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Biothiols, such as cysteine (Cys), glutathione (GSH), and homocysteine (Hcy), play crucial roles in various physiological processes and serve as biomarkers for oxidative stress and redox homeostasis. Their structural similarities, however, pose significant challenges in selective detection and quantification, limiting the availability of suitable probes. Here, we report the design and synthesis of a novel ratiometric fluorescent sensor based on a seleno-BODIPY (Se-BODIPY) derivative, enabling rapid discrimination and quantification of Cys, Hcy, and GSH with low detection limits (Cys = 0.8 μM, Hcy = 20.4 μM, and GSH = 35.9 μM) via fluorescence. The probe exhibits high selectivity towards these biothiols over 11 amino acids, operating through dual-mode detection (absorption and emission spectra) with a visible color change from blue to orange (Cys/Hcy) or pink (GSH) in a turn-on fluorescence process. Notably, the distinct reaction mechanisms between Se-BODIPY and GSH versus Cys/Hcy lead to a more prominent blue shift for Cys/Hcy, facilitating their differentiation. Kinetic studies further differentiate Cys from Hcy, with the BODIPY reacting much faster with Cys than the latter. The effectiveness of the sensor was demonstrated in quantifying biothiols in urine samples, providing a non-invasive method with high recovery rates. Additionally, its incorporation into paper strips allows detection of biothiols in water samples via visible and UV light-induced color changes, indicating its potential for solid-state detection without organic solvents.

There is an urgent need to develop sensitive, non-invasive biomarkers that can track airway inflammatory activity for patients with cystic fibrosis (CF). Urinary glutathione sulfonamide (GSA) levels correlate well with GSA levels in BAL samples and other markers of neutrophilic inflammation, suggesting that this biomarker may be suitable for tracking disease activity in this population. We recruited 102 children (median 11.5 years-old) and 64 adults (median 32.5 years-old) who were admitted to hospital for management of an acute pulmonary exacerbation and/or eradication of infectious agents such as Pseudomonas aeruginosa or Staphylococcus aureus. Our aim was to explore how urinary GSA levels changed across admission timepoints. Urine samples were collected at admission and discharge, and GSA measured by liquid chromatography with mass spectrometry. Paired admission-discharge results were compared using Wilcoxon signed-rank test. Paired admission-discharge samples were available for 53 children and 60 adults. A statistically significant difference was observed between admission-discharge for children and adults. Spearman's correlation analysis identified a correlation between urinary GSA levels and sex and S. aureus infection for children only. Our preliminary findings suggest that urinary GSA is responsive to the resolution of an acute pulmonary exacerbation and therefore warrants further studies in this population., Competing Interests: Declaration of competing interest T.L. Blake holds a Children's Hospital Foundation ECR Fellowship for salary support and an Australian CFA Research Trust Innovation Grant for unrelated research. P.D. Sly holds NHMRC Investigator and Clinical Trials & Cohort Study grants. Authors C.E. Wainwright and S.C. Bell have received institutional payments for chairing and speaking at educational events (Vertex Pharmaceuticals). C.E. Wainwright also declares participating in steering committee and advisory board sessions for Vertex Pharmaceuticals. N. Dickerhof holds a Sir Charles Hercus Research Fellowship from the Health Research Council of New Zealand. Remaining authors I. Andersen, D.W. Reid B.R. Smith and A.J. Kettle declare that they have no conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

Humans are chronically exposed to furan, a potent liver toxicant and carcinogen that occurs in a variety of heat-processed foods. Assessment of human exposure based on the furan content in foods is, however, subject to some uncertainty due to the high volatility of furan. Biomarker monitoring is thus considered an alternative or complementary approach to furan exposure assessment. Previous work suggested that urinary furan metabolites derived from the reaction of cis-2-butene-1,4-dial (BDA), the reactive intermediate of furan, with glutathione (GSH) or amino acids may serve as potential biomarkers of furan exposure. However, some metabolites were also reported to occur in urine of untreated animals, indicating either background contamination via animal feed or endogenous sources, which may limit their suitability as biomarkers of exposure. The overall aim of the present study was to accurately establish the correlation between external dose and concentration of furan metabolites in urine over time and to discriminate against endogenous formation and furan intake via feed. To this end, the furan metabolites GSH-BDA (N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine), NAcLys-BDA (R-2-(acetylamino)-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid), NAcCys-BDA-NAcLys (N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine) and NAcCys-BDA-NAcLys sulfoxide (N-acetyl-S-[1-[5-(acetylamino)-5-carboxypentyl]-1H-pyrrol-3-yl]-L-cysteine sulfoxide) were simultaneously analyzed by stable isotope dilution ESI-LC-MS/MS as unlabeled and [ 13 C 4 ]-furan dependent metabolites following oral administration of a single oral dose of isotopically labelled [ 13 C 4 ]-furan (0.1, 1, 10, 100 and 1000 µg/kg bw) to male and female F344/DuCrl rats. Although a linear correlation between urinary excretion of [ 13 C 4 ]-furan-dependent metabolites was observed, analysis of unlabeled NAcLys-BDA, NAcCys-BDA-NAcLys and NAcCys-BDA-NAcLys sulfoxide revealed substantial, fairly constant urinary background levels throughout the course of the study. Analysis of furan in animal feed excluded feed as a source for these background levels. GSH-BDA was identified as the only furan metabolite without background occurrence, suggesting that it may present a specific biomarker to monitor external furan exposure. Studies in humans are now needed to establish if analysis of urinary GSH-BDA may provide reliable exposure estimates., (© 2024. The Author(s).)

A dual-emission ratio-fluorescent sensing nanohybrid based on Radix Hedysari green-synthesized carbon quantum dots (CDs) and glutathione-functionalized gold nanoclusters (GSH-AuNCs) had been developed for the determination of cefodizime sodium (CDZM). The designed fluorescence nanohybrid had two significant fluorescence emission peaks at 458 nm and 569 nm when excited at 360 nm, which was attributed to the CDs and GSH-AuNCs. With the addition of CDZM, the fluorescence at 458 nm was slightly weakened while the fluorescence at 569 nm was enhanced obviously. Based on the relationship between the I 569 /I 458 fluorescence intensity ratio and the concentration of CDZM, the designed nanohybrid exhibited a good linearity range of 1.0-1000.0 μM and the limit of detection (LOD) was 0.19 μM. The method was finally applied in the detection of CDZM in urine, showing the potential applications in complicated biological samples.

Aminothiols are closely related to chronic kidney disease, but little is known regarding levels of related aminothiols in the urine of immunoglobulin A vasculitis with nephritis (IgAVN) patients. Herein, a well-defined core-shell Zr-based metal-organic framework (Zr-MOF) composite SiO 2 @50Benz-Cys was constructed as a mercury ion affinity material via a solvent-assisted ligand exchange strategy for the selective extraction and enrichment of low-concentration aminothiols in IgAVN patient urine. SiO 2 @50Benz-Cys was competent to enrich the total glutathione (GSH) and total homocysteine (Hcy) in virtue of the excellent affinity after chelation with mercury ions. The extraction efficiencies were closely related to the pH, dithiothreitol amount, and the dose of functional Zr-MOF. Coupled with HPLC-MS/MS in optimized conditions, GSH and Hcy were determined with low detection limits of 0.5 and 1 nmol L -1 , respectively. The recoveries of GSH and Hcy for the urine sample at three spiked levels were in the range of 85.3-105% and 79.5-103%, which showed good precision and accuracy. Benefiting from the matrix interference elimination in the process of extraction, the simultaneous detection of aminothiols in the urine of the healthy group and immunoglobulin A vasculitis (IgAV) and IgAVN patients was successfully carried out, suggesting that the Zr-MOF and the robust method together provided a potential application in the analysis of urinary biomolecules. The analysis of variance (ANOVA) showed that the levels of GSH and Hcy had significant differences between the patients and the control. This work is very valuable as it provides a better understanding of concentration alterations of GSH and Hcy in urine involved with IgAVN for clinical research.

Tuberculosis (TB) is a serious infectious disease with high infection and mortality rates. 5%-10% of the latent tuberculosis infections (LTBI) are likely to develop into active TB, and there are currently no clinical biomarkers that can distinguish between LTBI, active TB and other non-tuberculosis populations. Therefore, it is necessary to develop rapid diagnostic methods for active TB and LTBI. In this study, urinary metabolome of 30 active TB samples and the same number of LTBI and non-TB control samples were identified and analyzed by UPLC-Q Exactive MS. In total, 3744 metabolite components were obtained in ESI- mode and 4086 in ESI + mode. Orthogonal partial least square discriminant analysis (OPLS-DA) and hierarchical cluster analysis (HCA) showed that there were significant differences among LTBI, active TB and non-TB. Six differential metabolites were screened in positive and negative mode, 3-hexenoic acid, glutathione (GSH), glycochenodeoxycholate-3-sulfate, N-[4'-hydroxy-(E)-cinnamoyl]-l-aspartic acid, deoxyribose 5-phosphate and histamine. The overlapping pathways differential metabolites involved were mainly related to immune regulation and urea cycle. The results showed that the urine metabolism of TB patients was disordered and many metabolic pathways changed. Multivariate statistical analysis revealed that GSH and histamine were selected as potential molecular markers, with area under curve of receiver operating characteristic curve over 0.75. Among the multiple differential metabolites, GSH and histamine changed to varying degrees in active TB, LTBI and the non-TB control group. The levels of GSH and histamine in 48 urinary samples were measured by ELISA in validation phase, and the result in our study provided the potential for non-invasive biomarkers of TB., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A source of functional food can be utilized from a source that might otherwise be considered waste. This study investigates the hypocholesterolemic effect of defatted dabai pulp (DDP) from supercritical carbon dioxide extraction and the metabolic alterations associated with the therapeutic effects of DDP using 1 H NMR urinary metabolomic analysis. Male-specific pathogen-free Sprague-Dawley rats were fed with a high cholesterol diet for 30 days to induce hypercholesterolemia. Later, the rats were administered with a 2% DDP treatment diet for another 30 days. Supplementation with the 2% DDP treatment diet significantly reduced the level of total cholesterol (TC), triglyceride, low-density lipoprotein (LDL), and inflammatory markers (C-reactive protein (CRP), interleukin 6 (IL6) and tumour necrosis factor-α (α-TNF)) and significantly increased the level of antioxidant profile (total antioxidant status (TAS), superoxide dismutase (SOD), glutathione peroxide (GPX), and catalase (CAT)) compared with the positive control group (PG) group ( p < 0.05). The presence of high dietary fibre (28.73 ± 1.82 g/100 g) and phenolic compounds (syringic acid, 4-hydroxybenzoic acid and gallic acid) are potential factors contributing to the beneficial effect. Assessment of 1 H NMR urinary metabolomics revealed that supplementation of 2% of DDP can partially recover the dysfunction in the metabolism induced by hypercholesterolemia via choline metabolism. 1 H-NMR-based metabolomic analysis of urine from hypercholesterolemic rats in this study uncovered the therapeutic effect of DDP to combat hypercholesterolemia.

Chromophobe renal cell carcinoma (chRCC) and renal oncocytoma are closely related, rare kidney tumors. Mutations in complex I (CI)-encoding genes play an important role in dysfunction of the oxidative phosphorylation (OXPHOS) system in renal oncocytoma, but are less frequently observed in chRCC. As such, the relevance of OXPHOS status and role of CI mutations in chRCC remain unknown. To address this issue, we performed proteome and metabolome profiling as well as mitochondrial whole-exome sequencing to detect mitochondrial alterations in chRCC tissue specimens. Multiomic analysis revealed downregulation of electron transport chain (ETC) components in chRCC that differed from the expression profile in renal oncocytoma. A decrease in mitochondrial (mt)DNA content, rather than CI mutations, was the main cause for reduced OXPHOS in chRCC. There was a negative correlation between protein and transcript levels of nuclear DNA- but not mtDNA-encoded ETC complex subunits in chRCC. In addition, the reactive oxygen species scavenger glutathione (GSH) was upregulated in chRCC due to decreased expression of proteins involved in GSH degradation. These results demonstrate that distinct mechanisms of OXPHOS exist in chRCC and renal oncocytoma and that expression levels of ETC complex subunits can serve as a diagnostic marker for this rare malignancy. SIGNIFICANCE: These findings establish potential diagnostic markers to distinguish malignant chRCC from its highly similar but benign counterpart, renal oncocytoma., (©2020 American Association for Cancer Research.)

Furan is a liver toxicant and carcinogen that occurs in heat-processed foods. Due to its volatility, analysis of furan in food does not provide reliable estimates of exposure. Biomarker-based approaches offer the opportunity to more accurately assess human exposure, but a correlation between concentrations of potential biomarkers of furan exposure and external dose has not been established. Bioactivation of furan and subsequent reaction of cis-2-butene-1,4-dial (BDA) with cellular nucleophiles gives rise to a range of metabolites that may serve as biomarkers of furan exposure. In this study, N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-L-cysteinylglycine cyclic sulfide, a mono-glutathione adduct of BDA (GSH-BDA), and R-2-acetylamino-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)-1-hexanoic acid, an adduct of BDA with N α -acetyl-L-lysine (NAcLys-BDA), were synthesized and analysed by LC-MS/MS in urine of rats treated with furan at 0, 0.1, 0.5 and 2.0 mg/kg bw for 5 and 28 days. GSH-BDA and NAcLys-BDA were both excreted in a dose-related manner. 24 h excretion rates ranged between 0.6 and 1.1% of the administered dose for GSH-BDA, and 1.4-2.1% for NAcLys-BDA. In contrast to GSH-BDA, NAcLys-BDA was also present in urine of controls, suggesting either endogenous formation or background exposure. Overall, the close correlation between urinary furan metabolites and external dose provides experimental support for biomarker-based approaches to monitor human exposure to furan., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A microfluidic paper-based analytical device integrating carbon dot (CDs) is fabricated and used for a fluorometric off-on assay of biothiols. Vinyl sulfone (VS) click immobilization of carbon dots (CDs) on paper was accomplished by a one-pot simplified protocol that uses divinyl sulfone (DVS) as a homobifunctional reagent. This reagent mediated both the click oxa-Michael addition to the hydroxyl groups of cellulose and ulterior covalent grafting of the resulting VS paper to NH 2 -functionalized CDs by means of click aza-Michael addition. The resulting cellulose nanocomposite was used to engineer an inexpensive and robust microfluidic paper-based analytical device (μPAD) that is used for a reaction-based off-on fluorometric assay of biothiols (GSH, Cys, and Hcy). The intrinsic blue fluorescence of CDs (with excitation/emission maxima at 365/450 nm) is turned off via the heavy atom effect of an introduced iodo group. Fluorescence is turned on again due to the displacement of iodine by reaction with a biothiol. The increase in fluorescence is related to the concentration over a wide range (1 to 200 μM for GSH and 5-200 μM for Cys and Hcy, respectively), and the assay exhibits a low detection limit (0.3 μM for GSH and Cys and 0.4 μM for Hcy). The method allows for rapid screening and can also be used in combination with a digital camera readout. Graphical abstract Schematic representation of a μPAD based on click immobilized carbon dots and used for a reaction-based fluorometric off-on assay of biothiols. The intrinsic blue fluorescence of carbon dots is turned off via the heavy atom effect of an introduced iodo group and turned on by the displacement of this atom by reaction with a biothiol.

Dual-emission carbon dots were synthesized by one-pot hydrothermal pyrolysis of citric acid and polyethyleneimine in the presence of rhodamine B at 160 °C for 5 h. The carbon dots have an average diameter of 2.51 nm with rhodamine moiety on their surface. Two emission bands centered at 447 and 581 nm are exhibited in their fluorescence spectra excited at 360 nm, and the former is sensitive while the latter is insensitive to Hg 2+ and pH. Glutathione (GSH) can recover the fluorescence quenched by Hg 2+ . Therefore, the dual-emission carbon dots were developed as a fluorescent ratiometric probe employing the ratio of the two intensities at 447 and 581 nm (R I447/I581 ) as the signal for the determinations of pH, Hg 2+ , and GSH. In the range of 5.0-10.0, a good linear relationship between R I447/I581 and pH was built with a regression equation of R I447/I581  = 11.95-0.56 pH (R 2  = 0.998). In the range from 0.0 to 8.0 μM, an excellent linear relationship between R I447/I581 and the concentration of Hg 2+ was obtained with a calibration equation of R I447/I581  = 6.2317-0.4458c (R 2  = 0.995) and a limit of detection (LOD) of 0.24 μM. In the range from 1.0 to 10.0 μM, a linear equation, R I447/I581  = 1.9133-0.4157c (R 2  = 0.995), was calibrated between R I447/I581 and the concentration of glutathione with a LOD of 0.27 μM. The recoveries for the determinations of Hg 2+ and GSH in real samples were in the ranges of 94.6 to 103.8% and 94.3 to 104.2%, respectively. Graphical abstract Dual-emission carbon dots achieved by luminescence center modulation within one-pot synthesis for a fluorescent ratiometric probe of pH, Hg 2+ , and glutathione.

In this work, a simple device for extraction glutathione (GSH) in biological fluids using a hybrid monolithic material within a polypropylene syringe is developed. For this purpose, glycidyl methacrylate-based monolith was firstly prepared within this housing material, and the polymer was modified with different ligands (ammonia, cysteamine and cystamine). The resulting materials (containing amine or thiol groups, respectively) were then functionalized with gold nanoparticles (AuNPs). The hybrid material that gave the largest AuNPs coverage was selected as solid-phase (SPE) sorbent and several variables affecting the extraction recovery of this compound were investigated. Under optimal conditions, GSH was quantitatively retained at pH 6.0, and then it was desorbed with aqueous dithiothreitol solution and determined, after derivatization with o-phthaldialdehyde, via reversed-phase LC with fluorometric detection. The limit of detection was ca. 1.5 ng mL -1 , and the reproducibility between extraction units was below 8% (expressed as relative standard deviation), which demonstrates the robustness of the method. The developed material was also applied for the extraction of GSH in saliva and urine samples yielding recoveries ranging from 86 to 105%., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ochratoxin A (OTA) is a nephrotoxic mycotoxin with nephrocarcinogenic potential found in a broad spectrum of food commodities. The mode of action of this compound, as well as its metabolism, is still not fully understood. To determine whether the conjugation of OTA with glutathione plays an important role in human OTA metabolism, an ochratoxin-glutathione conjugate (OTB-GSH), as well as the corresponding urinary metabolite ochratoxin-N-acetyl-L-cysteine (OTB-NAC), were synthesized and their structures confirmed by NMR spectroscopy. By means of synthesized stable isotope-labeled d 5 -OTB-GSH and d 5 -OTB-NAC references, a sensitive HPLC-MS/MS method has been developed and applied for the screening of human urine samples. OTB-NAC could be detected in 11 of the analyzed 18 urine samples and was quantified in 5 urine samples in the range between 0.023 and 0.176 ng mg -1 creatinine. OTB-GSH has not been detected in the urine samples. In OTB-NAC positive samples, this metabolite contributed to a comparable concentration range to the total OTA excretion as the parent compound. This is the first direct analysis of an OTA phase 2 metabolite in humans.

Environmental and endogenous electrophiles cause tissue damage through their high reactivity with endogenous nucleophiles such as DNA, proteins, and lipids. Protection against damage is mediated by glutathione (GSH) conjugation, which can occur spontaneously or be facilitated by the glutathione S-transferase (GST) enzymes. To determine the role of GST enzymes in protection against electrophiles as well as the role of specific GST families in mediating this protection, we exposed mutant mouse lines lacking the GSTP, GSTM, and/or GSTT enzyme families to the model electrophile acrylamide, a ubiquitous dietary contaminant known to cause adverse effects in humans. An analysis of urinary metabolites after acute acrylamide exposure identified the GSTM family as the primary mediator of GSH conjugation to acrylamide. However, surprisingly, mice lacking only this enzyme family did not show increased toxicity after an acute acrylamide exposure. Therefore, GSH conjugation is not the sole mechanism by which GSTs protect against the toxicity of this substrate. Given the prevalence of null GST polymorphisms in the human population (approximately 50% for GSTM1 and 20-50% for GSTT1), a substantial portion of the population may also have impaired acrylamide metabolism. However, our study also defines a role for GSTP and/or GSTT in protection against acrylamide mediated toxicity. Thus, while the canonical detoxification function of GSTs may be impaired in GSTM null individuals, disease risk secondary to acrylamide exposure may be mitigated through non-canonical pathways involving members of the GSTP and/or GSTT families., Competing Interests: The authors have declared that no competing interests exist.

Biothiols play important roles in regulating redox balance in biological systems, but their discrimination is challengeable. In this work, a colorimetric nanosensing array for biothiols was established, which was composed of gold nanorods (AuNRs) and metal ions (Hg 2+ , Pb 2+ , Cu 2+ , Ag + ). By employing label-free AuNRs as the colorimetric probe, and the color and spectral changes of AuNRs as the output signal, principal component analysis (PCA) was applied to processing the signal and generating a clustering map. Due to the different binding affinity between biothiols and metal ions, AuNRs exhibited a unique pattern to form a fingerprint-like colorimetric array, which was able to discriminate five biothiols by the naked eyes. This strategy combines PCA and sensor array to achieve rapid and accurate discrimination and detection of biothiols. In addition, the method shows the great potential in analysis of biothiols in human urine samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The aim of this study was to identify biomarkers of zoledronate-induced nephrotoxicity and to further characterize the mechanisms underlying this process by analyzing urinary metabolites. Twenty-four rats were randomly divided into four groups containing four (two control groups) or eight rats (two zoledronate groups) per group. The rats were injected intravenously with saline or zoledronate (3 mg/kg) singly (single, 3 weeks) or repeatedly eight times (3 weeks/time, 24 weeks). Serum blood urea nitrogen, serum creatinine, creatinine clearance, and kidney injury observed by hematoxylin and eosin and immunohistochemical staining were changed only in the repeated zoledronate group (3 mg/kg, 3 weeks/time, 24 weeks). Urinary levels of S-adenosylmethionine, S-adenosylhomocysteine, l-cystathionine, l-γ-glutamylcysteine, and glutathione related to glutathione metabolism and fumaric acid and succinic acid related to the tricarboxylic acid cycle in the zoledronate-treated group (3 mg/kg, 3 weeks/time, 24 weeks) were significantly lower than those in the control group, suggesting that zoledronate may cause cellular oxidative stress. Besides, urinary levels of uracil and uridine related to pyrimidine metabolism also decreased after zoledronate treatment (3 mg/kg, 3 weeks/time, 24 weeks), while the levels of hypoxanthine related to purine metabolism, histamine related to histamine metabolism, and several amino acids were significantly increased. Moreover, zoledronate-induced enhanced oxidative stress and histamine overproduction were confirmed by reactive oxygen species (ROS) and histamine measurement in a human proximal tubular cell line. Taken together, zoledronate-induced nephrotoxicity may be attributed to it inducing perturbations in glutathione biosynthesis and the tricarboxylic acid cycle, further causing ROS overproduction, oxidative stress, and cellular inflammation, thereby leading to nephrotoxicity.

Biomonitoring of methylene diphenyl diisocyanate (MDI) in urine may be useful in industrial hygiene and exposure surveillance approaches toward disease (occupational asthma) prevention and in understanding pathways by which the internalized chemical is excreted. We explored possible urine biomarkers of MDI exposure in mice after respiratory tract exposure to MDI, as glutathione (GSH) reaction products (MDI-GSH), and after skin exposure to MDI dissolved in acetone. LC-MS analyses of urine identified a unique m/ z 543.29 [M + H] + ion from MDI-exposed mice but not from controls. The m/ z 543.29 [M + H] + ion was detectable within 24 h of a single MDI skin exposure and following multiple respiratory tract exposures to MDI-GSH reaction products. The m/ z 543.29 [M + H] + ion possessed properties of dilysine-MDI, including (a) an isotope distribution pattern for a molecule with the chemical formula C 27 H 38 N 6 O 6 , (b) the expected collision-induced dissociation (CID) fragmentation pattern upon MS/MS, and (c) a retention time in reversed-phase LC-MS identical to that of synthetic dilysine-MDI. Further MDI-specific Western blot studies suggested albumin (which contains multiple dilysine sites susceptible to MDI carbamylation) as a possible source for dilysine-MDI and the presence of MDI-conjugated albumin in urine up to 6 days after respiratory tract exposure. Two additional [M + H] + ions ( m/ z 558.17 and 863.23) were found exclusively in urine of mice exposed to MDI-GSH via the respiratory tract and possessed characteristics of previously described cyclized MDI-GSH and oxidized glutathione (GSSG)-MDI conjugates, respectively. Together the data identify urinary biomarkers of MDI exposure in mice and possible guidance for future translational investigation.

Background & Aims: Physical inactivity is associated with lean body mass wasting, oxidative stress and pro-inflammatory changes of cell membrane lipids. Alkalinization may potentially counteract these alterations. We evaluated the effects of potassium bicarbonate supplementation on protein kinetics, glutathione status and pro- and anti-inflammatory polyunsaturated fatty acids (PUFA) in erythrocyte membranes in humans, during experimental bed rest., Methods: Healthy, young, male volunteers were investigated at the end of two 21-day bed rest periods, one with, and the other without, daily potassium bicarbonate supplementation (90 mmol × d -1 ), according to a cross-over design. Oxidative stress in erythrocytes was evaluated by determining the ratio between reduced (GSH) and oxidized glutathione (GSSG). Glutathione turnover and phenylalanine kinetics, a marker of whole body protein metabolism, were determined by stable isotope infusions. Erythrocyte membranes PUFA composition was analyzed by gas-chromatography., Results: At the end of the two study periods, urinary pH was 10 ± 3% greater in subjects receiving potassium bicarbonate supplementation (7.23 ± 0.15 vs. 6.68 ± 0.11, p < 0.001). Alkalinization increased total glutathione concentrations by 5 ± 2% (p < 0.05) and decreased its rate of clearance by 38 ± 13% (p < 0.05), without significantly changing GSH-to-GSSG ratio. After alkalinization, net protein balance in the postabsorptive state improved significantly by 17 ± 5% (p < 0.05) as well as the sum of n-3 PUFA and the n-3-to-n-6 PUFA ratio in erythrocyte membranes (p < 0.05)., Conclusions: Alkalinization during long-term inactivity is associated with improved glutathione status, anti-inflammatory lipid pattern in cell membranes and reduction in protein catabolism at whole body level. This study suggests that, in clinical conditions characterized by inactivity, oxidative stress and inflammation, alkalinization could be a useful adjuvant therapeutic strategy., (Copyright © 2018 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Introduction: Few studies have investigated the role of exposure to metals and metal mixtures on oxidative stress in the general population., Objectives: We evaluated the cross-sectional association of urinary metal and metal mixtures with urinary oxidative stress biomarkers, including oxidized to reduced glutathione ratio (GSSG/GSH), malondialdehyde (MDA), and 8‑oxo‑7,8‑dihydroguanine (8-oxo-dG), in a representative sample of a general population from Spain (Hortega Study)., Methods: Urine antimony (Sb), barium (Ba), cadmium (Cd), chromium (Cr), cobalt (Co), copper (Cu), molybdenum (Mo), vanadium (V) and zinc (Zn) were measured by ICPMS in 1440 Hortega Study participants., Results: The geometric mean ratios (GMRs) of GSSG/GSH comparing the 80th to the 20th percentiles of metal distributions were 1.15 (95% confidence intervals [95% CI]: 1.03-1.27) for Mo, 1.17 (1.05-1.31) for Ba, 1.23 (1.04-1.46) for Cr and 1.18 (1.00-1.40) for V. For MDA, the corresponding GMRs (95% CI) were 1.13 (1.03-1.24) for Zn and 1.12 (1.02-1.23) for Cd. In 8-oxo-dG models, the corresponding GMR (95% CI) were 1.12 (1.01-1.23) for Zn and 1.09 (0.99-1.20) for Cd. Cr for GSSG/GSH and Zn for MDA and 8-oxo-dG drove most of the observed associations. Principal component (PC) 1 (largely reflecting non-essential metals) was positively associated with GSSG/GSH. The association of PC2 (largely reflecting essential metals) was positive for GSSG/GSH but inverse for MDA., Conclusions: Urine Ba, Cd, Cr, Mo, V and Zn were positively associated with oxidative stress measures at metal exposure levels relevant for the general population. The potential health consequences of environmental, including nutritional, exposure to these metals warrants further investigation., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Oxidative stress markers are usually measured in plasma, a stable environment for biomarkers. Blood collection is invasive, but the use of alternative biofluids is limited, due to high variability. In this study, we aimed to establish reference values for oxidative stress markers in plasma, urine and saliva of adult, healthy mice and to identify some sources of variability. Samples were obtained from 41 female and 37 male adult, healthy mice of the CD-1 strain, aged 95-480 days, weighing 21-55 grams. Reference ranges of TBARS (thiobarbituric acid reactive substances), AOPP (advanced oxidation protein products), fructosamine, GSH/GSSG (reduced and oxidized glutathione) ratio, TAC (total antioxidant capacity), and FRAP (ferric reducing antioxidant power) were measured in plasma and urine, and TBARS, GSH/GSSG ratio, TAC and FRAP in saliva, using standard spectrophotometric and fluorometric methods. Salivary GSH/GSSG and urinary AOPP were higher in females. Urinary fructosamine, GSH/GSSG and FRAP were higher in males. Urinary TAC and FRAP negatively correlated with age, and urinary GSH/GSSG positively correlated with weight. We determined that urine and saliva can be obtained non-invasively from mice, in sufficient amounts for reliable oxidative status assessment. Further studies are needed to uncover whether these biofluids reflect systemic oxidative status in diseases.

Oxidative stress is suggested to play an important role in several pathophysiological conditions. A recent study showed that decreasing 5-oxoproline (pyroglutamate) concentration, an important mediator of oxidative stress, by over-expressing 5-oxoprolinase, improves cardiac function post-myocardial infarction in mice. The aim of the current study is to gain a better understanding of the role of the glutathione cycle in a mouse model of myocardial infarction by establishing quantitative relationships between key components of this cycle. We developed and validated an LC-MS method to quantify 5-oxoproline, L-glutamate, reduced glutathione (GSH) and oxidized GSH (GSSG) in different biological samples (heart, kidney, liver, plasma, and urine) of mice with and without myocardial infarction. 5-oxoproline concentration was elevated in all biological samples from mice with myocardial infarction. The ratio of GSH/GSSG was significantly decreased in cardiac tissue, but not in the other tissues/body fluids. This emphasizes the role of 5-oxoproline as an inducer of oxidative stress related to myocardial infarction and as a possible biomarker. An increase in the level of 5-oxoproline is associated with a decrease in the GSH/GSSG ratio, a well-established marker for oxidative stress, in cardiac tissue post-myocardial infarction. This suggests that 5-oxoproline may serve as an easily measurable marker for oxidative stress resulting from cardiac injury. Our findings show further that liver and kidneys have more capacity to cope with oxidative stress conditions in comparison to the heart, since the GSH/GSSG ratio is not affected in these organs despite a significant increase in 5-oxoproline., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We report a new approach to create metal-binding site in a series of metal-organic frameworks (MOFs), where tetratopic carboxylate linker, 4',4'',4''',4''''-methanetetrayltetrabiphenyl-4-carboxylic acid, is partially replaced by a tritopic carboxylate linker, tris(4-carboxybiphenyl)amine, in combination with monotopic linkers, formic acid, trifluoroacetic acid, benzoic acid, isonicotinic acid, 4-chlorobenzoic acid, and 4-nitrobenzoic acid, respectively. The distance between these paired-up linkers can be precisely controlled, ranging from 5.4 to 10.8 Å, where a variety of metals, Mg 2+ , Al 3+ , Cr 3+ , Mn 2+ , Fe 3+ , Co 2+ , Ni 2+ , Cu 2+ , Zn 2+ , Ag + , Cd 2+ and Pb 2+ , can be placed in. The distribution of these metal-binding sites across a single crystal is visualized by 3D tomography of laser scanning confocal microscopy with a resolution of 10 nm. The binding affinity between the metal and its binding-site in MOF can be varied in a large range (observed binding constants, K obs from 1.56×10 2 to 1.70×10 4  L mol -1 ), in aqueous solution. The fluorescence of these crystals can be used to detect biomarkers, such as cysteine, homocysteine and glutathione, with ultrahigh sensitivity and without the interference of urine, through the dissociation of metal ions from their binding sites., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Several diseases are associated with perturbations in redox signaling and aberrant hydrogen sulfide metabolism, and numerous analytical methods exist for the measurement of the sulfur-containing species affected. However, uncertainty remains about their concentrations and speciation in cells/biofluids, perhaps in part due to differences in sample processing and detection principles. Using ultrahigh-performance liquid chromatography in combination with electrospray-ionization tandem mass spectrometry we here outline a specific and sensitive platform for the simultaneous measurement of 12 analytes, including total and free thiols, their disulfides and sulfide in complex biological matrices such as blood, saliva and urine. Total assay run time is < 10 min, enabling high-throughput analysis. Enhanced sensitivity and avoidance of artifactual thiol oxidation is achieved by taking advantage of the rapid reaction of sulfhydryl groups with N-ethylmaleimide. We optimized the analytical procedure for detection and separation conditions, linearity and precision including three stable isotope labelled standards. Its versatility for future more comprehensive coverage of the thiol redox metabolome was demonstrated by implementing additional analytes such as methanethiol, N-acetylcysteine, and coenzyme A. Apparent plasma sulfide concentrations were found to vary substantially with sample pretreatment and nature of the alkylating agent. In addition to protein binding in the form of mixed disulfides (S-thiolation) a significant fraction of aminothiols and sulfide appears to be also non-covalently associated with proteins. Methodological accuracy was tested by comparing the plasma redox status of 10 healthy human volunteers to a well-established protocol optimized for reduced/oxidized glutathione. In a proof-of-principle study a deeper analysis of the thiol redox metabolome including free reduced/oxidized as well as bound thiols and sulfide was performed. Additional determination of acid-labile sulfide/thiols was demonstrated in human blood cells, urine and saliva. Using this simplified mass spectrometry-based workflow the thiol redox metabolome can be determined in samples from clinical and translational studies, providing a novel prognostic/diagnostic platform for patient stratification, drug monitoring, and identification of new therapeutic approaches in redox diseases., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

This report outlines a highly sensitive and facile electrochemiluminescence (ECL) sensing platform based on a novel high-quantum-yield Au-nanocluster (AuNC) probe for glutathione (GSH) detection. Owing to the prominent quenching effect of GSH on the ECL of the AuNCs, the proposed ECL nanosensor showed a wide response to GSH in the ranges of 1.0 × 10 -9 -1.0 × 10 -5 M and 1.0 × 10 -5 -1.0 × 10 -1 M and a low detection limit of 3.2 × 10 -10 M. In addition, the proposed system exhibited good selectivity for GSH in the presence of other chemical/biological interferences. Moreover, since no further functionalization of AuNC-based sensor interface was necessary, together with the stability, high sensitivity and selectivity of the proposed nanosensor, this convenient approach was able to successfully detect GSH in both of human urine samples and blood samples with excellent recoveries, which indicated its promising application under physiological conditions. Of significant importance is that this study not only helps in gaining a better understanding of the applicability of the ECL properties of AuNCs, but also provides a new avenue for the design and development of ECL sensors based on the novel high-quantum-yield AuNC-based probe and other functional-metal-based NC probes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Application of metabolic phenotyping could expand the pathophysiological knowledge of mucopolysaccharidoses (MPS) and may reveal the comprehensive metabolic impairments in MPS. However, few studies applied this approach to MPS., Methods: We applied targeted and untargeted metabolic profiling in urine samples obtained from a French cohort comprising 19 MPS I and 15 MPS I treated patients along with 66 controls. For that purpose, we used ultra-high-performance liquid chromatography combined with ion mobility and high-resolution mass spectrometry following a protocol designed for large-scale metabolomics studies regarding robustness and reproducibility. Furthermore, 24 amino acids have been quantified using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Keratan sulfate, Heparan sulfate and Dermatan sulfate concentrations have also been measured using an LC-MS/MS method. Univariate and multivariate data analyses have been used to select discriminant metabolites. The mummichog algorithm has been used for pathway analysis., Results: The studied groups yielded distinct biochemical phenotypes using multivariate data analysis. Univariate statistics also revealed metabolites that differentiated the groups. Specifically, metabolites related to the amino acid metabolism. Pathway analysis revealed that several major amino acid pathways were dysregulated in MPS. Comparison of targeted and untargeted metabolomics data with in silico results yielded arginine, proline and glutathione metabolisms being the most affected., Conclusion: This study is one of the first metabolic phenotyping studies of MPS I. The findings might help to generate new hypotheses about MPS pathophysiology and to develop further targeted studies of a smaller number of potentially key metabolites., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Few studies to date have investigated the antioxidant nutrients such as vitamin C (ascorbic acid), vitamin E (α-tocopherol), retinol and carotenoids in plasma from patients with pulmonary disease in Japan. To clarify the role of antioxidant nutrients such as vitamin C, vitamin E, retinol and various carotenoids in plasma of Japanese patients with chronic obstructive lung diseases (COPD), asthma-COPD overlap syndrome (ACOS) and/or bronchial asthma (BA), we compared to healthy elderly controls., Methods: Ascorbic acid (AA), carotenoids (lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene and lycopene), retinol and α-tocopherol levels in plasma were determined by using a high performance liquid chromatography. Reduced glutathione (GSH), oxidised glutathione (GSSG) in whole blood and urinary 8-OHdG were also determined., Results: Plasma AA level of COPD subjects was significantly lower than that of healthy elderly people. Conversely, ACOS and BA subjects showed no significant difference from healthy elderly people. Moreover, plasma lycopene and total carotenoid levels and GSH content in blood were significantly lower in COPD subjects than these in healthy elderly people. However, other redox markers such as GSSG, GSH/GSSG ratio and urinary 8-OHdG found no significant differences between COPD, ACOS and BA compared to healthy elderly people., Conclusions: These results suggested that COPD of Japanese patients may develop partly because of oxidative stress derived from a shortage of antioxidant nutrients, especially of AA and lycopene, as well as GSH while this may not be the case in both ACOS and BA., (© 2016 John Wiley & Sons Ltd.)

The simultaneous electrochemical quantification of oxidized (GSSG) and reduced glutathione (GSH), biomarkers of oxidative stress, is demonstrated in biological fluids. The detection was accomplished by the development of a modified carbon electrode and was applied to the analysis of biological fluids of model organisms under oxidative stress caused by lead intoxication. Nanocomposite molecular material based on cobalt phthalocyanine (CoPc) and multiwalled carbon nanotubes functionalized with carboxyl groups (MWCNT f ) was developed to modify glassy carbon electrodes (GCE) for the detection of reduced and oxidized glutathione. The morphology of the nanocomposite film was characterized by scanning electron microscopy (SEM) and profilometry. The electrochemical behavior of the modified electrode was assessed by cyclic voltammetry (CV) to determine the surface coverage (Γ) by CoPc. The electrocatalytic behavior of the modified electrode toward reduced (GSH) and oxidized (GSSG) forms of glutathione was assessed by CV studies at physiological pH. The obtained results show that the combined use of CoPc and MWCNT f results in an electrocatalytic activity for GSH oxidation and GSSG reduction, enabling the simultaneous detection of both species. Differential pulse voltammetry reveals detection limits of 100 μM for GSH and 8.3 μM for GSSG, respectively. The potential interference from ascorbic acid, cysteine, glutamic acid, and glucose was also studied, and the obtained results show limited effects from these species. Finally, the hybrid electrode was used for the determination of GSH and GSSG in rat urine and plasma samples, intoxicated or not by lead. Both glutathione forms were detected in these complex biological matrixes without any pretreatment. Our results portray the role of GSH and GSSG as markers of oxidative stress in live organisms under lead intoxication.

The concentration of L-cysteine (Cys) and glutathione (GSH) is closely related to the critical risk of various diseases. In our study, a new rapid method for the determination of Cys and GSH in water and urine samples has been developed using a fluorescent probe technique, which was based on crystal violet (CV)-functionalized CdTe quantum dots (QDs). The original QDs emitted fluorescence light, which was turned off upon adding CV. This conjugation of CV and QDs could be attributed to electrostatic interaction between COO - of mercaptopropionic acid (MPA) on the surface of QDs and N + of CV in aqueous solution. In addition, Förster resonance energy transfer (FRET) also occurred between CdTe QDs and CV. After adding Cys or GSH to the solution, Cys or GSH exhibited a stronger binding preference toward Cd 2+ than Cd 2+ -MPA, which disturbed the interaction between MPA and QDs. Thus, most MPA was able to be separated from the surface of QDs because of the participation of Cys or GSH. Then, the fluorescence intensity of the CdTe QDs was enhanced. Good linear relationships were obtained in the range of 0.02-40 μg mL -1 and 0.02-50 μg mL -1 , and the detection limits were calculated as 10.5 ng mL -1 and 8.2 ng mL -1 , for Cys and GSH, respectively. In addition, the concentrations of biological thiols in water and urine samples were determined by the standard addition method using Cys as the standard; the quantitative recoveries were in the range of 97.3-105.8%, and relative standard deviations (RSDs) ranged from 2.5 to 3.7%. The method had several unique properties, such as simplicity, lower cost, high sensitivity, and environmental acceptability. Graphical abstract Crystal violet-functionalized CdTe quantum dots for detecting L-cysteine and glutathione with switch-on fluorescent strategy.

An indirect and sensitive electrochemical method for the determination of reduced glutathione (GSH) was developed by using poly(thionine) (PTH) as a mediator in the presence of Fenton-type reaction on the electrode surface in this work. Cyclic voltammetry and the adsorption of Cu 2+ were employed in sequence to fabricate a Cu-PTH modified electrode, which was characterized by scanning electron microscopy, XPS and electrochemical measurements. Hydroxyl radicals that were derived from the Fenton-type reaction between Cu 2+ and H 2 O 2 could effectively oxidize PTH, leading to the great enhancement of the cathodic peak current of the dye polymer in the cyclic voltammetric scan. The electroreduction of PTH on the electrode surface was found to be inhibited in the presence of GSH. Under the optimized conditions, the cathodic peak current change was found to be proportional to the logarithm of the GSH concentration from 10nM to 1mM with a detection limit of 2.5nM estimated at a signal-to-noise ratio of 3. The proposed electrochemical sensor was successfully employed for the determination of GSH in real samples with satisfactory results., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aim: To identify metabolic signatures in urine samples from healthy and inflammatory bowel disease (IBD) children., Methods: We applied liquid chromatography and gas chromatography coupled to targeted mass spectrometry (MS)-based metabolite profiling to identify and quantify bile acids and host-gut microbial metabolites in urine samples collected from 21 pediatric IBD patients monitored three times over one year (baseline, 6 and 12 mo), and 27 age- and gender-matched healthy children., Results: urinary metabolic profiles of IBD children differ significantly from healthy controls. Such metabolic differences encompass central energy metabolism, amino acids, bile acids and gut microbial metabolites. In particular, levels of pyroglutamic acid, glutamic acid, glycine and cysteine, were significantly higher in IBD children in the course of the study. This suggests that glutathione cannot be optimally synthesized and replenished. Whilst alterations of the enterohepatic circulation of bile acids in pediatric IBD patients is known, we show here that non-invasive urinary bile acid profiling can assess those altered hepatic and intestinal barrier dysfunctions., Conclusion: The present study shows how non-invasive sampling of urine followed by targeted MS-based metabonomic analysis can elucidate and monitor the metabolic status of children with different GI health/disease status., Competing Interests: Conflict-of-interest statement: FPM, SPG, JPG are employees of the Nestlé Group.

Arsenic exposure is considered a major environmental threat to human health. It is already known that high-level arsenic exposure has adverse effects on human health. Since the pregnant women are known to be more susceptible to some chemical exposures than ordinary people, the understanding regarding the health effects of low-level arsenic exposure on pregnant women is critical and remains unclear. The aim of this study is to investigate the urinary metabolic changes of pregnant women exposed to low-dose arsenic, and to identify biomarkers from metabolomics analysis. Urine samples of 246 pregnant women were collected in the first trimester of pregnancy and were divided into three groups based on the tertile distribution of urinary arsenic concentrations which were determined using inductively coupled plasma mass spectrometry (ICP-MS). Changes in the metabolite profile were measured using ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF MS). Arsenic- related metabolic biomarkers were investigated by comparing the samples of the first and third tertiles of arsenic exposure classifications using a partial least-squares discriminant model (PLS-DA). Nine urine potential biomarkers were putatively identified, including LysoPC (14:0), glutathione, 18-carboxy-dinor-LTE4, 20-COOH-LTE4, cystathionine ketimin, 1-(beta-d-ribofuranosyl)-1,4-dihydronicotinamide, thiocysteine, p-cresol glucuronide and vanillactic acid. The obtained results showed that environmental arsenic exposure, even at low levels, could cause metabolite alterations in pregnant women which might be associated with adverse health outcomes. This is the first report on metabolic changes in pregnant women for arsenic exposure. The findings may be valuable for the arsenic risk assessment for pregnant women., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Tetrachloroethylene (perchloroethylene; PERC) is a high-production volume chemical and ubiquitous environmental contaminant that is hazardous to human health. Toxicity attributed to PERC is mediated through oxidative and glutathione (GSH) conjugation metabolites. The conjugation of PERC by glutathione-s-transferase to generate S-(1,2,2-trichlorovinyl) glutathione (TCVG), which is subsequently metabolized to form S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC) is of special importance to human health. Specifically, TCVC may be metabolized to N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine (NAcTCVC) which is excreted through urine, or to electrophilic metabolites that are nephrotoxic and mutagenic. Little is known regarding toxicokinetics of TCVG, TCVC, and NAcTCVC as analytical methods for simultaneous determination of these metabolites in tissues have not yet been reported. Hence, an ultra-high-performance liquid chromatography electrospray ionization tandem mass spectrometry-based method was developed for analysis of TCVG, TCVC, and NAcTCVC in liver, kidneys, serum, and urine. The method is rapid, sensitive, robust, and selective for detection all three analytes in every tissue examined, with limits of detection (LOD) ranging from 1.8 to 68.2 femtomoles on column, depending on the analyte and tissue matrix. This method was applied to quantify levels of TCVG, TCVC, and NAcTCVC in tissues from mice treated with PERC (10 to 1000 mg/kg, orally) with limits of quantitation (LOQ) of 1-2.5 pmol/g in liver, 1-10 pmol/g in kidney, 1-2.5 pmol/ml in serum, and 2.5-5 pmol/ml in urine. This method is useful for further characterization of the GSH conjugative pathway of PERC in vivo and improved understanding of PERC toxicity.

Background: Although reduced glutathione (rGSH) is decreased in obese mice and humans, block of GSH synthesis by buthionine sulfoximine (BSO) results in a lean, insulin-sensitive phenotype. Data is lacking about the effect of BSO on GSH precursors, cysteine and glutamate. Plasma total cysteine (tCys) is positively associated with stearoyl-coenzyme A desaturase (SCD) activity and adiposity in humans and animal models., Objective: To explore the phenotype, amino acid and fatty acid profiles in BSO-treated mice., Design: Male C3H/HeH mice aged 11 weeks were fed a high-fat diet with or without BSO in drinking water (30 mmol/L) for 8 weeks. Amino acid and fatty acid changes were assessed, as well as food consumption, energy expenditure, locomotor activity, body composition and liver vacuolation (steatosis)., Results: Despite higher food intake, BSO decreased particularly fat mass but also lean mass (both P<0.001), and prevented fatty liver vacuolation. Physical activity increased during the dark phase. BSO decreased plasma free fatty acids and enhanced insulin sensitivity. BSO did not alter liver rGSH, but decreased plasma total GSH (tGSH) and rGSH (by ~70%), and liver tGSH (by 82%). Glutamate accumulated in plasma and liver. Urine excretion of cysteine and its precursors was increased by BSO. tCys, rCys and cystine decreased in plasma (by 23-45%, P<0.001 for all), but were maintained in liver, at the expense of decreased taurine. Free and total plasma concentrations of the SCD products, oleic and palmitoleic acids were decreased (by 27-38%, P <0.001 for all)., Conclusion: Counterintuitively, block of GSH synthesis decreases circulating tCys, raising the question of whether the BSO-induced obesity-resistance is linked to cysteine depletion. Cysteine-supplementation of BSO-treated mice is warranted to dissect the effects of cysteine and GSH depletion on energy metabolism., Competing Interests: AE and HR are inventors on a patent application for use of BSO as an anti-obesity agent. The authors confirm that this does not alter their adherence to PLOS ONE policies on sharing data and materials. All other authors reported no conflict of interest.

Diosbulbin B (DIOB), a furanoid, is a major constituent of herbal medicine Dioscorea bulbifera L. Exposure to DIOB caused liver injury in humans and experimental animals. The mechanisms of DIOB-induced hepatotoxicities remain unknown. The present study demonstrated that DIOB induced hepatotoxicities in a time- and dose-dependent manner in mice. H&E stained histopathologic image showed the occurrence of necrosis in the liver obtained from the mice treated with DIOB at dose of 200 mg/kg. Pretreatment with KTC protected the animals from hepatotoxicities and hepatic GSH depletion induced by DIOB, increased area under the concentration-time curve of blood DIOB, decreased urinary excretion of GSH conjugates derived from DIOB, and increased urinary excretion of parent drug. Pretreatment with BSO exacerbated DIOB-induced hepatotoxicities. In order to define the role of furan moiety in DIOB-induced liver toxicities, we replaced the furan of DIOB with a tetrahydrofuran group by chemical hydrogenation of the furan ring of DIOB. No liver injury was observed in the animals given the same doses of tetrahydro-DIOB. The furan moiety was essential for DIOB-induced hepatotoxicities. The results implicate the cis-enedial reactive metabolite of DIOB was responsible for the observed toxicities. The observed modest depletion of hepatic GSH in DIOB-treated animals suggests the actions of one or more reactive metabolites, and the hepatic injury observed could be due at least in part to reactions of these metabolites with crucial biomolecules. Cytochrome P450 3A enzymes are implicated in DIOB-induced hepatotoxicities by catalyzing the formation of the reactive metabolite of DIOB.

Background: The effect of liver resection on acetaminophen metabolism and whether it is affected by residual liver volume is poorly understood., Methods: We investigated the effects of liver resection on acetaminophen metabolism in a single centre, prospective observational, case-control study of inpatients. Patients undergoing liver resection were administered therapeutic post-operative acetaminophen. Glutathione and urinary acetaminophen metabolites were measured over the first three post-operative days and compared between patients with low (Group A) and high (Group B) residual liver volume., Results: 41 patients (41% female, median age 62 [IQR 53-72] years) were included. Mean urinary cysteine levels increased significantly from post-operative day 1 to 2 (578.0 mg/day 95% CI 478.9-677.1 vs. 775.4 mg/day, 95% CI 625.7-925.1; p=0.03). Group A (n=11) had significantly higher median levels of cysteine (day 1, 464.3 mg/day [IQR 355.6-582.0]; day 3, 717.6 mg/day [IQR 423.5-1104.0]) compared to Group B (n=11): day 1, 545.4 mg/day (IQR 346.9-843.5); day 3, 508.1mg/day (IQR 390.8-788.4; p=0.048). No significant difference was observed in glutathione or 5-oxoproline levels between the groups., Conclusion: Low residual liver volume results in altered acetaminophen metabolism, however, no evidence of glutathione deficiency was observed. Therapeutic acetaminophen is safe after major liver resection provided liver function is adequate., (Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.)

Ag-ZnO nanoplates and 2-chlorobenzoyl ferrocene were synthesized and used to construct a modified carbon paste electrode. The electrooxidation of glutathione at the surface of the modified electrode was studied. Under the optimized conditions, the square wave voltammetric (SWV) peak current of glutathione increased linearly with glutathione concentrations in the range of 5.0 × 10(-8) to 2.0 × 10(-4)M with sensitivity of 0.659 μA μM(-1) and method detection limit of 20.0 nM was obtained for glutathione. The prepared modified electrode exhibits a very good resolution between the voltammetric peaks of glutathione and tryptophan which makes it suitable for the detection of glutathione in the presence of tryptophan in real samples., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The clinical significance of high levels of homocysteine in autism spectrum disorder (ASD) is unknown. An experimental study was conducted in order to evaluate the concentration of homocysteine in children with ASD and typically developing children and to analyse any relationships with the severity of core symptoms of ASD and other clinical features (drugs, co-morbidities, gender, age, diet). Core symptoms of autism were evaluated by DSM-IV criteria. Homocysteine, glutathione, methionine, 3-nitrotyrosine were measured in urine. The increase in homocysteine concentration was significantly and directly correlated with the severity of the deficit in communication skills, but was unrelated to deficit in socialisation or repetitive/restricted behaviour. Urinary homocysteine concentration may be a possible biomarker for communication deficits in ASD and a potential diagnostic tool useful to evaluate new treatment options since no treatment for core symptoms of ASD are available., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Although mechanistically linked to disease, cellular molecules damaged by endogenous processes have not emerged as significant biomarkers of inflammation and disease risk, due in part to poor understanding of their pharmacokinetic fate from tissue to excretion. Here, we use systematic metabolite profiling to define the fate of a common DNA oxidation product, base propenals, to discover such a biomarker. Based on known chemical reactivity and metabolism in liver cell extracts, 15 candidate metabolites were identified for liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) quantification in urine and bile of rats treated with thymine propenal (Tp). Analysis of urine revealed three metabolites (6% of Tp dose): thymine propenoate and two mercapturate derivatives of glutathione conjugates. Bile contained an additional four metabolites (22% of Tp dose): cysteinylglycine and cysteine derivatives of glutathione adducts. A bis-mercapturate was observed in urine of untreated rats and increased approximately three- to fourfold following CCl4-induced oxidative stress or treatment with the DNA-cleaving antitumor agent, bleomycin. Systematic metabolite profiling thus provides evidence for a metabolized DNA damage product as a candidate biomarker of inflammation and oxidative stress in humans.

Unlabelled: Quercetin exerts vasodilator, antiplatelet and antiproliferative effects and reduces blood pressure, oxidative status and end-organ damage in hypertensive humans and animal models. We hypothesized that oral quercetin might induce vasodilator effects in humans and that they might be related to the deconjugation of quercetin-3-O-glucuronide (Q3GA)., Design: double blind, randomized, placebo-controlled trial. Fifteen healthy volunteers (26±5 years, 6 female) were given a capsule containing placebo, 200 or 400mg of quercetin in random order in three consecutive weeks. At 2h a dose-dependent increase in Q3GA was observed in plasma (∼0.4 and 1μM for 200 and 400mg, respectively) with minor levels of quercetin and isorhamnetin. No changes were observed in blood pressure. At 5h quercetin induced and increase in brachial arterial diameter that correlated with the product of the levels of Q3GA by the plasma glucuronidase activity. There was an increase in urinary levels of glutathione but there was no increase in nitrites plus nitrates. Quercetin and isorhamnetin also relaxed human umbilical arteries in vitro while Q3GA was without effect. In conclusions, quercetin exerts acute vasodilator effects in vivo in normotensive, normocholesterolemic human subjects. These results are consistent with the effects being due to the deconjugation of the metabolite Q3GA., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Molecularly imprinted polymer (MIP) particles for glutathione were synthesized through iniferter-controlled living radical precipitation polymerization (IRPP) under ultraviolet radiation at ambient temperature. Static adsorption, solid-phase extraction, and high-performance liquid chromatography were carried out to evaluate the adsorption properties and selective recognition characteristics of the polymers for glutathione and its structural analogs. The obtained IRPP-MIP particles exhibited a regularly spherical shape, rapid binding kinetics, high imprinting factor, and high selectivity compared with the MIP particles prepared using traditional free-radical precipitation polymerization. The selective separation and enrichment of glutathione from the mixture of glycyl-glycine and glutathione disulfide could be achieved on the IRPP-MIP cartridge. The recoveries of glutathione, glycyl-glycine, and glutathione disulfide were 95.6% ± 3.65%, 29.5% ± 1.26%, and 49.9% ± 1.71%, respectively. The detection limit (S/N=3) of glutathione was 0.5 mg·L(-1). The relative standard deviations (RSDs) for 10 replicate detections of 50 mg·L(-1) of glutathione were 5.76%, and the linear range of the calibration curve was 0.5 mg·L(-1) to 200 mg·L(-1) under optimized conditions. The proposed approach was successfully applied to determine glutathione in spiked human urine samples with recoveries of 90.24% to 96.20% and RSDs of 0.48% to 5.67%., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Low-molecular-weight biothiols such as homocysteine, cysteine, and glutathione are metabolites of the sulfur cycle and play important roles in biological processes such as the antioxidant defense network, methionine cycle, and protein synthesis. Thiol concentrations in human plasma and blood are related to diseases such as cardiovascular disease, neurodegenerative disease, and cancer. The concentrations of homocysteine, cysteine, and glutathione in plasma samples from healthy human subjects are approximately in the range of 5-15, 200-300, and 1-5 μM, respectively. Glutathione concentration in the whole blood is in the millimolar range. Measurement of biothiol levels in plasma and blood is thought to be important for understanding the physiological roles and biomarkers for certain diseases. This review summarizes the relationship of biothiols with certain disease as well as pre-analytical treatment and analytical methods for determination of biothiols in human plasma and blood by using high-performance liquid chromatography and capillary electrophoresis coupled with ultraviolet, fluorescence, or chemiluminescence detection; or mass spectrometry., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Apocynin (4'-hydroxy-3'-methoxyacetophenone) is the most commonly used NADPH oxidase (Nox) inhibitor. However, its application raises serious controversies, as the compound has been reported to reveal some prooxidative effects. The aim of this study was to elucidate apocynin action on glutathione, the main intracellular antioxidant, metabolism in kidneys of Zucker diabetic fatty (ZDF) rat, a well established model of diabetes type 2. Additionally, apocynin effects were compared with those of melatonin. The experiments were performed on five groups of animals: (1) untreated lean (?/+) ZDF rats, (2) ZDF ?/+ rats treated with apocynin (2 g/l) in drinking water, (3) untreated obese diabetic (fa/fa) ZDF rats, (4) ZDF fa/fa rats treated with apocynin (2 g/l) in drinking water, and (5) ZDF fa/fa rats treated with melatonin (20 mg/l) in drinking water. After 8weeks of the treatment, the following parameters were measured in kidneys: NADPH oxidase activity, the rate of hydroxyl free radicals (HFR) production, GSH and GSSG content and the activities of the enzymes of glutathione metabolism: γ-glutamylcysteine synthetase (GCS), glutathione reductase (GR) and glutathione peroxidase (GPx). Compared to ?/+ controls, ZDF fa/fa rats exhibited increased Nox activity, accelerated HFR generation and dramatically lowered GSH/GSSG ratio accompanied by increased GPx and diminished GCS activities. In case of diabetic animals, apocynin treatment resulted in attenuation of both Nox activity and HFR production, restoration of control GSH/GSSG ratio (due to both an increase in GSH and a decline in GSSG content), normalization of GPx activity and a slight increase in GCS activity. Similar observations were made upon melatonin application to ZDF fa/fa rats. Thus, it is concluded that, in the diabetic model studied, apocynin extends a beneficial effect on renal glutathione homeostasis. The mechanism of this phenomenon involves attenuation of glutathione peroxidase activity, which is overstimulated under conditions of oxidative stress accompanying diabetes., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Biologically active low-molecular-mass thiols, mainly including glutathione (GSH), cysteine (Cys), homocysteine (Hcy), and cysteinylglycine (Cys-Gly), are important physiological components in biological fluids, and their analytical methods have gained continuous attention over recent years. We developed and validated a novel HPLC method for the quantification of GSH, Cys, Hcy, and Cys-Gly in human plasma, urine, and saliva using 4-chloro-3,5-dinitrobenzotrifluoride as the derivatization reagent. Analyses were linear from 0.15 to 500 μM with the coefficient regression range of 0.9987-0.9994. Detection limits ranged from 0.04 to 0.08 μM (S/N=3). The developed method was applied to quantification of four thiols in human biological fluids collected from five donors with the concentration range of 2.50-124.25 μM, 0-72.81 μM, and 0-4.25 μM for plasma, urine, and saliva, respectively. The present method seemed to be an attractive choice for the determination of thiols in plasma, urine, and saliva.

Aim: The therapeutic options available to treat neonatal pain are limited, and one alternative for nonopioid systemic treatment is paracetamol. However, pharmacokinetic data from prolonged administration of intravenous paracetamol in neonates are limited. The aim of this study was to present pharmacokinetics after multiple dose of intravenous paracetamol in very preterm infants of <32 weeks' gestation., Methods: Fifteen very preterm infants received five, six-hourly doses of intravenous paracetamol (7.5 mg/kg). Blood samples were taken to measure paracetamol, glutathione and hepatic function, together with urine samples for paracetamol metabolites., Results: A two-compartment pharmacokinetic model gave the best fit for all individual patients and resulted in a predictable pharmacokinetic profile. The estimated pharmacokinetic population parameters were volume of distribution 0.764 ± 0.225 L/kg, elimination rate constant (ke ) 0.117 ± 0.091/h and intercompartment rate constants k12 0.607 ± 0.734/h and k21 1.105 ± 0.762/h., Conclusion: Our study found that multiple doses of intravenous paracetamol resulted in a predictable pharmacokinetic profile in very preterm infants. Increases in postmenstrual age and weight were associated with increased clearance. No evidence of hepatotoxicity was found., (©2014 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.)

The catalyzed luminol chemiluminescent reaction has received a great amount of attention because of its high sensitivity and low background signal which make the reaction an attractive analytical chemistry tool. The present study, introduces the beneficial catalytic effects of dinuclear Cu(II) complex [Cu2L2(TAE)]X2, where TAE=tetraacetylethane; L=N,N(')-dibenzylethylenediamine and X=ClO4 on the luminol chemiluminescent reaction as a novel probe for the determination of glutathione (GSH) and L-cysteine (CySH) in human serum and urine. The [Cu2L2(TAE)]X2 has exhibited highly efficient catalytic activity of luminol CL as an artificial peroxidase model at pH as low as 7.5 in water in the presence of H2O2⋅GSH and CySH can induce a sharp decrease in CL intensity from the [Cu2L2(TAE)]X2-catalyzed luminol system. Under the selected experimental conditions, a linear relationship was obtained between the CL intensity and the concentrations of GSH and CySH in the range of 1.0×10(-7)-1.0×10(-4) M, with detection limits (S/N=3) of 2.7×10(-8) and 6.8×10(-8) M and RSD<4.2% (n=7) for GSH and CySH, respectively., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Exercise has been consistently used as an oxidant stimulus in redox biology studies. However, previous studies have focused on group differences and did not examine individual differences. As a result, it remains untested whether all individuals experience oxidative stress after acute exercise. Therefore, the main aim of the present study was to investigate whether some individuals exhibit unexpected responses after an acute eccentric (i.e., muscle-damaging) exercise session. Ninety eight (N = 98) young men performed an isokinetic eccentric exercise bout with the knee extensors. Plasma, erythrocytes and urine samples were collected immediately before and 2 days post-exercise. Three commonly used redox biomarkers (F2-isoprostanes, protein carbonyls and glutathione) were assayed. As expected, the two oxidant biomarkers (F2-isoprostanes and protein carbonyls) significantly increased 2 days after exercise (46% and 61%, respectively); whereas a significant decrease in glutathione levels (by -21%) was observed after exercise. A considerable number of the participants exhibited changes in the levels of biomarkers in the opposite, unexpected direction than the group average. More specifically, 13% of the participants exhibited a decrease in F2-isoprostanes and protein carbonyls and 10% of the participants exhibited an increase in glutathione levels. Furthermore, more than 1 out of 3 individuals exhibited either unexpected or negligible (from 0% to ± 5%) responses to exercise in at least one redox biomarker. It was also observed that the initial values of redox biomarkers are important predictors of the responses to exercise. In conclusion, although exercise induces oxidative stress in the majority of individuals, it can induce reductive stress or negligible stress in a considerable number of people. The data presented herein emphasize that the mean response to a redox stimulus can be very misleading. We believe that the wide variability (including the cases of reductive stress) described is not limited to the oxidant stimulus used and the biomarkers selected.

Hydroxytyrosol (HT), one of the major polyphenols present in olive oil, is known to possess a high antioxidant capacity. The aim of the present study was to investigate dose dependent (0, 1, 10 and 100 mg/kg) alterations in the metabolism of HT in rats since it has been reported that metabolites may contribute to biological effects. Special attention was paid to the activation of the semiquinone-quinone oxidative cycle and the formation of adducts with potential deleterious effects. Thus, we developed a novel analytical methodology to monitor the in vivo formation of the HT mercapturate, N-acetyl-5-S-cysteinyl-hydroxytyrosol in urine samples. Biomarkers of hepatic and renal toxicity were evaluated within the dose range tested. Following HT administration, dose-dependent effects were observed for the recovery of all the metabolites studied. At the lowest dose of 1 mg/kg, the glucuronidation pathway was the most relevant (25-30%), with lower recoveries for sulfation (14%), while at the highest dose of 100 mg/kg, sulfation was the most prevalent (75%). In addition, we report for the first time the formation of the mercapturate conjugate of HT in a dose-dependent manner. The biochemical data did not reveal significant toxic effects of HT at any of the doses studied. An increase in the GSH/GSSG ratio at the highest dose was observed indicating that the products of HT autoxidation are counteracted by glutathione, resulting in their detoxification. These results indicate that the metabolic disposition of HT is highly dependent on the dose ingested., (Copyright © 2013. Published by Elsevier Ltd.)

The occurrence of several polyunsaturated testosterone related compounds (including 4,6-androstadien-3,17-dione and 4,6-androstadien-17β-ol-3-one) in urine after alkaline treatment of the sample has been recently reported. Although several experiments seem to indicate that they are testosterone metabolites, their origin is still unknown. In this study, it is demonstrated that these metabolites are produced from the degradation of cysteine conjugates. Several testosterone metabolites conjugated with cysteine have been synthesized and characterized by NMR techniques. Their detection in human urine has been performed by LC-MS/MS. The acquisition of several transitions in the SRM mode and the comparison between ion ratios and retention times allowed for the unequivocal confirmation of the presence of cysteine conjugates in urine. The analysis of urine samples collected after testosterone administration confirmed that synthesized cysteine conjugates are testosterone metabolites. The fact that these conjugates result in polyunsaturated compounds in urine after alkaline treatment was demonstrated by fraction collection and alkaline treatment of each fraction. Besides, the presence of these metabolites was also confirmed in human plasma. The formation of these metabolites implies an unreported metabolic biotransformation: 6,7-dehydrogenation as phase I metabolism followed by conjugation with glutathione and subsequent transformation to cysteine conjugates. Finally, the existence of similar metabolites for cortisol and progesterone was also confirmed by LC-MS/MS indicating that the presented metabolic pathway is not exclusively active in androgens, but common to progestagens and glucocorticoids., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A chemically modified electrode was prepared by incorporating p-aminophenol into multiwall carbon nanotubes paste matrix. Cyclic voltammetry, square wave voltammetry, double potential step chronoamperometry, and electrochemical impedance spectroscopy were used to investigate the electrochemical behaviour of glutathione at the chemically modified electrode prepared. According to the results, p-aminophenol multiwall carbon nanotubes paste electrode (p-APMWCNTPE) showed high electrocatalytic activity for glutathione oxidation, producing a sharp oxidation peak current at about +0.285 vs Ag/AgCl reference electrode at pH 5.0. Chronoamperometry was also used to determine glutathione's catalytic rate constant and diffusion coefficient at p-APMWCNTPE. The square wave voltammetric peak current of glutathione increased linearly with glutathione concentration in the range of 2.0 × 10(-7) - 1.0 × 10(-4) mol L(-1) with a detection limit of 9.0 × 10(-8) mol L(-1). The method was also successfully employed as a selective, simple, and precise method for the determination of glutathione in haemolysed erythrocyte, tablet, and urine samples., (Copyright © 2011 John Wiley & Sons, Ltd.)

Objective: To determine the plasma and urine levels of antioxidants and oxidative stress biomarkers in subjects with spinal cord injury (SCI) the first year after injury., Design: Descriptive 1-year follow-up study., Setting: Rehabilitation and research center., Participants: SCI subjects (n=37; age range, 18-70 y) consecutively enrolled within the first month after injury. A healthy, able-bodied control group (n=346) was also included., Interventions: Not applicable., Main Outcome Measures: Blood and urine levels of antioxidants and oxidative stress biomarkers were measured at inclusion and after 3 and 12 months postinjury., Results: One month after injury, the plasma antioxidants (total and oxidized glutathione and 6 different carotenoids and α-tocopherol) were reduced by 19% to 71% among the SCI subjects compared with the controls. The redox potential was reduced by 7% among the SCI subjects. The oxidative stress biomarker urinary 8-epi prostagladin F2α (PGF2α) increased to 161% in the SCI subjects compared with the controls. After 3 and 12 months, most of the antioxidant biomarkers were still significantly reduced compared with the controls, while urinary 8-epi PGF2α had increased to 208% compared with the controls., Conclusions: The levels of antioxidants were significantly lower, while the marker of oxidative stress was higher in the SCI subjects compared with the controls. This observation demonstrates that SCI patients experience increased oxidative stress and reduced antioxidant defense the first year after injury. Our findings warrant intervention studies where SCI patients receive dietary antioxidant support as part of their rehabilitation., (Copyright © 2012 American Congress of Rehabilitation Medicine. Published by Elsevier Inc. All rights reserved.)