THE SYNTHESIS OF RADIOACTIVE CHOLESTEROL

AND FATTY ACIDS IN VITRO*

II. A FURTHER STUDY OF PRECURSORS

BY ROSCOE 0. BRADY, J. RABINOWITZ, J. VAN BAALEN,

AND SAMUEL GURIN
(From the Department of Physiolo,ical Chemistry and the Endocrine Section,
Department of Medicine, School of Medicine, University
of Pennsylvania, Philadelphia, Pennsylvania)

(Received for publication, July 3, 1951)

Although it is now abundantly clear that mammalian liver can synthe-

size cholesterol from precursors capable of giving rise to 2-carbon frag-
ments, recent evidence has been obtained (1) suggesting that aceto-
acetate may be incorporated into cholesterol without appreciable cleavage
into 2-carbon units.
The suggestion that a 4-carbon precursor may be involved in this bio-
synthesis has prompted a further study of C”-labeled 4-carbon compounds
with the hope that more information on the nature of such a precursor
might be obtained. In addition, a number of non-labeled compounds
have also been investigated to determine whether or not they could in-
hibit or depress the incorporation of labeled acetate into cholesterol dur-
ing incubation with rat liver slices. When such an effect was observed,
the substance was labeled with Cl4 and studied further.
As a consequence of the finding that acetoacetate may represent an
important intermediate in the biosy&hesis of cholesterol, it follows that
any isolated tissue capable of forming cholesterol should also be able to
synthesize acetoacetate. Evidence supporting the validity of this con-
cept is presented.

EXPERIMENTAL

Complete details of the preparation of tissue slices, buffers, incubation

period, and the gas phase employed have been previously reported (1, 2).
After incubation, the fatty acids and cholesterol were purified as de-
scribed (1, 2).
Organic Syntheses-Carboxyl-labeled vinylacetic acid was prepared from
C140Zby carbonation of allylmagnesium chloride according to the direc-
* Aided by grants from the American Cancer Society administered by the Com-
mittee on Growth of the National Research Council and from the National Insti-
tutes of Health, United States Public Health Service. The radioactive compounds
were obtained on allocation from the United States Atomic Energy Commission.

137

This is an Open Access article under the CC BY license.

138 RADIOACTIVE CHOLESTEROL. II

tions of Gilman and McGlumphy (3). The final product was fractionally
distilled and the portion distilling from 70-105” at 60 mm. collected;
yield 28 per cent.
Aldol containing Cl4 equally distributed among all 4 carbon atoms was
prepared from an aqueous solution of doubly labeled acetaldehyde by
means of dilute aqueous KOH (4). After extraction with ether, the aldol
was recovered in 34 per cent yield, following several fractional distillations
at 9 to 10 mm. (bath temperature 60-90’).
Completely labeled crotonaldehyde was prepared in 67 per cent yield
from radioactive aldol (described above) by slow distillation in the pres-
ence of iodine (5). The distillate was fractionated and the portion dis-
tilling between 90-105” collected.
Ace&n-A new and convenient synthesis of C4-labeled acetoin was
developed by condensation of lactonitrile with radioactive methylmagne-
sium iodide and is described here in detail. Gauthier (6) has previously
reported an analogous reaction of lactonitrile with ethylmagnesium bro-
mide.
To 2.13 gm. of lactonitrile (0.03 mole) in a 3-necked flask equipped
with a large Herschberg stirrer, reflux condenser, and dropping funnel was
added 0.033 mole of non-labeled methylmagnesium iodide dissolved in
25 ml. of ether. The mixture was stirred vigorously until evolution of
methane ceased, at which time a similar amount of C14-labeled methyl-
magnesium iodide in ether was added slowly. When the addition was
complete, the double magnesium salt separated out as a gray precipitate.
Vigorous stirring and gentle warming were continued for another hour.
The reaction mixture was chilled and decomposed with a minimal quan-
tity of saturated aqueous sodium chloride, followed by dropwise addition
of concentrated HCl to pH 2. The ether layer was removed (without
shaking) and the aqueous layer continuously extracted with ether under
Nz. The combined ether extracts were dried over anhydrous MgSO4 and
some anhydrous Na&Oa. The ether was evaporated under NZ and the
remaining oil (again under NJ fractionally distilled. With the micro
distillation equipment employed and vigorous sweeping with Nt, the major
portion of the material was found to distil at 13&140” (from literature,
b.p. 140-144”). A sample of redistilled authentic acetoin showed the
same boiling point range upon distillation under the same conditions.
The yield, based on the lactonitrile, was 65 to 75 per cent. It is obvious
that acetoin labeled in other positions can similarly be made from labeled
lactonitrile prepared from labeled acetaldehyde or cyanide.
Carboxyl-Labeled Orsellinic Acid&-Orcin aldehyde was prepared by the
Gattermann and Kijbner procedure from labeled HCN and orcinol (7).
 BRADY, R.iBINOWITZ, VAN BAALEN, AND GURIN 139

The resulting compound was converted to orsellinic acid by the method

described by Hoesch (8); m.p. 173-175”, decomposed.
Non-Labeled Compounds-The sodium salt of acetyl acetaldehyde was
prepared by the method of Claisen and Stylos (9). Lu-Acetyl-@-methyl-
diethyl glutarate was synthesized by the procedure of Burton and Ingold
(10); b.p. 148-150’ at 10 mm. (Y, ru’-Diacetyl-/3-methyldiethyl glutarate
was prepared as described by Horning (11) ; m.p. 75-77.5”. Diethyl-
acetyl succinate was obtained from the Monsanto Chemical Company
and purified by vacuum distillation; b.p. 169” at 15 mm. l-carbethoxy-
2-methyl-4,6-cyclohexanedione was prepared from ethyl crotonate and
sodium ethylate by the procedure of Schilling and Vorliinder (12) ; m.p.
89-90”.

Results
Crotonate depressed to a very small extent the incorporation of radio-
active acetate into cholesterol and long chain fatty acids. It was accord-
ingly assumed that this substance is probably not an important inter-
mediate in these biosynthetic processes, and no further experiments were
carried out with this compound. Vinylacetic acid, on the other hand, was
more effective in the screening test and was accordingly labeled with C14.
The amount of Cl4 incorporated into cholesterol of fatty acids proved to
be much less than that obtained with the radioacetate controls (Table I,
Experiments 1 and 2).
Although non-labeled crotonaldehyde produced a marked diminution
in the incorporation of labeled acetate, radioactive crotonaldehyde itself
was very poorly utilized (Table I, Experiments 3 and 4). The effect of
crotonaldehyde may accordingly be ascribed to its inhibiting action. It
should be emphasized that the concentration of the crotonaldehyde em-
ployed was low enough to permit normal tissue respiration.
Labeled aldol, when compared with radioacetate controls, is as effi-
ciently incorporated into cholesterol as is acetoacetate. Since a sig-
nificant incorporation into long chain fatty acids was also observed, it is
assumed that aldol may, to somel extent, be cleaved by liver slices into
2-carbon fragments (Table I, Experiments 5 and 6). When compared
with the radioacetate controls, aldol is more efficiently incorporated into
cholesterol than into long chain fatty acids. It is therefore likely that
aldol is also converted to an appreciable extent into &hydroxybutyrate
or acetoacetate. This is in agreement with the early work of Parnas
(13) and of Lieben and Ehrlich (14).
Because it had been previously shown that acetaldehyde is more effi-
ciently converted into fatty acids and cholesterol than is acetate (l), it
 TABLE I
Incorporation of Labeled Substrates into Long Chain Fatty Acids and Cholesterol by Rat Liver Slices c.L
Recovered cholesterol Recovered fatty acids %
Activity I T
Erperi Substrate
merit Substrate COlICen-
tration :holestero ncorporated Substrate
No. Total per 10 mg. per mm
dministered iigitonide cholesterol fatty ac13
digitonide
--
Y c.p.m.cr C.Q.?lB. C.).rn. .$.rn. c,
m. m&T. w
w. c? ml. E
CH,: CHCHLY400Na 0.004 3,000 7,800 4.9 18 0.3 50 4 2.1
CHPOONa 0.005 6,@3(’ 10,500 5.0 271 4.2 41 31 16.0 ?J
CHS: CHCHdFOONa 0.004 3,000 8,600 5.1 26 0.4 44 5 2.6 El
CHdFOONa 0.005 6,oC’C’ 10,500 5.6 260 3.7 44 30 16.0 :
C”H&“H : C”HC”H0 0.002 2,0@3 2,700. 5.5 0 0 44 1 0.7 2
CH3C”OOKa 0.005 ‘3,@3f3 10,500 5.0 240 3.5 49 34 18.0
CFH&‘“H : C”HC”H0 0.002 2,700 5.1 2
2,ooo 33 0.7
CH$POONa 0.005 g,@Jo 16,900 5.6 702 7.3 G
C”H~CPHOHC”H&“HO 0.002 3,800 4,100 3.2 44 0.8 43 13 5.3
CH$.YOOKa 0.005 g,ooo 16,900 3.2 168 2.7 48 107 38.0 2
C~‘H~C~~HOHC’dH&l’HO 0.002 3,800 4,100 5.8 356 2.0 59 18 7.6 3
CH8CL400Ka 0.005 g,ooo 16,900 7.8 1140 5.3 50 74 26.0
8
C”H,COCHOHCHs 0.002 ‘39’300 g,ocJo 9.8 240 0.8 46 0 0 r
“ 0.005 6,600 10.2 62 0.2 44 0 0
=,m
CHdYOONa 0.005 10,ooo 18,000 7.0 980 5.8 42 59 18.0 z
8 C”H&OCHOHCHs 0.002 63’333 g,ooo 10.4 290 0.9 35 5 1.6
‘I 0.004 10.3 317 1.0 0
f3,‘33fJ 18,000 33 0
CH&“OO?r’a 0.005 10,ooa 18,000 10.1 10.0 39 44 13
9 HC”OO?ia 0.008 15,000 42,000 7.5 0 40 0 0
‘I 0.004 15,000 21,000 41 0 0
10 HC”H0 0.004 12,000 9,600 7.0 35 0.4 41 0 0
HCHO 0.004
“ + CHd.YOONa 0.005 16,900 11.8 1664 8.8
9,600
11 HWHO 0.004 12,000 9,600 4.0 a 0
CHdYOONa 0.005 9,600 16,900 4.1 237 3.0
 BRADY, RABINOWITZ, VAN BAALEN, AND QURIN 141

was thought worth, while to investigate the action of acetoin. Although

this substance was found to reduce significantly the incorporation of
acetate in the screening tests, Cl’-labeled acetoin was poorly utilized by
liver slices for the biosynthesis of either cholesterol or fatty acids.
Neither radioactive formate nor formaldehyde is converted to fatty
acids or cholesterol to any significant extent. This finding renders un-
likely the hypothesis of Miescher and Wieland (15) that the biosynthesis
of cholesterol may occur via condensation of acetoacetate with formalde-
hyde.
A number of miscellaneous compounds were screened and found to
have no significant diluting effect upon the incorporation of radioacetate
into cholesterol. Among them were l-carbethoxy-Z-methyl-4,6-cyclohex-

TABLE II
Conversion of CH&“OONa to Acetoacelate* by Human Adrenal and
Rabbit Testicular Tissue

Experiment No. Tissue Recovered acetaacetate, activity of

carbonyl C

c.p.m. par min. per rn#. c

1 Human adrenal 3.9 x lo”
2 “ “ 1.4 x l(r
. 3 Rabbit testis 3.3 x 1W

The acetate wae present at a final concentration of 0.005 M. The specific activity
of the carboxyl carbon of acetate was 3 X 10” c.p.m. per mg. of C.
* Carrier acetoacetate was present in the incubating medium at 0.01 M concen-
tration.

anedione, cu-acetyl-/3-methyldiethyl glutarate, a, cY’-diacetyl+methyldi-

ethyl glutarate, and diethylacetyl succinate. Carboxyl-C”-orsellinic acid
was algo found not to be incorporated into cholesterol by liver slices.
Acetoaeetate Format& in Extrahepatic Tissues--Slices of human ad-
renal tissue were incubated with carboxyl-Cl’-acetate in the presence
of a bank of non-labeled acetoacetate. The medium was treated with
copper lime, and the acetoacetate of the supernatant fluid decarboxylated
with HgSO4 and H&30’ in the usual manner. The mercury-acetone com-
plex was recovered, washed, and dissolved in dilute HCl and the acetone
distilled into a receive? containing DenigBs’ reagent. Only the radioactiv-
ity of the acetone fraction is reported. It may be seen that in the adre-
nals there is significant conversion of acetate into acetoacetate (Table
II). Similarly, rabbit testis was found to be capable of synthesizing
acetoacetate. The latter experiment confirms the report of Jowett and
Quastel (16) that testis is capable of forming ketone bodies.
142 RADIOACTIVE CHOLESTEROL. II

DISCUSSION

Of all the 4-carbon precursors, aldol appears to be the only one which
is as effective as acetoacetate as a precursor of cholesterol. Since, unlike
acetoacetate, it may be cleaved to a significant extent into 2-carbon frag-
ments prior to incorporation into cholesterol, it is difficult to assess its
importance as a precursor.
Crotonate has been excluded as an important intermediate of both
oxidation (17-19) and synthesis of butyrate (20). While vinylacetic acid
is believed to be more closely related to the intermediate involved in the
oxidation and synthesis of butyrate (19, 20), the results reported here
indicate that it is not an important intermediate in the biosynthesis of
either cholesterol or fatty acids.
The belief that acetoacetate may play an important role in the bio-
synthesis of cholesterol is strengthened by the observations that tissues
such as adrenals and testis, which are known to be capable of synthesizing
cholesterol (21, 22), are also able to synthesize acetoacetate. It is hoped
that further work will demonstrate that every isolated tissue capable of
synthesizing choksterol w-ill also have the ability to form acetomtate. It is
not surprising that acetoacetate has heretofore not been found in such
tissues, since the amount accumulating may be small. Furthermore,
there is good evidence that extrahepatic tissues are able to oxidize*aceto-
acetate efficiently, which suggests that these tissues probably have the
ability to cleave acetoacetate into active 2-carbon fragments.
Finally, the authors are well aware not only of the limitations of the
slice technique employed in these experiments, but of objections that
can be raised to the use of esterified compounds in such studies. The
compounds were all water-soluble in the concentrations employed, and,
on the basis of known liver esterase activity (23), it was felt that they
should be investigated. It is, of course, clear that negative results do not
have the significance of positive findings in these studies. However,
since the biosynthesis of cholesterol and fatty acids has not been ac-
complished in cell-free preparations, it was hoped that the screening tech-
nique supplemented with studies of C14-labeled precursors would furnish
some clues regarding the nature of these biosynthetic processes.

SUMMARY

C14-labeled vinylacetic acid, crotonaldehyde, aldol, acetoin, and orsellinic

acid have been prepared and their conversion to cholesterol and fatty
acids by liver slices studied. None of these substances appears to be an
important precursor, with the exception of aldol which is probably con-
verted partly into acetoacetate and to some extent into 2-carbon frag-
 BRADY, RABINOWITZ, VAN BAALEN, AND GURIN 143

ments. Labeled formate and formaldehyde are not significantly con-

verted by liver slices to cholesterol or fatty acids.
Human adrenal slices, as well as rabbit testis, are capable of synthesizing
acetoacetate from labeled acetate.
A new synthesis of CP-labeled acetoin is described.

The authors wish to express their gratitude to Miss Rita Kresge, Mr.
Frederick Blake, and Dr. Norman Abrahamsen for their capable assistance
in the preparation of labeled vinylacetic acid, crotonaldehyde and aldol,
and orsellinic acid, respectively, and to Dr. H. A. Zintel for the human
adrenal tissue.

BIBLIOGRAPHY

1. Brady, R. O., and Gurin, S., J. Biol. Chem., 189,371 (1951).

2. Brady, R. O., and Gurin, S., J. BioE. Chem., 186, 461 (1950).
3. Gilman, H., and McGlumphy, J. H., Bull. Sot. chim., 43, 1322 (1928).
4. Hammarsten, H., Ann. Chem., 421, 293 (1920).
5. Hibbert, H., J. Am. Ckem. Sot., 37, 1748 (1915).
6. Gauthier, D., Corn@. rend. Acad., 162, 1100 (1911).
7. Gattermann, L., and Kiibner, M., Ber. them. Ges., 3, 279 (1899).
8. Hoesch, K., Ber. them. Ges., 46, 886 (1913).
9. Claisen, L., and Stylos, N., Ber. them. Gee., 21, 1144 (1888).
10. Burton, H., and Ingold, C. K., J. Chem. Sot., 2022 (1929).
11. Horning, E., J. Org. Chem., 9, 547 (1944).
12. Schilling, R., and Vorlilnder, D., Ann. Chem., 308. 184 (1899).
13. Parnas, J., Biockem. Z., a8, 274 (1910).
14. Lieben, F., and Ehrlich, G., Biochem. Z., 198, 317 (1928).
15. Miescher, K., and Wieland, P., Helu. chim. acta, 35, 1847 (1950).
16. Jowett, M., and Quastel, J. H., Biochem. J., 29, 2181 (1935).
17. Jowett, M., and Quastel, J. H., Biochem. J., 29, 2143 (1935).
18. Lipmann, F., and Perlmann, G. E., Arch. Biockem., 1, 41 (1942-43).
19. Kleinzeller, A., Biochem. J., 37, 678 (1943).
20. Stadtman, E. R., and Barker, H. A., J. Biol. Chem., 181, 221 (1949).
21. Srere, P. A., Chaikoff, I. L., and Dauben, W. G., J. Biol. Chem., 178, 829 (1948).
22. Srere, P. A., Chaikoff, I. L., Treitman, S. S., and Burstein, L. S., J. Biol. Chem.,
182, 629 (1950).
23. Sobotka, H., and Glick, D., J. Biol. Chem., 106, 199 (1934).