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Total low-molecular-weight antioxidants as a summary parameter, quantified in biological samples by a chemiluminescence inhibition assay

Abstract

Aerobic metabolism requires a complex antioxidative system to balance reactive oxygen species (ROS). When in excess, ROS disrupt cellular activities and molecular structures. Owing to the variety of ROS, there are different antioxidative activities that require various tests for their detection. The so-called 'total antioxidative capacity' inhibition assay presented in this paper can be used to quantify the overall activity of low-molecular-weight antioxidants (AOs) in biological samples. The assay is based on enhanced horseradish peroxidase–catalyzed luminol chemiluminescence. It can be fine-tuned so that the biological samples meet the requirements of the light detector. A detailed protocol describing all relevant parameters is provided. The procedure is quick, inexpensive and reproducible. The assay can be used with diverse biological materials such as plant extracts and blood plasma. Hence, it is applicable to fields as diverse as crop breeding, medical diagnostics or food sciences. The time needed for the assay depends on the number of samples and their AO content. The protocol takes one working day to complete when five samples with five replicates are measured sequentially.

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Figure 1: The enhanced HRP cycle.
Figure 2: TAC assay procedure and data evaluation.
Figure 3: On a log-log scale, recovery time and amount of a pure antioxidant are linearly correlated.
Figure 4: TAC of different molecular-weight fractions (whole sample, filtrate and dialysate) from biological materials.
Figure 5: Changes in TAC in Lepidium sativum during abiotic stress treatment (colored columns) and after recovery (gray columns).
Figure 6: TAC of cell culture medium.

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Acknowledgements

We thank L. Shaw (Kiel) for critically reading the paper. We also thank S. Vollbehr, G. Weppner and S. Anderson for technical assistance; J. Scheller (Institute for Biochemistry, University of Kiel) for providing HepG2 cells; and A. Scheidig (Structural Biology Group, Kiel) and U.-P. Hansen (Biophysics Group, Kiel) for their generous support. We gratefully acknowledge the financial support of the Deutsche Forschungsgemeinschaft (Grant no. PL253/5), as well as access to the core facilities of the Zentrum für Biochemie und Molekularbiologie, Christian-Albrechts-Universität, Kiel.

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L.S. carried out the experiments reported in the main paper, performed data processing and participated in amending the draft. C.P. conceived of the protocol, carried out the experiments shown in the supplementary information and wrote the paper. Both authors approved the final version.

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Correspondence to Christoph Plieth.

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Supplementary Fig. 1

Decay of the antioxidative capacity of pure antioxidants (PDF 15 kb)

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Saleh, L., Plieth, C. Total low-molecular-weight antioxidants as a summary parameter, quantified in biological samples by a chemiluminescence inhibition assay. Nat Protoc 5, 1627–1634 (2010). https://doi.org/10.1038/nprot.2010.120

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