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A two-dimensional (2D) HPLC system focusing on the determination of phenylalanine (Phe) enantiomers in mammalian physiological fluids has been developed. ᴅ-Phe is indicated to have potential values as a disease biomarker and therapeutic molecule in several neuronal and metabolic disorders, thus the regulation of ᴅ-Phe in mammals is a matter of interest. However, the precise determination of amino acid enantiomers is difficult in complex biological samples, and the development of an analytical method with practically acceptable sensitivity, selectivity and throughput is expected. In the present study, a 2D-HPLC system equipped with a reversed-phase column in the 1st dimension and an enantioselective column in the 2nd dimension has been designed, following the fluorescence derivatization of the target amino acid enantiomers with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The analytical method was validated using both plasma and urine samples, and successfully applied to human, rat and mouse fluids. Trace levels of ᴅ-Phe were determined in the plasma, and the %ᴅ values were around 0.1% for all species. In the urine, relatively large amounts of ᴅ-Phe were observed, and the %ᴅ values for humans, rats and mice were 3.99, 1.76 and 5.25%, respectively. The relationships between the enzymatic activity of ᴅ-amino acid oxidase (DAO) and the amounts of intrinsic ᴅ-Phe have also been clarified, and high ᴅ-Phe amounts were observed (around 0.3% in the plasma and around 50% in the urine) in the DAO deficient rats and mice., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

D-serine is present in the vertebrate retina and serves as a coagonist for the N-methyl-D-aspartate (NMDA) receptors of ganglion cells. Although the enzyme D-amino acid oxidase (DAO) has been implicated as a pathway for d-serine degradation, its role in the retina has not been established. In this study, we investigated the role of DAO in regulating D-serine levels using a mutant mouse line deficient in DAO (ddY/DAO(-)) and compared these results with their wild-type counterparts (ddY/DAO(+)). Our results show that DAO is functionally present in the mouse retina and normally serves to reduce the background levels of D-serine. The enzymatic activity of DAO was restricted to the inner plexiform layer as determined by histochemical analysis. Using capillary electrophoresis, we showed that mutant mice had much higher levels of D-serine. Whole cell recordings from identified retinal ganglion cells demonstrated that DAO-deficient animals had light-evoked synaptic activity strongly biased toward a high NMDA-to-AMPA receptor ratio. In contrast, recordings from wild-type ganglion cells showed a more balanced ratio between the two receptor subclasses. Immunostaining for AMPA and NMDA receptors was carried out to compare the two receptor ratios by quantitative immunofluorescence. These studies revealed that the mutant mouse had a significantly higher representation of NMDA receptors compared with the wild-type controls. We conclude that 1) DAO is an important regulatory enzyme and normally functions to reduce D-serine levels in the retina, and 2) D-serine levels play a role in the expression of NMDA receptors and the NMDA-to-AMPA receptor ratio.

D-Amino acids are stereoisomers of L-amino acids. They are often called unnatural amino acids, but several D-amino acids have been found in mammalian brains. Among them, D-serine is abundant in the forebrain and functions as a co-agonist of NMDA receptors to enhance neurotransmission. D-Amino-acid oxidase (DAO), which degrades neutral and basic D-amino acids, is mainly present in the hindbrain. DAO catabolizes D-serine and, therefore, modulates neurotransmission. In the brains of mutant mice and rats lacking DAO activity, the amounts of D-serine and other D-amino acids are markedly increased. Mutant mice manifested behavioral changes characteristic of altered NMDA receptor activity, likely due to increased levels of D-serine. D-Serine and DAO have been demonstrated to play important roles in cerebellar development and synaptic plasticity. They have also implicated in amyotrophic lateral sclerosis and pain response. There have also been several lines of evidence correlating DAO with schizophrenia. Taken together, the experiments indicate that D-amino acids and DAO have pivotal functions in the central nervous system.

For elucidation of the regulation mechanisms of intrinsic amounts of D-serine (D-Ser) which modulates the neuro-transmission of N-methyl-D-aspartate receptors in the brain, mutant animals lacking serine racemase (SRR) and D-amino acid oxidase (DAO) were established, and the amounts of D-Ser in the tissues and physiological fluids were determined. D-Ser amounts in the frontal brain areas were drastically decreased followed by reduced SRR activity. On the other hand, a moderate but significant decrease in D-Ser amounts was observed in the cerebellum and spinal cord of SRR knock-out (SRR(-/-)) mice compared with those of control mice, although the amounts of D-Ser in these tissues were low. The amounts of D-Ser in the brain and serum were not altered with aging. To clarify the uptake of exogenous D-Ser into the brain tissues, we have determined the D-Ser of SRR(-/-) mice after oral administration of D-Ser for the first time, and a drastic increase in D-Ser amounts in all the tested tissues was observed. Because both DAO and SRR are present in some brain areas, we have established the double mutant mice lacking SRR and DAO for the first time, and the contribution of both enzymes to the intrinsic D-Ser amounts was investigated. In the frontal brain, most of the intrinsic D-Ser was biosynthesized by SRR. On the other hand, half of the D-Ser present in the hindbrain was derived from the biosynthesis by SRR. These results indicate that the regulation of intrinsic D-Ser amounts is different depending on the tissues and provide useful information for the development of treatments for neuronal diseases.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder involving an extensive loss of motoneurons. Aberrant excitability of motoneurons has been implicated in the pathogenesis of selective motoneuronal death in ALS. D-serine, an endogenous coagonist of N-methyl-D-aspartate receptors, exacerbates motoneuronal death and is increased both in patients with sporadic/familial ALS and in a G93A-SOD1 mouse model of ALS (mSOD1 mouse). More recently, a unique mutation in the D-amino acid oxidase (DAO) gene, encoding a D-serine degrading enzyme, was reported to be associated with classical familial ALS. However, whether DAO affects the motoneuronal phenotype and D-serine increase in ALS remains uncertain. Here, we show that genetic inactivation of DAO in mice reduces the number and size of lower motoneurons with axonal degeneration, and that suppressed DAO activity in reactive astrocytes in the reticulospinal tract, one of the major inputs to the lower motoneurons, predominantly contributes to the D-serine increase in the mSOD1 mouse. The DAO inactivity resulted from expressional down-regulation, which was reversed by inhibitors of a glutamate receptor and MEK, but not by those of inflammatory stimuli. Our findings provide evidence that DAO has a pivotal role in motoneuron degeneration through D-serine regulation and that inactivity of DAO is a common feature between the mSOD1 ALS mouse model and the mutant DAO-associated familial ALS. The therapeutic benefit of reducing D-serine or controlling DAO activity in ALS should be tested in future studies.

A fully automated two-dimensional HPLC system combining a microbore-ODS column and a narrowbore-enantioselective column was designed and validated, and the amounts of D-serine (D-Ser) and D-alanine (D-Ala) in various tissues and physiological fluids of Long-Evans agouti/SENDAI (LEA/Sen) rats lacking D-amino-acid oxidase (DAO) were determined. Intra- and inter-day precision was less than 4.3% and accuracy ranged between 99.9 and 104%. LEA/Sen rats were reported to lack DAO in their kidneys and expected to be a novel mutant animal lacking DAO, however, the amounts of D-amino acids in the LEA/Sen rats have not been investigated. In the present study, the intrinsic amounts of D-Ser and D-Ala, which are neuromodulators of the N-methyl-D-aspartate (NMDA) receptors, were determined in seven brain tissues, four peripheral tissues, plasma and urine of the LEA/Sen rats and compared to those of the control (Wistar and SD) rats having normal DAO activity. The levels of D-Ser in the tissues and physiological fluids of the LEA/Sen rats were significantly higher than those of the Wistar and SD rats except for the frontal brain regions. Concerning D-Ala, the amounts in the tissues and physiological fluids of the LEA/Sen rats were drastically increased compared to those of the Wistar and SD rats. These results indicate that the intrinsic amounts of D-Ser and D-Ala in the tissues of rats are regulated by DAO, and that LEA/Sen rats would be useful for the study of NMDA receptor-related diseases in which DAO is implicated., (Copyright © 2010 Elsevier B.V. All rights reserved.)

D-amino acid oxidase (DAO), an enzyme that degrades d-serine, has been suggested as a susceptibility factor for schizophrenia. Here we sought to understand more about the behavioral consequence of lacking DAO and the potential therapeutic implication of DAO inhibition by characterizing a mouse strain (ddY/DAO(-)) lacking DAO activity. We found that the mutant mice showed enhanced prepulse inhibition responses (PPI). Intriguingly, DAO-/- mice had increased sensitivity to the PPI-disruptive effect induced by the competitive NMDA antagonist, SDZ 220-581. In the 24-h inhibitory avoidance test, DAO-/- mice were not different from DAO+/+ mice during the recall. In Barnes Maze, we found that DAO-/- mice had a shortened latency to enter the escape tunnel. Interestingly, although these mice were hypoactive when tested in a protected open field, they showed a profound increase of activity on the edge of the unprotected open field of the Barnes Maze even with the escape tunnel removed. This increased edge activity does not appear to be related to a reduced level of anxiety given that there were no significant genotype effects on the measures of anxiety-like behaviors in two standard animal models of anxiety, elevated plus maze and novelty suppressed feeding. Our data suggest that DAO-/- mice might have altered functioning of NMDARs. However, these results provide only modest support for manipulations of DAO activity as a potential therapeutic approach to treat schizophrenia., (Copyright © 2010 Elsevier B.V. All rights reserved.)

D-amino acid oxidase (DAO) catalyzes oxidative deamination of D-amino acids. Since D-amino acids are considered to be rare in eukaryotes, physiological function of this enzyme has been enigmatic for a long time. Mutant mice lacking DAO were found, and their strain was established. The urine of the mutant mice contained large amounts of D-amino acids. D-Amino acids were also present in their organs and blood. The origin of these D-amino acids was pursued. The results indicate that one of the physiological functions of DAO is the metabolism of D-amino acids of internal and external origin. A large amount of D-serine is shown to exist in the brain of mammals. It binds to the coagonist-binding site of N-methyl-D-aspartate (NMDA) subtype of glutamate receptors and enhances the neurotransmission. DAO metabolizes this D-serine and, therefore, modulates neurotransmission. Mutant mice displayed phenotypes resulting from the enhanced NMDA receptor function. Recent studies have shown that DAO is associated with schizophrenia. Mutant mice were resistant to the drugs which act on NMDA receptors and elicit schizophrenia-like symptoms. Recently, mutant rats lacking DAO have also been found. They were free from D-serine-induced nephrotoxicity, indicating involvement of DAO in this toxicity. The mutant mice and rats lacking DAO would be useful for the elucidation of the physiological functions of DAO and the etiology of neuronal diseases associated with DAO.

Unidirectionally chiral inversion of N(G)-nitro-D-arginine (D-NNA) to its L-enantiomer (L-NNA) occurred in rats, and it was blocked markedly (ca. 80%) by renal vascular ligation, and entirely (100%) by the D-amino acid oxidase (DAO) inhibitor sodium benzoate, suggesting that renal DAO is essential for the inversion. However, the doses of sodium benzoate administrated were extremely high (e.g., 400 mg/kg) due to its low potency. It is thus possible that sodium benzoate-mediated blockade of D-NNA inversion might be due to its nonspecific (or non-DAO-related) effects. In addition, after D-NNA was incubated with the pure enzyme of DAO in vitro without tissue homogenates, L-NNA was not produced, even though D-NNA was disposed. We propose that this occurred because D-NNA was first converted to its corresponding alpha-keto acid by DAO and then to L-NNA by transaminase(s); however, there was no direct evidence for this process. The goal of this study is to further elucidate the process of D-NNA chiral inversion both in vivo and in in vitro tissue homogenates by comparing mutant ddY/DAO(-/-) mice that lack DAO activity entirely compared to normal ddY/DAO(+/+) mice and Swiss mice. Furthermore, the ability to produce L-NNA from D-NNA-corresponding alpha-keto acids (N(G)-nitroguanidino-2-oxopentanoic acid) produced by porcine kidney-derived DAO (pkDAO) was also studied in the DAO inhibitor-pretreated rats. We found that D-NNA chiral inversion occurred in Swiss mice and ddY/DAO(+/+) mice both in vivo and in in vitro kidney homogenates, but not in ddY/DAO(-/-) mice, correlated to their DAO activities. The alpha-keto acid (N(G)-nitro-guanidino-2-oxopentanoic acid) from D-NNA was able to produce L-NNA, and subsequent vasoconstriction and pressor responses. These results indicate that the role of renal DAO is indispensible but insufficient for chiral inversion of D-NNA and other neutral and polar D-amino acids, and unidentified aminotransferase(s) are involved in a subsequent mechanism for the process of chiral inversion.

Two-dimensional-HPLC procedures have been established for the sensitive and selective determination of D-serine (D-Ser) and D-alanine (D-Ala), and their amounts in the tissues and physiological fluids of mice with various D-amino-acid oxidase (DAO) activities have been demonstrated. These two D-amino acids are modulators of the N-methyl-D-aspartate receptor mediated neurotransmission, and the alterations in their amounts following the changes in the DAO activity are matters of interest. After pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the D-amino acids were determined by the 2D-HPLC system with fluorescence detectors. As the first dimension, a microbore-monolithic-ODS column (750 mm x 0.53 mm I.D.) was adopted and a self-packed narrowbore-Pirkle type enantioselective column (Sumichiral OA-2500S, 250 mm x 1.5 mm I.D.) was selected for the second dimension. The lower limits of quantitation of D-Ser and D-Ala were 500 amol, and the within-day and day-to-day precisions were less than 6.8%. Using these methods, the amounts of D-Ser and D-Ala in 6 brain tissues, 4 peripheral tissues, serum and urine of mice having various DAO activities were determined; the amounts of these D-amino acids were drastically increased with a lowering of the DAO activity except for the cases of D-Ser in the frontal brain regions. The present micro-2D-HPLC procedures are powerful tools for the determination of small amounts of D-Ser and D-Ala in mammalian samples, and the obtained results would be useful for developing novel drugs that modulate the DAO activity, such as DAO inhibitors, against neuronal diseases.

D-amino-acid oxidase (DAO) is known to be associated with schizophrenia. Since the expression of DAO gene had been reported to be very low in LEA rats, we examined LEA/SENDAI rats in detail. These rats did not have DAO activity, enzyme protein or mRNA encoding this enzyme. Sequencing of the 5'-upstream region of the DAO gene revealed the deletion of one triplet in the 15 TAA repeats approximately 700-bp upstream of the transcription start point. A 1.3-kb upstream fragment containing the TAA repeats and the transcription start point was inserted into a reporter vector and was transfected into COS-1, NRK-52E and CCL-PK1 cells. Although the fragments containing 15 or 14 repeats had high promoter activity, the fragment containing 13 repeats had very weak activity. Electrophoretic mobility-shift assays showed that the nuclear extracts from COS-1 and COS-7 cells had proteins that bound to the oligonucleotides containing the TAA repeats. These results suggest that the TAA repeats are important for expression of the DAO gene. The LEA/SENDAI rats lacking DAO would be a useful tool for the investigations aimed at the elucidation of the relationships between this flavoenzyme and schizophrenia.

A rapid genotyping method of D-amino acid oxidase (DAO), an enzyme that catalyzes the oxidative degradation of most of the D-amino acids in mammals, has been established. This method employs a one-step PCR, restriction enzyme digestion and rapid microchip electrophoresis (MCE), and the DAO genotype of the living individual mice was definitely determined within a day by clearly separating the 95 and 107 bp Hpa II digested DNA fragments. For verification of the method, the DAO activity in the kidney of individual mice was also determined, and the obtained values completely matched the estimated genotypes (DAO(+/+), DAO(+/-), and DAO(-/-)). The intrinsic amounts of D-Pro in the serum and kidney of mice with three DAO genotypes were compared for the first time, and demonstrated that the D-amino acid amounts in the DAO(+/+) mice (1.93 +/- 0.66 nmol/mL serum, not detectable in the kidney) and DAO(+/-) mice (1.50 +/- 0.24 nmol/mL serum, not detectable in the kidney) were almost the same. The present method should be a powerful tool to establish various pathologic-model animals under the complete care of their intrinsic DAO activity, which are useful for the screening of D-amino acids having physiological activity and/or diagnostic values.

(1). We investigated the effects of inhibiting D: -amino-acid oxidase (DAO) activity on nociceptive responses through the use of mutant ddY/DAO(-) mice, which lack DAO activity, and through the application of a selective inhibitor of DAO, sodium benzoate, in the tail flick test, hot-plate test, formalin test, and acetic acid-induced writhing test. (2). Compared with normal ddY/DAO+ mice, ddY/DAO(- )mice showed significantly prolonged tail withdrawal latency in the tail flick test and licking/jumping latency in the hot-plate test, as well as significantly reduced duration of licking/biting in the late phase of the formalin test and the number of abdominal writhing in the acetic acid-induced writhing test. (3). In addition, we investigated the effects of sodium benzoate in Kunming mice having normal DAO activity. (4). Intravenous administration of sodium benzoate (400 mg/kg) significantly inhibited pain responses of the late phase of the formalin test and abdominal writhing responses in the acetic acid-induced writhing test, with no effects on the early phase flinch responses in the formalin test, nociceptive responses in the tail flick test, or hot-plate test. (5). These results suggest that DAO acts as a pro-nociceptive factor in pain, particularly chronic pain, transmission and modulation, and may be a target for pain treatment.

The behavioral effects induced by methamphetamine (5.0 mg/kg) were compared in the mutant mice lacking d-amino acid oxidase activity and normal mice. The mutant mice exhibited marked decline in the methamphetamine-induced stereotypy compared to the normal mice, whereas the mutant mice displayed a drastic augmentation in the locomotor activity evoked by methamphetamine compared to the normal mice. Because the d-serine levels in the brain of the mutant mice are significantly higher than those in the normal mice, the enhanced d-serine in the brain of the mutant mice could antagonize the methamphetamine-induced stereotypy via the N-methyl-d-aspartate receptors.

To mutant ddY/DAO(-) mice lacking D-amino-acid oxidase activity and normal ddY/DAO(+) mice, five D-amino acids (D-Asp, D-Ser, D-Ala, D-Leu and D-Pro) were orally administered for two weeks, and the D-amino acid levels were examined in seven brain regions. The levels of D-Asp markedly increased in the pituitary and pineal glands in both strains. In the ddY/DAO(+) mice, the levels of the other D-amino acids did not significantly change in most of the brain regions. While in the ddY/DAO(-) mice the levels of D-Ser significantly increased in most of the brain regions except for the cerebrum and hippocampus. The levels of D-Ala and D-Leu increased in all regions but the levels of D-Pro did not significantly change. The same five D-amino acids were intravenously injected into Wistar rats and the D-amino acid levels in their brains were examined for 60 min after the administration. The levels of D-Asp markedly increased in the pineal gland 3 min after the administration, while the levels of D-Ser, D-Ala, and D-Pro increased both in the pineal and pituitary glands, the levels of D-Leu increased in all brain regions. These results are useful for the elucidation of the origins and regulation of D-amino acids in the mammalian body.

1. Many studies have shown that administration of d-3, 4-dihydroxyphenylalanine (D-dopa) produces contralateral rotation in hemi-parkinsonian animals comparable to L-dopa, with less potency and slower onset. It was postulated that D-dopa was converted to L-dopa to produce these effects. 2. To investigate the postulated chiral inversion of D-dopa to L-dopa and the related mechanism, an enantiomeric separation method for D- and L-dopa using HPLC was first established. Then, rat kidney homogenates containing D-dopa or L-dopa were incubated and subjected to HPLC to detect traces of respective enantiomer generation. The mechanism of the chiral inversion of d-dopa was explored by direct measurement of the production of L-dopa in kidney homogenates. D-dopa incubations containing different concentrations of an inhibitor of D-amino acid oxidase (DAAO) and an inhibitor of dopa transaminase were measured for L-dopa generation using HPLC. The role of DAAO in the chiral inversion of D-dopa to L-dopa was further investigated by using purified DAAO and mutant ddY/DAAO- mouse kidney lacking DAAO activity. 3. In rat kidney homogenate, D-dopa was, indeed, converted to L-dopa, whereas L-dopa was not converted to D-dopa. Sodium benzoate, a selective inhibitor of DAAO, blocked L-dopa generation in a concentration-dependant manner. In contrast with kidney homogenates of wild-type ddY/DAAO+ mice, those of mutant ddY/DAAO- mice lacking DAAO activity did not convert D-dopa to L-dopa unless exogenous DAAO protein was added. Conversely, when carbidopa, an inhibitor of dopa transaminase, was added to the homogenates, significant inhibition of L-dopa production was noted. 4. These results prove the proposal that d-dopa undergoes unidirectional chiral inversion and further suggest that D-dopa is first oxidatively deaminated by DAAO to its alpha-keto acid and then transaminated by dopa transaminase to L-dopa.

Using a column-switching HPLC system combining a micro-ODS column and a chiral column, the amounts of D-proline (D-Pro) have been determined in 18 tissues, plasma and urine of mice. To avoid the enzymatic degradation of D-amino acids in vivo, a mutant mouse strain lacking D-amino acid oxidase activity (ddY/DAO(-) mouse) was used. In the brain, relatively large amounts of D-Pro were observed in the anterior pituitary, posterior pituitary and pineal glands. In the peripheral tissues, the amounts of D-Pro were high in the pancreas and kidney. Above all, it is surprising that the ddY/DAO(-) mice excreted large amounts of D-Pro in their urine (433 nmol/mL, 20 times that of L-Pro). The origin of D-Pro has also been investigated. By comparing germ-free mice and gnotobiotic mice, intestinal bacteria were shown to have no effect on the urinary D-Pro amount. Concerning the dietary origin, a notable amount of D-Pro was still excreted in the urine after starvation for 4 days, suggesting that some of the D-Pro is produced in the mice. Age-dependent changes in the urinary D-Pro amount have also been investigated from the postnatal 1st month up to 12 months, and ddY/DAO(-) mice were found to excrete large amounts of D-Pro in the urine constantly throughout their lives.

D-amino acid oxidase (DAO) degrades D-serine, a co-agonist at the NMDA receptor (NMDAR). Hypofunction of the NMDAR has been suggested to contribute to the pathophysiology of schizophrenia. Intriguingly, DAO has been recently identified as a risk factor for schizophrenia through genetic association studies. A naturally occurring mouse strain (ddY/DAO-) has been identified which lacks DAO activity. We have characterized this strain both behaviorally and biochemically to evaluate DAO as a target for schizophrenia. We have confirmed that this strain lacks DAO activity and shown for the first time it has increased occupancy of the NMDAR glycine site due to elevated extracellular D-serine levels and has enhanced NMDAR function in vivo. Furthermore, the ddY/DAO- strain displays behaviors which suggest that it will be a useful tool for evaluation of the clinical benefit of DAO inhibition in schizophrenia.

D-serine is nephrotoxic in rats. Based on circumstantial evidence, it has been suspected that D-amino-acid oxidase is involved in this nephrotoxicity. Since we found that LEA/SENDAI rats lacked D-amino-acid oxidase, we examined whether this enzyme was associated with D-serine-induced nephrotoxicity using the LEA/SENDAI rats and control F344 rats. When d-propargylglycine, which is known to have a nephrotoxic effect through its metabolism by D-amino-acid oxidase, was injected intraperitoneally into the F344 rats, it caused glucosuria and polyuria. However, injection of d-propargylglycine into LEA/SENDAI rats did not cause any glucosuria or polyuria, indicating that D-amino-acid oxidase is definitely not functional in these rats. D-serine was then injected into the F344 and LEA/SENDAI rats. It caused glucosuria and polyuria in the F344 rats but not in the LEA/SENDAI rats. These results indicate clearly that D-amino-acid oxidase is responsible for the D-serine-induced nephrotoxicity.

We evaluated the role of D-amino-acid oxidase on spatial learning and long-term potentiation (LTP) in the hippocampus, since this enzyme metabolizes D-amino-acids, some of which enhance the N-methyl-D-aspartate receptor functions. The Morris water maze learning and the LTP in the CA1 area of the hippocampal slice were observed in wild-type mice and mutant mice lacking D-amino-acid oxidase. The mutant mice showed significantly shorter platform search times in the water maze and significantly larger hippocampal LTPs than the wild-type mice. These results suggest that the abundant D-amino-acids in the mutant mouse brain facilitate hippocampal LTP and spatial learning.

The determination of small amounts of D-amino acids in mammalian tissues is still a challenging theme in the separation sciences. In this review, various gas-chromatographic and high-performance liquid chromatographic methods are discussed including highly selective and sensitive column-switching procedures. Based on these methods, the distributions of D-aspartic acid, D-serine, D-alanine, D-leucine and D-proline have been clarified in the mouse brain. As the regulation mechanisms of D-amino acid amounts in mammals, we focused on the D-amino-acid oxidase, which catalyzes the degradation of D-amino acids. Using the mutant mouse strain lacking D-amino-acid oxidase activity, the effects of the enzymatic activity on the amounts and distributions of various D-amino acids have been investigated.

The behavioral effects produced by MK-801 (0.4 mg/kg) were compared in mutant DAO-/- mice lacking D-amino acid oxidase activity and normal DAO+/+ mice. Mutant mice display marked diminution of stereotypy and ataxia induced by MK-801 compared to normal mice. Because the D-serine level in the brain of mutant mice is significantly higher than that of normal mice, the elevated D-serine in the brain of mutant mice could antagonize MK-801-induced stereotypy and ataxia.

d-Amino acids are now recognized to be widely present in mammals. Renal d-amino-acid oxidase (DAO) is associated with conversion of d-amino acids to the corresponding alpha-keto acids, but its contribution in vivo is poorly understood because the alpha-keto acids and/or l-amino acids formed are indistinguishable from endogenous compounds. First, we examined whether DAO is indispensable for conversion of d-amino acids to their alpha-keto acids by using the stable isotope tracer technique. After a bolus intravenous administration of d-[(2)H(7)]leucine to mutant mice lacking DAO activity (ddY/DAO(-)) and normal mice (ddY/DAO(+)), elimination of d-[(2)H(7)]leucine and formation of alpha-[(2)H(7)]ketoisocaproic acid ([(2)H(7)]KIC) and l-[(2)H(7)]leucine in plasma were determined. The ddY/DAO(-) mice, in contrast to ddY/DAO(+) mice, failed to convert d-[(2)H(7)]leucine to [(2)H(7)]KIC and l-[(2)H(7)]leucine. This result clearly revealed that DAO was indispensable for the process of chiral inversion of d-leucine. We further investigated the effect of renal mass reduction by partial nephrectomy on elimination of d-[(2)H(7)]leucine and formation of [(2)H(7)]KIC and l-[(2)H(7)]leucine. Renal mass reduction slowed down the elimination of d-[(2)H(7)]leucine. The fraction of conversion of d-[(2)H(7)]leucine to [(2)H(7)]KIC in sham-operated rats was 0.77, whereas that in five-sixths-nephrectomized rats was 0.25. The elimination behavior of d-[(2)H(7)]leucine observed in rats suggested that kidney was the principal organ responsible for converting d-leucine to KIC.

The nucleotide sequence of cDNA that encodes hamster d-amino-acid oxidase (DAO) was determined. The cDNA consisted of 1,590 nucleotides and a poly(A) tail. It had an open reading frame for a protein consisting of 346 amino acid residues. The number of the amino acid residues is the same as that of the rat DAO. However, the hamster DAO has one residue more than mouse DAO and one residue less than human, pig, rabbit, and guinea pig DAOs. Amino acid sequence of the hamster DAO was highly similar to those of mouse and rat DAOs: 89% and 88% of the amino acid residues were identical between the hamster and mouse DAOs and between the hamster and rat DAOs, respectively. The homology was slightly less between the hamster DAO and the human (81%), pig (78%), rabbit (78%), or guinea pig DAO (82%). It has been proposed that the mouse and rat DAOs lack an amino acid residue corresponding to the 25th residue of the DAOs of other mammals. However, a detailed comparison of the amino acid sequences as well as the underlying nucleotide sequences by inclusion of the hamster ones revealed that the rodent DAOs does not lack the 25th, but the 27th residue.

A new procedure to accurately measure a trace amount of d-proline in biological samples has been developed. This D-amino acid was derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and was determined by a column-switching HPLC system, a combination of a micro-ODS column and a chiral column. The detection limit for D-proline spiked in a mouse cerebrum sample is 1 fmol (injection amount, S/N = 3). Within-day precision and day-to-day precision obtained for spiked d-proline (10 fmol) are 2.14 and 5.35% (RSD), respectively. Using the new method, the amount of free D-proline in eight brain regions and sera of mutant ddY/DAO- mice, lacking D-amino acid oxidase activity, and control ddY/DAO+ mice was determined. The amount of free D-leucine was also investigated. The amount and distribution of D-proline in the brains of ddY/DAO+ mice and ddY/DAO- mice are almost the same, and relatively high amounts of D-proline have been observed in the pituitary gland and in the pineal gland. On the other hand, the amount of D-leucine is different between the two strains. In the brains of ddY/DAO+ mice, a relatively high amount of D-leucine has been observed in the pineal gland compared with other regions. In the brains of ddY/DAO- mice, D-leucine amounts are approximately 10 times higher than those obtained in ddY/DAO+ mice and regional difference has not been observed, while the amounts of L-proline and L-leucine are not significantly different between the two strains. In the serum, the amounts of both free D-proline and d-leucine are significantly higher in the ddY/DAO- mice than those obtained in ddY/DAO+ mice., (Copyright 2001 Academic Press.)

A simple and precise method for the simultaneous determination of free D-aspartic acid, D-serine and D-alanine in mouse brain tissues was established, using a reversed-phase HPLC system with widely used pre-column derivatizing reagents, o-phthaldialdehyde and N-t-butyloxycarbonyl-L-cysteine. With the present method, the contents of these three D-amino acids in hippocampus, hypothalamus, pituitary gland, pineal gland and medulla oblongata as well as cerebrum and cerebellum of mutant mice lacking D-amino-acid oxidase activity were determined and compared with those obtained for control mice. In both mice, extremely high contents of D-serine were observed in forebrain (100-400 nmol/g wet tissue), and the contents were small in pituitary and pineal glands. While, D-serine contents in cerebellum and medulla oblongata of mutant mice were about ten times higher than those in control mice. In contrast, D-alanine contents in mutant mice were higher than those in control mice in all brain regions and serum.

Formalin-induced nociceptive behaviors and N-methyl-D-aspartate (NMDA) subtype glutamate receptor-mediated excitatory synaptic transmission were analyzed in mutant mice lacking D-amino-acid oxidase, which catalyzes the oxidative deamination of D-amino acids. The second phase of the formalin-induced licking response, a part of which is known to be mediated by NMDA receptors in the spinal cord, was significantly augmented in mutant mice. NMDA receptor-mediated excitatory postsynaptic currents recorded from spinal cord dorsal horn neurons by tight-seal whole-cell methods were significantly potentiated in mutant mice. The present observations provide another line of evidence that D-serine functions as an endogenous coagonist at the glycine site of NMDA receptors, and raise the possibility that D-amino-acid oxidase exerts a neuromodulatory function by controlling the concentration of D-serine in the central nervous system.

A cell line (R-Y121B.DF) has been established from a cell line (R-Y121B) derived from a rat hepatoma line (H4-II-E). The R-Y121B.DF cells have been continuously cultured in a serum-free modified Eagle's minimum essential medium in which L-phenylalanine was replaced by D-phenylalanine. They had D-amino-acid oxidase (DAO) activity which is essential for the growth in the medium containing D-amino acids. The enzyme activity of the R-Y121B.DF cells was approximately one-fourth of that of the rat liver. Northern hybridization using a DAO cDNA probe detected a hybridizing signal in the R-Y121B.DF cells and the rat liver but not in the parental R-Y121B and H4-II-E cells. Reverse transcription-polymerase chain reaction using DAO-specific primers amplified a DNA fragment of the expected size in the R-Y121B.DF cells but not in the R-Y121B and H4-II-E cells. This fragment was confirmed to be DAO cDNA by nucleotide sequencing. Western blotting showed that DAO protein was present in the R-Y121B.DF cells and the rat liver but not in the R-Y121B and H4-II-E cells. Southern hybridization showed that the DAO gene structure was not different among the R-Y121B.DF cells, R-Y121B cells, H4-II-E cells, and the rat liver. These results indicate that the R-Y121B.DF is a unique cell line which proliferates in the medium containing D-phenylalanine and explicitly expresses DAO. This line is useful for the study of DAO in vitro.

A part of D-amino-acid oxidase gene was amplified in the human and mouse by polymerase chain reaction. The amplified fragments were ligated to plasmids and then cloned. The plasmids containing the parts of D-amino-acid oxidase gene were biotinylated and hybridized to human and mouse metaphase chromosomes. The chromosomal slides were treated with fluorescein isothiocyanate (FITC)-conjugated avidin. The hybridized signals were amplified with biotinylated anti-avidin antibody and FITC-avidin. The chromosomes were counter-stained with diamidino-phenylindole for assignment of the signal to a specific band. Using this fluorescence in situ hybridization (FISH), D-amino-acid oxidase gene was assigned to human chromosome 12q23-24.1 and mouse chromosome 5E3-F. Since these regions are syntenic between human and mouse, the present results indicate that the locus for this enzyme has been conserved through evolution.

When D-propargylglycine was injected intraperitoneally into mice, polyuria, glycosuria, and aminoaciduria were observed as has been previously reported in rats. The urine of the mice treated with D-propargylglycine contained twice as much protein as that of the control mice. Polyacrylamide gel electrophoresis showed a new protein of approximately 62 kDa in the urine of the D-propargylglycine-treated mice. Protein sequencing revealed that this protein was serum albumin. Since the above-mentioned symptoms suggested dysfunction of the renal proximal tubules, the activity of urinary N-acetyl-beta-D-glucosaminidase, a marker enzyme of injury to the proximal tubules, was measured. The urinary enzyme activity was 2.6 times higher in the D-propargylglycine-treated mice than in the control mice. Light- and electron-microscopy showed degenerative and necrotic cells in the straight part of the proximal tubules of the treated mice. However, none of these symptoms was observed in D-propargylglycine-treated mutant mice, lacking D-amino-acid oxidase. These results indicate that D-propargylglycine itself is not nephrotoxic but its metabolite produced by the D-amino-acid oxidase reaction is nephrotoxic and injures proximal tubular cells, resulting in an impairment of the reabsorption of water, glucose, amino acids, and proteins.

The nucleotide sequence of cDNA that encodes guinea pig D-amino-acid oxidase (DAO) was determined. The cDNA consisted of 1,399 nucleotides and a poly(A) tail. The cDNA encodes 347 amino acid residues. In contrast to the hamster, rat, and mouse DAOs, guinea pig DAO had the 25th amino acid residue. The homology in amino acid sequences between the guinea pig DAO and the rodent DAOs was not high in comparison to the homology in amino acid sequences between the guinea pig DAO and DAOs of humans, pigs and rabbits. The phylogenetic position of the guinea pig varied depending on the source of sequences (amino acids or nucleotides) and the methods of phylogenetic tree construction. These results suggest that the guinea pig is not a simple rodent.

Nucleotide sequence of cDNA encoding rat D-amino-acid oxidase (DAO) was determined. Two species of DAO mRNA were present in rat kidney, liver, and brain. They were probably produced by alternative splicing. Rat DAO cDNA encodes 346 amino acid residues, indicating that rat DAO is an intermediate form between mouse DAO (345 amino acids) and DAOs (347 amino acids) of human, rabbit, and pig. Deduced amino acid sequence indicates 93% identity between rat and mouse DAO. Northern hybridization and western blotting supported the sequence data.

Since there are conflict reports on the presence of D-amino-acid oxidase in the mouse liver, this problem was examined. D-Amino-acid oxidase activity was not detected in the homogenates of the mouse liver, lung, or heart, whereas it was detected in the homogenates of the mouse kidney and brain. Western blotting showed that a protein which reacted with the antiserum against pig D-amino-acid oxidase was present in the homogenates of the mouse kidney and brain but not in those of the liver or heart. Northern hybridization using a D-amino-acid oxidase cDNA probe detected a hybridizing signal in poly(A)+ RNAs extracted from the mouse kidney and brain but not in those from the liver, heart, or lung. Reverse transcription-polymerase chain reaction using three primer pairs always amplified D-amino-acid oxidase cDNA fragments of expected sizes in the mouse kidney and brain but very rarely did so in the liver, heart, or lung. The results indicate that D-amino-acid oxidase is not present in the mouse liver in a measurable amount.

D-Alanine was administered orally to mutant mice lacking D-amino acid oxidase (EC 1.4.3.3). The mice had free access to drinking water containing 0.5% D- or L-alanine or 0.1% D-alanine for 2 weeks. The mice were then killed, and levels of the D- and L-enantiomers of free alanine, serine, proline, glutamate, and aspartate were determined in serum, liver, kidney, cerebrum, and cerebellum tissues. D-Alanine content increased by 60-fold (liver) to 110-fold (serum, brain), although the L-alanine level did not change. The increase of serum and brain D-alanine concentrations in animals fed 0.5% D-alanine was approximately five times more than that in animals fed 0.1% D-alanine, ie, the increase was roughly D-alanine dose-dependent in these tissues. The increase due to 0.5% D-alanine administration was reduced by 50% 17 hours after administration of D-alanine was stopped. Administration-induced increases in D-alanine levels in the cerebrum and cerebellum were not less than those in the serum, suggesting that D-alanine passed the blood-brain barrier quite freely. In the liver but not in other tissues, there were slight increases in D-serine and D-proline levels after administration of D-alanine. Administration of D-alanine produced no alterations in free glutamate and aspartate levels. No D-enantiomers of alanine, serine, proline, glutamate, or aspartate were detected in the liver and kidney tissue proteins of any animals, even in the mutant mice that received 0.5% D-alanine.

Urine of mutant ddY/DAO- mice lacking D-amino-acid oxidase contained 13 times more D-alanine than that of normal ddY/DAO+ mice. Because D-alanine is a component of bacterial cell walls, the possibility that the urinary D-alanine came from intestinal bacteria was examined. In ddY/DAO- mice that were made germ free at birth and reared in a germ-free environment, the quantity of urinary D-alanine was found to be at a low level comparable to that of the normal mice. When these germ-free mice were made gnotobiotic by inoculation with gram-negative bacteria (Escherichia coli and Bacteroides vulgatus), the urinary D-alanine increased to a high level. When these gnotobiotic mice were further inoculated with gram-positive bacteria (Bifidobacterium longum and Eubacterium aerofaciens), the urinary D-alanine increased further. These results indicate that most of the urinary D-alanine of the conventionally reared ddY/DAO- mice is of gastrointestinal bacterial origin.

We have examined whether D-amino acid oxidase (DAO) regulates free D-serine content using mutant ddY/DAO- mice lacking DAO activity. We find that the content of D-serine in the serum and cerebellum of mutant mice is much higher than that of normal mice, whereas a slight but significant difference in the cerebral D-serine level is observed between the two strains. These results suggest that, although DAO may participate in the catabolism of D-serine in the cerebellum and periphery, there appears to be other mechanisms for catabolism of endogenous D-serine in the brain.

Mutant ddY/DAO- mice lacking D-amino-acid oxidase (DAO) activity were examined for the cause of their lack of enzyme activity. Total RNA was extracted from the kidney of the ddY/DAO- mice and cDNA was synthesized. After cDNA encoding DAO was amplified by the polymerase chain reaction it was cloned into a plasmid and sequenced. Comparison of the DAO cDNA sequence with that of normal BALB/c mice revealed the presence of a single-base substitution (G----A) which causes a Gly-181----Arg substitution in the middle of the enzyme molecule. The mutant DAO cDNA was inserted into an expression vector and was expressed in transfected COS-1 cells. The transfected cells synthesized the DAO mutant protein, but they did not show DAO activity. In contrast, when cells were transfected with an expression vector carrying wild-type DAO cDNA, where the substituted base-pair was replaced by a normal base-pair, they showed DAO activity. These results indicate that the single base-pair substitution is the cause of the loss of DAO activity in the ddY/DAO- mice.

D-amino acid oxidase (DAAO) activity was not altered in the liver and kidney by oral administration of D-alanine to adult mice. The enzyme was apparently not induced by the enteric microflora either, since the enzyme activity in the liver and kidney of germ-free mice was not different from that of specific-pathogen-free mice. The times of appearance of DAAO activity and of free D-amino acids in the kidney were elucidated using suckling mice. DAAO activity started to increase 7 days after birth, and reached almost the adult level by 28 days. The content of free neutral D-amino acids also increased with age, in a similar fashion. A possible conclusion is that the enzyme activity normally increases during this period, to eliminate the free D-amino acids which have increased with age in the suckling mice. Consequently, the administration of D-alanine had no further effect in increasing enzyme activity.

1. Mutant mice lacking D-amino-acid oxidase activity were examined as to whether they possessed the enzyme protein. 2. Immunoblotting using an antibody against hog kidney D-amino-acid oxidase showed that kidney homogenates of the mutant mice as well as that of the normal mice had proteins reactive to the antibody. 3. Peroxisomal proteins of the kidney cells of the mutant mice were not different from those of the normal mice. 4. The peroxisomes of the mutant mice possessed a protein reactive to the antibody in the immunoblotting whose size was the same as the D-amino-acid oxidase protein present in the peroxisomes of the normal mice. 5. These results suggest that the mutant mice synthesize the D-amino-acid oxidase protein and integrate it into peroxisomes, though it is a nonfunctional enzyme.

Urine from mutant mice which lack D-amino-acid oxidase contained a large amount of D-alanine. The alanine content was not changed by changes in the dietary composition or by starvation. However, oral administration of an antibiotic, amoxicillin, decreased the urinary alanine to the normal level. These results suggest that the D-alanine is not of dietary origin but of intestinal bacterial origin.

A mutant mouse strain ddY/DAO- lacks D-amino acid oxidase activity and accumulates free neutral D-amino acids in its tissues. In this study, D-aspartate oxidase activity and D-aspartate content in the tissues of these mutant mice were compared with those of normal mice. No significant difference was observed, indicating that the metabolism of acidic D-amino acids was unaffected in the mutant.

Thin-layer chromatography and amino acid analysis showed that mutant (ddY/DAO-) mice lacking D-amino-acid oxidase activity excreted about 3.5 times more methionine in urine than did normal (ddY/DAO+) mice. High-performance liquid chromatography using a chiral column showed that approximately 82% of urinary methionine of the ddY/DAO- mice had the D-configuration. Analysis revealed that the mouse diet used contained 0.04% free methionine and that approximately 46% of methionine was the D-isomer. When the ddY/DAO- mice were given a diet containing a low level of supplementary DL-methionine or a diet without D-methionine, they excreted the normal levels of methionine. These results indicate that the ddY/DAO- mice were unable to metabolize D-methionine and excrete it in urine.

D-Amino acid oxidase activity in the kidney homogenates of mice of seven strains was measured to search for a mutant for this enzyme. There was a consistent sex difference in the enzyme activity in these strains: male mice showed higher levels of the enzyme activity than females. In contrast to other strains, some mice of the ddY strain did not possess enzyme activity. This trait was inheritable, and a mouse stock without enzyme activity (DAO-) was established. The allele (Dao-1c) carried by the DAO- mice was recessive and behaved as a single autosomal gene in inheritance. Heterozygous mice for this gene (Dao-1+/Dao-1c) showed nearly half the enzyme activity of the wild-type homozygotes (Dao-1+/Dao-1+), suggesting that Dao-1c is a null allele and that there is a gene dosage effect on the enzyme activity.

Thin-layer chromatography and amino acid analysis showed that urine of mutant ddY/DAO- mice lacking D-amino-acid oxidase activity contained more serine, proline, alanine and methionine than that of normal ddY/DAO+ mice. Among these four, an increase in alanine was conspicuous. However, the urinary levels of 11 other amino acids and glucose were not different between the ddY/DAO- and ddY/DAO+ mice. Amino acid analysis showed that the plasma levels of serine, proline and methionine were not elevated in the ddY/DAO- mice, though a slight increase in alanine was observed. Genetic crosses showed that aminoaciduria and lack of D-amino-acid oxidase activity were concomitantly transmitted as a set through generations. These results indicated that the lack of enzyme activity caused a specific renal aminoaciduria. Whether this enzyme merely diminishes the D-amino acid load presented for reabsorption, or actually participates catalytically in the reabsorption process, remains undetermined.

A simple and rapid method to screen for mutant mice (Mus musculus) lacking D-amino-acid oxidase activity has been devised. Mice were given water containing small amounts (0.02%) of either D-methionine or D-phenylalanine. Urinary levels of the D-amino acid were examined using thin-layer chromatography. Some mice excreted substantial amounts of the D-amino acid through the urine. None of them had detectable D-amino-acid oxidase activity.

Mutant ddY/DAO- mice lacking D-amino-acid oxidase were examined for their ability to utilize D-phenylalanine in place of its L-isomer. Chemically defined milk powder devoid of phenylalanine was used as a basal diet. Adult ddY/DAO- mice and normal ddY/DAO+ mice having D-amino-acid oxidase lost weight every day when fed this diet. However, they maintained their weight when fed the milk powder diet supplemented with 0.33% L-phenylalanine. Although adult ddY/DAO+ mice maintained their weight when fed the milk powder diet supplemented with 0.33% D-phenylalanine, ddY/DAO- mice lost weight while feeding on this diet, which indicated that the ddY/DAO- mice could not utilize D-phenylalanine in place of its L-isomer. Both ddY/DAO- and ddY/DAO+ mice maintained their weight when feeding on the milk powder diet enriched with 0.41% sodium phenylpyruvate, which suggested to us that the ddY/DAO- mice had the ability to invert this alpha-keto acid to L-phenylalanine. Therefore, these results indicate that D-amino-acid oxidase is indispensable for D-amino acid utilization. This is consistent with the concept that D-amino acids are oxidatively deaminated to the corresponding alpha-keto acids by D-amino-acid oxidase and that these keto intermediates are then asymmetrically reaminated to their L-amino acids.