EFFECT OF ETHIONINE ON THE FREE AMINO ACIDS

IN THE RAT*

BY CHUNG WU AND JESSE L. BOLLMAN

(From the Section of Biochemistry, Mayo Clinicand ilfayo Foundafion,t Rochester,
Minnesota)

(Received for publication, April 12, 1954)

It has been reported that ethionine, when injected intrapcritoneally,

causes inhibition of proteiu synthesis (2). It,s incorporation into body
proteins (3) in the rat has also been shown. Infiltration of the liver with

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fat (4, 5), pancreatitis (6, 7), and renal necrosis (8) have also been observed
as a result of the injection of ethioninc. A close relationship between these
histochemical changes and interference with protein metabolism by ethi-
onine has been suggested (9, 10). Hence it was decided to determine how
ethionine affects the metabolism of free amino acids, since the latter de-
pends upon the state of protein synthesis or degradation in the body (11).
The effect of ethionine on the concentrations of free amino acids in plasma
and tissues and their excret,ion in the urine is reported in this study.

EXPERIMENTAL

Albino rats of the Sprague-Dawley strain weighing 130 t’o 160 gm. were
maintained on Archer chow pellets. All animals were fasted for 18 hours
before the start of the experiments and thereafter. Water was available
at all times. The rats were anesthetized at the beginning of fasting to
allow insertion of plastic tubing through the abdominal wall into the uri-
nary bladder and ligation of the urethra. They were then confined to
individual cages according to their .sizes, which prevented them from dis-
lodging the tubing. The urine was allowed to drain out through the tub-
ing as soon as it reached the bladder. Such catheterization of the urinary
bladder permits the accurate collection of urine samples at any desired
intervals.
At the end of the 18 hour fast, the rats were injected intraperitoneally
with 6 ml. of the amino acid solutions in a single dose (Table I). To the
controls 6 ml. of sodium bicarbonate solution at pH 7.4 were similarly
given. Except in one experiment, in which the excretion of urinary CX-
* A preliminary report of this work was presented at the Forty-fifth annual meet-
ing of the American Society of Kological Chemists at Atlantic City, New Jersey,
April 12 to 16, 1954 (1).
t The Mayo Foundation is a part of the Graduate School of the University of
Minnesota.
673
674 EFFECT OF KTHIONINE ON AMIh’O ACIDS

amino nitrogen at intervals following the injection of DL-ethionine into five

female rats was being studied, animals of all experiments were killed with
ether 6 hours after the respective solutions were injected. Blood samples
were drawn by cardiac puncture. The liver and kidneys were immediately
excised, frozen in dry ice, and pulverized. Urine samples collected during
the experimental periods were frozen at - 10”.
The preparation of plasma and tissue extracts and urine dialysates and
the determination of free amino acids in these extracts and dialysates by
paper chromatography have been described previously (11). Total (Y-
amino nitrogen was determined by the copper method (12). Ethionine
a-amino nitrogen was calculated from ethionine determined by paper chro-

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matography.

TABLE I
Animals Used and Amino Acids Injected

No. of rats Sex Amino acid solution injected’ Concentration, mg.

a-amino N per ml.
_ _---_--

4 M. None
5 F. “
3 M. nL-Ethioninet 1.72
F. “ 1.72
12
5 M. uL-Methioninet 1.72
4 F. “ + DL-ethionine 1.72 each

* All amino acid solulions were adjusted to pII 7.4 with sodium bicarbonate.
t Nutritional Biochemicals Corporation.
$ General Biochemicals, Incorporated.

Results
The effect of intraperitoneally administered ethionine on t.he rate of uri-
nary excret.ion of a-amino nitrogen of female rats is shown in Fig. 1. The
values of a-amino nitrogen in the urine samples, obtained bctwcen two
successive collections divided by the number of hours elapsed, were used.
Both total and ethionine a-amino nitrogen reached their peaks 4 hours
after the injection, but the highest excretion of non-ethionine a-amino
nitrogen took place 2 hours later. At the end of 24 hours following the
injection, a little more than one-third of the ethionine administered had
appeared in the urine (Fig. 2).
The data concerning the changes in individual free amino acids in plasma,
liver, and kidneys induced by administrat,ion of ethionine are shown in
Tables IT, III, and IV, respectively. Values in Tables III and IV were
calculated on a wet weight basis. There was no significant difference in
 C. WU AND J. L. BOLLMr\N 675

the values between male and female rats serving as the controls or receiving
ethionine. Hence the values given in these two instances were the aver-
ages of both sexes.

2
800
r
600
t

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2 4 6 8 10 12 14 18 24
Time in hours
FIG. 1. The urinary excretion of or-amino nitrogen following intraperitoneal in-
jection of 10.32 mg. of a-amino nitrogen of nn-ethionine into female rats. Non-
ethionine a-amino nitrogen (long dash curve) was calculated by obtaining the differ-
ence between total (solid curve) and ethionine (short dash curve) a-amino nitrogen.

40

30

20

10

b
I I I , I I I I
Qi 0
2 4 6 0 10 12 14 18 24

Time in hours
Fxo. 2. Per cent of dose excreted in the urine of female rats following intrapcri-
toneal injection of 10.32 mg. of a-amino nitrogen of nn-ethionine.
 676 EFFECT OF ETHIONINE ON AMINO ACIDS

The injection of methionine caused little change in the concentrat.ions of

free amino acids. The concentrations of alanine and cystine generally
appeared to increase, while those of glycine and swine were decreased in

TABLE II
Effccf of Eihionine on Concentration (Mg. per 100 Ml.) of Free Amino Acids and
Related Compounds in Ral Plasma

Amino acid injected

Compound
Methionine Ethionine
__- ..~ ~-__

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Alanine. 4.0 f 0.4* 4.7 f 0.5*
Cystine.......... 1.6 h 0.1 2.0 f 0.1
Dthionine. 67.9 f 6.7
Glutamic acid.. . 0.82 f 0.04 I.2 f 0.1 1.2 f 0.1
Glutamine. 10.4 f 0.4 9.6 rt 0.9 19.1 f 2.2
Glycine. . 2.7 f 0.1 2.4 f 0.3 3.3 f 0.2
Methioninc . 1.4 f 0.1 42.2 f 3.3 1.4 f 0.2
Serine . 1.6 f 0.1 2.6 f 0.2 3.0 f 0.3
Taurinc.. 6.2 rt 0.7 8.8 f 1.4 4.5 f 0.6

* The standard error of the mean.

TABLE III
h’$ect of Ethionine otl C’oncenlration (iilg. per 100 Gm.) of Fret Amino Acids and
Related Compounds in Rut Liver
--___ -~-__-
I Amino acid injected
Compound
None Methionine Ethionine

Alanine........................ 13.4 f 1.3* 19.3 f 1.2* 17.7 f 0.9*

Cystine........................ 1.7 f 0.3 4.7 f 0.3 8.0 f 0.5
E:t.hionine. 121 f 10.4
Glutamic acid.. 25.8 f 1 .O 25.4 f 2.0 43.0 f 2.5
Glutamine. _ 66.8 f 3.6 6S.7 f 4.9 73.9 f 5.5
Glycinc........................ 21.4 f 1.3 11.5 f 1.2 23.6 f 1.9
hlcthionine 1.9 f 0.2 39.8 f 5.3 4.4 f 0.9
Serine......................... 7.1 f 0.7 4.5 f 0.7 6.3 f 0.9
Taurine. 51.2 f 3.3 14s f 2.7 38.0 f 1.1
.- -L ____--
* The standard error of the mean.

the liver. These changes could be due to the conversion of methionine to

some amino acids on the one hand, and to the prevention of the capture of
some other amino acids by the cells as a result of a high level of methionine
on the other hand (13).
The injection of ethionine resulted in an increase in nearly all the amino
 C. WU AND J. L. BOLLMAN G77

aeids determined, and in many cases the increase was greater than when
methionine was injected. The largest change was observed in the plasma,

TABLE IV
E.fect of Ethionine on Concentration (Mg. per 100 Gm.) of Free Amino Acids alld
Related Compounds in Rat Kidneys
___-___
Amino acid injected
Compound - -
Methionine
-I-
Alanine. ......... 8.7 f 0.4* 12.3 f 1.4* 9.8 f l.l*
Cystine. ......... 15.2 f 1.9 15.4 f 0.8 22.3 f 1.6

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Ethionine. ....... 81.6 zk 3.2
Glutamic acid .... 73.0 f 3.5 69.7 f 6.2 81.2 i 1.4
Glutamine. ...... 11.5 f 0.9 8.9 f 0.7 12.5 f 1.2
Glycine. ......... 31.3 * 2.2 36.4 f 2.2 36.4 f 3.9
Methionine. ..... . 2.3 f 0.2 54.0 f 7.5 2.1 f 0.2
Serine ........... . 7.7 * 0.4 11.9 f 2.0 11.4 f 1.3
Taurine .......... . 73.1 f 5.1 144 f 10.6 46.4 f 1.9
-
* The standard error of the mean.

TABLE V
Effect of Ethionine on Concentration of Free Amino Acids and Related Compounds in
iat Urine*
-
Amino acid injected
-
Compound Ethionine
NOW 1 Methionine I----- hfethionine
+ ethionine
- I I Male

Alanine. .. . 0.03 0.06 O.OG -I ---

0.17 0.14
Cystinc. 0.04 0.14 0.15 0.31 0.33
Ethionine.. 3.37 22.53 14.97
Glutamic acid.. 0.05 0.05 0.08 1.13 0.19
Glutamine. .. 0.08 0.18 0.30 0.93 0.16
Glycine 0.10 0.16 0.17 2.78 0.93
Mcthionine <0.04 1.33 <0.04 <0.04 9.33
Serine . 0.06 0.09 0.11 0.75 0.31
Taurine. . 0.89 5.92 3.96 4.85 2.73

* All the values are expressed as mg. excreted in the first 6 hours following the
injection.

in which the concentrations of all amino acids except mcthionine were

significantly increased. On the other hand, the least change was found in
the kidneys, in which the concentrations of most amino acids, except cys-
tine and serine, remained unchanged. Only cystine showed appreciable
678 EFFECT OF ETHIONINE ON AMINO ACIDS

increase in all tissues. The content of methionine was increased only in

the liver.
The marked increase in the urinary excretion of free amino acids follow-
ing the injection of ethionine is shown in Table V and Fig. 3. The excre-
tion of methionine in the control and ethionine-injected rats was so low
that no accurate determination could be made, and in these instances only

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FIG. 3. Chromatograms of free amino acids in the urine of control (a) and ethio-
nine-injected (b) female rats. Aliquots corresponding to 1.25 and 0.20 per cent
volume of the 24 hour urine samples of (a) and (b), respectively, were applied on
sheets at Point X. For chromatographic procedure, see the text. 1, phenylalanine;
S, leucine-isoleucine; 5, valine; 4, proline; 5, arginine-lysine; 7, tyrosine; 8, alanine;
9, threonine; 10, glutamine; 11, taurine; id, glycine; IS, serine; i4, glutamic acid; 16,
aspartic acid; 18, histidine; 19, glutathione; 27, cysteic acid (from cystine) ; $9, ethio-
nine sulfoxide (from ethionine).

the upper limit of methionine was indicated. Contrary to the situation in

the plasma or tissues, in which no difference in response to ethionine be-
tween the sexes could be demonstrated, the excretion of free amino acids,
including ethionine itself, was many times greater in female rats than in
male rats after injection of ethionine. This finding is in parallel with that
of infiltration of the liver with fat in female rats (5). Furthermore, si-
multaneous injection of methionine on an approximately equimolar basis
greatly reduced the effect of ethionine. With greater ratio of methionine
to ethionine, the increased urinary excretion of free amino acids induced
 C. W-U AND J. L. HOLLMAN 670

by ethionine may be completely prevent’ed. It should be pointed out that

urinary excretion of methionine was much less when met,hionine was in-
jected alone than when it was injected together with ethionine. This fact
may be taken to mean that et8hionine impairs the utilization of methioninc
by the body and consequently causes a greater excret,ion of t,he latter. In-
hibition of the incorporation of methionine into t,issue proteins by ethionine
has already been noted (2).
Comment
The data on the urinary excretion of ethionine observed in this study are
in good agreement with those of Shen and Lewis (14) and of Levine and

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Tarver (3), if account is taken of a difference in the species of animals used
by the former and of a tremendous difference in dosage by the latter. The
increase in the urinary excretion of non-cthionine a-amino nitrogen follow-
ing administration of ethionine may explain the observation by Hoover
and associates (15), who reported that rats receiving an ethionine diet ex-
creted large quantities of non-urea nitrogen.
Inasmuch as no conversion from ethionine to other amino acids can be
anticipated, the increase in t,he concentration of free amino acids in plasma,
tissues, and urine following administration of ethionine can result only
from an acceleration of protein catabolism or an inhibition of protein syn-
thesis, or both. However, the data obtained in this study with fasted
rats in which the rate of protein synthesis has been kept at a minimum
suggest that an accelerated breakdown of tissue proteins is chiefly respon-
sible for the immense increase in the urinary output of free amino acids.
Since ethionine has been shown to cause renal necrosis in both male and
female rat,s (8), the possibility that impairment of tubular reabsorption may
increase the concentration of free amino acids in the urine cannot be ex-
cluded. However, histologic changes in the kidneys do not appear to be
the primary factor, because, if they were the primary factor, no difference
in response to ethionine in the urinary pattern of free amino acids between
the sexes should be expected. The fact that such a difference does exist
may, nevertheless, be explained on the basis of a difference of sex hormones.
Androgens are known to have a nitrogen-sparing action (1G). The lower
urinary excretion of free amino acids by male rats could be due 60 the coun-
teraction of androgens to minimize the rate of protein breakdown induced
by ethionine.
The diminution of the effect of ethionine by methionine is another il-
lustrat,ion of the specific antagonism between these two amino acids. How-
ever, the effect on increasing the urinary out’put of free amino acids is not
limited to ethionine alone, since, on inject,ion of B-2-thienyl-Dr.-alanine,
which has been shown to be an antimetabolite of phenylalanine in the rat
080 EFFECT OF F,THIONINE ON AMINO ACIDS

(17, 18), similar effects were observed.’ An increase in the excretion of

other amino acids, following the injection of r-aminobutyric and cu-amino-
isobutyric acids to the rat, has also been reported (19).
It is shown in this study that the urinary excretion of taurinc was con-
siderably increased by the injection of either met.hionine or ethionine.
Whether ethionine could contribute any of its sulfur in the synthesis of
taurine is unknown. Until this could be demonstrated, the increase in the
urinary output of taurine following administration of ethionine could best
be explained on the basis of the conversion from cystine (or cyst&e), of
which the formation was increased as a result of the accelerated protein
catabolism. An increase in the formation and excretion of taurine in rats

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during a fast has been reported (11).
SUMMARY

Intraperitoneal injection of DL-ethionine resulted in an increase in the

concentrations of free amino acids in plasma and tissues of both male and
female rats. In the urine, the increase was very marked in female rats
but less so in male rat,s. The effect of ethionine was diminished by the
simultaneous injection of m-methionine. Some of the implications of these
findings are discussed.
BIBLIOGRAPHY

1. Wu, C., Federation Proc., 13, 325 (1954).

2. Simpson,M. V., Farber, E., and Tarver, H., J. Biol. Chem., 182, Sl (1950).
3. Levine, hf., and Tarver, Il., J. Riol. Chem., 192, 835 (1951).
4. Farber, E., Simpson,M. V., and Tarvcr, II., J. RioZ.Chem., 182, 91 (1950).
5. Jensen, D., Chaikoff, I. l,., and Tarver, H., J. Biol. Chem., 192, 395 (1951).
6. Farber, E., and Popper, II., Proc. Sot. Exp. Biol. and Med., 74, 838 (1950).
7. Goldberg, R. C., Chaikoff, I. I,., and Dodge, A. H., Proc. Sot. Exp. Biol. and
Med., 74, 869 (1950).
8. Wachstein, M., and Meisel, E., Proc. Sot. Exp. Biol. and Med., 77, 648 (1951).
9. Farber, E., Koch-Weser, D., and Popper, H., Endocrinology, 48, 205 (1951).
10. De Almeida, A. I,., and Grossman, M. I., Gastroenlcrology, 20, 554 (1952).
11. wu, c., J. Biol. Chem., 207,775 (1954).
12. Woiwod, A. J., Biochem. J., 45, 412 (1949).
13. Christensen, H. N., Streicher, J. A., and Elbinger, IL. IX., J. Biol. Chem., 172,
515 (1948).
14. Shen, C.-W., and Lewis, H. 13., J. BioZ. Chem., 166, 115 (1946).
15. Hoover, C., Swanson, P., and Stewart, II. M., Federation Proc., 8, 386 (1949).
16. Kochakian, C. D., Vitamins and Hormones, 4, 255 (1946).
17. Ferger, M. F., and du Vigneaud, V., J. Biol. Chem., 179, 61 (1949).
18. Garst, R. G., Campaigne,E., and Day, H. G., J. Biot. Chem., 180, 1013 (1949).
19. Lang, K., and Oster, H., Biochem. Z., 324,443 (1953).

1 Wu, C., unpublished data.

EFFECT OF ETHIONINE ON THE FREE
AMINO ACIDS IN THE RAT
Chung Wu and Jesse L. Bollman
J. Biol. Chem. 1954, 210:673-680.

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