There is an increasingly growing interest in identifying biomarkers for diseases, in which oxidative stress is involved For many years, GSH has been measured by several analytical methods. Each method has its advantages and limitations and may serve a particular need in analysis ED is an attractive alternative method for electroactive species detection, due to its inherent advantages of simplicity, ease of miniaturisation, high sensitivity and relatively low cost.
The aim of this study was to determine the GSH:GSSG ratio in the blood serum of paediatric cancer patients to use this ratio as a potential marker of oxidative stress. Other chemicals were purchased from Sigma-Aldrich unless otherwise stated.
Stock standard solutions of the thiols 1 mg. Working standard solutions were prepared daily by diluting the stock solutions. All solutions were filtered through 0. Each chamber contains four analytical cells and one analytical cell contains two referent hydrogen-palladium , as well as two counters and porous graphite working electrodes. The ED is situated in the thermostated control module. Other conditions were optimised and are described later. Accuracy was evaluated by comparing the estimated concentration with the known concentrations of both thiols.
Written informed patient consent was obtained from the patients. Subjects ranged between 1 and 10 years of age. The blood samples were collected before chemotherapy and radiotherapy. Primarily, it was necessary to optimise the separation and subsequent ED in order to achieve the required accuracy and sensitivity for the determination of GSH and GSSG in real blood serum samples.
Therefore, we focused on studying the influence of flow rate, concentration of components of the mobile phase, elution and applied potential of the working electrodes on GSH and GSSG signals. The mobile phase flow rate is an important parameter influencing the electrochemical response of the detector. This is probably caused by reducing the time-concentration of the analyte on the electrode surface.
Although a lower flow rate may not be significantly affected by resolution, it may extend the period of separation, which is critical for analysing a large number of clinical samples. Achieving an optimal resolution is crucial for simultaneous separation of analytes. In order to separate all determined substances in the system with reversed-phase, a gradient with the increasing content of organic solvent is required. Since the electrochemical determination of substances contained in the sample requires the presence of an electrolyte, we examined the effect of the organic solvent methanol on the electrochemical response of analytes.
A marked decline of GSSG signal was also observed. A significant tailing of peaks was observed during the elution of compounds with higher retention under these conditions. Therefore, we optimised the increasing content of methanol with respect to the sensitivity of the analysis. Sensitivity of the electrochemical detector may be more influenced by factors including the type of electrolyte in the mobile phase, concentration, pH and, in particular, applied potential. TFA was used as an ion-pair reagent, which provides the best separation conditions in the parameters mentioned above, and at a concentration of 80 mM it is also an extremely suitable electrolyte for the detection of thiols.
Tested potentials were , , , , , , , , and 1, mV. This is probably due to the requirement for greater power for partial dissociation of the -S-S- group on the surface of the working electrode, in comparison to the relatively easily accessible -SH moiety of GSH. We observed the highest signals for both compounds when a potential of —1, mV was applied, which is evident from the HDVs showed in Fig.
Based on the HDV results we were able to evaluate that the best glutathione detection was achieved when a potential of mV was applied to the working electrodes. After identifying the optimal separation and detection conditions, the calibration curves for GSH and GSSG were measured within the concentration range of 0. Overlay of HPL chromatograms is shown in Fig. Prior to chromatographic analysis, precipitation of proteins with TFA to avoid excessive clogging of filters and precolumns, which protect the separation column from contaminations, was required.
The proteins may interfere with detected substances and the obtained chromatograms may be extremely difficult to analyse. The tryptophan radical can react with ROS to form tryptophan hydroperoxide, which then rearranges to N -formylkynurenine NFK and kynurenine.
Protein carbonylation is the irreversible oxidative modification of side chains of proline, histidine, arginine, lysine and threonine. The result is the formation of reactive carbonyls and usually leads to the inactivation of protein function through unfolding and an increased susceptibility to degradation; global levels of carbonylation can be detected using a number of assays.
Derivatisation of carbonyl groups with 2,4-dinitrophenylhydrazine DNPH allows spectroscopic detection, or immunodetection using DNPH-specific antibodies and subsequent tandem MS can identify specifically oxidized proteins, e. Improvements could be made with greater diversity of cellular targeting of chemical probes and biosensors, e.
Subcellular estimation of ascorbate levels uses different approaches. Histochemical labelling with silver nitrate enables detection by microscopy, with a degree of specificity if conducted under cold and acidic conditions.
Finally, in vivo real time imaging of ascorbate has recently been achieved using a selective fluorescent probe, made of silicon phthalocyanine SiPc and two 2,2,6,6-tetramethylpiperidinyloxy TEMPO radicals, albeit in an animal model and yet to be tested in plants. However, all in vitro methods have their own drawbacks and limitations, notably their limited specificity and alteration of the glutathione pool during extract preparation and mixing from subcellular compartments.
In situ detection and estimation of glutathione, particularly at the subcellular level, provides new insights into its role in responses to cell stress. Monochlorobimane and monobromobimane fluorescent dyes have been used to report glutathione in trichoblast root hair cells , atrichoblasts, and nuclei and cytosol of different plant cells.
Another physical method of measuring O 2 concentration is through the use of probes that exploit O 2 -quenching of molecular luminescence. Like the Clarke electrodes, these probes have also successfully measured gaseous and dissolved O 2 concentrations in a range of plant tissues including generation of O 2 maps in seeds and measurement of O 2 dynamics in deepwater rice and seagrass.
The signal can also be impacted by chlorophyll fluorescence and incident light. Soluble luminescent indicators could be very useful, particularly if they could enter cells to measure intracellular O 2 concentrations. Towards this aim, Pt II —tetra-pentafluorophenyl-porphyrin has been encapsulated in polystyrene microbeads and injected into plant cells where, despite an overlap in fluorescent spectra from the probe and chlorophyll, multi-frequency phase modulation allowed determination of O 2 concentration, albeit without subcellular resolution.
Gold nanoparticles have indeed been shown able to enter plant tissue via roots and translocate to stems and leaves, entering cells by active transport mechanisms. The use of genetically-engineered fluorescent proteins for O 2 measurement is challenging, particularly in plant cells, because of the confounding challenges of chlorophyll fluorescence and the fact that commonly used GFP-derivatives require O 2 to form a chromophore.
Recently, however, an O 2 -independent green fluorophore UnaG, previously used in tumour cells was shown to fluoresce in anoxic Arabidopsis protoplasts. There is significant scope for an improved and expanded toolkit; while it is not yet possible to discriminate different ROS signals with the temporal and spatial resolution required to really understand their specific cause and effects, recent developments show continuing promise.
Intracellular fluorescent biosensors, despite their limitations, are favourable with respect to specificity and subcellular targeting. There is also considerable scope for ROS-activated chemical probes to be developed with improved characteristics.
Overall, continued investment is required to generate advanced chemical and biological probes for ROS and redox measurement in plants; particularly desirable are direct ROS-sensing probes that can rapidly elicit a response in order to understand cellular ROS dynamics.
We encourage the chemical biology community to take on this challenge! DOI: Received 31st March , Accepted 28th June Abstract Reactive oxygen species ROS are produced throughout plant cells as a by-product of electron transfer processes.
Tetrazolium dyes are frequently used in situ , e. Hydrogen peroxide. As the most stable of the ROS, a relatively high number of methods and probes exist to detect H 2 O 2 in plants. Luminol 3-amino-phthal-hydrazide is oxidized by peroxidase in the presence of H 2 O 2 to yield blue luminescence, however, the signal is quenched by many cellular components.
Singlet oxygen. In addition, the presence of 1 O 2 scavengers like histidine can reduce the degree of fluorescence quenching observed. When the anthracene reacts with 1 O 2 it forms an endoperoxide, which prevents this quenching activity, resulting in green fluorescence upon light excitation Fig. Spin traps. From the perspective of measuring ROS, the advantage of EPR is that it allows the detection of short-lived highly reactive cellular ROS radicals through the use of spin-traps.
Spin probes. Spin probes, or labels, are an alternative to spin traps for use in plant tissues, and work in the opposite fashion to spin trapping. Intrinsically, spin probes are relatively stable paramagnetic species but are converted to diamagnetic species upon reaction with free radicals, thus resulting in a decrease in their EPR signals.
Example of spin probes which have been used in plants are nitroxyl-based probes 82 such as 5-SASL 5-doxyl-stearic acid, Fig. Usefully, as the SASL probe is lipophilic, 84 it can easily attach to the lipid phase of membrane vesicles, which is the major site of free radical production in cells. Real-time imaging of the intracellular glutathione redox potential. Methods 5 , — Fan, Y. Monitoring redox dynamics in living cells with a redox-sensitive red fluorescent protein.
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