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

A significant decrease in methylene blue reducing power of plasma from patients with malignan t neoplastic disease was previously reported (1). At tha t time it was suggested that change in a reducing group of the albumin molecule was a likely source of this alteration. Similar conclusions were reported also by Savignac and associates (7) as the result of analogous studies. In an a t t empt to evaluate the effect of the sulfhydry l group on the reduction of methylene blue, a s tudy was under taken with various compounds of known -SH and S-S structures. In addition, an a t t empt was made to establish a s tandard method of calibration of various lots of methylene blue, so tha t more uniform results would be possible in the plasma reducing test. Glutathione, cysteine hydrochloride and methionine were made up in equimolar solutions (0.0325 M) in distilled water. One cc. of glutathione was added to 0.2 cc. of 0.13 per cent methylene blue in a Wasserman tube. Similarly, 0.2 cc. of methylene blue (0.13 per cent) was added to cysteine and to methionine. The tubes were immersed in a boiling water bath and observed for time of complete decolorization. The tube containing methionine and methylene blue failed to show any change in color in spite of continued boiling for an hour and a half. On the other hand, complete decolorization was noted in the tubes containing cysteine HC1 and glutathione in 6.0 and 15 minutes, respectively. An a t t empt was then made to evaluate changes in the reducing time with varying concentrations of cysteine and glutathione. Thus 1 cc. of varying concentrations of cysteine HC1 was mixed with 0.2 cc. of methylene blue (0.13 per cent) and the time noted for complete decolorization. Equimolar solutions ofglutathione were treated in a similar fashion. The values obtained are indicated in Fig. 1. The results indicate a linear relationship between the cysteine concentration and the reducing power, and a definite limiting value of cysteine concent rat ion for reduction of the methylene blue. The reactions with glutathione are similar, but the reactivi ty is less than half that of the cysteine. I t is noteworthy also that the resultant leuco mixture did not revert back to colored methylene blue on cooling, as was the case with methylene blue reduction by plasma. Similar relationships were investigated between cysteine and different concentrations of methylene blue. As seen in Fig. 2, similar curves are obtained, but the position of the curve on the graph varies with the concentration of the methylene blue used. I t should be noted that there is no appreciable difference in the reducing time of methylene blue on varying the concentrations between 0.10 per cent and 0.2 per cent, although 0.08 per cent shows a decided difference. Analogous findings were obtained on mixing similar concentrations of methylene blue with a plasma sample. The following reducing times were obtained when a plasma sample was used to reduce the methylene blue solutions: