Characterization of Urinary Degradation Products Derived from Type I Collagen

Abstract

The heterogeneity of urinary degradation products of C-terminal telopeptides derived from the ␣1 chain of human type I collagen was investigated and characterized. The urinary fragments characterized in this study consisted of two cross-linked (X) amino acid sequences derived from the C-terminal telopeptide (␣1) of type I collagen. Fragments containing the sequence EXAH-DGGR, with a DG site being either nonisomerized (AspGly) or ␤-isomerized (␤Asp-Gly), were identified. Pyridinoline was detected among the pyridinium crosslinks, but there was a dominance of deoxypyridinoline and a cross-link containing pyridinoline having a molecular weight identical with that of galactosyl pyridinoline. A nonfluorescent cross-link was also found. The concentration of fragments derived from the C-terminal telopeptide region of type I collagen containing the sequence Asp-Gly (␣CTX) and/or ␤Asp-Gly (␤CTX) was measured by enzyme-linked immunosorbent assays in urine and in collagenase digests of trabecular and cortical bone of young and old origin. It was shown that the urinary ratio between such fragments, ␣CTX/␤CTX, was higher in children compared with adults and that the ratio decreased with increasing age of bone. The results indicated that the C-terminal telopeptide fragments derived from type I collagen excreted into urine originated mainly from bone. In conclusion, it is demonstrated for the first time that the C-terminal telopeptide ␣1 chain of type I collagen contains an Asp-Gly site prone to undergo ␤-isomerization and that the degree of ␤-isomerization of this linkage apparently increases with increasing age of bone. These findings indicate that the ratio ␣CTX/␤CTX might be clinically important in diagnosing metabolic bone diseases.The rate of bone turnover has for years been estimated by measurement of different markers reflecting bone formation and bone resorption in urine and blood (1). The traditional bone-resorption markers are urinary calcium and hydroxyproline. Hydroxyproline, however, is not specific for bone (2), and its urinary excretion is influenced by diets rich in hydroxyproline (3), renal clearance, and metabolization (4, 5).The pyridinium cross-links pyridinoline (Pyr) 1 and deoxypyridinoline (Dpyr) are trifunctional cross-links that together with other di-, tri-, and tetrafunctional cross-links stabilize the collagen structure within the extracellular matrix (6). Pyr is found mainly in bone and cartilage (7,8) but also in collagen from most other connective tissues, whereas Dpyr is present mainly in bone and dentin (7). Although the pyridinium cross-links are not entirely bone-specific, they appear to be superior markers of bone resorption compared with hydroxyproline (1). The pyridinium cross-links occur at two intermolecular sites, one linking two C-terminal telopeptides to residue 87 of the triple helix ␣1 or ␣2 chain and the other linking two N-terminal telopeptides to residue 930 of the triple helix ␣1 chain (9). The C-terminal telopeptide ␣1 chain of type I collagen consists of a 26-amino acid sequen...

Bone Resorption in Post-menopausal Women with Normal and Low BMD Assessed with Biochemical Markers Specific for Telopeptide Derived Degradation Products of Collagen Type I

“…However, the ␣/␤ CTx ratio was similar in normal, osteopenic, and osteoporotic postmenopausal women, suggesting that decreased half-life of bone collagen reflected in the elevated ␣/␤ CTx ratio is a common feature occurring in the menopause, when the turnover rate is increased [34,35,37]. When the ␣/␤ CTx ratio was corrected to reflect the actual molar ratios of the two forms, the ratio in pre-and postmenopausal women was 0.65 and 0.85, respectively.…”

“…These two assays employ different antibodies: the RIA assay is specific for the authentic ␣L form of the CTx epitope, whereas the antibody employed in the ELISA is specific for the isomerized ('aged') ␤L form [34]. Thus it is possible to calculate the ratio between the ␣L and the ␤L form of CTx, and this ratio has been suggested to provide an estimate of the 'age' of the resorbed bone [34,35].…”

“…It may be considered surprising that no significant correlation was found between ␣/␤ CTx ratio and age. However, it has been shown that collagen type I isomerization/racemization have reached equilibrium in adult individuals [34,35]. After 20-30 years the ␣/␤ CTx ratio remains relatively constant and reflects the age of the resorbed collagen rather than the age of the individual.…”

“…Clinical information may also be derived from biochemical marker data is an assessment of the average 'age' of the resorbed bone. ␤-Aspartate isomerization, which occurs as a slow spontaneous reaction in the C-telopeptide of type I collagen, has been suggested to provide a measure of the bone 'age' [34,35]. C-telopeptide fragments measured in the urine CrossLaps (CTx) radioimmunoassay (RIA) are in the authentic ␣L form characteristic of newly synthesized collagen molecules [34,36].…”

“…␤-Aspartate isomerization, which occurs as a slow spontaneous reaction in the C-telopeptide of type I collagen, has been suggested to provide a measure of the bone 'age' [34,35]. C-telopeptide fragments measured in the urine CrossLaps (CTx) radioimmunoassay (RIA) are in the authentic ␣L form characteristic of newly synthesized collagen molecules [34,36]. The corresponding fragments measured in the urine CTx ELISA, as well as in the serum CTx assay, are in the isomerized ␤L form, and thus these molecules are 'older' than the molecules measured in the RIA [35].…”