Reiko Koga

HPLC analysis of naturally occurring free D-amino acids in mammals.

D-amino acids are currently recognized as naturally occurring physiologically active substances and biomarkers in mammals. The progress of analytical technologies, mostly high resolution chromatographic or electrodriven separation methods, has significantly contributed to the advances in D-amino acid research in real biological matrices. In this review, we would like to describe the D-amino acid research, from the discovery of appreciable amounts of free D-amino acids in mammals to the current metabolomics study focusing on amino acid enantiomers. The liquid phase enantioselective analytical methods utilized for the determination of D-amino acids in mammals including human beings will be discussed.

Changes in d-aspartic acid and d-glutamic acid levels in the tissues and physiological fluids of mice with various d-aspartate oxidase activities

D-Aspartic acid (D-Asp) and D-glutamic acid (D-Glu) are currently paid attention as modulators of neuronal transmission and hormonal secretion. These two D-amino acids are metabolized only by D-aspartate oxidase (DDO) in mammals. Therefore, in order to design and develop new drugs controlling the D-Asp and D-Glu amounts via regulation of the DDO activities, changes in these acidic D-amino acid amounts in various tissues are expected to be clarified in model animals having various DDO activities. In the present study, the amounts of Asp and Glu enantiomers in 6 brain tissues, 11 peripheral tissues and 2 physiological fluids of DDO(+/+), DDO(+/-) and DDO(-/-) mice were determined using a sensitive and selective two-dimensional HPLC system. As a result, the amounts of D-Asp were drastically increased with the decrease in the DDO activity in all the tested tissues and physiological fluids. On the other hand, the amounts of D-Glu were almost the same among the 3 strains of mice. The present results are useful for designing new drug candidates, such as DDO inhibitors, and further studies are expected.

Enantioselective two-dimensional high-performance liquid chromatographic determination of N-methyl-d-aspartic acid and its analogues in mammals and bivalves

A validated and fully automated chiral 2D-HPLC system was developed for the simultaneous determination of N-methyl-d-aspartic acid (NMDA) analogues by combining a long microbore-monolithic ODS column (0.53 mm i.d.× 1,000 mm) and narrowbore-enantioselective columns (1.5mm i.d.×150 or 250 mm). The target analytes, enantiomers of N-methylaspartic acid (NMA) and N-methylglutamic acid (NMG), were precolumn-derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and detected by their fluorescence. The values of the lower limit of quantification for these enantiomers were 2.5 fmol. In the tissues and plasma of rats, neither NMA nor NMG were detected. On the other hand, in the mantle and foot of Scapharca broughtonii, a large amount of NMDA was present (170.1 and 43.5 nmol/g), and the enantiomers of NMG were also observed. Meretrix lusoria contained NMDA (29.3 nmol/g) and NMLG (13.8 nmol/g), and Ruditapes philippinarum contained only NMLG (2.6 nmol/g). The obtained results were confirmed using three different enantioselective columns and also using a 2D-HPLC-MS/MS system. These results indicated that neuroactive d-amino acid, NMDA, and its analogues were present in animals, and their physiological significance is expected to be clarified.

Enantioselective determination of citrulline and ornithine in the urine of d-amino acid oxidase deficient mice using a two-dimensional high-performance liquid chromatographic system

Two-dimensional high-performance liquid chromatographic (2D-HPLC) and 2D-HPLC-mass spectrometric (2D-HPLC-MS) systems have been designed and developed for the determination of the citrulline (Cit) and ornithine (Orn) enantiomers. Several d-amino acids have already been identified as novel physiologically active molecules and biomarkers, and the enantioselective evaluation of the amounts, distributions and metabolisms of non-proteinogenic amino acids gain as well increasing interest. In the present study, highly selective analytical methods were developed using a capillary monolithic ODS column (0.53mm i.d. x 1000mm) for the reversed-phase separation of the target analytes from the matrix compounds in the first dimension, and a narrowbore-Pirkle type enantioselective column, KSAACSP-105S (1.5mm i.d. x 250mm), was used for the enantiomer separation in the second dimension. The amino acids were analyzed after pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and detected by the fluorescence detector and MS. The systems were applied to the urine of d-amino acid oxidase (DAO) deficient B6DAO- mice and control C57BL mice to evaluate the presence and metabolism of the Cit and Orn enantiomers in mammals. As a result, all of the 4 target enantiomers (d-Cit, l-Cit, d-Orn, l-Orn) were found in the urine of both strains. The %D value of Cit (d-Cit/Cit×100) increased about 3-fold in the urine of the DAO deficient mice and that of Orn also tended to increase with the DAO deficiency. These results were definitely confirmed by a 2D-HPLC-MS detection system. Further investigations about the biological significance of these d-isomers are currently ongoing.

Mouse D-amino-acid oxidase: Distribution and physiological substrates

d-Amino-acid oxidase (DAO) catalyzes the oxidative deamination of d-amino acids. DAO is present in a wide variety of organisms and has important roles. Here, we review the distribution and physiological substrates of mouse DAO. Mouse DAO is present in the kidney, brain, and spinal cord, like DAOs in other mammals. However, in contrast to other animals, it is not present in the mouse liver. Recently, DAO has been detected in the neutrophils, retina, and small intestine in mice. To determine the physiological substrates of mouse DAO, mutant mice lacking DAO activity are helpful. As DAO has wide substrate specificity and degrades various d-amino acids, many d-amino acids accumulate in the tissues and body fluids of the mutant mice. These amino acids are d-methionine, d-alanine, d-serine, d-leucine, d-proline, d-phenylalanine, d-tyrosine, and d-citrulline. Even in wild-type mice, administration of DAO inhibitors elevates D-serine levels in the plasma and brain. Among the above d-amino acids, the main physiological substrates of mouse DAO are d-alanine and d-serine. These two d-amino acids are most abundant in the tissues and body fluids of mice. d-Alanine derives from bacteria and produces bactericidal reactive oxygen species by the action of DAO. d-Serine is synthesized by serine racemase and is present especially in the central nervous system, where it serves as a neuromodulator. DAO is responsible for the metabolism of d-serine. Since DAO has been implicated in the etiology of neuropsychiatric diseases, mouse DAO has been used as a representative model. Recent reports, however, suggest that mouse DAO is different from human DAO with respect to important properties.

Chapter 6 – Amino Acid and Bioamine Separations

Abstract Liquid chromatographic methods for the determination of amino acids and bioamines are outlined and discussed. In Section 4.2 , direct separation methods for amino acids with postcolumn derivatization or MS/MS detection are described. In Section 4.3 , typical indirect methods for amino acids are summarized. In this section, precolumn derivatization reagents for UV-VIS detection, fluorescence detection, and MS/MS detection are explained. Enantioselective analytical methods for amino acids are also described in Section 4.4 . Direct and indirect separation methods for bioamines are summarized in Sections 4.5 and 4.6 .

Amino acid and bioamine separations

Liquid chromatographic methods for the determination of amino acids and bioamines are outlined and discussed. In Section 4.2, direct separation methods for amino acids with postcolumn derivatization or MS/MS detection are described. In Section 4.3, typical indirect methods for amino acids are summarized. In this section, precolumn derivatization reagents for UV-VIS detection, fluorescence detection, and MS/MS detection are explained. Enantioselective analytical methods for amino acids are also described in Section 4.4. Direct and indirect separation methods for bioamines are summarized in Sections 4.5 and 4.6.

Determination of d-Amino Acids and Their Distribution in Mammals

d-Amino acids are the enantiomers of l-amino acids. Although the l-forms are predominantly present in life systems in the free form and also in the protein-bound form, the optically inversed d-forms were hardly found especially in higher animals. This is due to the very low amounts of these d-amino acids, and the determination is frequently disturbed by large amounts of l-amino acids and uncountable numbers of peptides and amino compounds. However, due to the recent progress in analytical technologies, several d-amino acids have been found in mammals including humans. In this chapter, the analytical methods for determining these d-amino acids and their distribution in mammals are described.