An isotope dilution method for determining the absolute amounts and specific activities of proline and hydroxyproline in tissues.

Abstract A method is described for estimating the absolute amounts and the specific activities of proline and hydroxyproline in tissue collagen, following the in vivo administration of H 3 -labelled proline. The method depends on an isotope dilution procedure, C 14 -labelled imino acids being added to tissue hydrolysates and recoveries, on chemical analysis, computed by reference to calibration curves prepared with a series of standard imino acid solutions. The chemical analysis is adapted from that of Peterofsky and Prockop and preliminary experiments for establishing the optimal conditions for each individual step in the separation, appropriate to this type of biological material, are described. Some cross-contamination between the “assay fractions” results in small over-estimates of the specific activities of both imino acids and of the absolute concentration of proline, but appropriate correction factors can be applied. Rat skin collagen contains 1·5 times as much proline as hydroxyproline; the specific activities obtained 48 hr after injection of H 3 -labelled proline are approximately equal.