The biochemical mechanism accounting for the connective tissue abnormalities in homocystinuria was explored by examining the effects of various amino acids known to accumulate in the plasma of patients with this disease on cross-link formation in collagen. Neutral salt solutions of purified, rat skin collagen, rich in cross-link precursor aldehydes, were polymerized to native type fibrils by incubating at 37°C in the presence of homocysteine, homocystine, or methionine. After the polymerization was completed, each sample was examined for the formation of covalent intermolecular cross-links, assessed indirectly by solubility tests and directly by measuring the cross-link compounds after reduction with tritiated sodium borohydride and hydrolysis.
Collagen solutions containing homocysteine (0.01 M-0.1 M) failed to form insoluble fibrils. Furthermore, much less of the reducible cross-links, Δ6,7 dehydrohydroxylysinonorleucine, Δ6,7 dehydrohydroxylysinohydroxynorleucine, and histidino-dehydrohydroxymerodesmosine were formed in the preparations containing homocysteine as compared with the control and the samples containing methionine or homocystine. The content of the precursor aldehydes, α-aminoadipic-δ-semialdehyde (allysine) and the aldol condensation product, was also markedly diminished in tropocollagen incubated with homocysteine. It is concluded that homocysteine interferes with the formation of intermolecular cross-links that help stabilize the collagen macromolecular network via its reversible binding to the aldehydic functional groups.
Analysis of the collagen cross-links in skin biopsy samples obtained from three patients with documented homocystinuria showed that the cross-links were significantly decreased as compared with the age-matched controls, supporting the tentative conclusions reached from the in vitro model studies. In addition, the solubility of dermal collagen in non-denaturing solvents was significantly increased in the two patients examined, reflecting a functional defect in collagen cross-linking. Although the concentration of homocysteine used in this study to demonstrate these effects in vitro is clearly higher than that which is observed in homocystinuric's plasma, the data do suggest a possible pathogenetic mechanism of connective tissue defect in homocystinuria.