SERUM DIAGNOSIS OF ROUS'S CHICKEN SARCOMA, BASED ON CHEMICAL METHODS CASIMIR FUNK Introduction. Serum diagnosis of tumors is, at present, in a preliminary stage. During the last few years there have been described several methods which are mostly based on biological properties of serum. None of these methods, however, has given satisfactory results. Among the procedures tried by the author were the method of Freund, the meiostagmin-reaction of Ascoli, and the optical method of Abderhalden. In view of the failure of these biological tests to give a reliable method for tumor diagnosis, it seemed advisable to ascertain whether purely chemical methods would serve better for this purpose. This investigation was conducted with serum of chickens inoculated with Rous's chicken sarcoma. The large amount of serum obtainable from these animals was a great advantage for our preliminary studies. The tumor used being very malignant, the animals died in a few weeks. One would therefore expect the chemical changes to have been very pronounced. In the first part of the work, the serum was precipitated with absolute alcohol; and the amount of precipitate, and its nitrogen and phosphorus contents, were estimated. The filtrate was analyzed in the same way. Throughout the whole inquiry, normal and tumor sera were treated simultaneously. The results tend to show that the tumor serum was poorer in proteins and phosphorus. Better results were obtained by analyzing serum itself. In this case the proportions of nitrogen, phosphorus, sulfur, chlorin, aminonitrogen, and sugar, and the molecular depression, were taken into account. Of 22 tumor sera, 20 gave practically identical results. Tumor serum was, as a rule, poorer in nitrogen, phosphorus and sulfur than normal, though richer in amino-nitrogen; the molecular depression was greater in normal serum. Sugar was determined in serum which had been kept, for some time, in an incubator; hence the figures are not exact. There was, perhaps, more sugar in tumor serum, but not enough cases were investigated to warrant a definite conclusion. Rolly and Oppermann found, in the few cases at their disposal, an increase of sugar in the plasma.1 The two sera which gave contradictory results were, strangely enough, taken from animals in which inoculation occurred much earlier than in other cases. Whether these results were due to the resistance of the animal to tumor growth, or to the weakness of the tumor, must be left for further study to determine. For these two sera the tumors were very small and entirely encapsulated. In summarizing the results the author feels justified in concluding that in the case of Rous's sarcoma, chemical analysis gives much more reliable results than other diagnostic methods. Several objections can, however, be put forward. The most important of these is that Rous's chicken sarcoma differs in many respects from other tumors; it is regarded by Rous himself as being of infective origin. In a few instances rat serum was used of rats inoculated with Jensen's sarcoma. In each case 5-6 rats were bled and the bloods combined, so that the figures in the Tables represent good averages. Here, too, the differences were very much of the same order as for chicken sera. This matter awaits further study. A second objection, no less important, is the fact that the same chemical differences may be found in other pathological conditions. The animals regarded as normal were brought up in town, and were kept for a long time in the laboratory. Everybody who has worked with fowls knows that, under such conditions, fowls do not develop normally. Incidentally, a few cases of avian tuberculosis were investigated among the non-tumor animals and normal figures were obtained. We see, from the results in the accompanying Tables, that the size of the tumor does not seem to have an effect on the data, though possibly the length of the "inoculation period" played a part. This point will be studied in the near future. The chlorin content was found to be practically the same in tumor and normal sera, and therefore can be disregarded. Although one is practically able to diagnose Rous's chicken sarRolly and Oppermann: Biochem. Zeit., 1913, xlviii, p. 471. coma from chemical data for the serum, it does not follow that analogous results can be obtained for human serum. Our study will be extended in this direction as soon as further clinical material is available. By using micro-methods, the amounts of blood to be taken can be very greatly diminished. Very interesting, also, will be the analysis of serum in pregnancy, where differences in other directions may be revealed. The method is slightly inconvenient because normal serum must be taken as a control. From the figures obtained it is evident that, by working in pairs (one normal and one tumor serum), both results were relatively either high or low. This was due very likely to the fact that the birds were bled completely, and practically the whole serum was used for analysis. TABLE I Data pertaining to the alcoholic extraction method on fowls Experimental. 1. The method used was as follows: The blood was taken from pairs (one normal and one tumor animal), and was obtained by cutting the throat of the animal. Both bloods were left for the same length of time in an incubator. The separated serum was weighed and precipitated with 10 times its weight of alcohol. Both precipitates were left in the same desiccator and weighed, and an aliquot part taken for nitrogen and phosphorus estimations. The filtrate was diluted, with the alcoholic washings of the precipitate, to 250 cc.; 100 cc. were taken for each determination. In estimating small amounts of phosphorus, in a total volume of 50 c.c., by the method described in Hoppe-Seyler-Tierfelder, precipitation frequently failed to occur but could be obtained at greater dilutions. These experiments (Table 1) show a larger quantity of alcoholic precipitate for normal animals, a corresponding increase in nitrogen, and a slight decrease in phosphorus. In the alcoholic filtrate for tumor animals, there were increases in nitrogen and phosphorus. The experiments were deficient, however, in the fact that, by cutting the throats, the contents of the crops possibly contaminated the blood. All the subsequent experiments on fowls were made with blood from the heart, taken by means of a canula, with the animal under anesthesia. TABLE 2 Data pertaining to the alcoholic extraction method on rats 2. There were eight rats with Ehrlich's sarcoma and six normal ones in this series (Table 2). The method was that described above (1). TABLE 3 Data pertaining to the alcoholic extraction method on fowls 5N13.. R5.. 0.315 0.0063 0.0154 0.0113 2.32 2.91 0.407 0.0078 0.0219 0.0134 Aver. R.. 3.78 0.505 0.0116 0.0243 0.0181 Ν.. 3.99 0.559 0.0102 0.0220 0.0172 |