Journal of Chromatography B (v.879, #29)

This review highlights recent progresses in capillary electrophoresis (CE) analysis of amino acid enantiomers in the last decade. Various chiral selectors including cyclodextrins (CDs), bile salts, crown ethers, cinchona alkaloids, metal–chiral amino acid complexes, macrocyclic antibiotics and proteins have been employed to separate amino acid enantiomers. In the CE analysis of amino acids, the selection of the separation mode is one of the most important issues to obtain good resolution of target enantiomers. Among several separation modes, CD-modified capillary zone electrophoresis (CD-CZE), CD electrokinetic chromatography (CDEKC), micellar EKC (MEKC), CD-modified micellar electrokinetic chromatography (CD-MEKC), capillary electrochromatography (CEC), ligand-exchange CE (LE-CE), and nonaqueous CE (NACE) have been employed to the chiral analysis of amino acids. More than 160 published research articles collected from SciFinder Scholar databases from the year 2001 described the enantioseparations of amino acids by capillary-based electrophoresis. This review provides a comprehensive table listing the CE analysis of amino acid enantiomers with categorizing by the separation modes.
Keywords: Enantioseparation; Amino acids; Capillary electrophoresis; Electrokinetic chromatography; Capillary electrochromatography; Cyclodextrins;

Poly-γ-glutamate, a nylon-like polyamide material typically consisting of both enantiomers of glutamate, exhibits reasonable biodegradability and its multi-functionality is attracting particular attention. Thus, its industrial application as a versatile and chiral polymer is in increasing demands. Poly-γ-glutamate is presently synthesized using several biocatalysts (e.g., bacterial cells), but the uncontrollable structural diversity of such biosynthesized materials is an area of concern. This review highlights analytical approaches of interest to assure the functional and structural reproducibility of poly-γ-glutamate.
Keywords: Poly-γ-glutamate; Quantification; Molecular size; Stereochemistry;

Experimental strategies for the analysis of d-amino acid containing peptides in crustaceans: A review by Daniel Soyez; Jean-Yves Toullec; Nicolas Montagné; Céline Ollivaux (3102-3107).
Detection of d-amino acids in natural peptides has been, and remains a challenging task, as peptidyl isomerization is a peculiar and subtle posttranslational modification that does not induce any change in primary sequence or in physicochemical properties of the molecule such as molecular mass or pI. Therefore, the presence of a d-amino acid residue in a peptide chain is generally transparent to classical methods of peptide analysis (electrophoresis, chromatography, mass spectrometry, molecular biology). In this article, we will review the various experimental strategies and analytical techniques, which have been used to characterize and to study d-amino acid containing peptides in crustaceans.
Keywords: Isomerization; d-Amino acid containing peptides; Mass spectrometry; Chromatography; Crustaceans;

It was long believed that d-amino acids were either unnatural isomers or laboratorial artifacts and that the important functions of amino acids were exerted only by l-amino acids. However, recent investigations have shown that a variety of d-amino acids are present in mammals and that they play important roles in physiological functions in the body. Among the free d-amino acids that have been identified in mammals, d-aspartate (d-Asp) has been shown to play a crucial role in the neuroendocrine and endocrine systems as well as in the central nervous system. Here, we present an overview of recent studies of free d-Asp, focusing on the analytical methods in real biological matrices, expression and localization in tissues and cells, biological and physiological activities, biosynthesis, degradation, cellular transport, and possible relevance to disease. In addition to frequently used techniques for the enantiomeric determination of amino acids, including high-performance liquid chromatography and enzymatic methods, the recent development of analytical methods is also described.
Keywords: d-Aspartate; Localization; Biosynthesis; Degradation; Cellular transport; d-Amino acid;

The historical development of the enantioseparation of derivatized α-amino acids by high-resolution capillary gas chromatography on chiral stationary phases derived from α-amino acid-derivatives and modified cyclodextrins is described. The pioneering work emerging from Emanuel Gil-Av and his associates at the Weizmann Institute of Science is reviewed. A bridge to more recent developments is spanned aimed at helping to select appropriate tools for contemporary chiral α-amino acid analysis by gas chromatography in different research areas.
Keywords: α-Amino acids; Derivatization; Gas chromatography; Enantioseparation; Enantioselectivity; Enantiomer labelling; Chiral stationary phases; Chirasil-Val; Chirasil-Dex; Racemization;

d-Amino acids in aged proteins: Analysis and biological relevance by Noriko Fujii; Yuichi Kaji; Norihiko Fujii (3141-3147).
Homochirality is essential for life. l-Amino acids are exclusively used as substrates for the polymerization and formation of peptides and proteins in living systems. However, d-amino acids, which are enantiomers of l-amino acids, were recently detected in various living organisms in the form of free d-amino acids and d-amino acid residues in peptides and proteins. In particular, d-aspartyl (Asp) residues have been detected in various proteins from diverse tissues of elderly individuals. Here, we describe three important aspects of our research: (i) a method for detecting d-β-Asp at specific sites in particular proteins, (ii) a likely spontaneous mechanism by which Asp residues in proteins invert and isomerize to the d-β-form with age under physiological conditions, (iii) a discussion of factors that favor such a reaction.
Keywords: d-Amino acid; d-Aspartate; Aging; Lens; Crystallin; Skin; Oxidative stress; CML; Racemization; Isomerization; UV B irradiation;

The present paper describes an updated knowledge and status on Marfey's reagent (MR), 1-fluoro-2,4-dinitrophenyl-5-l-alanine amide (FDNP-l-Ala-NH2). The reagent is used for pre-column derivatization of amino acids followed by HPLC separation of the diastereomers so formed. Emphasis is put on the design and application of structural variants which are synthesized by introducing different (other than l-Ala-NH2) l- and d-amino acid amides and amino acids in the 1,5-difluoro-2,4-dinitro benzene (DFDNB) moiety, as the chiral auxiliary. Advantages, disadvantages, the required precautions and suitability of the approach for the separation of multi component mixtures of dl-amino acids are assessed. Use of two dimensional (2D) techniques, in particular online HPLC in combination with various mass spectrometry techniques is discussed as well as methods designated ‘advanced Marfey's method’ and ‘C3 Marfey's method’. Application of MR and its variants for the determination of the stereochemistry of protein and non-protein amino acids in bioactive natural products isolated from living organisms (bacteria including blue-green algae, filamentous fungi, plants, marine sponges, invertebrates and vertebrates), in physiological samples including human beings, and in biologically relevant synthetic peptides are presented. In an outlook future applications are envisaged.
Keywords: Chiral derivatizing reagents; 1-Fluoro-2,4-dinitophenyl; Natural and synthetic peptides; d-Amino acids;

d-Amino acid metabolism in mammals: Biosynthesis, degradation and analytical aspects of the metabolic study by Hiroko Ohide; Yurika Miyoshi; Rindo Maruyama; Kenji Hamase; Ryuichi Konno (3162-3168).
It was believed for long time that d-amino acids are not present in mammals. However, current technological advances and improvements in analytical instruments have enabled studies that now indicate that significant amounts of d-amino acids are present in mammals. The most abundant d-amino acids are d-serine and d-aspartate. d-Serine, which is synthesized by serine racemase and is degraded by d-amino-acid oxidase, is present in the brain and modulates neurotransmission. d-Aspartate, which is synthesized by aspartate racemase and degraded by d-aspartate oxidase, is present in the neuroendocrine and endocrine tissues and testis. It regulates the synthesis and secretion of hormones and spermatogenesis. d-Serine and d-aspartate bind to the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors and function as a coagonist and agonist, respectively. The enzymes that are involved in the synthesis and degradation of these d-amino acids are associated with neural diseases where the NMDA receptors are involved. Knockout mice for serine racemase and d-aspartate oxidase have been generated, and natural mutations in the d-amino-acid oxidase gene are present in mice and rats. These mutant animals display altered behaviors caused by enhanced or decreased NMDA receptor activity. In this article, we review currently available studies on d-amino acid metabolism in mammals and discuss analytical methods used to assay activity of amino acid racemases and d-amino-acid oxidases.
Keywords: Serine racemase; d-Amino-acid oxidase; Aspartate racemase; d-Aspartate oxidase; d-Serine; d-Aspartate;

d-Serine is a unique endogenous substance enriched in the brain at the exceptionally high concentrations as a free d-amino acid in mammals throughout their life. Peripheral tissues and blood contain low or trace levels of the d-amino acid. In the nervous systems, d-serine appears to act as an intrinsic coagonist for the N-methyl-d-aspartate type glutamate receptor (NMDA receptor) based upon the following characteristics: (i) d-serine stereoselectively binds to and stimulates the glycine-regulatory site of the NMDA receptor consisting of GRIN1/GRIN2 subunits more potently than glycine with an affinity and ED50 at high nanomolar ranges, (ii) the selective elimination of d-serine in brain tissues attenuates the NMDA receptor functions, indicating an indispensable role in physiological activation of the glutamate receptor, and (iii) the distribution of d-serine is uneven and closely correlated with that of the binding densities of the various NMDA receptor sites, and especially of the GRIN2B subunit in the brain. Moreover, d-serine exerts substantial influence on the GRIN1/GRIN3-NMDA and δ2 glutamate receptor. In the brain and retina, metabolic processes of d-serine, such as biosynthesis, extracellular release, uptake, and degradation, are observed and some candidate molecules that mediate these processes have been isolated. The fact that the mode of extracellular release of d-serine in the brain differs from that of classical neurotransmitters is likely to be related to the detection of d-serine in both glial cells and neurons, suggesting that d-serine signals could be required for the glia–synapse interaction. Moreover, the findings from the basic experiments and clinical observations support the views that the signaling system of endogenous free d-serine plays important roles, at least, through the action on the NMDA receptors in the brain wiring development and the regulation of higher brain functions, including cognitive, emotional and sensorimotor function. Based upon these data, aberrant d-serine–NMDA receptor interactions have been considered to be involved in the pathophysiology of a variety of neuropsychiatric disorders including schizophrenia and ischemic neuronal cell death. The molecular and cellular mechanisms for regulating the d-serine signals in the nervous system are, therefore, suitable targets for studies aiming to elucidate the causes of neuropsychiatric disorders and for the development of new treatments for intractable neuropsychiatric symptoms.
Keywords: Brain; d-Serine; N-methyl-d-aspartate receptor; Glutamate neurotransmission; Neuropsychiatric disorders; Synapse–glia interaction;

Simultaneous two-dimensional HPLC determination of free d-serine and d-alanine in the brain and periphery of mutant rats lacking d-amino-acid oxidase by Yurika Miyoshi; Kenji Hamase; Tadashi Okamura; Ryuichi Konno; Noriyuki Kasai; Yosuke Tojo; Kiyoshi Zaitsu (3184-3189).
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.
Keywords: d-Amino-acid oxidase; d-Serine; d-Alanine; LEA/Sen rat; 2D-HPLC; Enantiomer separation;

An enzymatic assay system of d-amino acids was established using the d-amino acid oxidase of Schizosaccharomyces pombe. In this method, the enzyme converts the d-amino acids to the corresponding α-keto acids, which are then reacted with 1,2-diamino-4,5-methylenedioxybenzene (DMB) in an organic solvent. The resultant fluorescent compounds are separated and quantified by high-performance liquid chromatography (HPLC). Use of an organic solvent following the α-keto acid modification with DMB prevents the non-enzymatic deamination of l-amino acids, which are generally present at much higher concentrations than d-amino acids in biological samples. With this method, d-Glu, d-Asn, d-Gln, d-Ala, d-Val, d-Leu, d-Phe, and d-Ile can be quantified in the order of micromolar, and other d-amino acids except d-Asp can be assayed within a sensitivity range of 50–100 μM. The established enzymatic method was used to analyze the d-amino acid contents in human urine. The concentration of d-Ser obtained using this enzymatic method (223 μM) was in good agreement with that obtained using the conventional HPLC method (198 μM). The enzymatic method also demonstrated that the human urine contained 5.45 μM of d-Ala and 0.91 μM of d-Asn. Both d-amino acids were difficult to be identified using the conventional method, because the large signals from l-amino acids masked those from d-amino acids. The enzymatic method that we have developed can circumvent this problem.
Keywords: d-Amino acid; d-Amino acid assay; d-Amino acid oxidase; α-Keto acid; 1,2-Diamino-4,5-methylenedioxybenzene (DMB);

Simultaneous determination of d-aspartic acid and d-glutamic acid in rat tissues and physiological fluids using a multi-loop two-dimensional HPLC procedure by Hai Han; Yurika Miyoshi; Kyoko Ueno; Chieko Okamura; Yosuke Tojo; Masashi Mita; Wolfgang Lindner; Kiyoshi Zaitsu; Kenji Hamase (3196-3202).
For a metabolomics study focusing on the analysis of aspartic and glutamic acid enantiomers, a fully automated two-dimensional HPLC system employing a microbore-ODS column and a narrowbore-enantioselective column was developed. By using this system, a detailed distribution of d-Asp and d-Glu besides l-Asp and l-Glu in mammals was elucidated. For the total analysis concept, the amino acids were first pre-column derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) to be sensitively and fluorometrically detected. For the non-stereoselective separation of the analytes in the first dimension a monolithic ODS column (750 mm × 0.53 mm i.d.) was adopted, and a self-packed narrowbore-Pirkle type enantioselective column (Sumichiral OA-2500S, 250 mm × 1.5 mm i.d.) was selected for the second dimension. In the rat plasma, RSD values for intra-day and inter-day precision were less than 6.8%, and the accuracy ranged between 96.1% and 105.8%. The values of LOQ of d-Asp and d-Glu were 5 fmol/injection (0.625 nmol/g tissue). The present method was successfully applied to the simultaneous determination of free aspartic acid and glutamic acid enantiomers in 7 brain areas, 11 peripheral tissues, plasma and urine of Wistar rats. Biologically significant d-Asp values were found in various tissue samples whereas for d-Glu the values were very low possibly indicating less significance.
Keywords: d-Aspartic acid; d-Glutamic acid; 2D-HPLC; Enantiomer separation;

A rapid and sensitive microchip electrophoresis (MCE) method with laser induced fluorescence (LIF) detection has been developed for the quantification of d-tyrosine (Tyr) in biological samples. The assay was performed using a MCE-LIF system with glass/poly(dimethylsiloxane) (PDMS) hybrid microchip after pre-column derivatization of amino acids with fluorescein isothiocyanate (FITC). Chiral separation of the derivatives was achieved by cyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) using γ-CD as chiral selector in the running buffer. d/l-Tyr enantiomer was well separated in less than 140 s. The limit of detection (S/N  = 3) was 3.3 × 10−8  M. Using the present method, d-Tyr level in human plasma was found to vary significantly from normal humans to patients suffering from renal failure.
Keywords: Microchip electrophoresis; Chiral separation; d-Tyrosine; Human plasma; Renal failure;

Similar to l-tryptophan (l-Trp), d-Trp can be converted to unique metabolites in the mammalian body. In the present study, the difference in the plasma half-life (t 1/2) between Trp enantiomers was investigated by following the alterations in the plasma concentration of d- or l-Trp after intraperitoneal (i.p.) administration of each enantiomer to male Sprague–Dawley rats (100 mg/kg). The investigation was performed using reversed-phase high-performance liquid chromatography (HPLC) and pre-column fluorescence derivatization with a chiral fluorescent labeling reagent, R(−)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (R(−)-DBD-PyNCS). The t 1/2 value of d-Trp was significantly smaller than that of l-Trp, suggesting that d-Trp was eliminated from the plasma more rapidly than l-Trp. In addition, a significant increase in the plasma concentration of l-Trp was observed following administration of d-Trp, whereas no d-Trp was detected after l-Trp administration. Furthermore, the increase in the plasma concentration of l-Trp was significantly suppressed by pretreatment with an inhibitor of d-amino acid oxidase (DAAO), 3-methylpyrazole-5-carboxylic acid, which suggests that DAAO was involved in the conversion of d-Trp to l-Trp in vivo.
Keywords: d-Tryptophan; l-Tryptophan; R(−)-DBD-PyNCS; Rat; Fluorescence; HPLC;

Determination of time-dependent accumulation of d-lactate in the streptozotocin-induced diabetic rat kidney by column-switching HPLC with fluorescence detection by Mei-Hsiang Lin; Hsiang-Yin Chen; Tzu-Hsin Liao; Tzu-Chuan Huang; Chien-Ming Chen; Jen-Ai Lee (3214-3219).
For better understanding the complete metabolism and the physiological role of d-lactate, the concentrations of d-lactate in the serum, liver and kidney of normal and diabetic rats were determined by our established column-switching HPLC method with pre-column fluorescence derivatization. Eight-week-old male Sprague–Dawley rats were administered with streptozotocin (STZ) (80 mg/kg) or citrate buffer intraperitoneally. The tissues were then removed and homogenized after 4, 8, 12 and 16 weeks of drug administration, respectively. The homogenates were centrifuged at 1200 ×  g for 10 min, then the supernatants were derivatized with a fluorescent reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ), separated on an ODS column followed by a Chiralpak AD-RH chiral column for enantioseparation. The results showed that the d-lactate content elevated in all the 3 examined tissues under diabetic stages. In addition, d-lactate concentrations in rat kidney were accumulated significantly and time-dependently in diabetic groups after receiving STZ for 4, 8, 12 and 16 weeks (2.99, 13.11, 18.19, 23.23 vs. 0.79 μmol/mg protein as control group). Moreover, the kidney of induced 12-week diabetic rat renal showed some histological changes of progressive diabetic nephropathy. The results suggest that d-lactate may be used as a marker of diabetic nephropathy.
Keywords: d-Lactate; l-Lactate; Diabetic rat; Nephropathy; Streptozotocin; Methylglyoxal;

The resolution of free dl-amino acids in human nail was carried out by combination of the R(−)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(−)-DBD-PyNCS] derivatives and UPLC–ESI-TOF-MS. The reaction of the reagent with amino acids effectively proceeds at 55 °C for 20 min in the presence of 1% triethylamine (TEA) to produce the corresponding diastereomers. Each pair of the resulting derivatives was efficiently separated by a gradient program (a mixture of H2O and CH3CN containing 0.1% formic acid (HCOOH) or 5 mM CH3COONH4 and CH3CN) using a reversed-phase ACQUITY UPLC™ BEH C18 (1.7 μm, 100 mm × 2.1 mm i.d.) column and sensitively detected by TOF-MS. The detection limits (S/N = 3) of the TOF-MS were 1.0–750 fmol, respectively. A good linearity was achieved from the calibration curves, which was obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), i.e., 6-aminohexanoic acid, versus the injected amounts of each amino acid (r 2  > 0.996), and the intra-day and inter-day assay precisions were less than 8.93%. The derivatives of the free dl-amino acids in human nail were successfully identified by the proposed procedure. As we know, for the first time, these five kinds of d-amino acids, which were d-Ala, d-Val, d-Pro, d-Ile and d-Leu, were found from human nail samples. Fifteen kinds of l-amino acids were also recognized from human nails. Using these methods, the amounts of dl-amino acids in the nails of healthy volunteers and diabetic patients were determined. When comparing the index from diabetic patients to those from healthy volunteers, there is no significant difference in the content of the l-amino acids in the nails. However, a statistically significant (P  < 0.01) correlation was observed between the d/l-amino acid concentration ratios (Ala, Val, Ile, Leu). Therefore, because the proposed method provides a good mass accuracy and the trace detection of the dl-amino acids in human nails, this analytical technique could be a noninvasive technique to assist in the diagnosis and assessment of disease activity in diabetic patients.
Keywords: Human nail; d-Amino acids; Diabetes; Diastereomer separation; Derivatization; 4-(3-Isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole; Ultra-performance liquid chromatography; Time-of-flight mass spectrometry;

Determination of d-aspartate N-methyltransferase activity in the starfish by direct analysis of N-methyl-d-aspartate with high-performance liquid chromatography by Kimihiko Shibata; Noriko Sugaya; Wakana Ono; Katsumasa Abe; Shouji Takahashi; Yoshio Kera (3229-3234).
We describe a method for the detection and quantification of d-aspartate N-methyltransferase activity. The enzyme catalyzes the S-adenosyl-l-methionine-dependent N-methylation of d-aspartate to form N-methyl-d-aspartate (NMDA). NMDA is detected directly by high-performance liquid chromatography (HPLC) of their (+)- and/or (−)-1-(9-fluorenyl)ethyl chloroformate fluorescent derivatives. The NMDA production in the assay mixture is linearly proportional to the incubation time and the amount of tissue homogenate. Using a 10 min incubation time, the method allows detection of the enzyme activity below 10 fmol/min. It can be used to analyze kinetic behavior and to quantify the enzyme from a wide variety of organisms.
Keywords: N-Methyl-d-aspartate; d-Aspartate N-methyltransferase; S-Adenosyl-l-methionine:d-aspartate N-methyltransferase; Starfish;

Analyzing the d-amino acid content in biological samples by engineered enzymes by Luca Frattini; Elena Rosini; Loredano Pollegioni; Mirella S. Pilone (3235-3239).
The aim of our present research is to produce mutant forms of d-amino acid oxidase from Rhodotorula gracilis in order to determine d-amino acid content in different biological samples. During the past few years, our group has produced yeast d-amino acid oxidase variants with altered substrate specificity (e.g., active on acidic, or hydrophobic, or on all d-amino acids) both by rational design and directed evolution methods. Now, the kinetic constants for a number of amino acids (even for unnatural ones) of the most relevant d-amino acid oxidase variants have been investigated. This information constitutes the basis for considering potential analytical applications in this important field.
Keywords: Substrate specificity; d-Amino acids; Detection; Flavoproteins; Oxidases; Mutagenesis;

The major soluble eye lens protein, αA-crystallin, has a very long half-life. Thus, many post-translational modifications, including stereoinversion, have been found in its constituent amino acids. We determine the rates of β-linkage isomerization, which is the main reaction through the formation of a succinimide intermediate, of specific Asp residues of recombinant human αA-crystallin protein by simple RP-HPLC method. Kinetic analyses of the β-linkage isomerization were performed on the three Asp residues of αA-crystallin, 58Asp, 84Asp, and 151Asp, because the d/l ratios of both the 58Asp and 151Asp residues were higher than 1.0 in the αA-crystallin isolated from aged human eye lens. The β-linkage isomerizations of both the 58Asp and 84Asp residues were suppressed in the recombinant protein by approximately 0.4–0.5 times compared to those in the synthetic peptide below 50 °C, whereas the isomerization of the 151Asp residue occurred at the same rate for the whole protein and synthetic fragmentary peptide. The suppression of 58Asp isomerization in the recombinant protein relaxed to some extent when the αA-crystallin protein was incubated at a high temperature. The far-UV CD spectra showed that the secondary structure of the protein was partially disordered at temperatures greater than 60 °C in the recombinant αA-crystallin protein. These results suggest that the 58Asp residue was restrained from forming the succinimide intermediate by the higher order structure of the αA-crystallin protein, and that the structural environment around the 151Asp residue of the αA-crystallin was similar to that of the synthetic fragmentary peptide with respect to succinimide formation. The difference in the influence of the secondary structure of the αA-crystallin protein inverts the order of the succinimide formations of the 58Asp and 151Asp residues in the recombinant protein as compared with the order in the synthetic fragmentary peptides.
Keywords: Stereoinversion; Succinimide formation; Secondary structure; Diastereomer; CD spectrum;

Engineering the substrate specificity of Alcaligenes d-aminoacylase useful for the production of d-amino acids by optical resolution by Shigekazu Yano; Hiroyuki Haruta; Takuya Ikeda; Takeshi Kikuchi; Masahiro Murakami; Mitsuaki Moriguchi; Mamoru Wakayama (3247-3252).
d-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (AxD-NAase) offers a novel biotechnological application, the production of d-amino acid from the racemic mixture of N-acyl-dl-amino acids. However, its substrate specificity is biased toward certain N-acyl-d-amino acids. To construct mutant AxD-NAases with substrate specificities different from those of wild-type enzyme, the substrate recognition site of the AxD-NAase was rationally manipulated based on computational structural analysis and comparison of its primary structure with other d-aminoacylases with distinct substrate specificities. Mutations of amino acid residues, Phe191, Leu298, Tyr344, and Met346, which interact with the side chain of the substrate, induced marked changes in activities toward each substrate. For example, the catalytic efficiency (k cat/K m) of mutant F191W toward N-acetyl-d-Trp and N-acetyl-d-Ala was enhanced by 15.6- and 1.5-folds, respectively, compared with that of the wild-type enzyme, and the catalytic efficiency (k cat/K m) of mutant L298A toward N-acetyl-d-Trp was enhanced by 4.4-folds compared with that of the wild-type enzyme. Other enzymatic properties of both mutants, such as pH and temperature dependence, were the same as those of the wild-type enzyme. The F191W mutant in particular is considered to be useful for the enzymatic production of d-Trp which is an important building block of some therapeutic drugs.
Keywords: d-Amino acids; Optical resolution; Alcaligenes; d-Aminoacylase; 3D model structure;

Simultaneous determination of selenomethionine enantiomers in biological fluids by stable isotope dilution gas chromatography–mass spectrometry by Takehisa Matsukawa; Hiroshi Hasegawa; Yoshihiko Shinohara; Jun Kobayashi; Atsuko Shinohara; Momoko Chiba; Kimiyoshi Ichida; Kazuhito Yokoyama (3253-3258).
A method for the stereoselective determination of d- and l-enantiomers of selenomethionine in mouse plasma was developed using gas chromatography–mass spectrometry with selected-ion monitoring (GC–MS-SIM). dl-[2H3, 82Se]selenomethionine was used as analytical internal standard to account for losses associated with the extraction, derivatization and chromatography. Selenomethionine enantiomers in mouse plasma were purified by cation-exchange chromatography using BondElut SCX cartridge and derivatized with HCl in methanol to form methyl ester followed by subsequent N-acylation with optically active (+)-α-methoxy-α-trifluoromethylphenylacetyl chloride to form diastereomeric amide. Quantification was performed by SIM of the molecular-related ions of the diastereomers on the chemical ionization mode. The intra- and inter-day precision for d- and l-selenomethionine spiked to mouse plasma gave good reproducibility with relative standard deviation of 3% and 3% for d-selenomethionine and 6% and 3% for l-selenomethionine, respectively. The estimated amounts were in good agreement with the actual amounts spiked, the intra- and inter-day relative error being 5% and 2% for d-selenomethionine and 2% and 1% for l-selenomethionine, respectively. The present method is sensitive enough to determine pharmacokinetics of selenomethionine enantiomers.
Keywords: Selenomethionine; GC–MS; Stable isotope; Chiral separation;

High-performance liquid chromatography analysis of naturally occurring d-amino acids in sake by Yoshitaka Gogami; Kaori Okada; Tadao Oikawa (3259-3267).
We measured all of the d- and l-amino acids in 141 bottles of sakes using HPLC. We used two precolumn derivatization methods of amino acid enantiomer detection with o-phthalaldehyde and N-acetyl-l-cysteine, as well as (+)-1-(9-fluorenyl)ethyl chloroformate/1-aminoadamantane and one postcolumn derivatization method with o-phthalaldehyde and N-acetyl-l-cysteine. We found that the sakes contained the d-amino acids forms of Ala, Asn, Asp, Arg, Glu, Gln, His, Ile, Leu, Lys, Ser, Tyr, Val, Phe, and Pro. We were not able to detect d-Met, d-Thr d-Trp in any of the sakes analyzed. The most abundant d-Ala, d-Asp, and d-Glu ranged from 66.9 to 524.3 μM corresponding to relative 34.4, 12.0, and 14.6% d-enantiomer. The basic parameters that generally determine the taste of sake such as the sake meter value (SMV; “Nihonshudo”), acidity (“Sando”), amino acid value (“Aminosando”), alcohol content by volume, and rice species of raw material show no significant relationship to the d-amino acid content of sake. The brewing water (“Shikomimizu”) and brewing process had effects on the d-amino acid content of the sakes: the d-amino acid contents of the sakes brewed with deep-sea water “Kaiyoushinosousui”, “Kimoto yeast starter”, “Yamahaimoto”, and the long aging process “Choukijukusei” are high compared with those of other sakes analyzed. Additionally, the d-amino acid content of sakes that were brewed with the adenine auxotroph of sake yeast (“Sekishoku seishu kobo”, Saccharomyces cerevisiae) without pasteurization (“Hiire”) increased after storage at 25 °C for three months.
Keywords: d-Amino acid; Sake; Chiral derivatization; HPLC; Lactic acid bacteria; Yeast;

d-Asp: A new player in reproductive endocrinology of the amphibian Rana esculenta by Franca Raucci; Maria Maddalena Di Fiore (3268-3276).
We investigated the involvement of d-Aspartic acid (d-Asp) on ovarian and testicular morphology of the green frog, Rana esculenta, and its effect on the testosterone production. The study has been performed throughout the reproductive cycle. In both ovary and testis a substantial amount of d-Asp is endogenously present and its concentration varies as function of reproduction. In the frog, d-Asp content is differently correlated with gonadal and plasmatic levels of testosterone, depending on the sex. In fact, the amount of the d-Asp is inversely linked with that of the testosterone in the ovary, while this correlation directly matched in the testis. In vivo short-term experiments, consisting of a single intra-peritoneal injection of d-Asp (2.0 μmol/g body weight), demonstrated that the enantiomer is significantly accumulated by both the ovary and testis, reaching after 3 h the highest uptake and thereafter decreasing to baseline values within 24 h. Furthermore, d-Asp influences the synthesis and/or the release of testosterone, causing a decrease of its level in the female, and an increase in the male, respectively. In vivo long-term experiments, d-Asp, chronically administered to the frogs of both sexes, enhances the maturation of both gonads, determining in the oocytes an higher accumulation of carbohydrate yolk plates in the ooplasm, and stimulating the spermatogenesis in the testis. Taken altogether, our results show that d-Asp operates differently in female and male frog gonads, indicating that it has different targets in the reproductive machinery depending on the sex.
Keywords: d-Aspartic acid; Testosterone; Seasonal breeders; Oocyte; Testis; Frog;

N-Carbamoyl-β-alanine amidohydrolase from Agrobacterium tumefaciens C58: A promiscuous enzyme for the production of amino acids by A.I. Martínez-Gómez; M. Andújar-Sánchez; J.M. Clemente-Jiménez; J.L. Neira; F. Rodríguez-Vico; S. Martínez-Rodríguez; F.J. Las Heras-Vázquez (3277-3282).
The availability of enzymes with a high promiscuity/specificity relationship permits the hydrolysis of several substrates with a view to obtaining a certain product or using one enzyme for several productive lines. N-Carbamoyl-β-alanine amidohydrolase from Agrobacterium tumefaciens (Atβcar) has shown high versatility to hydrolyze different N-carbamoyl-, N-acetyl- and N-formyl-amino acids to produce different α, β, γ and δ amino acids. We have calculated the promiscuity index for the enzyme, obtaining a value of 0.54, which indicates that it is a modestly promiscuous enzyme. Atβcar presented the highest probability of hydrolysis for N-carbamoyl-amino acids, being the enzyme more efficient for the production of α-amino acids. We have also demonstrated by mutagenesis, modelling, kinetic and binding experiments that W218 and A359 indirectly influence the plasticity of the enzyme due to interaction with the environment of R291, the key residue for catalytic activity.
Keywords: Binding; Thermodynamics; Circular dichroism; Promiscuity; Elasticity; N-Carbamoyl-β-alanine amidohydrolase;

The distribution of d-amino acids was examined on several tissues of kuruma prawn Marsupenaeus japonicus. d-Alanine was found in all tissues, and the ratio of d-alanine to total alanine ranged from 18.7 to 43.7% depending on the tissues. Of these tissues, muscle, heart, and gill contained a relatively large amount of d-alanine. Nervous tissue and eye, on the other hand, contained a large amount of d-aspartate. d-Glutamate was specifically detected in testis. The percentage of d-glutamate to total glutamate was over 50% in testis, suggesting the existence of the biosynthetic enzyme in this tissue. The changes of alanine racemase activity were determined in the muscle and hepatopancreas of M. japonicus before and after molting. The activity after molting increased twice in the muscle. On the other hand, it was not changed in the hepatopancreas. These data suggest that d-alanine plays an important role in the muscle during ecdysis. However, the free d-alanine level in the muscle was not changed significantly before and after ecdysis. From these data, several d-amino acids are considered to be utilized in some essential physiological phenomena in the different tissues of the prawn.
Keywords: d-Alanine; Alanine racemase; d-Glutamate; d-Amino acid; Aquatic invertebrate; Testis; Marsupenaeus japonicus;

Reaction pathway of tryptophanase-catalyzed l-tryptophan synthesis from d-serine by Akihiko Shimada; Haruka Ozaki; Takeshi Saito; Noriko Fujii (3289-3295).
Tryptophanase, l-tryptophan indole-lyase with extremely absolute stereospecificity, can change the stereospecificity in concentrated diammonium hydrogenphosphate solution. While tryptophanase is not inert to d-serine in the absence of diammonium hydrogenphosphate, it can undergo l-tryptophan synthesis from d-serine along with indole in the presence of it. It has been well known that tryptophanase synthesizes l-tryptophan from l-serine through a β-substitution mechanism of the ping-pong type. However, a metabolic pathway of l-tryptophan synthesis from d-serine has remained unclear. The present study aims to elucidate it. Diammonium hydrogenphosphate plays a role in the emergence of catalytic activity on d-serine. The salt gives tryptophanase a small conformational change, which makes it possible to catalyze d-serine. Tryptophanase-bound d-serine produces l-tryptophan synthesis by β-replacement reaction via the enzyme-bound aminoacrylate intermediate. Our result will be valuable in studying the origin of homochirality.
Keywords: Stereospecificity; Diammonium hydrogenphosphate; Tryptophanase; d-Serine; l-Tryptophan synthesis; Origin of homochirality;

Identification of novel mammalian phospholipids containing threonine, aspartate, and glutamate as the base moiety by Taketo Omori; Ai Honda; Hisaaki Mihara; Tatsuo Kurihara; Nobuyoshi Esaki (3296-3302).
In this study, we showed the occurrence of phosphatidyl-l-threonine (PThr), phosphatidyl-l-aspartate (PAsp), and phosphatidyl-l-glutamate (PGlu) in rat brain. Analyses using an HPLC–ESI-MS and an amino acid analyzer showed the presence of l-threonine, l-aspartate, and l-glutamate in the acid-hydrolysates of phospholipids from porcine cerebrum, rat cerebrum, and rat liver. Results of ESI-MS/MS analyses with neutral loss scanning and product ion scanning suggest the presence of PThr-(18:0, 18:1), PThr-(18:0, 22:6), PAsp-(18:0, 18:1), PAsp-(18:0, 22:6), PGlu-(18:0, 18:1), and PGlu-(18:0, 22:6) in rat brain. This is the first study to identify 2 novel phospholipids, PAsp and PGlu, with a carboxylate–phosphate anhydride bond, in living organisms.
Keywords: Aspartate; Glutamate; Threonine; Phospholipids; Mammal; Carboxylate–phosphate anhydride;

UV B-irradiation enhances the racemization and isomerizaiton of aspartyl residues and production of Nɛ-carboxymethyl lysine (CML) in keratin of skin by Yuhei Mori; Kenzo Aki; Katsunori Kuge; Shingo Tajima; Natsuko Yamanaka; Yuichi Kaji; Naoki Yamamoto; Ryoji Nagai; Hanako Yoshii; Norihiko Fujii; Masami Watanabe; Tadatoshi Kinouchi; Noriko Fujii (3303-3309).
UV-B irradiation is one of the risk factors in age-related diseases. We have reported that biologically uncommon D-β-Asp residues accumulate in proteins from sun-exposed elderly human skin. A previous study also reported that carboxymethyl lysine (CML; one of the advanced glycation end products (AGEs)) which is produced by the oxidation of glucose and peroxidation of lipid, also increases upon UV B irradiation. The formation of D-β-Asp and CML were reported as the alteration of proteins in UV B irradiated skin, independently. In this study, in order to clarify the relationship between the formation of D-β-Asp and CML, immunohistochemical analysis using anti-D-β-Asp containing peptide antibodies and anti-CML antibodies was performed in UV B irradiated mice. Immunohistochemical analyses clearly indicated that an anti-D-β-Asp containing peptide antibody and anti-CML antibody reacted at a common area in UV B irradiated skin. Western blot analyses of the proteins isolated from UV B irradiated skin demonstrated that proteins of 50–70 kDa were immunoreactive towards antibodies for both D-β-Asp containing peptide and CML. These proteins were identified by proteomic analysis as members of the keratin families including keratin-1, keratin-6B, keratin-10, and keratin-14.
Keywords: UV B irradiation; Racemization; D-Aspartic acid; Skin; Advanced glycation end products (AGEs); Keratin;

Unusual amino acid residues such as l-β-aspartyl (Asp), d-α-Asp, and d-β-Asp have been detected in proteins and peptides such as α-crystallin in the lens and β-amyloid in the brain. These residues increase with age, and hence they are associated with age-related diseases. The enzyme protein d-aspartyl (l-isoaspartyl) O-methyltransferase (PIMT) can revert these residues back to the normal l-α-Asp residue. PIMT catalyzes transmethylation of S-adenosylmethionine to l-β-Asp and d-α-Asp residues in proteins and peptides. In this work, the substrate recognition mechanism of PIMT was investigated using docking and molecular dynamics simulation studies. It was shown that the hydrogen bonds of Ser60 and Val214 to the carboxyl group of Asp are important components during substrate recognition by PIMT. In addition, specific hydrogen bonds were observed between the main chains of the substrates and those of Ala61 and Ile212 of PIMT when PIMT recognized l-β-Asp. Hydrophobic interactions between the (n  − 1) residue of the substrates and Ile212 and Val214 of PIMT may also have an important effect on substrate binding. Volume changes upon substrate binding were also evaluated in the context of possible application to interpretation of size exclusion chromatography data.
Keywords: Protein d-aspartyl (l-isoaspartyl) O-methyltransferase; Molecular dynamics; Docking; β-Aspartic acid; d-Amino acid;

A rapid and comprehensive analytical method for d- and l-enantiomers of proteinogenic amino acids was developed using ultra-high performance liquid chromatography (UHPLC) equipped with a circular dichroism (CD) detector. Pre-column derivatization reagents were examined for enhanced sensitivity and selectivity for UV and CD detection: 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was selected. The method, using a CD detector, does not require separation of optical isomers on a column to calculate the enantio ratio (%D) using the g-factor value and produces a simple chromatogram in comparison to other reported methods. Using this advantage, combined with UHPLC technology, analysis time for the derivatized proteinogenic amino acids was within 5.5 min. The UV detection limit was 4.9–23 pmol/injection and the CD detection limit was 11–64 pmol/injection. The method was applied to the analysis of d- and l-amino acids in food samples. d-Ala, d-Asp, d-Glu and d-Ser were detected at high concentrations in some Japanese black vinegars, fermented milks and yogurts. The results were identical to the results determined by the OPA method. We suggest the UHPLC–CD method would be useful in screening the d-amino acid content of foods and in helping to clarify the importance and reason for the presence of d-amino acids in foods.
Keywords: d-Amino acids; Circular dichroism; UHPLC; Enantio ratio;

Rapid determination of free d-serine with chicken d-serine dehydratase by Chihiro Suzuki; Masahito Murakami; Hirokazu Yokobori; Hiroyuki Tanaka; Tetsuo Ishida; Kihachiro Horiike; Yoko Nagata (3326-3330).
We have developed a simple, rapid, and inexpensive method of measuring the concentration of intrinsic free d-serine in tissue samples. This method uses chicken d-serine dehydratase in an enzymatic reaction to produce pyruvate, which is detected spectrophotometrically. Pyridoxal 5′-phosphate (PLP), a cofactor of d-serine dehydratase, increased pyruvate formation by 28%. The presence of Zn2+ or ethylenediaminetetraacetic acid (EDTA) did not have any effect on pyruvate formation under the present assay conditions. In addition, this method was not affected by the presence of a large excess of l-serine, nor by the presence of tissue extracts, and accurately determined concentrations of 2–30 μM (200 pmol–3 nmol) of d-serine. The entire assay requires only 60 min. With this method, we determined the concentration of d-serine in various silkworm tissues. The results were in agreement with high performance liquid chromatography measurements. We found high concentrations of d-serine in silkworm larvae at day 3 of the fifth instar; specifically, 509 nmol g−1 wet tissue in the midgut, 434 nmol g−1 in the ovary, and 353 nmol g−1 in the testis.
Keywords: d-Serine; d-Serine dehydratase; Colorimetric analysis;

Enantioselective determination of 3-hydroxybutyrate in the tissues of normal and streptozotocin-induced diabetic rats of different ages by Wei-Yu Hsu; Chen-Yi Kuo; Takeshi Fukushima; Kazuhiro Imai; Chien-Ming Chen; Pen-Yuan Lin; Jen-Ai Lee (3331-3336).
l-3-Hydroxybutyrate (3HB) and d-3HB are enantiomers that exist in various rat tissues, and the ratio of the 2 compounds is of importance since it may affect glucose utilization in cardiomyocytes. In this study, we determined the concentrations of l-3HB and d-3HB in the tissues of normal and streptozotocin (STZ)-induced diabetic rats of different ages by column-switching high-performance liquid chromatography using a fluorescence detection system. In normal rats, the levels of l-3HB peaked at 8 weeks of age in the cerebrum, liver, spleen, lung, kidney, adrenal gland, and heart and then decreased afterwards. The concentrations of l-3HB were the highest in the heart, with 26.24 ± 13.74 μmol/mg protein. In addition, there was an increase in the levels of (d  +  l)-3HB, d-3HB, and l-3HB in the tissues of diabetic rats with time, whereas the ratios of l-3HB to (d  +  l)-3HB declined (46.44% vs. 21.03%, P  < 0.05, in heart tissue after 24 weeks of STZ treatment). Both the concentration and the ratio of l-3HB may be associated with disease conditions, and the determination of l-3HB may help clarify the role of l-3HB under physiological and pathological conditions.
Keywords: d-3-Hydroxybutyrate; l-3-Hydroxybutyrate; Enantiomer; Column-switching HPLC; Diabetes;

Molecular dynamics simulations of amyloid β1–42 containing d-aspartic acid residues were performed using several continuous solvent models to investigate the usefulness of simulation methods for d-amino acid-containing proteins and peptides. Normal molecular dynamics simulations and replica exchange molecular dynamics simulations, which are one of the generalized-ensemble algorithms, were performed. Because the β-structure contents of amyloid β1–42 peptides obtained by replica exchange molecular dynamics simulations with Onufriev–Bashford–Case generalized Born implicit solvent were qualitatively consistent with experimental data, replica exchange molecular dynamics rather than other methods appeared to be more reasonable for calculations of amyloid β1–42 containing d-aspartic acid residues. Computational results revealed that peptides with stereoinversion of Asp23 tend to form β-sheet structures by themselves, in contrast to the wild-type peptides that form β-sheet structures only after aggregation. These results are expected to be useful for computational investigations of proteins and peptides such as prediction of retention time of peptides and proteins containing d-aspartic acid residues.
Keywords: Molecular dynamics; Implicit solvent; Secondary structure; Amyloid β; Aspartic acid;

Effect of d-aspartate uptake on uncoupling protein-3 and α-tubulin expressions in rat Harderian gland by Alessandra Santillo; Lavinia Burrone; Rosalba Senese; Federica Cioffi; Antonia Lanni; Gabriella Chieffi Baccari (3344-3348).
Although d-aspartate (d-Asp) has been recognized as having an important physiological role within different organs, high concentrations could elicit detrimental effects on those same organs. In this study, we evaluated the oxidative stress response to d-Asp treatment in rat Harderian gland (HG) by measuring total cellular hydroperoxide levels. Further, we examined the effect of d-Asp uptake on the expression of the mitochondrial uncoupling protein-3 (UCP3), β-actin, and α-tubulin. In rat HG, elevated levels of d-Asp significantly increased hydroperoxide production. This phenomenon was probably due to d-Asp uptake as well as lipid and porphyrin increased levels. Higher UCP3 levels and lower α-tubulin expression were also observed after d-Asp treatment. On the contrary, β-actin expression was unchanged. Given the possible role of UCP3 in lipid handling, the higher expression of mitochondria UCP3 protein in d-Asp-treated HG may reflect a major need to export excessive amounts of hydroperoxides deriving from a greater fatty acid flux across these organelles and higher mitochondrial porphyrin levels. Moreover, abundance of hydroperoxides in d-Asp treated rat HG could determine the decrease of α-tubulin expression. Thus, our findings indicate that a high concentration of d-Asp is critical in initiating a cascade of events determined by oxidative stress.
Keywords: d-Aspartate; Harderian gland; UCP3; Tubulin; Hydroperoxides; Oxidative stress;

Substrate stereoselectivity of mammalian d-aspartyl endopeptidase by Tadatoshi Kinouchi; Norihiko Fujii; Noriko Fujii (3349-3352).
The formation and accumulation of d-aspartate residue (d-Asp) in proteins caused by oxidative stress leads to dysfunction and/or denaturation of proteins, and is consequently responsible for aging-related misfolding diseases such as cataracts, prion disease, and Alzheimer's disease. We sought to identify that an unknown protease selectively degrades the noxious d-Asp-containing protein, namely d-aspartyl endopeptidase (DAEP), and finally purified it from the inner mitochondrial membrane of mouse liver. In order to analyze the substrate stereoselectivity of DAEP, we synthesized a peptide corresponding to 55–65 (Thr-Val-Leu-Asp-Ser-Gly-Ile-Ser-Glu-Val-Arg) of human αA-crystallin and its corresponding diastereoisomers in which l-α-Asp was replaced with l-β-, d-α- or d-β-Asp residue at position 58. Following incubation of that peptide with purified DAEP, it was only degraded at d-α-Asp58, independent of ATP or NAD. This result indicates that DAEP stereoselectively recognizes and degrades its substrate at the internal d-α-Asp residue. DAEP therefore seems to physiologically serve as the quality control system against the noxious d-Asp-containing protein in the long life span of mammals.
Keywords: Peptidase; Racemization; d-Aspartate; d-Amino acid; Aging; Protein-misfolding disease;