Evidence for a deficiency in inter-molecular cross-linking in collagen fibrils isolated from rheumatoid joints.

Collagen fibrils can be isolated from mature uncalcified connective tissue by a preliminary treatment with crude bacterial a-amylase. This type of collagen fibril has been described as Nishihara collagen (Steven, 1964). Amino acid analyses of Nishihara collagen preparations indicate that the fibrils are a highly purified form of collagen, containing less of the impurities which are often found in trace amounts in preparations of acid soluble collagen (Steven, in preparation). Electron microscopic examination of reconstituted acid soluble collagen fibrils and Nishihara collagen fibrils indicate that both are made up of collagen molecules (tropocollagen monomers) polymerized laterally and endto-end. Reconstituted collagen fibrils are held together by non-covalent bonds, e.g. ionic, hydrogen, and hydrophobic bonds. However, Nishihara collagen fibrils have in addition a second type of stabilizing force, covalent cross-linkages between monomers, which confer stability on the fibril towards physical and biological destruction. Nishihara collagen provides us with purified samples of naturally-occurring collagen at the fibrillar level. Nishihara collagen fibrils isolated from rheumatoid knee joints were previously demonstrated to be more susceptible to proteolytic digestion by pronase than were collagen fibrils isolated from normal synovia (Steven, 1965). Chemical studies (Steven, 1966) have indicated the presence of at least two distinct types of covalent intermolecular crosslinkages in mature collagen fibrils. One type of cross-linkage is destroyed by hydroxylamine and may be considered to be "ester-like" and a second type is destroyed in cold alkali. It was therefore of special interest to compare this system of intermolecular cross-linking bonds in normal and rheumatoid collagen fibrils. The present work outlines the results of this study without giving all the chemical evidence which was necessary to prove the presence of two or more types of cross-linkages in mature collagens. Experimental Conditions Synovia were removed post mortem from the knee joints of five subjects with no known history of connective tissue disease. Samples, examined by Dr. J. Ball, were shown to be histologically normal. Six rheumatoid patients underwent synovectomy and the synovium plus some adhering capsule was used in each case as the source of the collagen fibrils. Tissue samples from these patients were examined histologically and all six subjects were confirmed to be typical rheumatoid cases. The tissues were defatted with acetone and the collagen fibrils isolated by the Nishihara technique. Preliminary studies indicated that, after treatment with hydroxylamine, both normal and rheumatoid collagens retained enough resistant cross-linkages to prevent the solubilization of 70 to 80 per cent. of the original collagen fibrillar nitrogen during thermal denaturation. Thus hydroxylamine degradation could not distinguish between normal and rheumatoid collagen fibrils. On the other hand alkaline degradation was found to be much more effective on collagen from rheumatoid tissue than on normal synovial collagen. The alkaline degradation technique is based on the procedure described by Hey and Stainsby (1965). The collagen fibrils prepared by the Nishihara technique were suspended in 2N. NaOH at 40 C. for 6 days. The reaction mixture was then brought to pH 7 with N acetic acid and allowed to equilibrate at room temperature for 1 hour. The suspended fibrils were then placed in a boiling water bath for 1 hour. The amount of nitrogen in the soluble gelatin produced during boiling and also the amount of nitrogen in the insoluble residue were determined by microkjeldahl nitrogen analysis. The amount of total nitrogen in the fibrils initially was obtained by summation of the nitrogen values obtained for the soluble and insoluble fractions. The results are presented as the amount of total nitrogen in the insoluble residue expressed as a percentage of the total nitrogen content initially present in the intact collagen fibrils.