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

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Highlights

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    d-Asp and d-Glu were determined in whole body of DDO-KO mice.

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    d-Asp were drastically increased with the decrease in the DDO activity.

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    The amounts of d-Glu were almost the same between DDO-KO and control mice.

Abstract

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.

Introduction

It has been believed that a large proportion of amino acids in higher animals were l-enantiomers, and the antipodes, d-amino acids, were hardly present. However, based on the recent progress in the analytical sciences and technologies, various d-amino acids, such as d-serine (d-Ser) [1], [2], [3], d-aspartic acid (d-Asp) [4], [5], [6], [7], d-alanine (d-Ala) [8], [9], [10], [11] and d-glutamic acid (d-Glu) [12], [13] were widely found in mammals including human beings, and they are now thought to be the candidates of novel physiologically active substances and/or biomarkers [14], [15]. Among these d-amino acids, acidic d-amino acids, d-Asp and d-Glu, are attracting attention in the fields of endocrinology and neuroscience. d-Asp was reported to play significant physiological roles in the hormonal synthesis and secretion in various endocrine tissues (e.g., the pineal gland, testis, pituitary gland and adrenal gland) [16], [17], [18], [19], [20], and both d-Asp and d-Glu are reported to regulate neuronal transmission [21], [22], [23].

Both of d-Asp and d-Glu are metabolized by d-aspartate oxidase (DDO, EC 1.4.3.1) in mammals including human beings [24], [25]. DDO is a peroxisomal flavoprotein, which was firstly reported in 1949 [26], that stereospecifically catalyzes the oxidative deamination of acidic d-amino acids. The high activity of DDO is mainly observed in the kidney, liver and brain of mammals and especially concentrated in nerve cells [25], [27]. In addition to the functions in the endocrine and neuroendocrine systems, d-Asp could be a potent antipsychotic drug in the brain for the treatment of N-methyl-d-aspartate (NMDA) receptor-related diseases [28], [29]. Therefore, to develop novel drug candidates controlling the amounts of d-Asp and d-Glu via regulation of the DDO activity, such as DDO inhibitors, the changes in the intrinsic amounts of d-Asp and d-Glu in various tissues by changing the DDO activities are expected to be clarified.

The aim of the present study is to investigate the intrinsic amounts of d-Asp and d-Glu in the brain and peripheral tissues of DDO+/+, DDO+/− and DDO−/− mice. Recently, significant elevations of d-Asp in the cerebral cortex, cerebellum, kidney, spleen, adrenal gland and testis of DDO-deficient mice have been reported [30], [31], [32]. However, for the clinical applications controlling the intrinsic amounts of these acidic d-amino acids by changing the DDO activity, such as the use of DDO inhibitors, the amounts in specific brain areas, various peripheral tissues and physiological fluids in the mice having various DDO activities are matters of interest. In the present study, the amounts of Asp and Glu enantiomers in 6 brain tissues, 11 peripheral tissues, plasma and urine of the DDO+/+, DDO+/− and DDO−/− mice were determined by the highly selective two-dimensional micro-HPLC system.

Section snippets

Materials

Enantiomers of aspartic acid and HPLC grade acetonitrile (MeCN) were obtained from Nacalai Tesque (Kyoto, Japan). Enantiomers of glutamic acid, HPLC grade methanol (MeOH), trifluoroacetic acid (TFA), citric acid monohydrate and boric acid were obtained from Wako Pure Chemical Industries (Osaka, Japan). The fluorescence derivatizing reagent, 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was purchased from Tokyo Kasei (Tokyo, Japan). Water was purified using a Milli-Q gradient A 10 system (Merck

Determination of d-Asp and l-Asp in the tissues and physiological fluids of DDO+/+, DDO+/− and DDO−/− mice

To clarify the alteration of the d- and l-Asp amounts by changing the DDO activity, we raised the DDO+/+, DDO+/− and DDO−/− mice and the Asp enantiomers were determined in 6 brain areas, 11 peripheral tissues and 2 physiological fluids. The DDO−/− mice are the knock-out mice lacking the DDO protein, while the DDO+/+ mice are the wild type mice having normal DDO activity. The DDO+/− mice have half the DDO activity compared to that in DDO+/+ mice. As the target brain areas, pineal gland, cerebral

Conclusion

Using the two-dimensional HPLC system combining reversed-phase and enantioselective columns, d-Asp and d-Glu in the tissues and physiological fluids of mice were successfully determined with no severe interference by intrinsic substances. In the mice lacking the DDO activity (DDO−/− mice), the amounts of d-Asp drastically increased compared to those in the mice with a normal DDO activity (DDO+/+ mice). On the other hand, the amounts of d-Glu were not changed by a decrease in the DDO activity,

Acknowledgements

This work was partly supported by JSPS KAKENHI Grant Number 25293007 and 26860023, Japan. The authors thank Shiseido Co., Ltd., (Tokyo, Japan) for their technical supports. The authors also thank Professors Solomon H. Snyder (The Johns Hopkins University School of Medicine), Stephen G. Young, Anne Beigneux (University of California) and their co-workers for their kindly providing the DDO-KO mouse materials and suggestions.

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