Electrochemical monitoring of native catalase activity in skin using skin covered oxygen electrode (original) (raw)
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Microchimica Acta
Elevated amounts of reactive oxygen species (ROS) including hydrogen peroxide (H2O2) are observed in the epidermis in different skin disorders. Thus, epidermal sensing of H2O2 should be useful to monitor the progression of skin pathologies. We have evaluated epidermal sensing of H2O2 in vitro, by visualising H2O2 permeation through the skin. Skin membranes were mounted in Franz cells, and a suspension of Prussian white microparticles was deposited on the stratum corneum face of the skin. Upon H2O2 permeation, Prussian white was oxidised to Prussian blue, resulting in a pattern of blue dots. Comparison of skin surface images with the dot patterns revealed that about 74% of the blue dots were associated with hair shafts. The degree of the Prussian white to Prussian blue conversion strongly correlated with the reciprocal resistance of the skin membranes. Together, the results demonstrate that hair follicles are the major pathways of H2O2 transdermal penetration. The study recommends th...
Acidic catalase in human skin in vivo: a new marker of permanent damage
Melanoma Research, 2009
Malignant melanoma incidence is increasing rapidly in Western countries. Its prevention requires a deep knowledge of the biological basis of the neoplasm leading to the identification of new biological risk markers. In in-vitro and ex-vivo models we demonstrated that catalase was modified not only in its activity but also in its charge properties after ultraviolet A irradiation through pheomelanin. Here we focus on the electrophoretic behaviour of catalase in the human skin in vivo, in association with cutaneous phototype. Zymographic analysis of the enzyme on skin biopsies from Caucasian population (phototype I-IV), collected from the trunk in autumn-winter, to exclude possible influences of an acute photoexposure, evidenced a protein doublet, representing the coexistence of two active isoforms of catalase with different charge properties. In the skin from low-phototype subjects, the percent contribution of the more acidic component of the doublet was prevalent, inversely correlated with total melanin concentration in hair, and associated with a high number of melanocytic nevi. In summary, this study shows for the first time the existence of an acidic catalase in association with clinically defined risk characteristics in low phototype skin in vivo, contributing to the knowledge of a new biochemical marker of cutaneous photosusceptibility. Melanoma Res 19:372-378 c 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins.
Exploration of the global antioxidant capacity of the stratum corneum by cyclic voltammetry
Journal of Pharmaceutical and Biomedical Analysis, 2006
OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible. This is an author-deposited version published in: http://oatao.univ-toulouse.fr/ Eprints ID : 3078 To link to this article : URL : http://dx.( 2006) Exploration of the global antioxidant capacity of the stratum corneum by cyclic voltammetry.
Noninvasive Potentiometric Method of Determination of Skin Oxidant/Antioxidant Activity
IEEE Sensors Journal, 2012
The article proposes theoretical and experimental justification for using potentiometry as a new noninvasive method of evaluating antio xidant/oxidant state (oxidative stress) of a skin. Since the inductor of o xidative stress is an overall deficit of electrons accessible to cells, electrochemical methods of evaluating this parameter are naturally considered as fully corresponding with the nature of the phenomenon. A mathematical model is proposed that describes the processes determining analytical signals in the 'skin-gel-electrode' system. The signal occurs as a result of interaction between antioxidants/oxidants diffusing from the epidermis of the skin into the gel and the mediator system o xidised or reduced component introduced into the gel. The in formation source with regard to AOA/OA (antio xidant/oxidant activity) is the electrode potential shift. It occurs when the gel co mes into contact with the skin. The series of potential determining substance-time dependences was obtained as a result of numerical simu lation and experiment. Typical relationships between different parameters (chemical reaction rate, gel layer thickness, time) and concentration equal to antio xidant or o xidant activity, (A OA or OA) were found. An agreement between the calculated and experimental data was obtained. Findings analysis enables to forecast features of the experimental relations and provided an opportunity to choose experimental conditions ensuring the most reliable results.
Study of catalase electrode for organic peroxides assays
Bioelectrochemistry, 2002
The catalytic activity of immobilized catalase (EC 1.11.1.6) for two model peroxide compounds (dibenzoyl peroxide and 3chloroperoxibenzoic acid) in a non-aqueous medium was used to prepare an organic-phase enzyme electrode (OPEE). The enzyme was immobilized within a polymeric film on spectrographic graphite. The amperometric signal of the enzyme electrode in substrate solutions was found to be due to the reduction of oxygen generated in the enzyme layer. The electrode response is proportional to peroxide concentrations up to about 40 AM within the potential range from À 450 to À 650 mV (vs. Ag/AgCl), and the response time is at most 90 s. The enzyme electrode retains about 35% of its initial activity after a 3-week storage at room temperature. D
Journal of Investigative Dermatology, 2006
The harmful effects of UVA radiation (320-400 nm) on the skin have been related to the generation of reactive oxygen species. Pheomelanin, the pigment characteristic of fair-skinned individuals, amplifies these effects. In vitro, in the presence of photosensitizing agents, UVA light produces singlet oxygen, which reacts with several targets. We have investigated a possible correlation between melanin-type and the antioxidant defense system after UV, focusing on the activities of superoxide dismutase and catalase, which correlated with the phototype of epidermal reconstructs. UVA was more effective than UVB in damaging these enzymatic activities, especially catalase. Furthermore, UVA irradiation induced a free-radical-mediated damage in the cells, leading to an oxidation of cell proteins. On catalase, synthetic pheomelanin amplified this effect on specific targets, such as residues of tryptophan and methionine. UVA irradiation of low phototype reconstructed epidermis and of U937 through synthetic pheomelanin induced a modification in the electrophoretic properties of native catalase, which was counteracted by histidine, a quencher of singlet oxygen. These results demonstrate that pheomelanin could act as a photosensitizing agent, following UVA irradiation, inducing charge modifications of native catalase, by a mechanism involving singlet oxygen or its downstream products.
Journal of Investigative Dermatology, 2006
The human epidermis is especially vulnerable to oxidative stress, which in turn leads to oxidation of important antioxidant enzymes, other proteins, and peptides. Molecular dynamic computer modelling is a new powerful tool to predict or confirm oxidative stress-mediated structural changes consequently altering the function of enzymes/proteins/peptides. Here we used examples of important epidermal antioxidant enzymes before and after hydrogen peroxide (H 2 O 2)-mediated oxidation of susceptible amino-acid residues (i.e. tryptophan, methionine, cysteine, and selenocysteine), which can affect enzyme active sites, cofactor binding, or dimerization/tetramerization domains. Computer modelling predicts that enzyme active sites are altered by H 2 O 2-mediated oxidation in thioredoxin reductase (TR) and acetylcholinesterase (AchE), whereas cofactor nicotinamide adenine dinucleotide phosphate (reduced form) binding is affected in both catalase and TR but not in glutathione peroxidase. Dimerization is prevented in catalase. These structural changes lead to impaired functionality. Fourier transform-Raman-and Fluorescence spectroscopy together with enzyme kinetics support the results. There are limitations of modelling as demonstrated on the AchE substrate-binding domain, where the computer predicted deactivation, which could not be confirmed by enzyme kinetics. Computer modelling coupled with classical biochemical techniques offers a new powerful tool in cutaneous biology to explore oxidative stress-mediated metabolic changes in the skin.
Analytica Chimica Acta, 2019
Antioxidants are important to protect and maintain biological barriers, such as the skin. Antioxidant effects are often assessed using clinical trials, however these tests are costly and time consuming. In this work we introduce a skin membrane-covered oxygen electrode (SCOE) as an in vitro tool for monitoring H 2 O 2 and antioxidant reactions in skin. The SCOE gives amperometric response to H 2 O 2 concentrations down to 0.05 mM. More importantly, the electrode allows measurements of polyphenol penetration and reaction with H 2 O 2 in skin. Measurements with SCOE show that lipophilic polyphenols such as quercetin, piceatannol, resveratrol, and plant extract from Plantago major impose their antioxidant effect in skin within 2-20 min. Rutin is however too hydrophilic to penetrate into stratum corneum and therefore cannot deliver its antioxidant effect during similar time interval. The measurements are interpreted considering polyphenol partition-penetration through stratum corneum and the reaction with the H 2 O 2-catalase system in the skin. The contribution of other enzymes cannot be ruled out and will be addressed in the future.
Archives of Dermatological Research, 1998
The purpose of this study was to elucidate the differential contribution of catalase and glutathione peroxidase (GSH-Px) to H 2 O 2 scavenging in cultured human dermal fibroblasts. Responses of the cells in terms of both enzyme activities were examined by using two sorts of inhibitors, 3-amino-1H-1,2,4-triazole (AT) for catalase and DL-buthionine-[S, R]-sulfoximine (BSO) for GSH-Px, under exposure to H 2 O 2 or ultraviolet (UV) B radiation. AT treatment resulted in a decrease in H 2 O 2 scavenging activity, while BSO treatment did not affect H 2 O 2 scavenging. When fibroblasts were exposed to a low concentration of H 2 O 2 (100 µM). AT treatment resulted in a significant decrease in cell survival, but BSO treatment did not affect survival. At higher concentrations of H 2 O 2 ranging from 500 µM to 1 mM, BSO-treated fibroblasts showed reduced survival. In addition, AT treatment was much more cytotoxic in the presence of UVB than BSO treatment. The intracellular levels of H 2 O 2 in fibroblasts treated with AT or BSO were also determined. BSO-treated cells showed similar H 2 O 2 levels to control cells, but the intracellular H 2 O 2 levels of AT-treated fibroblasts were 1.4-fold higher than found in control cells. These results with human dermal fibroblasts indicate that catalase acts as a primary defence against oxidative stress from exogenous or endogenous H 2 O 2 at low concentrations. In contrast, GSH-Px helps protect the cell from damage during exposure to high concentrations of H 2 O 2 .