The Citric Acid Content of Tumor Tissue and
of Tumor-bearing Rats*
FRANCESL. HAVEN,PH.D., CHALLISSRANDALL,M.S., ANDW. R. BLOOR,PH.D.
(From the Department of Biochemistry,
School of Medicine and Dentistry,
Unirersity of Rochester, Rochester, New York)
A high content of citric acid has been shown by tissue into necrotic and non-necrotic portions was
Dickens (1) to be characteristic of rapidly growing carried out by careful dissection. All tissues were
and regenerating tissue. Embryonic tissue, tissue ground with sand and extracted with 10 per cent
of the newborn, precancerous tissues, and a wide trichloracetic acid solution until extraction was
variety of tumors of animals and of man were complete. Kidneys, plasma, and occasionally blood
found by him to be high in citric acid. Since the from two animals carefully matched in bodynon-necrotic tissue of Crocker mouse sarcoma 180 weight and tumor age and size were pooled, in
contained more citric acid than did the necrotic order to obtain samples sufficiently large for
tissue, Dickens concluded that the high citric acid analysis.
content was a property of the tumor tissue itself
All citric acid determinations were made by the
and not the result of a change, such as calcification method of Pucher, Sherman, and Vickçry (2),
accompanying the necrosis.
modified as previously reported (3). Among
The experiments described in this paper were numerous substances tried by Pucher and others,
undertaken to determine the citric acid content of the only substance found to enhance the color
the transplantable tumor, Walker carcinoma 256, given by citric acid in this method is ß-hydroxyand of various tissues of rats bearing this tumor. butyric acid In view of this fact, determinations
In some cases the whole tumor was analyzed, and ,of j3-hydroxybutyric acid were carried out by
in others the necrotic and non-nccrotic portions of Behre's (4) method on samples of necrotic and
the same tumor were analyzed separately. Citric non-necrotic tumor tissue and on kidneys of
acid was determined on liver, kidney, spleen, tumor-bearing animals and gave colors equivalent
whole blood, and blood plasma of tumor-bearing
to a maximum of only 0.2 per cent of the total
rats and was compared with values obtained on amount of citric acid found. Therefore, /3-hydroxythe corresponding tissues of rats in which the butyric acid does not contribute to the values re
tumor did not grow after transplantation (non- ported as citric acid.
take rats).
RESULTS AND DISCUSSION
MATERIALS AND METHODS
The citric acid content of whole tumors and of
Young male rats of the Wistar strain, main
tained on Purina Fox Chow and water, were used non-necrotic and necrotic portions of other tumors
throughout these experiments. Tumor tissue was is shown in Table 1. The values for the citric acid
transplanted subcutaneously into the groin by the content of whole tumors ranged from 8.6 to 19.5
trocar method. The majority of the tumors made mg. per 100 gm., with a median value of 12.3 mg.
up from 10 to 35 per cent of the total weights per 100 gm. of fresh tissue. These values are simi
when the animals were anesthetized, bled, killed, lar to those reported by Dickens (1) for two
and the specified tissues removed. If liver was to Walker 256 tumors. Non-necrotic portions of tu
be analyzed, the animals were deprived of food mor contained from 3.4 to 10.0 mg. of citric acid
per 100 gm., with a median value of 5.8 mg. per
about 18 hours previously. Blood was obtained
100 gm., while necrotic portions of the same tu
under ether anesthesia, either from the abdominal
mors
contained from 20.0 to 70.4 mg. of citric acid
aorta or by heart puncture. Both whole blood and
per 100 gm., with a median value of 47.5 mg. per
plasma were extracted with 10 per cent trichloroacetic acid solution. Relative separation of tumor 100 gm. The markedly higher content of citric
acid in the necrotic center of the tumor than in the
* This investigation was supported by a grant from the
non-necrotic periphery is diametrically opposed
Donner Foundation. Presented in part at the Thirty-eighth
to the findings of Dickens (1) on Crocker mouse
Annual Meeting of the American Association for Cancer Re
sarcoma 180.
search, Inc., at Chicago, May, 1947.
90
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1949 American Association for Cancer Research.
HAVENet al.—Citric Acid in Tumors and Ratti
The citric acid content of the non-necrotic pe
ripheral tumor tissue was inversely proportional to
the size of the tumor, as shown in Fig. 1. This
finding constitutes further evidence that the
composition of the tumor is not always constant,
a fact previously reported by Mider et al. (5). The
relationship between the citric acid concentration
in the peripheral tissue and the size of the tumor
might be the result of differences in oxygen tension
between small and large tumors.
Since the separation of tumor tissue into
necrotic and non-necrotic portions was not abso
lute, the citric acid in the non-necrotic portion
91
forming citric acid, determinations of the activity
of aconitase, the enzyme which catalyzes the con
version of cis-aconitic to citric acid, were carried
out essentially by the method of Johnson (6). The
fresh tissues were homogenized in the buffer, and
aconitase activity was determined immediately on
aliquots of the homogenate. The final concentra
tion of cis-aconitic acid in the incubation mixture
was 0.04 M, a concentration which was found
sufficiently high for maximum activity of the
enzyme.
Aconitase activity was present in peripheral
tumor tissue, as shown in Table 2, but was not
TABLE1
CITRICACIDCONTENT
OFWALKER
TUMOR
256
N'o. OF
TUMOH8
Periphery
Center
/
Whole tumor
21
18
CITRICACID
(MO/100OMWETWT.)
Range
Median
( 3.4-10.0
5.8
\20.0-70.4
47.5
8.6-19.5
12.3
TUMORWEIGHT
BANGE
(at,.)
21.2-67.6
13.4-62.0
TABLE2*
THEACONITASE
ACTIVITY
OFPERIPHERAL
AND
CENTRAL
TUMOR
TISSUE
Mg. dry tissue
per ml. of incu-
Tumor tissue
Peripheral
Central
batton mixture
2.5
2.1
2.8
2.7
3.0
3.«
Mg. citric acid
formed per ml. of
incubation mixture
0.34
0.31
0.51
0.47
0.35
0.51
Qcilr.1.
15.9
17 2
21.3
22.3
13.7
16.1
3.0
4.8
5.4
FIG. 1.—The citric acid content (mg/100 gm wet weight)
of peripheral
tumor tissue plotted against the tumor per
centage of body-weight. The line is based on average values.
0
0
0
U
0
0
* Incubation mixtures were prepared by combining 3 ml. of 0.13f
phosphate buffer of pH 7.4; 1 nil. of O.Õil/ci.s-acouiticacid neutralized in icecold water with solid NaHCOs; and 1 ml. of the tissue homogenate in
0.1 J/ phosphate buffer of pH 7.4. Incubation was carried out for 1 hour
at 40°C. under anaerobic conditions.
might be attributed to contamination of the
sample with necrotic tissue high in citrate. This
possibility is unlikely for several reasons. Citric
acid was found in pooled samples of metastatic
growth known to be relatively free from necrosis.
Moreover, if citric acid of the periphery were due to
chance contamination, it is extremely unlikely
that the regular relationship shown in Fig. 1 would
be obtained. Citric acid seems to be a metabolic
product of actively growing tumor tissue.
The high content of citric acid in the center of
the tumor might be due to (a) increased formation
or decreased oxidation of citric acid within the
center or (6) accumulation of citric acid formed in
the periphery. In order to find out whether the
tissue in the center of the tumor is capable of
found in the tissue from the center of the tumor.
Therefore, one can assume that citrate is not
formed in the center of the tumor through the
action of aconitase. Instead, the citric acid formed
in the periphery may have accumulated in the
center and been held there, perhaps in combina
tion with some cation which would remove it from
active metabolism. The rapid growth of the pe
riphery and the poor blood supply of the center,
which would foster anaerobic conditions, would
also contribute to the accumulation of citric acid
in the center.
Values for the citric acid content of organs of
tumor-bearing rats and of rats in which the tumor
did not take are shown in Table 3. In general, the
average values for citric acid were higher in the
TUMOR
% BODY WEIGHT
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Cancer Research
organs of animals with tumor. The exception
presented by whole blood is not surprising, since
Gathe and Xygaard (7) showed that citrate added
to whole blood remained in the plasma. On the
other hand, plasma of animals with tumor was
higher in citrate than was the plasma of "nontake" rats.
Two explanations can be offered for the higher
citrate content of the organs of tumor-bearing rats.
The citrate formed in the tumor in large quanti
ties or oxidized slowly might be carried in the
3. The citric acid content of the peripheral
tumor tissue was inversely proportional to the
size of the tumor.
4. Plasma, liver, kidney, and spleen of rats
bearing tumor were higher in citric acid than were
similar tissues of "non-take" rats.
REFEKENCES
1. DICKENS,F. The Citric Acid Content of Animal Tissues,
with Reference to Its Occurrence in Bone and Tumor.
Biochem. J., 35:1011-1023, 1941.
TABLE 3
CITRICACIDCONTENTOFRATTISSUES
(Mg/100 Gm Wet Weight)
NOS-TAKES
TuMOB ANIMALS
No. of
No.
ofrata2020522137No.
ofanalyses1010192110Mean4.984.866.114.416.81Range3.43-6.534.19-5.624.05-
No. of
TISSUEWhole
blood*Plasma*KidneyLiverSpleen*
6.544
7.836.46-12.403.51-10.476.06-10.94
80-
Mg/100 ml.rats2226502921analyses1112212910Mean4.636.298.885.978.23Range3.67-
plasma and accumulate in the liver, kidney, and
spleen. Or, in line with the many observations on
the chemistry of the tumor-bearing host assembled
by Greenstein (8), the presence of neoplasm in the
animal may have caused the various organs to
assume "chemically neoplastic qualities," result
ing in an increased formation or decreased utiliza
tion of citrate.
SUMMARY
1. Walker 256 rat tumors contained more citric
acid in the nongrowing center than in the growing
periphery.
2. Aconitase activity was present in the periph
eral tissue but was completely absent from the
center.
2. PUCKER,G. W., SHERMAN,C. C., and VICKERY,H. B.
A Method To Determine Small Amounts of Citric Acid in
Biological Material. J. Biol. Chem., 113:235-245, 1936.
3. HAVEN,F. L., and RANDALL,C. The Urinary Excretion of
Citrate in Uranium-poisoned Rats. J. Biol. Chem., 176:
737-744, 1948.
4. BEHRE,J. A. A Modified Salicylaldehyde Method for the
Determination of Acetone Bodies in Blood and Urine. J.
Biol. Chem., 136:25-34, 1940.
5. MIDER, G. B., TESLUK,H. and MOBTON,J. J. Effects of
Walker 256 on Food Intake, Body Weight, and Nitrogen
Metabolism of Growing Rats. Acta cancro!, (in press).
6. JOHNSON,W. A. Aconitase. Biochem. J., 33:1046-1053,
1939.
7. GATHE,F., and NTGAARD,K. K. The Distribution of Cit
rates in Citrated Whole Blood. Skandinav. Arch. f. Physiol.,
83:199-200, 1940.
8. GREENSTEIN,J. P. Biochemistry of Cancer, chaps, ix and
x. New York: Academic Press, Inc., 1947.
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The Citric Acid Content of Tumor Tissue and of Tumor-bearing
Rats
Cancer Res 1949;9:90-92.
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