Isoaspartyl bond formation within N-terminal sequences of collagen type I: implications for their use as markers of collagen degradation

An ELISA was developed for the measurement of N-telopeptides of the α2(I) collagen chain containing an isomerized Asp-Gly bond (β-peptide) using polyclonal antibodies raised against the synthetic peptide. The presence of this isomerized form in bone was confirmed by positive immunostaining of sections from human femoral head. The ELISA was used to measure isomerized peptide in both human bone digests and urine samples, showing that an isoaspartyl rearrangement occurs in the Asp-Gly sequence at the N-terminus of the α2(I) chain in an analogous fashion to that found in the C-terminal telopeptide of the α1(I) chain of collagen. Using this assay in conjunction with a monoclonal antibody ELISA to the non-isomerized α2(I) N-telopeptide (α-peptide), ratios of isomerized to normal peptides were estimated in the bone and urine samples. Urinary α2(I) N-telopeptides showed a higher degree of isomerization than the peptides derived from a human bone digest. This is possibly due to relative enrichment of the isoaspartyl-bonded peptide during metabolic processing due to the proximity of the isoaspartyl bond to a cross-link site. Urinary concentrations of isomerized and normal peptides were determined in normal adults, children, post-menopausal control subjects and subjects with osteoporosis. A lower ratio of β-peptide to α-peptide was observed in children's urine, indicative of a higher rate of bone metabolism allowing less time for the isomerization to occur. No significant differences were found between the post-menopausal control and osteoporotic populations although the trends observed supported the hypothesis that a lower degree of isomerization may be associated with faster bone turnover.