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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 CsOsterols,
lanosterol and agnosterol, are natural constituents of liver, though in very
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-
R. B. CLAYTON AND K. BLOCH 307
TABLE I
Alumina Chromatography of Unsaponifiable Fraction from Liver Homogenates
after Incubation with Acetate-l-04
TABLE II
Chromatographic Separation of “Isocholesterol” and Cholesterol
The volume for Fractions 1 to 12 was 10 ml.
-
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.
310 SYNTHESIS OF LANOSTEROL AND AGNOSTEROL
TABLE III
Chromatogram of Unsaponijiable Fraction from Liver Homogenates after
Incubation with and without Carrier ‘LIsocholesterol”
- -7
Ex eriment A
“Isocho Pesterol”-treated
Fraction homogenate
No. Solvent ‘OlUltlf
Total Appearance Total Appearance
ictivit: activity
_-
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
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-
SUMMARY
BIBLIOGRAPHY
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