Application of periodate oxidation to the histochemical identification of acidic polysaccharides

Abstract

This thesis is concerned with the application of periodate oxidation to the histochemical identification of the acidic polysaccharides (glycosaminoglycans) of connective tissues, especially cartilage. When fresh frozen sections of bovine nasal septal cartilage are given prolonged exposure to sodium periodate solution a great deal of basophilic sulphated material is lost. This was characterised by staining with Alcian blue in 0.40M MgC12 to be mainly chondroitin sulphates. The loss was shown to be due specifically to the periodate ion and most occurs during the first hour of exposure of sections. Two main sites of action of periodate in cartilage were considered. These are the network of collagen fibrils and the interfibrillar proteoglycans. Periodate was shown to cause great decreases in viscosity for proteoglycan and glycosaminoglycuronan solutions. Propan-1-ol, a presumed free-radical scavenger, was found to diminish loss of stainable material from cartilage sections and the rate of decrease of viscosity of glycos-aminoglycuronans in periodate solution. It is proposed that free-radicals are present in periodate solution and that they cause depolymerization of glycosaminoglycuronans, thus facilitating their loss by diffusion. In addition, periodate causes a general loosening-up of the matrix of collagen fibres. Methods of preventing the loss from cartilage sections were investigated. Formaldehyde fixation had a little effect. Cetylpyridinium, a precipitant for acidic polymers such as chondroitin sulphates, was much more effective. Treating sections with solutions of low pH produced a fixation which could not be reversed. The use of free-radical scavengers was considered. It was not possible to determine the effect of cetyl-pyridinium complex formation upon the rate of periodate oxidation of glycosaminoglycuronans by performance of model experiments, with these substances spotted on stripe of 'paper'. Ordinary cellulose paper was periodate reactive, cellulose acetate paper gave anomalous results and polysaccharide would not remain on glass-fibre paper when exposed to periodate solution.