Radioautography of rat incisor dentin as a continuous record of the incorporation of a single dose of 3H-labeled proline and tyrosine

Abstract

After injection of labeled precursors such as 3H-proline or 3H-tyrosine into rats, the incisor dentin contains a continuous and stable record of precursor incorporation into labeled proteins. This record was visualized and quantitated with radioautography in order to evaluate the quantitative changes in enamel where newly secreted proteins randomize with older proteins and both are eventually lost. Up to 4 hours after injection, the pulse-dose was incorporated as a highly labeled band of predentin. The band was entirely within calcified dentin at 2 days and was further removed from new predentin by 4 and 8 days. Dentin which formed proximal to the heavily labeled band contained an amount of radioactivity reflecting the level of labeled precursor available at that time. A standardizing factor for experimental error was obtained by quantitating the reaction in the heavily labeled band, and a post-pulse incorporation factor was determined from the amount of radioactivity added per day as weakly labeled dentin. The variation within the heavily labeled band was assumed to reflect experimental error. The number of grains in the bands were averaged from 4 hours to 8 days to give the standardizing factor. This was multiplied by the ratio of enamel to dentin counts in the same section to obtain a corrected enamel count. In this way the coefficient of variation was improved from a high of 17.2% in uncorrected enamel counts to 2.4% in corrected counts. The post-pulse incorporation factor was higher with tyrosine than with proline. With proline it amounted to 5% increase per day from 1 to 4 days and 2.5% per day from 4 to 8 days after injection. In addition, with 3H-proline the incorporation into predentin increased from 30 minutes to 4 hours. With tyrosine, the counts increased from 30 minutes to 1 hour, but decreased by nearly one third from 1 to 4 hours. This was interpreted as a loss of short-lived matrix proteins including procollagen peptides produced during conversion from procollagen to tropocollagen in the predentin.