Nonisotopic evaluation of collagen in fibroblasts cultures

A method for the evaluation of collagen concentrations in the medium of fibroblasts in culture was developed. Collagen was precipitated with other proteins by addition of ethanol and hydrolyzed by 6 M HCl. The primary amino acids of the hydrolyzate were reacted with o-phthalaldehyde (OPA) and secondary amino acids (Pro, Hyp) were derivatized with 9-fluorenylmethyl-chloroformate (FMOC-Cl). The mixture was separated by isocratic HPLC on a reverse-phase column. FMOC-derivatives were detected by fluorometry, whereas OPA-derivatives were not. This method is suitable for the monitoring of collagen metabolism in fibroblast cultures exposed to various effectors.