THE CARBOHYDRATE METABOLISM OF TUMORS.

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1 THE CARBOHYDRATE METABOLISM OF TUMORS. II. CHANGES IN THE SUGAR, LACTIC ACID, AND CO COMBINING POWER OF BLOOD PASSING THROUGH A TUMOR. BY CARL F. CORI AND GERTY T. CORI. (From the State Institute for ihe Study of lllalignant Disease, Bufalo.) (Received for publication, May 13, 1925.) It is the object of this pafer to adduce further evidence that Warburg s experiments on tumor tissue in vitro are also valid for in vivo conditions.? he work of Warburg, Posener, and Negelein (1) has already been discussed in the first paper (2) of this series, where it was shown that certain experiments of Warburg on surviving tumor tissue could be duplicated on tumorbearing animals. The following point seemed to require further investigation. According to Warburg, tumor cells produce in 1 hour an amount of lactic acid that is equivalent to about 2 per cent of their fresh weight. Yet, tumors of starving animais were found to contain, as an average, only per cent of lactic acid. How could this discrepancy be explained? Some experimental proof could be advanced that the low lactic acid content of the tumors was due to the fact that up to a certain limit an excess of lactic acid can be completely eliminated into the blood stream. It was thought that this question could be definitely settled by comparing the lactic acid content of blood that passed through a tumor with blood that has passed through normal tissues. The analysis of the systemic blood would be less favorable for the following reasons. An increase in the lactic acid content of the systemic blood could only be expected if the tumor constituted a large enough percentage of the body weight. The ability of the liver and the muscles to take up lactic acid from the blood is well known. Unless an amount of lactic acid constantly enters the blood, which surpasses the capacity of these organs to 397

2 398 Changes in Blood Passing through a Tumor withdraw it, the lactic acid content of the blood will remain normal. It has been shown previously (2) that glucose administration raises the lactic acid content of the tumor to to per cent. Under these conditions, an increase of the lactic acid content of the systemic blood could be demonstrated in two cases, where the tumor constituted 13.7 and 22 per cent of the body weight respectively. Four analogous results have since been obtained. Due to the lack of suitable tumor material, the experiments are not numerous enough to warrant publication. The first observations of the lactic acid content of blood that has passed through a tumor were made on a patient, who had a large sarcoma on the forearm. The vein, leaving the tumor, could easily be punctured. Since human tumors of such a favorable location are of a rare occurrence, it was found necessary to make the further experiments on animal tumors. Use was made of the Rous chicken sarcoma, which grows to a large size when transplanted into the musculature of the wing. By analyzing blood that was drawn from the normal wing and from the tumorbearing wing, observations of the changes in the composition of blood passing through a tumor could be made. EXPERIMENTAL. Plymouth Rock chickens were used. The transplantations were made by injecting fresh emulsions of tumor cells in salt solution into the musculature of the fore limb. In about 3 weeks, the tumors were of the desired size of about a hen s egg. These tumors always showed a more or less necrotic area in the center. The axillary veins of both sides were exposed and freed from the surrounding tissues shortly before the experiment was started and great care was taken to prevent any flapping of the wings. If the chickens did not remain perfectly quiet they were given a long period of rest before the blood was collected. Due to the position of the veins, blood could not be drawn simultaneously from both sides. Control experiments were therefore made, in which blood drawn alternately from the two sides was analyzed for sugar and lactic acid. If 2 to 3 cc. of blood were taken from each side, the time interval was about 2 to 3 minutes and

3 C. F. Cori and G. T. Cori it made no marked difference from which side the blood was taken first. If 6 to 8 cc. of blood were drawn from each side, the time interval was longer and the sugar concentration of the blood that was drawn last tended to be a few mg. higher. The lactic acid concentration, however, remained uninfluenced. It seemed advisable to alternate the side from which the blood was taken first on the different chickens and this course was followed in our experiments. Generally, only 2.5 to 3 cc. of blood were drawn from each side, which sufficed for quadriplicate sugar and lactic acid determinations. If duplicate CO2 analyses were intended, 6 cc. of blood had to be collected. In several experiments, in order to check the results of the first blood sampling, a small amount of blood for sugar determinations was taken, 1 or 2 hours later, in the reversed order. Precautions against glycolysis were not necessary, since the blood was analyzed as soon as it was drawn. The method for blood sugar was that of Hagedorn and Jensen (3) which seemed well adapted for our purpose, since it allowed very accurate determinations with 0.1 cc. of blood. Since the blood corpuscles of chickens sediment extremely rapidly, special attention had to be paid to a thorough mixing of the blood. The lactic acid was determined by the Clausen (4) H&SO4 procedure. This method has already been used on former occasions and certain precautions, which are not described in Clausen s paper were found of importance for the outcome of the results. The concentrated sulfuric acid should be added to the solution containing the lactic acid very slowly and under constant cooling. Care should be taken that the two fluids do not mix before aeration is started. Generally, four determinations were run simultaneously. The aeration tubes leading into the lactic acidsulfuric acid mixture should be of as small and as equal diameter as possible. The aeration should be maintained at a rather slow and very uniform rate. Compressed air was found to serve this purpose better than a suction pump. The decrease in the volume of the reaction mixture should not be too large and should be the same in all tubes. If an excess of iodine is used for the decomposition of the free bisulfite, the following titration of the bound bisulfite gives too low results. The greater part of the unbound bisulfite was removed with 0.1 N iodine, for the endpoint, how

4 400 Changes in Blood Passing through a Tumor ever, 0.01 N iodine was carefully added until a faint blue tc starch was obtained. The COz analyses were made according to the technique of Van Slyke and Stadie (5), using the fine bore type of the Van Slyke apparatus. For the determination of the COzcombining power the plasma was saturated with alveolar air. The blood for the COzcontent was collected under oil and the plasma was separated under the precautions outlined by Cullen (6). TABLE I. Comparison of the Sugar, Lactic Acid, and CotCombzning Power in the Blood of the Right and Left Wing Vein of Normal Chickens. Chicken Average.. No. Right m 1. Blood Left rein. m * * sugar. Difference. m I $ $6 1 I * After subcutaneous injection of glucose. _ Right mg. Blood lactic acid. Left m Difference. mc $ $ COzcnmbining power of plasma. Right I ml. per 1: cent _ Left Difference. _ 01. pn 001. pm cent cent 41.2 $ o $ $ Table I shows the results that were obtained when the blood from the right and left wing vein of normal chickens was compared. The blood of the right vein contained, as an average, the same amount of sugar, 0.1 mg., less of lactic acid and showed after saturation with alveolar air, 0.3 volume per cent less of COz than the blood of the left If blood that had passed through a tumor was compared with normal venous blood, the results were entirely different (Table II). The blood from the tumorbearing wing contained, as an average, 23 mg. less of sugar and 16.2 mg. more of lactic acid, and showed after saturation with alveolar air 3.7 volumes per cent less of COz than the blood that was

5 C. F. Cori and G. T. Cori 401 drawn from the normal wing. In addition, the CO2 content of blood drawn under oil has been determined (Table III). The blood from the tumorbearing wing showed, as an average, a TABLE Comparison of Sugar, Lactic Acid, and CO*Combining Power in the Venous Blood of the TumorBearing Wing and the Normal Wing. II. Blood sugar Blood lactic acid. COrcombining power of plasma. Chicken No ~ 1 orma Differ 7orms.l Differ Tumor ence. ence. _ m3. ml. m3. ma. mg. vol. per w7. cent 01. pn cent Difference * * * * : * t $ * $ * $ Average ;, * After subcutaneous injection of glucose. t Chicken moribund. Died lfi hours after blood sampling. d. per cent 0.7 volume per cent lower CO2 content than the blood from the normal wing. The experiments on the patient referred to in the introduction are recorded in Table IV and show that the blood

6 402 Changes in Blood Passing through a Tumor coming from a human tumor also contains less sugar and more lactic acid than blood that has passed through normal tissue. It should be noted that the tumor of this patient had been treated with xrays 1 month previously. The response of the tumor to the treatment, however, was not very marked. TABLE Comparison of the CO2 Content of Blood Drawn under Oil from the Tumor Vein and the Normal Vein. III. Chicken No. co2 content of plasma. Tumor Normal Difference. ml. per cat vol. per cent rd. pm cent Average TABLE Comparison of the Sugar and Lactic Acid Content in the Venous Blood from the TumorBearing Arm and the Normal Arm of a Patient with a Sarcoma on the Forearm. Tumor Blood sugar. Blood lactic acid. nw Average.., z 2 IV. RWIX3&S. 2 _~ ml/. ma. ma. ml. w O Fasting hr. after5ogm.glucose $6.5 Fasting hr. after 50 gm. ghxyse. 1 I I 50 L DISCUSSION. The experiments in Table II support our previous conception that the lactic acid content of tumors remains low, because the excess that is produced in the tumors is eliminated into the

7 C. F. Cori and G. T. Cori blood stream. The blood drawn from the vein of the tumorbearing wing contained, in the majority of the cases, decidedly more lactic acid than the venous blood of the normal wing. One chicken, which is not recorded in Table II, was negative upon repeated examination. This could be traced to an almost complete necrosis of the tumor. The agreement of the lactic acid content of the venous blood from the right and left wing vein of normal chickens (Table I) shows that the method is accurate enough to regard even small differences in the lactic acid content as positive. The increase in the lactic acid content of the blood from the tumor vein was in most cases far beyond the error of the method. The lactic acid in Clausen s method, as in other similar methods, is first converted into acetaldehyde. The latter is then distilled over into a sodium bisulfite solution. Yet, several other substances, as for instance acetone bodies, yield bisulfite compounds. There could have been such substances in the blood that has passed through a tumor, which would have been determined as lactic acid because of their bisulfitebinding properties. The isolation of the lactic acid as the zinc salt did not seem an accurate enough procedure with the small amounts of blood available. It was argued that if more lactic acid entered the blood on the tumor side than on the normal side differences in the COZcombining power of the plasma should be detected, since the excess of acid on the tumor side would bind part of the available base. Table II shows that the plasma from the tumor side, after saturation with alveolar air, yielded as an average, 3.7 volumes per cent less of CO2 than the plasma from the normal side. This was not due to a difference in the CO2 content, since blood collected under oil and centrifuged under the necessary precautions showed only 0.7 volume per cent less of CO2 in the plasma from the tumor side than from the normal side, as is illustrated in Table IV. It is, therefore, demonstrated by an independent, though indirect method, that more acid is contained in the blood that has passed through a tumor than in blood that has passed through normal tissue. A calculation will show that the agreement between the two methodsis satisfactory. The experiments 1 It was found in several experiments with chicken s blood that lactic acid determinations in whole blood and in plasma gave almost identical values.

8 404 Changes in Blood Passing through a Tumor in Table II, in which both the lactic acid and the CO*combining power have been determined, show that the blood of the tumor vein contained, as an average, 16.3 mg. more of lactic acid and 3.5 volumes per cent less of CO2 than the blood of the normal 1 molecule of lactic acid displaces 1 molecule of COB 3.5 cc. of CO2 at 0 C. and 760 B. weigh 6.87 mg. and 90.1 X 6.87 correspond to or 14.0 mg. of lactic acid. It can, 44 therefore, be concluded that most of the lactic acid that leaves the tumor is in the free state. The strong glycolysis that is taking place in the tumor tissue sets up a constant demand for sugar. This explains why the blood sugar in the tumor vein was decidedly lower than in the normal In three experiments in Table II, the difference was not very marked. In two of these cases, a rather iarge amount of blood was drawn in order to include C02combining and CO2 content determinations and the blood from the tumor side was drawn last. We are able to confirm the previous observations of Tadenuma, Hotta, and Homma (7), who noted a diminution of the sugar content of the blood drawn from the vein of a tumorbearing wing. Other substances than sugar were not determined by these authors. The observations that were made on the chicken sarcoma could be duplicated on a human tumor (Table IV). This seems of importance since the chicken sarcoma has a unique position among all other malignant tumors. It is in all probability caused by a filtrable virus. Warburg (8) mentioned in a recent communication that the chicken sarcoma showed a glycolysis of a similar magnitude to that of other malignant tumors. The results recorded in this paper are valid for malignant tumors in general and they are another confirmation of Warburg s in vitro experiments under in vivo conditions. SUMMARY. 1. On comparing the sugar and lactic acid contents and the COzcombining power of the plasma of blood drawn from the right and left wing vein of normal chickens, identical values were obtained.

9 C. F. Cori and G. T. Cori 2. If a tumor was growing on one wing, the blood that had passed through the tumor contained, as an average, 23 mg. less of sugar and 16.2 mg. more of lactic acid than the blood that had passed through the tissues of the normal wing. 3. The plasma obtained from the blood of the tumor side showed upon saturation with alveolar air 3.7 volumes per cent less of CO2 than that of the normal side. The plasma of blood that had been collected under oil contained as an average only 0.7 volume per cent less of COz when compared with that of the normal side. We are under obligation to Dr. James B. Murphy from The Rockefeller Institute, who kindly supplied us with the chicken sarcoma powder. BIBLIOGRAPHY. 1. Warburg, O., Posener, K., and Negelein, E., Biochem. Z., 1924, clii, Cori, C. F., and Cori, G. T., J. Biol. Chem., 1925, lxiv, Hagedorn, H. C., and Jensen, N. B., Biochem. Z., 1923, cxxxv, Clausen, S. W., J. Biol. Chem., 1922, lii, Van Slyke, D. D., and Stadie, W. C., J. Biol. Chem., 1921, xlix, Cullen, G. E., J. Biol. Chem., 1922, Iii, Tadenuma, K., Hotta, S., and Homma, J., Biochem. Z., 1923, cxxxvii, Warburg, O., Klin. Noch., 1925, iv, 534.

10 THE CARBOHYDRATE METABOLISM OF TUMORS: II. CHANGES IN THE SUGAR, LACTIC ACID, AND CO2COMBINING POWER OF BLOOD PASSING THROUGH A TUMOR Carl F. Cori and Gerty T. Cori J. Biol. Chem. 1925, 65: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at ml#reflist1