An improved method for the determination of gamma-carboxyglutamic acid in proteins, bone, and urine

A method of high-performance liquid chromatographic separation of the fluorescence derivative of gamma-carboxyglutamic acid (Gla) is presented. Alkaline hydrolysates of protein samples were reacted with o-phthalaldehyde in the presence of ethanethiol for 2 min, and the fluorescence derivative of gamma-carboxyglutamic acid was resolved from the other amino acids by a short column packed with silica-based anion exchanger under isocratic conditions. By this method, as low as 200 fmol of gamma-carboxyglutamic acid can be quantitatively analyzed within 10 min. The method presented here shortened the analysis time for Gla and was at least 10 times more sensitive than the method we described previously (Anal. Biochem. 117, 259-265, 1981). The application of this method to the formic acid-soluble or insoluble gamma-carboxyglutamic acid-containing proteins in chicken bone and the concomitant increase of gamma-carboxyglutamic acid content in chicken bone with age are reported.