Alterations in D-amino acid levels in the brains of mice and rats after the administration of D-amino acids.

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.

Determination of free D-proline and D-leucine in the brains of mutant mice lacking D-amino acid oxidase activity.

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.

Determination of free D-aspartic acid, D-serine and D-alanine in the brain of mutant mice lacking D-amino acid oxidase activity.

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.

Determination of minute amounts of D-leucine in various brain regions of rat and mouse using column-switching high-performance liquid chromatography.

A highly sensitive method for the determination of minute amounts of D-Leu in biological samples was developed. For accurate and sensitive determination, a column-switching system using a micro ODS column and a chiral column was adopted. After pre-column derivatization of D- and L-Leu with NBD-F, the derivatives of the enantiomers were purified on a micro ODS column as a DL mixture. The eluted DL-Leu was then introduced to the chiral column, and each enantiomer was determined. The calibration curve for D-Leu, which was constructed by adding known amounts of D-Leu to a rat hippocampus, was linear from 1 to 1000 fmol (r>0.999), and the detection limit of added D-Leu was 1 fmol (S/N=5). Within-day and day-to-day precisions of D-Leu determination using the same homogenate of rat hippocampus were 5.11 and 5.25% (RSD), respectively. The content of D-Leu in rat hippocampus was 0.69 nmol/g wet tissue (the percentage of D-enantiomer for total Leu was 0.97%), which was consistent with the reported value. The distribution of D-Leu in mouse brain was also investigated, and the presence of D-Leu in various regions of the mammalian brain was first observed.

Determination of pineal melatonin by precolumn derivatization reversed-phase high-performance liquid chromatography and its application to the study of circadian rhythm in rats and mice.

Determination of minute amounts of endogenous melatonin in rat and mouse pineal gland was performed using an RP-HPLC system. Melatonin was separated following precolumn derivatization and determined with a fluorescence detector at the emission wavelength of 380 nm with the excitation at 245 nm. The calibration curve of melatonin constructed by adding known amounts of melatonin to the homogenates of mouse pineal gland was linear over the range of 1-500 fmol (injection amount/20 microl). The detection limit of added melatonin was 1 fmol (S/N = 5). Repeatability and day-to-day precision for the melatonin spiked sample of mouse pineal gland was 4.0 and 3.8% (RSD), respectively. Using the present method, circadian changes of melatonin content in rat (Wistar) and mouse (C3H) pineal gland were determined. In addition, a minute amount of melatonin in ddY mouse pineal gland was determined, because pineal melatonin of many inbred mouse strains has been reported to be lower than the detection limit.

Alteration in the D-amino acid content of the rat pineal gland under anesthesia.

In a previous report (Hamase, K. et al., Biochim Biophys Acta 1134: 214-222 (1997)), we showed that the rat pineal gland contains D-leucine (D-Leu) as well as D-aspartic acid (D-Asp). In this communication we report alterations in the content of these D-amino acids during anesthesia. The D-Asp content was significantly increased from 2.8 to 5.0, 4.8 and 5.8 nmol/pineal gland by administration of ether, urethane and pentobarbital, respectively. In contrast, the D-Leu content was decreased by administration of urethane or pentobarbital. The D-Leu content decreased from 4.2 to 2.2 pmol/pineal gland 4 hours after administration of urethane, although the content remained unchanged until 1.5 hours after administration. The content of the L-enantiomers of these amino acids were not affected by anesthesia. The urethane-induced decrease in D-leucine content was almost completely suppressed by a beta-agonist, (-)-isoproterenol, whereas the agonist itself had no effect.

Simple and rapid analytical method for carbapenems using capillary zone electrophoresis.

A simple and rapid analytical method for carbapenems using high-performance capillary electrophoresis is described. All therapeutic carbapenem injections in Japan (imipenem, panipenem and meropenem) and four other beta-lactams (piperacillin, cefotiam, cefotaxime, latamoxef) were separated and determined with good repeatability in about 10 min using simple free zone capillary electrophoresis. The electrophoresis buffer was 100 mM phosphate buffer of pH 8.0, and a fused-silica capillary of 25 microm I.D. and 47 cm length was adopted. The present method was successfully applied to monitor the degradation of carbapenems under various conditions (at various temperatures or in coexistence with other drugs prescribed in the case of methicillin-resistant Staphylococcus aureus).

Sensitive determination of melatonin by precolumn derivatization and reversed-phase high-performance liquid chromatography.

A sensitive determination method for melatonin was developed. Melatonin was derivatized under alkaline conditions in the presence of hydrogen peroxide. The resultant fluorophore was excited at 247 nm and the emission wavelength was 384 nm. The Stokes shift was 137 nm, which was longer than that of melatonin itself (lambda ex 280 nm, lambda em 330 nm). The melatonin derivative was separated by reversed-phase HPLC in about 15 min and the calibration curve was linear from 500 amol to 5 pmol (r > 0.999) with the detection limit of 500 amol (S/N = 5). The sensitivity of this method was about ten times higher than that of previous methods. Both the day-to-day precision and within-day precision were about 5%, and the derivative of melatonin in the aqueous solution was stable for more than 10 days. This method was successfully applied to the determination of melatonin in rat pineal gland.

Regional distribution and postnatal changes of D-amino acids in rat brain.

Regional distribution of D-amino acids in rat brain was studied by the modified highly sensitive analytical method which was previously developed. The method includes fluorogenic derivatization of each amino acid, isolation of each amino acid by reverse-phase HPLC, followed by enantiomeric separation with Pirkle-type chiral stationary phases. D-Amino acid contents were determined in the cerebrum, cerebellum, hippocampus, medulla oblongata, pituitary gland and pineal gland. D-Aspartic acid was observed in the pineal gland (3524 +/- 263 nmol/g, data are for male rats of 6 weeks of age) and the pituitary gland (80.5 +/- 9.0 nmol/g). D-Serine was found in various regions of the brain except for the cerebellum and medulla oblongata. D-Alanine was observed exclusively in the pituitary gland (25.9 +/- 4.4 nmol/g), whereas D-leucine was found in the pineal gland (3.4 +/- 0.4 nmol/g) and the hippocampus (1.6 +/- 0.07 nmol/g). No other D-amino acids were detected in the brain. The contents of D-aspartic acid in the pituitary gland and D-serine in the pineal gland were higher in female rats. In contrast the contents of D-alanine in the pituitary gland and D-leucine in the pineal gland and the hippocampus were higher in males. Postnatal changes of D-aspartic acid and D-leucine in the pineal gland and D-alanine in the pituitary gland were also investigated. The results described in this paper suggested that distinct regulatory mechanisms exist for individual D-amino acids in the corresponding region of rat brain.

Analytical chemistry and biochemistry of D-amino acids.

The methodologies for the analysis of D-amino acids in biological materials have been reviewed, including the use of enzymes, gas and liquid chromatography with chiral stationary phases and diastereomer derivatization with chiral reagents followed by GC or HPLC separation. The distribution of D-amino acids in the body, their origin, metabolism and possible roles in human diseases are discussed.