The long-term objective is to ascertain the effects of disease [systemic (diabetes) and local (inflammation)] and chemotherapeutic treatments (ascorbate and minocycline) on collagen metabolism in gingiva, these two perturbations serving as models for understanding the deleterious effects of periodontal disease on oral tissues. Towards this objective, sophisticated methodology will be used to """"""""dissect"""""""" the effect of these diseases (and treatments) on different components of collagen metabolism, including synthesis, intracellular procollagen degradation, production (the amount of collagen actually formed in the tissue), accretion, and degradation. Until recently, methods were either too tedious or inexact to allow for such determinations, but the pool-expansion approach corrects for these deficiencies. This method involves the injection of excess unlabeled amino acid (proline) along with the radiolabeled precursor ([3H]proline) and accomplishes the following: maintains a constant (or measurable) source of radiolabeled precursor; eliminates differences in proline pools between different tissues and between one tissue under different states; and allows for a calculation of the pivotal component of collagen metabolism, namely, collagen production.
The specific aims are as follows: (1) fractional rate of collagen production (and synthesis); (2) intracellular procollagen degradation: extent, relationship to collagen hydroxylation, and characterization of degradation products; (3) effects on the components of collagen metabolism in streptozotocin-induced diabetic rats; (4) in ascorbate and minocycline-treated diabetic rats; and (5) in antigen-antibody-induced inflamed gingiva of control and ascorbate- and minocycline-treated rats. The methods for achieving these objectives are as follows: fractional rate of collagen production--specific radioactivity of [3H]hydroxyproline in the TCA-insoluble fractions of gingiva/specific radioactivity of tissue free [3H]proline; intracellular procollagen degradation--the amount of TCA-soluble [3H]hydroxyproline/total [3H]hydroxyproline in the tissue; relationship to hydroxylation--the % of hydroxyproline/hydroxyproline + proline, unlabeled and radiolabeled, in isolated alpha chains of gingival collagen; characterization of degradation products--column chromatography of [3H]hydroxyproline-containing material in TCA-soluble fractions. Overall, this should be the most definitive study of collagen metabolism in gingiva of healthy, diseased, and chemotherapeutically-treated diseased rats.
