Collagen fragments in urine derived from bone resorption are highly racemized and isomerized: a biological clock of protein aging with clinical potential

Fragments of the alpha 1 C-terminal telopeptide of type I collagen containi ng the sequence AHDGGR(1209-1214) (CTx) can be measured in urine as an inde x of bone resorption, We report here that these molecules undergo racemizat ion and isomerization of Asp(1211) in vitro and in vivo, generating a mixtu re of four isomers: the native peptide form (alpha L), an isomerized form c ontaining a beta-Asp bond (beta L), a racemized form containing a D-Asp res idue (alpha D) and an isomerized/racemized form (beta D). To study these re actions at this specific site in collagen, we have employed four immunoassa ys, each specific for one of the isoforms, and developed HPLC methods for t heir separation. The kinetics of these reactions were studied in vitro unde r physiological conditions by incubation of synthetic AHDGGR hexapeptide or mineralized bone collagen. Reactions were found to be strongly shifted tow ards the beta-Asp forms and slightly in favour of the D-enantiomeric forms. CTx isomers were measured in human urine and in enzymic digests of bovine bone collagen. The results indicated that the extent of racemization and is omerization were correlated with the age and turnover of collagen, The rati os between the native and age-related forms of CTx were elevated in urine f rom patients with Paget's disease or osteoporosis as compared with that fro m healthy adults. The alpha L/alpha D CTx ratio had the highest discriminat ory power (T-score = 23.2; P < 0.0001 and T-score = 1.5; P < 0.0001 for Pag et's disease and osteoporosis respectively). In conclusion, these findings indicate that an assessment of CTx ratios in urine may provide an estimate of bone turnover, aiding in the diagnosis of metabolic bone diseases.