Radiochemical purity of [14C]-proline:implications for measurement of intracellular collagen degradation.

Measurement of intracellular degradation of newly synthesized collagen requires use of radiochemically pure [14C]-proline. It is particularly important that levels of contaminating hydroxy-[14C]-proline or other radioactive species that migrate with hydroxyproline on cation-exchange resins be very low. The objective of this study was to determine how far the levels of these impurities could be reduced. Commercially available [14C]-proline was subjected to preparative ion-exchange chromatography. The procedure was capable of adequately separating authentic hydroxyproline from proline, however, upon rechromatography of the "purified" isotope on an analytic resin it was found that background radioactivity in the hydroxyproline region could not be reduced below approximately 500 parts per 10(6) parts of [14C]-proline. The contaminants appear to be generated during the procedure itself, suggesting that further efforts to purify the material would be unproductive. The background level can be a limiting factor in attempting to measure degradation in systems that synthesize very small amounts of collagen.