Analysis of hydroxyproline by high performance liquid chromatography and its application to collagen turnover studies in bone cultures.
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 less than 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.
Rydziel
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S
Department of Medicine, St. Francis Hospital and Medical Center, Hartford, CT 06105.
Canalis
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E
D000818:Animals; D001842:Bone and Bones / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000378:metabolism*; D006909:Hydroxyproline / Q000032:analysis* / Q000378:metabolism; D010281:Parathyroid Hormone / Q000494:pharmacology; D051381:Rats; D012886:Skull / Q000166:cytology / Q000187:drug effects; D014316:Tritium
D010281:Parathyroid Hormone; D014316:Tritium; D003094:Collagen; D006909:Hydroxyproline