14-diene. With hexane-benzene (60 : 40) as the eluting solvent, fractions 4.4 ml in volume were collected. Aliquots were taken for assay of radioactivity and sterol content. Most (63%) of the radioactivity was recovered in Fractions 6 through 14, corresponding to the mobility of saturated and mono-

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unsaturated (As, As(14), A7, and AS) C27 steryl acetates in this system. The contents of these fractions were pooled and applied to an alumina-Super-Gel-silver nitrate column (100 X 1 cm). With hexane-benzene (90: 10) as the eluting solvent, fractions 2.6 ml in volume were collected. The resulting chromatogram indicated that most (97.2y0) of the recovered radioactivity had the same chromatographic mobility as cholesteryl acetate. Ap- proximately 2.1 and 0.5y0 of the radioactivity was eluted as two peaks with the expected mobilities of 3@acetoxy-cholest-7-ene and 3&acetoxy-cholest-8(14)-ene, respectively. The contents of Fractions 30 through 35, corresponding to the expected mobility of 3/3-acetoxy-cholest-8(14)-ene, were pooled and subjected to gas-liquid radiochromatographic analysis on a &F-l column, along with unlabeled 3fi-acetoxy-cholestane and 3@-acetoxy- cholest-8(14)-ene. The resulting chromatogram showed that virtually all of the radioactivity had the same retention time as authentic 3fl-acetoxy-cholest-8(14)-ene. The contents of Frac- t.ions 53 through 62 from the alumina-Super Cel-silver nitrate column, corresponding to the expected mobility of 3fi-acetoxy- cholest-7-ene, were pooled and subjected to gas-liquid radiochro- matographic analysis on a QF-1 column along with carrier 3p- acetoxy-cholest-7-ene and 3fi-acetoxy-cholest-8-ene. Virtually all of the radioactivity had the same chromatographic mobility as a.uthentic 3P-acetoxy-cholest-7-ene.

The sterols recovered from the second aerobic incubation were applied t,o an alumina-Super Cel-silver nitrate column (50 X 1 cm) along with unlabeled cholesta-7,14-dien-3P-01. With chlo- roform-acetone (98:2) as the eluting solvent, fractions 1.7 ml in volume were collected and aliquots were taken for assay of radio- activity and sterol content. The resulting chromatogram showed that most of the radioactivity (84%) had the expected mobility of cholesteryl acetate (center of peak at Fraction 63). Identification of this labeled material as cholesteryl acetate was made by purification by way of the dibromide after the addition of unlabeled cholesteryl acetate. The specific activity before and after this purification was 1.075 x lo5 cpm per mg and 1.070 x 10; cpm per mg, respectively. Approximately 4% of the

radioactivity recovered from t.he alumina-Super Cel-silver nitrate column was eluted in Fractions 21 through 40, corresponding to the expected location of CZ7 monounsaturated sterols, such as cholest-8(14)-en-3P-01, cholest-8-en-3@-01, and cholest-7-en-30-01. The contents of these fractions were pooled, treated with acetic anhydride and pyridine, and the resulting labeled acetates were applied to an alumina-Super Ccl-silver nitrate column (100 x 1 cm) along wit,h unlabeled 3fl-acetoxy-cholest-8(14)-ene, 3/3- acetoxy-cholest-7-ene, and cholesteryl acetate. With hexane- benzene (90 : 10) as the eluting solvent, fractions 3.0 ml in volume were collected and aliquots were taken for assay of radioactivity and stcrol content. Most (73%) of the radioactivity was asso- ciated with 36.acetoxycholest-7-ene. Approximately 17 % of the radioactivity was associated with 3P-acetoxy-cholest-8(14)- ene. The latter material was diluted with unlabeled, authentic 3~.acetoxy-cholest-8(14)-ene and subjected to repeated crystal- lization. No significant change in the specific activity of the crystals was observed after two crystallizations from acetone- water and two crystallizations from methanol (Table I).

The sterols recovered from the incubation of the [3&H] cholesta-7,14-dien-3P-ol with the boiled enzyme preparation were treated with acetic anhydride and pyridine, and the result- ing acetates were applied to a Silica Gel G-Super Cel-silver nitrate column (50 x 1 cm) along with unlabeled 3P-acetosy-cholesta-

TABLE I Cocrystallization of acetate derivative of isolated [3a-3H]cholest-

8(14)-en-S&o1 (derived from [3cr-3H]cholesta-7,14-dien-3p-ol) with authentic 3@-acetoxy-choles-8(14)-ene

I. Sample from aerobic incubation

Initial.. After one recrystallization from ace-

tone-water. After two recrystallizations from ace-

tone-water. After one recrystallization from meth-

anol............................. After two recrystallizations from

methanol...........................

II. Sample from anaerobic incubation

Initial.......... ., After one recrystallization from ace-

tone-water. After two recrystallizations from ace-

tone-water. After one recrystallizat,ion from meth-

anol................................ After two recrystallixations from

methanol.

pecific activit) of crystals

CPMW

2190

21GO

2150

2110

2160

pecific activit> of crystal

cPm/m&T

3400

3450

3290

3310

3350

- is

_-

,s

-

pecific activity of mother

liquor

CPdW

3380

2230

2180

2200

pecific activity of mother

liquor

3360

3330

3380

3360

7,14-diene. With hexane-benzene (90 : 10) as the eluting solvent, fractions 4.4 ml in volume were collected. The resulting chro- matogram indicated that all of the recovered radioactivity had the same mobility as the incubated substrate.

Incubations of [Sa-31J]Cholesta-7, 14.&en-S&o1 with Rat Liver Homogenate Preparation Under Anaerobic Conditions-[3&H] Cholesta-7,14-dien-3fl-ol (200 pg, 2.74 x lo7 cpm) in propylene glycol (0.4 ml) was incubated with 100 ml of a 10,000 x g super- natant fraction of a rat liver homogenate for 3 hours at 37” in a helium-filled desiccator over alkaline pyrogallol as described previously (8). The sterols (98% recovery of incubated radio- activity) were isolated from the saponified incubation mixture as described previously. The labeled sterols were acetylated with acetic anhydride and pyridine (99% recovery) as described pre- viously, and the labeled acetates were applied to a Silica Gel G- Super Cel-silver nitrate column (100 x 1 cm) along with unla- beled 3@-acetoxy-cholesta-8,14-diene (5.1 rug). With a mixture of hexane and benzene (60:40) as the eluting solvent, fractions 3.0 ml in volume (30 min per fraction) were collected. Choles- teryl acetate and monoene sterol precursors of cholesterol were eluted in Fractions 21 through 35. The contents of these frac- tions (2.2 x 10’ cpm) were pooled, and one-half of this material was applied to an alumina-Super Cel-silver nitrate column (100 x 1 cm) along with unlabeled 30.acetoxy-cholcst-8(14)-ene and 30.acetoxy-cholest-7-ene. With a mixture of hexane-benzene (90 : 10) as the eluting solvent, fractions 2.2 ml in volume were collected and aliquots were taken for assay of sterol content and radioactivity. The resulting chromatogram is shown in Fig. 4. Little radioactivity ( < 0.3 To) was associated chromatographically with cholesteryl acetate. Nest of the radioactivity (-98.3%)

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6742 Sterol Synthesis Vol. 246, No. 22

FRACTION NUMBER

FIG. 4. Alumina-Super Cel-silver nitrate column chromato- graphic analysis of acetate derivatives of monounsaturated sterols recovered after incubation of [30r-3H]cholesta-7, 14-dien-30-01 under anaerobic conditions. O----O, radioactivity; A-A, steryl acetate, measured calorimetrically. The Jirst peak is due to 30-acetoxy-cholest-8(14)-ene, the second peak is due to 3fi- acetoxy-cholest+ene, and the third peak is due to cholesteryl acetate.

Lr 0 25 35 45

TIME (MINUTES 1

FIG. 5. Gas-liquid radiochromatographic analysis of labeled acetate (with chromatographic mobility of 3&acetoxycholest- 8(14)-ene.on an alumina-Super Cel-silver nitrate column) derived from incubation of 13&Hlcholesta-7.14-dien-3&ol with rat liver homogenate preparation under anaerobic conditions. The $rst muss peak is due to added authentic 3p-acetoxy-cholest-8(14)-ene, and the secorrd peak is due to added authentic cholestanyl acetate. The analysis was made on an 8 foot 3% &F-l on Gas-chrom Q column at a column temperature of 220” and a flow rate of 60 ml per min as described previously (11).

showed the chromatographic mobility of 30.acetoxy-cholest-7- ene. Approximately 1.4y0 of the recovered radioactivity had the same chromatographic mobility as 3P-acetoxy-cholest-8(14)- ene (Fractions 31 through 35). The contents of these fractions were pooled, diluted with authentic 3/%acetoxy-cholest-8(14)-ene, and subjected to repeated crystallization. The specific activities of the crystals and mother liquors were essentially the same through two recrystallizations from acetone-water and two re- crystallizations from methanol (Table I).

The remainder of the contents of Fractions 21 through 35 from the Silica Gel G-Super Cel-silver nitrate column (1.1 x lo7 cpm) was applied to an alumina-Super Cel-silver nitrate column as described above, but without the addition of unlabeled carriers. The elution profile was identical with that shown in Fig. 4. The contents of Fractions 33 through 36, corresponding to the mo- bility of 3P-acetoxy-cholest-8(14)-ene, were pooled and subjected to gas-liquid radiochromatographic analysis on a 3’$& &F-l col- umn, along with authentic unlabeled 3/%acetoxycholest,-8(14)- ene and 3p-acetoxy-cholestane. Virtually all of the eluted radio-

TIME (: MINUTES )

FIG. 6. Gas-liquid radiochromatographic analysis of labeled acetate (with chromatographic mobility of 3&acetoxy-cholest-7- ene on an alumina-Super Cel-silver nitrate column) derived from incubation of [3a-3H]cholesta-7,14-dien-3.B-o1 under anaerobic conditions. The first mass peak is due to added authentic 38- acetoxy-cholest-8-ene, and the second peak is due to added authen- tic 3@-acetoxy-cholest-7-ene. The column and the operating conditions employed were the same as outlined in the legend to Fig. 5.

activity showed the same retention time as Sfl-acetoxy-cholest- 8(14)-ene (Fig. 5). The contents of Fractions 59 through 64, corresponding to the expected mobility of 3&acetoxy-cholest-7- ene, were pooled and subjected to gas-liquid radiochromato- graphic analysis on 30/, &F-l column along with unlabeled 3/% acetoxy-cholest-8-ene and 3/%acetoxy-cholest-7-ene. Essentially all of the radioactivity showed the same mobility as authentic 3/%acetoxy-cholest-7-ene (Fig. 6). The contents of Fractions 55 through 58 (on the proximal side of the 3P-acetoxy-cholest-7-ene peak and corresponding to the expected mobility of 3@-acetoxy- cholest-8-ene) were pooled and similarly analyzed by gas-liquid radiochromatography. Virtually all of the radioactivity showed the same mobility as authentic 3fi-acetoxy-cholest-7-enc. Little or no ( <0 to 0.1%) radioactivity was associated with S&ace- toxycholest-8-ene.

Incubation of [Soc-3H]ChoZesta-8, i&lien-J/Sol with blvashed Microsomal Enzyme System Under Anaerobic Conditions-Mi- crosomal preparations from rat liver catalyze the conversion of A*-sterols to A7-sterols (7-9). The reaction proceeds under an- aerobic conditions and requires no cofactors (7-9). Catalysis of the reduction of the Ar4-double bond of A7 *r4- and A* v %terols by rat liver microsomes has been reported to be dependent on the presence of reduced nicotinamide adenine dinucleotide phos- phate (3, 16). Incubation of labeled cholesta-8,14-dien-3P-ol with washed microsomes under anaerobic conditions should per- mit detection of possible catalysis by rat liver microsomes of the conversion of a A* *r4-sterol to a A7 sr4-sterol.

The 10,000 x g supernatant fraction of rat liver (90 g) was isolated as described previously (lo), except that the homogeniza- tion buffer (0.1 M potassium phosphate, pH 7.4) contained MgCle (5 x lop3 M). and MnClz (5 X low4 M). A portion (120 ml) of the 10,000 x g supernatant w-as recentrifuged for 60 min at 105,000 x g. The resulting pellet was suspended in the buffer (120 ml), and the resulting suspension was recentrifuged at 105,000 x g for 60 min. The washed microsomes were sus- pended in the buffer (120 ml). An aliquot of this suspension (12.5 ml) was diluted to 16 ml with the buffer and incubated for 3 hours at 37” with [30(-3H]cholesta-8, 14-dien-30-01 (128 pg, 1.62 x lo7 cpm) in propylene glycol (0.2 ml) under anaerobic condi- tions. The sterols were recovered from the saponified incubation mixture and treated with acetic anhydride and pyridine as de-

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Issue of November 25, 1971 R. N. Lutslcy, J. A. Martin, and G. J. Schroepjer, Jr. 6743

wril----- made in studies of the metabolism of [3ar-3H]cholesta-8, 14.dien- 36-01 in which significant amounts of the labeled cholest-8-en-3/3- 01 were detected in addition to the A7- and A8(r4)-sterols (2). It

15 is also noteworthy that although the amounts of radioactivity

i

associated with cholest-8(14)-en3P-ol after incubation of the labeled A7J4-sterol w-ere small, much more radioactivity was consistently recovered in the A 8(%sterol with this substrate than in the case where [3a-3H]cholesta-8, 14-dien-30-01 was used as the substrate.

We have also investigated the convertibility of cholesta-8,14- diem3&ol to cholesta-7,14-diem3P-01. Microsomal prepara- tions from rat liver catalyze the efficient conversion of a number

I I! of A%terols to the corresponding A7-sterols (7-9). The reaction

! I t ,s.-. proceeds under anaerobic conditions and occurs in the absence

20 40 60 80 120 of added cofactors (7-9). Enzymatic reduction of the Ala-double FRACTION NUMBER bond of A8J4- and ArJQterols has been reported to be dependent

FIG. 7. Silica Gel G-Super Cel-silver nitrate column chro- upon the presence of reduced nicotinamide adenine dinucleotide

matographic analysis of acetate derivatives of sterols recovered phosphate (3, 16). Preparations of washed microsomes of rat after incubation of [3cQH]cholesta-8,14-dien-3p-ol with washed liver were, therefore, incubated with [3a-3H]cholesta-8, 14-dien- rat liver microsome preparation under anaerobic conditions. O---O, radioactivity; A--A, 3P-acetoxy-cholesta-7,14-diene,

30-01 under anaerobic conditions in the absence of added reduced

measured calorimetrically. nicotinamide dinucleotide phosphate in an attempt to demon- strate the formation of labeled cholesta-7,14-dien-3P-01. By

scribed previously. A portion of the labeled acetylated sterols means of a method (described herein) which permits the chro-

(4.2 x lo6 cpm) was applied to a Silica Gel G-Super Cel-silver matographic separation of 3/3-acetoxy-cholesta-8,14-diene and

nitrated column (50 x 1 cm) along with unlabeled cholesteryl 3fl-acetoxy-cholesta-7,14-diene, no significant conversion of the A*JJ-sterol to the A7J4-sterol could be demonstrated.

acetate and 3fl- acetoxy-cholesta-7,14-diene. With hexane- benzene (70 :30) as the eluting solvent, fractions 5.6 ml in volume

The efficient formation of cholesterol from cholesta-7, lCdien-

were collected. Aliquots were taken for assay of radioactivity 3fi-01 in rat liver homogenate preparations suggests the considera-

and sterol content. The resulting chromatogram (Fig. 7) indi- tion of a possible intermediary role of ArJ4-sterols in the bio-

cates a clear separation of the bulk of the radioactivity (center synthesis of cholesterol. In such considerations, it is important

of peak at Fraction 90) from the authentic 3fi-acetoxy-cholesta- to note that the isolation of a A7J4-sterol from the tissues of

7,14-diene (center of peak at Fraction 67). Little or no radioac- higher animals or the formation of such a sterol from a precursor

tivity is associated with the A7J4-sterol. A very small amount with some status as an intermediate in cholesterol biosynthesis

of radioactivity was eluted in Fractions 11 through 13, corre- (such as mevalonic acid or squalene) has not been reported.

sponding to the mobility of C& saturated and monounsaturated However, it is important to note that little or no attention has

(As, Ase4), A7, and A”) steryl acetates in this system and suggests been directed towards this matter and that the present report

that the washed microsomes were not completely free of reduced describes for the first time a method which permits the chroma-

nicotinamide adenine dinucleotide phosphate. A small but sig- tographic separation of a A8J4-sterol from a A7J4-sterol. The

nificant amount of radioactivity was eluted in Fractions 26 availability of this method will permit further experiments di-

through 30. The nature of this material is not known. rected toward the isolation of a A7J4-sterol from tissues and

To confirm these findings the entire experiment was repeated studies of the possible mode of origin of such a sterol. Applica-

under conditions similar to those described above (except that tion of this chromatographic method has permitted the demon-

the homogenization buffer contained no MnC& or MgClJ. The stration that cholesta-8,14-dien-3P-ol undergoes little, if any,

results were essentially the same as in the first experiment. enzymatic conversion to cholesta-7,14-dien-3fi-ol under condi-

Moreover, in this case, the washed microsomal preparation was tions which allow the facile conversion of a number of AQterols

shown to be active (65% conversion) in the catalysis of the to the corresponding A7-sterols. It, therefore, appears unlikely

incorporation of the label of [3a-3H]cholest-8-cn-3/3-ol into cho- that A7J4-sterols are formed by a direct isomerization of the

lest-7-en-3/3-01. A8-bond of the corresponding A8J4-sterols. As noted below, it is possible that a A7J4-sterol could arise directly from a decar-

DISCUSSION boxylation of a A’-32steroidal acid.

The results described herein demonstrate the efficient incor- The removal of the three “extra” methyl groups of lanosterol

poration of the label of [3ac-3H]cholesta-7, 14-dien-3P-ol into (4,4,14cr-trimethyl-cholesta-8,24-dien-3P-01) has been the sub-

cholesterol in rat liver homogenate preparations incubated under ject of a number of investigations. The early studies of Olson,

aerobic conditions. Under these conditions, the incorporation Lindberg, and Bloch (1) indicated the formation of 3 moles of

of the label into cholest-8(14)-en-3/3-ol and cholest-7-en-3P-ol was carbon dioxide per mole of cholesterol formed from lanosterol, a

also observed. Incubation of [30L-3H]cholesta-7, 14-dien-3P-ol finding indicating that the ultimate fate of each of the extra

with rat liver homogenate preparations under anaerobic condi- methyl groups of lanosterol is carbon dioxide. The removal of

tions yielded labeled cholest-8(14)-en-3P-ol and cholest-7-en-3P- these methyl groups has been considered to proceed by way of 01. The formation of labeled cholest-8en3@ol from this sub- an initial oxygen-dependent hydroxylation followed by dehydro- stratc could not be detected. This is in contrast to the findings genations (17, 18), or oxidations or both (19) to yield the corre-

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6744 Xterol Synthesis Vol. 246, No. 22

sponding aldehydes and carboxylic acids. In the case of the removal of the methyl group at carbon atom 14 of a cholesterol precursor with a A*-nuclear double bond, hydroxylation and subsequent conversion to the carboxylic acid would yield a Aa-32- steroidal acid, a /3, y-unsaturated acid. Among many possible schemes for the decarboxylation of a A*-32steroidal acid, two general schemes have been given serious consideration. The first of these involves the formation of a A804)-sterol as the initial product of the decarboxylation. Sterols with a A8(i4)-double bond have been shown to occur in nature (11, 20-22), and the convertibility of As(%terols to cholesterol in intact rats and in rat liver homogenate preparations has been demonstrated (11, 23-26). Detailed studies of the metabolism of cholest-8(14)-en- 3p-01 have been initiated. The enzymatic formation of cholesta- 8,14-dien-3fl-ol (26) and cholest-7-en-3fi-ol from this sterol has been demonstrated and shown to be dependent on the presence of molecular oxygen (11, 26). Since molecular oxygen is not required for the demonstration of the enzymatic conversion of cholesta-8,14-dien-3P-ol to cholest-8en-30-01 and cholest-7-en- 3fi-01 (a), the oxygen-dependent step in the over-all conversion of cholest-8(14)-en-3@-ol to cholest-7-en-3fi-ol appears to be localized to the reaction(s) involved in the formation of the cholesta-8,14-dien-30-01. The possible intermediary role of a A@,r*)-15-hydroxysterol in this process has been suggested by the efficient enzymatic conversion of cholest-8(14)-en-3/3,15ol-diol and cholest-8(14)-en-3/?, 15P-diol to cholesta-8,14-dien-30-01 (26, 27). This general scheme, therefore, involves, as key points, the initial formation of a A8(%terol with subsequent formation of a A*~%terol.

A second suggested sequence involves the formation of a Assir- sterol as the initial product of the enzymatic decarboxylation of a As-32.steroidal acid. At the present time, insufficient informa- tion is available to assess the importance of these two general schemes.

A point of some importance relative to the matter of the possi- ble origin of A7s%terols arises when the same considerat,ions are applied to the case of the removal of the methyl group of A7- I4oc-methyl precursors of cholesterol. The presence of 4,4,14a- trimethyl-cholesta-7,24-dien-3&ol in tissues has been reported (28-29). Moreover, no detectable enzymatic isomerization of the As-double bond of 14c+methyl-AQterols to the corresponding A’-sterols has been reported (30). The enzymatic convertibility of 14ol-methyl-AT-sterols to cholesterol is well documented (28, 31, 32). In the removal of the methyl group of a 14ar-methyl- A?-sterol, the reactions noted above would lead to the formation of a AT-32.steroidal acid, also a 0, y-unsaturated acid. By the first of the two general schemes noted above, enzymatic decar- boxylation would lead to formation of a A8(i4)-sterol. It is im- portant to note that the enzymatic formation of 4,4-dimethyl- cholest-8(14)-en-30-01 from 4,4-dimethyl-14ol-hydroxymethyl- cholest-7-en-30-01 and 4,4-dimethyl-14a-formyl-cholest-7-en-3P- 01 has been reported (24, 33). By the second of the two general schemes, enzymatic decarboxylation of a A7-32.steroidal acid would lead directly to the formation of a A7s%terol. Further studies are in progress t,o test this possibility.

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Barry N. Lutsky, J. A. Martin and G. J. Schroepfer, Jr.-ol in Rat Liver Homogenate Preparationsβ-Cholesta-7,14-dien-3

α-ol and 5β-Cholesta-8,14-dien-3αStudies of the Metabolism of 5

1971, 246:6737-6744.J. Biol. Chem. 

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