Identification of the uronic acid from oestriol 'monoglucuronide.'.

In 1936 Cohen & Marrian isolated from human pregnancy urine an amorphous water-soluble substance containing about 50% of combined oestriol. From its elementary composition and properties this substance appeared to consist largely of an oestriol monoglucuronide. Subsequently (Cohen, Marrian & Odell, 1936) this substance yielded a crystalline sodium salt, which, on the basis of its elementary composition and a strongly positive Tollens naphthoresorcinol reaction, appeared to be nearly pure sodium oestriol glucuronidate. In the course of this work it was shown that the uronic acid moiety is attached to the oestriol by a glycosidic linkage involving the potential aldehyde group ofthe former, and the C-16 or C-17 hydroxyl group of the latter. No clear-cut evidence was obtained, however, that the uronic acid is indeed glucuronic acid. In view of the lack of specificity of the Tollens reaction for glucuronic acid (Mandel & Neuberg, 1908; Dische, 1946) it was felt that a further investigation of the identity of the uronic acid component should be undertaken. The identification of uronic acids is difficult, and the choice of method was restricted by the small quantity of purified oestriol 'monoglucuronide' which could be prepared. Unequivocal methods such as the isolation of D-glucurone as used by Pryde & Williams (1933) in the case of borneol glucuronide, or the preparation of the p-toluidine complex of ammonium glucuronidate (Smith & Williams, 1949) are not recommended for work with small quantities of glucuronide (Williams, 1949). The method ofLohmar, Dimler, Moore & Link (1942) involving oxidation ofa uronic acid to a dicarboxylic acid, with subsequent identification of the latter as the dibenziminazole derivative, is open to the criticism that it will not distinguish between D-glucuronic and L-guluronic acids as both of these acids give D-glucosaccharic acid on oxidation. The method has however been employed by Levvy (1948) working with small quantities of menthyl glucuronide, and although Bernhauer & Irrgang (1935) believe that they have demonstrated the production of L-guluronic acid by the action of certain bacteria on glucose, this acid has not so far been found to be produced by animal organisms. It therefore appeared that this method would provide a means of identifying the uronic acid moiety of oestriol 'monoglucuronide' with reasonable certainty.