Methods for measuring hydroxyproline and estimating in vivo rates of collagen synthesis and degradation

A major pathogenic feature of fibrotic diseases is the excessive and disorganized deposition of collagens. This results from changes in both synthetic and degradative pathways. The posttranslational hydroxylation of proline to hydroxyproline and the relative abundance of hydroxyproline in collagens, compared with other proteins, facilitates its use as a relatively specific marker of collagen content and metabolism. This chapter describes in vivo methods for the estimation of collagen synthesis and degradation rates, as well as the proportion of newly synthesised collagen rapidly degraded intracellularly. These pathways have all been shown to play important roles in the altered deposition of collagen associated with the development of fibrosis. The methods are based on the incorporation of radiolabeled proline into collagen as hydroxyproline and its measurement in intact protein and collagen breakdown products. In addition, an accurate and highly sensitive high-performance liquid chromatography method is described for the measurement of hydroxyproline for in vivo and in vitro studies.