Download A Study of Free Amino Acids and of Glutamine

A Study of Free Amino Acids and of Glutamine
Synthesis in Tumor-bearing Rats*
CHUNG Wu
(Department
of Internal Medicine,
AND JERE M. BAIJER
University of Michigan
Medical School, Ann Arbor, Mich.)
SUMMARY
The concentrations
have
been
of some eight free amino acids in the plasma, liver, and muscle
determined
in male
rats
bearing
tumors
of different
sizes
(Walker
car
cinoma 256) and in their pair-fed controls. No change in the concentrations
of free
amino acids was found in the plasma or tissues when tumors were of small size. As the
tumors grew larger, the concentrations
of most free amino acids determined were in
creased in the plasma and liver and decreased in the muscle. Glutamine was the only
compound studied whose concentration was consistently decreased in the plasma, liver,
and muscle.
Increased excretion of the nonprotein-bound
form of all amino acids determined
was observed
in the urine
of tumor-bearing
rats.
The
excretion
of free amino
acids
could be shown to be increased also, if the quantities excreted daily were expressed as
a function of carcass weight rather than total body weight (carcass plus tumor).
Both glutamine synthetase and glutamyltransferase
activities in the liver of tumor
bearing rats were decreased when the tumors were still very small, and no further de
crease was noted as the tumors grew larger. However, the ratio of the synthetase
activity to the transferase activity was less in the tumor-bearing
rat than in the con
trol rat.
Even to gross inspection,
ing in the advanced
stages
a characteristic
of cancer
in both
find
while Sassenrath
man
characteristic
tern of plasma,
@ndanimals is a loss in mass of the body's protein
stores,
especially
from muscle.
These
have been put on a more quantitative
-studies
by Mider
and
associates
small
observations
basis in
(13, 19), which
showed that the demands for nitrogen by a rapidly
growing malignant tumor exceed that available
from dietary sources alone. As a result, a portion
of the nitrogen needed for tumor growth is drawn
from the protein stores of the host (10). Under
these circumstances, it would seem likely that the
amino acid metabolism of the host would be dis
turbed. Information
on this point, however, has
not been clear or consistent.
For instance,
Levy
et
at. (11) observed an elevated free amino acid con
•centration in the liver of rats with large tumors,
* This
investigation
was
supported
in
part
by
a
tumors.
and Greenberg
change
(17) found
in the free amino
acid
no
pat
liver, and muscle of rats bearing
This
raises the question
of whether
the effects of cancer on the free amino acid concen
tration may not be related to tumor size. Also, in
the study of Levy et at. (11) no regulation of die
tary intake was imposed on the control animals in
the experiment in which liver free amino acids of
the tumor-bearing
animals were studied. This
would seem to be necessary, since diminished food
intake and inanition frequently occur in animals
bearing malignant tumors, especially in the later
stages of tumor growth, and it is known that the
concentration
of free amino acids can be affected
by dietary factors (9, 23, 26). It was felt, therefore,
that the effects of tumor growth on the free amino
research
grant (C-1719) from the National Cancer Institute,
U.S.
Public Health Service, and by allocations from an Institutional
Cancer Research Grant to the University of Michigan from
the American Cancer Society.
Receivedforpublication
December 14,1959.
acid concentration of the host should be reinvesti
gated by the pair-feeding technic, so that any ob
served changes in free amino acids might be re
lated to the stage of tumor growth. The first part
of the present report is concerned with the results
of such an investigation.
848
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WU AND BAUER—Amino
Acids
Inthecourseofthisstudyit wasfoundthatthe
concentration
of glutamine was consistently de
creased in the plasma and tissues of tumor-bearing
rats. The important role played by glutamine in
protein synthesis of the Ehrlich ascites carcinoma
(15) and the high requirement
for this amino
amide in tissue cultures of different malignant
acid
cell
types (4) indicate a special function for glutamine
in growth. Therefore,
the enzymes glutamine
syn
thetase (5, 20) and gbutamybtransferase
(@, 22) in
the liver of tumor-bearing animals were studied in
an effort to explain the changes seen in glutamine
concentration.
These findings constitute the latter
part of this report.
MATERIALS
AND METHODS
Care of animal&.—Twoseries of experiments
were done, 1 year apart.
rats of the Wistar
strain
In the first series male
(Carwortli
Farms),
with
body weights of 200-300 gm. at the beginning of
the experiment, were used. In the second series
Sprague-Dawley
rats weighing 100—ISOgm. mi
tinily were employed. As far as the data obtained
in this study are concerned, these two series of ex
periments gave essentially the same results. Hence,
for presentation the results have been treated as a
single group.
@
The animals
on a balanced
were first fed ad libitum
diet' containing
849
Synthesis
Tissue preparaiion.—Pairs of animals were sac
nificed under ether anesthesia at various stages of
tumor growth. The abdominal wall was opened.
While the heart was still beating, blood was col
lected from the abdominal aorta in a hepaninized
syringe.
The liver and the gastrocnemius
muscles
of both legs were excised, weighed, and immedi
ately frozen in liquid nitrogen. The frozen tissues
were lyophilized
by a freer.@dry technic.
tissues were weighed again and passed
Wiley mill. The dry, palvonized
in a deep freeze until
analyses
The dried
through a
tissues were kept
were made.
Ana1@&a1 methods.—
a) Paper chromatography of amino -acids: The
procedure
described
below is similar to the one
published
elsewhere
(26), except
that
the extrac
tion technic has been improved and another sol
vent system has replaced 2,4-lutidine to avoid the
Gifensive
odor
of the
latter
solvent.
Extraction
of amino acids from tissues with ethanol was
found satisfactory for subsequent paper -chroma
tography. No desalting was necessary, and the
amino acid color spots developed with ninhydrin
appeared well defined on the chromatograms.
Ap
proximately
200 mg. of dry liver or muscle
were
extracted with 10 ml. of 80 per cent ethanol. The
extraction with occasional shaking was completed
@5 in hour -at room temperature.
In a preliminary
experiment, the same values were obtained by a
ithihydrin colorimetric method (27) for the amino
weeks before the experiment started. After the
acids extracted
from a sample of muscle after
initial feeding period, the animals were paired by ethanolic extraction for periods of @,
1, 2, 4, 10, and
body weight and relative growth rate. Into one 24 hours.
The
ethanolic
extract
was
dialyzed
per cent
chemicals
semi-synthetic
-and Glutamine
“vitamin free― casein (Nutritional
Blo
Corporation)
for a period of about 2
animal of each pair the Walker carcinoma 256 was
transplanted
subcutaneously
near the right groin
by trocar technic. The other animal of the pair,
against
individual
stainless steel metabolism
mob was used as a urine preservative.
intakes were recorded.
Body weights
arm of a machine that inverted the tube repeatedly
at.a slow speed. At the-end of dialysis, the bag with
its contents
was removed from the tube and dis
3 times
its volume
of 80 per cent ethanol
for about 4 hours at 6°C. with cellulose casing
(Nojax, Visking Company) as the dialyzing mem
which served as a control, received only a sterile
brane. The dialysis was carried out by tying the
trocar puncture. The control animal was pair-fed
lower end of the cellulose easing into a knot, intro
with the tumor-bearing
animal on the semi-syn
ducing the extract to be dialyzed into the casing,
thetic diet throughout
the entire experimental
pe
and tying the upper end -of the casing. The dialyz
nod. Water was available
ad libitum at all times.
ing bag was placed in a tube containing a measured
In thoseexperiments
in whichurinecollectionsvolume of 80 per cent ethanoL The tube was
were made regularly, the animals were housed in tightly stoppered
and mounted
on the rocking
cages. Thy
Daily food
were deter
mined, and in vivo tumor weights were estimated
(18) every
4th day.
‘Thediet contained: 25 per cent vitamin-free casein, 7.9
per cent corn oil, 0.1 per cent cod liver oil, 4 per cent Hubbell
Mendel-Wakeman salt mixture, 40 per cent starch, 22 per cent
sucrose, and 1 per cent vitamin mixture. The vitamin mixture
containedthe followingvitaminsin mg/gm: thiaminehydro
chloride,
1.0;pyridoxinehydrochloride,
14);ribOflavin,
2.0;
inositol, 5.0; niacin, 19.0; calcium pantothenate, 10.0; choline
chloride, 100.0; p-amino-benzoic acid, 50.0; menadione, 0.05;
biotin,0.1;folioacid,0.1;and sufficient
quantityof sucrose
to make I gin.
carded. That the dialysis in an alcoholic solution
was satisfactory
is shown by an ‘experiment in
which a solution of leucine in 80 per cent ethanol
was dialyzed
as described
for various
intervals
of
time and the dialysates were used to determine
leucine. It was found that dialysis for 2 hours was
adequate, and rprobonged dialysis up to 24 ‘hours
did not change the leucine value in the dialysate.
The recovery of heucine in the dialysate was found
to be 106 per cent, based<m the assumption that at
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850
Cancer Research
equilibrium
was
the
the concentration
same
inside
and
of the amino acid
outside
the
dialyzing
membrane (7). For chromatography
the dialysate
of a tissue extract was dried in vacuo, and the
residue was taken up in a small volume of 10 per
cent 2-propanol. Aliquots equivalent to 10 mg. of
dry liver or muscle were used.
Plasma and unhydrolyzed
urine were desalted
(21) with Dowex 2-x8 (100—200mesh). The elu
ates were dried in vacuo, and the residue was dis
solved
in small volumes
of 10 per cent 2-propanol.
Aliquots of these concentrates in 2..propanol were
used for the determination
of free amino acids.
When
the nonprotein-bound
were determined,
lyzed
against
for 16 hours
hydrolyzed.
tial because
amounts of
dialysate
amino
acids
of urine
the urine sample was first dia
3 times
its volume
of distilled
water
at 6°C., and the dialysate was then
Dialysis before hydrolysis was essen
contamination
of the urine with small
dietary casein was unavoidable. The
was hydrolyzed
by reiluxing
in 6 N HC1
for 24 hours. The hydrolysate was dried in vacuo to
remove HC1. The residue was dissolved in water,
neutralized
treated
if necessary,
similarly
desalted,
and
to the unhydrolyzed
thereafter
urine.
Whatman paper #2 (28.5 X 46.3 cm.). The ascend
ing technic with the frame of Datta et a!. (3) was
employed to prepare two-dimensional
chromato
grams. A mixture of liquefied phenol (Gilt Label,
and water
(80 : 20) containing
0.01
per cent 8-hydroxyquinoline
and ammonia vapor
were used to develop the first dimension. The sec
ond solvent system consisted of 1-butanol, 88 per
cent formic acid, and water (75:15:10). The de
velopment of the chromatograms
with ninhydrin
and subsequent elution and measurement
of the
color were the same as described previously (26).
The reliability
of the entire procedure was
tested by adding a mixture of known amino acids
to the ethanolic extracts of muscle and liver. The
extracts were dialyzed and chromatographed,
and
the amino acids were determined
as mentioned
above. The recovery
values for alanine, aspartic
acid, glutamic acid, glutamine, glycine, serine, and
threonine were found to be 86.6, 107.4, 96.9, 88.8,
98.2, 101.0, and 96.0 per cent, respectively. The
amino acids used as the standards were purchased
from the Nutritional Biochemicals Corporation.
b) Total free amino acids: Aliquots of the dry
liver and muscle tissues were used to determine
a-amino N by the ninhydrin-C02
method (8).
c) Glutamine
synthetase
and glutamyltrans
ferase: Glutamine
synthetase
activity
in a system
consisting
of liver
was assayed
homogenate
taming 20 mg. of lyophilyzed
methyl)
aminomethane
mmoles ; monosodium
adenosine
triphosphate.
con
1960
liver; tris (hydroxy
buffer of
i@-gluthmate,
pH 7.5, 1.5
0.5 mmole;
adenosine triphosphate,
adjusted to pH
mmole ; MgCl,, 0.2 mmole ; cysteine, 0.2
hydroxylamine
. @H,SO4,adjusted to pH
mmole ; and water to a final volume of 4.5
control flask contained all constituents
Incubations
7.0, 0.1
mmole;
7.0, 0.5
ml. The
except
were carried
out under oxygen at 36°C. for 80 minutes in a
Dubnoff shaking incubator. At the end of the in
cubation period, 0.5 ml. of 50 per cent trichboro
acetic acid was added to each flask. The contents
of each flask were filtered, and 3 ml. of the filtrate
were used for the determination
of @y-glutamyl
hydroxamic
acid (12). i@-7-Glutamythydroxamic
acid, which was synthesized according to Roper
and Mcllwain (16), was used as a standard.
Glutamyltransferase
activity
was assayed
as
follows : Into a small Erlenmeyer
flask were added
liver homogenate
containing
10 mg. of lyophilyzed
liver; phosphate
buffer, pH 6.7, 0.02 mmole;
adenosine triphosphate,
adjusted to pH 6.7, 0.002
mmole; i-glutamine, 0.25 mmole; hydroxylamine.
4H@SO4, adjusted
Aliquots
of the 2-propanol
concentrates
of tis
sue, plasma,
or urine were applied
to sheets of
Mallinckrodt)
Vol. 20, July
to pH 6.7, 0.5 mniole;
MnCl2,
0.02 mmole; and water to 4.5 ml. The control flask
contained no i@-glutamine and the hydroxamic
acid synthesized was negligible. The flasks were
incubated at 36°C. for 30 minutes in a Dubnoff
shaking incubator.
The rest of the procedure
was
the same as for the determination
of glutamine
synthetase
described
activity.
were found
Liver
powders
to have
prepared
as
lost no activity
of
either enzyme after 2 years of storage.
RESULTS
For the purpose of presenting a large number of
data in a concise and intelligible form, it was found
necessary to group them according to the size of
tumor,
expressing
the tumor
weight
as a per cent
of the total body weight. The tumors were divided
on the basis of mass into three groups, namely
(a) “small,―
constituting less than 10 per cent of
total body weight, (b) “medium,―
greater than 10
per cent but less than 80 per cent, and (c) “large,―
greater than 30 per cent and up to 50 per cent.
Such
a classification
is arbitrary,
but
it appears
useful in summarizing the data and relating the
biological events of tumor growth to the biochemi
cal findings.
Plasma amino acid2.—Table 1 shows the
changes in some eight amino acids in the blood
plasma of tumor-bearing
rats. These eight amino
acids constitute about two-thirds of the total free
amino acids of normal rat plasma (26). It is ap
parent that no change in the concentration of free
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851
Wu ANDBAuErt—Amino
AcidsandGlutamineSynthesis
vidual free amino acids with the effects of tumor
growth on the total free amino acid concentration
of liver and muscle, the free a-amino nitrogen of
these tissues was also determined.
In the liver
(Table 2), no significant difference in the total free
amino acids between the tumor-bearing
and con
trol animals was ever found. Yet, a definite in
crease in the concentrations
of aspartic acid,
serine, and threonine and a significant decrease in
glutamine concentration can be seen in those ani
mals with medium-sized tumors. The failure of the
total free amino acid data to show any significant
change in this instance is probably due to the fact
that on a molar basis the increase in the concen
trations of aspartic acid, serine, and threonine was
largely compensated for by a decrease in the con
centration of glutamine. On the other hand, in the
liver of rats with large tumors the concentrations
of all amino acids determined, with the exception
of glutamine, were not changed. The difference ob
served here in the liver between animals with me
dium-sized tumors and those with large ones may
reflect the varying anabolic activity of this tissue
amino acids occurred with tumors of only medium
size. However, when tumors had reached large
proportions,
the plasma
concentrations
of aspartic
and glutamic acids, serine, and tyrosine were sig
nificantly increased. The only compound studied
which showed a decrease in concentration
was
glutamine. White et a!. (25) also reported an in
crease in plasma glutamic acid in tumor-bearing
rats but observed no change in plasma glutamine.
The difference with respect to changes in plasma
glutamine
between
the
observation
made
in the
present study and that of White et al. (25) is prob
ably related to tumor size, because in the early
stages
of tumor
growth
there
may
not
be a de
crease in plasma glutamine level. It is interesting
to note, also, that these authors did record a very
significant decrease in plasma glutamine level in
male rats bearing methylcholanthrene-induced
tu
mors but not in female rats bearing the same tu
mor.
Tissue amino acids.—-Tables 2 and 3 show the
effects of tumor growth on the concentrations
of
free amino acids of liver and muscle, respectively.
In order to compare the changes seen in the mdi
in the course
of tumor
growth.
TABLE 1
FREE AMINO ACIDS IN PLASMA OF TUMOR-BEARING RATs
All values are expressed as zmoles/100 ml of plasma.
SIZEMediumLargeAlanineControl
Cou@oviwTYPE
ANIMALTUMOR
91.0±23.6Aspartic
Tumor-bearing75.2
acidControl
6.8@Glutamic
Tumor-bearing4.4
(61.8— 97.8)* (3)t
87.6 (78.6—103.2)76.4±40.5@
5.6
(2.6—5.5)(3)
(3.4— 7.3)7.5
acidControl
47.6±[email protected]
(26.6- 31.3) (2)
39.4 (38.1— 40.8)34.0±
5.5@GlycineControl
Tumor-bearing30.8
(24.0—39.7) (3)
20.6 (14.4— 25.0)30.1±
60.0±17.3SerineControl
Tumor-bearing45.4
53.4(45.3— 62.6)52.0±
9.5@ThreonineControl
Tumor-bearing50.5
50.5(44.7—
57.1)44.8±
(7)
± 3.0 (6)
12.8±
9.5 (7)
9.6(6)
17.8±
(42.6—48.0) (3)
14.7 (7)
(41.9—65.6) (3)
61.4)(3)
60.1±16.0TyrosineControl Tumor-bearing46.2(37.8—43.7(38.6— 48.7)39.5±10.1
Tumor-bearing15.5
* The
@
two
values
in
the
parentheses
indicate
( 9.4—
21.0)(3)
15.5 (11.6- 22.1)12.7±
the
9.5 (7)
63.8±
(7)
6.1 (6)
16.6± 3.9@
ranges.
t The number in the parenthesesindicates the number of pairs of animals used.
The value after the ±sign showsthe standard deviation from the mean.
§
The differencein the mean valuesbetweenthe experimentaland control groups is significantby
the “t―
test at a 0.05 level.
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-
TABLE
2
FREE AMINo AcIDs IN LIVER OF TUMOR-BEARING RATS
In this and the followingtable, all values are expressedas @moles/gm
of dry tissue.
SIZESmallMediumLargeAlanine
COMPOUNDTYPE
ANIMALTUMOR
2.3*(8)t
Asparticacid
Glutamicacid
2.2± 0.6(8)
3.2± 1.4
2.3± 0.8(10)
4.1± 0.2@
2.3±0.7 (9)
3.0±1.6
Control
4.1± 1.5(8)
Tumor-bearing
4.6± 2.0
4.0± 1.6(8)
4.9± 2.2
3.9±1.9(8)
4.1±2.0
15.3± 3.9(9)
9.0± 4.9@
14.3±2.5(9)
6.8±2.1@
Glutamine
Control
Tumor-bearing
15.5± 3.4(7)
16.0± 3.6
Glycine
Control
Tumor-bearing
7.1± 1.2(8)
8.8± 3.3
Serine
Control
2.0± 0.8(7)
Threonine
Totalfreeamino
acidsControl
91.0±6.9*
See
footnote
(8)
7.5±3.8
Control
Tumor-bearing
11.2± 3.3
2.4(8)
7.0± 3.8
Tumor-hearing
-
8.0± 3.3(10)
8.0± 3.3
6.3±1.5
6.1±2.3
(9)
(7)
2.0± 0.8(10)
2.9± 1.1
2.5±1.2
Tumor-bearing
6.9± 3.0@
2.9±1.4
Control
Tumor-bearing
1.1± 0.2(6)
1.9± 1.2
1.8± 0.5(7)
4.0± 1.3@
1.7±0.4 (8)
2.6±1.6
98.4±12.4(6)
92.0±7.0(6)
Control
91.9±10.0(8)
Tumor-bearing10.1±
87.2±12.79.2±
@,Table
103.9±17.47.8±1.7
1.
t See footnote t@Table 1.
@
See footnote §,Table 1.
TABLE S
RATSCoiipouxnTYPE
FREE AMINO ACIDS AND TAIJRINE IN MUSCLE OF TUMOR-BEARING
SIZESmallMediumLargeAlanine
ANIMALTUMOR
1.2*(l0)t
Glutamicacid
Glutamine
9.5± 1.6
7.6± 1.4
7.4±1.8
Control
Tumor-bearing
2.1± 0.6(10)
2.0± 0.6
2.0± 0.7(9)
1.3± 0.3@
2.4±0.7 (10)
1.6±0.6@
10.8± 2.6 (10)
9.6± 2.6
11.2± 1.2 (9)
7.8± 1.8@
Control
Tumor-bearing
7.5± 1.5(10)
8.0± 1.8(9)
8.9±2.3(10)
7.8± 2.0
8.5± 2.7
6.4±%.1@
Control
2.8± 0.6(10)
2.9± 0.6
3.6± 0.8(9)
4.0±1.4
3.6± 0.9
2.7±1.0@
2.1± 1.3(10)
2.0± 0.6
2.7± 1.2(9)
2.3±0.5(10)
1.6±0.4@
Tumor-bearing
73.4±10.4 (9)
74.6±10.9
79.8±10.9 (6)
85.0±12.9
71.7±7.1 (6)
62.6±8.7
Control
56.1± 6.9(9)
56.6± 6.7(6)
59.3±4.6(6)
Tumor-bearing8.8±
58.2± 6.68.2±
51.8± 7.58.1±1.2(10) 48.4±3.7@
Control
Tumor-bearing
Glycine
Serine
Tumor-bearing
Control
Threonine
Tumor-bearing
Control
Taurine
Totalfreeamino
acidsControl
* See
footnote
1.6(9)
Tumor-bearing
@,Table
2.2± 0.8
11.6±1.8
(10)
8.2±S.1@
(10)
1.
t See footnote t, Table 1.
See footnote §,Table 1.
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Wu ANDBAUER—Amino
Acids and Glutamine Synthesis
In the muscle (Table 3), in contrast
decrease
in the concentration
of total
Urinary amino acids.—The influence of malig
to liver, a
nant growth on the excretion of amino acids in the
urine was investigated next. The data in Table 4
are expressed as the quantities of dialyzable amino
acids excreted per day by control and tumor-bear
ing animals, which, under the conditions of pair
feeding,had very similartotalbody weights
throughout the entire experimental period. When
the data are expressed in this way, it can be seen
that the daily excretion of free amino acids in the
urine showed no consistent change. However, if
the data had been expressed as a function of
free amino
acids was found. This decrease was evident, how
ever, only when the tumors had attained a large
size. The trend toward a lowered level of individ
ual free amino acids in the muscle of the tumor
bearing animals can be seen, however, at an earlier
stage
of tumor
growth.
Thus,
glutamic
acid
and
glutamine
were significantly
decreased in the
muscle of rats with medium-sized tumors. As tu
mor growth
cine, serine,
continued,
the concentrations
of gly
and threonine
were also lowered. It is
TABLE
DIALYZABLE
AMINO
853
4
ACIDS IN URINE
OF TUMOR-BEARING
RATS
The values are expressed as @imoles/day.
SIZESmall
(1Q)*Medium
COMPOUNDTYPE
ANIMALTUMOR
(3)FreeBoundFreeBoundFreeBoundAlanine
Asparticacid
Tumor-bearing
3.8
7.7
4.3
10.3
2.4
19.1
Control
3.9
11.4
Tumor-bearing
3.6
11.8
4.9
1.7
10.1
21.5
2.9
3.6
11.0
41.6
10.0
14.0
22.6
24.8
11.4
25.9
Control
Tumor-bearing
Glutamicacid
(10)Large
11.2
9.1
17.8
48.0
26.8
43.8
Control
1.0
0.7
0.5
Tumor-bearing
1.0
1.6
0.7
Glycine
Control
Tumor-bearing
9.2
7.9
46.3
45.5
6.9
6.1
33.8
61.8
4.4
4.3
35.6
65.1
Serine
Control
Tumor-bearing
3.8
3.0
8.3
7.5
3.3
2.5
5.7
11.8
2.3
2.7
3.7
22.0
Control
4.0
Tumor-bearing4.7
3.58.9
4.2
4.55.2
3.6
4.34.4
Glutamine
ThreonineControl
* The
number
in
the
parentheses
indicates
the
number
of
urine
samples
employed
3.0
5.93.1
to
obtain
2.6
1.53.6
the
averaged
2.4
4.3
values
indicated for that group.
interesting
to note
that,
despite
its known
meta
bolic relationships with other amino acids deter
mined, the concentration
of alanine remained
unchanged in the plasma, liver, and muscle of the
host regardless of the size of tumor. Similarly, the
tumor did not affect the concentration
of free
taurine, which was present abundantly
in the
muscle. At this point, attention should be called to
the unique effect of tumor growth on the metabo
lism of glutamine. It was the only compound ex
amined which showed consistently a decrease in
concentration
in plasma, liver, and muscle of the
tumor-bearing
rats. The possible significance of
thu finding will be considered in more detail in the
“Discussion.―
residual carcass weight (total body weight less tu
mor weight), it can be shown that the excretion of
nearly
every
free
amino
acid
determined
have been increased in the tumor-bearing
especially
those with
spects it would appear
would
animals,
large tumors.
In some re
more valid to compare the
data on the basis of carcass weight rather than to
tal body weight, since a given mass of tumor and
of carcass would hardly be expected to exert simi
lar metabolic effects on the excretion of free amino
acids in the urine. Even with the data expressed as
they are, it is interesting to observe that the non
protein-bound
(conjugated)
form of all amino
acids determined was excreted in greater quanti
ties as the tumors grew larger (Table 4). This form
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854
Vol. @20,
July 1960
Cancer Research
of the amino
acids was calculated
by difference
and of glutamyltransferase
in the liver. A few per
tinent facts can be discerned from the data in this
be
tween the total dialyzable amino acids (free plus
bound) and the free amino acids as determined by
paper chromatography.
The increase in the excre
tion of bound amino acids became highly sig
nificant after the tumors had attained a medium
table.
size.
Enzymes in glutamine metabolis-m.-—Thecon
sistent
plasma,
lowering
of glutamine
concentration
in
liver, and muscle of the tumor-bearing
TABLE
COMPARISON
OF THE ACTIVITIES
OF GLUTAMINE
.
.
numberTumor
weight
per
cent*(1)
the
activities
of both
SYNTRETASE
RATS
Synthetase
Transferase
activityt(S)
activityt.
Ratio
Tumor-bearing3.10.86
0.26II.Control
0.432.83
1.650.30
Tumor-bearing7.20.98
0.28III.Control
0.563.26
2.000.30
0.28IV.Control
Tumor-bearing18.40.94
0.582.50
2.060.38
0.532.80
1.830.36
0.562.98
1.500.36
0.29VII.Control
Tumor.bearing41.90.96
0.462.80
1.590.34
0.29VIII.Control
Tumor-bearing44.11.02
0.402.80
1.390.36
0.28Walker
Tumor-bearing44.91.01
0.562.68
1.990.38
Tumor-bearing23.21.02
0.29V.Control
0.37VI.Control
Tumor-bearing28.61.06
AND
IN THE LIVER
OF TUMOR-BEARING
Pair
that
5
OF GLUTAMYLTRANSFERASE
(1)/(5)I.Control
It is apparent
synthetase and transferase were appreciably re
duced in tumor-bearing rats. The reduction was as
great in an animal with a very small tumor as in
one with a large tumor. This indicates that the
tumor produced a very early effect on the reduc
tion of these two enzyme activities. Moreover, al
though this effect persisted during the course of
carcinoma 2560.070.280.25
* Tumor
weight
expressed
as
a percentage
of
the
total
body
weight.
t Expressed as @imoles
of L--y-glutamylhydroxamic acid synthesized per
10 mg. of dry tissue in 30 minutes.
rats
suggests
that
might be impaired,
the synthesis
of this compound
and a knowledge of the activ
ity of glutamine
synthetase
in the liver would
therefore
be of interest. Since this enzyme system
also catalyzes
an analogous
reaction in which hy
droxylamine
replaces ammonia
(20) and since the
hydroxamic
acid formed can be readily deter
mined, the synthesis of glutamyihydroxamic
acid
rather than glutamine itself was investigated. In
addition, a closely related glutamyl transfer reac
tion (6, 22), which synthesizes L-'y-glutamylhy
droxamic acid from frglutamine
and hydroxyl
amine, was also studied.
is made of the activities
In TableS,
a comparison
of glutamine
synthetase
tumor growth, it was not enhanced as the meta
bolic load of the tumor on its host was progres
sively increasing.
Under the described conditions for assaying the
activities
of these two enzyme
systems
the average ratio of the synthetase
transferase
activity
was found
in the liver,
activity
to the
to be 0.32, with
a
range from 0.26 to 0.38. Of course, no significance
can be attached to the absolute value of this ratio,
for it is conceivable that under differently defined
conditions of assay, different values will be ob
tained. However,
stant relationship
it is important
that a fairly con
between the activities of the two
enzyme systems did exist under a given set of as
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Wu
@D
BAUER—Amino Acids and Glutamine Synthesis
very small tumors to those of enormous size. Sec
ond, the effects that changes in dietary intake
might have on the metabolism of free amino acids
were compensated for by pair-feeding of the con
trol to the tumor-bearing
animals. It is believed
that the data obtained under these two conditions
not only will show at what stage of growth the
tumor begins to exert its effect on the metabolism
of free amino acids of the host, but also will give
an accurate account of this effect. Sassenrath and
Greenberg (17) reported no characteristic changes
in the free amino acids in rats bearing the Walker
carcinoma 256. It should be emphasized, however,
that their observations were made on animals with
very small tumors. Their results can be compared,
therefore, with only the smallest group of tumors
in the present study, in which no change in any
say conditions. This indicates that with these two
enzyme systems the activity of one may be used as
a valid index of the activity of the other. However,
when the ratio values of each pair of animals are
compared, the ratio for the tumor-bearing
animal
is less than that for the control animal in seven out
of eight cases. This shows that the synthetase ac
tivity was affected to a greater extent by tumor
growth
than
the
transferase
activity.
Further
more, it is apparent from Table S that Walker car
cinoma 956 possessed very little activity of either
enzyme when compared with liver on a dry weight
basis. Here the synthetase activity was found to be
one-fourth as great as the transferase activity.
In order to assure the reproducibility
of the
data in Table 5, the activity of glutamyltrans
ferase in the liver of a larger group of rats bearing
TABLE
GLUTAMYLTRANSFERASE
855
6
ACTIVITY
TUMOR-BEARING
IN THE LIVER OF
RATS
All values are expressed as j@molesof L--y-glutamylhydroxamic acid synthesized/lO
mg dry liver in 30 minutes.
SIZESmall
TYPE
ANIMALTUMOR
LargeControl
Medium
Tumor-bearing2.33±0.48*(7)t
1.53±0.25@
* See
footnote
@,Table
2.36±0.16(7)
1.44±0.29t
2.27±0.38(6)
1.26±0.32@
1.
t See footnote t@Table 1.
See footnote
§,Table 1.
tumors of varying size and of their respective pair
fed controls was determined. These data, which
are summarized in Table 6, confirm the findings
mentioned above. (a) The activity of glutamyl
transferase
was decreased
in the
liver
of experi
mental animals early in the course of tumor growth
(most tumors being less than 5 per cent of total
body weight), when no detectable decrease in the
glutamine levels of tissues or plasma had occurred.
(b) The decrease was not significantly greater in
animals with large tumors than in those with small
ones, indicating that the tumor produced a very
early and continuing effect on the reduction of the
transferase activity.
DISCUSSION
The present study differs from previously re
ported work on the metabolism of free amino acids
in tumor-bearing
animals in two respects. First,
the effects of Walker carcinoma 256 on the free
amino acid concentrations
of the host were re
corded for various stages of tumor growth, from
free amino acid determined was observed. Levy et
a!. (11) observed a definite increase in the concen
trations of free glycine, methionine, serine, and
threonine in the liver of rats bearing large tumors
(Jensen sarcoma). The results in the present study
indicate, however, that, while some amino acids
were increased in the liver of rats with medium
sized tumors, none showed an increased concentra
tion in the liver of animals bearing large tumors.
From the data presented, it appears that the
concentrations
of the free amino
acids in the plas
ma and liver of the tumor-bearing
animal were in
creased at the expense of muscle proteins as the
wasting
process
progressed
during
tumor
growth.
It may be suggested further that, during the active
growth of the tumor, its demands for free amino
acids are so great that the muscle tissue surrenders
its own free amino acid pool to the plasma and ul
timately to the tumor. As has been shown, the con
centrations of most free amino acids in the muscle
were decreased in the tumor-bearing rat. Likewise,
the nonprotein-bound
amino acids present in in
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Cancer Research
856
creased
amounts
in the tissues
(1) and urine of the
tumor-bearing
rat must be regarded as intermedi
ates of proteolysis rather than of protein synthesis.
In the face of diminishing muscle mass and, hence,
decreasing protein content, the ability of the
catabolic machinery of the host to maintain higher
than control levels of free amino acids in plasma
and liver is very interesting and suggests that there
may be an increased activity of proteolytic en
zymes in the muscle tissue of the tumor-bearing
rat. The failure of muscle to show an increased
concentration of free amino acids, despite obvious
net protein catabolism in this tissue of the tumor
bearing
animal,
may
indicate
an
associated
de
crease in amino acid uptake. The work of Norberg
and Greenberg (14) showing diminished incorpora
tion of labeled glycine into muscle protein of tu
mor-bearing mice would support this view.
An interesting observation made in this study is
concerned with the metabolism of glutamine. Al
though fasting has been shown to result in a de
crease of the glutamine levels in plasma and tissues
of the rat (26), it is unlikely that this was the cause
of lowered glutamine concentration
in the tumor
bearing animal. Certainly increased destruction
through deamidation, increased utilization for pro
tein synthesis, increased excretion due to impair
ment of tubular reabsorption, and decreased syn
thesis
may
be considered.
Increased
deamidation
seems improbable, since White et at. (25) have
shown that there was no change in the glutaminase
activity in tumorous rats. Examination
of the
urinary data does not appear to favor the view
that glutamine
was excessively excreted. The
amount of free glutamine in the urine was too
small to account for the fall in plasma and tissue
glutamine levels. The available evidence points to
an increased demand for glutamine for the synthe
sis of tumor protein (15). This is particularly true
since, as has been shown in this study, the ability
of tumor tissue itself to synthesize glutamine was
quite limited. Despite the increased demands for
glutamine as a result of tumor growth, the gluta
mine synthetase activity in the liver of the host
was decreased. This appears to be a paradoxical
situation. Of course, no direct evidence was pro
vided in this study to show that the synthesis of
glutamine in vivo paralleled the synthetase activity
as measured in @,itro,since it is possible that the
remaining enzyme activity would be commensu
rate with the normal demand for glutamine as has
been observed in other enzyme systems (2, 24).
Whether it is still adequate to meet the increased
demand for glutamine in the presence of a rapidly
growing malignant tumor is open to question. It
can be noted that the synthetase activity was al
Vol. 20, July
ready significantly
barely
palpable
1960
decreased while the tumor was
and at a time when no decrease
in
the glutamine concentration
of tissues or plasma
could be detected. It appears that the remaining
enzyme activity was adequate to maintain normal
levels of glutamine
in the plasma
and tissues
while
the tumor was small and presumably
its need for
glutamine
grew larger
could be met. As the tumor
and its demand for glutamine became more in
tense, the remaining enzyme activity could not
synthesize
enough
glutamine
to fulfill the require
ments for normal metabolism and for neoplastic
growth. Under these circumstances the size of the
glutamine pool of the organism was perforce re
duced. Hence, the decrease in the level of gluta
mine in plasma and tissues of the host appears to
result from an increased demand for this corn
pound by the tumor-host system and an inability
of the system to meet this demand by synthetic
processes.
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A Study of Free Amino Acids and of Glutamine Synthesis in
Tumor-bearing Rats
Chung Wu and Jere M. Bauer
Cancer Res 1960;20:848-857.
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