The purpose of this study is to investigate possible mechanisms underlying ag associated changes that have been observed in collagen crosslinking in a previously funded study. There is evidence that two mechanisms may be operative in introducing such changes: 1. Available data suggest that a pool of collagen, exists that is not subject turnover; it is hypothesized that such a pool, which may increase in size over time, undergoes progressive molecular changes over the lifetime of the animal. 2. There is evidence that collagen synthesized later in life may be structurally different from collagen synthesized earlier. In this proposal both in vivo and in vitro techniques will be used to investigate these intertwined issues. To investigate turnover, an in vivo labelling protocol conducted in rats will be used to test the hypothesis that there is a pool of collagen (operationally defined as that collagen containing intermolecular, difunctional crosslinks derived from Isyyl oxidase-generated aldehydes) that is as old as the animal. Lifetime labelling studies performed thus far have not sen able to distinguish between different collagen pools at the molecular level; such properties as solubility characteristics are generally used to infer structural aspects. In the present study it is proposed to track collagen pools directly through labelling of crosslinks. To investigate the second possible mechanism, concerning differences in collagen synthesized by older and younger animals, both fibroblast and organ cultures from rats and monkeys of different ages will be used to analyze collagen biosynthesis in vitro. Such parameters as rate of biosynthesis, collagen type ratios, crosslink production, extent of lysine hydroxylation and enzymatic and nonenzymatic glycosylation will be measured. Rats and monkeys were selected as examples of a short lived and long-lived species, as previous data suggest that collagen may age differently in rats than in humans. Possible mechanisms underlying any observed changes will be examined in logical sequence. Precise and rapid HPLC techniques have been developed for analysis of collagen crosslinks. Unfractionated tissue hydrolysates may be analyzed directly at earlier time points in the in vivo labelling study, when specific activity of the labelled crosslinks is still relatively high. At later time points, when specific activity drops due to growth and presence of labelled collagen, tissue hydrolysates are prepared for analysis by gel filtration.
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