334 ALBERT DORFMAN AND SARA SCHILLER 



for the delayed wound healings and demineralization of bone typical of 

 Cushing's disease. 



Investigation of the metabolism of acid mucopolysaccharides in alloxan 

 diabetes indicated a decreased capacity to metabolize mucopolysaccharides 

 and a restoration of the defect toward normal by the administration of 

 insulin [47]. The rate of turnover of polysaccharides of partly fasted 

 animals, which served as controls for the weight loss of the diabetic rats, 

 was not different from that found in normal rat skin. 



The influence of hypophysectomy [48], growth hormone, and thy- 

 roxine have also been investigated but these studies are difficult to interpret 

 since changes in pool size occurred during the course of the experiments. 

 Turnover studies depend upon the maintenance of a steady state or 

 constant pool size ; differences in rates of synthesis are difficult to define 

 if a reasonably constant pool size does not obtain. 



The information derived from turnover experiments suggested that 

 marked variations in the concentrations of mucopolysaccharides might 

 occur. It therefore became important to devise methods for the quantita- 

 tive determination of acid mucopolysaccharides in tissues. 



By the application of relatively simple procedures it is now possible to 

 perform such analyses. The method, which is published elsewhere in 

 detail [49], depends upon the solubilization of tissues with papain, followed 

 by further deproteinization with trypsin and trichloroacetic acid. The 

 partly purified polysaccharide preparation is separated on the basis of the 

 differential solubility of complexes formed with cetyl pyridinium chloride 

 (CPC). In the presence of 0-03 5-0 -040 m NaCl all the polysaccharides are 

 quantitatively precipitated by CPC providing Celite is added. When the 

 resultant precipitate is extracted with varying concentrations of NaCl 

 sharp separation of three fractions is obtained. The CPC-hyaluronic acid 

 complex is solubilized by 0-4 M NaCl in o- 1% CPC, while the complexes 

 of chondroitin-sulphuric acids-A, -B, -C, and heparin monosulphuric 

 acids are solubilized by i -2 M NaCl in o-i^',, CPC. Least soluble is the 

 complex of heparin which is solubilized in 2 • i m NaCl. Further separation 

 of these fractions can be achieved by subsequent chromatography on 

 Dowex 1x2, chloride. 



Table II illustrates a typical separation of a preparation of rat skin. 

 The small amount of colour found in tubes i and 2 is due to interference 

 by protein with the carbazole reaction and does not indicate significant 

 loss of mucopolysaccharide. In the case of rat skin the fraction obtained 

 with 1-2 M NaCl contains chondroitin sulphuric acids, but in other 

 tissues heparin monosulphuric acid may be obtained in this fraction. 



The validity of this method has now been established by a variety of 

 prodedures. Table III indicates the recovery of polysaccharides added to 

 rat skin in duplicate experiments. Excellent recovery and reproducibility 



