Sulfhydryl Reduction of Methylene Blue with Reference to Alterations in Malignant Neoplastic Disease

Sulfhydryl Reduction of Methylene Blue With Reference to Alterations in Malignant Neoplastic Disease

Maurice M. Black, M. D.

(From the Department of Biochemistry, New York Medical College, New York 29, N. t;., and the Brooklyn Cancer Institute, Brooklyn 9, N. Y.) (Received for publication May 8, 1947)

A significant decrease in methylene blue re- reactivity is less than half that of the cysteine. It is ducing power of plasma from patients with malig- noteworthy also that the resultant leuco mixture nant neoplastic disease was previously reported did not revert back to colored methylene blue on (1). At that time it was suggested that change in a cooling, as was the case with methylene blue re- reducing group of the albumin molecule was a duction by plasma. likely source of this alteration. Similar conclusions Similar relationships were investigated between were reported also by Savignac and associates (7) cysteine and different concentrations of methylene as the result of analogous studies. blue. As seen in Fig. 2, similar curves are obtained, In an attempt to evaluate the effect of the sulf- but the position of the curve on the graph varies hydryl group on the reduction of methylene blue, a with the concentration of the methylene blue used. study was undertaken with various compounds of It should be noted that there is no appreciable known -SH and S-S structures. In addition, an difference in the reducing time of methylene blue attempt was made to establish a standard method on varying the concentrations between 0.10 per of calibration of various lots of methylene blue, so cent and 0.2 per cent, although 0.08 per cent shows that more uniform results would be possible in the a decided difference. plasma reducing test. Analogous findings were obtained on mixing Glutathione, cysteine hydrochloride and meth- similar concentrations of methylene blue with a ionine were made up in equimolar solutions (0.0325 plasma sample. The following reducing times were M) in distilled water. One cc. of glutathione was obtained when a plasma sample was used to reduce added to 0.2 cc. of 0.13 per cent methylene blue in the methylene blue solutions: a Wasserman tube. Similarly, 0.2 cc. of methylene Methylene blue, Reducing time, blue (0.13 per cent) was added to cysteine and to per cent minutes methionine. The tubes were immersed in a boil- 0.08 5.5 ing water bath and observed for time of complete 0.13 8 decolorization. 0.20 8 The tube containing methionine and methylene blue failed to show any change in color in spite of The curves depicted in Fig. 2 possess an additional continued boiling for an hour and a half. On the feature which merits attention in relation to the other hand, complete decolorization was noted in results obtained with plasma, namely, the sharp the tubes containing cysteine HC1 and glutathione break in the curves occurring after the methylene in 6.0 and 15 minutes, respectively. blue reducing time of 15 minutes. A similar break An attempt was then made to evaluate changes is found at 15 minutes when the occurrence of in the reducing time with varying concentrations various reducing times of numerous plasma sam- of cysteine and glutathione. Thus 1 cc. of varying ples from cancer patients is plotted. These figures concentrations of cysteine HC1 was mixed with 0.2 are based on 109 plasma samples whose reducing cc. of methylene blue (0.13 per cent) and the time times fell between 13 and 25 minutes. As with the noted for complete decolorization. Equimolar solu- cysteine, apparently, a point is reached beyond tions ofglutathione were treated in a similar fashion. which decrease in reducing activity is attended by The values obtained are indicated in Fig. 1. The marked change in the reducing time; in short, a results indicate a linear relationship between the steeper rate of change. cysteine concentration and the reducing power, In view of the parallel results obtained with and a definite limiting value of cysteine concen- cysteine HC1 and plasma, the cysteine solutions tration for reduction of the methylene blue. The may be used to test the reactivity of different lots reactions with glutathione are similar, but the of methylene blue. Concentrations between 0.1

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35 Incomplete Incomplete 35, reduction reduction Incomplete reduction t 30 30- E " \ I.- 25 25. x

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m 15 *= 15. D (i) --... II] $ le- e :S x - Glutothione ~"~"0.-...r O ~ Gysteine HGI 2

,'.o ~o 3.o ,b 2'.0 3'.0 4'.0 Moles x I0 "s Moles x I0 "s GysteineHCI Fig. I- Reduction of methylene blue (Icc. of 0.13%) by FIG?-.- Reduction of methylene blue (0.2cc.) of varied concentration cysteine H CI and glutothione 35-

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Methylene Blue Reducing Time (minutes) Fig.3.- Distribution of reducing time values in 109 cancer cases per cent and 0.2 per cent would seem to be most injections. Similar results were obtained by in- efficacious, since variations in this zone produce jection" of 25 to 50 mgm. of cysteine HC1 although minimal changes in reducing time. Thus, a con- the initial reaction did not occur. It should be centration of 0.13 per cent methylene blue has been mentioned that although the symptomatic im- adopted in evaluating the reducing power of plasma. provement might be great, there was no apparent The apparent decrease in sulfhydryl activity in effect on the growth of the neoplasm. plasma of patients with malignant neoplastic disease suggested therapeutic trial of sulfhydryl DISCUSSION compounds on such patients. Glutathione in doses The reduction of methylene blue by cysteine of 50 to 100 mgm. was injected intravenously in and glutathione and not by methionine is an in- several patients with varying types of malignant dication that, in these compounds at least, a free neoplastic diseases. A reaction of chills and fever -SH bond is required. The more prolonged time occurred in most cases in about 30 minutes and found for glutathione (15 minutes) as compared lasted from 20 minutes to an hour. This was with cysteine HC1 (6 minutes) would seem to in- usually replaced by a sense of well being and relief dicate that the availability or reactivity of the from previous symptomatic complaints, viz. pain, -SH bond may be altered by its location in the asthenia etc. Beneficial effects usually lasted molecule. Thus in cysteine the -SH is terminal several days and could be regained by repeated and presumably unhindered in its reactivity. In

the case of the glutathione, the internal location 2. The reaction with cysteine HC1 is more rapid of the -SH bond seems to decrease its reactivity. (6.0 minutes) than the reaction with equimolar The reversible decrease in reducing power of glutathione (15 minutes). plasma associated with malignant neoplastic dis- 3. The reaction between cysteine and methylene ease might be explained on the basis of changes in blue shows a linear relationship, which may be used the spatial configuration of the albumin molecule. for calibration of different lots of methylene blue. Such changes would be readily reversible and 4. A possible relationship between the spatial would not necessitate changes in amount of total configuration of the albumin molecule and the de- protein of -SH bonds. This is of importance since crease in reducing power of plasma in the presence the observed decrease in reducing power is not of malignant neoplastic disease is discussed. correlated with changes in concentration of plasma 5. Effects of the administration of cysteine and proteins. glutathione on patients with malignant neoplastic The observation that glutathione and cysteine were often efficacious in relieving the symptomatic diseases are reported. complaints of patients with malignant neoplastic disease finds some parallel in the literature. Thus, ACKNOWLEDGEMENT Stern and Wilheim (8), in reviewing the relation of sulfhydryl compounds to life processes, point out Appreciation is expressed to Dr. I. S. Kleiner, Professor that the growth inhibition of normal animals treat- of Biochemistry at New York Medical College, and to Dr. ed with various organic carcinogens could be can- H. Bolker, Pathologist, of Brooklyn Cancer Institute, for their interest and criticism. celled by increased quantities of sulfhydryl compounds in the diet. This is interpreted as being due to utilization of sulfhydryl compounds in detoxifi- REFERENCES cation of the carcinogens and also the competitive utilization of -SH groups by neoplastic tissue itself, 1. BLACK, M. M. Changes in the Reducing Power of leaving relative deficiencies of these indispensible Serum or Plasma of Patients with Malignant Neo- plastic Disease. Cancer Research, 7:321-325. 1947. requisites for growth, enzyme activity and metab- 2. BRUNSCHWIG, A., ARNOLD, J., and EDGECOMB, J. olism. Therapeutic use of cysteine and glutathione Stimulation and Retardation of Neoplastic Growth also has been suggested by the observation of tu- by Sulfhydryl Compounds. Cancer Research, 6: mor growth inhibition in some neoplasms (2, 3, 5), 560-563. 1946. although no such effect was obtained in others (4). 3. CARR,J. L. Action of Compounds Related to Cysteine The data presented in this and our previous on the Regression of Jensen's Rat Sarcoma. Proc. study would seem to point to the importance of Soc. Exper. Biol. &Med., 35:341-342. 1936. the sulfhydryl group as a factor in the over-all 4. CARR, J. L., CONNOR, C. L., and GINZTON, L. L. Some Experiments with Cysteine Hydrochloride in redox substrate of the body rather than as a specific the Treatment of Animal Tumors. Am. J. Cancer, tumor-inhibiting agent. One might consider this 34:428-430. 1938. group analagous to a buffer in acid-base systems. 5. CONNOR, C. L., CARR, J. L., and GINZTON, L. Cys- Loss of such control would lead to suboptimal sulf- teine in Jensen's Sarcoma. Proc. Soc. Biol. & Med., hydryl potentials with attendant alterations in 34:374-376. 1936. function of various enzyme systems. The report 6. HIRSHFELD, S., DUBOFF, G., and WEST, P. M. Demonstration of an Enzyme-Inhibiting Factor in by Hirshfeld, Duboff, and West (6) of an inhibitory the Serum of Cancer Patients ( A Preliminary action of serum of cancer patients on the tyrosine- Study). Cancer Research, 6:57-61. 1946. tyrosinase system might be explained on the basis 7. SAVIGNAC, R. J., GANT, J. C., and SIZER, I. W. Re- of such alteration in optimal -SH potential. ducing Properties of Serum from Malignant and Nonmalignant Patients and from Normal Individ- SUMMARY uals. A. A. A. S. Research Conference on Cancer. 1944. pp. 241-252. 1. Methylene blue is reduced by boiling with 8. STERN, K., and WILHEIM, R. The Biochemistry of cysteine HC1 and with glutathione but not with Malignant Tumors. Brooklyn, N. Y.: Reference methionine. Press. 1943, pp. 401-402.

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Maurice M. Black

Cancer Res 1947;7:592-594.

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