Substituted kynurenic acid derivatives as fluorophore-based probes for D- and L-amino acid oxidase assays and their in vitro application in eels.

High levels of D-amino acid oxidase (DAO) are associated with neurological and psychiatric disorders, while L-amino acid oxidase (LAO) exhibits antimicrobial and antitumor properties. The enzymatic conversion of the non-fluorescent kynurenine (KYN) into the endogenous weak fluorescent kynurenic acid (KYNA) by the action of DAO has previously been reported. However, the fluorescence of KYNA can be improved by changing the substituents on the aromatic rings. In this study, we prepared different 6-phenyl-substituted KYNA derivatives and investigated their fluorescence properties. Among them, 2-MePh-KYNA showed the maximum fluorescence quantum yield of 0.881 at 340 nm excitation and 418 nm emission wavelengths. The effects of solvent properties (dielectric constant, pKa, viscosity, and proticity) on the fluorescence intensity (FLI) of the KYNA derivatives were explored. The FLI of 2-MePh-KYNA was significantly large in protic solvents. Subsequently, 2-MePh-D-KYN and 2-MePh-L-KYN were prepared with high enantiopurity (>99.25%) for the enzymatic conversion. 2-MePh-D-KYN exhibited high sensitivity (∼19 times that of a commercial DAO substrate and ∼60 times that of the previously reported MeS-D-KYN) and high selectivity, as it was not cross-reactive towards LAO, while 2-MePh-L-KYN was also converted into 2-MePh-KYNA by LAO. Furthermore, the 2-MePh-D-KYN probe successfully detected DAO in eel liver, kidney, and heparin-anticoagulated plasma in the in vitro study.

Imatinib induces diastolic dysfunction and ventricular early-repolarization delay in the halothane-anesthetized dogs: Class effects of tyrosine kinase inhibitors.

Imatinib has been reported to induce heart failure and/or QTc prolongation. To better understand their underlying mechanisms, we assessed its effects on cardiohemodynamic, electrocardiographic and echocardiographic variables along with biomarkers of myocardial damage. Imatinib mesylate in doses of 1 and 10 mg/kg was intravenously administered to the halothane-anesthetized beagle dogs (n = 4). Effects of imatinib on each phase of isovolumetric contraction, ejection, isovolumetric relaxation and filling were studied, whereas its electrophysiological effects on early and late repolarization were analyzed by measuring J-Tpeak and Tpeak-Tend, respectively. The low and high doses of imatinib provided peak plasma concentrations of 3.23 and 17.39 µg/mL, reflecting clinically-relevant and supratherapeutic concentrations, respectively. Neither lethal ventricular tachyarrhythmia nor cardiohemodynamic collapse was observed. Imatinib decreased amplitude of peak -dP/dt, indicating suppression of isovolumetric relaxation, whereas no significant change was detected in the other phases. Imatinib prolonged QTc and J-Tpeakc without altering Tpeak-Tend, indicating increase of net inward current, which leads to intracellular Ca2+ overload. Thus, imatinib suppressed ventricular active relaxation and early repolarization, which may suggest the association of mitochondrial dysfunction-associated inhibition of ATP production. Since those findings were also reported for dasatinib, sunitinib and lapatinib, they could be common cardiac phenotype of tyrosine kinase inhibitors in vivo.

The difference of chows affects mouse physiological conditions.

Acetaminophen-induced liver injury in mice is a model system of human acetaminophen overdose and oxidative stress in vivo. The system is technically established, and we usually obtain severe liver damage in the treated mice; however, it is possible that the degree of liver damage is affected by the type of chow fed to mice. Thus, in this experiment, we investigated the effect of different chows on mice by comparing acetaminophen-induced liver damage, liver antioxidant level, and serum amino-acid concentrations. The results showed that differences in chows, even standard ones, affected mouse physiological conditions, with the response to oxidative stress greatly affected.

Increased Indoleamine 2,3-Dioxygenase Levels at the Onset of Sjögren's Syndrome in SATB1-Conditional Knockout Mice.

Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by dysfunction of salivary and lacrimal glands, resulting in xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). Autoantibodies, such as anti-SSA and anti-SSB antibodies, are hallmarks and important diagnostic factors for SS. In our previous study, we demonstrated that SS-like xerostomia was observed in SATB1 conditional knockout (SATB1cKO) mice, in which the floxed SATB1 gene was specifically deleted in hematopoietic cells as early as 4 weeks of age. In these mice, autoantibodies were not detected until 8 weeks of age in SATB1cKO mice, although exocrine gland function reached its lowest at this age. Therefore, other markers may be necessary for the diagnosis of SS in the early phase. Here, we found that mRNA expression of the interferonγ (IFN-γ) gene and the IFN-responsive indoleamine 2,3-dioxygenase (IDO) gene is upregulated in the salivary glands of SATB1cKO mice after 3 and 4 weeks of age, respectively. We detected l-kynurenine (l-KYN), an intermediate of l-tryptophan (l-Trp) metabolism mediated by IDO, in the serum of SATB1cKO mice after 4 weeks of age. In addition, the upregulation of IDO expression was significantly suppressed by the administration of IFN-γ neutralizing antibodies in SATB1cKO mice. These results suggest that the induction of IFN-dependent IDO expression is an initial event that occurs immediately after the onset of SS in SATB1cKO mice. These results also imply that serum l-KYN could be used as a marker for SS diagnosis in the early phases of the disease before autoantibodies are detectable.

LC-MS/MS Analysis of Thiol-Containing Amino Acids in Exosomal Fraction of Serum.

It has been suggested that thiol-containing amino acids could be used as biomarkers for diseases associated with oxidative stress. We investigated the thiol-containing amino acids, homocysteine (Hcy), cysteine (Cys), glutathione (GSH) and γ-glutamylcysteine (γ-GluCys), in commercial human serum by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after precolumn derivatization with 4-fluoro-7-sulfobenzofurazan. This method was applied to determine the composition of thiol-containing amino acids in exosomes prepared from the serum. Hcy, Cys, GSH and γ-GluCys could be detected in the exosomal fraction, and the ratio of each thiol-containing amino acid was similar to those in the corresponding native serum. Cys (94.76%) was most enriched in the exosomal fraction, followed by GSH (2.97%), γ-GluCys (1.59%) and Hcy (0.68%). These findings suggest that thiol-containing amino acids, Hcy, Cys, GSH and γ-GluCys, are included in exosomes in human serum.

Association of Dermatomyositis Sine Dermatitis With Anti-Nuclear Matrix Protein 2 Autoantibodies.

Importance: Reports on dermatomyositis (DM) sine dermatitis (DMSD) are scarce, and the concept of the disease has not been widely accepted. Objective: To confirm the existence of DMSD, determine its prevalence, and characterize its serologic features. Design, Setting, and Participants: This is a cohort study that reviewed clinical information, laboratory data, and muscle pathology slides from January 2009 to August 2019. We further assessed the follow-up data of 14 patients with DMSD. The median (interquartile range) follow-up period was 34 (16-64) months. Muscle biopsy samples, along with clinical information and laboratory data, were sent to a referral center for muscle diseases in Japan for diagnosis. Of patients whose myopathologic diagnosis was made at the National Center of Neurology and Psychiatry between January 2009 and August 2019, 199 patients were eligible for inclusion. These patients underwent full investigation for DM-specific autoantibodies (against transcriptional intermediary factor γ, Mi-2, melanoma differentiation-associated gene 5, nuclear matrix protein 2 [NXP-2], and small ubiquitin-like modifier activating enzyme ); however, 17 patients were excluded because their muscle fibers did not express myxovirus resistance protein A, a sensitive and specific marker of DM muscle pathology. Main Outcomes and Measures: Diagnosis of DMSD was based on the absence of a skin rash at the time of muscle biopsy. Results: Of the 182 patients, 93 were women (51%) and 46 were children (25%) (<18 years). Fourteen patients (8%) had DMSD and none were clinically diagnosed with DM. Among the 14 patients with DMSD, 12 (86%) were positive for anti-NXP-2 autoantibodies, while the remaining 2 were positive for anti-transcriptional intermediary factor γ and anti-Mi-2 autoantibodies, respectively. Only 28% of patients (47 of 168) with a skin rash were positive for anti-NXP-2 autoantibodies, indicating a significant association between anti-NXP-2 autoantibodies and DMSD (86% [12 of 14] vs 28% [47 of 168]; P < .001). This association was also supported by multivariable models adjusted for disease duration (odds ratio, 126.47; 95% CI, 11.42-1400.64; P < .001). Conclusions and Relevance: Dermatomyositis sine dermatitis does exist and accounts for 8% of patients with DM confirmed with muscle biopsy. Dermatomyositis sine dermatitis is significantly associated with anti-NXP-2 autoantibodies, which contrasts with anti-MDA5 DM, which is typically clinically amyopathic in presentation. It is essential to distinguish DMSD from other types of myositis because DM-specific therapies that are currently under development, including Janus kinase inhibitors, may be effective for DMSD.

An Adult Case of Herpes Simplex Virus-associated Granulomatous Encephalitis.

Brain granuloma occurs under certain conditions. Herpes simplex virus (HSV) causes granulomatous encephalitis in children; however, it has been rarely reported in adults. A 74-year-old man with a history of herpes simplex encephalitis suffered recurrent seizures. Brain magnetic resonance imaging revealed a mass lesion and resection was performed. A polymerase chain reaction using a brain biopsy specimen was positive for HSV DNA; thus, the patient was diagnosed with HSV-associated granulomatous encephalitis. After administering acyclovir, the patient showed improvement. HSV can cause granulomatous encephalitis in adults, and acyclovir can be used for its treatment.

Succinimidyl (3-[(benzyloxy)carbonyl]-5-oxo-1,3-oxazolidin-4-yl)acetate on a triazole-bonded phase for the separation of dl-amino-acid enantiomers and the mass-spectrometric determination of chiral amino acids in rat plasma.

Changes in the levels of amino-acid enantiomers are associated with some serious diseases; consequently, amino acid monitoring in peripheral blood can be used to diagnose and predict the onset of disease. Herein, we report the design and synthesis of a new chiral derivatization reagent, namely succinimidyl (4S)-(3-[(benzyloxy)carbonyl]-5-oxo-1,3-oxazolidin-4-yl)acetate ((S)-COXA-OSu), for the separation of dl-amino-acid enantiomers. The usefulness of (S)-COXA-OSu was examined as a derivatization reagent for LC-MS/MS following certification of its total optical purity (>99%). The enantiomeric separations of amino-acid derivatives tagged with the reagent were examined using a triazole-bonded phase. (S)-COXA-OSu enabled the simultaneous enantiomeric separation of more than 40 α-amino acids. (S)-COXA-amino-acid derivatives were efficiently converted into their product ions, from which formaldehyde (CH2O) was eliminated [M-30] from the oxazolidinone moiety of COXA by collision-induced dissociation during LC-MS/MS. Limits of detection were in the 0.0138-0.518 pmol/injection range. For precise and accurate quantitation, we synthesized and used a stable-isotope-labeled (S)-COXA-OSu that was used as an internal standard in LC-MS/MS-determination experiments. Finally, changes in plasma amino-acid levels in rats, following administration of S-methyl-l-cysteine, an alanine-serine-cysteine transporter-1 (Asc-1) inhibitor, were successfully detected by LC-MS/MS using (S)-COXA-OSu.

Amino acid analyses of the exosome-eluted fractions from human serum by HPLC with fluorescence detection.

OBJECTIVES: Amino acid levels in serum or plasma are used for early detection and diagnosis of several diseases. The objective of this study was to analyze amino acid levels in serum exosomes, which have not been previously reported. DESIGN AND METHODS: We investigated the amino acid composition of exosomes from human serum using HPLC with fluorescence detection. RESULTS: The composition ratios of His, Arg, Glu, Cys-Cys, Lys, and Tyr were significantly increased in the exosomes compared with those in the corresponding native serum. d-Ser, an endogenous co-agonist of the N-methyl-d-aspartate receptor, was also enriched in the exosome-eluted fraction. CONCLUSIONS: Our results suggest that certain amino acids are enriched in the exosome-eluted fraction from human serum. These differences could have future diagnostic potential.

Liquid chromatography-mass spectrometry with triazole-bonded stationary phase for N-methyl-D-aspartate receptor-related amino acids: development and application in microdialysis studies.

We aimed to monitor changes in the levels of amino acid neurotransmitters or neuromodulators simultaneously at the synaptic clefts of experimental animals. We developed a method for the simultaneous determination of the levels of amino acids, such as D-Ser, Gly, and L-Glu, which were involved in neurotransmission via the N-methyl-D-aspartate (NMDA) receptor, and other protein-constituted amino acids in a rat brain microdialysis (MD) sample. We used a liquid chromatography-mass spectrometry (LC-MS)/MS device equipped with a triazole-bonded column. The determination was achieved without using stable isotope-labeled compounds. We instead used suitable amino acid analogues as internal standards (ISs). We examined various analyte-IS combinations to improve reproducibility. We found a positive correlation (r = 0.720, **p < 0.0001) between relative standard deviation (%) of the area ratio and the analyte-IS retention time differences. Using the proposed method, we were able to accurately analyze trace amounts of amino acids in MD samples using ISs that were structurally similar to the analytes. Furthermore, we observed that the peripheral administration of S-methyl-L-cysteine, which was an inhibitor of the amino acid transporter Asc-1, caused some amino acid level changes in the rat brain. The proposed LC-MS/MS method can be applied in vivo to study the effects of novel therapeutic agents with monitoring the levels of amino acid neuromodulators, such as Glu, Gly, GABA, and D-Ser, in the brain. Graphical abstract LC-MS/MS analysis of amino acid enantiomers in microdialysis samples from rat striatum using triazole-bonded stationary phase.

Determination of sex-based differences in serum γ-linolenic [corrected] acid and dihomo-γ-linolenic [corrected] acid using gas chromatography-mass spectrometry.

Because serum unsaturated fatty acids can provide useful information on disease diagnosis, the simultaneous determination of several fatty acids in small volumes of human serum would be beneficial for clinical applications. In the present study, serum fatty acids were extracted with n-heptane/chloroform from 10µL of serum collected from 26 healthy Japanese subjects (11 men, ages 23-37 years; 15 women, ages 18-37 years) after deproteinization with perchloric acid, derivatization to their methyl ester using p-toluenesulfonic acid as an acid catalyst, and subsequent separation and measurement by gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode. Nine types of fatty acids (palmitoleic acid [PLA], oleic acid [OA], linoleic [corrected] acid [LA], γ-linolenic acid [GLA], α-linolenic acid [ALA], dihomo-GLA [DGLA], arachidonic acid [AA], eicosapentaenoic acid [EPA], and docosahexaenoic acid [DHA]) were analyzed in the serum within 35 min by GC-MS. The concentrations of these fatty acids in serum ranged from 3.64±0.38µM (GLA) to 413±26.3 µM (LA). Among these nine fatty acids, GLA and DGLA levels were significantly lower in women than in men (p=0.0034 and 0.0012, respectively), suggesting that there may be sex-based differences in the biosynthetic production or metabolic processes of GLA and DGLA in humans.

Anti-proliferative effect of Fe(III) complexed with 1-(2-hydroxy-3-methoxybenzaldehyde)-4-aminosalicylhydrazone in HepG2 cells.

We previously developed a chelating ligand, 1-(2-hydroxy-3-methoxybenzaldehyde)-4-aminosalicylhydrazone (HMB-ASH), which can chelate Fe(III) to form a complex. The HMB-ASH-Fe(III) complex exhibits a dose-dependent anti-proliferative effect in HepG2 cells, whereas the ligand, HMB-ASH, and Fe(III) alone had no considerable effect. The HMB-ASH-Fe(III) complex was composed of Fe(III):HMB-ASH (1:2), as determined by high-performance liquid chromatography with high-resolution mass spectrometry. The IC50 value was approximately 20 µM, which was comparable to those of the anti-cancer drugs oxaliplatin (OXP) and etoposide (ETP) under the same conditions. Similar to OXP and ETP, HMB-ASH-Fe(III) induced apoptosis in HepG2 cells, as revealed by terminal deoxynucleotidyl transferase fluorescein-12-dUTP nick end labeling assay.

Quantitative analyses of schizophrenia-associated metabolites in serum: serum D-lactate levels are negatively correlated with gamma-glutamylcysteine in medicated schizophrenia patients.

The serum levels of several metabolites are significantly altered in schizophrenia patients. In this study, we performed a targeted analysis of 34 candidate metabolites in schizophrenia patients (n = 25) and compared them with those in age- and gender-matched healthy subjects (n = 27). Orthogonal partial least square-discriminant analysis revealed that complete separation between controls and patients was achieved based on these metabolites. We found that the levels of γ-glutamylcysteine (γ-GluCys), linoleic acid, arachidonic acid, D-serine, 3-hydroxybutyrate, glutathione (GSH), 5-hydroxytryptamine, threonine, and tyrosine were significantly lower, while D-lactate, tryptophan, kynurenine, and glutamate levels were significantly higher in schizophrenia patients compared to controls. Using receiver operating characteristics (ROC) curve analysis, the sensitivity, specificity, and the area under curve of γ-GluCys, a precursor of GSH, and D-lactate, a terminal metabolite of methylglyoxal, were 88.00%, 81.48%, and 0.8874, and 88.00%, 77.78%, and 0.8415, respectively. In addition, serum levels of D-lactate were negatively correlated with γ-GluCys levels in patients, but not in controls. The present results suggest that oxidative stress-induced damage may be involved in the pathogenesis of schizophrenia.

HPLC-fluorescence determination of thiol compounds in the serum of human male and female subjects using HILIC-mode column.

High-performance liquid chromatography-fluorescence detection using a hydrophilic interaction chromatography-mode column (ZIC®-HILIC) was used to determine four kinds of thiol compounds in human serum. Sera were obtained from 34 subjects for this study (17 male subjects aged 22-38 years and 17 female subjects aged 18-38 years). Serum cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine, derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate, were separated on the ZIC®-HILIC column and quantified. The serum concentrations of cysteine, cysteinylglycine, glutathione and γ-glutamylcysteine were 226 ± 4.7, 23.4 ± 1.3, 3.7 ± 0.2 and 3.2 ± 0.1 µm, respectively. In addition, the concentrations of serum thiol compounds from male subjects were significantly higher than those of the female subjects (p < 0.05).

Diminished emotional sweating in patients with limbic encephalitis.

OBJECTIVE: Sweating on the palms of the hands and soles of the feet, so-called emotional sweating, is considered to be mediated by the limbic system, including the amygdala and anterior cingulate cortex. To reveal involvement of the limbic system in emotional sweating, we evaluated emotional sweating on the palms in patients with limbic encephalitis. METHODS: Sweat and skin vasoconstriction responses to arousal stimuli were recorded on the palms of 7 patients with limbic encephalitis caused by viral infection (n=3) or immune-mediated encephalitis (n=4). All patients had amnesia, and magnetic resonance imaging revealed mesial temporal lobe lesions, including those on the amygdala, in 6 of these patients. RESULTS: Sweat responses were absent or markedly reduced in patients with limbic encephalitis compared to normal controls following deep inspiration (p<0.05), mental arithmetic (p<0.01), exercise (p<0.05), and tactile stimulation (p<0.01). Skin vasoconstriction responses in these patients were also impaired, but the extent of such impairment was mild compared to that of the sweating reductions. CONCLUSION: Sweating on the palm was significantly impaired in patients with mesial temporal lesions. Sweating on the palm could be a useful index of limbic function.

Analysis of hydroxyl radical-induced oxidation process of glucagon by reversed-phase HPLC and ESI-MS/MS.

Structural modification of a polypeptide hormone, glucagon, by a hydroxyl radical in vitro was investigated by reversed-phase high-performance liquid chromatography (RP-HPLC), and the oxidized site of glucagon was detected by electrospray ionization tandem mass spectrometry (ESI-MS/MS). It was shown that (27)methionine (Met) was oxidized to (27)Met sulfoxide by hydroxyl radical, and the production rate of (27)Met sulfoxide was faster than that by hydrogen peroxide. In addition, production of (27)Met sulfoxide enantiomer was confirmed by RP-HPLC analysis. cAMP production in a HepG2 cell induced by (27)Met sulfoxide glucagon was reduced to approximately 75% as compared with that induced by the native form of glucagon.

Suppression of thiol exchange reaction in the determination of reduced-form thiols by high-performance liquid chromatography with fluorescence detection after derivatization with fluorogenic benzofurazan reagent, 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate and 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole.

The derivatization of the reduced-form thiols with SBD-F (7-fluoro-2,1,3-benzoxadiazole-4-sulfonate) and ABD-F (4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole) was studied. The yields of the derivatives of the reduced-form thiols (cysteine, homocysteine, reduced-form glutathione) with SBD-F at 60 degrees C for 45 min in the borate buffer (pH 9.3) were significantly decreased in the presence of the oxidized-form thiols (cystine, homocystine, oxidized-form glutathione) because of the thiol exchange reaction between the reduced-form and the oxidized-form thiols. The use of ABD-F at low temperature enabled the suppression of these thiol exchange reactions, and the recommended conditions were below 5 degrees C for 90 min in borate buffer (pH 9.3). These results suggest that ABD-F is a preferred derivatization reagent for the accurate determination of the reduced-form thiols in samples containing the oxidized-form thiols. In addition, it was also suggested that the derivatization of the reduced-form thiols should also be performed at low temperature when derivatization reagents such as o-phthalaldehyde (OPA) and monobromobimane (BrB) are used.

Steroidal glycosides from the underground parts of Solanum sodomaeum.

A new steroidal glycoside has been isolated from the underground parts of Solanum sodomaeum L., along with seven known steroidal glycosides. Their chemical structures were determined on the basis of spectroscopic data and chemical evidence, and the structure of one known pregnane type glycoside was corrected. In addition, their antiproliferative activity against human promyelocytic leukemia (HL-60) cells was investigated, and five compounds exhibited stronger activity than cisplatin.

Development of an HPLC-fluorescence determination method for carboxylic acids related to the tricarboxylic acid cycle as a metabolome tool.

We report the simultaneous determination of the carboxylic acids related to the tricarboxylic acid (TCA) cycle, which plays an important role in producing adenosine triphosphate (ATP) and generating energy in mitochondria. Seven carboxylic acids from the TCA cycle, and pyruvic acid and 2-methylsuccinic acid, as an internal standard, were derivatized with a fluorescent reagent for carboxyl groups, 4-N,N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ), in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 4-N,N-dimethyaminopyridine as the coupling reagents, at 60 degrees C for 120 min. Subsequently, the excess DBD-PZ was removed efficiently using a cation-exchange cartridge, SDB-RPS (Empore). These fluorescent derivatives were separated well from each other on an octadecyl silica column (TSKgel ODS-80Ts, 250 x 4.6 mm, i.d.) with an eluent of acetonitrile-water containing 1% formic acid at a flow rate of 0.8 mL/min, and were detected fluorometrically at 560 nm, with excitation at 450 nm. The validation data were satisfactory in the range of 2.5-100 microm citric acid, isocitric acid, 2-oxoglutaric acid, succinic acid and fumaric acid. The detection limit (S/N = 3) for citric acid was 2 fmol on the column. The structures of these derivatives were confirmed by high-performance liquid chromatography-mass spectrometry, which proved that their carboxylic groups were completely labeled with DBD-PZ, except for oxaloacetic acid. This HPLC method was successfully applied to the analysis of TCA cycle metabolites in rat urine. The method will also be useful for metabolome research, such as for target analyses of metabolites with carboxyl groups, not only in urine but also in cells and organs.