A study of the mechanism of the methylene blue test for bile pigment in urine; preparation of a compound of methylene blue and bilirubin.

According to Franke (l), the occurrence of a green color following addition of solutions of methylene blue to fluids containing bilirubin first was described by Chetchowski in 1897. It has provided the basis for a simple method of testing for bile pigment in urine and recent reports have reaffirmed its value for this purpose (2-5). The explanation for the color effect has long been a subject of controversy. The belief that the green color is caused by the blending of the blue and yellow of the two pigments has been prevalent (6-9). However, Franke maintained that a chemical reaction was responsible. We have investigated the mechanism of the formation of the green color and have found it to be both a color blending and an actual chemical combination of the bilirubin and the methylene blue. Changes occurring in the absorption spectra of the two pigments when mixed and the formation of a green precipitate under certain conditions of concentration and pH demonstrated conclusively that a chemical reaction takes place. However, this accounts for only a small part of the observed optical effect, the mixing of the blue and yellow pigments apparently being responsible for most of the green color.