尿中の遊離γ-カルボキシグルタミン酸定量のためのHPLCによる改良法

Abstract

A rapid and sensitive high performance liquid chromatographic (HPLC) method for the determination of free γ-carboxyglutamic acid (Gla) in urine using precolumn fluorescent derivatization with OPA/ET reagent was previously developed. In the present study, we improved the method for quantitative analysis of urinary free Gla. 1) Fluorescent strength of OPA/ET reagent reached a maximum level after 3 days from the preparation of the reagent. Then the fluorescent strength was maintained at least after 3 weeks by the addition of ethanthiol every 3 days. 2) An urine sample was diluted by 20 to 60-folds and a 2.5μl aliquot of the diluted urine sample containing about 1 to 2 pmol of Gla was injected into the ChemcoPak Liquid Chromatography Columns packed with Nucleosil 5SB. The mobile phase consisted of 0.12 M sodium citrate buffer (pH 5.28) and acetonitrile in the ratio 60:40. Under these conditions, Gla peak appeared in a retention time of about 7 to 10 minutes and was completely resolved from the other amino acids. Since the peak disappeared after the sample was subjected to decarboxylation treatment, the peak was confirmed to contain only Gla. 3) This method gave a linear standard curve in a range of 0.10-150pmol and allowed quantitative analysis of Gla in an amount as low as 0.1pmol. We used the standard curve in a range of 0.10-4.0pmol for urinary Gla analysis. This is a sensitive and simple assay of free Gla in urine which was subjected to only dilution without further treatment.