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When high-density, cartilage proteoglycan (and the lower molecular weight mucopolysaccharides derived from it by papain or trypsin digestion) is centrifuged to equilibrium in complex gradients of CsCl and guanidinium chloride, the peak density is markedly and reproducibly decreased. Hyaluronic acid, a less dense macromolecule, is also decreased in density by the presence of guanidinium ion in the gradient and the extent of the decrease is directly related to the concentration of the guanidinium ion in the starting solution. Other cations, such as Li + or Ca 2+ , also produce marked, concentration-dependent, decreases in the density of hyaluronate. The effect is complicated by the fact that the equilibrium distribution of the added cation does not follow the CsCl gradient, but forms a reverse gradient. The density shift must involve an interaction of the added cation with the charged groups of the mucopolysaccharide polyanion since the added cations have no effect on uncharged polysaccharides such as dextran or Ficoll. Furthermore, it seems unlikely that the primary effect could be caused by increased hydration due to increased water activity since centrifugation in Cs 2 SO 4 (which reduces the density of nucleic acids by increasing their hydration) leads to substantial decreases in density of all polysaccharides including the uncharged dextran and Ficoll. Although the density of proteins, such as albumin and lysozyme, is not appreciably affected by guanidinium ion; proteoglycan and isolated keratan sulfate fractions of high protein content are markedly decreased in density by guanidinium ion. Such density shifts of the components of proteoglycan must be considered in any attempt to interpret experiments which study disaggregation phenomena in CsCl/guanidinium chloride gradients.

A systematic study has been made of the types and quantities of acid mucopolysaccharides isolated from a variety of tissues and fluids. Identification of the polysaccharides has been based on the precipitation of the polysaccharides as calcium salts, on analysis, on optical rotation and on digestibility by testicular and bacterial hyaluronidases. On this basis, six distinct mucopolysaccharides have been established: hyaluronic acid, chondroitin, the chondroitin sulfates designated as A, B and C, and keratosulfate. Additional mucopolysaccharides have been demonstrated in amyloid tissue and, in aorta, which are resistant to testicular hyaluronidase and contain D-glucosamine, a uronic acid and varying amounts of sulfate. In addition, incompletely sulfated chondroitin sulfates A or C have been obtained by fractional elution from an ion exchange resin. The tissues have been divided into seven groups on the basis of the mucopolysaccharides isolated from them, as follows: 1. (1) Vitreous humor, synovial fluid, filterable fowl tumors, liposarcoma: hyaluronic acid only. 2. (2) Cartilage, adult bone, chondrosarcoma, chordoma: chondroitin sulfate A and/or C. 3. (3) Umbilical cord, loose connective tissue of electric eel, fibroblasts grown in tissue culture: hyaluronic acid as the major with chondroitin sulfate C as a minor component. 4. (4) Pig skin, ligamentum nuchae (the latter contains chondroitin sulfate A in addition): chondroitin sulfate B and hyaluronic acid as major components. 5. (5) Adult connective tissue, tendon, heart valve: chondroitin sulfate B and C with hyaluronate as minor component. 6. (6) Cornea: keratosulfate, chondroitin and chondroitin sulfate A. 7. (7) Incompletely characterized fractions (amyloid, bovine aorta, calf bone). From calf bone, chondroitin sulfate A and C and a large incompletely sulfated fraction were isolated and, in addition, a minor fraction of keratosulfate. From bovine aorta, a major fraction with the properties of chondroitin sulfate A was isolated, accompanied by hyaluronate, chondroitin sulfate B and a variety of glucosamine-containing fractions of varying degrees of sulfation. These latter fractions are believed to be related to heparin. All normal tissues investigated appear to contain two or more mucopolysaccharides in contrast to tumors of mesodermal origin which produce a single type of mucopolysaccharide.

1. 1. ChS-B occurs in bullskin mainly in the middle and bottom layers. 2. 2. The fraction designated as ChS-B probably is a mixture of at least two isomeric mucopolysaccharides, the major component, the chondroitin sulfate B proper, containing iduronic acid, and the other containing glucuronic acid. Both are resistant to testicular hyaluronidase. 3. 3. The chemically desulfated product of chondroitin sulfate A was hydrolyzed by testicular and bacterial hyaluronidases analogous to their action on hyaluronate and chondroitin from cornea, while the desulfated products of chondroitin sulfate B remained resistant to the two hyaluronidases. 4. 4. Chondroitin sulfate B appears to be associated with the coarse bundle type of collagen fibers.

Summary1. From the urine of 4 out of 5 patients with gargoylism, a mixture of mucopolysaccharides was isolated which could be identified as ChS-B and heparitin sulfate—with the former predominating. The urine of one patient, and the liver of another, yielded only heparitin sulfate. 2. The type of polysaccharide excreted by these patients had no obvious correlation with the severity of the disease or the postulated mode of inheritance suggested by the history. 3. It is suggested that gargoylism represents an overproduction of certain mucopolysaccharides due to a genetically determined error of differentiation of fibroblasts.