TI. ABSORPTION SPECTRA OF FRACTIONATED CANCER AND NORMAL HUMAN BLOOD PLASMA

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1 SPECTROGRAPHIC STUDIES OF CANCER AND NORMAL BLOOD PLASMA TI. ABSORPTION SPECTRA OF FRACTIONATED CANCER AND NORMAL HUMAN BLOOD PLASMA RACHEL G. FRANKLIN, EDWARD B. SANIGAR, AND ALEXANDER J. ALLEN (From the Biochemical Research Foiindation of The Franklin Institute, Philadelphia, Pa., Dr. Ellice McDonald, Director) INTRODUCTION In the experiments on the absorption of normal and cancer rat plasma fractionated with zinc hydroxide powder, described in the preceding paper ( I), it was found that the absorption curves of normal rat plasma treated with zinc hydroxide powder were practically identical between the wavelengths of 2900 A and 3400 A while those of cancer rat plasma, similarly treated, deviated from the normal, the deviation being roughly proportional to the size of the tumor growth. In the present study, on human plasma, the same technic was employed to determine whether a similar difference exists. RESULTS Absorption curves typical of those obtained are shown in Fig. 1; these were obtained from a healthy subject, Case 26. Curve A is that of undiluted plasma; curve B of plasma diluted five times; curve C of plasma diluted five times and treated with 0.1 gm. of zinc hydroxide powder per 5 C.C. of diluted plasma, and curve D of plasma diluted twenty times. Such determinations were made on 12 cancer and 7 normal plasmas. A careful examination of these curves was made to discover any possible correlation between the shape of the curves and the pathological condition of the subject. The height of the band at approximately 2780 A, which is a rough measure of the protein content, varied over a wide range in both cancer and normal plasmas, and nothing peculiar to the cancerous condition could be detected. The extinction coefficients in the region of 2900 A to 3100 A of plasma after treatment with 0.1 gm. of zinc hydroxide powder (curve C) gave no consistent values for these human specimens as was the case for rats (1). The ratio of the maximum extinction coefficient of the plasma treated with zinc hydroxide powder (curve C) to that of the untreated plasma of the same dilution (curve B) for the 2800 A band was, however, found to be greater for normal plasmas than for cancer plasmas. The work, therefore, was continued along this line. The results of tests on a total of 20 normal and 26 cancer plasmas are shown in Table I. Since the exact height of the maximum is difficult to determine, as it is an extrapolated point, the ratios were calculated at 2700 A. Fig. 2 is a graphic representation of these results with the ratios as ordinates. It will be noted that in general the ratios for normal plasmas are 0.47 or 30 1

2 ~ 3 02 RACHEL G. FRANKLIN, EDWARD B. SANIGAR AND ALEXANDER J. ALLEN TABLE I: Extinction Coefiicients, at ii, of Human Rlood Plasma (.<lx! NO Average.. - I Jn treated oo Treat etl IZatio _-.... Case NO It sn 59 ~- ~ Averngc IJntreated no Treated I h t io greater while the values for the cancer plasmas are 0.47 or less. Case 8, one of skin cancer, has an abnormal ratio for a cancer case. The value 0.59 brings it into the normal range, but the similarity of plasma from people having skin cancer to that from healthy persons has been previously noted (2). Case 50, a healthy person, fell within the cancer range, with a ratio of 0.39 when first examined, but after several months a second determination gave a value of. In Cases 2 and 17 observations were repeated after an interval of several days, and while the values show some change, they are, nevertheless, well within the normal range. In Case 7 there was edema of the retina and in Case 12 chronic asthma, but both gave normal values. Attempts to correlate age, sex, blood group, blood ph, condition of tumor, and previous treatment of the patient with these ratios gave negative results. It was thought that some knowledge as to the nature of this difference between cancer and normal plasma could be gained by comparison with the precipitation of proteins by another fractionating agent, such as ammonium sulfate. This agent does not absorb ultraviolet light in the region studied and gives filtrates of a suitable dilution for use with the present method. Accordingly, from a number of samples of both cancer and normal plasma the protein fractions were successively precipitated by ammonium sulfate, using the method described in Hawk and Bergheim's Practical Physiological Chemistry

3 SPECTROGRAPHIC STUDIES OF BLOOD PLASMA. I1 303 (31, and the absorption spectra of the filtrates were determined. At the same time, whenever possible, different amounts of protein were removed from the plasma by increasing weights of zinc hydroxide powder, and the absorption spectra were taken. The ratios were calculated as before. The results for fractionation by ammonium sulfate are shown in Table I1 and in Fig. 3. It will be observed that in almost every case the ratios for the cancer plasma are smaller than for the normal plasma. At the foot of Table I1 are values representing the decrease in the average ratios due to the removal of each of the protein fractions. While these values do not represent actual percentages of the total protein, since the extinction coefficients Fic. 1. TYPICAL ABSORPTION CURVES OF WHOLE AKD FRACTIOXATED HUMAS BLOOD PLASMAS A. Whole undiluted plasma. B. Whole plasma dillited 5 times. C. Plasma diluted 5 times and fractionated with 0.1 gm. zinc hydroxide per 5 C.C. of diluted plasma. D. Whole plasma diluted 20 times. of the various proteins differ, they are, nevertheless, an indication that the amounts of fibrinogen, euglobulin, and pseudoglobulin are greater in cancer plasma than in normal plasma. The results obtained with the use of zinc hydroxide powder were calculated in the same way as for the precipitation with ammonium sulfate and are shown in Table I11 and in Fig. 4. Here also the ratios for the cancer plasma are smaller than for the normal plasma, and the proteins most easily removed by zinc hydroxide powder are present in greater amounts in cancer plasma than in normal plasma. The percentage of proteins removed by 46 per cent saturation of ammonium sulfate (the fibrinogen-globulin fractions j is approximately the same as that removed by 0.1 gm. of zinc hydroxide powder.

4 ~ ~ -~~ 0.64 T.1 J~.E I J : Precipitation by Ammonium Sulfate (Extinction Coefficients at 2700 A) Case No. I'lasma minus Fibrinogen 25% Satn. Ratio I'lasma minus Fibrinogen and Euglobulin 33 % Satn. - Iiatio Plasma minus Fibrinogen, Euglobulin and Pseudoglobulin ~ 46 % Satn. Ratio -. ~ Aver'ige 0.92 I Average _. ~ ~ ~ Ikcrease in Average lintio dire to Precipitation by Ammonium Sulfate of Fibrinogen Euglobulin Pseudoglobulin Normals Cancers.....O ('ase No. ~ ~ ~ Iiritreated - _ ~- _ ~ 0.05 gm Ratio 0.10 gni. Ratio Average ~ Normal I Increnient Decrease in Average liatio due to Treatment by Zinc Hydroxide 0.05 gm gm gru. Normals.....O Cancers

5 SPECTROGRAPHIC STUDIES OF BLOOD PLASMA. I1 305 The mechanism of the removal of the proteins by these two agents is probably different. Svedberg (4, 5) and Block (6) have concluded that the proteins probably undergo a change on fractionation by such agents as ammonium sulfate. Their behavior on removal by zinc hydroxide powder is not known, but from these and other experiments, it appears that there is a certain amount of preferential adsorption, that is, fibrinogen and globulin are removed in greater proportions than albumin by smaller weights of zinc hy-.6 5 X x NORMAL 0 CANCER.SO YX 0 FIG. 2. RATIOS OF EXTINCTION COEFFICIENTS OF FRACTIONATED PLASMA TO WHOLE PLASM AT 2700.& x = Normal plasma o == Cancer plasma droxide powder. On this assumption our results could be explained as due to an increase in the percentage of fibrinogen and globulin of cancer plasma.' In this case the difference between the cancer and normal plasmas observed on treatment with zinc hydroxide powder would not be specific for cancer, since an increase in the fibrinogen-globulin content is a condition known to accompany many diseases (7). It is possible, however, that there may be an actual qualitative difference in these proteins and that cancer plasma may contain some substance similar to ordinary fibrinogen or globulin not present in normal plasma. Work on this problem is in progress in our laboratory.

6 0 I I I I I 0 40 x SATU~RAT~ON AMMONIUM SULFATE FIG. 3. RATIO or EXTINCTION COLITICILNIS OP PLASMA FRACTIONATED WITII AMMOXIUM SULFATE TO WHOLE PLASMA AT 2700 A x = Normal plasma o == Cancer plasma 00 I I I 05.I0 15 WEIGH'T ZINC HYDROXIDE IN GRAMS FIG. 4. htio OF EXTINCTION COEFPICItiNTS OF PLASMA FRACTIONATED WITII ZINC HYDROXIDE POWDER AT 2700 A x == Normal plasma o = Cancer plasma 306

7 SPECTROGRAPHIC STUDIES OF BLOOD PLASMA. I1 307 SUMMARY 1. By the use of spectroscopic methods normal and cancer human blood plasmas have been found to fractionate differently. When blood plasma from normal and cancer-bearing individuals was treated with zinc hydroxide powder, the decrease in the absorption of the protein band was proportionately greater for cancer plasma than for normal plasma. The ratio of the extinction coefficient at 2700 A of the treated plasma to that of the untreated plasma was calculated for 20 normal and 26 cancer cases, and with very few exceptions the values were 0.47 or greater for the normal plasma and 0.47 or less for the cancer plasma. 2. The amount of protein removed from 5 C.C. diluted plasma (diluted 5 times) by 0.1 gm. of zinc hydroxide powder was comparable to that precipitated by 46 per cent saturation of ammonium sulfate (Le., the fibrinogenglobulin fractions). The difference in the fractionation of cancer and normal plasmas may be explained, therefore, by the assumption that there is an increase in the fibrinogen-globulin content in cancer. 3. A comparison of methods of fractionation gave evidence that zinc hydroxide powder may have some selective action in removing proteins. 4. Significant differences in the absorption curves do not occur in the whole blood plasma. The authors wish to express their appreciation to Dr. Ellice McDonald for his help and encouragement, and to Miss J. Schoonover and members of the staff of the Philadelphia General Hospital for their cooperation in obtaining blood for this work. BIBLIOGRAPHY 1. ALLEN, A. J., FRANKLIN, R. G., AND SANIGAR, E. G.: Am. J. Cancer 27: 296, WOODWARD, G., SCHOONOVER, J., FRY, E., TORRANCE, E., AND MCDONALD, E.: J. Lab. & Clin. Med. 16: 701, HAWK AND BERGHEIM : Practical Physiological Chemistry, 10th Edition, P. Blakiston s Sons and Co., Philadelphia, Pa., 1927, p SVEDBERG, T.: Nature 128: 999, SVEDBERG, T., AND SJ$GREN, B.: J. Am. Chem. Soc. 52: 2855, BLOCK, R. J.: J. Biol. Chem. 103: 261, KAMINER, G. : Die Biochemie des Karzinoms, Abhandlungen aus der Gesamtgebiet der Medizin, Julius Springer, Wien, 1926.