Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

Measurement of glutathione (GSH) and glutathione disulfide (GSSG) is a crucial tool to assess cellular redox state. Herein we report a direct approach to determine intracellular GSH based on a rapid chromatographic separation coupled with acidic potassium permanganate chemiluminescence detection, which was extended to GSSG by incorporating thiol blocking and disulfide bond reduction. Importantly, this simple procedure avoids derivatisation of GSH (thus minimising auto-oxidation) and overcomes problems encountered when deriving the concentration of GSSG from 'total GSH'. The linear range and limit of detection for both analytes were 7.5 × 10(-7) to 1 × 10(-5) M, and 5 × 10(-7) M, respectively. GSH and GSSG were determined in cultured muscle cells treated for 24 h with glucose oxidase (0, 15, 30, 100, 250 and 500 mU mL(-1)), which exposed them to a continuous source of reactive oxygen species (ROS). Both analyte concentrations were greater in myotubes treated with 100 or 250 mU mL(-1) glucose oxidase (compared to untreated controls), but were significantly lower in myotubes treated with 500 mU mL(-1) (p < 0.05), which was rationalised by considering measurements of H(2)O(2) and cell viability. However, the GSH/GSSG ratio in myotubes treated with 100, 250 and 500 mU mL(-1) glucose oxidase exhibited a dose-dependent decrease that reflected the increase in intracellular ROS.     

Dual-stimuli-sensitive poly(ortho ester disulfide urethanes)-based nanospheres with rapid intracellular drug release for enhanced chemotherapy

Herein, new poly(ortho ester disulfide urethanes) (POEDU) and poly(ortho ester urethanes) (POEU) were successfully synthesized via polycondensation between active esters of 1,6-hexandiol (HD) and dual-stimuli-sensitive ortho ester disulfide diamine or pH-senstive ortho ester diamine. The corresponding POEDU and POEU nanospheres were easily fabricated using an oil-in-water emulsion technique. In vitro degradation experiments indicated that POEDU nanospheres degraded faster than POEU nanospheres in mildly acidic and reductive environments. Doxorubicin (DOX) as a model antitumor drug was successfully incorporated into these nanospheres to give DOX-loaded nanoparticles (POEDU-DOX and POEU-DOX). In vitro drug release studies showed that release of DOX from dual-stimuli-sensitive POEDU-DOX was accelerated compared with release from the pH-sensitive POEU-DOX under DL-dithiothreitol (DTT) and mildly acidic conditions. In addition, in vitro uptake and cytotoxicity assays revealed that POEDU-DOX exhibited more efficient antitumor effect than POEU-DOX did against both two-dimensional (2D) cells and three-dimensional (3D) multicellular tumor spheroids (MCTS). Finally, in a mice H22 tumor model, POEDU-DOX exhibited preferable antitumor capability. In conclusion, the pH and redox dual-stimuli-sensitive POEDU nanospheres can be superior drug carriers for cancer treatment.     

A rapid and precise method for determination of D/H ratios of plant methoxyl groups

Stable hydrogen isotope ratio measurements of specific plant components are increasingly used in numerous fields of research, including sample origin verification and climate research. A recently suggested method with considerable potential in this context is the D/H isotope ratio (δ²H value) analysis of plant matter methoxyl groups. The method entails ether or ester cleavage with hydriodic acid (HI) to form the gaseous compound methyl iodide (CH₃I) and measurement of the δ²H value of this gas. Here we describe a method for the rapid and precise δ²H analysis of plant matter methoxyl groups using gas chromatography/pyrolysis/isotope ratio mass spectrometry (GC/P/IRMS). The conditions for sample preparation were investigated for isotope discrimination effects, the GC conditions were optimized, the reproducibility of the measurement of standards was studied, and the precision of the method was defined. The reproducibility of δ²H values determined for a CH₃I standard on 20 consecutive measurements was found to be 2‰. The method was also tested on four methoxyl‐rich plant components: vanillin, lignin, wood and pectin. The analytical precision obtained, when expressed as the average standard deviation for these compounds, was better than 1.6‰. The described method is rapid, allowing preparation and analysis of a sample within 1 h, and produces accurate and reproducible isotopic measurements. Copyright © 2008 John Wiley & Sons, Ltd.     

An optimized method for determination of intracellular glutathione in mouse macrophage cultures by fluorimetric high-performance liquid chromatography

We have optimized a method for the determination of intracellular glutathione by high-performance liquid chromatography, using fluorimetric detection. To minimize artifacts and provide an accurate determination of intracellular glutathione, cell extracts were prepared using extraction conditions specifically designed to inhibit autoxidation and enzymatic degradation of glutathione. The sensitivity of the method was enhanced by adjusting the dansyl chloride derivatization reaction with regard to parameters such as pH, reaction time and dansyl chloride concentration. Both oxidized and reduced forms of glutathione were quantified using the refined method in extracts of oxidatively stressed J774A.1 mouse macrophage cells and reflected an expected shift in cellular redox status.     

The rapid determination of silver to silver plus copper ratios in coins using 14 MeV neutron activation analysis

A non-destructive method for the analysis of the silver to silver plus copper ratio in coins using 14 MeV neutron activation analysis has been studied. The mass of silver to the mass of copper is obtained by measuring the 0.511 MeV annihilation radiation emitted by⁶⁴Cu and¹⁰⁶Ag at two separate times after irradiation. A series of British silver three-penny pieces dated 1891–1941 were analysed and agreed well with the mint assay. The interference from the presence of zinc in some coins was investigated.     

Chip-based CE for rapid separation of 8-aminopyrene-1,3,6-trisulfonic acid (APTS) derivatized glycans

Fluorescently labeled carbohydrates released from glycoproteins were separated using a commercially available microfluidic chip electrophoresis system. While the instrumentation was primarily designed for DNA analysis it was found that the application base can be easily expanded using the development software provided by the manufacturer. The carbohydrates were released by enzymatic digestion (PNGase F) from glycoproteins present in human plasma after boronic acid - lectin affinity enrichment. After fluorescent labeling with 8-aminopyrene-1,3,6-trisulfonic acid the carbohydrates were separated based on capillary gel electrophoresis mechanism and detected by a fluorescence detector using a blue (470 nm) LED. The separation was completed in 40 s in a microfluidic channel of 14 mm length. Glucose ladder carbohydrate oligomers differing by one glucose unit were baseline separated up to a 20-mer with the main limitation being the detection sensitivity. As expected, the observed resolution in these experiments did not approach that of standard CE with 20 times longer separation distance; however, the chip-based analysis excelled in the speed of the separation. Similar electrophoretic profiles of glycans released from plasma glycoproteins were obtained using a standard CE equipment with 35 cm separation length and microfluidic chips with a separation distance of only 14 mm.     

Membrane electrodes for the determination of glutathione

Four glutathione (GSH)-selective electrodes were developed with different techniques and in different polymeric matrices. Precipitation-based technique with bathophenanthroline-ferrous as cationic exchanger in polyvinyl chloride (PVC) matrix was used for sensor 1 fabrication. β-Cyclodextrin (β-CD)-based technique with either tetrakis(4-chlorophenyl)borate (TpClPB) or bathophenanthroline-ferrous as fixed anionic and cationic sites in PVC matrix was used for fabrication of sensors 2 and 3, respectively.β-CD-based technique with TpClPB as fixed anionic site in polyurethane (Tecoflex) matrix was used for sensor 4 fabrication. Linear responses of 1 × 10⁻⁵ to 1 × 10⁻⁴ M and 1 × 10⁻⁶ to 1 × 10⁻³ M with slopes of 37.5 and 32.0 mV/decade within pH 7-8 were obtained by using electrodes 1 and 3, respectively. On the other hand, linear responses of 1 × 10⁻⁵ to 1 × 10⁻² and 1 × 10⁻⁵ to 1 × 10⁻³ M with slopes of 47.9 and 54.3 mV/decade within pH 5-6 were obtained by using electrodes 2 and 4, respectively. The percentage recoveries for determination of GSH by the four proposed GSH-selective electrodes were 100 ± 1, 100.5 ± 0.7, 100 ± 1 and 99.0 ± 0.8% for sensors 1, 2, 3 and 4, respectively. Determination of GSH in capsules by the proposed electrodes revealed their applicability for determination of GSH in its pharmaceutical formulations. Also, they were used to determine GSH selectively in presence of its oxidized form (GSSG). Sensor 4 was successfully applied for determination of glutathione in plasma with average recovery of 100.4 ± 1.11%. The proposed method was compared with a reported one. No significant difference for both accuracy and precision was observed.     

Centri-voltammetric determination of glutathione

Centri-voltammetry is a method for concentrating an analyte on an electrode with the aid of a centrifuge. It can be performed in the presence or the absence of a solid carrier/support. This is followed by a voltammetric (analytical) scan. Specifically, we describe here an application of the technique to the determination of glutathione (GSH). Silica gel is used as the carrier precipitate to which gold nanoparticles were added in order to improve accumulation as a result of their strong affinity for thiols. Voltammetry was performed with a carbon paste electrode modified with multi-wall carbon nanotubes. The response to GSH is linear in the 25 and 800 μM concentration range (the correlation coefficient being 0.9915) and the relative standard deviation is 3.40 % (at 250 μM of GSH and n?=?6). The procedure was successfully applied to the determination of GSH in wine and in synthetic plasma using the standard addition method. The recoveries are 100.8 % and 100.0 %, respectively.

A convenient technique for determination of reactivity ratios

A new technique to determine the reactivity ratios for monomers is described. It is based on vapor-phase chromatography and can be used for any volatile monomer. A new simplified equation was deduced which permits the direct use of the retention times to obtain the pair r₁r₂. The method is suggested for use in determination of the molar ratios of the chemical units in the polymer chain, regardless of chemical composition or constitution. The simplicity of this technique allows its use for monomer–polymer systems composed of any number of volatile monomers. The validity of this method was checked by using the pair methyl methacrylate–styrene. This technique is also being applied to the study of kinetics in polymerizations, since it allows the measurements of several samples taken from a single experiment.     

A ratiometric fluorescent probe for real-time monitoring of intracellular glutathione fluctuations in response to cisplatin

Real-time imaging of fluctuations in intracellular glutathione (GSH) concentrations is critical to understanding the mechanism of GSH-related cisplatin-resistance. Here, we describe a ratiometric fluorescence probe based on a reversible Michael addition reaction of GSH with the vinyl-functionalized boron-dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene or BODIPY) 1. The probe was applied for real-time monitoring of the fluctuations in GSH levels in cells under cisplatin treatment. Notably, in cellular cisplatin-sensitive A549 cells, GSH concentrations rose until cell death, while in cisplatin-resistant cell lines, GSH levels first rose to the maximum then fell back to the initial concentration without significant apoptosis. These results indicate that different trends in GSH fluctuation can help distinguish cisplatin-resistant from cisplatin-sensitive cells. As such, this study has shown that probe 1 may potentially be used for real-time monitoring of intracellular GSH levels in response to therapeutics.

Real-time imaging of intracellular glutathione in response to cisplatin by a ratiometric fluorescent probe reveals that the different trends in intracellular GSH levels is crucial in distinguishing cisplatin-resistant from cisplatin-sensitive cells.     

Peroxidase-catalysed luminol chemiluminescence method for the determination of glutathione

A luminol chemiluminescence (CL) method was developed for the determination of glutathione (GSH). GSH was indirectly determined by measuring the amount of hydrogen peroxide formed during the Cu(II)-catalysed oxidation of GSH with oxygen. The amount of hydrogen peroxide formed was continuously measured using the Arthromyces ramosus peroxidase-catalysed luminol CL reaction. The CL intensities at maximum light emission were linearly correlated with the concentration of GSH over the range 7.5 x 10(-7)-3.0 x 10(-5) M. The detection limit for GSH was about 10 times better than that of the spectrophotometric method using Ellman reagent.     

Palladium(II) diamine complex induces reduction of glutathione disulfide

Reaction of [Pd(1R,2R-diaminocyclohexane)Cl(2)] with the oxidised form of the tripeptide glutathione ([gamma]-l-glutamyl-l-cysteinyl-glycine) in aqueous solution leads to reductive cleavage of the disulfide bond.     

A gamma-spectrometric method for the determination of plutonium isotope ratios

A gamma-spectrometric method using an intrinsic high resolution germanium detector has been developed for the determination of isotope ratios of plutonium from samples in solution form. The method is based on the assay of low energy gamma-rays of²³⁸Pu,²³⁹Pu,²⁴⁰Pu and²⁴¹Pu and does not require the use of branching intensities or the knowledge of detection efficiencies for different gamma rays. Since low energy gamma-rays are used, the effect of²⁴¹Am has also been studied. It is found that results are not affected up to 0.5 wt% of²⁴¹Am in plutonium samples. An accuracy of 3% is achievable in the determination of²⁴⁰Pu/²³⁹Pu and²⁴¹Pu/²³⁹Pu atom ratios as demonstrated by carrying out measurements on isotopic standards of plutonium.     

Methodology for rapid tritium determination in urine

Summary The present work proposes a methodology for a rapid determina-tion of the tritium content in urine. The urine is treated with active carbon and tritium is determined by liquid scintillation counting. Different kinds of active carbon were experimented for the metabolites removal from urine samples. The counting efficiency was calculated by a quench curve using yellow food dye as a quenching agent. It was obtained a procedure which maintained a good reproducibility, low uncertainty and detection limit of 7 Bq/l (10 ml of urine, 120 minutes of counting time and efficiency of 23.9%).     

Disposable chemiluminometric sensor for rapid determination of EDTA

A new chemiluminescence-based disposable sensor for EDTA is described. The EDTA-sensitive element is a transparent bilayer membrane containing Ru(Bipy)₃ ²⁺ complex and Ce(IV), respectively, on a polyester support. The sample is injected by means of a syringe, acting pneumatically, into a 10 mm cell containing the CL membrane and placed in the external holder of a luminometer. The composition of the membrane and reaction conditions have been adjusted to obtain adequate sensitivity and selectivity. The disposable sensor responds to EDTA rapidly over a dynamic range from 6.6 × 10⁻⁶ M to 7.0 × 10⁻⁴ M, with sensor-to-sensor reproducibility around 6% as RSD (relative standard deviation) at the medium level of the range. The performance of the optical disposable sensor was tested for the analysis of EDTA in ophthalmic collyrium and different types of sauce samples, validating the results against a reference procedure. The sensing approach presented here demonstrates the feasibility of chemiluminescent disposable sensors for quantitative analysis with good features.     

A rapid spectrophotometric method for determination of piperazine

A spectrophotometric method has been developed for the determination of piperazine and its salts (citrate, phosphate, and tartrate) without prior separation, based on the interaction of piperazine or any of its salts with phenothiazine and N-bromosuccinimide in aqueous methanol. The products exhibit absorption maxima at 448, 595 and 645 nm. Measurements are made at 595 nm. Beer's law is obeyed in the concentration range 0.5-5 mug/ml for piperazine salts and 0.5-3 mug/ml for piperazine hexahydrate. The method is rapid, simple and successful for analysis of some pharmaceutical preparations.     

Fluorescent strip sensor for rapid determination of toxins

Here, we report a simple fluorescent strip sensor based on aptamer-quantum dots technology that can meet toxin monitoring demands using ochratoxin A (OTA) as a model toxin. The limit of the detection (LOD) for the fluorescent strip was 1.9 ng mL(-1), while the time needed for the detection is only 10 min; this conforms to the standards of World Health Organization (WHO) or better. Overall functional parameters are also better than the analogous characteristics of gold nanoparticle strips. High selectivity was maintained as well, making them suitable for the samples with complex solution composition.     

Ratios of biliary glutathione disulfide (GSSG) to glutathione (GSH): a potential index to screen drug-induced hepatic oxidative stress in rats and mice

Hepatotoxicity of drug candidates is one of the major concerns in drug screening in early drug discovery. Detection of hepatic oxidative stress can be an early indicator of hepatotoxicity and benefits drug selection. The glutathione (GSH) and glutathione disulfide (GSSG) pair, as one of the major intracellular redox regulating couples, plays an important role in protecting cells from oxidative stress that is caused by imbalance between prooxidants and antioxidants. The quantitative determination of the GSSG/GSH ratios and the concentrations of GSH and GSSG have been used to indicate oxidative stress in cells and tissues. In this study, we tested the possibility of using the biliary GSSG/GSH ratios as a biomarker to reflect hepatic oxidative stress and drug toxicity. Four compounds that are known to alter GSH and GSSG levels were tested in this study. Diquat (diquat dibromide monohydrate) and acetaminophen were administered to rats. Paraquat and tert-butyl hydroperoxide were administered to mice to induce changes of biliary GSH and GSSG. The biliary GSH and GSSG were quantified using calibration curves prepared with artificial bile to account for any bile matrix effect in the LC–MS analysis and to avoid the interference of endogenous GSH and GSSG. With four examples (in rats and mice) of drug-induced changes in the kinetics of the biliary GSSG/GSH ratios, this study showed the potential for developing an exposure response index based on biliary GSSG/GSH ratios for predicting hepatic oxidative stress.