Abstract
The purpose of the present study was to employ two methods—square wave voltammetry (SWV) performed on screen printed sensors and ferric reducing antioxidant power (FRAP)—as suitable tools for the assay of low-molecular-weight antioxidants (LMWAs). LMWAs were assayed by both methods and the resulting data were statistically compared. Plasma samples from five Cinereous vultures accidentally intoxicated with lead were used to represent real biological matrices with different levels of LMWAs. Blood was collected from the birds prior to and one month after treatment with Ca-EDTA. SWV resulted in two peaks. The first peak, with the potential value of 466 ± 15 mV, was recognized as ascorbic and uric acids, while the second one (743 ± 30 mV) represented glutathione, tocopherol, ascorbic acid and in a minor effect by uric acid, too. Contribution of individual antioxidants was recognized by separate assays of LMWA standards. Correlation between peaks 1 and 2 as well as the sum of the two peaks and FRAP was analysed. While peak 1 and the sum of peaks were in close correlation to FRAP results (correlation coefficient of 0.97), the relation between peak 2 and FRAP may be expressed using a correlation coefficient of 0.64. The determination of thiols by the Ellman assay confirmed the accuracy of SWV. Levels of glutathione and other similar structures were stable in the chosen model and it may be concluded that SWV is appropriate for assay of LMWAs in plasma samples. The methods employed in the study were advantageous in minimal sample volume consumption and fast acquisition of results.
Highlights
Low-molecular-weight antioxidants (LMWAs) are a wide group of quite small molecules providing electrons to oxidizing agents which protect other molecules from oxidation in this way
Trolox®, reduced glutathione, ascorbic acid and uric acid (Sigma-Aldrich, Prague Branch, Czech Republic) were assayed as the standard physiological antioxidants expected to be present in blood and plasma samples
The position of peaks was investigated in order to obtain standard values of redox peaks on the used screen printed electrodes and 1 mM antioxidants solutions in phosphate buffered saline (PBS)
Summary
Low-molecular-weight antioxidants (LMWAs) are a wide group of quite small molecules providing electrons to oxidizing agents which protect other molecules from oxidation in this way. Apart from LMWAs of non-enzymatic nature, the endogenous antioxidant defences include enzymatic antioxidants critical for the control of reactive-molecular-species-mediated oxidative damage of biomolecules. Enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) may be mentioned as typical examples of enzymatic antioxidants [2,3]. It is common for the enzymatic antioxidants be assayed using standard biochemical procedures [4]. LMWAs are assayed using different analytical methods for functional groups in the antioxidant molecule and/or its redox power
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