Quantitative Colorimetric Assay of Acid Mucopolysaccharides

In 1946 Hale (1) described a histochemical method for the detection of acid polysaccharides in animal tissues, based on the ability of these compounds to bind ferric ions, which were subsequently detected as Prussian blue. It has been possible to adapt this histochemical method to the quantitative determination of acid mucopolysaccharides on a macro or micro scale. The major modification of the original method involves the measurement of the iron bound by the mucopolysaccharides by an adaptation of Lyons’ (2) thioglycolic acid method. This change was necessitated by the low solubility of Prussian blue in aqueous solutions. Reagents 1. Acetate buffer. 0.2 M sodium acetate-acetic acid (pH 6) to which NaCl is added to give a concentration of 0.15 M. 2. Acid mucopolysaccharide solution. 50 mg. of chondroitin sulfate’ or hyaluronic acid* (each as a salt) are dissolved in 100 ml. of the acetate buffer. 3. Ferric chloride reagent. containing 0.648 gm. of FeC13.6H20, 1 gm. of mannitol, and 12 mg. of ascorbic acid in 100 ml. of water. After 24 hours this reagent remains stable for at least, 1 week, if kept in a dark bottle at 20”. 4. 2 per cent (volume per volume) aqueous solution thioglycolic acid. 5. Concentrated nitric acid, sp. gr. 1.42. 6. Concentrated ammonium hydroxide, sp. gr. 0.90. Macromethod-Into each test-tube3 calibrated at 5 ml. is delivered either a 1 ml. aliquot of solution containing from 50 to 500 y of acid mucopoly-