Summary
We describe a high performance liquid chromatography (HPLC) technique for separating and quantitating hydroxyproline in calvarial cultures. Using a reverse-phase Nova-Pak C18 column and a 140 mM sodium acetate, 0.05% triethylamine (TEA), 6% acetonitrile solvent system, we obtained a complete separation of hydroxyproline. Recovery of added standards ranged from 89 to 103% and intraassay variability was <8%. [3H]hydroxyproline measurements were used to examine changes in collagen turnover in rat calvariae labeled with [3H]proline and “chased” in the presence of 10 mM unlabeled proline. The addition of parathyroid hormone (PTH) during a 24–48 hour “chase” period increased the release of acid-soluble [3H]hydroxyproline into the culture medium, indicating an increase of fully degraded collagen. This method offers a sensitive and reproducible technique for monitoring changes in bone matrix degradation and in studying agents that modify this process.
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Rydziel, S., Canalis, E. Analysis of hydroxyproline by high performance liquid chromatography and its application to collagen turnover studies in bone cultures. Calcif Tissue Int 44, 421–424 (1989). https://doi.org/10.1007/BF02555971
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DOI: https://doi.org/10.1007/BF02555971