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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.)

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.)

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.)

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.)

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.)

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.)

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.)

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.)

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.)

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.)

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.)

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.)

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.)

Our recent study demonstrates the synthesized triangular gold nanoparticles (AuNPs) by trisodium citrate reduction of HAuCl(4) in the presence of nonionic fluorosurfactant (FSN) could display stronger catalytic activity towards luminol-chemiluminescence (CL) than spherical AuNPs. Ultratrace aminothiols may cause a great decrease in CL intensity of the triangular AuNPs-luminol CL system. In this article, we utilize the as-prepared triangular AuNPs as novel postcolumn CL reagents to explore a simple high-performance liquid chromatography (HPLC)-CL method for the determination of low molecular weight aminothiols (i.e., cysteine, homocysteine, glutathione, cysteinylglycine and glutamylcysteine). The as-prepared triangular AuNPs were easier to synthesize, stable at a wider pH range and high ionic strength, and highly selective and sensitive towards reduced aminothiols. The detection limits at a signal-to-noise ratio of 3 for cysteine, homocysteine, glutathione, cysteinylglycine and glutamylcysteine were 0.016, 0.08, 0.1, 0.04 and 0.1pmol, respectively. Recoveries from spiked urine and plasma samples were 95.7-104.3%. The applicability of the proposed method has been validated by determining these low molecular weight aminothiols in human urine and plasma samples with satisfactory results, and thus it will have great potential application in clinical diagnosis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Here we report a new isotopic pair of derivatization reagents, ω-bromoacetonylquinolinium bromide (BQB) and d(7)-ω-bromoacetonylquinolinium bromide (d(7)-BQB). BQB and d(7)-BQB both rapidly and selectively reacted with thiols in acidic medium within 3min with the aid of a microwave. Reduced thiols and total thiols in urine were labeled with BQB and d(7)-BQB, respectively. The BQB- and d(7)-BQB-labeled urine samples were then mixed and separated on a HILIC (hydrophilic interaction chromatography) column followed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection. The new strategy, which we have named isotope differential derivatization, allows us to simultaneously determine thiols and oxidized thiols in a single run. Compared with positive mode ESI detection of unlabeled thiols, the positive mode ESI-MS signal intensities of BQB-labeled thiols were found to increase by 10-, 20-, and 40-fold for cysteine (Cys), homocysteine (HCys), and glutathione (GSH), respectively (unlabeled N-acetylcysteine (Nac) is difficult to detect by ESI-MS in positive mode due to its low ionization efficiency). The detection limits calculated at a signal-to-noise ratio of 3 were found to be 8.02, 1.56, 0.833, and 3.27nmol/L for Cys, HCys, Nac, and GSH, respectively. Recoveries of thiols and disulfides from spiked urine samples were between 80% and 105%. The method was successfully used to determine thiols and oxidized thiols in urine samples of 25 healthy volunteers., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The widely used analgesic-antipyretic drug acetaminophen (APAP) is known to cause serious liver necrosis at high doses in man and experimental animals. For studies of toxic processes, 1H NMR spectroscopy of biofluids allows monitoring of endogenous metabolite profiles that alter characteristically in response to changes in physiological status. Herein, a 1H NMR metabolomics approach was applied to the investigation of APAP toxicity in rats and the effect of phenobarbital (PB) on APAP-induced hepatotoxicity. Metabolite differences due to hepatotoxicity were observed in 1H NMR spectra of serum and urine, and enhanced APAP hepatotoxicity by pretreatment with PB was clearly shown by a principal components analysis of the spectral data. NMR spectra of APAP-dosed rat urine provided profiles of APAP-related compounds together with endogenous metabolites. By comparison of endogenous and APAP-related metabolite spectra with those from rats pretreated with PB, it was possible to show the importance of oxidative metabolism of APAP to N-acetyl-p-benzoquinone, an essential step in APAP hepatotoxicity.

In this article, we report a simple method for selective enrichment of aminothiols using Tween 20-capped gold nanoparticles (AuNPs) prior to capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF). Compared to citrate-capped AuNPs, Tween 20-capped AuNPs exhibit the ability to disperse in a highly saline solution and selectively extract aminothiols through the formation of Au-S bonds. After extraction and centrifugation, 1mM thioglycollic acid (TGA) was utilized to remove aminothiols that attached to the NP surfaces. After a solution of 8.0 mL aminothiols were extracted using 2x AuNPs (200 microL), the extracted aminothiols derivatized with o-phthalaldehyde at pH 12.0 were detected by CE-LIF. As a result, the limits of detection at a signal-to-noise ratio of 3 for homocysteine (HCys), glutathione (GSH), and gamma-glutamycysteine (Glu-cys) are 4013.2, 79.8, and 382.8 pM, respectively. The use of this probe provided approximately 11-, 282-, and 21-fold sensitivity improvements for HCys, GSH, and Glu-cys, respectively. A practical analysis of HCys, GSH, and Glu-cys in human urine sample has been accomplished by this present method.

After exhaustive exercise, intravenous or oral glutamine promoted skeletal muscle glycogen storage. However, when glutamine was ingested with glucose polymer, whole-body carbohydrate storage was elevated, the most likely site being liver and not muscle, possibly due to increased glucosamine formation. The rate of tricarboxylic acid (TCA) cycle flux and hence oxidative metabolism may be limited by the availability of TCA intermediates. There is some evidence that intramuscular glutamate normally provides alpha-ketoglutarate to the mitochondrion. We hypothesized that glutamine might be a more efficient anaplerotic precursor than endogenous glutamate alone. Indeed, a greater expansion of the sum of muscle citrate, malate, fumarate and succinate concentrations was observed at the start of exercise (70% VO2(max)) after oral glutamine than when placebo or ornithine alpha-ketoglutarate was given. However, neither endurance time nor the extent of phosphocreatine depletion or lactate accumulation during the exercise was altered, suggesting either that TCA intermediates were not limiting for energy production or that the severity of exercise was insufficient for the limitation to be operational. We have also shown that in the perfused working rat heart, there is a substantial fall in intramuscular glutamine and alpha-ketoglutarate, especially after ischemia. Glutamine (but not glutamate, alpha-ketoglutarate or aspartate) was able to rescue the performance of the postischemic heart. This ability appears to be connected to the ability to sustain intracardiac ATP, phosphocreatine and glutathione.

Styrene (S) has been shown to be responsible for neurotoxic effects, including behavioural changes and neuroendocrine disturbances. The initial step of S metabolism is conversion to styrene 7,8-epoxide (SO), which is present in two enantiomeric forms [(R)(+)-SO and (S)(-)-SO]; this electrophilic intermediate is considered to be directly responsible for most toxic effects of S. The major urinary metabolites derived from the biotransformation of SO in man are mandelic acid (MA) and phenylglyoxylic acid (PGA). In rats an alternative pathway has been demonstrated, which involves the conjugation of SO to glutathione (GSH), leading to the excretion of two specific mercapturic acids, N-acetyl-S-(-(1-phenyl-2-hydroxyethyl)-cysteine [M1] and N-acetyl-S-(2-phenyl-2-hydroxy-ethyl)-cysteine [M2]; a close relationship has been found between exposure to S and urinary excretion of M1 and M2 in rats. As a consequence of the chiral nature of SO, both M1 and M2 consist of two diastereoisomers (M1-'R', M1-'S', M2-'R' and M2-'S'). Early reports have shown that the conversion of S to mercapturic acids is much lower in man (below 1% of the absorbed dose) than in rats (about 10%). We propose an analytical method for the determination of urinary M1 and M2 in man, which involves a urine clean-up by a chromatographic technique with a short reversed-phase pre-column; purified samples are then deacetylated with porcine acylase and deproteinized by centrifugal ultrafiltration. A derivatization is then performed with o-phthaldialdehyde and 2-mercaptoethanol and the fluorescent derivatives are separated on a reversed-phase analytical column. The mobile phase consists of acetate buffer and methanol mixed at variable proportions, the fluorescence detector is set at 330 nm (exc.) and 440 nm (em.). M1-'S' and M1-'R' are separated (retention times = 52.8 and 73.7 min, respectively) while the diastereoisomers of M2 coelute as a single peak at 70.5 min. The detection limit is about 7 micrograms/l, the coefficients of variation are below 7% and the error percentages are less than 6%. The method was applied to 25 urine samples from workers exposed to S: significant correlations were found between mercapturic acids and MA and PGA, the best correlation being between M2 and PGA (r = 0.79). Urine samples form unexposed subjects showed no detectable amounts of the analytes. A high stereoselectivity is shown by the enzymes involved in the metabolism of S to mercapturic acids: M1-'S', which derives from (S)-SO, is excreted in much higher amounts than M1-'R', which derives from (R)-SO.

The amperometric detection of thiols following their separation by reversed-phase chromatography and reaction into a post-column mercury carrier stream is shown to be a sensitive method when using a tungsten wire sensor as the working electrode in a three-electrode flow cell. Five organic thiols (cysteine, homocysteine, reduced glutathione, D,L-penicillamine and 2-mercaptopropionic acid) and thiourea could be separated within approx. 18 min. The analytical performance is comparable, and stability superior, to chemically modified electrodes previously reported.

Methods have been adapted for the high-performance liquid chromatographic (HPLC) analysis of urinary paracetamol metabolites on radial compression columns. Enhanced resolution and decreased analysis time were two major advances. Various modifications to existing methods were made to counter the effect of the different C18 surface. Thus in ion suppression HPLC the addition of triethylamine at pH 3.0 (phosphate buffer) was necessary to block residual hydroxyl sites, while in ion-pair HPLC a higher tetrabutyl-ammonium hydroxide concentration of 0.01 M at pH 5.0 was used to enhance selectivity. The methods were successfully applied to the study of the metabolism of paracetamol, its glutathione conjugate and 3-thiomethylparacetamol in Sprague-Dawley rats. 3-Thiomethyl-paracetamol sulphoxide and its glucuronide and sulphate conjugates were shown to be metabolites of both 3-thiomethylparacetamol and paracetamol. 3-Thiomethylparacetamol sulphate was unresolved from the sulphates of paracetamol and 3-methoxyparacetamol in ion-pair HPLC. This raises a previously unrecognised problem in which the peak normally attributed to paracetamol sulphate contains metabolites arising from an oxidative metabolic pathway. Elevated levels of 3-methoxyparacetamol conjugates were found in human overdose urine and to some extent in analgesic nephropathy.

Rats which had received N-hydroxy-2-acetylaminofluorene (N-OH-AAF) excreted two glutathione conjugates in the bile. Pretreatment with the sulphotransferase inhibitor pentachlorophenol reduced the amounts excreted by 50% suggesting that these conjugates were formed in vivo from the highly reactive N-O-sulphate ester of N-OH-AAF (AAF-N-sulphate). In order to identify these biliary conjugates synthetic GSH conjugates were prepared by the reaction of N-acetoxy-AAF, (a model compound for AAF-N-sulphate), with GSH. Four conjugates were isolated and characterized by ultraviolet, proton magnetic resonance, mass spectroscopy and by the determination of their amino acid composition. These conjugates were identified as 1-, 3-, 4- and 7-(glutathion-S-yl)-N-acetyl-2-aminofluorene, respectively. The two biliary conjugates were identified as 1- and 3-(glutathion-S-yl)-N-acetyl-2-aminofluorene.

In spite of the importance of glutathione (GSH) in the detoxification of toxic metabolites of drugs, virtually nothing is known about the regulation of hepatic GSH homeostasis in man. In order to estimate the turnover of hepatic GSH and to assess the effect of different doses of acetaminophen (paracetamol) on the synthesis of GSH in man, [3H]cystine and varying doses of acetaminophen were administered to healthy volunteers, and the time course of the specific activity of the cysteine moiety of N-acetylcysteinyl-acetaminophen excreted in urine was followed. The fractional rate of turnover of the tracer in N-acetylcysteinyl-acetaminophen increased significantly from 0.031 +/- 0.007 h-1 after doses of acetaminophen ranging from 50 to 300 mg to 0.045 +/- 0.011 and 0.121 +/- 0.027 h-1 following 600 and 1200 mg of acetaminophen, respectively. The data indicate that therapeutic doses of acetaminophen markedly stimulate the rate of turnover of the pool of cysteine available for the synthesis of GSH, most likely due to an increased rate of synthesis of GSH which is required to detoxify the toxic metabolite of acetaminophen. Patients who are not able to respond to a similar demand on their stores of GSH by increasing the synthesis of GSH may be at higher risk of developing hepatic injury from drugs that require GSH for their detoxification.

To study the glutathione conjugation of alpha-bromoisovalerylurea in the rat in vivo, a reversed-phase liquid chromatographic assay of the thioether metabolites in bile and urine was developed. Since alpha-bromoisovalerylurea has a chiral centre, two diastereomeric glutathione conjugates (in bile) and two diastereomeric mercapturates (in urine) can be expected. The separation characteristics of these metabolites and the corresponding cysteine conjugates were investigated. Whereas all thioether metabolites could be separated in one run, optimal separation of the diastereomers required different mobile phases for the glutathione conjugates (in bile) and the mercapturates (in urine). The glutathione conjugates were analysed with the ion-pairing agent sodium decanesulphonate in the mobile phase, but the mercapturates were analysed without an ion-pair-forming agent. For detection, on-line generation of a constant bromine level (100%) was used; bromine-reactive compounds result in a decrease of the amperometric response from the 100% baseline. This technique could be used in continuous automated operation and required little clean-up of the sample. Thus, the diastereomeric glutathione conjugates and mercapturates were quantified in rat bile and urine samples, respectively, by direct injection of the (centrifuged and diluted) samples on the column. The limit of determination of the respective metabolites was 9 and 2.6 ng in bile and urine, respectively. Incubation mixtures of alpha-bromoisovalerylurea with a rat liver cytosolic fraction or with isolated rat hepatocytes were chromatographed after deproteinization with a double volume of methanol. The limit of determination of the diastereomeric glutathione conjugates in the deproteinized incubation samples was 2.0 ng.