THE EFFECT OF ETHIONINE ON TRANSMETHYLATION

FROM METHIONINE TO CHOLIKE AND CREATINE

IN VIVO”

BY SOFIA SIMMONDS, ELIZABETH B. KELLER, JOSEPH P. CHANDLER,

AND VINCENT DU VIGNEAUD
(From the Department of Biochemistry, Cornell University Medical College,
New York City)

(Received for publication, October 20, 1949)

In order to determine the specificity of the methyl group of methionine

in the conversion of methionine to cystine in. vivo, Dyer (1) tested ethionine

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(X-ethylhomocysteine) to see whether it would replace cystine in the diet.
She found that ethionine, in contrast to homocystine and methionine,
would not support the growth of young rats on a diet devoid of cystine
and containing suboptimal amounts of methionine. It was concluded,
therefore, that deethylation of ethionine to yield homocysteine did not
occur in the rat. Further evidence in support of this conclusion was
presented by Shen and Lewis (Z), who found that the rabbit excreted
the sulfur of ethionine largely as extra neutral sulfur.
Dyer (1) also observed that supplementation of a 5 per cent casein
diet with 0.54 per cent ethionine produced a marked toxicity in rats. The
food consumption of animals on the ethionine-containing diet dropped
to about two-thirds of that found with the unsupplemented 5 per cent
casein diet; animals receiving ethionine lost weight and died after about
10 days, while animals on the unsupplemented diet neither gained nor
lost weight. At autopsy, however, no gross pathology due to the toxic
effects of ethionine was observed. The toxicity of ethionine could be
overcome by including a molar equivalent of methionine in the diet.
In this connection, it is of interest that ethionine has been found to inhibit
the growth of Escherichia coli (3, 4) and Plasmodium know& (5), and the
growth inhibition for both of these organisms could be reversed by methi-
onine. Although these data do not provide any clue to the mechanism of
the action of ethionine, its behavior as a metabolic antagonist may be
attributed to interference with some phase of the metabolism of methionine.
Farber, Simpson, and Tarver (6) have reported that ethionine appar-
ently interferes with protein synthesis, since experiments both in vivo
and in vitro showed that ethionine inhibited the incorporation into liver
*The authors wish to express their appreciation to the Lederle Laboratories
Division, American Cyanamid Company, for a research grant which has aided
greatly in this work.
191
192 ETHIONINE AND TRANSMETHYLATION

protein of S35 from labeled methionine and Cl4 from labeled glycine. Ex-
periments in vivo with both amino acids indicated that additional methi-
onine would reverse the inhibition. These authors also observed that
ethionine inhibited the conversion of methionine to cystine and that
this inhibition, also, could be reversed by additional methionine. They
suggested that ethionine probably prevented the demethylation of methi-
onine. Stekol and Weiss (7) recently have shown that choline as well
as methionine reverses the growth inhibition caused by ethionine in the
rat.
The investigation described in the present paper was designed to de-
termine whether ethionine interferes with the transmethylation reactions

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involved in the formation of choline and creatine. Methionine labeled
with deuterium in the methyl group was administered to adult rats and,
after a suitable period, the deuterium content of the body choline and
creatine was determined as a measure of the extent of transmethylation.
Two experiments were carried out, each with a group of four rats. In each
experiment, two animals were given ethionine in addition to the labeled
methionine, and two animals served as controls, receiving only labeled
methionine. Since it was known that the amount of deuteriomethyl
transferred from dietary methionine to tissue choline and creatine in a
given time is dependent upon the amount of deuteriomethionine ingested
(8), the food intake of each group of rats was controlled by the paired
feeding technique.
Preliminary feeding experiments to determine the level at which ethio-
nine may be incorporated in the diet without seriously reducing the food
consumption of test animals showed that when a basal diet containing
0.7 per cent methionine was supplemented with 0.7 per cent ethionine
the daily food intake dropped from over 10 gm. to 1 to 2 gm. per day in
3 days. However, when the ethionine supplement was reduced to 0.5
per cent, the daily food intake dropped only to 7 to 8 gm. The latter
diet, in which t,he molar ratio of ethionine to methionine is about 2~3,
was used for the feeding experiments with labeled methionine. In view
of the fact that Farber et al. (6) have found that ethionine interferes with
the conversion of methionine to cystine in vivo, it should be noted that
the synthetic diet employed in our experiments contained 0.5 per cent
cystine.
At the end of the feeding period, the rats were sacrificed, and choline
and creatine were isolated from the tissues of each animal and analyzed
for deuterium. The data showed that the presence of ethionine in the
diet reduced the amount of choline synthesized from dietary methionine
by about 20 per cent. On the other hand, the amount of creatine syn-
thesized from dietary methionine was not decreased by the presence of
ethionine in the diet,.
 SIMMONDS, KELLER, CHANDLER, AND DU VIGNEAUD 193

EXPERIMENTAL

Feeding of DL-Ethionine and Deuterio-DL-methionine-The four adult male

rats in each group were fed ad libitum a choline-free amino acid diet for
a control period of 8 days, at the end of which time they had regained their
initial weight. The percentage composition of this basal diet was as fol-
lows: amino acid mixture (9, 10) 21.3, L-cystine 0.5, Dr.-methionine 0.7,
sucrose 53.5, hydrogenated vegetable oil 19, corn oil 1, and Osborne and
Mendel’s salt mixture (11) 4. The fat-soluble vitamins were included in
this diet in the following amounts per 100 gm. of diet: 4000 U. S. P. units
of vitamin A, 400 U. S. P. units of vitamin D, 1 mg. of a-tocopherol acetate,
0.1 mg. of Z-methyl-l ,4-naphthoquinone. The B vitamins were given

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twice a day as a supplement. Each rat received daily 20 y each of thiamine
TABLE I
Food Consumption and Weight Changes

Control period Experimental period

Group No. Rat No. Initial
Food and final Diet Food Final
/ intake intake weight
weight
.!. -I- _ .-__
I:m. per daj km. :m. per da) m.
14.0 260-254 Ethionine 8.1 242
12.4 266-273 “ 8.0 254
11.6 270-272 Control 8.0 252
“
12.9 250-247 8.1 232
11.4 244-246 Ethionine 7.7 235
14.0 236-260 “ 7.6 238
10.5 232-239 Control 7.6 230
10.7 253-254 “ 7.6 256
-

hydrochloride, riboflavin, pyridoxine hydrochloride, and nicotinic acid, 0.2

mg. of calcium dl-pantothenate, and 25 mg. of choline-free ryzamin-B (9).
During the subsequent experimental period, two rats in each group
were given 0.5 per cent nn-ethionine (I), which replaced an equivalent
amount of sucrose in the basal diet, and all the rats received in place of
the non-isotopic methionine 0.7 per cent deuterio-nn-methionine (12))
cont.aining 89.4 atom per cent deuterium in the methyl group.
During the experimental period, the food intake of all rats in each
group was limited, by the paired feeding technique, to the amount eaten
by the rat with the lowest food consumption. The food consumption and
weight changes of the rats during both the control and experimental periods
are given in Table I. It will be noted from the data in Table I that the
presence of the ethionine in the diet decreased the food consumption from
over 10 gm. per day to about 8 gm. per day. With Group 1 the experi-
mental period lasted 9 days, and with Group 2, 14 days.
194 ETHIONINE AND TRANSMETHYLATION

Creatine was isolated from the carcass of each rat as creatinine potas-
sium picrate (12). Analysis of the recrystallized picrates for creatinine
with the Jaffe color reaction indicated that they were pure within the limits
of accuracy of the calorimetric method. Choline was isolated from each
rat carcass as the choline chloroplatinate (12). The chloroplatinat.es
from Group 1 were recrystallized several times from alcohol-water mixtures,
and those from Group 2 were recrystallized three times from water. The
platinum determinations for each of the isolated chloroplatinates agreed
with the theoretical values.
The creatine and choline derivatives were analyzed for deuterium and
the deuterium in the methyl group was calculated on the assumption that

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TABLE II
Choline and Creatine Synthesis in Presence and Absence of Dietary Ethionine
Per cent of choline
methyl derived from
dietary methionine*
-.
1 1 Ethionine t 7.20 zk 0.10
2 “ 6.85 f 0.18 8.66 It 0.10
3 Control 5.50 f 0.18 10.5 dz 0.08
4 “ 5.68 f 0.18 10.4 f 0.07
2 5 Ethionine 14.45 f 0.31 14.65 f 0.14
6 I‘ 12.80 f 0.36 14.82 f 0.16
7 Control 12.89 f 0.36 18.81 f 0.16
8 “ 12.44 f 0.36 18.76 f 0.16
-
* Atom per cent deuterium in methyl groups of isolated compound
x 100.
Atom per cent deuterium in methyl group of methionine fed
t Samplelost.

all the deuterium in the compounds was present in the methyl group
(12). The results are recorded in Table II.
SUMMARY

nn-Ethionine (S-ethyl-nn-homocysteine) was fed to adult male rats at

the level of 0.5 per cent in a synthetic amino acid diet containing 0.7
per cent deuterio-nn-methionine. The deuteriomethyl content of the tis-
sue choline and creatine was determined and compared with that of choline
and creatine isolated from pair-fed controls which received labeled methio-
nine but no ethionine. It was observed that ethionine decreased the
amount of transmethylation from methionine to choline by about 20 per
cent. The amount of creatine synthesized from dietary methionine was
not decreased by the presence of ethionine in the diet.
 SIMMONDS, KELLER, CHANDLER, AND D’ZT VIGNEAUD 195

The authors wish to express their appreciation to Mrs. Josephine T.

Marshall for the analyses reported in this paper and to Miss Miriam L.
Thompson for her assistance with some of the isolations.
BIBLIOGRAPHY

1. Dyer, H. M., J. Biol. Chem., 134, 519 (1938).

2. Shen, C.-W., and Lewis, H. B., J. Biol. Chem., 166, 115 (1946).
3. Harris, J. S., and Kohn, H. I., J. Pharmacol. and Exp. Therap., 73, 383 (1941).
4. Roblin, R. O., Jr., Lampen, J. O., English, J. P., Cole, Q. P., and Vaughan, J.
R., Jr., J. Am. Chem. Sot., 67, 290 (1945).
5. McKee, R. W., and Geiman, Q. M., Federation Proc., 7, 172 (1948).
6. Farber, E., Simpson, M., and Tarver, H., Federation Proc., 8, 196 (1949).

Downloaded from http://www.jbc.org/ by guest on October 9, 2020

7. Stekol, J. A., and Weiss, K., J. Biol. Chem., 179, 1049 (1949).
8. Cohn, M., Simmonds, S., Chandler, J. P., and du Vigneaud, V., J. BioZ. Chem.,
162, 343 (1946).
9. du Vigneaud, V., Chandler, J. P., Moyer, A. W., and Keppel, D. M., J. BioZ.
Chem., 131, 57 (1939).
10. du Vigneaud, V., Kilmer, G. W., Rachele, J. R., and Cohn, M., J. BioZ. Chsm.,
166, 645 (1944).
11. Osborne, T. B., and Mendel, L. B., J. BioZ. Chem., 37, 572 (1919).
12. du Vigneaud, V., Cohn, M., Chandler, J. P., Schenck, J. R., and Simmonds, S.,
J. BioZ. Chem., 140, 625 (1941).
 THE EFFECT OF ETHIONINE ON
TRANSMETHYLATION FROM
METHIONINE TO CHOLINE AND
CREATINE IN VIVO
Sofia Simmonds, Elizabeth B. Keller, Joseph
P. Chandler and Vincent du Vigneaud
J. Biol. Chem. 1950, 183:191-195.

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