THE CARBOHYDRATE METABOLISM OF TUMORS.

I. THE FREE SUGAR, LACTIC ACID, AND GLYCOGEN CONTENT OF MALIGNANT TUMORS.

l3y CARL F. CORI AND GERTY T. CORI.

(From the State Institute for the Study of Malignant Disease, Buffalo.)

(Received for publication, February 4, 3923.)

Warburg, Posener, and Kegelein (1) discovered the important fact that surviving tumor tissue showed an unusually large glyco- lytic power when compared with other tissues under identical ex- perimental conditions. All other tissues including embryonic tissue showed some glycolysis in the absence of oxygdn, but practically none when oxygen was present, while the glycolytic power of tumor cells was only slightly decreased by the presence of oxygen. This unique behavior of tumor tissue among all other tissue, namely to produce more lactic acid from glucose than can be removed by oxidation, induced Warburg to compare the metabolism of the tumor cells with that of fermenting yeast. Warburg also adduced evidence that the glycolysis was bound to the structure and did not take place in the liquid medium of the cells, which made it very probable that the energy derived from the splitting of glucose into lactic acid was utilized by the tumor cells; in other words, that the phenomena observed in vitro were also taking place in vivo. The experiments reported in this paper bring forward some evidence that tumor tissue in the living animal splits large amounts of glucose into lactic acid. Our first csperimems dealt with the free sugar content of tumors under various conditions and it was only after WTarburg’s work came to our notice that lactic acid determinations were included.

EXPERIMENTAL.

1. Methods.

The analyses were performed on spontaneous mammary car- cinoma of the mouse, on two types of transplantable mouse car-

II

by guest on Decem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

12 Cwbohydrate Metabolism of Tumors. I

cinema, on Jensen rat sarcoma, and on two human tumors. Many tumors had to be discarded on account of necrosis or cystic degen- eration. As a rule only such tumors were used for analysis as were entirely solid and of even white appearance upon a macroscopic examination. Even small necroses were easily detected by their yellowish tinge and by their soft consistency. Micro- scopic examination of these tumors showed t’hat they were mainly composed of tumor cells and that connective tissue formed only a small percentage of t’he tumor mass.

The fret sugar (2), lactic acid (3), and total carbohydrate (4) content of the tumors were determined by methods which have hecn carefully studied on other tissues and which have been described previously. The free sugar was extracted from the maccratcd tumors with boiling water, the proteins were precipi- tated with colloidal iron, and the sugar, aft,er removal of inter- fering substances with Lloyd’s reagent, was determined by the IIagedorn and Jensen (5) method. The lactic acid was deter- minctl in the filt,ratc from Schenk’s precipitation after removing mercury by II,S and the glucose and other interfering substances by t’hc Palkowski-Van Slyke procedure. The lactic acid in the final filtrate was then determined by the Clauscn (6) I-I&3O.1 met’hod. In some instances the same Schenk filtrate that was used for the lactic acid determination was also analyzed for inor- ganic phosphates by the Briggs (7) modification of the Bell and noisy method. If a separate piece of the tumor was analyzed for inorganic phosphates, it was treated exactly as for the lactic acid determination with the exception of the Salkowski-Van Slyke precipitation. Organic phosphates of the so called lactacidogen fraction and the lact,ic acid maximum were obtained by incubat- ing macerated tumor tissue with 1 per cent KaHC03 for 2 hours .at 37°C. Glycogen was determined by Pflueger’s (8) simplified method. For blood sugar IIagedorn and Jensen’s (5) procedure was used.

2. Free Sugar in Tumor Tissue.

It was found previously (2) that the water used for the extrac- Lion of the tissue sugar dissolves also a number of other substances, as uric acid, creatinine, and crcatinc, which have reducing proper- ties. These interfering substances, which were responsible for

by guest on Decem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

C. F. Cori and G. T. Cori 13

the “rest reduction” could easily be removed by shaking out with Lloyd’s reagent. There was a considerable “rest reduction” in water extracts of muscle, kidney, and brain, while none was found in water extracts of the liver. Tumor tissue also shows a high “rest reduction” as can bc seen from Table I.

Table II summarizes the values for the free sugar content of malignant tumors, which were obtained at a normal or slightly increased blood sugar level of the tumor-bearing animals. It should be noted that t.here is no marked differencein thefrccsugar cont,cnt of carcinoma on the one hand and sarcoma on the other hand. Table III, in which a comparison is made between the free

T:\BLB I.

Effecf of Shnlcirig Out uilh Lloyfl’ J Rengenl on lhc 12educing Power 0.f K’nter E.rlrac1.s of 7’wnor l’issuc.

____- __ -~ Free sugar in tumor.

/ .~__ -__ _- Diminution in reducing power by treatment with Lloyd’s reagent,: 37

per cent.

sugar content of tumor t’issue with that of other tissues of the mouse, shows that the lowest free sugar content is found in the tumors. Since this applies to tumors of different histological origin, namely to carcinoma and to sarcoma, it seems very prob- able that a very low free sugar content is characteristic of malig- nant tumors in general.

In view of the low free sugar content of tumors at a normal blood sugar level it seemed of interest to study the permeability of tumor tissue for glucose at blood sugar levels higher and lower than normal. An increase in blood sugar was produced by the administration of glucose or by epinephrine while the blood sugar

by guest on Decem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

14 Carbohydrate Metabolism of Tumors. I

TABLE II.

Normal Free Sugar Content of Mouse Carcinoma and Jensen Rat Sarcoma.

Length of time of fasting. Weight of tumor. Blood sugar. Free sugar in tumor.

hrs. QW%.

0 2.73 0 3.73 1 2.56 1 1.50 3 1.04 3 0.64 3 1.58 3 1.37 4 1.78

14 3.40 15 0.97 15 1.45 17 0.68 17 0.22 20 0.71 24 0.25

Average.........................

* Jensen rat sarcoma. t Spontaneous mouse carcinoma. $ Transplanted mouse carcinoma.

_-

_-

per cent

0.166 0.175 0.130 0.173 0.126

0.127 0.117 0.110 0.130 0.105 0.126

0.135

TABLE III.

per cent

0.042* 0.053t 0.050t 0.044* 0.051t 0.053t 0.0461 0.0541 0.052t 0.039* 0 .oe5t 0.036t 0.059t 0.047t 0.06tt 0.069$

0.851 -

Comparison of the Free Sugar Content of Jensen Rat Sarcoma and of Mouse Carcinoma with the Free Sugar Content of Other Tissues of the Mouse.

Type of ‘tissue. Remarks.

per cent hrs.

Jensen rat sarcoma.. . . . 0.041 1 Average of 3 experiments. Mouse carcinoma.. . . . . 0.053 10 “ “ 13 “ Liver*. . . . . . . . . . . . . . . . 0.357 1 “ “ 15 “

0.198 19 “ “ 10 “ Muscle*. . . . . . . . . . . . . . . 0.079 20 “ “ 8 “ Kidney*. . . . . . . . . . . . . . . . 0.126 19 “ “ 10 “ Brain*. . . . . . . . . . . . . . . . . . 0.059 18 “ “ 4 “

* Calculated from previous experiments (2).

by guest on Decem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

C. I?. Cori and G. ‘I’. Cori 15

was lowered by insulin. Table IV contains the data on the effect of glucose and Table V on the influence of cpinephrine administra- tion. It wiI1 be noted that with increasing blood sugar conccn-

TABLE IT’.

EJect oj Glucose Administration on the Free Sugar Concentration of the Tumors.

100 mg. of glucose were given intraperitoneally to mice fasted previously for 17 to 24 hours and weighing from IS to 25 gm. The tumors were analyzed 15, 30, 60, 90, and 120 minutes after the glucose injection.

15 min. 30 min. 60 min. 90 min. 120 min.

Average.....0.541

TABLE I’.

Egect ojEpincphrine on the Free Sugnr Concentration of the Tumors.

0.05 mg. of epinephrine was injected subcutaneously and the tumors acre analped 15, 30, 60, and 150 minutes 1:tber.

-.~ ~-

15 rrlin. 30 min. 60 min. 150 min.

-___. per cm t

0.19S 0.276 0.298 0.312

Average.. . . .0.2X

tration the free sugar content of the tumors was also increased. Thus 15 minutes after the glucose injection the free sugar content of the tumors was raised four and one-half times, 30 minutes after

by guest on Decem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

16 Carbohydrate Metabolism of Tumors. I

the injection five times, while 60 and 90 minutes after the glucose injection with the return of the blood sugar to normal, the free sugar concentration of t.he tumors also approached the normal. These results show that tumor tissue is very easily permeable to glucose at higher blood sugar levels. The determination of the free sugar concentration is, of course, not a quantitative measure, but only a rough estimate of permeability, since simultaneously with the intake of glucose by the tissues, there is also a removal of glucose, the magnitude of which is not taken into account. A lowering of the blood sugar level by insulin (Table VI) caused only a very slight diminution of the free sugar content of the tumors.

TABLE VI.

InfIzcence of Insulin on the Free Sugar Content of Tumors.

0.16 unit of insulin was injected intraperitonenlly. -._.-

Blood sugar Free sugar in tumor.

per cm1 per cent

0.016 0.029 0.032 0.059 0.037 0.056 0.075 0.038

Average. . ...0.040 0.045

S. Lactic Acid in Tumors.

The work of Warburg made it desirable to obtain accurate data on the lactic acid content of tumors under normal conditions. Only a limited number of analyses could be performed, since it was difficult to obtain tumors of a sufficiently large size which were entirely free from necrosis or cysts.

From the experiments of Warburg it may be calculated that tumor cells in the presence of oxygen, in a Ringer solution con- taining 0.04 per cent glucose, form in 1 hour an amount of lactic acid equivalent to about 1 per cent of their fresh weight. Yet the results of Table VII and the comparison of the lactic acid values in Table VIII indicate clearly that the tumors have a very low lactic acid content under normal conditions. This was rather surprising and there seemed to be only two explanations possible. Either the strong glycolytic power of tumor cells

by guest on Decem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

C. F. Cori and G. T. Cori 17

described by Warburg was only an in vitro reaction due to the more or less abnormal conditions to which the tumor cells were subjected, or, if t,he glycolysis was of the same magnitude in viva as in vitro the lactic acid was carried away by the blood

TABLE VII.

*Vormal Lactic Acid Cordent of Mouse Cnrcinomn rind Jensen Rat Sarcoma.

Length of time of fasting.

hrs. ml.

0 1.20 0 3.72 0 1.61 1 2.56 1 0.78 2 1.33 2 1.72

14 3.74 17 1.93

\Yeight of tumor.

per cent

0.053

0.050

0.040 0.035

Averagc............................................

* Spontaneous mouse carcinoma. t Jensen rat sarcoma. 1 Metastatic mammary carcinoma (human).

- Lactic acid in tumor.

per cent 0.022* 0.041t 0.060* 0.013* 0.015* 0.022* 0.054* 0.04st 0.0361

0.034

TABLE VIII.

Comparison of the Lactic Acid Content of Mouse Carcinoma and Jensen Rat Sarcoma with the Lactic Acid Content of Other Tissues of the Mouse.

Mouse carcinoma.. Jensen rat sarcoma.. Liver* . . . . . . . . . . . . . . . . . .

Mrmle*. . .

Remarks.

I I

per cent hrs.

0.031 o--2 Average of 6 experiments 0.044 O-14 ‘( ‘( 2 ((

0.051 o-2 ‘I “ 15 “ 0.011 17-22 “ “ 10 “ 0.112 1-28 “ “ 16 “

* Calculated from previous experiments (3).

stream as fast as it was formed. Warburg found that the rate of glycolysis of surviving tumor tissue was increased with increasing glucose concentration. Thus raising the sugar con- centration from 0.04 to 0.2 per cent nearly doubled the rate of

by guest on Decem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

18 Carbohydrate Metabolism of Tumors. I

glycolysis, while just above 0.2 per cent glucose the rate reached its maximum. Since the normal free sugar concentration of the tumors was, as an average, only 0.051 per cent (Table II) it seemed possible that the rate of glycolysis was not sufficiently large to produce an accumulation of lactic acid in the t,umors. The fact that the free sugar content of the tumors could be raised to 0.2 per cent by the administration of glucose (Table IV) made it pos- sible to increase the lactic acid production in the tumors to such an extent that an accumulation of lactic acid occurred. This is illustrated in Table IX. .4ftcr glucose administration the lactic acid content of the tumors was four times higher than normal. In con- trast to this behavior of tumor tissue the lactic acid content of the liver was not altered by glucose injections (Table X). Such a high lactic acid content of the tumors should lead Do an increase in the lactic acid concentration of the systemic blood, provided the tumor constitutes a sufficient~ly large percentage of the body weight. Our preliminary data recorded in Experiments 1 to 7 (Table IX) seem to point in this direction. Thus glucose administration raised the blood lactic acid of the tumor-bearing animals to 0.080 and 0.075 per cent respectively, while seven suitable control ani- mals showed lactic acid values ranging from 0.017 to 0.034 per cent. The values for the inorganic and organic phosphorus in tumors, recorded in Experiments 1 to 7, Table IX, are also of a preliminary character, since they are not numerous enough to warrant definite conclusions.

Effect of Glwose Administralion on the Lacfic Acid Concentrtrliorr in Tumors.

~. _.. -- Esperimcnt No. mxxl S11”3i-. Free sug:m i II tulnor.

per cc XL per cent

1 0.224 O.lG4 2 0 .‘a0 0.2.55 3 0.250 4 0 .3GG O.lS3 5 O.liO G 0.215 7 0.106 0.14.5

Alverage 0.190

I -1 -...-~~

pu ccn 1

0.117 O.lGO O.lM 0.141 0.0% 0.090 0.1%

0.137 .--

by guest on Decem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

C. I;‘. Cori and G. T. Cori

Experiment I.--Rat with a very large sarcoma. Weight of rat 234.5 gm. Weight of tumor 32 gm. or 13.7 per cent of the body weight. 2.37 gm. of glucose were given by stomach tube and the rat was killed lf hours later. The blood contained 0.085 per cent of lactic acid. The tumor contained 0.265 per cent of glycogen, 0.029 per cent of inorganic phosphates, and 0.154 per cent of organic phosphates (lactacidogen fraction) and gave a lactic acid maximum (in 1 per cent NaHC08) of 0.184 per cent.

Experiment d.--Mouse with very large spontaneous tumor. Weight of mouse 39.6 gm. Weight of tumor 3.74 gm. or 22 per cent of the body weight. 400 mg. of glucose were given intrnperitoncally and the mouse was killed 19 hours later. The blood contained 0.075 per cent of lactic acid. The tumor contained 0658 per cent of total carbohydrates, 0.033 per cent of inorganic phosphates, and 0.151 per cent of organic phosphates (lact- acidogen fraction) and gave a lactic acid maximum (in 1 per cent NaHCO,) of 0.352 per cent.

Experiment S.---Aiouse with spontaneous tumor. Weight of mouse 29.1 gm. Weight of tumor 1.3 gm. or 4.46 per cent of the body weight. 200 mg. of glucose were given intraperitoneally and the mouse was killed after 45 minutes.

Experiment Q.-Mouse with spontaneous tumor. Weight of mouse 30.3 gm. Weight of tumor 1.1 gm. or 3.67 per cent of the body weight. 140 mg. of glucose were injected intraperitoneally and the mouse was killed after 1 hour.

Experi?nent S.-Mouse with spontaneous tumor. Weight of tumor 1.23 gm. Injected with 100 mg. of glucose and killed after 1 hour.

Experiment 6.-Mouse with spontaneous tumor. Weight of mouse 32.1 grn. Weight of tumor 2.2 gm. or 6.8 per cent of the body weight. 200 mg. of glucose were injected subcutaneously and the mouse was killed after 30 minutes.

Experiment 7.-Patient with a metastasizing melano-carcinoma, Blood sugar before, 0.127 per cent. Blood lactic acid before, 0.022 per cent. 100 gm. of glucose were given per OS. 1 hour later blood sugar 0.166 per cent, blood lactic acid 0.039 per cent. 2 hours after the glucose ingestion a metastatic growth, located in the axilla, was removed under local anes- thesia and glycolysis immediately checked by freezing with compressed COP. The tumor contained 0.852 per cent of total carbohydrates, 0.033 per cent of inorganic phosphates, and 0.156 per cent of organic phosphates (lactacidogen fraction) and gave a lactic acid maximum (in 1 per cent NaHC03) of 0.419 per cent.

4. Glycogcn in Tumors.

That malignant tumors contain considerable amounts of glyco- gen has long been known, cspecjally through the microchemical methods of pathologists. Our data show likewise that this reserve carbohydrate is abundantly present in tumors. Glucose ingestion

by guest on Decem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

20 Carbohydrate IMetabolism of Tumors. I

seems to increase the glycogen content of the tumors as can be judged from the glycogen and lactic acid maximum values given in Experiments 1 to 7, Table IX.

TABLE X.

Effect of Glucose Administration on the Lactic Acid Content of the Mouse Liver.

The mice were fasted from 1 to 3 hours prior to the experiments. 100 mg. of glucose were given intrapcritoneally and the mice killed 1 hour after the injection.

per cent

0 391 0.310 0.282 0.300

Average..........................

Lactic acid in liver.

per cent

0.060 0.038 0.034 0.070 0.041

0.048

Average for control mice 0.051 (see Table VIII).

TABLE XI.

Glycogen Content of Mouse Curcinorne and Jensen Rat Sarcoma.

Length of time of fasting. Weight of tumor. Blood sugar. Glycogen in tumor.

- -..~- hrs. gm. per cent pei- cent 1 2.94 0.126 0.176* 1 1.87 0.141 0.1s3* 2 6.14 0.152 0.223* 4 3.62 0.149 0.171* 4 6.27 0.168 0.303*

14 23.5 0.094 0.122t -

Average............................................ 0.196

* Spontaneous mouse carcinoma. t Jensen rat sarcoma.

It seems very difficult to demonstrate an increased lactic acid production in the tissues of the living animal, since up to a cer-

by guest on Decem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

C. F. Cori and G. T. Cori 21

tain limit an excess of lactic acid that is formed in the tissues is eliminated into the blood stream. Some evidence for this was found in former experiments (3). During insulin convulsions or after epincphrine injections a strong increase in the lactic acid content of the blood was noted, while muscles and liver, which were undoubtedly the source of this increase, showed a normal lactic acid content. For these reasons the low lactic acid content of tumors of fasting animals must not be taken as a contradiction of Warhurg’s experiments. It was shown that after glucose ad- ministration t.he lactic acid content of the tumor tissue was strongly increased. The reason for this seems to be that the rate of gly- colysis of the tumor Cssue in the living animal is dependent on the free sugar concentrat.ion. Tumors of fasting animals showed a low free sugar concentration. Rft.er glucose inject.ion the free sugar concentration was strongly increased and this resulted in such an excess of lactic acid production that t,he lact,ic acid could not completely be carried away by the blood stream. These findings in the living animal were considered to be analogous to Warburg’s in vitro experiments in which the rate of glycolysis of surviving tumor tissue was increased with increasing glucose concentration. Our experiments bring forward some evidence that the important results of Warburg on the metabolism of surviving tumor tissue arc also valid for in vivo conditions.

SUMMARY.

1. Spontaneous and transplanted mouse carcinoma and Jensen rat sarcoma showed at a normal blood sugar level of the tumor- bearing animals a free sugar content of 0.051 per cent (average of 16 experiments) and a lactic acid content of 0.034 per cent (aver- age of 9 experiments).

2. After glucose administration the free sugar concentration of these tumors rose to 0.228 and 0.254 per cent (average of 4 experi- ments each) and the lactic acid concentration to 0.137 per cent (average of 7 experiments).

3. The glycogen content of six individual tumors was as an average 0.196 per cent.

by guest on Decem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

22 Carbohydrate Metabolism of Tumors. I

We wish to thank Dr. Woglom of the Cancer Institute of Colum- bia University for supplying us with the transplantable Jensen rat sarcoma and Mr. Marsh of this Institute for the mice with spontaneous tumors.

BIBLIOGRAPHY.

1. Warburg, O., Posener, K., and Negelein, E., Biochem. Z., 1924, clii, 309. 2. Cori, C. F., and Cori, G. ‘I’., J. Pharmacol. and Ezp. Therap., 192425,

xxiv, 465. 3. Cori, C. F., J. Biol. Chem., 1925, lxiii, 253. 4. Cori, C. F., J. Pharmacol. and Exp. Therup., 1925, xxv, 1. 5. Hagedorn, II. C., and Jensen, N. B., Biochem. Z., 1923, cxxxv, 46. 6. Clausen, S. R., J. Biol. Chem., 1922, Iii, 263. 7. Briggs, A. P., J. Biol. Chem., 1924, lix, 255. 8. Pfliiger, E., in Abderhnlden, E.. Handbuch dcr biochemischen Arbeits-

methoden, Berlin and Vienna, 1910, ii, 1070.

by guest on Decem

ber 5, 2020http://w

ww

.jbc.org/D

ownloaded from

Carl F. Cori and Gerty T. CoriCONTENT OF MALIGNANT TUMORS

LACTIC ACID, AND GLYCOGENOF TUMORS: I. THE FREE SUGAR,

THE CARBOHYDRATE METABOLISM

1925, 64:11-22.J. Biol. Chem. 

  http://www.jbc.org/content/64/1/11.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  #ref-list-1

http://www.jbc.org/content/64/1/11.citation.full.htmlaccessed free atThis article cites 0 references, 0 of which can be by guest on D

ecember 5, 2020

http://ww

w.jbc.org/

Dow

nloaded from