BIOLOGICAL SYNTHESIS OF LANOSTEROL AND AGNOSTEROL*

BY R. B. CLAYTON AND KONRAD BLOCH

(From the Converse Memorial Laboratory, Harvard University, Cambridge, Massachusetts)

(Received for publication, May 23, 1955)

There is considerable experimental evidence in support of the view that, in the biogenesis of cholesterol, squalene is an intermediate (1, 2). It has been suggested (3) that the cyclization of the aliphatic triterpenoid hy- drocarbon to the tetracyclic ring system of the steroids occurs in a manner which would also rationalize the structure of lanosterol, a naturally occur- ring 4,4’, 14-trimethylcholestane derivative (4). In view of its structural relationship to squalene on the one hand and to cholesterol on the other, it has become of interest to investigate the possible role of lanosterol, or of related structures having 30 carbon atoms, as intermediates in the bio- synthesis of cholesterol. That the synthesis of lanosterol from acetate takes place in mammalian tissues has now been demonstrated. Moreover, as will be shown in the accompanying paper (5), lanosterol serves as a precursor of cholesterol.

At the start of this work, since pure lanosterol was not available to us, tentative information on the biogenesis of C& sterols was obtained by the use of natural “isocholesterol” as carrier. The “isocholesterol” of wool fat, the most abundant source of lanosterol, is known (6) to consist of the four components (Fig. l), lanosterol, dihydrolanosterol, agnosterol, and di- hydroagnosterol, in variable proportions, with lanosterol as the major con- stituent. From this mixture pure lanosterol can be isolated only by very tedious procedures (6,7).

For the demonstration of lanosterol biosynthesis, rat liver homogenates prepared according to the method of Bucher (8) were incubated with sodium acetat,e-l-Cl4 and the unsaponifiable fractions combined with small amounts of “isocholesterol” as carrier. Careful chromatography of the resulting mixture demonst,rated that a small but significant percentage of the total Cl4 in the unsaponifiable material remained associated with the “isocholesterol” fraction. When rat liver homogenates were incubated un- der similar conditions but with prior addition of suspensions of “isocho- lesterol,” the incorporation of acetate carbon into this fraction was mark-

* Supported by grants-in-aid from the Life Insurance Medical Research Fund, and from the Milton Fund of Harvard University. Presented in part at the meeting of the American Society of Biological Chemists, at San Francisco, April 12-15, 1955.

305

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306 SYNTHESIS OF LANOSTEROL AND AGNOSTEROL

edly enhanced. This procedure was therefore adopted for the preparation of labeled material in quantities sufficient for further chemical and biologi- cal experiments.

Subsequently, lanosterol (A sJ4-lanostadienol) was isolated from a yeast concentrate and the Cr4-“isocholesterol” diluted with this pure sterol. By suitable chemical reactions it was shown that lanosterol was in fact the major radioactive component of the biosynthetic product. Evidence for the biosynthesis of agnosterol in the same liver system was also obtained. Hitherto the presence of lanosterol in liver tissue has not been observed. The experiments reported here clearly indicate that at least two CsO sterols, lanosterol and agnosterol, are natural constituents of liver, though in very small amounts.

Ho-&!+ ,&&P III IV

FIG. 1. Constituents of wool fat sterol mixture (isocholesterol). I, lanosterol; II, dihydrolanosterol; III, sgnosterol; IV, dihydroagnosterol.

EXPERIMENTAL

Materials

“Isocholesterol” was supplied generously by Eli Lilly and Company. Lanosterol (m.p. 140-141’) was isolated by chromatography from a con- centrate of the unsaponifiable fraction of yeast from which most of the ergosterol had been removed previously by crystallization (9). The yeast concentrate was kindly provided by Professor L. Ruzicka of Zurich. Gen- erous samples of agnosterol and dihydroagnosterol were made available to us by Professor D. H. R. Barton, Birkbeck College, London. The di- phosphopyridine nucleotide (DPN) was a product of the Pabst Brewing Company, 95 per cent purity. The alumina was Merck, chromatographic grade of activity II. All solvents used in the chromatographic procedures were anhydrous.

Rat Liver Homogenates-Female rats weighing approximately 100 gm. were killed by a blow on the head and the livers homogenized according to the procedure of Bucher (8) in a 0.08 M phosphate buffer (pH 7.4) con-

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R. B. CLAYTON AND K. BLOCH 307

taining nicotinamide (0.03 M) and magnesium chloride (0.0048 M). The supernatant fluid obtained after homogenization and centrifugation at low speed (700 X g) was divided into 2 ml. aliquots. In control experiments 0.2 ml. of 0.01 M DPN and 0.2 ml. of 0.1 M sodium acetate-l-C4 were added to each aliquot. For experiments involving additions of carrier sterols to the homogenates prior to incubation, suspensions of the steroids were pre- pared in the following manner. The sterol (10 mg.) was ground to a thin paste with the minimum of Bucher phosphate buffer containing 0.5 per cent bovine serum albumin. The paste was then diluted to 2.0 ml. with the same 0.5 per cent serum albumin buffer solution and transferred to a Raytheon magnetostriction oscillator in which it was subjected to an oscil- lation frequency of 9 kc. for 30 to 90 minutes, depending upon the time required to reduce the bulk of the solid to a satisfactory emulsion. Sus- pensions prepared in this way were generally found to remain stable at 0” for 24 hours and could be stored in the frozen state indefinitely. The con- centration of sterols could be assumed to be approximately 5 mg. per ml. 0.1 ml. of this suspension was added to each 2 ml. of homogenate. When the homogenates were incubated with 3 times the amount of sterol sus- pension, essentially the same results were obtained.

Incubations were carried out in a Dubnoff shaker in an atmosphere of oxygen at 37” for 5 hours. After incubation the homogenates were sapon- ified with 30 per cent potassium hydroxide at 25” for 12 hours and the unsaponifiable fractions extracted with petroleum ether. Total Cl4 in- corporation was measured by plating aliquots as infinitely thin samples and counting in a gas flow counter.

Incorporation of Acetate into Lanosterol in Control Experiments--The following is one of several preliminary experiments which showed that the unsaponifiable fraction contained in small percentage a labeled substance which was chromatographically inseparable from “isocholesterol.” 6 ml. of homogenate prepared as described above, and incubated with sodium acetate-l-C4 (0.025 mc. per mmole), yielded 1.8 mg. of unsaponifiable material containing a total of 4700 c.p.m. 10 mg. of “isocholesterol,” m.p. 137-140”, and 10 mg. of pure cholesterol were added and the mixture rechromatographed on alumina to give the fractions in Table I.

Fractions 6 and 7, 7.5 mg., corresponded approximately in both weight and melting point with the added carrier “isocholesterol.” They were therefore presumed to consist mainly of this material. For the purpose of washing out, these fract,ions were combined with 10 mg. of pure cholesterol and again chromatographed on 3 gm. of alumina (Table II). Fractions 4 to 6, 6.1 mg., contained a total of 46 c.p.m. or 7.5 c.p.m. per mg. After recrystallization from methanol, these combined fractions gave 4 mg. of material, m.p. 135-140°, with a specific activity of 6.5 c.p.m. per mg.

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308 SYNTHESIS OF LANOSTEROL AND AGNOSTEROL

The cholesterol fractions of the second chromatogram (Fractions 8 to 11, Table II) contained no detectable radioactivity.

Incubations with Carrier “Isocholesterol’‘-40 ml. of rat liver homogenate prepared as described were divided into twenty aliquots of 2 ml., and 0.2 ml. of 0.1 M sodium acetate-l-U4 (1 mc. per mmole) was added to each. Two flasks were taken as controls (Experiment B), and to each of the re-

TABLE I

Alumina Chromatography of Unsaponifiable Fraction from Liver Homogenates after Incubation with Acetate-l-04

Fraction No.

1

2

3

4 5 6 7 8 9

10 11 12 13 14 15 16 17

Solvent

Petroleum ether, b.p. 60-68”

Benzene-petroleum ether 1:4

Benzene-petroleum ether 2:3

Benzene Et,her-benzene 1:50

“ 1:20 “ 1:20 “ 1:20 “ 1:9 “ 1:9 “ 1:9 “ 1:9 “ 1:9 “ 1:9 “ 1:4

Ether Methanol-ether 2:3

Volume

?l!l.

30

10

10 10 30 30 30 10 10 10 10 10 30 10 20 20 20

Weight of fraction

Total activity

mg. c.p.nt.

0.2 25 Oil

0.1 25 0.1 13 0.8 49 3.5 39 4.0 57 0.9 40 1.2 109 1.2 277 1.5 377 2.0 361 4.0 830 1.6 173 0.8 186 0.3 74 0.2 190

Gum “

Semisolid White solid 134-139

‘I “ 135-138 “ “ 125135

Semisolid 115-130 White solid 130-135

“ “ 135145 “ “ 140-148 ‘I “ 135-145 “ “ “ “ 130-142

White semisolid

Appearance; m.p

“C.

maining eighteen flasks (Experiment A) was added 0.1 ml. of a suspension of “isocholesterol” (5 mg. per ml.). After incubation, the homogenates were saponified and the unsaponifiable fractions extracted with petroleum ether, yielding a total of 3 mg. from the combined controls and 27 mg. from the homogenates treated with “isocholesterol.” The total activities esti- mated from infinitely thin aliquots were 154,000 and 1,250,OOO c.p.m., re- spectively.

The combined unsaponifiable fractions of the controls were mixed with 5 mg. of cholesterol and 5 mg. of lanosterol; those of the “isocholesterol”- treated preparations were not further diluted. The two batches of un-

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R. B. CLAYTON AND K. BLOCH 309

saponifiable material were now chromatographed in parallel on columns of 10 gm. of alumina, the fractions being identified according to their appear- ance and levels of activity, as seen in Table III. For purposes of direct comparison of Experiments A and B, the total Cl4 activities for Experiment A in Table III were divided by 9 because the scale of Experiment A was 9 times that of the control Experiment B. Fractions 6 to 13 from Experi- ment A combined weighed 5.1 mg. and had a specific activity of 24,000

TABLE II

Chromatographic Separation of “Isocholesterol” and Cholesterol

The volume for Fractions 1 to 12 was 10 ml.

Fraction No. Solvent

1 Benzene 2 Ether-benzene 1:20 3 “ 1:20 4 “ 1:20

5 “ 1:20

6 “ 1:20

7 “ 1:20 8 I‘ 1:20

9 “ 1:20

10 ‘I 1:20

11 C‘ 1:4

12 “ 1:4

-7-

- -

Weight of fraction

WC.

0.7 2.2

2.3

1.6

0.9 1.1

2.4

3.7

2.6

0.2

r

. -

-

l3tal activity

C.).rn.

6 13

18

15

6 0.5

0

0

0

0

-

_-

-

Appearance; m.p.

“C.

White solid “ “

133-139 White solid 133-139 White solid 134-139 White solid

I‘ “

135-147 White solid 140-14s White solid 145-148 White solid 144-148

c.p.m. per mg. After dilution with 5 mg. of pure lanosteroll and crystal- lization from methanol, 8.2 mg. of needles were obtained which were further diluted to 82 mg. by addition of pure lanosterol. One recrystallization now gave 73.5 mg. of needles, m.p. 140-141”, having 1300 c.p.m. per mg. From the proportion of carrier lanosterol added, assuming no loss of ac-

* The material termed “pure lanosterol” was shown to absorb 1 mole of hydrogen and therefore consisted entirely of material with the unsaturated side chain. Care- ful ultraviolet absorption measurements revealed the presence of a maximum of 1.5 per cent of substances such as agnosterol, having the A’s9 structures; otherwise the physical properties of the material were those recorded in the literature for pure lanosterol.

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310 SYNTHESIS OF LANOSTEROL AND AGNOSTEROL

tivity on crystallization, the calculated specific activity of this material is 1200 c.p.m. per mg.

To obtain sufficient lanosterol for chemical characterization, this labeled

TABLE III

Chromatogram of Unsaponijiable Fraction from Liver Homogenates after Incubation with and without Carrier ‘LIsocholesterol”

Fraction No.

-

_-

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17

18 19 20 21 22 23 24

Hexane Benzene Ether-benzene 1: 99

“ 1:99 “ 1:99 “ 1:99 ‘( 1:99 “ 1:99 “ 1:99 I‘ 1:99 “ 1:99 “ 1:99 ‘I 1:99 “ 1:99 ‘I 1:99 “ 3:97 “ 3:97 “ 1:9 “ 1:9 “ 1:9 “ 1:4 “ 1:4 “ 1:4 “ 1:4

Ether “

25 26 27 28

Solvent

“

Ether-methanol 1: 1

-7

‘OlUltlf

ml.

30 30 30 30 30 10 10 10 10 10 10 10 10 20 30 20 30 20 20 20 20 20 20 20 20 20 40 40

Ex eriment A “Isocho esterol”-treated P

homogenate

Total ictivit: Appearance

c.p.nr.

1370 1550 200 650 330 740

2500 2340 2660 2440 1660 500 570 160 500 180 700

2500 9000 6000 8500 7200 3200 2600 1640 1330 2600 4500

Oil Gum

Trace solid White “

“ “ “ “ “ “ “ “

Trace “ “ “ “

gum

Trace gum “ solid

White “ “ “ “ “ “ “ “ “ “ “ “ L‘ “ “ “ “

Trace “ ‘I “

-

Total activity

c.pm.

128

684 160 370 260

48 100 78

136 126 136 104 156 370 490 110

1,900 2,100 7,800 8,000

21,000 15,400 14,500 3,400 2,100

636 12,000

Appearance

Oil Gum

Trace solid White “

“ “ ‘I “ “ “ “ “

Trace “ “

gum “ “

Trace gum “ solid

White “ “ “ “ “ I‘ “ “ “ “ 1‘ “ “ “ “

Trace solid ‘1 ‘I

material was subjected to further dilutions and recrystallizations: (1) 50 mg. of lanosterol (1300 c.p.m. per mg.) were diluted to 500 mg. with pure lanosterol. Crystallization from acetone-methanol gave 460.3 mg. of lanos- terol, the specific activity of which, by combustion and counting as in- finitely thick samples of barium carbonate, was 97 c.p.m. (2) The fore- going material was diluted with its own weight of pure lanosterol and again

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R. B. CLAYTON AND K. BLOCH 311

recrystallized to yield 776 mg. of lanosterol, m.p. 140-141“. The specific activity as infinitely thick samples of barium carbonate was now 48.8 c.p.m. The chemical work reported below was carried out with this prod- uct.

Chemical Characterization of Labeled Lanosterol-The specific activities of the lanosterol derivatives described in this section are expressed as counts per minute of infinitely thick samples of barium carbonate.

Dehydration and Rearrangement of Ring A-Lanosterol (48.8 c.p.m.) was hydrogenated under neutral conditions in ethyl acetate solution in the presence of a platinum catalyst to give dihydrolanosterol, m.p. 143-146” (10). Treatment of this product with phosphorus pentachloride in anhy- drous petroleum ether, followed by chromatography and recrystallization from acetone, gave the known isopropylidene rearrangement product (V, Fig. 2), m.p. 140-143”, [C-C], +65’; specific activity, 51 c.p.m. Previously reported (lo), m.p. 143-144”, [ar], +67’.

S/3-Acetoxylanost-8-ene-7,ll -dione2-Dihydrolanosteryl acetate (48.8 c.p.m.) was oxidized with chromic acid to give 3@-acetoxylanost-8-ene-7,l l- dione (VII, Fig. 2), m.p. 156-158’, previously reported (6), m.p. 156.5- 158.8”; specific activity, 50 c.p.m.

Degradation of Side Chain of Lanosterol-Lanosterol (48.8 c.p.m.) was converted to the 3p-24,25-trio1 (II, Fig. 2) by treatment with osmium tetroxide, according to the method of Wieland and Benend (11). The trio1 was obtained as plates, m.p. 167-177”, [a], +45”; specific activity, 45 c.p.m. Oxidation of the trio1 was carried out by a modification of the method de- scribed by the above authors. The foregoing trio1 (250 mg.) was dissolved in the minimum of glacial acetic acid, and 375 mg. of lead tetraacetate (1.5 moles per mole of sterol), also dissolved in the minimum of acetic acid, were added. Nitrogen was passed through the solution for 20 hours at 25” and the issuing gas passed through two traps connected in series and cooled in liquid nitrogen. The traps were periodically detached and their contents washed out with 50 ml. quantities of Van Slyke mercuric sulfate reagent (12) for the precipitation of acetone. These solutions were refluxed for 1 hour to precipitate the acetone-mercuric sulfate complex. From the combined precipitates (300 mg., 60 per cent of the theory), ace- tone was regenerated by boiling with 10 per cent hydrochloric acid and reprecipitated as the mercuric sulfate complex (170 mg.). This material was subjected to wet combustion by the method of Van Slyke and Folch (13), and the carbon dioxide precipitated as barium carbonate; specific activity, 34 c.p.m. Assuming by analogy with the data on acetate utili- zation for cholesterol (14) that only the carbonyl carbon of the acetone contains Cl4 and that lanosterol is derived from 18 methyl and 12 carboxyl

2 Experiments carried out by Dr. T. Lyssy in this laboratory.

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312 SYNTHESIS OF LANOSTEROL AND AGNOSTEROL

carbon atoms of acetic acid, the theory requires 48.8 X 30 / 12 X 3 = 41 c.p.m.

ZIP-Acetoxylanost-8-en-%$-a&-2.5 hours after the beginning of the above treatment with lead tetraacetate, a crystalline, precipitate of 3P-acetoxy- lanost-S-en-24-al (III, Fig. 2) was separated from the solution by filtra- tion, and the filtrate returned to the reaction vessel in which the removal of acetone in a nitrogen stream was continued. The aldehyde (100 mg.) melted with decomposition over the range, 155-185”. Recrystallization from chloroform and acetic acid gave prisms, m.p. 210-220” (decomposi- tion), [(Y]~ +57”. It readily gave a yellow 2,4-dinitrophenylhydrazone and

AcO

VII 101 (100) IV III 96 (94)

,Me -0 = qMe

70 (84)

& \ j v 104 (100)

FIG 2. Isotope concentrations in lanosterol and derivatives. The calculated values are given in parentheses.

showed absorption bands in the i&a-red at 2540 cm.-l (C-H stretching of aldehyde), a strong band at 1720 cm.-1 (C=O of both the aldehyde and acetoxy groups), and a broad band at 1250 cm.-’ (ester -C-O-). No hydroxyl band was present. Found, C 78.78, H 10.79; C&HMO~ requires C 78.6, H 10.47. The specific activity of the compound as an infinitely thick sample of BaC03 was 47 c.p.m.

The Cl4 analyses of the various degradation products of lanosterol are summarized in Fig. 2. Values of the specific activities of the derivatives are recalculated and compared with an assigned value of 100 for that of the lanosterol.

Incubation with Various Components of “Isocholesterol”-Incubation and chromatographic procedures were the same as those described above. The levels of acetate incorporation into the lanosterol fraction of controls and

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R. B. CLAYTON AND K. BLOCH 313

of homogenates incubated with either natural or reconstituted “isocholes- terol” were compared with those obtained on treatment of the homogenates with suspensions of the various individual components of wool fat sterol. Suspensions of synthetic “isocholesterol” were prepared by mixing 45 parts of lanosterol, 45 parts of dihydrolanosterol, 5 parts of agnosterol, and 5 parts of dihydroagnosterol. In all the experiments given in Fig. 3, the total quantity of sterol added to the incubation mixtures was 1.5 mg. per 2 ml. of homogenate.

Isolation of Agnosteryl Acetate from Labeled “Isocholesteryl” Acetate- “Isocholesterol” obtained by incubation of a homogenate with carrier

FIG. 3 FIG. 4

FIG. 3. Enhancement of acetate incorporation into “isocholesterol” in liver ho- mogenates in the presence of carrier sterols. The heights of the bars represent the ratio of total counts per minute in the “isocholesterol” fraction obtained from incu- bations with carrier sterol, to those obtained in control experiments. I, natural “isocholesterol;” II, reconstituted “isocholesterol;” III, lanosterol; IV, dihydro- lanosterol; V, agnosterol; VI, lanosterol, 4 parts, plus agnosterol, 1 part.

FIG. 4. Isolation of agnosteryl acetate by crystallization of “isocholesteryl” ace- tate.

“isocholesterol” as described above was diluted and acetylated to yield 7.5 gm. of “isocholesteryl” acetate, having 50 c.p.m. per mg. as an infinitely thin sample. Crystallization of this material from ethyl acetate, according to Windaus and Tschesche (7), results in an enrichment of agnosterol in the less soluble fraction. In the present experiments pure agnosteryl acetate could be obtained after six crystallizations, provided that crystallization was allowed to take place slowly. At each stage the content of agnosteryl acetate was determined by measuring the light absorption at 244 rnK, and the radioactivity determined by counting aliyuots as infinitely thin sam- ples. The final product was pure agnosteryl acetate, m.p. 175-179”; cZd4 = rnp 18,000 (15); specific activity as infinitely thin aliquots, 40 c.p.m. per mg. The variation of specific activity with intensity of light absorption al 244 rnp is given in Fig. 4.

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314 SYNTHESIS OF LANOSTEROL AND AGNOSTEROL

DISCUSSION

When liver homogenates are incubated with labeled acetate, about two- thirds of the Cl4 in the unsaponifiable fraction is ordinarily recovered as cholesterol. Attempts to account for the remainder of the radioactivity in the form of known steroids have hitherto been largely unsuccessful. In regard to sterols which are known to accompany cholesterol in animal tis- sues (cholestanol (16), A’-cholestenol (17), 7-dehydrocholesterol (IS), 3@- 5oc-dihydroxy-6-ketocholestanol (19), and 3/3-5c+6/Strihydroxycholestane (20))) no evidence exists that they are in any large measure responsible for the remainder of the activity. In the course of the present work, the un- saponifiable fractions after incubation of the homogenates with labeled acetate were carefully chromatographed and it was invariably found that the major part of the radioactivity was associated with four well defined fractions. Two principal fractions, less polar than cholesterol, could be eluted in turn with petroleum ether and benzene. By analogy with in viva experiments (1) and those employing the perfused liver (21), part of the activity in the petroleum ether eluate may be expected to reside in squa- lene. The benzene fraction remains completely unidentified. The radio- activity associated with this fraction was found to travel chromatographi- tally with ambrein, but chemical evidence indicates that the two materials are not identical. After elution of cholesterol, another fraction of high specific activity emerges from the column with ether-methanol mixtures.

After it had been ascertained that the components of “isocholesterol,” though inseparable from each other, could be chromatographically sepa- rated from cholesterol, it became possible to show the presence of a fifth fraction, slightly less polar than cholesterol. This “lanosterol” fraction under the conditions of the control experiments was very small, both in weight and in CY content, and in fact its presence could be demonstrated only by chromatography of the unsaponifiable material with lanosterol or “isocholesterol” added as carrier.

The data of Tables I and III demonstrate that in the control experi- ments not more than 1 per cent of the total Cl4 content of the unsaponifiable fraction could be attributed to lanosterol or other components of “iso- cholesterol.” It was, however, clearly shown by a chromatographic wash- ing out procedure (Table II) that the radioactivity of the lanosterol fraction was not due to contamination by cholesterol.

On the assumption that lanosterol might be an intermediate with a high rate of turnover, it was considered likely that newly synthesized lanosterol could be trapped and the yields of Cl4 increased by the presence of carrier during the incubation. Data such as those in Table III, Experiment A, illustrate that a considerable enhancement of the incorporation of Cl4 into the lanosterol fraction does indeed result from the addition of carrier “iso-

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R. B. CLAYTON AND K. BLOCH 315

cholesterol” to the homogenate prior to incubation. In the particular ex- periment cited, the Cl4 content of the lanosterol fraction accounted for 10 per cent of the total in the unsaponifiable fraction, corresponding to a 30- fold increase compared with the control.

Since the “isocholesterol” used as a carrier consisted of four Go sterols which were inseparable by our chromatographic procedure, it was clearly possible for any or all of the components to contain C14. In the expecta- tion that one of the radioactive sterols was lanosterol, the labeled “iso- cholesterol” (Fractions 6 to 13, Table III) was diluted with pure lanosterol and recrystallized several times without change of specific activity. This material was converted to derivatives which were chosen to demonstrate,

as far as possible, that the radioactivity was associated with a compound which had the typical structural features of lanosterol. These derivatives were (1) the C&T aldehyde produced together with acetone by oxidative degradation of the side chain, (2) the 7,11-diketone formed on oxidation of dihydrolanosteryl acetate, and (3) the isopropylidene rearrangement product obtained on dehydration of dihydrolanosterol with PC15. Through all of these transformations the radioactivity of the starting material was retained (Fig. 2).

The specific activities of the C&T aldehyde and acetone (III, Fig. 2) indicated that all the radioactivity of the starting material resided in substances unsaturated in the 24:25 position. If compounds having a saturated side chain such as dihydrolanosterol or dihydroagnosterol had contributed to any appreciable extent to the radioactivity of the starting material, the Cl4 content of these degradation products would have been proportionately reduced.3 The concomitant dehydration and rearrange- ment of ring A to give isopropylidene derivatives of the type V (Fig. 2) are characteristic of the 3/%hydroxy-4,4’-dimethyl sterols and triterpenes. From the formation of this well characterized derivative without alteration of specific activity, it can therefore be concluded that this moiety also is present in the radioactive starting material. The 7,11-diketo-A* com- pound (VII, Fig. 2) is an expected product of the chromic acid oxidation of the A*-&0 sterols, but is also formed from the corresponding A7sg-dienes under similar conditions (6). The unaltered radioactivity in this deriva- tive therefore does not distinguish between lanosterol and agnosterol as the radioactive substance. The relative isotope concentrations of these two components will be discussed below.

3 In a preliminary experiment the Cao sterols with saturated side chains were chem- ically separated from the labeled “isocholesterol” and shown to account for a maxi- mum of 5 per cent of the radioactivity in the mixture. Since the content of saturated sterols in “isocholesterol” is 40 per cent, their specific activity is calculated to be at most 12.6 per cent of that of the total mixture.

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316 SYNTHESIS OF LANOSTEROL AND AGNOSTEROL

In experiments involving carrier dilution, contamination by substances of closely similar structure cannot be rigidly excluded. However, the close correspondence between the experimental and calculated values for the specific activities of all the derivatives makes it highly improbable that appreciable radioactivity resides in compounds other than lanosterol or agnosterol.

Evidence that, under the conditions of our experiments, both the lanos- terol and the agnosterol became labeled is presented in Fig. 4. Crystal- lization of a large batch of labeled “isocholesteryl” acetate (e244 = 2000; specific activity, 50 c.p.m. per mg.) yielded a few mg. of pure agnosteryl acetate (~24~ = 18,000; specific activity 40 c.p.m. per mg.). These light absorption values show that at the start the “isocholesteryl” acetate con- tained a total of 11 per cent of substances with the 7,9-diene structure which would include both agnosteryl acetate and dihydroagnosteryl ace- tate. The relative proportions of these two components cannot be as- certained, but it may be assumed for the present purpose that they occur in equal amounts. Hydrogenation data obtained with the “isocholesteryl” acetate used in this experiment indicate the presence of a total of 60 per cent material having an unsaturated side chain. Hence, if 5.5 per cent of the total mixture is agnosteryl acetate, the lanosteryl acetate comprises 54.5 per cent, and since the dihydro compounds contain no more than 5 per cent of the total radioactivity, the specific activity of lanosterol may be calculated to be 83 c.p.m. per mg.4 or slightly more than twice that of the agnosterol.

It will be noted from Fig. 4 that during the enrichment of agnosteryl acetate on fractional crystallization the specific activity does not decline steadily but undergoes a marked increase during the initial steps. In ac- counting for this effect it must be remembered that “isocholesterol” is a mixture of four components. The first stages of crystallization of the ace- tates could result in the progressive removal of the dihydro compounds which, though present to the extent of 40 per cent of the total weight, ac- count for no more than 5 per cent of the total radioactivity.

In the initial experiments carrier “isocholesterol,” the only source of lanosterol then available to us, was added to the homogenates prior to in- cubation, for the purpose of trapping labeled lanosterol. The success of t,his procedure in producing a marked enhancement of the acetate incor- poration into the “isocholesterol” fraction has already been noted. How- ever, when lanosterol itself was used subsequently for the same purpose, it failed to reproduce the results obtained with “isocholesterol.” Negative

4 If the value assigned to the agnosterol content of the original mixture is varied from 1 to 11 per cent, the calculated value for the specific activity of lanosterol would vary but slightly, namely from 86 to 80 c.p.m. per mg.

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R. 13. CLAYTON AND Ii. BLOCII 217

results were also obtained with dihydrolanosterol and agnosterol (Fig. 3). On the other hand, a “synthetic isocholesterol, ” reconstituted from the four known components in the proportions in which they seemed most likely to occur in our natural “isocholesterol,” duplicated the results given by the natural mixture. It thus seems improbable that the enhancement is due to some hitherto unrecognized component of natural “isocholesterol,” and this conclusion is supported by the finding that suspensions containing both lanosterol and agnosterol give a degree of enhancement comparable to that obtained with the four-component mixture. No explanation can at present be given for the synergistic action of lanosterol and agnosterol, but it, is worth noting that these are the only two members of the “isocholes- terol” group of sterols which became significantly labeled in the system studied. Further experiments to investigate the effect of other combina- tions of wool fat sterols are in progress.

Evidence for the conversion of lanosterol to cholesterol is presented in the succeeding paper (5). As an intermediate in cholesterol biogenesis, lanosterol would be expected to show the same distribution pattern of the carboxyl carbons of acetate as cholesterol itself. The isopropyl moiety of lanosterol isolated from the present experiments had a specific activity of 34 c.p.m., in reasonable agreement with the predicted value. A value in accord with current theory has also been obtained for carbon atom 11 of the C& structure,’ and hence it is probable that the carboxyl carbon atoms of acetate are distributed in lanosterol as they are in cholesterol.

SUMMARY

The unsaponifiable fraction of rat liver obtained on incubation of the homogenized tissue with radioactive acetate has been shown to contain two radioactive C&o sterols, lanosterol and agnosterol. When carrier “iso- cholesterol” is present during incubation, a much greater proportion of the radioactivity in the unsaponifiable fraction can subsequently be isolated in the lanosterol fraction.

The identity of the labeled products with lanosterol and agnosterol was established by preparation of several derivatives characteristic for this type of sterol. In the course of these transformations the Cl* content of the CZO sterols remained unaltered.

A markedly increased yield of labeled lanosterol can be obtained by the incubation of liver homogenates in the presence of either “isocholesterol” from wool fat or of a synthetic isocholesterol reconstituted from the four pure components known to occur in the natural mixture. This enhancing effect is also shown by a mixture of lanosterol and agnosterol, but not by any single constituent of “isocholesterol.”

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318 SYNTHESIS OF LANOSTEROL AND AGNOSTEROL

BIBLIOGRAPHY

1. Langdon, R. G., and Bloch, K., J. Biol. Chem., 200, 135 (1953). 2. Langdon, R. G., and Bloch, K., J. BioZ. Chem., 200, 129 (1953). 3. Woodward, R. B., and Bloch, K., J. Am. Chem. SOL, ‘76,2023 (1953). 4. Voser,.W., Mijovic, M. V., Heusser, H., Jeger, O., and Ruzicka, L., Helv. chim.

acta, 36, 2414 (1952). 5. Clayton, R. B., and Bloch, K., J. Biol. Chem., 218, 319 (1956). 6. Ruzicka, L., Rey, E., and Muhr, A. C., Helv. chim. acta, 27, 472 (1944).. 7. Windaus, A., and Tschesche, R., 2. physiol. Chem., 190, 51 (1930). 8. Bucher, N. L. R., J. Am. Chem. Sot., 76, 498 (1953). 9. Wieland, H., Pasedach, H., and Ballauf, A., Ann. Chem., 629, 68 (1937).

10. Ruzicka, L., Montavon, M., and Jeger, O., Helv. chim. acta, 31, 818 (1948). 11. Wieland, H., and Benend, W., 2. physiol. Chem., 274, 215 (1942). 12. Van Slyke, D. D., J. Biol. Chem., 83, 415 (1929). 13. Van Slyke, D. D., and Folch, J., J. BioZ. Chem., 136, 509 (1940). 14. Little, H. N., and Bloch, K., J. BioZ. Chem., 183, 33 (1950). 15. Ruzicka, L., Denss, R., and Jeger, O., Helv. chim. acta, 29, 204 (1946). 16. Schoenheimer, R., Behring, H., Hummel, R., and Schindel, L., 2. physiol. Chem.,

192, 73 (1930). 17. Fieser, L. F., J. Am. Chem. Sot., 73, 5007 (1951). 18. Windaus, A., and Stange, O., 2. physiol. Chem., 244, 218 (1936). 19. Schwenk, E., Werthessen, N. T., and Rosenkrantz, H., Arch. Biochem. and Bio-

phys., 37, 247 (1952). 20. Fieser, L. F., and Bhattacharyya, B. K., J. Am. Chem. Sot., 76, 4418 (1953). 21. Schwenk, E., Todd, D., and Fish, C. A., Arch. Biochem. and Biophys., 49, 187

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R. B. Clayton and Konrad BlochLANOSTEROL AND AGNOSTEROL

BIOLOGICAL SYNTHESIS OF

1956, 218:305-318.J. Biol. Chem. 

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