Induced connective tissue metabolism in vivo: reutilization of pre-existing collagen.

Light and electron microscopy of resorbing connective tissues have shown that the collagen fibers and fibrils disappear. It is usually presumed that resorbed collagen is degraded proteolytically to hydroxyproline-containing peptides and to free amino acids, including hydroxyproline. We wish to present evidence that collagen can be resorbed and then reutilized without such breakdown. Urinary excretion of hydroxyproline in peptides has been regarded as an index of collagen catabolism in vivo because collagen is the only source of significant amounts of hydroxyproline.1 2 It has been suggested that a relatively constant fraction (20%) of the hydroxyproline from degraded collagen is excreted as peptides in urine.2' 3 However, there is considerable evidence that under some conditions collagen is resorbed and the process is accompanied by minimal increases of excretion of peptide hydroxyproline. It has been observed that with increased bone resorption occurring in clinical4 and experimental5 hyperparathyroidism, there was increased urinary excretion of calcium unaccompanied by an increased excretion of peptide hydroxyproline. Likewise, in studies on the postpartum involution of the rat6 and human uterus,7 where massive resorption of collagen occurs within 1-8 days after parturition, a significant increase in urinary excretion of peptide hydroxyproline was not observed. These observations caused us to postulate that fibrous collagen might be depolymerized to a soluble subunit which may be reutilized without breakdown to free amino acids or low-molecular-weight peptides.5 7 If collagen can be reutilized, it should be possible to demonstrate the contribution of pre-existing connective tissues to newly formed connective tissues by labeling the pre-existing collagen with radioactive proline before induction of the synthesis of new tissue. New connective tissues can be induced in animals by the subcutaneous injection of carrageenan,8 or by subcutaneous implantation of polyvinyl sponges.9 These techniques permit separation of newly formed collagen from pre-existing collagen and thus allow study of the new tissue without contamination with "old" tissue. This approach necessitates a conlsideratiolL of the possible pathways of reutilization10 that may occur after labeling an intact animal with radioactive proline. Radioactivity in new tissues could arise from de novo synthesis using amino acid or peptide residues derived from either collagen or noncollagenous labeled protein. Alternately, reutilization could occur by the depolymerization (disaggregation) of collagen fibrils to soluble molecular units or subunits and subsequent extracellular repolymerization to fibrous collagen. Since the hydroxylation of proline to hydroxyproline during collagen synthesis can be inhibited by vitamin C deficiency in ViVo8' 11 and in vitro,12' 13 the possibility of a reaggregation pathway can be tested in prelabeled guinea pigs. If reutilization occurred by reaggregation of pre-existing labeled collagen subunits, the collagen