The measurement of glucuronide synthesis by tissue preparations.

To measure the ability of surviving tissue slices to form glucuronides of compounds such as menthol and phenol, Lipschitz & Bueding (1939) determined ether-soluble compounds giving the Tollens colour reaction for glucuronic acid. Whilst this procedure yielded valuable information in their hands, it is inconvenient for routine purposes, and the lack of specificity in the colour reaction can be a serious disadvantage. It must also be borne in mind that not all glucuronides are as ether-soluble as that derived from menthol. De Meio & Arnolt (1944), in their study of phenol conjugation by tissue slices, assumed the figure for combined phenol given by the method of Theis & Benedict (1924) to represent phenylsulphuric acid and phenylglucuronide. As shown below, it is doubtful whether the results of De Meio & Arnolt (1944) have any bearing on the problem of glucuronide synthesis. It was considered that the study of the biological mechanism of glucuronide formation required a sensitive reaction, specific for a glucuronide in the presence of a large excess ofthe hydroxy compound. Following a suggestion made by Dr R. T. Williams (private communication), the possibilitywas investigated of determining o-aminophenyl-fl-D-glucuronide (Williams, 1943) in the presence offree o-aminophenol by the reaction described by Bratton & Marshall (1939) for the estimation of sulphonamides. If the reaction was carried out according to their directions, in strongly acid solutions, the bluish pink given by the glucuronide was slow to develop, and when it had reached its full intensity an appreciable colour of similar shade was seen in parallel experimentswith the free phenol. By careful control of pH it was found possible, not only to shorten the period for complete colour development with the glucuronide, but to eliminate interference by free o-aminophenol. To avoid having to adjust the pH of the solution after removal of proteins, methods of protein precipitation at the pH chosen for colour development were studied. Application of the complete procedure to the measurement of glucuronide synthesis by mouse-liver slices is described below.