THE CYTOLOGIC DEMONSTRATION OF β-GLUCURONIDASE EMPLOYING NAPHTHOL AS-BI GLUCURONIDE AND HEXAZONIUM PARAROSANILIN; A PRELIMINARY REPORT

The preparation of a new substrate for β-glucuronidase, naphthol AS-BI β-d-glucuronide, has been described. The potassium salt of naphthol AS-BI and acetobromomethyl glucuronate were reacted in ethanol. Unreacted anilide was removed and deacetylation and deesterification were carried out with barium hydroxide. The barium salt of naphthol AS-BI glucuronide was separated and was then converted to free acid. The compound has an elemental analysis which agrees with the theoretical one. It has also been possible to devise a satisfactory cytochemical technique for the in situ demonstration of β-glucuronidase employing sodium salt of naphthol AS-BI glucuronide and hexazonium pararosanilin. The optimal staining reaction was obtained with 0.25 mM substrate and 1.8 mM diazo reagent at pH 5.2 in 20 to 30 minutes at 37°C for rat liver and kidney. The brilliant red dye was visualized at the site of enzyme activity mostly as discrete granules. A brief discussion concerned the evaluation of the present method as a cytochemical technique for the demonstration of β-glucuronidase.