High-performance liquid chromatographic quantitation of collagen biosynthesis in explant cultures

Abstract

The capacity of lung explant cultures to synthesize collagen can be estimated by determining the content of [3H]hydroxyproline in protein following incubation with [3H]proline. The technique requires acid hydrolysis followed by quantitative separation of hydroxyproline from proline for scintillation counting and is often restricted to methods that can accommodate large samples because of relatively low specific radioactivity. A method which is useful for such samples, providing rapid separation of nonderivatized amino acids by ion-exchange HPLC, is described here. The HPLC system employs an HPX-87C cation-exchange column in 10 mM calcium acetate, pH 5.5, at 85 degrees C. Under isocratic conditions hydroxyproline is completely resolved from proline with quantitative recovery of the 3H cpm applied to the column. Large amounts of material, equivalent to at least 150 mg wet wt of lung, can be applied without affecting resolution or recovery, and samples can be injected at intervals as short as 40 min. This method was used to study collagen biosynthesis in a model of pulmonary fibrosis induced in rabbits by the tumor-promoting agent, phorbol myristate acetate (PMA), and provides information concerning total protein synthesis as well as production of collagen. The data show a doubling in the rate of collagen production in lung explants prepared from animals treated with PMA compared with explants from control animals.