THE TITRATION AND BIOLOGICAL ASSAY OF VITAMIN C IN TUMOR msue 1

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1 THE TITRATION AND BIOLOGICAL ASSAY OF VITAMIN C IN TUMOR msue 1 K K. MUSULIN, ETHEL SILVERBLATT, A\D C G KING (Fi om the DrpnutniPnt of Chiwzctrv, Unzveydy of Pzttthurgh, Pz~lchi~glz) AND GLADYS E WOODWARD (Froin thf Hiorhenizrnl RrJecirch Foundation, Frnnklin Inctittrtp, Phliidelphia) Considerable progress has been made in quantitative studies of the organic metabolites and enzymes of tumor tissue (1, 2), but the quantity of vitamin C (ascorbic acid) in such tissue has remained uncertain (3, 4). In view of the intimate relationship between vitamin C and other factors in general tissue respiration (5) and the need for both qualitative and quantitative characterization of the biochemical processes in tumors, it is of evident importance to check carefully the best chemical methods of analysis against a biological assay to differentiate or identify the indophenol-reducing substance or substances in a standard type of tumor. The present investigation has included: (a) a series of chemical analyses for ascorbic acid in Philadelphia No. 1 sarcoma, supplemented by similar tests on other tumors; (0) a comparison of the oxidative destruction of ascorbic acid by means of the Tauber and Kleiner specific enzyme (6) with the loss of titration value; (c) an animal assay to measure the true vitamin C value of standard tumors under conditions which would indicate an interference due to toxicity of the tumors fed, followed by titration of the vitamin reserves in the tissues of the animals at autopsy. Tissur Studied: Philadelphia No. 1 sarcoma was grown under carefully controlled conditions in albino rats. When about three weeks old the tissue was removed and packed in dry ice until used for analysis and feeding, the interval of storage being one to five days. The type of tumor used had a relatively high reducing value (7), and at the above age there was seldom a marked degree of necrosis. Tissues showing a marked tendency toward necrosis were discarded or the necrotic area was removed. There was practically no loss in titration value during the few days which intervened between excision of the tissue and completion of the tests on any one lot. Fresh tumor was removed for study two or three times each week. Titration Technic: Approximately 10 gm. of frozen tissue was crushed daily in a glass mortar, the latter being packed in dry ice to prevent thawing and oxidation before the sample could be thoroughly ground. Aliquot portions were then further ground in 8 per cent trichloro-acetic acid containing 2 per cent metaphosphoric acid for titration with 2, 6-dichlorophenol-indo- 1 A summary of this paper was presented at the meeting of the Fedcratioii of American Societic3 for Experimental Biology, Washington, D. C., April 26, Contribution No 315 from the Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennwlvania Received for publication Map

2 708 R. R. MUSULIN, E. SILVERBLATT, c. G. KING, G. E. WOODWARD phenol, using the general technic previously described by Bessey and King (8). Parallel titrations were made with 0.01 N iodine to provide a measure of other reducing substances in addition to ascorbic acid (particularly sulfhytfryl groups). Assay Mrthod: The major portion of the finely ground sample was then made up to volume as an aqueous emulsion in specially distilled water and fed to the assay animals from graduated pipettes as quickly as possible. Similar aliquot portions of such suspensions were used for enzyme studies. The quantity given to each animal was based upon the titration value obtained for each lot just before feeding. The curative type assay was used (9), in which guinea-pigs were given the standard Sherman diet (10) supplemented with green foods until normal growth and health had been demonstrated and their average weight had Vitamin source Tairr.i.: I : Aioloeical Assay qf I itnmin C in Tumor Tissue ~- - Feed i 11 g level No. of animals initial weight final weight gain * Scurvy score * l ii I ni in sol ui ion ruin or Vit I 111 i 11 solution \ ii,~niin solution and iunior Control 011 b.isal diet Control on bnsal diet Illgm. / clay oo.oo _ gm gm gln. -lo+ 6-32f19 59&1 I 3x f.3 5.9f.4 1.2f.3 1.4f * The probable error is calcul,ited as outlined in Sherman: Chemistry of Food and Nutrition, hl,icmill,in Co., 1932, reached about 325 gm. They were then depleted of their vitamin C reserves by being kept on the basal diet until a moderate decline in body weight was induced (thirteen days). Quantitative feeding tests were then begun with carefully matched groups of animals and continued over a period of twentyone days. The test feeding levels, 0.25 and 0.50 mg. per day, were low cnough to provide a sensitive response to differences in terms of either body weight or scurvy score. The scurvy score is generally more regular and more specific than the weight change, because the latter is subject to rapid variation in mildly scorbutic animals. I~SCUSSION The results of the assay, given in Table I, indicate clearly that the titration with 2, 6-dichlorophenol-indophenol provides a good index of the quantity of vitamin C in this type of tumor tissue. The parallel response in both scurvy score and body weight of the animals receiving 0.50 mg. of vitamin per day in pure solution and as estimated in tumor suspension (0.25 mg.) plus vitamin solution (0.25 mg.), and the similar concordance for groups receiving 0.25 mg. of vitamin either in pure solution or in tumor tissue, gives evidence that the titration value was, within the limits of assay measurement, a true index of the ascorbic acid content of the tissue.

3 ~.~~ ~.- TITRATION AND ASSAY OF VITAMIN C IN TISSUE 709 The results further indicate that such a level of tumor feeding was not markedly injurious to the animals. Had this not been true, there would have been greater disagreement between the growth response and degree of scurvy in the two groups. In necrotic or other low-vitamin tissue (note Table 111), or with a longer test period, the probability of encountering assay-interference due to toxicity would obviously be greater. The scurvy scores showed closer agreement than the weight changes, even with the small amount of tumor fed in the present study. Titration of the livers and adrenals (Table 11) of assay animals at the end of the test period provided a further check on the true vitamin intake, and again the data showed good agreement on equivalent levels. It is very unlikely that non-vitamin reducing material, if present in rat tumors, would be redistributed and stored in the tissues of guinea-pigs on a quantitative level with the vitamin... - ~ _ ~ Test level._. Vit am i ti Tuni or Vita ni i n Vi taniin Vi tanii n solution source solution solution solution Negative and tumor 1.OO controls nigni. nigni. nigm nigm. -~_-~ -~ mgni. ~ -- Livers Adrennls < TABLE 111: Comparison of Iodine and Dye Titration (mgrn. Ascorbic Acid per gm. Tissue) Type Condition Phi1,idelphia No. 1 sarcoma Very little necrosis Large liver carcinoma Severe necrosis Benign uterine tunior Slight necrosis Human Enzyme Tests: In order to check further the accuracy of the indophenol titration as a measure of the ascorbic acid content of the tissue, we prepared and used both an alcoholic extract and a purified preparation of the ascorbic acid oxidase described by Tauber and Kleiner. Through the courtesy and generous cooperation of these investigators, we were privileged to make further tests with a purified preparation and an alcoholic extract prepared in their own laboratory. A summary of these tests is given in Table IV. It is evident that the enzyme catalyzed the oxidation of the major portion of the indophenolreducing material present. The approach toward a zero titration value was not as rapid in the tissue emulsion as in simple vitamin solutions, but the agreement in activity and specificity of the independent enzyme preparations provided valuable corroborative data in support of the assay and direct titration data. It appears reasonably certain that approximately 90 per cent of the indophenol-reducing material in this type of tissue is ascorbic acid. Fur-

4 710 R. R. MUSULIN, E. SILVERBLATT, c. G. KING, G. E. WOODWARD thcr enzymic tests may establish the value with greater precision. Beyond the one hour period there was a further, but slower, decrease in the dye titration value which would indicate even less than 10 per cent non-vitamin dye-reducing material, but this point should be checked further before a conclusion is reached. The iodine titration value, beyond the indophenol value, was not significantly affected during the interval of enzyme and air exposure which resulted in destruction of the indophenol-reducing material. As a further check on the enzyme and titration experiments, and to provide comparative data for another type of tissue, several clinical tumors were compared with the rat tumors. The results (given in Table IV) were comparable with those obtained previously for the Philadelphia No. 1 sarcoma. The slower rate of oxidation in the clinical tumors was in part due to the difficulty of grinding. T~\III,I~, JV: Destruction of KrdzicinR Material by Means of the Tauher-Kleiner Sp~ c Enzyme - ~ Tissue I hiladelphia No. 1 s irconi.1 Philadelphia No. 1 s;momi (control) Iknign tumor fknign tunior Benign tumor (control) Source of enqme T.-K 0 T.- K. Prepared by authors 0 SUMMARY Driginal itration value (mgrn. xr gm.) Titration due after 1 hour (ingm. per gin.) _ ~ Amount destroyed (per cent) _- Three-weeks rat tumors, Philadelphia No. 1 sarcoma, relatively free from necrotic areas and high in ascorbic acid content, were kept in dry ice for intervals of one to five days without a significant loss in indophenol titration value. Finely crushed samples were titrated chemically by the method of Uessey and King, modified by adding 2 per cent metaphosphoric acid, and fed in standard suspension to guinea-pigs in quantities corresponding to 0.25 and 0.50 mg. of vitamin C per day. The group receiving 0.50 mg. of vitamin solution was matched against a group receiving 0.25 mg. of vitamin per day in a tumor emulsion supplemented by 0.25 mg. per day in a vitamin solution. The growth response and degree of protection from scurvy agreed with the titration value within the limits of biological assay, in comparison with animals receiving a standard solution of the pure vitamin. At the end of the assay period the vitamin C content (dye-reducing value) of the adrenals and livers was the same on equivalent feeding levels. The indophenol titrations of both rat and clinical tumors were further checked by destruction of the ascorbic acid with the specific oxidase described by Tauber and Kleiner. In this case the dye titration, but not the iodine titration, approached zero at a rate corresponding approximately with vitamin C oxidation.

5 TITRATION AND ASSAY OF VITAMIN C IN TISSUE 71 1 BIBLIOGRAPHY 1. HOLMES, BARBARA E.: Annual Review of Biochemistry, 1935, ed. by J. M. Luck, Stanford University Press, Stanford. 2. MAVER, M. E.. AND VOEGTLIN, C.: Am. J. Cancer 25: 780, BOYLAND, E.: Biochem. J. 27: 802, HARRIS, L. J.: Nature 2: 605, KING, C. G.: Physiol. Rev. 16: 238, TAUBER, H., AND KLEIKER, I. S.: J. Biol. Chem. 108: 563, WOODWARD, G. E.: J. Biol. Chem. 109: 1, 1935; Biochem. J. 29: 2405, BESSEY, 0. A., AND KING, C. G.: J. Biol. Chem. 103: 687, HARRIS, L. J., AND RAY, S. H.: Biochem. J. 27: 580, SHERMAN, H. C.. LAMER, V. K., AND CAMPBELL, H. L.: J. Am. Chem. SOL 44: 165, 1922.