Determination of collagen and protein turnover by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the determination of tissue collagen and protein synthesis rates in vivo, together with an index of collagen degradation. The technique utilises post-column reaction with 7-chloro-4-nitrobenzofurazan (NBDCl), which shows higher reactivity towards the secondary amino acids, proline and hydroxyproline, and also exploits differences in absorbance and fluorescence spectra to avoid interference by primary amino acids, including cysteine which reacts rapidly with NBDCl. The relative benefits of using fluorescence or absorbance detection are discussed. A detailed description is given of the steps involved in sample preparation and data for five normal tissues in the mouse are presented using fluorescence detection.