THE METABOLISM OF ACETONE IN THE INTACT RAT*

BY WARWICK SAKAMI AND JEAN M. LAFAYE

(From the Department of Biochemistry, School of Medicine, Western Reserve
University, Cleveland, Ohio)

(Received for publication, May 31, 1951)

Two pathways of acetone utilization in the rat have been indicated.

One mechatim involves its carboxylation to acetoacetate (1) ; the second,
the production of “formate”’ from acetone methyl carbon (2). This
formate has been postulated to arise by a 2- and l-carbon cleavage in
which “acetate”’ is also formed.
In the present investigation, the possibility of a direct oxidation of
acetone to a 3-carbon intermediate of glycolysis, such as pyruvate, has
been considered. In previous studies (2), methyl-labeled acetone was
found to introduce 04 into the glycogen with higher activity in the 3,4-
than in the 2,5-carbons. This aspect of the isotope distribution was
originally considered to have been produced by CO2 fixation, although
the 3 ,Ccarbon labeling was somewhat higher than had been anticipated
for this mechanism. An alternative explanation was the additional oc-
currence of a major conversion of acetone to “pyruvate”,2 a pathway
which would raise the activity of the 3,4-carbons (and of the 1,6) relative
to that of the 2,5.
C”Hs. CO. C”H, + C”H,. CO +C”OOH -+ 1,6 and 3,4 > 2,5-Cl’-glucose

To test this hypothesis, 2-Cl’-acetone has been administered to rats and

the distribution of isotope in the liver glycogen determined. Metabolism
of acetone by the carboxylation and cleavage mechanisms would introduce
labeling solely into the 3,4 positions (3).
CH,.C”O.CH1 + CO, + CH*.C”O*CH~.COOH -J CH,.C1’OOH + 3,4-Cl’-glucose

CH*.C”O.CHs + “CHa.V400H + “HCOOH” -+ 3,4-Cl’-glucose

If, in addition to these pathways, the direct oxidation of acetone to “pyru-

l Presented at the Thirty-fifth annual convention of the Federation of American
Societies for Experimental Biology in April, 1951 (Federation Proc., 10, 241 (1951)).
Aided by a grant from the American Cancer Society, recommended by the Com-
mittee on Growth of the National Research Council, and by the Elisabeth Severance
Prentiss Fund of Western Reserve University.
The radiocarbon used in these experiments was obtained on allocation from the
United States Atomic Energy Commission.
1 “Formate” and “acetate” are used to indicate l- and 2-carbon particles, either
formate and acetate themselves or intermediates of their metabolism.
* “Pyruvate” is used to indicate a 3-carbon intermediate of glycolysis.
199

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

200 ACETONE METABOLISM

vate” contributed to acetone utilization, the 1,6- and 2,5-carbons would

also be labeled, with higher activity in the 2,5 than in the 1,6 positions,
the contribution of acetone by this mechanism being similar to that of
2-carbon-labeled lactate (4).
CH,.C”O.CH, + “CH8.CP0.COOH” -+ 2,5 > 1,6-CP-glucose
The relative activity of the 1,2,5,6- and 3,4-carbons would depend on the
relative quantitative importance of the “pyruvate”-forming and 3,4-car-
bon-labeling mechanisms. In agreement with this hypothesis of a direct
oxidation of acetone to “pyruvate,” the liver glycogen has been found to
be totally labeled, with higher activity in the 2,5- than in the 1,6-carbons.
To study the extent to which acetone participates as an intermediate
of acetoacetate oxidation, 1 ,3-C14-acetoacetate was administered to rats
under the conditions of the acetone experiment and the labeling of the
liver glycogen, determined. The carbonyl isotope of the acetoacetate did
not follow the pathway of the acetone carbonyl carbon into the 1,6- and
2,5carbons of the liver glycogen, but was almost entirely incorporated
into the 3,4 positions.

EXPERIMENTAL

The 2-Ci4-acetone and 1 ,3-C14-acetoacetate employed in these experi-

ments were labeled with activities of 3.7 X lo6 and 1.62 X lo6 c.p.m. per
mg. of labeled carbon, respectively. Acetone was prepared from carboxyl-
labeled acetate by the procedure described for C14-methyl-labeled acetone
(2) ; acetoacetate was synthesized by the method employed for the cor-
responding W-labeled compound (5).
Six male albino rats were fasted for 24 hours, after which they weighed
from 110 to 130 gm. each. They were given a dose of 2.52 ml. of 1.35 M
glucose by stomach tube and 1.00 ml. of 0.29 M 2-C14-acetoneor 0.26 M
1,3-C”-sodium acetoacetate solution subcutaneously per 100 gm. Four
rats received the acetone and two the acetoacetate. The acetoacetate-
treated animals and two of these receiving acetone were kept in metabo-
lism cages for the collection of respiratory COZ.
The Cl4 activities of consecutive hourly samples of the respiratory CO,
are shown in Table I. During the 4 hours of the experiment 27 per cent
of the activity administered as acetone and 62 per cent of that adminis-
tered as acetoacetate were recovered in the CO*. The rate of excretion
of acetone radioactivity as PO2 increased throughout the experimental
period, whereas that of the acetoacetate was highest in the 1st hour and
rapidly diminished.
After 4 hours, the livers were removed under sodium amytal anesthesia
and pooled. Glycogen was isolated by the method of Good, Kramer,
and Somogyi (6) and purified by reprecipitating it twice from LiCl solu-
 W. SAKAMI AND J. M. LAFAYE 201

tion by means of alcohol. The glycogen was hydrolyzed by heating it on

a steam bath for 3 hours with 1 N HzSOp and subjected to the “bacterial
degradation” procedure of Wood, Lifson, and Lorber (7). The results
of the glucose degradations are shown in Table II. The Cl* of carbonyl-
labeled acetone was introduced into all of the carbons of the glycogen with
higher labeling in the 2,5- than in the 1 ,Bcarbons. The Cl* of carboxyl-
TABLE I
Ezcretion of Cl4 in Respiratory CO, after Administration of CW’orbonyl-Labeled
Acetone* and Cl’-Carbozyl- and Carbonyl-Labeled Acetoacetate

I Activity of respiratory Cot

I Specific activity I Total activity

Acetone Acetoacetate
-
hr. C.).rn. per ?mg. c c.p.m. per mg. c c.p.m. c.p.m.
1st 596 4980 96,700 1,090,000
2nd 1020 2020 219,000 398,000
3rd 1490 650 240,000 100,ooo
4th 1800 308 255,000 45,600

Total...................................... 811,000 1,630,OOO

* The total activities administered as acetone and acetoacetate were 2.97 X 106
and 2.63 X 1Oa c.p.m. respectively.

TABLE II
Distribution of Isotope in Liver Qlycogen after Administration of C”-Carbonyl-Labeled
Acetone and C”-Carbonyl- and Carboxyl-Labeled Acetoacetate

Labeling of carbon fractions. activity in c.p.m. per mg. C

Compound administered
1.6’ I 2,5* I 3.4’

Acetone ........................... 180 233 278

Acetoacetate ...................... 22 26 1260

* Position of the glucose unit of the glycogen.

and carbonyl-labeled acetoacetate was introduced largely into the 3,4-
carbons. A small amount of activity was present in the 1,6- and 2,5-
carbon fractions. The activity in the 1,6 positions may be largely an
artifact of the degradation procedure (8).
DISCUSSION

The introduction of the Cl* of carbonyl-labeled acetone into the 1,6-

and 2,8carbons of the liver glycogen cannot be accounted for either by
carboxylation of acetone to acetoacetate or its cleavage to “acetate” and
202 ACETONE METABOLISM

“formate.” The data are in agreement with the occurrence of an addi-

tional pathway via the direct oxidation of acetone to a 3-carbon inter-
mediate of glycolysis, in that the 2,5-carbons are more active than the
1,6. Under this interpretation of the data, comparison of the amount of
isotope in the 1,6- and 2,5-carbons, introduced by the oxidation of ace-
tone to “pyruvate,” with that in the 3,4-positions, introduced by all
mechanisms, indicates that “pyruvate” formation is a major mechanism
of acetone utilization. This is consonant with the higher activity in the
1,6 and 3,4 positions than in the 2,5 found with methyl-labeled acetone
(2). The occurrence of the additional pathway accounts for the activity
of the 3,4-carbons, without alteration of the distribution of labeling in
the 1,6 and 2,5 positions (1,6 > 2,5) produced by the cleavage reaction
w
The data of this study do not, however, conclusively prove the exist-
ence of an additional pathway of acetone catabolism in that the mecha-
nism of “formate” production may not have been correctly formulated.
Some intermediate compound other than “acetate” and capable of intro-
ducing isotope from the acetone 2-carbon into the 1,6- and 2,5-carbons
of the liver glycogen might be generated in the process of “formate”
formation. In this case, it would be necessary to assume that the ex-
tensive formation of “acetate” from acetone which has been observed by
Zabin and Bloch (9) and by Price and Rittenberg (10) occurs with this
intermediate or is produced by the carboxylation of acetone to acetoace-
tate. The physiological importance of the latter mechanism has not been
demonstrated; in Plaut and Lardy’s experiments in vitro (1) the carboxyl-
ation was rather slow, but this is also true of the rate of acetone metabo-
lism in the intact animal (10, 2).
No direct evidence concerning the significance of acetone utilization is
yet available. It appears from these experiments that it is not normally
of importance as a mechanism of the utilization of acetoacetate per se.
Neither the methyl or methylene (2) nor the carbonyl carbon of aceto-
acetate has been found to follow the metabolic pathways of the corre-
sponding carbons of acetone. However, the present experiment may re-
flect liver metabolism more than that of other organs of the body. It is
possible that acetoacetate is produced in nascent rather than free form
and that this intermediate is metabolized via an active acetone deriva-
tive. The CO*-carboxyl exchange of acetoacetate in incubating liver ho-
mogenates has been shown to be enhanced by the addition of pyruvate,
butyrate, caprylate, and certain other metabolites, but not of acetoace-
tate (1, 11). The contingency that the isopropyl group of leucine is
utilized via an acetone derivative and is normally of importance as a
source of “formate” also merits further study. In this connection, it has
 IV. S?\KAMI AND J. M. LAFAYE 203

been reported that the isopropyl group of isovaleric acid yields a 3-carbon
particle which, like acetone, is at least in part carboxylated to acetoace-
tate and apparently considerably more rapidly than is acetone per se (11,
12).

SUMMARY

C”-Carbonyl-labeled acetone has been administered to rats and the

distribution of activity in the liver glycogen determined. All of the car-
bons were labeled, with higher activity in the 2,5 than in the 1,6 posi-
tions. This indicates the existence of a pathway of acetone metabolism
other than carboxylation to acetoacetate or cleavage to “acetate” and
“formate.” A mechanism involving the direct conversion of acetone to
a 3-carbon intermediate of glycolysis has been postulated.

BIBLIOGFtAPHY

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(1948).
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