A quantitative cytochemical assay for osteoclast acid phosphatase activity in foetal rat calvaria

Abstract

Acid phosphatase activity is prominent in osteoclasts (bone resorbing cells) and has been implicated in the process of bone resorption, although its precise role is not understood. To study the distribution and activity of this enzyme, a quantitative cytochemical method has been developed using undecalcified fresh frozen sections of foetal rat calvariae. Sections were allowed to react with 3 mM naphthol ASBI phosphate at pH 5.0, and the reaction was stopped by rinsing in ice-cold tap water containing 50 mM sodium fluoride. The reaction product was post-coupled to Fast Garnet at 4 degrees C. The absorbance of areas of reaction product in the cytoplasm was measured using scanning and integrating microdensitometry. The initial velocity rate was maintained for up to 2 min at pH 5.0 with a substrate concentration of 3 mM and a section thickness of 5 micron. Under these conditions reaction product was localized to osteoclasts and the surface of bone matrix beneath these cells. Activities in osteoblasts and chondrocytes were negligible. Osteoclastic acid phosphatase was almost totally inhibited by 10 mM fluoride and reduced by 70% by 100 mM tartrate.