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Acrolein (ACR), methylglyoxal (MGO), and glyoxal (GO) are a class of reactive carbonyl species (RCS), which play a crucial role in the pathogenesis of chronic and age-related diseases. Here, we explored a new RCS inhibitor (theanine, THE) and investigated its capture capacity on RCS in vivo by human experiments. After proving that theanine could efficiently capture ACR instead of MGO/GO by forming adducts under simulated physiological conditions, we further detected the ACR/MGO/GO adducts of theanine in the human urine samples after consumption of theanine capsules (200 and 400 mg) or green tea (4 cups, containing 200 mg of theanine) by using ultraperformance liquid chromatography-time-of-flight-high-resolution mass spectrometry. Quantitative assays revealed that THE-ACR, THE-2ACR-1, THE-MGO, and THE-GO were formed in a dose-dependent manner in the theanine capsule groups; the maximum value of the adducts of theanine was also tested. Furthermore, besides the RCS adducts of theanine, the RCS adducts of catechins could also be detected in the drinking tea group. Whereas, metabolite profile analysis showed that theanine could better capture RCS produced in the renal metabolic pathway than catechins. Our findings indicated that theanine could reduce RCS in the body in two ways: as a pure component or contained in tea leaves.

Purpose: This study aims to compare the fecal metabolome in post pull-through HD with and without HAEC patients and healthy young children using nuclear magnetic resonance (NMR) spectroscopy., Methods: Fresh fecal samples were collected from children under 5 years of age in both post-pull-through HD patients and healthy Thai children. A total of 20 fecal samples were then analyzed using NMR spectroscopy., Results: Thirty-four metabolites identified among HD and healthy children younger than 5 years were compared. HD samples demonstrated a significant decrease in acetoin, phenylacetylglutamine, and N-acetylornithine (corrected p value = 0.01, 0.04, and 0.004, respectively). Succinate and xylose significantly decreased in HD with HAEC group compared to HD without HAEC group (corrected p value = 0.04 and 0.02, respectively). Moreover, glutamine and glutamate metabolism, and alanine, aspartate, and glutamate metabolism were the significant pathways involved, with pathway impact 0.42 and 0.50, respectively (corrected p value = 0.02 and 0.04, respectively)., Conclusion: Differences in class, quantity, and metabolism of protein and other metabolites in young children with HD after pull-through operation were identified. Most of the associated metabolic pathways were correlated with the amino acids metabolism, which is required to maintain intestinal integrity and function., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Clonorchiasis is an important foodborne parasitic disease. However, eggs of Clonorchis sinensis (C. sinensis) cannot be detected in feces during biliary obstruction. Moreover, many diseases can cause biliary obstruction, such as gallstones, adenocarcinoma, cholangiocarcinoma and Ascaris lumbricoides infection. Therefore, it is of great significance to distinguish between patients of biliary obstruction and biliary obstruction with C. sinensis infection., Methods: A total of 48 biliary obstruction patients were enrolled, including 23 infected with C. sinensis (C. sinensis) (OB+C.s) and 25 non-infected subjects (OB). The bile samples were collected by endoscopic retrograde cholangiopancreatography and analyzed using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS). Additionally, multivariate statistical analysis methods were employed to identify differential metabolites. Next, bile amino acid levels were determined by targeted metabolomics analysis., Result: A total of 146 and 132 significant metabolites were identified in electrospray ionization (ESI)+ and ESI- modes, respectively. The levels of amino acids (asparagine, glutamate, ornithine) and polyamines (spermidine and spermine) were significantly changed. Targeted analysis showed that the levels of amino acids (such as L-arginine, L-glutamine, L-lysine, L-propionic, and L-tyrosine) were lower in OB+C.s patients compared to those in OB patients. Marked metabolic pathways were involved in "Glutathione metabolism", "Caffeine metabolism", "Alanine, aspartate and glutamate metabolism", "Arginine and proline metabolism", "Purine metabolism", "Beta-Alanine metabolism", and "D-glutamine and D-glutamate metabolism"., Conclusion: These results show that there were significant differences between OB+C.s and OB patients, especially in amino acids. The metabolic signature and perturbations in metabolic pathways may help to better distinguish OB+C.s and OB patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhang, Han, Jiang, Duan, Gao, Ding, Li, Sun, Hu, Zhang and Zhang.)

In light of the numerous studies identifying post-transcriptional regulators on the surface of the endoplasmic reticulum (ER), we asked whether there are factors that regulate compartment specific mRNA translation in human cells. Using a proteomic survey of spatially regulated polysome interacting proteins, we identified the glycolytic enzyme Pyruvate Kinase M (PKM) as a cytosolic (i.e. ER-excluded) polysome interactor and investigated how it influences mRNA translation. We discovered that the PKM-polysome interaction is directly regulated by ADP levels-providing a link between carbohydrate metabolism and mRNA translation. By performing enhanced crosslinking immunoprecipitation-sequencing (eCLIP-seq), we found that PKM crosslinks to mRNA sequences that are immediately downstream of regions that encode lysine- and glutamate-enriched tracts. Using ribosome footprint protection sequencing, we found that PKM binding to ribosomes causes translational stalling near lysine and glutamate encoding sequences. Lastly, we observed that PKM recruitment to polysomes is dependent on poly-ADP ribosylation activity (PARylation)-and may depend on co-translational PARylation of lysine and glutamate residues of nascent polypeptide chains. Overall, our study uncovers a novel role for PKM in post-transcriptional gene regulation, linking cellular metabolism and mRNA translation., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

1. A study was conducted to evaluate the developmental changes of protein and free amino acid concentrations in amniotic, allantoic fluids and yolk during the incubation period of broiler eggs.2. A total of 120 Cobb 500 fertile eggs were individually weighed and then placed in an incubator. On incubation day: 8, 11, 13, 14, 16, and/or 18, amniotic, allantoic fluids and yolk were collected from 20 eggs for analysis of protein content and free α-amino acid concentration in allantoic and amniotic fluids and yolk.3. The total protein concentration in amniotic fluid increased from d 11 of incubation, and reached a peak at d 16 (69.85 g/l; P < 0.01), then declined at d 18 (P < 0.05). The total protein concentration in allantoic fluid increased with age of the embryo (P < 0.01). Crude protein concentration in yolk decreased (P < 0.05) from d 0 to 8, then increased gradually from d 8, and reached a peak at d 16 (P < 0.05). The concentration of most free amino acids in amniotic and allantoic fluids and yolk was related to embryo weight. Amniotic fluid amino acids gradually increased from d 13 to 18, with arginine being the most abundant at d 11 and 14. Glutamate was the most predominant amniotic fluid amino acid at d 16 and 18. From d 13 to 18, the concentrations of most α-amino acids in allantoic fluid increased, and reached a peak at d 18 (aspartate, 373 μmol/l; asparagine, 519 μmol/l; glutamine, 1230 μmol/l; threonine, 537 μmol/l; citrulline, 112 μmol/l; arginine, 2747 μmol/l; alanine, 276 μmol/l; tyrosine, 330 μmol/l; tryptophan, 212 μmol/l; valine, 140 μmol/l; phenylalanine, 102 μmol/l; isoleucine, 92.39 μmol/l; lysine, 1088 μmol/l; P < 0.05). Glutamine was the second most abundant amino acid in allantoic fluid at d 13 and 18. Glutamate was the most abundant α-amino acids at d 8, and 13 in the yolk.4. These results demonstrated that the concentration of free α-amino acids in chicken embryo fluid was related to embryo weight. Arginine, glutamine and glutamate were abundant free α-amino acids in chicken embryo fluid, to support the higher rates of tissue protein synthesis and growth for the embryo.

With Zn deficiency increasing in the global population, functional plant food (including tea) can help to fill the nutrition gap that the main crops cannot meet. Glycinebetaine (GB), an important bioactive substance with a wide range of natural sources, has received limited attention towards its effects on Zn biofortification and the quality of tea. The Zn enrichment and metabolite responses of albino tea [cv. White leaf No. 1 (WL-1)] to the foliar application of GB, Zn, and their combination (Zn + GB) were investigated in a field experiment. The result indicated that the 100-buds weight, total N, Zn, Thea, and total amino acid content in the young leaves of WL-1 with Zn2 + GB2 treatment were significantly increased, whereas the Chla contents were decreased ( p < 0.05). The total catechins and CAF contents of Zn2 + GB2 treatment were lower than those of other treatments, with significance ( p < 0.05). Multivariate analysis and general quantitative analysis returned complementary results, revealing that Zn2 + GB2 treatment was better for the apparent and functional quality of WL-1. The more theanine and Zn, limited chlorophyll, catechin, and caffeine contributed to the quality improvement, as well as to maintaining the leaf albinistic characteristics, inhibiting astringency and bitterness, exerting flavor and umami, and improving the ultimate beneficial functions. The combined application of Zn and GB is a promising practice for Zn biofortification and for the quality improvement of tea, with spraying 750 L ha -1 of 2.0 g L -1 Zn fertilizer and 3.2 g L -1 GB mixture recommended.

Yunnan Province has a very wide diversity of tea germplasm resources. A variety of special tea germplasms with outstanding traits have been discovered, including tea germplasms with high anthocyanin content and low caffeine content. Albino tea cultivars generally have higher contents of theanine that contribute to the umami taste, and the quality of tea brewed from it is higher. The catechin index (CI), the ratio of dihydroxylated catechins (DIC) to trihydroxylated catechins (TRIC), is a crucial index of suitability for processing tea. In this study, the albino tea plant Menghai Huangye (MHHY) with yellow leaves was identified. Analysis of the biochemical components revealed that MHHY was enriched in theanine and the total catechins (TC) were lower than Yunkang 10 (YK10). In addition, the CI value of MHHY was extremely significantly higher than that of YK10. Metabolic profile of catechins and the related gene expression profile analysis found that the coordinated expression of the key branch genes F3'H and F3'5'Ha for the synthesis of DIC and TRIC in tea plant was closely related to the high CI and low TC of MHHY. Further analysis of the F3'H promoter showed that a 284-bp deletion mutation was present in the F3'H promoter of MHHY, containing the binding sites of the transcriptional repressor MYB4 involved in flavonoid metabolism, which might be an important reason for the up-regulated expression of F3'H in MHHY. Overall, this study provides a theoretical basis for understanding the characteristics of albino tea germplasm resources and efficiently utilizing high-CI tea germplasm resources., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Magnetic Resonance Spectroscopy (MRS) allows for the non-invasive quantification of neurochemicals and has the potential to differentiate between the pathologically distinct diseases, multiple sclerosis (MS) and AQP4Ab-positive neuromyelitis optica spectrum disorder (AQP4Ab-NMOSD). In this study we characterised the metabolite profiles of brain lesions in 11 MS and 4 AQP4Ab-NMOSD patients using an optimised MRS methodology at ultra-high field strength (7T) incorporating correction for T2 water relaxation differences between lesioned and normal tissue. MS metabolite results were in keeping with the existing literature: total N-acetylaspartate (NAA) was lower in lesions compared to normal appearing brain white matter (NAWM) with reciprocal findings for myo-Inositol. An unexpected subtlety revealed by our technique was that total NAA differences were likely driven by NAA-glutamate (NAAG), a ubiquitous CNS molecule with functions quite distinct from NAA though commonly quantified together with NAA in MRS studies as total NAA. Surprisingly, AQP4Ab-NMOSD showed no significant differences for total NAA, NAA, NAAG or myo-Inositol between lesion and NAWM sites, nor were there any differences between MS and AQP4Ab-NMOSD for a priori hypotheses. Post-hoc testing revealed a significant correlation between NAWM Ins:NAA and disability (as measured by EDSS) for disease groups combined, driven by the AP4Ab-NMOSD group. Utilising an optimised MRS methodology, our study highlights some under-explored subtleties in MRS profiles, such as the absence of myo-Inositol concentration differences in AQP4Ab-NMOSD brain lesions versus NAWM and the potential influence of NAAG differences between lesions and normal appearing white matter in MS., Competing Interests: Declaration of Competing Interest GT reported no declarations of interest.; YK reported no declarations of interest.; RM reported no declarations of interest.; SM reported receiving travel grants from Biogen, Novartis, Bayer, Merck & Co, Roche, and Almirall and honorarium from Biogen for advisory work.; AW reported no declarations of interest.; AC reported no declarations of interest.; RE reported no declarations of interest.; GDL is supported by the NIHR Biomedical Research Centre (BRC), Oxford and has research funding from the Oxford BRC, MRC(UK), UK MS Society, and National Health and Medical Research (Australia). GD has received travel expenses from Bayer Schering, Biogen Idec, Genzyme, Merck Serono, Novartis, American Academy of Neurology, and MS Academy, and honoraria as an invited speaker for Novartis, American Academy of Neurology, and MS Academy.; AW reported no declarations of interest.; MC reported no declarations of interest.; IT reported no declarations of interest.; JP reported no declarations of interest.; CS reported no declarations of interest.; UE reported no declarations of interest., (Copyright © 2021. Published by Elsevier Inc.)

L-theanine involves a great number of health benefits and dietary supplements containing this molecule are becoming increasingly popular. There is, therefore, a growing need to find ways to discriminate between natural L-theanine extracted from tea leaves and the cheaper, synthetic one obtained using specific bacterial enzymes. A first attempt of stable isotope ratio analysis characterization of the possible synthetic adulterant L-theanine (δ 13 C of -14.3 ± 1.5‰), obtained from vegetable substrates with C4 photosynthetic cycle, and of the more expensive natural L-theanine (δ 13 C of -24.4 ± 1.3‰), extracted from Camellia sinensis plants with C3 photosynthetic cycle, is reported here.

l-Theanine is the most popular nonprotein amino acid contained in tea leaves. It is one of the umami components of green tea, contributing to the unique flavor of tea. Because of its various health functions, l-theanine has been commercially developed as a valuable ingredient easily used for various applications in food and pharmaceutical industries. Nowadays, l-theanine is mass-produced by plant extraction, chemical synthesis, or enzymatic transformation in factories. This review embodies the available up to date information on the l-theanine synthesis metabolism in the tea plant as well as approaches to produce it, placing emphasis on the biotransformation of l-theanine. It also gives insight into the challenges of l-theanine production on a large scale, as well as directions for future research. This review comprehensively summarizes information on l-theanine to provide an approach for an in-depth study of l-theanine production.

Based on the abundance of taste compounds in leaves at different leaf positions on the same shoot, green tea made from one bud and one leaf, or even just one bud, has the best quality. To elucidate the mechanism underlying the regulation of the biosynthesis of these compounds, we profiled the metabolome, transcriptome, sRNA, degradome, and WGCNA using leaves from five leaf positions of shoots. Through this analysis, we found 139 miRNA-target pairs related to taste compound biosynthesis and 96 miRNA-target pairs involved in phytohormone synthesis or signal transduction. Moreover, miR166- HD-ZIP , miR169- NF-YA , IAA, ZA, ABA, and JA were positively related to the accumulation of gallated catechin, caffeine, and theanine. However, miR396- GRF , miR393- bHLH , miR156- SBP , and SA were negatively correlated with these compounds. Among these important pairs, the miR396- GRF and miR156- SBP pairs were further validated by using qRT-PCR, Northern blots, and cotransformation. This is the first report describing that miRNA-TF pairs and phytohormones might synergistically regulate the biosynthesis of taste compounds in the leaves of tea plants.

In plants, long-distance transport of chemicals from source to sink takes place through the transfer of sap inside complex trafficking systems. Access to this information provides insight into the physiological responses that result from the interactions between the organism and its environment. In vivo analysis offers minimal perturbation to the physiology of the organism, thus providing information that represents the native physiological state more accurately. Here we describe capillary microsampling with electrospray ionization mass spectrometry (ESI-MS) for the in vivo analysis of xylem sap directly from plants. Initially, fast MS profiling was performed by ESI from the whole sap exuding from wounds of living plants in their native environment. This sap, however, originated from the xylem and phloem and included the cytosol of damaged cells. Combining capillary microsampling with ESI-MS enabled targeted sampling of the xylem sap and single parenchymal cells in the pith, thereby differentiating their chemical compositions. With this method we analyzed soybean plants infected by nitrogen-fixing bacteria and uninfected plants to investigate the effects of symbiosis on chemical transport through the sap. Infected plants exhibited higher abundances for certain nitrogen-containing metabolites in their sap, namely allantoin, allantoic acid, hydroxymethylglutamate, and methylene glutamate, compared to uninfected plants. Using capillary microsampling, we localized these compounds to the xylem, which indicated their transport from the roots to the upper parts of the plant. Differences between metabolite levels in sap from the infected and uninfected plants indicated that the transport of nitrogen-containing and other metabolites is regulated depending on the source of nitrogen supply.

Carbon nanoelectrodes in the sub-micron range were modified with an enzyme cascade immobilized in a spatially separated polymer double layer system for the detection of glutamate at the cellular level. The enzyme cascade consists of glutamate oxidase (GlutOx) that was immobilized in a hydrophilic redox silent polymer on top of a horseradish peroxidase (HRP)/redox polymer layer. In the presence of O2, glutamate was oxidized under concomitant reduction of O2 to H2O2 at GlutOx. H2O2 is further reduced to water by means of HRP and electrons are shuttled via the redox polymer matrix that wires the HRP to the electrode surface, hence delivering a current response proportional to the glutamate concentration. The nanometer-sized sensors could be successfully used to measure glutamate release from primary mouse astrocytes in 10 mM HEPES buffer.

Introduction: Traumatic brain injury (TBI) is the most relevant external risk factor for dementia and a major global health burden. Mild TBI (mTBI) contributes to up to 90% of all TBIs, and the classification "mild" often misrepresents the patient's burden who suffer from neuropsychiatric long-term sequelae. Magnetic resonance spectroscopy (MRS) allows in vivo detection of compromised brain metabolism although it is not routinely used after TBI., Objective: Thus, we performed a systematic review and meta-analysis to elucidate if MRS has the potential to identify changes in brain metabolism in adult patients after a single mTBI with a negative routine brain scan (CCT and/or MRI scan) compared to aged- and sex-matched healthy controls (HC) during the acute or subacute postinjury phase (≤90 days after mTBI)., Methods: A comprehensive literature search was conducted from the first edition of electronic databases until January 31, 2020. Group analyses were performed per metabolite using a random-effects model., Results: Four and 2 out of 5,417 articles met the inclusion criteria for the meta-analysis and systematic review, respectively. For the meta-analysis, 50 mTBI patients and 51 HC with a mean age of 31 and 30 years, respectively, were scanned using N-acetyl-aspartate (NAA), a marker for neuronal integrity. Glutamate (Glu), a marker for disturbed brain metabolism, choline (Cho), a marker for increased cell membrane turnover, and creatine (Cr) were used in 2 out of the 4 included articles. Regions of interests were the frontal lobe, the white matter around 1 cm above the lateral ventricles, or the whole brain. NAA was decreased in patients compared to HC with an effect size (ES) of -0.49 (95% CI -1.08 to 0.09), primarily measured in the frontal lobe. Glu was increased in the white matter in 22 mTBI patients compared to 22 HC (ES 0.79; 95% CI 0.17-1.41). Cho was decreased in 31 mTBI patients compared to 31 HC (ES -0.31; 95% CI -0.81 to 0.19). Cr was contradictory and, therefore, potentially not suitable as a reference marker after mTBI., Conclusions: MRS pinpoints changes in posttraumatic brain metabolism that correlate with cognitive dysfunction and, thus, might possibly help to detect mTBI patients at risk for unfavorable outcome or posttraumatic neurodegeneration early., (© 2020 The Author(s) Published by S. Karger AG, Basel.)

Sweet aftertaste (Huigan) is a sensation perceived after drinking tea, and lasts in the mouth and throat, leading to salivation for an extended period of time. The study aimed to reveal the underpinning mechanisms of Huigan and the its influencing factors. Tribology approach was applied to this study in conjunction with sensory analysis and other physiology assessments. Tea compounds of commercial interest were selected for the study. Preliminary sensory tests of 24 subjects were carried out to evaluate the Huigan intensity of these tea compounds. Based on these tests, 12 subjects were selected and divided into two groups, each with 6 subjects: sensitive subjects and non-sensitive subjects. In vitro tribology measurements were made for samples prepared either prepared as 1:1 mixtures of tea compound solution and human whole saliva or expectorated tea compound solutions along with the saliva which were collected from the subjects after oral processing. The Huigan intensity perceived by the sensitive group was found to be highly correlated with the friction coefficient measured, especially at the sliding speeds lower than 0.5 mm/s., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Although specialized metabolite distributions in different tea (Camellia sinensis) tissues has been studied extensively, little is known about their within-tissue distribution owing to the lack of nondestructive methodology. In this study, desorption electrospray ionization imaging mass spectrometry was used to investigate the within-tissue spatial distributions of specialized metabolites in tea. To overcome the negative effects of the large amount of wax on tea leaves, several sample preparation methods were compared, with a Teflon-imprint method established for tea leaves. Polyphenols are characteristic metabolites in tea leaves. Epicatechin gallate/catechin gallate, epigallocatechin gallate/gallocatechin gallate, and gallic acid were evenly distributed on both sides of the leaves, while epicatechin/catechin, epigallocatechin/gallocatechin, and assamicain A were distributed near the leaf vein. L-Theanine was mainly accumulated in tea roots. L-Theanine and valinol were distributed around the outer root cross-section. The results will advance our understanding of the precise localizations and in-vivo biosyntheses of specialized metabolites in tea., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Tea provides a rich taste and has healthy properties due to its variety of bioactive compounds, such as theanine, catechins, and caffeine. Theanine is the most abundant free amino acid (40%-70%) in tea leaves. Key genes related to theanine biosynthesis have been studied, but relatively little is known about the regulatory mechanisms of theanine accumulation in tea leaves. Herein, we analyzed theanine content in tea ( Camellia sinensis ) and oil tea ( Camellia oleifera ) and found it to be higher in the roots than in other tissues in both species. The theanine content was significantly higher in tea than oil tea. To explore the regulatory mechanisms of theanine accumulation, we identified genes involved in theanine biosynthesis by RNA-Seq analysis and compared theanine-related modules. Moreover, we cloned theanine synthase (TS) promoters from tea and oil tea plants and found that a difference in TS expression and cis-acting elements may explain the difference in theanine accumulation between the two species. These data provide an important resource for regulatory mechanisms of theanine accumulation in tea plants.

Shaking is a critical process in the formation of oolong tea quality, although the metabolites and their changes in this sensitive process have not yet been determined. In this study, untargeted analysis based on ultrahigh-performance liquid chromatography/quadrupole-Orbitrap mass spectrometry was conducted to comprehensively profile metabolite changes in different cultivars. Theanine glucoside was identified for the first time in oolong tea. Hierarchical cluster analysis indicated that shaking caused major changes in metabolite levels in oolong tea. Seventy-one, 83 and 73 potential features showed significant differences between pre- and post-shaking samples for var. sinensiscv. "Zimudan", "Shuixian", and "Huangmeigui," respectively. Chemometrics analysis of the three cultivars led to the identification of 18 shared metabolites, including epigallocatechin gallate, phenylalanine, tryptophan, proline, and hydroxy-jasmonic acid, as potential markers. This study identified the metabolites that allow monitoring of tea quality formation during both processing and preservation, and it provides a novel strategy for data reduction in studies to discover key metabolites., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

N -carbamylglutamate (NCG), a synthetic analogue of N -acetylglutamate, is an activator of blood ammonia conversion and endogenous arginine synthesis. Here, we established an accurate quantitative determination of NCG in feeds, animal tissues, and body fluids using the high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The sample pretreatment procedures included extraction with 0.5% of formic acid in water/methanol (80/20, v / v ), and purification using an anionic solid phase extraction cartridge. Satisfactory separation of NCG was achieved in 20 min with the application of an Atlantis T3 column, and a confirmative detection of NCG was ensured by multiple reaction monitoring of positive ions. NCG spiked in feeds, tissues, and body fluids were evaluated in regard to linearity, sensitivity, recovery, and repeatability. Recoveries for different sample matrices were in the range of 88.12% to 110.21% with relative standard deviations (RSDs) less than 8.8%. Limits of quantification were within the range of 0.012 to 0.073 mg kg -1 and 0.047 to 0.077 μg mL -1 for solid and liquid samples, respectively. This study will provide a solid foundation for the evaluation of availability and metabolic mechanism of NCG in animals.

'Anji Baicha' (Camellia sinensis) is a low-temperature-sensitive tea variety. During the development of young shoots, the leaves of 'Anji Baicha' exhibit periodic albinism. The quality of 'Anji Baicha' is closely related to the color of the fresh leaves, with whiter leaves affording a higher amino acid content and superior sensory quality after processing. However, the metabolic mechanism of its quality formation is still unclear. In this study, we analyzed the metabolomic changes of young shoots of 'Anji Baicha' and screened for metabolic markers that may be involved in the periodic albinism. Positive- and negative-mode UPLC-QTOF-MS was applied to the metabolomic analysis of young leaves of 'Anji Baicha' during three developmental stages (i.e., the pre-albescent, albescent, and regreening stages). The results revealed significant differences in the metabolic profiles of the young leaves at the three stages. The differential metabolites were mainly related to the pathways of flavonoid, phenylpropanoid, and amino acid biosynthesis. The concentrations of several amino acids (primarily l-theanine, l-glutamate, N 2 -acetyl-l-ornithine, l-aspartic acid, d-proline, l-glutamine, l-leucine, and pyroglutamic acid) and 12-OPDA were significantly higher in the albescent stage. In contrast, during the albescent stages, the concentrations of several carbohydrates (d-fructose, β-d-galactopyranose, 3-O-fucopyranosyl-2-acetamido-2-deoxyglucopyranose, galactose-β-1, 4-xylose acetyl-maltose, and 2-fucosyllactose) were significantly lower. Moreover, catechins (mainly epigallocatechin and catechin derivatives), dimeric catechins (primarily proanthocyanidins), and flavonol and flavonol/flavone glycosides (mainly kaempferol, myricetin, quercetin, cyanidin, and delphinidin glycosides) were detected at the highest levels in the regreening or pre-albescent stages. The obtained results enhance the current understanding of the metabolic mechanisms of periodic albinism and quality development formation in 'Anji Baicha'. PRACTICAL APPLICATION: The obtained results not only provide information regarding differential metabolites but also advance the understanding of the mechanism of periodic albinism in 'Anji Baicha' at the metabolite level and open up new possibilities for the genetic improvement of tea cultivars., (© 2019 Institute of Food Technologists®.)

Insect attack is known to induce a high accumulation of volatile metabolites in tea ( Camellia sinensis). However, little information is available concerning the effect of insect attack on tea quality-related nonvolatile specialized metabolites. This study aimed to investigate the formation of characteristic nonvolatile specialized metabolites in tea leaves in response to attack by major tea insects, namely, tea green leafhoppers and tea geometrids, and determine the possible involvement of phytohormones in metabolite formation resulting from insect attack. Both tea green leafhopper and tea geometrid attacks increased the jasmonic acid and salicylic acid contents. The abscisic acid content was only increased under tea green leafhopper attack, perhaps due to special continuous piercing-sucking wounding. Tea green leafhopper attack induced the formation of theaflavins from catechins under the action of polyphenol oxidase, while tea geometrid attack increased the l-theanine content. Exogenous phytohormone treatments can affect the caffeine and catechin contents. These results will help to determine the influence of major tea pest insects on important tea quality-related metabolites and enhance understanding of the relationship of phytohormones and quality-related nonvolatile metabolite formation in tea exposed to tea pest insect attacks.

Large-leaf yellow tea (LYT) imparting unique toasty flavor is a traditional beverage in China. The volatile composition of LYT after full fire processing (FF-YT) was determined by different extraction methods. Steam distillation (SD), solid-phase microextraction (SPME) and headspace (HS) were applied for analysis of tea body notes, tea infusion and dry tea aroma, respectively. A total of 143 volatile compounds was identified by the three methods, of which SD-FF-YT, SPME-FF-YT, and HS-FF-YT extracts contained 100, 72, and 56 volatiles, respectively. Heterocyclics and aromatic compounds were the main volatile composition of LYT and might be contributors to its crispy-rice-like odor. l-Theanine was demonstrated to contribute to the formation of tea volatiles. N-Ethylacetamide, formed in the l-theanine only model thermal reaction, was probably involved in the LYT volatile formation. The current results provide new insight into l-theanine on aroma formation, although the detailed formation mechanism remains largely unknown., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

l-Theanine is a specialized metabolite in tea (Camellia sinensis) leaves that contributes to tea function and quality. Yellow tea leaves (albino) generally have higher l-theanine contents than green tea leaves (normal), but the reason is unknown. The objective of this study was to investigate why l-theanine is accumulated in yellow tea leaves. We compared original normal leaves (green) and light-sensitive albino leaves (yellow) of cv. Yinghong No. 9. The l-theanine content was significantly higher in yellow leaves than in green leaves (p ≤ 0.01). After supplementation with [ 2 H 5 ]-l-theanine, yellow leaves catabolized less [ 2 H 5 ]-l-theanine than green leaves (p ≤ 0.05). Furthermore, most plants contained the enzyme catalyzing l-theanine conversion to ethylamine and l-glutamic acid. In conclusion, l-theanine accumulation in albino-induced yellow tea leaves was due to weak l-theanine catabolism. The differential accumulation mechanism differed from the l-theanine accumulation mechanism in tea and other plants., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Use of covering cultivation to shade tea (Camellia sinensis L.) trees to produce high-quality, high-priced green tea has recently increased in Japan. Knowledge of shading effects on morphological and color traits and on chemical components of new tea shoots is important for product quality and productivity. We assessed these traits of tea shoots and their relationships under covering cultivation of various radiation intensities., Results: Leaf thickness, leaf mass per area, and leaf density of new tea leaves were smaller under covering culture than under open-field culture. SPAD values and chlorophyll contents were larger under covering culture than under open culture. The derived exponential equation for estimating chlorophyll contents from SPAD values was improved by considering leaf thickness. Covering culture decreased epicatechin and epigallocatechin contents, and increased theanine and caffeine contents. Principal component analysis on shoot and leaf traits indicated that leaf mass per area, chlorophyll, epicatechin, and epigallocatechin contents were strongly associated with shading effects., Conclusion: The morphological traits, color traits, and chemical components of new tea shoots and leaves varied depending on radiation intensity, shoot growth, and cropping season. These findings are useful for covering cultivation with high quality and high productivity in tea gardens. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

l-Theanine, the most abundant amino acid in tea, is widely believed to be associated with the tea taste, however, its contribution to the formation of tea aroma is still unknown. Volatiles were determined and nitrogen-containing compounds formed during manufacturing processes were quantified. Lower levels of total sugar and l-theanine were detected in the Oolong tea product undergoing full fire processing (FFOT) suggesting that l-theanine probably involved in the volatile formation during manufacturing processes. Methylpyrazine and 2,5-dimethylpyrazine, two newly formed compounds in FFOT, together with other volatiles were successfully detected in a model thermal reaction of d-glucose and l-theanine (GT-MTR) but not detectable in thermal reactions with single d-glucose (G-MTR) or l-theanine (T-MTR). The concentration of 2,5-dimethylpyrazine increased significantly by adding additional l-theanine to 2nd roasted tea. Our study demonstrated that l-theanine, at least partly, contributed to the formation of 2,5-dimethylpyrazine, a key roasted peanutty flavor in Oolong tea., (Copyright © 2018. Published by Elsevier Ltd.)

l-theanine (l-Th), a non-protein amino acid present in tea, is a valuable nutraceutical product with unique health benefits and used as an additive in food industry. l-Th enhances the umami taste but its use is limited due to its inadequate production. Different extraction approaches from tea shoots, chemical synthesis to microbial transformation have been tried to meet its demand. In vitro, in vivo as well as clinical studies have shown its positive effect in regulating CNS disorders. l-Th has become choice ingredient in CNS active products due to its anti-stress and neuroprotective role in dementias particularly in retrogression of Alzheimer's. l-Th biochemically modulates various anti-neoplastic agents by increasing their bioavailability in tumour cells. The review, is an effort to condense the recent research on l-Th highlighting its biological resource, plausible role in tea plant, production approaches, its physiological role on human health and future prospects., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background and Aim: Oral administration of cystine and theanine (CT) may modulate antioxidant glutathione (GSH) metabolism, thereby improving outcomes after gut ischemia reperfusion., Methods: Experiment 1: Institute of Cancer Research mice (n = 35) were assigned to a Vehicle (n = 11), a CT140 (n = 14), or a CT280 (n = 10) group. The CT140 and 280 groups were given CT at respective dosages of 140 and 280 mg/kg (cystine: theanine = 5: 2) once daily via gavage for 5 days. All mice underwent 75-min occlusion of the superior mesenteric artery (SMA). Survival after reperfusion was observed. Experiment 2: Mice (n = 67) were pretreated for 5 days (Vehicle: n = 24, CT280: n = 20, vehicle/sham: n = 23). The Vehicle and CT280 groups underwent 60-min SMA occlusion. Levels of GSH, the oxidized form of GSH, Glutathione-S-S-Glutathione (GSSG), and GSH-related amino acids (cysteine and glutamic acid) in the small intestine, and plasma cytokine (IL-6, IL-1β, TNFα) levels, were evaluated before (0 h), 3, 6, or 9 h after reperfusion., Results: Experiment 1: The CT280 group showed significantly better survival than the Vehicle group. Experiment 2: Gut GSSG, cysteine, and glutamic acid levels were higher in the CT280 than in the Vehicle group after reperfusion. Plasma IL-6 and TNFα levels rose more rapidly in the CT280 than in the Vehicle group., Conclusion: Oral administration of CT improves survival after gut I/R, possibly through the modulation of the GSH-redox cycle and cytokine responses., (© 2018 S. Karger AG, Basel.)

The non-essential amino acid glutamate participates in numerous metabolic pathways in the body. It also performs important physiologic functions, which include a sensory role as one of the basic tastes (as monosodium glutamate [MSG]), and a role in neuronal function as the dominant excitatory neurotransmitter in the central nervous system. Its pleasant taste (as MSG) has led to its inclusion as a flavoring agent in foods for centuries. Glutamate's neurotransmitter role was discovered only in the last 60 years. Its inclusion in foods has necessitated its safety evaluation, which has raised concerns about its transfer into the blood ultimately increasing brain glutamate levels, thereby causing functional disruptions because it is a neurotransmitter. This concern, originally raised almost 50 years ago, has led to an extensive series of scientific studies to examine this issue, conducted primarily in rodents, non-human primates, and humans. The key findings have been that (a) the ingestion of MSG in the diet does not produce appreciable increases in glutamate concentrations in blood, except when given experimentally in amounts vastly in excess of normal intake levels; and (b) the blood-brain barrier effectively restricts the passage of glutamate from the blood into the brain, such that brain glutamate levels only rise when blood glutamate concentrations are raised experimentally via non-physiologic means. These and related discoveries explain why the ingestion of MSG in the diet does not lead to an increase in brain glutamate concentrations, and thus does not produce functional disruptions in brain. This article briefly summarizes key experimental findings that evaluate whether MSG in the diet poses a threat to brain function., (© 2018 The Author(s) Published by S. Karger AG, Basel.)

We analyzed the changes of theanine content in postharvest tea leaves under high temperature (38 °C), low temperature (4 °C), and shading spreadings by using ultrahigh-performance liquid chromatography. The differentially expressed proteins (DEPs), CsFd-GOGAT and CsNADH-GOGAT, which are involved in theanine biosynthesis pathway, were identified from the corresponding proteome data. The protein-protein interactions of CsFd-GOGAT and CsNADH-GOGAT, CsTS1, or CsNiR were verified by yeast two-hybrid technology. The expression profiles of 17 genes in theanine metabolism, including CsFd-GOGAT and CsNADH-GOGAT, were analyzed by quantitative real-time polymerase chain reaction. The correlations between the dynamic changes of theanine content and expression profiles of related genes and DEPs were analyzed. This study preliminarily proved the importance of CsGOGAT in dynamic changes of theanine content in postharvest tea leaves during spreading.

Tea is one of the most widely consumed non-alcoholic beverages in the world next to water. It is classified as Camellia sinensis and non-Camellia sinensis (herbal teas). The common bioactive compounds found mainly in green teas are flavan-3-ols (catechins) (also called flavanols), proanthocyanidins (tannins) and flavonols. Black tea contains theaflavins and thearubigins and white tea contains l-theanine and gamma-aminobutyric acid (GABA), while herbal teas contain diverse polyphenols. Phytochemicals in tea exhibit antimicrobial, anti-diabetic and anti-cancer activities that are perceived to be helpful in managing chronic diseases linked to lifestyle. Many of these phytochemicals are reported to be biologically active when combined. Knowledge of the synergistic interactions of tea with other teas or herbs in terms of biological activities will be of benefit for therapeutic enhancement. There is evidence that various types of teas act synergistically in exhibiting health benefits to humans, improving consumer acceptance and economic value. Similar observations have been made when teas and herbs or medicinal drugs were combined. The aim of this review is to highlight potential beneficial synergies between combinations of different types of teas, tea and herbs, and tea and medicinal drugs. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Tea has been a very popular beverage around the world for centuries. The reason that it is delicious, enabling hydration, showing warming and relaxing effect can be mentioned why it is consumed so much in addition to its prominent health effects. Although the catechins and caffeine are the primary bioactive components that are related with the health effects of the tea, the health effects of theanine amino acid, which is a nonproteinic amino acid special to tea, has become prominent in recent years. It has been known that the theanine amino acid in tea has positive effects especially on relaxing, cognitive performance, emotional status, sleep quality, cancer, cardiovascular diseases, obesity, and common cold. The results of acute and chronic toxicity tests conducted on the safety of theanine express that L-theanine is reliable in general even if it is consumed too much with diet. However, it has not revealed a clear evidence-based result yet regarding theanine metabolism, health effects, and its safety. Within this frame, chemical structure of theanine, its biosynthesis, dietary sources, metabolism, health effects, and safety are discussed in present study.

This is the first study to use chemometric methods to differentiate among 21 cultivars of Camellia sinensis from China and between leaves harvested at different times of the year using 30 compounds implicated in the taste and quality of tea. Unique patterns of catechin derivatives were observed among cultivars and across harvest seasons. C. sinensis var. pubilimba (You 510) differed from the cultivars of C. sinensis var. sinensis, with higher levels of theobromine, (+)-catechin, gallocatechin, gallocatechin gallate and theasinensin B, and lower levels of (-)-epicatechin, (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG), respectively. Three cultivars of C. sinensis var. sinensis, Fuyun 7, Qiancha 7 and Zijuan contained significantly more caffeoylquinic acids than others cultivars. A Linear Discriminant Analysis model based on the abundance of 12 compounds was able to discriminate amongst all 21 tea cultivars. Harvest time impacted the abundance of EGC, theanine and afzelechin gallate., (Copyright © 2016. Published by Elsevier Ltd.)

Tea contains characteristic volatile organic compounds, polyphenols, caffeine and catechins, and is therefore among the most widely consumed beverages all over the world. In this study, fresh Jukro tea leaves collected from Damyang-gun (Jeollanam-do) at 40, 60 and 90day growth stages, were semi-fermented. The volatile organic compounds (VOCs) were extracted by simultaneous distillation-solvent extraction (SDE) and analyzed by gas chromatography/mass spectrometry (GC/MS). Catechins, caffeine and theanine were analyzed by high performance liquid chromatography (HPLC). A total of 159 VOCs were identified in the analyzed Jukro tea leaves. Comparatively, the increase in the concentrations of VOCs was high in 60day leaves. The content of catechins increased along the three growth stages, whereas caffeine, compared to 90day leaves, was higher for 40 and 60day leaves. Based on the results, the 60day leaves were found to be the most suitable and useful for making semi-fermented Jukro tea., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A kind of graphene oxide/silver (GO/Ag) nanocomposites with high Surface enhanced Raman scattering (SERS) activity were fabricated via a facile and green liquid phase reduction method. The synthesized materials were characterized in detail using various microscopic and spectroscopic techniques. In this method, the GO sheets worked as a holder which makes silver nanoparticles (AgNPs) aggregate to a particular morphology, and under a suitable dosage of silver ions, well-dispersed AgNPs on the surface of GO were obtained, which could generate more "hot spots" of SERS. Moreover, SERS technique based on the obtained GO/Ag nanocomposites was used as an effective way to detect l-Theanine. The detection limit was estimated to be as low as 10(-7)M, and a multivariate linear regression model for the concentration of l-Theanine was established. The optimal fitting equation is Y=5.6765+0.0307X1458-0.0267X1251., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Taste is a crucial factor of meat quality, and amino acids are important taste-active components in meat. Here, the effects of dietary lysine (Lys) content on taste-active components in meat, especially free glutamate (Glu), were investigated. Twenty-eight-day-old broilers (Gallus gallus) were fed diets with graded Lys content of 90% or 100% of the recommended Lys requirement, (according to the National Research Council, ) for 10 days. Free amino acid content in meat and sensory scores of meat soup were estimated. Free Glu content, the main taste-active component of meat, was significantly increased by a reduction of dietary Lys. Compared with the Lys 100% group (control), free Glu concentrations of meat were increased by 35.7% in the Lys 90% group (P < 0.05). In addition, free glycine, valine, isoleucine, leucine, histidine and threonine concentrations of meat were significantly increased in the Lys 90% group (P < 0.05). Sensory evaluation of meat soup made from the Lys 100% and 90% groups indicated different meat tastes. Sensory scores of taste intensity, umami and kokumi tastes were significantly higher in the Lys 90% group. These results suggest that a reduction of dietary lysine increased free glutamate content in meat and improved its taste., (© 2016 Japanese Society of Animal Science.)

An HPLC method with postcolumn derivatization was developed and validated for the determination of theanine content in tea dietary ingredients and supplements. A variety of common commercially available supplement forms such as powders, liquid tinctures, tablets, softgels, and gelcaps, as well as three National Institute of Standards and Technology Camellia sinensis Standard Reference Materials were investigated in the study. A simple extraction procedure using citrate buffer at pH 2.2 allowed for the analysis of theanine without additional cleanup or concentration steps, even at low ppm levels. Theanine was separated from other naturally occurring amino acids using a cation-exchange column and detected using a UV-Vis detector after derivatization with ninhydrin reagent. A single-laboratory validation demonstrated that specificity, accuracy, precision, and other method performance parameters have met the requirements set for theanine analysis by the AOAC Stakeholder Panel on Dietary Supplements.

Okara, or soybean residue, is a soy food processing by-product from the manufacture of soymilk and soybean curd (tofu). In this study, solid-state fermentation of okara was conducted over 5days using yeast Yarrowia lipolytica, and the changes in proximate composition, antioxidant capacity, non-volatiles and volatiles were investigated. Yeast metabolism of okara significantly increased the amounts of lipid, succinate and free amino acids and enhanced the antioxidant capacity. In particular, there was a marked increase in important umami tastants after fermentation, with 3-fold increase in succinate and a 20-fold increase in glutamate. The final fermented okara contained 3.37g succinate and 335mg glutamate/100g dry matter. Aldehydes and their derived acids in the fresh okara were catabolised by Y. lipolytica mainly to methyl ketones, leading to a reduced grassy off-odour and a slightly pungent, musty and cheese-like odour in the fermented okara. Amino acid-derived volatiles, such as 3-methylbutanal and 2-phenylethanol, were also produced. Overall, the okara fermented by Y. lipolytica had a greater amount of umami-tasting substances, a cheese-like odour, improved digestibility and enhanced antioxidant capacity. These changes highlight the potential of Yarrowia-fermented okara as a more nutritious, savoury food product or ingredient. Y. lipolytica was thus demonstrated to be suitable for the biovalorisation of this soy food processing by-product., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Pu-erh ripened tea is produced through a unique microbial fermentation process from the sun-dried leaves of large-leaf tea species (Camellia sinensis (Linn.) var. assamica (Masters) Kitamura) in Yunnan province of China. In this study, the changes of amino acid profiles during fermentation of Pu-erh tea were investigated, based on the improved HPLC-UV method with PITC pre-column derivatization for the simultaneous determination of twenty free amino acids. Results showed that aspartic acid, glutamic acid, arginine, alanine, theanine and tyrosine were the major amino acids in tea samples. Fermentation significantly influenced on the amino acid profiles. The total free amino acid contents significantly decreased during fermentation (p<0.05). Meanwhile, low amount of acrylamide were detected. Its concentration increased after 7-days' fermentation and then decreased gradually. The results provided the useful information for the manipulation of fermentation process according to the changes of amino acids and acrylamide contents in Pu-erh ripened tea., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Umami is a basic tastes, along with sweet, salty, bitter and sour, which is imparted by glutamate, one of the free amino acids in foods. Since its discovery of umami by a Japanese scientist in 1908, umami is now perceived globally a basic taste. Recent collaboration among chefs and researchers on traditional soup stocks showed a difference in taste profiles of Japanese soup stock 'dashi' and Western style soup stock. The free amino acids profile's in dashi and soup stock showed how Japanese have traditionally adopted a simple umami taste. The exchange of knowledge on cooking methods and diverse types of umami rich foods in different countries displays the blending of the culinary arts, food science and technology for healthy and tasty solutions. Since Japanese cuisine 'WASHOKU' was listed in the 'Intangible Heritage of UNESCO' in 2013, many people in the world now have great interest in Japanese cuisine. One of the unique characteristics of this cuisine is that 'dashi' is an indispensable material for cooking a variety of Japanese dishes. Many chefs from Europe, US and South America have come to Japan to learn Japanese cuisine in the last 10 years, and umami has become recognized as a common taste worldwide. Researchers and culinary professionals have begun to pay attention to the traditional seasonings and condiments rich in glutamate available throughout the world.

We propose a new approach of an indirect enzyme-linked immunosorbent assay (ELISA) for determination of D-glutamic acid (D-Glu) using a monoclonal antibody against D-glutamic acid (D-Glu-MAb), which recognizes D-Glu-glutaraldehyde (GA) molecule but not D-Glu molecule. Human serum albumin (HSA) was coated on an immunoplate and reacted with D-Glu via GA to produce D-Glu-GA-HSA conjugates in situ in the well to be recognized by D-Glu-MAb, which enabled the development of an indirect ELISA for the determination of free D-Glu. In this indirect ELISA, D-Glu can be specifically detected with limit of detection of 7.81 μ g/mL. Since anti-conjugate antibodies are often produced, even though anti-hapten antibodies are desired, this new approach could be very useful as an application of anti-conjugate antibodies to the development of quantitative analysis for detecting hapten.

Tests on physiological functions of umami have been actively conducted and a need recognized for a high-performance quantification device that is simple and cost-effective, and whose use is not limited to a particular location or user. To address this need, Ajinomoto Co. and Tanita Corp. have jointly been researching and developing a simple device for glutamate measurement. The device uses L-glutamate oxidase immobilized on a hydrogen peroxide electrode. L-glutamate in the sample is converted to α-ketoglutaric acid, which produces hydrogen peroxide. Subsequently, the electrical current from the electrochemical reaction of hydrogen peroxide is measured to determine the L-glutamate concentration. In order to evaluate its basic performance, we used this device to measure the concentration of L-glutamate standard solutions. In a concentration range of 0-1.0%, the difference from the theoretical value was minimal. The coefficient of variation (CV) value of 3 measurements was 4% or less. This shows that the device has a reasonable level of precision and accuracy. The device was also used in trial measurements of L-glutamate concentrations in food. There was a good correlation between the results obtained using the developed device and those obtained with an amino acid analyzer; the correlation coefficient was R=0.997 (n=24). In this review, we demonstrate the use of our device to measure the glutamate concentration in miso soup served daily at a home for elderly people, and other foods and ingredients.

In this study, we developed the stable isotope dilution hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC-MS/MS) technique for the accurate, reasonable and simultaneous quantification of glutamic acid (Glu), glutamine (Gln), pyroglutamic acid (pGlu), γ-aminobutyric acid (GABA) and theanine in mouse brain tissues. The quantification of these analytes was accomplished using stable isotope internal standards and the HILIC separating mode to fully correct the intramolecular cyclization during the electrospray ionization. It was shown that linear calibrations were available with high coefficients of correlation (r(2)  > 0.999, range from 10 pmol/mL to 50 mol/mL). For application of the theanine intake, the determination of Glu, Gln, pGlu, GABA and theanine in the hippocampus and central cortex tissues was performed based on our developed method. In the region of the hippocampus, the concentration levels of Glu and pGlu were significantly reduced during reality-based theanine intake. Conversely, the concentration level of GABA increased. This result showed that transited theanine has an effect on the metabolic balance of Glu analogs in the hippocampus., (Copyright © 2015 John Wiley & Sons, Ltd.)

A method for the quantitative determination of folates in rodent diets is very important for correct interpretation of folate intake during feeding trials, given the possible discrepancy between the actual folate concentration in the diet and that mentioned on the product sheet. Liquid chromatography tandem-mass spectrometry is the method of choice to differentiate and quantify the individual folate species present. This discrepancy may be accounted for by, e.g., inaccurate folic acid supplementation and/or the presence of endogenous reduced and substituted folates. We developed a method, validated based on FDA guidelines, that allows the measurement of added and endogenous folates by quantitative determination of 5 folate monoglutamates with linear ranges from 8 μg to 2 mg/kg feed. This information, combined with feed intake data, allows insight into the actual folate intake in animal feeding studies. The relevance of this method was illustrated by the analysis of several feed samples of varying composition, by the investigation of the effect of casein incorporation, and by evaluating the variability of the folate content between pellets and production batches.

This paper describes a systematic approach to overcoming challenges in developing a robust and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for reliable and precise determination of carglumic acid in human plasma. Sample extraction was tested on several reversed-phase solid-phase extraction (SPE) sorbents with different chemistries, such as hydrophobic C18, hydrophilic-lipophilic balance, and mixed-mode cation and anion exchange. The best recovery under the optimized extraction conditions was obtained with Oasis MAX (30 mg, 1cc) mixed-mode anion exchange (~ 50%) cartridge, compared to other sorbents from 100 μL plasma sample. Complete analytical separation of carglumic acid and carglumic acid-13C5 15N as an internal standard (IS) from endogenous plasma components was achieved on ACE 5CN (150 × 4.6 mm, 5 µm) column under isocratic conditions using acetonitrile:methanol (50:50, v/v) - 0.1% acetic acid in water [80:20, v/v] as the mobile phase. The deprotonated precursor → product ion transitions for carglumic acid (189/146) and IS (195/152) were monitored in the negative ionization mode on a triple quadrupole mass spectrometer. The regression curves were linear over a concentration range of 6.00-6000 ng/mL (r(2) ≥ 0.9987). Matrix effect was evaluated in terms of IS-normalized matrix factors, which ranged from 0.95 to 1.01 across four quality control levels. Intra- and inter-batch accuracy and precision, and the stability of carglumic acid in spiked plasma samples were assessed under different conditions. The method was applied to assess the pharmacokinetics of 100 mg/kg body weight carglumic acid in a healthy Indian subject., (Copyright © 2015 John Wiley & Sons, Ltd.)

Folates are essential micronutrients for animals as they play a major role in one carbon metabolism. Animals are unable to synthesize folates and obtain them from plant derived food. In the present study, a high performance liquid chromatography coupled to mass spectrometric (HPLC-MS/MS) method was developed for the high throughput screening and quantitative analysis of folate monoglutamates in tomato fruits. For folate extraction, several parameters were optimized including extraction conditions, pH range, amount of tri-enzyme and boiling time. After processing the extract was purified using ultra-filtration with 10 kDa membrane filter. The ultra-filtered extract was chromatographed on a RP Luna C18 column using gradient elution program. The method was validated by determining linearity, sensitivity and recovery. This method was successfully applied to folate estimation in spinach, capsicum, and garden pea and demonstrated that this method offers a versatile approach for accurate and fast determination of different folate monoglutamates in vegetables., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Amitraz is a formamidine pesticide that has been reported to be a neurotoxic compound that induces convulsions among other effects. Excitatory and inhibitory neurotransmission is mediated mainly by glutamate and GABA, respectively, so their alteration could be responsible for induction of seizures. In this regard, amitraz α2 adrenergic agonist action, which has been suggested as likely responsible for this effect, could alter these neurotransmitter systems and lead to seizure induction. Moreover, other amitraz mechanisms such as histamine H1 receptor inhibition could be involved. To confirm if amitraz disrupts glutamatergic/GABAergic transmission by these mechanisms, we evaluated, in primary hippocampal neurons, the effect of amitraz (0.01 μM to 100 μM) with or without the α2 adrenergic antagonist idazoxan (1 μM) and/or the H1 receptor agonist n-methylhistaprodifen (30 μM) co-treatment on 4-aminobutyrate aminotransferase, glutamate decarboxylase 65 (GAD 65), succinate-semialdehyde dehydrogenase and glutaminase gene expression and on glutamate and GABA levels after 24h treatment. We observed that amitraz disrupts glutaminase and GAD 65 gene expression, altering glutamatergic and GABAergic transmission. These effects were mediated partially by H1 and α2 receptors suggesting that other mechanisms could be involved. These data could help explain the mechanism by which amitraz induces seizures and provide a therapeutic strategy to protect against this effect in case of poisoning., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A stable isotope dilution LC-MS/MS method is the method of choice for the selective quantitative determination of several folate species in clinical samples. By implementing an integrated approach to determine both the plasma and red blood cell (RBC) folate status, the use of consumables and time remains limited. Starting from a single 300μl whole blood sample, the folate status in plasma and RBCs can be determined after separating plasma and RBCs and sequential washing of the latter with isotonic buffer, followed by reproducible lysis using an ammonium-based buffer. Acidification combines both liberation of protein bound folates and protein precipitation. Sample cleanup is performed using a 96-well reversed-phase solid-phase extraction procedure, similar for both plasma and RBC samples. Analyses are performed by UHPLC-MS/MS. Method validation was successfully performed based on EMA-guidelines and encompassed selectivity, carry-over, linearity, accuracy, precision, recovery, matrix effect and stability. Plasma and RBC folates could be quantified in the range of 1-150nmol/l and 5-1500nmol/l, respectively. This method allows for the determination of 6 folate monoglutamates in both plasma and RBCs. It can be used to determine short and long term folate status in both normal and severely deficient subjects in a single analytical sequence., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Caffeine (1,3,7-trimethyl xanthine) and theanine (γ-glutamyl-L-ethylamide) are the major nitrogen-containing secondary metabolites in tea leaves. The aim of the present study was to elucidate the relative concentration and amounts of these compounds and the de novo biosynthetic activity in different parts of tea seedlings grown for 27-, 106- and 205 days. The results indicated that caffeine and its biosynthetic activity occur only in leaves and stems, while theanine is distributed in all organs, including roots. The concentration of caffeine and theanine in leaves ranged from 0.3-1.1 mg N/g and 0.1-0.5 mg N/g fresh weight, respectively. A higher concentration of theanine was found in roots (0.5-1.1 mg N). The total amounts of theanine expressed as g N/seedling were 1.1-1.5 times higher than that of caffeine. The high biosynthetic activity of caffeine from NH4+ was found in young leaves during the first 106 days after germination. Theanine biosynthetic activity probably occurs in roots, since higher 15N atom% excess was observed in roots during the first 27 days. Theanine may be synthesized mainly in roots and translocated to leaves. The de novo biosynthesis of caffeine and theanine in tea seedlings and their accumulation and translocation are discussed.

Dicycloplatin is a new generation supramolecular platinum-containing anti-cancer drug. Due to its structure, it is difficult to differentiate dicycloplatin from physical mixtures of carboplatin and cyclobutane dicarboxylate, and confounding results may arise during drug characterization. To solve this problem, this study aims to provide a reliable and reproducible standard for the determination of dicycloplatin. A simple method for dicycloplatin quality control has been developed using X-ray powder diffraction (XRPD) and high performance liquid chromatography (HPLC). XRPD allowed the control of impurities and dissociation of the dicycloplatin active ingredient to less than 1%, and HPLC allowed the monitoring and control of the relative molar ratio of carboplatin and cyclobutane dicarboxylate within the purity range. The study proved for the first time that the dicycloplatin supramolecule is substantially different from a physical mixture of carboplatin and cyclobutane dicarboxylate.