Formation of proline metabolites in chick embryo bone: Interference with the measurement of free hydroxyproline by ion-exchange chromatography

Abstract

Metabolites of l-[ 14C]proline were found in the trichloroacetic acid-soluble fraction of 16-day-old chick embryo frontal bones. In several ion-exchange procedures these metabolites interfered with the analysis of hydroxyproline derived from the metabolic breakdown of collagen. The major metabolite was identified as glutamic acid by its chromatographic and crystallization properties. It was eluted from AG50 cation-exchange resin with 1.0 n HCL in the hydroxyproline region, but was separated from hydroxyproline on a DC-6A column in the amino acid analyzer. Another metabolite was identified as aspartic acid. It was not separated from hydroxyproline on either AG50 using 1 n HCL for elution or on DC-6A using 0.1 m sodium citrate, pH 3.25, for elution, but adequate separation was obtained by elution with 0.2 m sodium citrate buffer at pH 2.91. Formation of these metabolites was not related either to protein synthesis or proline hydroxylation. Therefore, it is possible to analyze for hydroxyproline accurately by using a separate unhydroxylated sample to correct for the presence of the metabolites. The formation of glutamic acid suggested that proline oxidase activity might be present in bone tissue, but none was detected using a sensitive radioisotopic assay. Although the amount of radioactivity found in the metabolites was 36% of the amount of [ 14C]proline incorporated into protein, no radioactive glutamic or aspartic acid was present in protein hydrolyzates. This observation suggests that the metabolites did not enter the major amino acid pool used for protein synthesis.