Limitation on sensitivity of measuring collagen degradation: studies with cultured liver cells

Intracellular degradation of newly synthesized collagen is studied by incubating cells with (14C)proline and measuring the hydroxy(14C)proline in a low molecular weight fraction relative to total hydroxy-(14C)proline synthesized. This communication describes a fundamental limitation on the precision with which the measurement can be made; the limitation derives from an irreducible uncertainty in estimating the background radioactivity contributed by the isotope used for metabolic labeling. The problem is illustrated by experiments with cultured liver cells. In rat hepatocytes, the hydroxy-(14C)proline in the low molecular weight fraction was barely detectable above background; the degradation was 29%, however the uncertainty was +/- 28%. In contrast, significant amounts of hydroxy(14C)proline were detected in fractions from human and rat hepatoma cells; and while the levels of degradation were approximately the same as in rat hepatocytes, the measurements could be made with substantially greater precision.