Isolation of Crystalline Urobilin from Human Urine

Abstract

Following the isolation of crystalline stercobilin from feces^{1, 2} attempts had been made to isolate the corresponding substance, urobilin, from urine. These were unsuccessful. The amalgam reduction of crystalline stercobilin did not yield mesobilirubinogen. 2 Neither did its oxidation with nitric acid yield methyl-ethyl-malein imid which under the same conditions and with the same amounts of substance is easily obtained from mesobilirubinogen. Urobilinogen from urine had already been shown to be identical with mesobilirubinogen, the latter 3 obtained by the amalgam reduction of bilirubin. These facts suggested that stercobilin from feces and urobilin from urine were probably not identical, although very similar. Newer evidence, however, indicates a different explanation of the above findings.

The complete separation of mesobiliviolin and stercobilin, as described in this issue, suggested the application of this principle to extracts of urines rich in urobilin. This has been done with the result that crystalline urobilin was readily obtained. Since both stercobilin and urobilin do not melt sharply, decomposing between 110-130°, other means of identification are necessary. Crystallo-graphic studies as well as measurements of the refractive indices of the two substances are under way. However, their other characteristics including solubilities, absorption spectra, Ehrlich reaction after amalgam reduction, and zinc salts are apparently identical. Preliminary studies of the crystals have shown that both exhibit parallel extinction. Comparative elementary analyses are being carried out.

The crystalline substance was obtained out of several liters of urine rich in urobilin by the following procedure: The urine was strongly acidified with acetic, and shaken out repeatedly with small amounts of chloroform until both fractions no longer showed green fluorescence with zinc acetate.