The primary functional role of collagen is as a supporting tissue and it is now established that the aggregated forms of the collagen monomers are stabilised to provide mechanical strength by a series of intermolecular cross-links. In order to understand the mechanical properties of collagen, it is necessary to identify and quantitatively determine the concentration of the cross-links during their changes with maturation, ageing and disease. These cross-links are formed by oxidative deamination of the ε-amino group of the single lysine or hydroxylysine in the amino and carboxy telopeptides of collagen by lysyl oxidase, the aldehyde formed reacting with a specific lysine or hydroxylysine in the triple helix. The divalent Schiff base and keto-amine bonds so formed link the molecules head to tail and spontaneously convert during maturation to trivalent cross-links, a histidine derivative and cyclic pyridinolines and pyrroles, respectively. These latter bonds are believed to be transverse inter-fibrillar cross-links, and are tissue rather than species specific. We describe the determination of these cross-links in detail. Elastin is also stabilised by cross-linking based on oxidative deamination of most of its lysine residues to yield tetravalent cross-links, desmosine and iso-desmosine, the determination of which is also described. A second cross-linking pathway occurs during ageing (and to a greater extent in diabetes mellitus) involving reaction with tissue glucose. The initial product glucitol-lysine can be determined as furosine and pyridosine, and determination of advanced glycation end-products believed to be cross-links, such as pentosidine, are also described.
