Studies of the metabolism of 5 -cholesta-8,14-dien-3 -ol and 5 -cholesta-7,14-dien-3 -ol in rat liver homogenate preparations.

Abstract [3α-3H]Cholesta-7,14-dien-3β-ol 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-3β-ol, and cholest-8(14)-en-3β-ol was shown. Under anaerobic conditions, labeled cholest-8(14)-en-3β-ol and cholest-7-en-3β-ol were formed. A method for the separation of the acetate derivatives of cholesta-8,14-dien-3β-ol, cholesta-7,14-dien-3β-ol, and 7-dehydrocholesterol has been described. Employing this method, the convertibility of labeled cholesta-8, 14-dien-3β-ol to cholesta-7,14-dien-3β-ol upon incubation with washed rat liver microsomes has been investigated. Significant conversion of the Δ8,14-sterol to the Δ7,14-sterol was not observed.

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.
Significant conversion 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 AsJ4diene system in the biosynthesis of cholesterol (l-6).
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 COIIversion 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,, gasliquid radiochromatographic analyses, thin layer radiochromatographic 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 chromatographic 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 homogenates of rat liver, incubation of sterols with homogenate preparations 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).
No absorption due to olefinic protons was observed. Two sharp absorption peaks at 8.0 7 to 8.1 7, corresponding 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.
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 analysis on a 30/, &F-l column.
The nuclear magnetic resonance spectrum indicated the presence of 2 olefinic protons (4.25 7 and 4.53 T) and disappearance 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.
The melting points of the two samples of the A7J4-steryl benzoate were not depressed upon admixture.
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.
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. 25,1971 B. N. Lutslcy, J. A. Martin, arm! G. J. Xchroepfer, Jr.
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 combined ether solutions were washed with water and dried over anhydrous sodium sulfate. Thin layer radiochromatographic 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.
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 separation of the AZz7-isomer from the A8,14-and A7*14-isomers. However, although a partial resolution of the A8,14-and A7v%somers has been observed on these columns, the separations have frequently 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 consideration. 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 lyophilized 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 approximately 5 p.s.i. of nitrogen.
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 Fractions 3.8 ml in volume were collected.
The flow rate was 0.19 ml per min.
Fractions 3.8 ml in volume (20 min per fraction) were collected.
were collected.
Approximately 567, of the radioactivity was recovered in Fractions 15 through 26, corresponding to the mobility of saturated and monounsaturated (A*, A*(14), A7, and A3) 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) along with unlabeled 3fl-acetoxycholest-8(14)-ene (10.8 mg) and 3fi-acetoxy-cholest-7-ene (3.8 mg). With hexane-benzene (9O:lO) as the eluting solvent, fractions 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 cholesteryl 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 3/3acetoxy-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 characterization 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 homogenate 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 isolated from the saponified incubation mixtures as described previously.
The sterofs recovered from the first aerobic incubation were treated with acetic anhydride and pyridine as described previously, 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-Issue of November 25, 1971 B. N. L&sky, J. A. Martin, and G. J. Xchroepfer, Jr. 6741 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 showed that virtually all of the radioactivity had the same retention time as authentic 3fl-acetoxy-cholest-8(14)-ene.
With chloroform-acetone (98:2) as the eluting solvent, fractions 1.7 ml in volume were collected and aliquots were taken for assay of radioactivity 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, 3/3acetoxy-cholest-7-ene, and cholesteryl acetate. With hexanebenzene (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.
No significant change in the specific activity of the crystals was observed after two crystallizations from acetonewater 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 resulting acetates were applied to a Silica Gel G-Super Cel-silver nitrate column (50 x 1 cm) along with unlabeled 3P-acetosy-cholesta- Initial.. After one recrystallization from acetone-water. After two recrystallizations from acetone-water. 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 supernatant 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 radioactivity) were isolated from the saponified incubation mixture as described previously.
The labeled sterols were acetylated with acetic anhydride and pyridine (99% recovery) as described previously, and the labeled acetates were applied to a Silica Gel G-Super Cel-silver nitrate column (100 x 1 cm) along with unlabeled 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.
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 A-A, steryl acetate, measured calorimetrically.
The Jirst peak is due to 30-acetoxy-cholest-8(14)-ene, the second peak is due to 3fiacetoxy-cholest+ene, and the third peak is due to cholesteryl acetate. Cel-silver nitrate column) derived from incubation of 13&Hlcholesta-7.14-dien-3&ol with rat liver homogenate preparation under anaerobic conditions. The $rst muss peak is due to added authentic 3p-acetoxy-cholest-8(14)-ene, and the secorrd peak is due to added authentic cholestanyl acetate. The analysis was made on an 8 foot 3% &F-l on Gas-chrom Q column at a column temperature of 220" and a flow rate of 60 ml per min as described previously (11).
The specific activities of the crystals and mother liquors were essentially the same through two recrystallizations from acetone-water and two recrystallizations from methanol (Table I). The remainder of the contents of Fractions 21 through 35 from the Silica Gel G-Super Cel-silver nitrate column (1.1 x lo7 cpm) was applied to an alumina-Super Cel-silver nitrate column as described above, but without the addition of unlabeled carriers. The elution profile was identical with that shown in Fig. 4. The contents of Fractions 33 through 36, corresponding to the mobility of 3P-acetoxy-cholest-8(14)-ene, were pooled and subjected to gas-liquid radiochromatographic analysis on a 3'$& &F-l column, along with authentic unlabeled 3/%acetoxycholest,-8(14)ene and 3p-acetoxy-cholestane.
The first mass peak is due to added authentic 38acetoxy-cholest-8-ene, and the second peak is due to added authentic 3@-acetoxy-cholest-7-ene.
The column and the operating conditions employed were the same as outlined in the legend to to the expected mobility of 3&acetoxy-cholest-7ene, were pooled and subjected to gas-liquid radiochromatographic 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@-acetoxycholest-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.

Microsomal
Enzyme System Under Anaerobic Conditions-Microsomal preparations from rat liver catalyze the conversion of A*-sterols to A7-sterols (7-9).
The reaction proceeds under anaerobic 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 phosphate (3,16). Incubation of labeled cholesta-8,14-dien-3P-ol with washed microsomes under anaerobic conditions should permit 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 homogenization 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 suspended in the buffer (120 ml). An aliquot of this suspension (12.5 ml) was diluted to 16 ml with the buffer and incubated for nicotinamide dinucleotide phosphate in an attempt to demonstrate 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 hexanebenzene (70 :30) as the eluting solvent, fractions 5.6 ml in volume The efficient formation of cholesterol from cholesta-7, lCdienwere collected. Aliquots were taken for assay of radioactivity 3fi-01 in rat liver homogenate preparations suggests the consideraand sterol content. The resulting chromatogram (Fig. 7) indi-tion of a possible intermediary role of ArJ4-sterols in the biocates 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 chromanicotinamide 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 dithrough 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. Applicaunder conditions similar to those described above (except that tion of this chromatographic method has permitted the demonthe 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 subcholesterol 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)  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 dehydrostratc could not be detected. This is in contrast to the findings genations (17, 18), or oxidations or both (19) to yield the corre-