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Open AccessArticle

Development of an Analytical Assay for Electrochemical Detection and Quantification of Protein-Bound 3-Nitrotyrosine in Biological Samples and Comparison with Classical, Antibody-Based Methods

1
Center for Cardiology, Department of Cardiology 1–Molecular Cardiology, University Medical Center, 55131 Mainz, Germany
2
Institute of Clinical Chemistry and Laboratory Medicine, Medical Center of the Johannes Gutenberg University, 55131 Mainz, Germany
3
Faculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia
4
Institute for Biological Research “Sinisa Stankovic”—National Institute of Republic of Serbia, University of Belgrade, 11000 Belgrade, Serbia
5
Partner Site Rhine-Main, German Center for Cardiovascular Research (DZHK), Langenbeckstr. 1, 55131 Mainz, Germany
*
Author to whom correspondence should be addressed.
Antioxidants 2020, 9(5), 388; https://doi.org/10.3390/antiox9050388
Received: 1 April 2020 / Revised: 30 April 2020 / Accepted: 2 May 2020 / Published: 6 May 2020
(This article belongs to the Special Issue Oxidative Stress, Classification and Quantitation)
Reactive oxygen and nitrogen species (RONS) cause oxidative damage, which is associated with endothelial dysfunction and cardiovascular disease, but may also contribute to redox signaling. Therefore, their precise detection is important for the evaluation of disease mechanisms. Here, we compared three different methods for the detection of 3-nitrotyrosine (3-NT), a marker of nitro-oxidative stress, in biological samples. Nitrated proteins were generated by incubation with peroxynitrite or 3-morpholino sydnonimine (Sin-1) and subjected to total hydrolysis using pronase, a mixture of different proteases. The 3-NT was then separated by high performance liquid chromatography (HPLC) and quantified by electrochemical detection (ECD, CoulArray) and compared to classical methods, namely enzyme-linked immunosorbent assay (ELISA) and dot blot analysis using specific 3-NT antibodies. Calibration curves for authentic 3-NT (detection limit 10 nM) and a concentration-response pattern for 3-NT obtained from digested nitrated bovine serum albumin (BSA) were highly linear over a wide 3-NT concentration range. Also, ex vivo nitration of protein from heart, isolated mitochondria, and serum/plasma could be quantified using the HPLC/ECD method and was confirmed by LC-MS/MS. Of note, nitro-oxidative damage of mitochondria results in increased superoxide (O2•–) formation rates (measured by dihydroethidium-based HPLC assay), pointing to a self-amplification mechanism of oxidative stress. Based on our ex vivo data, the CoulArray quantification method for 3-NT seems to have some advantages regarding sensitivity and selectivity. Establishing a reliable automated HPLC assay for the routine quantification of 3-NT in biological samples of cell culture, of animal and human origin seems to be more sophisticated than expected. View Full-Text
Keywords: oxidative stress; peroxynitrite; protein-bound 3-nitrotyrosine; HPLC with electrochemical detection; mitochondrial superoxide oxidative stress; peroxynitrite; protein-bound 3-nitrotyrosine; HPLC with electrochemical detection; mitochondrial superoxide
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Vujacic-Mirski, K.; Bruns, K.; Kalinovic, S.; Oelze, M.; Kröller-Schön, S.; Steven, S.; Mojovic, M.; Korac, B.; Münzel, T.; Daiber, A. Development of an Analytical Assay for Electrochemical Detection and Quantification of Protein-Bound 3-Nitrotyrosine in Biological Samples and Comparison with Classical, Antibody-Based Methods. Antioxidants 2020, 9, 388.

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