Short communication
Exploration of the global antioxidant capacity of the stratum corneum by cyclic voltammetry

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Abstract

Cyclic voltammetry is proposed as a new method for evaluating the antioxidant capacity of skin based on the reducing properties of low molecular weight antioxidants (LMWA). Experiments were performed simply by recording the anodic current at 0.9 V/SCE of a platinum microelectrode placed directly on the epidermis surface without any gel or water. This method ensured a direct, rapid (less than 1 min), reliable (accuracy 12%) and non-invasive measurement of the global antioxidant capacity of the stratum corneum with a high spatiotemporal resolution. At the same time, the pH of the skin surface was determined by recording the cathodic current at 0 V/SCE. Based on an exploratory study involving nine volunteer subjects, the evolution of the amperometric response of the microelectrode with time revealed a periodic modification of the redox properties.

Introduction

Oxidative stress has been one of the most widely studied biochemical processes for several years as it has been considered as an entity similar to the inflammation syndrome. It is involved in the aging process and is also the cause of cataracts, cancers, cardiovascular or degenerative diseases (Parkinson's, Alzheimer's) [1], [2]. Oxidative stress is the consequence of an imbalance between pro- and antioxidant species. The former essentially concerns highly reactive oxygen or nitrogen species (ROS or RNS), e.g., superoxide anion O2radical dot, hydrogen peroxide H2O2, hydroxyl radical OHradical dot, nitric oxide NO or peroxynitrite ONOO [3]. Naturally produced by the organism, these metabolites play a role in many physiological processes: cell respiration, neurotransmission, blood vessel dilatation, cytochrome P450-mediated or immune reactions [4], [5], [6]. Nevertheless, excessive production leads to the oxidation of lipids, proteins and nucleic acids, and contributes to the cellular dysfunction and death [7]. To avoid oxidative stress, cells are equipped with two major antioxidant defense systems. The first concerns enzymes which catalyze ROS or RNS degradation, such as superoxide dismutase, catalase or glutathione reductase. The second involves low molecular weight antioxidants (LMWA) like Vitamins A, C, E and glutathione, which reduce ROS and RNS by oxido-reduction reactions [8], [9]. Skin, which constitutes the interface between the human body and the environment, is the largest organ and its outermost barrier. It represents a major target of oxidative stress since it is continuously exposed to external oxidant aggressions, such as ultraviolet (UV) radiation, ozone or chemicals. Repeated exposure to such aggressions is responsible for skin damage and contributes to the development of cutaneous diseases like psoriasis and cancers [10]. The epidermis provides the first line of defense against exogeneous ROS by means of many antioxidant systems [11].

Numerous analytical techniques are available to evaluate oxidative stress namely, electron spin resonance, chromatography, spectroscopy or mass spectrometry [2], [12], [13], [14], [15]. All these techniques need expensive materials, involve complex protocols, often need tissue biopsy and provide delayed results. Electrochemistry has been considered for a few years as a promising alternative approach. Electrochemistry deals with electron transfer phenomena between an electrode and oxidized or reduced molecules. It therefore represents a suitable methodology to study the redox properties of solid, liquid or gaseous media and to detect oxidant and antioxidant species. Cyclic voltammetry was for example, tested to evaluate the global antioxidant capacity of real samples like wine, biological fluids or tissues [16], [17], [18]. The anodic part of the voltammogram provided information concerning the ability of LMWA to transfer electrons to an anode, and thus to act as reducing agents. Kohen recently applied this technique successfully to skin analysis [19], [20]; nevertheless, the method used suffered from two major drawbacks. Firstly, the analysis was either invasive because it involved skin homogenates, or indirect as it was performed in an electrolytic solution in contact with the skin surface. Secondly, the measurements used macroelectrodes with surface areas in the range of a square centimeter; they presented low sensitivity and did not allow localized measurements. The present paper shows preliminary works allowing the direct evaluation of the global antioxidant capacity of the stratum corneum. Cyclic voltammograms were performed directly on the skin surface without adding water or gel. Platinum disk microelectrodes with surface areas lower than 2 × 10−3 mm2 were used in order to perform rapid, sensitive, non-invasive and localized measurements on the skin surface. The accuracy of the amperometric signal was studied, as well as the evolution of the electrochemical response in time.

Section snippets

Chemicals

All chemicals were reagent grade and used as-received. Potassium ferricyanide K3[Fe(CN)6] and potassium chloride KCl were purchased from Acros Organics (Noisy Le Grand, France). Potassium dihydrogen phosphate KH2PO4 and dipotassium hydrogen phosphate K2HPO4 were purchased from Sigma–Aldrich (Lyon, France). Unless otherwise indicated, all solutions were prepared in phosphate buffer (KH2PO4/K2HPO4 0.1 mol L−1, pH 7.0).

Apparatus and material

The electrochemical experiments were carried out with a PAR model 283

Electrochemical determination of the electrode radius

The radius of the platinum disk microelectrodes was determined by plotting the current-potential curve in a 5 mmol L−1 deaerated ferricyanide solution. Fig. 1 shows the steady-state voltammogram corresponding to the electrochemical reduction of ferricyanide into ferrocyanide: Fe(CN)63− + e  Fe(CN)64−. The current generated at a microelectrode is dependent on its geometry. For a disk microelectrode, the limiting current is directly proportional to the disk radius r (cm) [22] : Ilim = 4nFrDC, where Ilim

Conclusion

Cyclic voltammetry allowed direct, rapid, non-invasive, precise and high spatiotemporal resolution measurements to be made on the skin surface by using ultramicroelectrodes. The anodic current recorded at 0.9 V/SCE on platinum was correlated to the global antioxidant capacity of the epidermis surface, whereas pH was determined simultaneously at 0 V/SCE. Involving a multipotentiostat allowed the validation of the measurement and to estimate the mean accuracy at 12%. Evolution of the anodic

Acknowledgments

The authors thank Stéphane Arbault (Ecole Normale Supérieure de Paris) for giving us experimental details concerning microelectrode fabrication and Emilie Pages and Jennifer Natan for the help in experimental works.

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