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      A specific immunoassay for monitoring human bone resorption: Quantitation of type I collagen cross-linked N-telopeptides in urine

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          Peptides of low molecular weight that contain pyridinoline cross-links were isolated from adolescent human urine. A fraction was selected that was enriched in the N-telopeptide-to-helix intermolecular cross-linking domain of bone type I collagen. Mouse monoclonal antibodies were generated against these urinary peptides conjugated to a carrier protein as immunogen. A high-affinity antibody was identified that specifically bound to the trivalent peptides derived from the N-telopeptide-to-helix pyridinoline cross-linking site in type I collagen of human bone. This was confirmed by the direct isolation from human bone collagen of similar fragments recognized selectively by the antibody. A sensitive inhibition ELISA was established on microtiter plates that could quantify the bone-derived peptides in human urine. The assay, which can be run directly on untreated urine, was thoroughly tested against samples from normal subjects and from patients with metabolic bone disease. For example, strong correlations with urinary hydroxyproline and total pyridinoline cross-links were found in patients with Paget's disease of bone. The method shows considerable promise as a rapid and specific index of human bone resorption rates, with greatly improved specificity and convenience over total pyridinoline analysis. Potential applications include the study of normal metabolism, the diagnosis and monitoring of bone disease, and evaluating the effectiveness of antiresorption therapies.

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          Most cited references 18

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          Cross-linking in collagen and elastin.

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            Quantitation of hydroxypyridinium crosslinks in collagen by high-performance liquid chromatography.

            An HPLC method for quantifying the 3-hydroxypyridinium crosslinks of collagen is described. It can be applied to crude hydrolysates of all types of connective tissue. Mineralized tissues can be hydrolyzed directly and analyzed without interference from the mineral ions. The hydroxylysyl (HP) and lysyl (LP) forms of hydroxypyridinium residue were resolved on a reverse-phase C18 column using a gradient of acetonitrile in water and 0.01 M n-heptafluorobutyric acid as an ion-pairing agent. The crosslinking amino acids were accurately quantified down to 2 PM (1 ng) injected, by detecting their natural fluorescence with a spectrofluorometer. Tissues in which hydroxypyridinium crosslinks were plentiful included all forms of cartilage, bone, dentin, ligament, tendon, fascia, intervertebral disc, lung, gut, cervix, aorta, and vitreous humor. Among normal tissues, LP, the minor form of the crosslink, was present in significant amounts relative to HP only in bone and dentin. Both crosslinks were essentially absent from skin, cornea, rat tail tendon, and basement membranes.
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              [7] Collagen cross-linking amino acids

               David Eyre (1987)

                Author and article information

                Journal of Bone and Mineral Research
                J Bone Miner Res
                November 1992
                March 03 2010
                : 7
                : 11
                : 1251-1258
                © 2010


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