Characterization of polychaetes inhabiting estuaries and inner bays by composition analysis of amino acids and lactate enantiomers.

In this study, we investigated the composition of free amino acids and lactate (Lac) in polychaetes in river estuaries and inner bays using chromatographic techniques. Both L-amino acids and D-amino acids (D-asparagine, D-alanine (D-Ala), D-serine, D-aspartic acid, and D-proline (D-Pro)) were detected, indicating that polychaetes contain some D-amino acids. Some polychaete species exhibited notable amino acid levels, such as glycine in Capitellidae sp. and Thelepus sp., D-Pro in Glycera sp., and ß-Ala in Scoletoma nipponica and Scoletoma sp.. High D-Lac levels were detected in Tylorrhynchus osawai and Hediste diadroma, (691 and 797 µmol/100 g-wet, respectively), with the D-form exceeding 98%. T. osawai was dominant in the upper tidal-sensitive zone, wherein other organisms were less abundant because of low salinity (3-8 PSU). Seasonal differences in the concentrations of components in T. osawai were observed, particularly a significant increase in D-Lac in the reproductive period. Notably, the D-Lac concentrations of T. osawai were higher upstream than downstream. Thus, D-Lac might be involved in strategies underlying adaptations to low salinity and reproductive activity. These results suggest that both the D-form of Lac and amino acids may play certain physiological roles in the life of polychaetes.

Evaluation of D-amino acid oxidase activity in rat kidney using a D-kynurenine derivative, 6-methylthio-D-kynurenine: An in vivo microdialysis study.

D-Amino acid oxidase (DAO), a D-amino acid metabolizing enzyme, is reportedly associated with the psychiatric disease schizophrenia, suggesting a role for DAO inhibitors in its treatment. We have previously reported that DAO catalyzes the conversion of nonfluorescent 6-methylthio-D-kynurenine (MeS-D-KYN) to fluorescent 5-methylthiokynurenic acid (MeS-KYNA) in vitro. The present study aimed to determine the potential of MeS-D-KYN in evaluating DAO activity in vivo using renal microdialysis technique in rats. Male Sprague-Dawley rats were subjected to linear microdialysis probe implantation in the left kidney. Continuous perfusion of MeS-D-KYN was maintained, and DAO activity in the kidney cortex was evaluated by measuring the MeS-KYNA content in the microdialysate. The microdialysate was collected every 30 min and analyzed by high-performance liquid chromatography with fluorescence detection, monitored at 450 nm with an excitation wavelength of 364 nm. A significant production of MeS-KYNA was observed during, but not before, infusion of MeS-D-KYN, indicating that this compound is not endogenous. MeS-KYNA production was suppressed by the co-infusion of DAO inhibitor, 5-chlorobenzo[d]isoxazol-3-ol (CBIO), suggesting that MeS-D-KYN was converted to MeS-KYNA by renal DAO. Moreover, oral administration of CBIO effectively suppressed DAO activity in a dose-dependent manner. DAO converted MeS-D-KYN to MeS-KYNA in vivo, suggesting the potential of this compound in evaluating DAO activity. The use of the renal microdialysis technique developed in this study facilitates the monitoring of DAO activity in live experimental animals.

Analyzing Citramalic Acid Enantiomers in Apples and Commercial Fruit Juice by Liquid Chromatography-Tandem Mass Spectrometry with Pre-Column Derivatization.

Optically active citramalic acid (CMA) is naturally present as an acidic taste component in fruits, such as apples. The absolute configuration of CMA in such fruits was investigated by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) following pre-column derivatization with a chiral reagent, benzyl 5-(2-aminoethyl)-3-methyl-4-oxoimidazolidine-1-carboxylate. The developed LC-MS/MS method successfully separated the enantiomers of CMA using an octadecylsilica column with a resolution and separation factor of 2.19 and 1.09, respectively. Consequently, the R-form of CMA was detected in the peel and fruit of three kinds of apple at concentrations in the 1.24-37.8 and 0.138-1.033 mg/wet 100 g ranges, respectively. In addition, R- CMA was present in commercial apple juice, whereas no quantity was detected in commercial blueberry, perilla, or Japanese apricot juice.

Determination of d- and l-Amino Acids in Garlic Foodstuffs by Liquid Chromatography-Tandem Mass Spectrometry.

Black garlic is currently attracting interest as a health food and constituent of commercial supplements; however, no data regarding the d-amino acids within black garlic have been reported. Therefore, the amino acid compositions of methanol extracts from fresh and black garlic were compared herein. We investigated the contents of the d- and l-forms of amino acids in commercial fresh, black, and freeze-dried garlic foodstuffs by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a pre-column chiral derivatization reagent, succinimidyl 2-(3-((benzyloxy)carbonyl)-1-methyl-5-oxoimidazolidin-4-yl) acetate. Several d-amino acids, namely, the d-forms of Asn, Ala, Ser, Thr, Glu, Asp, Pro, Arg, Phe, Orn, Lys, and Tyr, were observed in the methanol extract of black garlic, whereas only d-Ala was detected in that of fresh garlic foodstuffs. These data suggest that several d-amino acids can be produced during fermentation for preparing black garlic.

Preparation of imidazolidinone compounds as derivatization reagent for diastereomerization and chromatographic separation of chiral organic acids.

A chiral derivatization reagent for application in LC-tandem mass spectrometry (LC-MS/MS)-based detection, benzyl 5-(2-aminoethyl)-3-methyl-4-oxoimidazolidine-1-carboxylate (CIM-C2-NH2), which can react with the carboxyl group, was synthesized. Both chiral and non-chiral organic acids such as lactic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, acetic acid, succinic acid, tartaric acid, malic acid, and citramalic acid were derivatized with CIM-C2-NH2; their derivatives were analyzed by LC-MS/MS. We investigated the enantioseparation of chiral organic acids on an octadecylsilica column and obtained the resolution values within 1.31-2.19 with a mobile phase of H2O-CH3CN with the exception of malic acid. In contrast, no enantioseparation of the chiral organic acids was observed when benzyl 5-(aminomethyl)-3-methyl-4-oxoimidazolidine-1-carboxylate (CIM-C1-NH2), which bears fewer methylene units than CIM-C2-NH2, was used. In addition, the mass spectra of malic acid, tartaric acid, and succinic acid, which have two carboxyl groups, showed product ions of m/z 278, while those of other organic acids showed product ions of m/z 91, corresponding to the benzyl moiety. The proposed method was applied to analyze organic acids in commercial wine, and some organic acids such as d- and l-lactic acid were successfully detected.

A review of chromatographic methods for bioactive tryptophan metabolites, kynurenine, kynurenic acid, quinolinic acid, and others, in biological fluids.

Kynurenine (KYN) is synthesized from an essential amino acid, tryptophan, by tryptophan 2,3-dioxygenase or indoleamine 2,3-dioxygenase via N-formyl-KYN in vivo. Subsequently, KYN acts as a precursor of some neuroactive metabolites such as kynurenic acid, quinolinic acid, and an important enzyme co-factor, nicotine adenine dinucleotide. These metabolites of tryptophan are a part of the 'kynurenine pathway.' In addition, KYN functions as an endogenous ligand for the aryl hydrocarbon receptor, which acts as a transcription factor. The levels of tryptophan metabolites are important for the assessment of the stage of neurological disorders, and therefore have garnered significant interest for clinical diagnosis. In this review, the detection of kynurenine, kynurenic acid, quinolinic acid, and other tryptophan metabolites performed via chromatographic methods such as HPLC using UV absorbance, fluorescence, and chromatographic-mass spectrometric detection is summarized.

Increased inosine levels in drug-free individuals with at-risk mental state: A serum metabolomics study.

AIM: At-risk mental state (ARMS) has been recently attracting attention with respect to the improvement of the management and outcome of psychiatric diseases, such as schizophrenia. Since only a few studies have reported on biological alterations in ARMS, serum metabolomics was carried out in ARMS subjects and healthy controls using liquid chromatography with high-resolution mass spectrometry. METHODS: Serum samples were collected from ARMS subjects (n = 24; male: 12; female 12) and age- and sex-matched healthy controls (n = 23 male: 11, female: 12). After serum pre-treatment, liquid chromatography with high-resolution mass spectrometry was performed. Multivariate analyses, such as orthogonal partial least-squares discriminant and volcano plot analyses, were performed. RESULTS: Serum inosine, lactate, taurine, 2,3-dihydroxypropanoate and glutamate levels differed between the two groups. A significant increase in inosine levels was detected in the positive- and negative-ion modes; however, significant differences were not observed in the levels of other purine-related metabolites (hypoxanthine, xanthine and urate) between the two groups. CONCLUSION: Increased inosine levels may serve as biological markers for ARMS, in addition to alterations in the levels of lactate and certain amino acids.

Separation of vigabatrin enantiomers using mixed-mode chromatography and its application to determine the vigabatrin enantiomer levels in rat plasma.

Mixed-mode chromatography-comprising a mixed phase with reversed and ionic phases, enabling hydrophobic and ion-exchange interactions simultaneously-was applied to identify vigabatrin enantiomers by HPLC with pre-column fluorescence derivatization with 2,5-dioxopyrrolidin-1-yl (4-(((2-nitrophenyl)sulfonyl)oxy)-6-(3-oxomorpholino)quinoline-2-carbonyl)prolinate (Ns-MOK-(S)-Pro-OSu). The MOK-(S)-Pro-vigabatrin enantiomers were efficiently separated within 12 min (total analysis time per sample: 28 min, including washing and equilibrium time for the column). The mobile phase was H2O/CH3OH/10 mM ammonium formate (pH 2.0) (20/20/60, v/v/v). Column temperature was maintained at 60℃. The proposed HPLC method could successfully monitor plasma vigabatrin enantiomer levels in rats administered (±)-vigabatrin (50 mg/kg, p.o.).

Development of a Derivatization Reagent with a 2-Nitrophenylsulfonyl Moiety for UHPLC-HRMS/MS and Its Application to Detect Amino Acids Including Taurine.

Taurine (Tau) has some important ameliorating effects on human health and is present in bivalve. For the selective analysis of Tau with other amino acids, we designed a derivatization reagent, 2,5-dioxopyrrolidin-1-yl(4-(((2-nitrophenyl)sulfonyl)oxy)-6-(3-oxomorpholino)quinoline-2-carbonyl)pyrrolidine-3-carboxylate (Ns-MOK-ß-Pro-OSu). After derivatization with Ns-MOK-ß-Pro-OSu, amino acids with Tau in Japanese littleneck clams were determined through ultra-high-performance-liquid chromatography with high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) using an octadecyl silica column. We could detect 18 amino acids within 10 min. Tau, valine, glutamine, glutamic acid, and arginine in the clams were determined in the negative ion mode using the characteristic fragment ion, C6H4N1O5S, which corresponded to the 2-nitrobenzenesulfonylate moiety. The fragment ion, C6H4N1O5S, was recognized as a common feature regardless of the amino acid to be derivatized, and it was convenient for detecting amino acid derivatives with high selectivity and sensitivity. Therefore, highly selective quantification using UHPLC-HRMS/MS was possible using Ns-MOK-ß-Pro-OSu.

Development of derivatization reagents bearing chiral 4-imidazolidinone for distinguishing primary amines from other amino acids and application to the liquid chromatography-tandem mass spectrometric analysis of miso.

We designed and synthesized three novel derivatization reagents bearing chiral 4-imidazolidinone, namely succinimidyl 2-(3-((benzyloxy)carbonyl)-1-methyl, ethyl, and -phenyl-5-oxoimidazolidin-4-yl)acetates (CIMs), for use in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The CIMs were able to discriminate primary amines from other compounds such as secondary amines and phenols, based on their unique m/z reduction of precursor ion to form product ion in MS/MS. As amino acid derivatization reagents, the CIMs were compared in terms of enantioseparation of amino acid and detection sensitivity. CIMa-OSu with 1-methyl-5-oxoimidazolidinone moiety gave the best optical resolution and detection sensitivity among the CIM reagents. Next, we applied (R)-CIMa-OSu to determine amino acids in miso by LC-triple-quadrupole MS. The proposed method achieved simultaneous determination of 20 l-amino acids and two d-amino acids (d-alanine and d-serine) in the sample with a high sensitivity (limits of detection 5-238 fmol, signal-to-noise ratio 3.3). After derivatization with CIMa-OSu, it was possible to determine whether each peak in the chromatogram was a component of primary amine or not, by using a high-resolution orbitrap MS instrument.

Fluorimetric determination of the enantiomers of vigabatrin, an antiepileptic drug, by reversed-phase HPLC with a novel diastereomer derivatization reagent.

Herein, determination of an antiepileptic drug, (±)-vigabatrin (VB), was performed by reversed-phase HPLC with fluorimetric detection using a newly designed and synthesized fluorescence derivatization reagent, 2,5-dioxopyrrolidin-1-yl (4-{[(2-nitrophenyl)sulfonyl]oxy}-6-(3-oxomorpholino)quinoline-2-carbonyl)prolinate [Ns-MOK-(R)- or (S)-Pro-OSu]. During the derivatization of VB with Ns-MOK-(R)-Pro-OSu at 60°C, the nosyl (Ns) group, which was introduced to protect a phenolic hydroxy group, was released within 30 min to produce MOK-(R)-Pro-VB, which was detected fluorimetrically at 448 nm with an excitation wavelength of 333 nm. The VB enantiomers were separated on an octadecylsilica (ODS) column with a resolution value of 5.57, because Ns-MOK-(R)-Pro-OSu bears an optically active D-proline structure. A complete separation of MOK-(R)-Pro-(R)- and -(S)-VB enantiomers was achieved on the ODS column within 40 min using stepwise gradient elution, and the detection limits were ~0.80 and 0.37 pmol on the column, respectively. The proposed HPLC with fluorimetric detection method was successfully used for determining VB enantiomers in VB-spiked human serum following solid-phase extraction with an anion-exchange cartridge.

Serum d- and l-lactate, pyruvate and glucose levels in individuals with at-risk mental state and correlations with clinical symptoms.

AIM: Little information exists on the peripheral metabolite levels in individuals with at-risk mental state who meet the criteria for a high-risk state of psychosis. Here, we aimed to investigate serum levels of glucose, pyruvate and d- and l-lactate, which may act as a signalling molecule for learning and memory in neuronal cells. METHODS: High performance liquid chromatography or commercial kits were used to assess serum metabolites in individuals with attenuated psychosis symptoms of at-risk mental state (n = 24, men = 12) who were not receiving antipsychotics. The metabolite levels of these individuals were compared with those of age- and sex-matched healthy individuals (controls, n = 23, men = 11). Correlations between the metabolites and clinical symptoms of at-risk mental state were also examined. RESULTS: Individuals with at-risk mental state had higher serum glucose levels than did controls (P = 2.18 × 10-3 ), while no significant difference in pyruvate levels were observed between the groups. Individuals with at-risk mental state had significantly lower serum l-lactate levels than did controls (P = 6.31 × 10-5 ), while no differences in d-lactate levels were observed. Furthermore, a negative correlation was identified between serum l-lactate levels and Positive and Negative Syndrome Scale negative symptoms scores (r = -0.5651, P = 4.01 × 10-3 ) in individuals with at-risk mental state. CONCLUSIONS: We found that, compared with controls, individuals with at-risk mental state have reduced serum l-lactate levels, which may predate psychosis onset, and may be involved in the related negative symptoms.