Measurement of intracellular collagen degradation

Intracellular degradation of newly synthesized collagen is quantitated by incubating fibroblasts with ¹⁴C]proline and determining the percentage of total ¹⁴C]hydroxyproline that is present in a low molecular weight fraction. Several problems make this difficult. (1) Commercial ¹⁴C]proline is often contaminated with ¹⁴C]hydroxyproline and must be purified before use. (2) Salt and ¹⁴C]proline interfere with the determination of ¹⁴C]hydroxyproline in the low molecular weight fraction and must be removed by preparative ion-exchange chromatography. (3) Epimerization of trans- to cis-hydroxyproline during acid hydrolysis is variable and must be taken into account. (4) Loss of ¹⁴C]hydroxyproline during processing varies; ³H]hydroxyproline can be used as an internal measure of recovery, even though tritium may be lost during hydrolysis. An analytic cation-exchange resin is used for the final quantitation of ¹⁴C]hydroxyproline in the low and high molecular weight fractions. With these methods, degradation of newly synthesized collagen can be determined with a precision of ± 3%.