A simple method of isolation of crystalline stercobilin or urobilin from feces.

The methods described two decades ago (l-3) for the isolation of crystalline stercobilin or urobilin from feces or urine are relatively slow and cumbersome. It is often desirable to obtain a few mg of crystalline material quickly, either to permit determination of the preponderant type (whether stercobilin, urobilin, or d-urobilin) or calibration of a quantitative method. More recently, the studies of Shemin and associates (4-S), based on the essential nature of glycine and acetate in the biosynthesis of the heme pigments, have made it highly desirable to have at hand a simple method permitting rapid and repeated isolation of stercobilin from feces in order that serial observations of N16 content may be made, following administration of N16-glycine. Of special interest in this connection is the question of exact significance of the early appearance of N15 in the stercobilin molecule, a finding indicating that a certain fraction, varying under normal and pathological conditions, has a derivation other than the hemoglobin of red blood cells which have survived a normal life span (6, 7). Since determinations of the atom per cent excess of N15 in the mass spectrometer require but 10 mg. of pure substance, the method to be described in the following is particularly adapted to studies of this type. This method depends in considerable part on the dehydrogenation of stercobilinogen or urobilinogen with iodine, in petroleum ether solutions, as described in the preceding paper (9). It may be noted that Lichtenstein and Terwen, in 1925 (lo), used petroleum ether to extract a urobilinogen from an aqueous filtrate of a feces-ferrous hydroxide mixture. The petroleum ether was then allowed to stand in the light, with resultant formation and precipitation of an amorphous urobilin. The latter was not crystallized, but was evidently relatively pure. It probably consisted in the main of the levorotatory stercobilin. We have, in fact, been able to isolate crystalline sterco-