THE METABOLISM OF AMINO ACIDS.

BY JAMES MURRAY LUCK.

(From the Department of Chemistry, Stanford University, California.)
(Received for publication, December 29, 1927.)

It was shown by Seth and Luck (1) that the intestinal absorp-
tion of glycine and alanine by rabbits was followed by pronounced
aminoacidemia. Other amino acids when administered in similar
amounts provoked smaller increasesin the amino nitrogen content
of the blood. Glutamic acid and aspartic acid were absorbed

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with but a slight aminoacidemia.
The question then arose as to whether these striking differences
in behavior were due to equally profound differences in the rates
of absorption from the alimentary canal, in the rates of diffusion
into liver, muscle, and other tissues, and in the rates of metabolism
of the amino acids therein. It may be recalled that Bang (2)
demonstrated marked inequalities in the absorption of various
amino acids by isolated fragments of liver suspended in solutions
of these substances. A not unlikely explanation of the results of
Seth and Luck would seem to be that the glycine-alanine amino-
acidemia was due to slow diffusion of the substances into liver and
muscle, or a low rate of oxidation of the metabolites, or both. In
contrast, the failure of glutamic and aspartic acids to provoke an
appreciable increase in the amino acid content of the blood might
be attributed to their rapid absorption therefrom.
According to such a theory it might be expected that the inges-
tion of glycine and alanine would causebut a small increase in the
amino acid content of liver and muscle, or a slow conversion of the
amino nitrogen to urea. On ,the other hand glutamic acid might
be expected to increase more markedly the amino acid content of
liver and muscle or to lead to more rapid urea formation than is
observed with glycine and alanine. Finally one may advance the
view that the differences in the degree of aminoacidemia may be
due in part to specific differences in the excretion of amino acids
by the kidney.
13
14 Metabolism of Amino Acids
In this paper a report is given of changes in the amino acid con-
tent of blood, liver, and muscle following the oral administration
of amino acids. The object of the work was to determine the
relationship if any which existed between the degree of amino-
acidemia obtained and the concentration increase of the amino
acids in liver and muscle. The 6ndings proved to be of a somewhat
unexpected nature.
EXPERIMENTAL.

Male albino rats of 160 to 220 gm. in weight (except in Experi-

ments 55 and 58) were employed throughout. The actual weights
of the animals in gm. on the day of experiment were as follows:
Experiment 19, 160, 164, 180, 164; Experiment 21, 188,206, 188,

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196; Experiment 23,200, 180,188, 180; Experiment 25, 198,220,
188, 190; Experiment 27, 210 to 220; Experiment 29, 194, 190,
198, 199; Experiment 31,210 to 218; Experiment 33, 190 to 210;
Experiment 35, 200’ to 220; Experiment 39, 200 to 220; Experi-
ment 41, 190 to 210; Experiment 44, 160 to 170; Experiment 48,
200 to 210; Experiment 50,180 to 200; Experiment 52,155 to 160;
Experiment 55,120 to 151; Experiment 58,128 to 147.
The animals were maintained on a standard diet1 and fasted for
24 hours preceding an experiment. The animals were used in
groups of four or five. The experimental material was adminis-
tered by stomach tube (a No. 8 French catheter was used) after
which the animals were killed at intervals of approximately 0,
0.8, 2, 4, and 6 hours from the time of administration. The rats
were kilIed by stunning, and rapid incision of the thorax. The
blood which drained away was collected in a crucible containing
powdered potassium oxalate.2 The liver and 4 to 5 gm. of muscle
1 Cracked wheat 26, oatmeal 26, corn-meal 26, flaxseed meal 10, dried
whole milk (Klim) 5, alfalfa (leaves and blossoms) 5, bone meal 15, sodium
chloride 0.5,-with greenstufTs two or three times per week.
2 It was found necessary to stun the animal by a sharp blow in the mid-
cervical region. If the blow were received on the head, profuse bleeding
took place through the nose. Such blood was rejected in view of the
possibility that it might have come in contact with the mouth which would
probably contain drippings from the stomach tube. If the blow were
received in the thoracic region, intravascular blood clotting was observed
to proceed with such rapidity that only a drop or two of semiclotted blood
would drain from the incision.
 J. M. Luck 15

from the hind limbs were promptly excised and frozen by immer-
sion in liquid air. The amino nitrogen contained in these samples
was determined by the method described in the preceding paper
(3). The amino nitrogen content of the blood was determined by
the method of Folin (4).
The dicarboxylic acid fraction of hydrolyzed caseinogen, the
monoamino monocarboxylic acid fraction of hydrolyzed casein-
ogen, totally hydrolyzed egg albumin, dl-aspartic acid, d-glutamic
acid, glycine, and dhalanine, were used. The last four were ob-
tained from the Eastman Kodak Company. From acid-hydrolyzed
caseinogen the monoamino monocarboxylic acids were obt,ained
by the butyl alcohol extraction method of Dakin (5), and the
dicarboxylic acids by precipitation of the calcium salts (6) from

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the non-extractable portion. The residue represented the hexone
base fraction. The sulfuric acid-barium hydroxide method was
employed in hydrolysis of the egg albumin.
Each rat received 3 cc. of an aqueous, neutralized, solution of
the experimental substance equivalent in concentration to 0.2,
0.3, or 0.4 gm. of amino nitrogen per kilo.
It will be seen that by this procedure any questionable secondary
effects resulting from prolonged anesthesia and the use of surgical
methods are avoided. The animal is in a normal state throughout
the experiment. The metabolism of the amino acids proceeds
normally in every tissue. Within 2 minutes of the killing of the
animal the samples are frozen and postmortem autolytic changes
are prevented.
It is also apparent that the method of group experimentation
permits one to make a number of successive analyses at suitably
spaced intervals of time. This cannot be done by the single
animal method without employing anesthetics and resorting to
troublesome if not questionable operative means. It is evident
moreover if smooth continuous curves be obtained by the series
method when time is plotted against the tissue concentration of
the metabolite, that each animal serves as a confirmatory check
against its neighbors in the series. It is almost unnecessary to
point out that the use of small animals is no mean consideration
whep costly amino acids are to be administered.
16 Metabolism of Amino Acids

Amino N,
mg. per 100
cc. blood.

701 I 2I 3, 4I 5I
HOURS AFTER A LWIMIS TY?flT/oN
CHART 1A.

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Amino N,
mg. pe; 100
gm. liver.

HOURS AFTER ADH1IMiSTRRT/ON

CHART 1B.

Amino N,
mg. per 100
gm. muscle.

h’dURS /IfT,CR ADMftWSTRATlON

CHART 1C.
CHART 1. Experiment 19, water; Experiment 23, glycine; Experiment
27, dicarboxylic acids (m--- -•); Experiment 31, hexone bases
w- - -X) ; Experiment 35, monoamino acids (-e-*--m); Experiment
39, alanine (X -X).
 Amino N,
mg. per 100
cc. blood.

HOURS AFTER ADMINfSTRRTlON

CHART 2 A.

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HOURS Af=TEff ADMlN/STRAT/ON
CHART 2 B.

Amino N,
mg. per 100
gm. muscle.

I I 1 I I
1 2 3 9 5 6
HOURS AFTEU ADMIM..STRATION
CHART 2 C.
CHART 2. Experiment 21, water; Experiment 25, glycine; Experiment
29, dicarboxylic acids; Experiment 33, hexone bases; Experiment 41,
alanine; Experiment 44, totally hydrolyzed albumin; Experiment 48,
alanine.
17
 18 Metabolism of Amino Acids

Amino N,
mg. per 100
cc. blood.

HOURS AFTER AOMINISTR~TION

CHART 3 A.

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Ammo N,
mg. per 100
gm. liver.

HOURS AFTa!% ADMhWTRAT~OfV

CHART 3B.

Amino N,
mg. per loo
gm. muscle.

k/ffS AFTER AOMiNtsTRATiON

cIL4RT 3C.
CHART 3. Experiment 50, totally hydrolyzed albumin; Experiment 52,
aspartic acid; Experiment 55, monoamino acids; Experiment 58, glutamic
acid.
 J. M. Luck 19
RWAltS.

Amino Acid Content of Blood, Liver, and Muscle.-The accom-

panying curves are self-explanatory. Charts 1 A, 1 B, 1 C repre-
TABLE I.
Ammonia Content of Liver and Muscle.
-
Ammonia N, mg. per 100 gm. thue.

TiCBIle. Time after adminihation..

-
Oh?. g* id hrs.
-
W8ter. Mu&e. 15.0 12.5 12.1 11.9
Liver. 18.5 18.6 15.3 15.9

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Water. Muscle. 13.7 14.2 14.1 12.6
Liver. 17.4 16.1 17.7 17.2

Glycine, 0.19 gm. N per kilo. Muscle. 18.2 13.2 13.4

Liver. 19.5 16.9 16.2 17.3

Glycine, 0.38 gm. N per kilo. Muscle. 10.8 12.1

Liver. 14.6

Dicarboxylic acid fraction, Muscle. 12.5 14.9 13.6 12.9

0.20 gm. N per kilo. Liver. 18.7 21.6 22.5 19.7

Dicarboxylic acid fraction, Muscle. 12.9 11.6 12.6 13.6

0.40 gm. N per kilo. Liver. 16.3 19.6

Hexone base fraction, 0.20 Muscle. 11.9 11.1 14.0

gm. N per kilo. Liver. 14.3 17.3 15.7

Hexone base fraction, 0.80 Muscle. 12.6 14.4 14.9 14.2

gm. N per kilo. Liver. 18.4 20.6 20.6 22.0

Monoamino acid fraction, Muscle. 12.4 13.0 13.0 12.6

0.20 gm. N per kil’o. Liver. 16.9 16.5 17.9 16.0

Alanine, 0.20 gm. N per kilo. Muscle. 12.9 13.2 14.5 13.3
Liver. 17.1 18.9 17.8 14.5

Alanine, 0.40 gm. N per kilo. Muscle. 13.5 15.0

Liver. 16.2 16.9
-
sent the results of experiments in which the substances were ad-
ministered in quantities of 0.20 gm. of amino nitrogen per kilo
20 Metabolism of Amino Acids

of rats. In Charts 2 A, 2 B, 2 C the quantities were 0.40 gm. of

amino nitrogen, and in Charts 3 A, 3 B, 3 C, 0.30 gm. of amino
nitrogen per kilo. There are, however, the following exceptions:
Experiment No. gm. per kg.
23 0.19, amino N
25 0.38, “ “
33 0.80, total “
44 0.47, amino “

Ammonia Content of Liver and Muscle.-It proved convenient

in some of the experiments to determine the amounts of ammonia
evolved during the period of boiling in alkaline solution required
by this analytical method. The ammonia was collected in dilute
sulfuric acid and estimated by Nesslerizing. The results are

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presented in Table I.
The ammonia content of liver and muscle showed no appreciable
change or but slight increases following the absorption of amino
acids. The absolute ammonia values were invariably greater in
liver than in muscle, being 14.3 to 22.5 mg. per cent in liver and
10.8 to 15.0 mg. per cent in muscle. These values agree approxi-
mately with those reported by others (7). We are not satisfied,
however, that the methods employed in this work or in that of any
others known to the writer, give ammonia values of much signi-
ficance. It is probable (cf. Gad-Andresen, 1919; Warburg et al.,
1924; Meyerhof, 1925; Grafe, 1925) that the same scrupulous
care needs to be exercised in such estimations as is known to be
indispensable in determining the ammonia content of blood.

DISCUSSION.

The most striking result of these experiments is to be seen in the

absence of any appreciable increase in the amino nitrogen content
of muscle following the oral administration of amino acids.
To this generalization, glycine alone is exceptional. Following
the oral administration of this substance the amino nitrogen con-
tent of muscle increases slowly and uniformly, this increase pro-
ceeding at an unchanged rate after the maximum increases in the
blood and liver have been observed (Charts 2 A, 2 B, 2 C). What-
ever additional significance these relative rates of change may
have, certain it is that the accumulation of glycine in muscle
 J. M. Luck 21

proceeds much more slowly than it does in the liver. This, is

doubtless accountable in part by the method of administration,
-all of the absorbed amino acids having to pass through the liver
before entering the systemic circulation. It is not even im-
probable that the pronounced difference in the behavior of the
whole group of amino acids in liver and muscle is to be explained
similarly. We are examining this point experimentally by ad-
ministering these substances in other ways.
It is perhaps well to point out that the amino nitrogen increase
in muscle is not determined by the level of amino acids in blood.
This follows from the results obtained with alanine which caused
an equally marked aminoacidemia but no apparent increase in
muscle amino nitrogen. Nor is there any relationship between the

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amino acid increase in liver and the behavior in muscle. Totally
hydrolyzed egg albumin increased the amino nitrogen content of
the liver almost as greatly as did glycine, but resulted in no appre-
ciable change in the amino acid content of muscle (Charts 2 B,
2 C). The behavior of glycine in muscle is apparently specific for
that amino acid. It cannot be considered a representative mem-
ber of the products of protein hydrolysis and the deduction of
generalizations in protein metabolism from results obtained pri-
marily if not solely with glycine is to be cautioned against.
The remarkable differences in behavior of glycine and alanine
were quite unexpected. In view of their neighborly relationship
in a homologous series and the similar increases in the amino
nitrogen content of blood following their oral administration, it
was considered likely that they would induce the same measure
of change in liver and muscle. It is to be observed, however, that
while glycine induced a marked increase in the amino nitrogen
content of the liver, alanine caused but a slight and transient
increase. The contrast in their behavior in muscle has already
been pointed out. It is not likely, moreover, that results materi-
ally different would have attended the use of d-alanine instead
of the racemic mixture. For though it might be supposed that
d-alanine would be metabolized more or less rapidly than Lalanine,
it is very improbable that any pronounced increase in liver and’
muscle amino nitrogen caused by one isomer would be completely
nullified by the other. It is to be observed in this connection that
the naturally occurring isomer of glutamic acid behaved quali-
tatively in muscle like the racemic alanine (Charts 2 C, 3 C).
22 Metabolism of Amino Acids

It will have been noticed that though in muscle none of the

amino acids except glycine increased the amino nitrogen content
of that tissue, most of them elevated in some measure the amino
nitrogen content of the liver. This in itself throws little light on
the relative dominance of the roles of liver and muscle in protein
metabolism. As has already been mentioned, the mode of ad-
ministration of the experimental material almost certainly deter-
mines in part the muscle-liver picture. It might appear per-
missible, moreover, to regard muscle as being so efficient in the
metabolism of absorbed amino acids that it succeeds in main-
taining its amino nitrogen content at the normal level. This sort
of explanation is discredited by the vast amount of evidence, much
of it of great weight, concerning the locus of amino acid catabolism.

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Most of this regards but poorly the urea-forming power of muscle.
As for the anabolic change, the rapid synthesis of protein from the
absorbed material, it is difficult to see how this could proceed from
a single amino acid. Finally, a quite improbable explanation
would be to regard muscle as being impermeable to all amino acids
but glycine. One would then be driven to exercise unusual in-
genuity to account for the formation of the muscle proteins.
It is somewhat premature to advance an explanation for the
profound inequalities in the rates of increase of the various amino
acids in blood and liver. Additional information must first be
had concerning the excretion of these substances by the kidney,
their rates of oxidation, and the differences if any in their rates of
absorption from the alimentary canal. With respect to the last
mentioned point we have reason to believe that glycine, alanine,
glutamic acid, and aspartic acid are absorbed at much the same
rate from an isolated intestinal loop of the dog (1). It will also
be noticed that all of the amino acid preparations employed in this
work are readily soluble in water,-a consideration which is perti-
nent to the question of their absorption.
A discussion of these experiments cannot be complete without
reference to the closely related work of others.
It appears as a 6rst consideration that the amino acid content
of animal tissues fluctuates normally within rather narrow limits.
Fasting appears to be without effect, as demonstrated by Van
Slyke and Meyer (8) on dogs and by Mitchell, Nevens, and
Kendall (9) on the entire carcasses of rats after fasting for 19 to
 J. M. Luck 23

26 hours. We too have never observed any change in the amino

acid content of the whole animal or in the muscles and livers of
rats as a result of fasting 1 or 2 days. Buglia and Costantino
(10) in a few experiments on dogs observed slightly higher values
for the non-urea, non-protein nitrogen of muscle after fasting
periods of 12 to 25 days. It does not necessarily follow, however,
that the amino acids were similarly increased in quantity. The
analytical method employed by these investigators (desiccation
of the samples at 70-80” and formal titration of the extract) is also
open to criticism.
Neither do high nor low protein diets appear to alter very
markedly the concentration of amino acids in tissues. Mitchell,
Nevens, and Kendall report experiments of 11 to 48 days duration

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in which rats were maintained on nitrogen-free diets without
TABLE II.
Amino Nitrogen Content of a Foreign Group of Rats.

Bat No. I. Amino

MUSOlE.
N, mg. per 100 gm. tissue.

Liver.

1 78.9 70.9
2 76.0 66.5
3 72.6 63.7

change in the amino acid content of the animals. Kiech (unpub-

lished data) in this laboratory has observed that high and low
protein diets administered to rats for 2 day periods are productive
.of small but certain differences. Thus twenty-six rats which had
been on a starch-butter diet for 2 days contained an average of
47.4 mg. of amino nitrogen per 100 gm. of tissue. All values fell
between ,40.2 and 51.8. Similarly nine rats on a high protein
diet (87 per cent caseinogen) for 2 days gave corresponding values
of 50.6 (47.1 to 53.5).
There is, however, an indication as shown in Table II that some
factors presumably of dietary origin may influence profoundly
the amino acid content of liver and muscle. The group of rats
reported in Table II was of the same age, size, and sex as the
animals used in all the other experiments. They had been fasted
for 24 hours before analysis. They came from another colony and
24 Metabolism of Amino Acids

were related as second or third cousins to one section of our own

stock. They had, however, been maintained since weaning on a
very different and much more varied basal diet. This group, the
only foreign one examined, has been the only one to give abnormal
basal values. Mitchell (7) has also reported on adult rats which
gave, so it seems to us, unusually high amino acid values.
The amino acid content of muscle is increased following hepa-
tectomy (11).
Apart from changes induced by the ingestion of proteins or
their products of hydrolysis, the amino acid content of blood is
maintained at quite a constant level. In acute yellow atrophy of
the liver (12)) myelogenous leucemia (13-15)) possibly in pernicious
anemia (16)) in polycythemia (17), and in hydrazine poisoning

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(18), the amino acid content of the blood is greater than normal.
It is also probable that in disturbances of carbohydrate metabolism
of pancreatic origin (13, 19), the amino acid content of the blood
is altered.
Finally reference may be made to other experiments on the
effect of protein or amino acid administration on the amino acid
content of muscle and liver. The feeding of large quantities of
meat to dogs was found by Wishart (20) to be without influence
on the non-urea, non-protein nitrogen of the muscle. This agrees
with our own observations on the fate of ingested amino acids.
In the well known experiments of Van Slyke and Meyer (21),
alanine and hydrolyzed caseinogen were injected intravenously
in dogs. Cathcart (22) administered glycine to dogs by the same
means. Although the results of these experiments are in agree-
ment with oui- own in so far as they indicate changes of greater
magnitude and rapidity in the amino acid content of liver than in
muscle, they differ with respect to the absolute increases observed.
This is, however, to be expected in view of the different mode of
administration of the experimental materials, and the important
differences in subsequent treatment of the animal and analysis
of the tissue samples.
The interesting experiments of Lombroso, Artom, Paterni, and
Luchetti (23) on the entrance of amino acids into the perfused
.liver, muscle, and kidney are hard to interpret because of the con-
flicting results obtained with defibrinated blood and Ringer’s SOIU-
tion respectively.
 J. M. Luck 25
Experiments similar in method are now in progress to study the
entrance of the amino acids into liver and muscle after subcutane-
ous injection, and to determine the differences, if any, in their
rates of oxidation, in vivo.

Part of this work was done in the Biochemical Laboratory of the

University of Toronto.
SUMMARY.

1. When amino acids are administered to rats, per OS, and in

equimolecular amounts, increases of varying magnitude are ob:
served in the amino acid content of liver, but no appreciable
change, except with glycine, is observed in the amino acid content

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of muscle.
2. Although glycine and alanine increased in the same measure
the amino acid content of the systemic blood, the former provoked
a great increase in the amino nitrogen content of liver, while the
latter caused no significant change.
3. In most cases no appreciable changes were observed in the
ammonia content of liver and muscle.

BIBLIOGRAPHY.

1. Seth, T. N., and Luck, J. M., Biochem. J., 1925, xix, 366.
2. Bang, I., Biochem. Z., 1916, lxxiv, 278.
3. Luck, J. M., J. Biol. Chem., 1928, lxxvii, 1.
4. Folin, O., J. Biol. Chem., 1922, li, 377.
5. Dakin, H. D., Biochem. J., 1918, xii, 290.
6. Foreman, F. W., Biochem. J., 1914, viii, 463.
7. Sumner, J. B., J. Biol. Chem., 1916, xxvii, 95. Mitchell, H. H., J.
Biol. Chem., 1918, xxxvi, 501. Van Slyke, D. D., J. Biol. Chem.,
1913-14, xvi, 187. But cf. Gad-Andreaen, K. L., J. Biol. Chem.,
1919, xxxix, 267.
8. Van Slyke, D. D., and Meyer, G. M., J. BioZ. Chem., 1913-14, xvi, 231.
9. Mitchell, H. H., Nevens, W. B., and Kendall, F. E., J. BioZ. Chem.,
1922, lii, 417.
10. Buglia, G., and Costantino, A., Arch. ital. biol., 1913, lx, 51.
11. Bollman, L. J., Mann, F. C., and Magath, T. B., Am. J. Physiol.,
1926, lxxviii, 258.
12. Feigl, J., and Lute, H., Biochem. Z., 1917, lxxix, 162. Tileston, W.,
and Comfort, C. W., Jr., Arch. Znt. Med., 1914, xiv, 620. von Falken-
hausen, M. F., Arch. exp. Path. u. Pharmakol., 1924, ciii, 322.
‘26 Metabolism of Amino Acids
13. Okada, S., and Hayashi, T., J. Biol. Chem., 1922, Ii, 121.
14. Martin, C. L., and Denis, W., Am. J. Med. SC., 1920, clx, 223.
15. Sandiford, K., Boothby, W. M., and Giffin, H. Z., J. Biol. Chem., 1923,
Iv, p. xxiii.
16. Gorchkoff, M., Grigorieff, W., and Koutoursky, A., Compt. rend. Sot.
biol., 1914, lxxvi, 454.
17. Luck, J. M., unpublished data.
18. Lewis, H. B., and Iaume, S., J. Biol. Chem., 1926-27, lxxi, 33.
19. Wiechmann, E., Z. ges. erp. Med., 1924, xliv, 158. Wolpe, G., Miinch.
med. Woch., 1924, lxxi, 363.
20. Wishart, M. B., J. BioZ. Chem., 1915, xx, 535.
21. Van Slyke, D. D., and Meyer, G. M., J. BioZ. Chem., 1913-14, xvi,
197, 213.
22. Cathcart, G. D., Bioehem. J., 1916, x, 197.
23. Lombroso, U., Artom, C., Paterni, L., and Luchetti, Arch. ital. biol.,

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1915, lxiv, 165.
THE METABOLISM OF AMINO ACIDS
James Murray Luck
J. Biol. Chem. 1928, 77:13-26.

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