Development of a three-dimensional HPLC system for acidic amino acid enantiomers and determination of their amounts in mice lacking D-aspartic acid oxidase activity

Abstract

A highly-selective three-dimensional (3D) HPLC system has been designed/developed for the determination of acidic amino acid enantiomers, i.e., aspartic acid (Asp) and glutamic acid (Glu). The 3D-HPLC system consisted of a reversed-phase column (Singularity RP18, 1.0 ×250 mm) in the first dimension, a mixed-mode column (Singularity MX-001, 1.5 ×250 mm) in the second dimension and a Pirkle-type enantioselective column (Singularity CSP-011S, 1.5 ×250 mm) in the third dimension. By using this system, the amounts of Asp and Glu enantiomers in 6 tissues (cerebrum, heart, lung, liver, kidney and testis) and 2 physiological fluids (plasma and urine) were determined in the control C57BL/6J mice and B6DDO^-/- mice lacking D-aspartic acid oxidase activity. In the control C57BL/6J mice, a high amount of D-Asp (466.15 nmol/g) was observed in the testis, and relatively high amounts of D-Asp were observed in the cerebrum, lung, liver and kidney (21.40–92.87 nmol/g). In the heart and 2 physiological fluids, the amounts of D-Asp were low (trace–7.75 nmol/g or mL). In the B6DDO^-/- mice, the amounts of D-Asp drastically increased in all the tissues and physiological fluids. In contrast to the results of D-Asp, the amounts of D-Glu did not change depending on the alteration of the DDO activity, and the amounts were extremely low and not detectable in most of the tested tissues and physiological fluids. Although the amounts of D-Glu were low, it is noteworthy that clear localization in the testis (8.60–9.53 nmol/g) was demonstrated in both the C57BL/6J and B6DDO^-/- mice.