Simultaneous determination of the reducible and nonreducible cross-links of connective tissue. Analysis of mineralized and nonmineralized bone collagen.

Secondary amine cross-links occur in collagen and elastin from a number of tissue sources. Quantification of these cross-links by amino acid analysis is complicated by the problem of separating cross-links, which are often minor components, from the more common amino acids and also because relatively large amounts of a cross-link are required to determine a color factor. A specific radioactive labeling method has been developed and used to quantify cross-links in bone collagen. Primary amines such as lysine and hydroxylysine are first guanidinated with 3,5-dimethylpyrazole-1-carboxamidine nitrate (DMPC). Secondary amines, which are unreactive with DMPC, are then quantitatively cyanoethylated with [14C]acrylonitrile. This procedure can be used to detect any secondary amine cross-link, with higher sensitivity than ninhydrin analysis, in peptide form as well as in acid hydrolysates. It is applied here in conjunction with [3H]NaBH4 reduction to simultaneously quantify Schiff base cross-links and amounts of in vivo reduction of Schiff bases in mineralized versus nonmineralized bovine bone.