Simultaneous Electrochemical Speciation of Oxidized and Reduced

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Simultaneous electrochemical speciation of oxidized and reduced glutathione. Redox profiling of oxidative stress in biological fluids with a modified carbon electrode. Patricia M. Olmos Moya, Minerva Martinez-Alfaro, Rezvan Kazemi, Mario A. Alpuche-Aviles, Sophie Griveau, Fethi Bedioui, and Silvia Gutierrez-Granados Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01690 • Publication Date (Web): 30 Aug 2017 Downloaded from http://pubs.acs.org on September 1, 2017

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Analytical Chemistry

Simultaneous electrochemical speciation of oxidized and reduced glutathione. Redox profiling of oxidative stress in biological fluids with a modified carbon electrode. Patricia M. Olmos Moya,1 Minerva Martínez Alfaro,2 Rezvan Kazemi,3 Mario A. AlpucheAvilés,3* Sophie Griveau,4-7 Fethi Bedioui4-7 and Silvia Gutiérrez Granados.1* 1. Departamento de Química, Universidad de Guanajuato, Guanajuato, México. 2. Departamento de Farmacia, Universidad de Guanajuato, Guanajuato, México. 3. Department of Chemistry, University of Nevada, Reno, Nevada, 89557, USA. 4. Chimie ParisTech-PSL Research University, Unité de Technologies Chimiques et Biologiques pour la Santé, (UTCBS), 75005 Paris, France 5. INSERM U 1022, UTCBS, 75005 Paris, France 6. CNRS 8258, UTCBS, 75005 Paris, France 7. Université Paris Descartes-Sorbonne Paris Cité, UTCBS, 75006 Paris, France ABSTRACT The simultaneous electrochemical quantification of oxidized (GSSG) and reduced glutathione (GSH), biomarkers of oxidative stress, is demonstrated in biological fluids. The detection was accomplished by the development of a modified carbon electrode and was applied to the analysis of biological fluids of model organisms under oxidative stress caused by lead intoxication. Nanocomposite molecular material based on cobalt phthalocyanine (CoPc) and multiwalled carbon nanotubes functionalized with carboxyl groups (MWCNTf) was developed for modifying glassy carbon electrodes (GCE) for the detection of reduced and oxidized glutathione. The morphology of the nanocomposite film was characterized by scanning electron microscopy (SEM) and profilometry. The electrochemical behavior of the modified electrode was assessed by cyclic voltammetry (CV) to determine the surface coverage (Γ) by CoPc. The electrocatalytic behavior of the modified electrode towards reduced (GSH) and oxidized (GSSG) forms of glutathione was assessed by CV studies at physiological pH. The obtained results show that the combined use of CoPc and MWCNTf results in an electrocatalytic activity for GSH oxidation and GSSG reduction, enabling the simultaneous detection of both species. Differential pulse voltammetry allows obtaining detection limits of 100 µM for GSH and 8.3 µM for GSSG, respectively. The potential interference from ascorbic acid, cysteine, glutamic acid, and glucose was also studied, and the obtained results show limited effects from these species. Finally, the hybrid electrode was used for the determination of GSH and GSSG in rat urine and plasma samples, intoxicated or not by lead. Both glutathione forms were detected in these complex biological matrices without any preACS Paragon Plus Environment


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treatment. Our results portray the role of GSH and GSSG as markers of oxidative stress in live organisms under lead intoxication. Keywords: Cobalt Phthalocyanine, Glutathione, Carbon Nanotubes, Electrochemical Sensors, Toxicology, Oxidative stress

INTRODUCTION We present an electrochemical method to simultaneously measure the ratio of reduced and oxidized glutathione in model organisms under oxidative stress. Oxidative stress has been recognized as a factor in aging and lifespan1 and in several diseases,2 including schizophrenia.3 Oxidative stress has been defined as a disruption in the antioxidant defense against prooxidants like reactive oxygen species (ROS).4,5 The antioxidant cellular defense mechanism is governed significantly in vivo by reduced glutathione (GSH), which oxidizes continuously, generating disulfide glutathione (GS-SG).6 Therefore, more than 90 % of glutathione species is in the GSH form in healthy cells.7 GSH protects cells against oxidative damage by trapping ROS8 and is involved in conjugation reactions and in the transport of metal ions.9 Here we determine independently the concentrations of GSH and GSSG under oxidative stress caused by Pb intoxication. There is evidence implicating Pb exposure with pathological incidences, including documented renal10 and neurological11 dysfunctions in humans and studies with Wistar rats.12 There is an association between the extent of dysfunction of tissues and intracellular Pb bioaccumulation which leads to Pb-induced toxic manifestations associated with increased oxidative stress 12,13 which is the principal source of ROS and thus the stimulation of activity of antioxidants as GSH. Therefore, glutathione species, GSH and GSSG, were established to be markers of oxidative stress.7,8 Glutathione concentration in the organism is associated with several conditions14 and can be relevant in clinical diagnostics.15 The ratio of GSH/GSSG is not in equilibrium under oxidative stress,16 therefore, detection of GSH and GSSG is critical to understand physiological functions and the development of clinical diagnoses.7,13,17 Over the years, numerous enzymatic, spectrophotometric, fluorometric and electrochemical methods have been developed for the quantification of GSH, GSSG and total (tGSH) in biological samples.7,13,18-21 However, measurement protocols suffer from difficulties due to the auto-oxidation of GSH that generates GSSG which causes errors in the final quantification.22,23 The transformation of GSH occurs during sample collection,24 preparation,17,23 and analysis25,26 so derivatization strategies have been devised to allow speciation. 25-27

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The implementation of electrochemical methods for the determination of GSH, GSSG and tGSH at the clinical level can provide a suitable answer to the growing need for rapid, specific and inexpensive analysis.28 In general the current electrochemical methodologies for detection of GSH and GSSG with voltammetric technique have comparable performances regarding limit of detection29,30 but to the best of our knowledge, are used in conjunction with separation techniques to determine GSH/GSSG ratios.31-33 They show adequate analytical parameters in buffered solution and in some biological samples for the assessment of GSH.28,34,35 Few studies were reported in the literature on the simultaneous detection of GSH and other significant analytes. Cu hydroxide nanomaterials applied in conjunction with carbon ionic liquid have been proposed for the detection of GSH and GSSG in human plasma.36 However, the concentration of GSH and GSSH were measured in diluted human plasma, without induced oxidative stress, and without any comparison with other analytical techniques. Also, the simultaneous detection of GSH and GSSG using HPLC and electrochemical detection has been demonstrated for rat hepatocytes and cardiomyocytes.37 Finally, the simultaneous detection of GSH and acetaminophen has been also previously reported.38 We demonstrate in this study the simultaneous determination of GSH and GSSG without the use of a separation technique under conditions of oxidative stress. To do so, we develop a Cmodified electrode and demonstrate its application to determine the GSH/GSSG ratio in model organisms, under conditions of oxidative stress induced by Pb intoxication.39 We use a C-modified electrode with Co phthalocyanine for the direct determination of GSH and GSSG. Electrodes modified with metallopthalocyanines have been extensively developed for the electrochemical oxidation of a variety of biologically relevant thiols.31,33,40-44 They also have been reported as having substantial electrocatalytic activity for the electroreduction of disulfide GSSG at physiological pH.32,45,46 The electrocatalytic effect promoted by cobalt phthalocyanine, CoPc, can be enhanced and improved by incorporating carbon nanotubes. 47 The main contribution of carbon nanotubes in electroanalysis studies are attributed to their efficiency in charge transfer and giving greater electroactive area in such a way that the signals are significantly improved.28,48 In this study, we focus on the independent measurement of GSH and GSSG under conditions of oxidative stress in biological fluids without analytical separations. However, we demonstrate the application of our novel electrode in two types of biological fluids from model organisms, Wistar rats, under biologically relevant conditions. Our study includes the use of control samples without oxidative stress to validate the detection of GSH and GSSG simultaneously under conditions of Pb intoxication, that were previously demonstrated to cause oxidative stress.39 We will show that our technique does not suffer from interfering sensing of ascorbic acid, AA and cysteine, Cys, which are also involved in the chemical profiling of oxidative stress.. The ACS Paragon Plus Environment


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performance of the developed sensor was compared to the classical spectrophotometric assay for GSH. EXPERIMENTAL Chemicals. Cobalt(II) phtalocyanine (Co(II)Pc), reduced glutathione (GSH) γ-L-Glutamyl-LCysteinyl-glycine, oxidized L-glutathione, L- ascorbic acid, L- cysteine, L-glutamic acid, D (+) – glucose, Glycine, 1-5 µm and 15±5 nm diameter multiwalled carbon nanotubes were purchased from Sigma Aldrich and were analytical grade. Lead (II) thihydrate acetate analytic grade was from EMSURE, KCl and NaCl salts from Karal and KH2PO4, Na2HPO4, HNO3, DMF, and K4Fe(CN)6 from J. T. Baker. Purification and functionalization of MWCNT. The MWCNT were oxidized with nitric acid to remove metal impurities, increase MWCNT solubility and functionalize MWCNT with carboxylic groups, as previously reported.49,50 The procedure follows: 100 mg of MWCNT were added to 10 mL of 16 M HNO3 and sonicated for one hour. The suspension was then refluxed for 12 hours with constant stirring at a temperature of 80°C; then it was rinsed several times with water until reaching neutral pH. The MWCNT were centrifuged at 4500 rpm for 15 minutes to remove as much water as possible and immediately put to dry in a furnace at 70°C for 7 hours in air. These carbon nanotubes are named MWCNTf and were used in for the subsequent ink preparation. 49,50 Electrochemical experiments. The electrochemical studies were conducted with an Epsilon EC potentiostat/galvanostat or a CHI 660 Electrochemical Workstation (CH Instrument, Austin TX). A conventional three-electrode cell was used. A glassy carbon (diameter = 3 mm) was used as a working electrode (GCE), a platinum wire as an auxiliary electrode and an Ag/AgCl (3 M KCl) as a reference electrode. All potentials are given with respect to this Ag/AgCl reference. Unless otherwise stated, the electrochemical experiments were performed in a deaerated 0.1 M buffer phosphate solution at pH 7.4. Preparation of the modified electrode p The ink preparation was done in two steps: 1) the cobalt phthalocyanine mass was weighed for each of the concentrations (1, 5, 10 and 50 mM) then placed in a 5 ml flask to which approximately 500 μl of DMF (dimethyl formamide) were added to dilute the cobalt phthalocyanine complex and water to the volume mark; the mixture was placed in the ultrasonic bath for 5 min. 2) 1 mg of CNTMWf was placed into a 2 ml Eppendorf tube and 1 ml of the corresponding cobalt phthalocyanine solution was added. The suspension was placed in the ultrasonic bath for 30 min. GCE was freshly polished with alumina in decreasing sizes of 1, 0.3 and 0.05 m and finally rinsed with deionized water and sonicated in ethanol for 5 minutes. The electrodes were characterized by CV in 5 mM K4Fe(CN)6 in a range of -0.2 to 0.7 V potential at a scanning rate of 100 mV/s to obtain a CV characteristic of a ACS Paragon Plus Environment

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clean GCE. Then the electrode was modified by placing 10 µL of the suspension on the top of GCE substrate and dried in an oven at 80°C for 10 min to form the nanocomposite CoPc/MWCNTf on the GCE surface. The electrodes were stored in air at room temperature until they were used for the analysis of samples. Nanocomposite film characterization. The morphology and composition of the nanocomposite CoPc/MWCNTf on the surface of the GCE were characterized by scanning electron microscopy on a Hitachi model S-4700 type II equipped with an Oxford instrument and an EDXS detector. Film thickness was evaluated with a Stylus Profilometer Model D-100 (KLA Tecnor). Biological model. The protocol for the animal manipulation has been reported in detail by some of us.39 Briefly, three male Winstar rats were intoxicated for two weeks with a dose of 50 mg/kg of lead acetate. The Pb was administered orally (gavage tube feeding). This feeding should be done according to ethical standards and a specially trained technician supervised this activity at the University of Guanajuato. An early daily dose was administered daily, the dose was 50 mg of lead acetate/kg of weight, which amounts to 5 - 10 mg in an approximate volume of 1 ml. The rats were kept in their plastic cages, fed a commercial diet, and purified water supplied ad libitum, and were housed under a 12 h light-dark cycle. Lead contaminated waste was collected by specialized service. At the end of the 2 week treatment, 15 mL of urine samples were taken from each rat and immediately deoxygenated, sealed and centrifuged at 5000 rpm/10 min. The samples were refrigerated at 4°C for the following glutathione determinations. Also, blood samples were taken in tubes with EDTA to avoid coagulation, separating the plasma centrifuging the sample at a 3,500 rpm for 10 min; the samples are deoxygenated and refrigerated at 4°C for the following glutathione determinations.51 The samples were deoxygenated to minimize complications from the oxidation of GSH. The electrochemical experiments were performed in solutions purged from O2 at room temperature. In the optimized procedure, samples were diluted in PBS with a dilution factor 3 for plasma and 2 for urine because of the high viscosity of the biological samples. All measurements were performed at room temperature. Additional details are given in the supporting information. Spectrophotometric determination of GSH. We used a procedure based on the derivatization of GSH with N-ethylmaleimide (NEM).

The method is based on the rapid

quenching of free thiol groups by NEM. The detailed procedure has been reported elsewhere 23,25 Briefly, approximately 15 mL of urine and 5 mL of blood were obtained before and after lead intoxication. The samples were dropped into a tube (with EDTA in the blood case), and immediately centrifuged at 3500 rpm for 10 min; after rapid mixing, 3 mL of each sample was reacted with 150 µL 350 mM NEM. After 25 s of NEM treatment, both samples were diluted with buffer PBS 1:1 (v/v) and centrifuged for a second time at 3500 rpm for 5 min. Known ACS Paragon Plus Environment


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concentrations of GSH were added to PBS, blood and urine before NEM treatment. The method relies in the decrease of the UV-Vis absorbance at 300 nm, due to the change of double bond to single bond in the NEM molecule after derivatization with GSH. Calibration curves (0.5 – 7 mM) were prepared in triplicate. Additional details on the parameters of this method are given in the supporting information. RESULTS AND DISCUSSION Electrochemical characterization of the hybrid electrode: CoPc/MWCNTf/GC. The optimization of the modified electrodes is particularly important for elaborating electrochemical sensors. MWCNTf (1mg mL-1)-CoPc suspensions were prepared at different concentrations of the metallophthalocyanine (1, 5, 10, 50 mM) to yield nanostructured composites of different composition. This study is thus aimed at analyzing the correlation between the concentrations of CoPc and the amount of actives sites from electrochemical characterizations in buffer phosphate solution (PBS, pH 7.4). The electrochemical behavior of the obtained nanocomposite electrode evaluated by cyclic voltammetry is illustrated in Figure 1. The cyclic voltammograms exhibit a characteristic peak for a redox couple located at -0.60 V vs. Ag/AgCl 1M KCl that is assigned to the Co(II)/Co(I) reversible process of the adsorbed CoPc. This potential corresponds to the apparent formal potential which was reported from the same CoPc in basic and neutral aqueous solution .41,44,52 This peak shows a reversible process only for 1, 5 and 10 mM concentration of the complex (in the suspension). At higher concentrations, and notably at 10 mM, the peak current values are similar to that obtained at 5 mM. Above 10 mM, a substantial decrease of the peak current is observed probably because of the formation of precipitates of CoPc crystallites from the suspension over the GCE surface leading to non homogeneous partition of the complex. Another less defined peak (-0.170 V) of low intensity is also observed and can reflect changes in the double layer capacity in this potential region due to the presence of CoPc precipitates.32 The use of a dispersion of 1mg mL-1 MWCNTf and 1 mM CoPc was considered as optimal for this work based on a previous study on the effect of the film thickness (and thus larger number of embedded metallocomplexes) on the electrocatalytic activity of the modified electrodes towards thiol oxidation and disulfide reduction:

42

the experiments showed that thicker films do not

necessarily provide larger catalytic effect (in terms of current intensity) towards both thiol oxidation and disulfide reduction. We thus decided to use lower concentration of cobalt phthalocyanine. Additionally, with the lower amount of metallocomplex, decreases the capacitive current and minimizes the cost of the sensor. ACS Paragon Plus Environment

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From the intensity of the anodic peak current (Figure 1), the amount of the apparent coverage of the electroactive CoPc sites was estimated to  = 3.33 × 10-11 mol cm-2 (considering the geometrical area of the electrode).

I(A) 60 40 20 0 -20 PBS 1mM 5mM 10mM

-40 -60 -80 -1.0

-0.5

0.0

0.5

1.0

E/V vs Ag/AgCl Figure 1. Cyclic voltammograms collected at hybrid electrode CoPc-MWCNTf /GC for different concentrations of CoPc (1, 5, 10, 50 mmol L-1) in a phosphate buffer solution pH 7.4. Scan rate 100 mV s-1.

Physical characterization of the hybrid electrode CoPc/MWCNTf /GC. Figure 2A shows the SEM image of the nanocomposite CoPc/MWCNTf forming a porous coating on the glassy carbon surface. The EDS mapping (Figure 2B) shows that while there are some areas of higher Co content (bright dots in the false color image), it is homogenously distributed over the film indicating a good dispersion of CoPc onto the surface. X-ray dispersion energy spectrum (Figure 2C) shows that the elemental composition is constituted by C atoms (0.280 keV), O (0.527 keV), Co (0.790, 6.90 keV), Mo (2.30, 2.80 keV), and Fe (6.37 keV). These last two elements are impurities assigned to the synthesis of the commercial MWCNT that were not fully removed during the functionalization of MWCNT. 46



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Figure 2. (A) SEM image of the composite CoPc-MWCNT. (B) False color plot of Co mapping; (C) EDS analysis of the composite CoPC-MWCNT and composition of the elements detected on the modified electrode.

The morphology of the electrode is discussed in the supporting information. Figure S1 shows the profilometry results of CoPc/MWCNTf /GC electrode, before and after their electrochemical characterization in the presence of GSH. Based on these results, the mean thickness values are 2.2 and 1.7 µm, respectively. As the change in the thickness is minimal, one can conclude that the nanostructured adsorbed is stable during the electrochemical measurement and the required electrode manipulation. Electrocatalytic behavior of CoPc/MWCNTf /GC electrode towards GSH and GSSG. Figure 3 shows the forward voltammetric scan obtained with CoP/-MWCNTf adsorbed on a GC electrode in the presence of 3 mM GSH (Figure 3A) and 3 mM GSSG (Figure 3B) and the cyclic voltammograms in the presence of both of them (Figure 3C).


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For GSH in deaerated PBS solution (pH 7.4), during the positive potential scan, from -1.2 V to 0.4 V, a well-defined peak is observed at ca. 0.110 V.

This peak is assigned to the

electrocatalytic oxidation of GSH.32,53 at the modified electrode. The voltammetry performed in a solution of GSSG shows a reduction peak observed at ca -0.8 V, attributed to the electrocatalytic reduction of GSSG at the nanocomposite modified electrode.32 Note that the CVs in the absence of GSH and GSSG only show the redox process related couple Co(II)/Co(I)Pc. To further investigate GSH and GSSG in biological samples, experiments were performed with the mixture of both species (GSH/GSSG). Figure 3C shows the cyclic voltammogram of the nanocomposite electrode in an equimolar solution of GSH and GSSG (3 mmol L-1). The voltammetric peaks for the oxidation of GSH and the reduction of GSSG at ca. +0.1 V and -0.8 V, respectively, are both clearly visible.

Figure 3. Linear voltammograms of the modified electrode CoPc / MWCNT / GC in pH 7.4 PBS buffer, (A) 3 mM glutathione GSH, (B) 3 mM GSSG and (C) the mixture of both 3 mM GSH and GSSG. Scan rate 100 mV s-1. All studies were made under a nitrogen atmosphere.



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Detection and quantification of GSH and GSSG. Figure 4 shows the simultaneous detection of GSH and GSSG at different concentrations by DPV. In both cases, a constant increase in the peak current intensity as a function of concentration is observed. The peak at -0.10 V corresponds to the oxidation of the GSH and that at -0.72 V corresponds to the GSSG reduction. The peak related to the Co(II)/Co(I) redox process appears at ca. -0.5 V and shifts to positive direction in presence of GSH. Its intensity decreases with increased concentration of GSH/GSSG. The Co peak is expected to shift because Co is a mediator on the redox processes involved. The current is a function of the exchange rate of the Co redox centers with the analyte, e.g., GSH, and of the concentration of the analyte in solution. Therefore, changes in the Co2+/1+ peaks are expected because the oxidation of the surface confined species of CoPc is competing with electron exchange between the CoPc and the analyte in solution. Calibration curves are shown in Figure 4B and 4C for GSH and GSSG, respectively. The limits of detection LOD are 100 µM for GSH and 8.3 µM for GSSG while the limits of quantification LOQ are 280 µM for GSH, and 29 µM for GSSG. The stability of the sensor was checked by performing 150 measurements in presence of 500 µM GSH and 200 µM GSSG in phosphate buffer pH 7.4 and no change was observed in their response. When stored at ambient temperature, no significant difference in the results was observed for at least one month. The sensor also presents satisfactory reproducibility for the GSH and GSSG determinations. The relative standard deviation (RSD%) for ten determinations of 500 µM GSH and 200 µM GSSG was 1.5% and 1.7% respectively. Also, a series of 5 sensors were prepared in the same manner and tested in 500 µM GSH and 200 µM GSSG and responses with a relative standard deviation of 2.3% and 2.8% were obtained respectively. All these analyses show the good repeatability and stability of the sensor in synthetic solutions of both analytes.


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Figure 4. A) Differential pulse voltammetry analysis at CoPc/MWCNTf/GC electrode using various GSH (0.5 mM, 1 mM, 3 mM, 5 mM y 7 mM) and GSSG (150 µM, 500 µM, 1 mM, 2 mM, 3 mM) concentrations mixed in a synthetic solution. Pulse height 50 mV, width 2 mV B) GSH and C) GSSG calibration curves. DPV parameters: Pulse amplitude: 50 mV, Increment E: 4 mV, Pulse width: 50 ms, sample period: 1 line period, pulse period: 200 ms.

Feasibility study of the simultaneous detection of GSH and GSSG in biological samples. GSH and GSSG simultaneous analysis in Wistar rats urine and plasma samples were carried out to validate the use of the nanocomposite modified electrode in these complex matrices. The samples were spiked with known amount of GSH (0.5, 1, 3, 5 and 7 mM) and GSSG (0.15, 0.5, 1, 2 and 3 mM) and the detection limit (LOD) and quantification limit (LOQ) were determined in the same conditions by DPV as described above. Due to the high viscosity of the biological samples, these were diluted in PBS with a dilution factor 3 for plasma and 2 for urine. From the calibration curves obtained in LOD was determined using the LOD = 3 (noise) / slope of the linear regression equation, similarly, the LOQ was estimated using the equation LOQ = 10 (noise) / slope of the linear regression equation. The sensitivity was the slope of the calibration lines. Table 1 shows the obtained results for LOD, LOQ and sensitivities. They are similar to that obtained in deareated phosphate buffer solution (pH 7.4), showing that the performances of the sensor is not drastically affected by the matrix effects, on the timescale of the experiments. We note that there is a difference in the limit of detection and the sensitivity of GSH and GSSG. Although we have not ACS Paragon Plus Environment


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studied the differences in detail, we have shown that for the detection of GSSG in CV and pulsed voltammetry, the higher background currents complicate the measurement. Also, there could be kinetic difference in the redox mediation of the GSH and GSSG, as the peak currents, higher for GSH in both electroanalytical techniques for GSSG suggest. The result is that the LOD, LOQ and sensitivities are higher for GSH than for GSSG for buffer, blood and urine samples. Table 1. Results for the simultaneous detection of GSH and GSSG in PBS, Urine, and Plasma using DPV. Biomarker

LOD

LOQ

Sensitivity (µA/mM)

PBS

Urine

Plasma

PBS

Urine

Plasma

PBS

Urine

Plasma

GSH/ µM

100

130

220

280

320

470

9.7

9.2

6.4

GSSG/ µM

8.3

16.6

28.5

29

45

71

1.3

0.72

0.42

Interference study. The potential interference from ascorbic acid, glycine, cysteine, glutamic acid, and glucose was investigated, given their presence in biological samples of interest. Solutions of 2 mmol L-1 of each of these components were prepared under the same conditions as the mixture of 1 mmol L-1 GSH/GSSG. The sensor response was monitored against each substance individually and subsequently added to the mixture solution of GSH/GSSG in a 1:1 v/v. As it can be seen on Figure 5, when the potential sweep is performed between 0.2 V and 1.0 V, ascorbic acid and cysteine are oxidized at 0.55 V and 0.79 V, respectively. It is important to note that the signals corresponding to the reduction process of the GSSG (E = -0.75 V), the redox process of CoPc (E = -0.56 V) and the oxidation of GSH (E = -0.10 V) are still visible regardless of the presence of interferences. No electrochemical response was observed for glutamic acid and glucose. To avoid interference from ascorbic acid and cysteine, the anodic direction potential sweeps were made from -0.90 V to 0.20 V, and the cathodic direction sweeps were made from 0.40 V to -0.90 V.


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 20

GSH

GSSG

Ascorbic acid (AA) PBS pH 7.4 Cysteine (Cys) GSH+GSSG (synthetic sample) Wistar rats plasma Wistar rats urine

Cys

10

(II)

(I)

Co /Co

AA GSGS

0

GSH

-0.5

0.0

E (V) / Ag/AgCl

0.5

1.0

Figure 5. Study of main interfering analytes: ascorbic acid and cysteine in the electrochemical analysis of the GSH and GSSG using the modified electrode CoPc-MWCNTf/GC. The DPV curves show the GSSG reduction process to E= -0.750 V. Below in anodic direction, the cobalt phthalocyanine redox process; E = -0.560 V, the GSH oxidation; E = -0.100 V, the ascorbic acid oxidation; E = 0.550 V and the Cysteine oxidation; E = 0.790 V. DPV parameters as on Figure 4. Simultaneous Analyses of GSH and GSSG in Wistar Rat plasma and urine samples as biological matrixes in lead toxicity analyses. In the present study, the antioxidant biomarkers GSH and GSSG have been considered to evaluate the phenomenon of oxidative stress in Wistar rats. The analysis was carried out on three urine samples and three plasma samples each in triplicate and the results are presented as the mean ± SD of n independent experiments. For comparison, the samples were analyzed using spectrophotometric assay with NEM (N-Ethylmaleimide). The results obtained for the proposed electrochemical method and the spectroscopic allow reaching similar performances (Table 2), promoting a satisfactory level of reliability between the measurements. The data related to the effect of lead intoxication on the GSH and GSSG levels in the organism, pre- and posttreatment with lead acetate, are presented in Table 2. The mechanism of lead toxicity is not clearly understood,12 and in our samples, the independent evaluation of GSH and GSSG concentration shows a significant increase in GSH concentration. Also, the GSSG levels in both urine and plasma (post-intoxication) increase with respect to the values of the control samples (pre-intoxication). In general, the variation in GSH levels depends on the animal (living) organism under study, the ACS Paragon Plus Environment


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process of intoxication, and/or pathophysiology and on the type of biological fluid that is analyzed.12,18,54,55 In Wistar rats, it has been reported that under oxidative stress, GSH level increases in plasma,56 serum,57 skeletal muscle, 58 lumbosacral spinal cord,59 kidney60 and liver,58 while it decreases in sciatic nerve,61 liver,60,62 and brain.62 The increase of GSSG level can be connected with the cellular redox activity to protect the tissue against oxidative damage. Indeed, it was reported that GSSG level increases in plasma, lung and brain,

56

liver

56,62

and kidney.60

Thus the results obtained here, for each biomarker in post-intoxication samples can be considered as a consequence of exposure to lead. Exposure to Pb causes multiple pathologies including renal10 dysfunction, triggering oxidative stress and at the same time the stimulation of the antioxidant defense mechanism increasing the secretion of GSH which protects ROS cells and generated GSSG as a product is considered as a metabolite marker of cellular damage.12,54 The GSH/GSSG ratio is then considered as an important indicator that can be used to establish the oxidative status.59,63-65 Using an intoxidation model, Wistar rats have been intoxicated to generate oxidative stress. Previous studies have reported a decrease of GSH/GSSG ratio in plasma (control 10; post injury 5),66 liver (control 4.8; post-injury 2.9), brain (control 4.8; post-injury 2.9) and (control 4.5; post-injury 2.7),62 kidney (control 4 ; post-injury 1.5),60 and also lung (control 10.3; post-injury 7)56. Our results using the electrochemical sensor in plasma also show a decrease of GSH/GSSG ratio after lead intoxication (control 4.5; post-intoxication 2.6). This demonstrates the satisfactory application of the proposed simple electroanalytical method for the detection and quantification of GSH and GSSG and the GSH/GSSG ratio in plasma and urine samples of Wistar rats, which opens the possibility for use in future toxicological studies.


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Table 2. GSH and GSSG concentration values ± SD obtained by the proposed electroanalytical method. Also the variation in the GSH/GSSG ratio in Wistar rats’ plasma and urine analysis pre and post lead intoxication. GSH and GSSG concentration values Wistar rats plasma

SAMPLES

GS-H /mM  

GS-H /mM  

GS-SG /mM  

GSH/GSSG

Spectrophotometric assay

Plasma control Post-intoxication 1 Post-intoxication 2 Post-intoxication 3 SAMPLES

1.11 2.18 3.14 2.08

± 0.07 ± 0.02 ± 0.03 ± 0.02

1.92 2.98 3.79 2.56

± 0.18 ± 0.13 ± 0.15 ± 0.12

0.247 0.966 0.979 0.925

± 0.006 ± 0.002 ± 0.001 ± 0.001

GSH and GSSG concentration values Wistar rats urine GS-H /mM   GS-H /mM   GS-SG /mM  

4.5 2.3 3.2 2.2 GSH/GSSG

Spectrophotometric assay

Urine control Post-intoxication 1 Post-intoxication 2 Post-intoxication 3

0.13 ≈ LDa) 0.65 0.76 0.57

± 0.01

0.37

± 0.03

Simultaneous Electrochemical Speciation of Oxidized and Reduced

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