Improvement of glutathione production by a metabolically engineered Yarrowia lipolytica strain using a small-scale optimization approach.

OBJECTIVE: In this study, we aimed to maximize glutathione (GSH) production by a metabolically engineered Yarrowia lipolytica strain using a small-scale optimization approach. RESULTS: A three levels four factorial Box-Behnken Design was used to assess the effect of pH, inulin extract, yeast extract and ammonium sulfate concentrations on cell growth and to generate a mathematical model which predict optimal conditions to maximize biomass production and thus GSH titer. The obtained results revealed that only yeast and inulin extract concentrations significantly affect biomass production. Based on the generated model, a medium composed of 10 g/L of yeast extract and 10 g/L of inulin extract from Jerusalem artichoke was used to conduct batch cultures in 2 L bioreactor. After 48 h of culture, the biomass and the glutathione titer increased by 55% (5.8 gDCW/L) and 61% (1011.4 mg/L), respectively, as compared to non-optimized conditions. CONCLUSION: From the obtained results, it could be observed that the model established from small scale culture (i.e. 2 mL) is able to predict performance at larger scale (i.e. 2 L bioreactor, two orders of magnitude scale-up). Moreover, the results highlight the ability of the optimized process to ensure high titer of glutathione using a low-cost carbon source.

In Situ Ligand-Directed Growth of Gold Nanoparticles in Biological Tissues.

Fundamental understandings and precise control of nanoparticle growth in the complex biological environment are crucial to broadening their potential applications in tissue imaging. Herein, we report that glutathione (GSH), a widely used capping ligand for precise control of the size of gold nanoparticle (AuNP) down to single-atom level in test tubes, can also be used to direct the selective growth of the AuNPs in the mitochondria of renal tubule cells as well as hippocampus cells in the tissues. Precise control of this growth process can lead to the formation of both ultrasmall AuNPs with near-infrared luminescence and large plasmonic AuNPs. The observed selective growth of the AuNPs is likely due to unique GSH storage function of the mitochondria. Using a different ligand, ß-glucose thiol, we also found that the brush border of the intestine for glucose absorption became the major site for the growth of luminescent AuNPs. These findings suggest that selective growth of AuNPs in the biological tissues can indeed be directed with specific ligands, opening up a new avenue to tissue labeling and future development of artificial bionano hybrid systems.

Salt-effect enhanced hollow-fiber liquid-phase microextraction of glutathione in human saliva followed by miniaturized capillary electrophoresis with amperometric detection.

A hollow-fiber liquid-phase microextraction (HF-LPME) method was established for purification and enrichment of glutathione (GSH) in human saliva followed by a miniaturized capillary electrophoresis with amperometric detection system (mini-CE-AD). Based on regulating isoelectric point and increasing salt effect to modify donor phase, HF-LPME could provide high enrichment efficiency for GSH up to 471 times, and the extract was directly injected for mini-CE-AD analysis. The salt-effect enhanced HF-LPME/mini-CE-AD method has been successfully applied to saliva analysis, and acceptable LOD (0.46 ng/mL, S/N = 3) and recoveries (92.7-101.3%) could be obtained in saliva matrix. The sample pretreatment of this developed method was simple and required no derivatization, providing a potential alternative for non-invasive fluid analysis using portable instrument.

Altered oxidative stress markers in relation to T cells, NK cells & killer immunoglobulin receptors that are associated with disease activity in SLE patients.

Systemic Lupus Erythematosus is an autoimmune disease with symptoms pervasive to all organ systems. It affects more females as compared to males (in the ratio 9:1). Oxidative stress plays a major role in the pathogenesis of SLE and other autoimmune diseases. In order to understand the relationship between cell specific oxidative stress and the severity of SLE, this research study involving the estimation of intracellular ROS accumulation in T and NK cell was conducted on SLE patients of North Indian Population. At the same time, to estimate anti-oxidant defense, Keap1 and Nrf2 levels were estimated in these cell types. The relationship between the expression of Killer immunoglobulin receptors i.e., KIR2DL4 & KIR3DL1 and oxidative stress was also evaluated as these receptors are imperative for the function and self-tolerance of NK cells.Oxidative stress was raised along with Keap1 and Nrf2 in T and NK cell subsets in SLE patients. The expression of KIR2DL4 was raised and that of KIR3DL1 was reduced in the NK cells of patients. The intensity of change in expression and its significance varied among the subsets. Nrf2 expression was raised in these species against oxidative stress as the antioxidant defense mechanism pertaining to Keap1-Nrf2 pathway, but the adequacy of response needs to be understood in further studies. The expression of KIR2DL4 and KIR3DL1 varied among the patient and healthy controls and the expression of the latter was found to have a significant positive relationship with plasma Glutathione(reduced) concentration.

Optimization of background electrolyte composition for simultaneous contactless conductivity and fluorescence detection in capillary electrophoresis of biological samples.

In this article, optimization of BGE for simultaneous separation of inorganic ions, organic acids, and glutathione using dual C4 D-LIF detection in capillary electrophoresis is presented. The optimized BGE consisted of 30 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid, 15 mM 2-amino-2-hydroxymethyl-propane-1,3-diol, and 2 mM 18-crown-6 at pH 7.2 and allowed simultaneous separation of ten inorganic anions and cations, three organic acids and glutathione in 20 min. The samples were injected hydrodynamically from both capillary ends using the double-opposite end injection principle. Sensitive detection of anions, cations, and organic acids with micromolar LODs using C4 D and simultaneously glutathione with nanomolar LODs using LIF was achieved in a single run. The developed BGE may be useful in analyses of biological samples containing analytes with differing concentrations of several orders of magnitude that is not possible with single detection mode.

Mass Spectrometry Characterization of Thiol Conjugates Linked to Polyoxygenated Polyunsaturated Fatty Acid Species.

Radical mediated oxidation of polyunsaturated fatty acids (PUFA) is known to generate a series of polyoxygenated cyclic products (PUFA-On, n ≥ 3). Here, we describe the characterization of glutathione (GSH) conjugates bound to polyoxygenated docosahexaenoic (DHA-On, n = 3-9), arachidonic (ARA-On, n = 3-7), α-linolenic (ALA-O3), and linoleic (LA-O3) acid species. Similar conjugates were also characterized for N-acetylcysteine (NAC) and Cu,Zn-superoxide dismutase (SOD1). Extensive LC-MS/MS characterization using a synthetic α-linolenic hydroxy-endoperoxide (ALA-O3) derivative revealed at least two types of mechanisms leading to thiol adduction: a mechanism involving the nucleophilic attack by thiolate anion on 1,2-dioxolane to form a sulfenate ester-bonded conjugate and a mechanism involving cleavage of the dioxolane to form a α,ß-unsaturated carbonyl followed by the Michael addition reaction. Finally, we detected a GSH conjugate with hydroxy-endoperoxide derived from linoleic acid (LA-O3) in mice liver. In summary, our study reveals the formation of a series of thiol conjugates that are bound to highly oxygenated PUFA species. GSH conjugates described in our study may potentially play relevant roles in redox and inflammatory processes, especially under high oxygen tension conditions.

A Fluorescence Inner-Filter Effect Based Sensing Platform for Turn-On Detection of Glutathione in Human Serum.

A novel turn-on fluorescence assay was developed for the rapid detection of glutathione (GSH) based on the inner-filter effect (IFE) and redox reaction. Molybdenum disulfide quantum dots (MoS2 QDs), which have stable fluorescent properties, were synthesized with hydrothermal method. Manganese dioxide nanosheets (MnO2 NSs) were prepared by exfoliating the bulk δ-MnO2 material in bovine serum albumin (BSA) aqueous solution. The morphology structures of the prepared nanoparticles were characterized by transmission electron microscope (TEM). Studies have shown that the fluorescence of MoS2 QDs could be quenched in the presence of MnO2 NSs as a result of the IFE, and is recovered after the addition of GSH to dissolve the MnO2 NSs. The fluorescence intensity showed a good linear relationship with the GSH concentration in the range 20⁻2500 µM, the limit of detection was 1.0 µM. The detection method was applied to the analysis of GSH in human serum samples. This simple, rapid, and cost-effective method has great potential in analyzing GSH and in disease diagnosis.

Polystyrene nanofibers capped with copper nanoparticles for selective extraction of glutathione prior to its determination by HPLC.

Polystyrene nanofibers were coated with copper nanoparticles (CuNPs) by a combination of electrospinning and in-situ reduction of Cu(II) using sodium borohydride as the reductant. The CuNPs on the nanofibers were characterized by energy dispersive spectrometry, scanning electron microscopy and transmission electron microscopy. A cartridge was packed with the nanofibers which then were activated with methanol and water. Glutathione (GSH) is found to quantitatively adsorbed by the packed cartridge at pH 3.0, and then can be desorbed with aqueous 2-mercaptoethanol and detected, after derivatization with ortho-phthalaldehyde, via high performance liquid chromatography with fluorometric detection. Under optimized conditions, the method has a 1.1 ng·mL-1 detection limit and a response that is linear in the 10-1000 ng·mL-1 GSH concentration range. The recoveries of GSH from artificial urine spiked at three levels (80, 400 and 800 ng·mL-1) are in the range of 94.6-98.6% with relative standard deviations (RSD) of <4.5% (n = 5). The method was applied to assessing the differences in urinary GSH between high-risk infants and healthy infants. The results show that the levels of GSH of normal infants are significantly higher than those of high-risk infants (P < 0.05). Graphical abstract Schematic of the preparation of CuNP-assembled nanofibers and the mechanism of extracting glutathione (GSH). GSH can be extracted by this material based on a strong interaction between the sorbent and GSH. This is attributed to the formation of Cu-S bonds between Cu and -SH.

A Novel Dicyanoisophorone-Based Ratiometric Fluorescent Probe for Selective Detection of Cysteine and Its Bioimaging Application in Living Cells.

A selective and ratiometric turn-on fluorescent probe was designed and synthesized by using a novel dicyanoisophorone-based derivative and acrylate moiety. The probe displayed high stability and good selectivity to cysteine (Cys) over homocysteine (Hcy) and glutathione (GSH). It also exhibited rapid response to Cys within 180 s. Most importantly, the fluorescence intensity ratio at 590 and 525 nm (I590/I525) was linearly dependent on the Cys concentration in the range from 0 to 40 µM and the detection limit calculated to be 0.48 µM. This probe was also applied for bioimaging of intracellular Cys in living HeLa cells with low cytotoxicity.

Rapid quantification of glutaminase 2 (GLS2)-related metabolites by HILIC-MS/MS.

Glutamine, glutamate and glutathione are key modulators of excessive oxidative stress in tumor cells. In this study, we developed a rapid and accurate HILIC-MS/MS method to simultaneously determine concentrations of cellular glutamine, glutamate and glutathione. A bared silica HILIC column was employed to analyze these polar metabolites. The LC-MS parameters were optimized to achieve high sensitivity and selectivity. The analysis can be completed within 4 min under optimal conditions. The method was validated in terms of accuracy, precision, and linearity. Intra-day (n = 9) precision was within 2.68-6.24% among QCs. Inter-day precision (n = 3) was below 12.4%. The method accuracy was evaluated by the recovery test, and the accuracy for three analytes were between 91.6 and 110%. The developed method was applied to study antioxidant function of GLS2 in non-small cell lung cancer cells. Changes in concentrations of glutamine, glutamate and glutathione revealed that the overexpression of GLS2 could effectively decrease oxidative stress. In summary, this study developed a rapid HILIC-MS/MS method for quantification of GLS2-related metabolites that could facilitate elucidation of the role of GLS2 in tumor development.

4-Hydroxybenzyl-substituted glutathione derivatives from Gastrodia elata.

Seven new 4-hydroxybenzyl-substituted glutathione derivatives (2-8), together with a known analogue (1), were isolated from the aqueous extract of Gastrodia elata Blume rhizomes. Their structures were determined by using spectroscopic and chemical methods. The absolute configurations of 1-8 were assigned by using Marfey's method, combined with comparing the NMR and CD spectroscopic data of sulfoxide moieties in 3-6 with those of S-(4-hydroxybenzyl)cysteine sulfoxide stereoisomers (9-12) synthesized as authentic samples. The configurations of 9-12 were confirmed by electronic CD calculations based on the quantum-mechanical time-dependent density functional theory. Furthermore, the structures of 1, 3, 5, 7, and 8 were verified by synthesis. Compound 3 was active against serum deprivation-induced PC12 cell damage and synthetic 9-14 exhibited activity against Fe(2+)-cysteine induced rat liver microsomal lipid peroxidation.

[ELEMENTS OF THE LOW MOLECULAR WEIGHT CHAIN OF ANTIOXIDANT DEFENSE IN TISSUES OF THE BLACK SEA MOLLUSC ANADARA KAGOSHIMENSIS BRUGÙIERE].

The content of reduced glutathione (GSH) and the activity of the coupled with it antioxidant enzymes - glutathione peroxidase and glutathione reductase as well the level of glucose, carbamide and amino acids were investigated in the hepatopancreas, gills and foot of the Black, Sea mollusk Anadara kagoshimensis. The highest content of GSH and the highest activity of glutathione peroxidase were found in mollusk foot, evidencing the active antioxidant role of glutathione played both within composition of this enzyme and independently. The maximal content of glucose, amino acids and carbamide was in the hepatopancreas and gills and the minimal - in the anadara's foot. The possible involvement and role of these low molecular weight antioxidants in the defense of mollusk tissues against action of free radical oxidation and in providing adaptation reactions of anadara in hypoxic habitats are considered. Key words: antioxidant complex, glutathione, glucose, carbamide, amino acids, anadara Anadara kagoshimensis, Black Sea.

A new BODIPY-based long-wavelength fluorescent probe for chromatographic analysis of low-molecular-weight thiols.

A new long-wavelength fluorescent probe, 1,7-dimethyl-3,5-distyryl-8-phenyl-(4'-iodoacetamido)difluoroboradiaza-s-indacene (DMDSPAB-I), was designed and synthesized for thiol labeling in high-performance liquid chromatography (HPLC). The excitation and emission wavelengths of DMDSPAB-I are 620 and 630 nm, respectively, with a high fluorescence quantum yield of 0.557, which is advantageous in preventing interference of intrinsic fluorescence from complex biological matrices and enabling high sensitivity HPLC. Based on DMDSPAB-I, a reversed-phase HPLC method was developed for measuring low-molecular-weight thiols including glutathione, cysteine, homocysteine, N-acetylcysteine, cysteinylglycine, and penicillamine. After the specific reaction of DMDSPAB-I with thiols, baseline separation of all six stable derivatives was achieved through isocratic elution on a C18 column within 25 min, with the limits of detection (signal-to-noise ratio = 3) from 0.24 nmol L(-1) for glutathione to 0.72 nmol L(-1) for penicillamine. The proposed method was validated in part by measuring thiols in blood samples from mice, with recoveries of 95.3-104.3%.

Simple enrichment of thiol-containing biomolecules by using zinc(II)-cyclen-functionalized magnetic beads.

A simple and efficient method based on magnetic-bead technology has been developed for the enrichment of thiol-containing biomolecules, such as l-glutathione and cysteine-containing peptides. The thiol-binding site on the bead is a mononuclear complex of zinc(II) with 1,4,7,10-tetraazacyclododecane (cyclen); this is linked to a hydrophilic cross-linked agarose coating on a particle that has a magnetic core. All steps for the thiol-affinity separation are conducted in aqueous buffers with 0.10 mL of the magnetic beads in a 1.5 mL microtube. The entire separation protocol for thiol-containing compounds, from addition to elution, requires less than one hour per sample, provided the buffers and the zinc(II)-cyclen-functionalized magnetic beads have been prepared in advance. The thiol-affinity magnetic beads are reusable at least 15 times without a decrease in their thiol-binding ability, and they are stable for six months at room temperature.

Photoelectrochemical detection of glutathione by IrO2-hemin-TiO2 nanowire arrays.

We have developed sensitive detection of glutathione using the IrO2-hemin-TiO2 nanowire arrays. Single-crystalline TiO2 nanowires are synthesized by a hydrothermal reaction, followed by surface functionalization of ~3 nm thick hemin and ~1-2 nm diameter IrO2 nanoparticles. The IrO2-hemin-TiO2 nanowire arrays offer much enhanced photocurrent with ∼100% increase compared to the pristine TiO2 nanowires and allow for label-free, real-time, sensitive photoelectrochemical detection of glutathione. The sensitivity achieved is ~10 nM in buffer, comparable to or better than most of the existing glutathione detection methods. Furthermore, cell extracts containing glutathione are robustly detected, with ~8000 cells/mL for HeLa cells and ~5000 cells/mL for human embryonic kidney 293T cells. This nanowire PEC sensor assay exhibits excellent selectivity and stability, suggesting a potential detection platform for analyzing the glutathione level in biosamples.

Separation of glutathione and its novel analogues and determination of their dissociation constants by capillary electrophoresis.

A CE protocol was developed to separate reduced glutathione and its four novel analogues UPF1 (Tyr(Me)-γ-Glu-Cys-Gly), UPF17 (Tyr(Me)-α-Glu-Cys-Gly), UPF50 (ß-Ala-His-Tyr(Me)-γ-Glu-Cys-Gly), and UPF51 (ß-Ala-His-Tyr(Me)-α-Glu-Cys-Gly), and their homo- and heterodimers by varying the ionic strength and/or pH of different BGEs. For the determination of dissociation constants (pK(a)) of the above-mentioned peptides the CE method was used. Effective electrophoretic mobilities of analytes were measured in the pH range 5.50-10.00 using optimized BGE with an ionic strength of 50 mM at 25°C. pK(a) values were calculated by fitting the experimental points to a suitable model with correlation coefficients higher than 0.99. The pK(a) values for imidazolyl, amino and thiol moieties of the analyzed peptides were in the range 5.94-6.29, 8.81-9.10, and 7.86-8.13, respectively.

Accurate quantification of the redox-sensitive GSH/GSSG ratios in the yeast Pichia pastoris by HILIC-MS/MS.

A novel method for the simultaneous quantification of both glutathione (GSH) and its oxidized form glutathione disulfide (GSSG) by hydrophilic interaction chromatography-MS/MS has been developed and is critically discussed. Internal standardization based on isotopically labeled standards for both analytes is an absolute prerequisite for accurate quantification of this redox pair. Hence, a highly efficient and selective miniaturized procedure for the synthesis of isotopically labeled GSSG from commercially available glutathione-(glycine-(13)C(2),(15)N) was established using H(2)O(2) as oxidant and NaI as catalyst. Moreover, a tool is presented to monitor and hence uncover artifactual GSSG formation due to oxidation of GSH during sample preparation, which is the main source of systematic error in GSSG analysis. For this purpose, we propose to monitor the oxidation product formed by reaction of naturally occurring GSH with the isotopically labeled GSH used as internal standard. For the determination of GSH/GSSG ratios in yeast, different extraction methods based on (1) hot extraction with aqueous, acidic, or organic solvents, (2) mechanical cell lysis, and (3) extraction at subambient temperature were investigated in terms of recovery, extraction efficiency, and artifactual formation of GSSG. Total combined uncertainties of as low as 25-30 % (coverage factor=2) for the determination of GSH/GSSG ratios without derivatization were made possible by the addition of the internal standards early in the analytical procedure (before extraction) and immediate analysis of the analytes.

A rapid and simple separation and direct detection of glutathione by gold nanoparticles and graphene-based MALDI-TOF-MS.

In this study, we present a rapid and simple method for the separation and direct detection of glutathione by combining gold nanoparticles and MALDI-TOF-MS with graphene as matrix. Gold nanoparticles enable the selective capture of thiol-containing compounds. Gold nanoparticles bound with analytes can be mixed with graphene matrix for direct analysis by MALDI-TOF-MS, which can avoid sample loss and contamination during transfer process. Compared with a conventional matrix, α-cyano-4-hydroxycinnamic acid, graphene exhibits an excellent desorption/ionization efficiency, thermal and mechanical properties. The use of graphene as matrix avoids the fragmentation of analytes. Stable analysis was achieved with less background interference even at the concentration of 0.625 ng/µL. To further confirm its efficiency, the optimized approach was applied to the separation and detection of glutathione in mouse liver extraction. This result showed the great potential of detection of biologically important thiols in biochemical and biomedical research.

Electrophoretic separations in poly(dimethylsiloxane) microchips using a mixture of ionic and zwitterionic surfactants.

The use of mixtures of ionic and zwitterionic surfactants in poly(dimethylsiloxane) (PDMS) microchips is reported. The effect of surfactant concentration on electroosmotic flow (EOF) was studied for a single anionic surfactant (sodium dodecyl sulfate, SDS), a single zwitterionic surfactant (N-tetradecylammonium-N,N-dimethyl-3-ammonio-1-propanesulfonate, TDAPS), and a mixed SDS/TDAPS surfactant system. SDS increased the EOF as reported previously while TDAPS showed an initial increase in EOF followed by a reduction at higher concentrations. When TDAPS was added to a solution containing SDS, the EOF decreased in a concentration-dependent manner. The EOF for all three surfactant systems followed expected pH trends, with increasing EOF at higher pH. The mixed surfactant system allowed tuning of the EOF across a range of pH and concentration conditions. After establishing the EOF behavior, the adsorption/desorption kinetics were measured and showed a slower adsorption/desorption rate for TDAPS than SDS. Finally, the separation and electrochemical detection of model catecholamines in buffer and reduced glutathione in red blood cell lysate using the mixed surfactant system were explored. The mixed surfactant system provided shorter analysis times and/or improved resolution when compared to the single surfactant systems.

Development of a high-performance liquid chromatography method for the simultaneous quantitation of glutathione and related thiols.

The development of drugs with the ability to increase the level of the antioxidant glutathione and related metabolites has become an important research area for many age-related diseases. Here we describe a high-performance liquid chromatography (HPLC) method that uses the thiol-specific, fluorogenic reagent 4-fluoro-7-aminosulfonylbenzofurazan (ABD-F) for the simultaneous determination of total glutathione (GSH), cysteine (Cys), cysteinylglycine (CysGly), and homocysteine (Hcys) in cell culture medium. ABD-F-labeled thiols were separated using an isocratic mobile phase consisting of 14% methanol and 86% 0.1M acetate buffer at pH4.0. The method was validated for linearity, accuracy, and intra- and interday precision, and the lower and upper limits of quantitation (LLOQ and ULOQ, respectively) were determined using a Dionex RF-2000 detector set to medium sensitivity. In addition, the suitability of N-acetylcysteine (NAC) as an internal standard was evaluated by external and internal standard calibration methods. Although both calibration methods showed acceptable linearity (correlation coefficients>0.99) and intra- and interday precision (relative standard deviations=10.2 and 6.6%, respectively), the external standard calibration method performed better in terms of accuracy (recovery=93.7-125%) and also had lower LLOQ values for all analytes (Cys=6.3µM, CysGly=0.8µM, Hcys=0.8µM, and GSH=1.6µM).