A method for the separation of acid mucopolysaccharides: its application to the isolation of heparin from the skin of rats.

Increased attention has been focused on the potential significance of the variations in the qualitative and quantitative composition of connective tissue ground substance. Changes in tissue content of acid mucopolysaccharides are difficult to define since existing quantitative methods are generally recognized as inadequate. Moreover, these methods are laborious and require large amounts of starting material. Such limitations in methodology impose restrictions on metabolic studies of connective tissues. The present communication describes a procedure for quantitative separation of small quantities of hyaluronic acid, chondroitinsulfuric acids, and heparin, and its application to a study of the mucopolysaccharides of skin. A previously published method for extraction of acid mucopolysaccharides from skin (2) was modified to incorporate Scott’s finding (3) that this tissue can be solubilized by activated papain at elevated temperature. The technique for separation was based on the differential solubility of cetylpyridinium complexes of acid mucopolysaccharides (3). Use of the quaternary ammonium ion for isolation and separation of acid mucopolysaccharides has been recently reviewed (4). Several procedures useful for fractionating mucopolysaccharides were enumerated. The method described in this paper is similar to one proposed by Scott. Gardellr reported that acid mucopolysaccharides able to form complexes with cetylpyridinium chloride were separated on a cellulose column by increasing concentrations of MgCl*. Since the column appeared to act merely as a supporting medium, it was considered probable that equivalent separation could be achieved by a simple process of solubility. The observation of Korn (5) that heparin, at a concentration as dilute as 25 pg per ml, was precipitated quantitatively as the cetyltrimethylammonium complex (providing an inert agent, Celite, was present) suggested that a simple separation procedure was feasible. The efficacy and reproducibility of the methods were studied with authentic samples of acid mucopolysaccharides from several sources and with rat skin.