TY  - JOUR
AU  - Vujačić-Mirski, Ksenija
AU  - Bruns, Kai
AU  - Kalinović, Sanela
AU  - Oelze, Matthias
AU  - Kröller-Schön, Swenja
AU  - Steven, Sebastian
AU  - Mojović, Miloš
AU  - Korać, Bato
AU  - Münzel, Thomas
AU  - Daiber, Andreas
PY  - 2020
UR  - https://www.mdpi.com/2076-3921/9/5/388
UR  - http://www.ncbi.nlm.nih.gov/pubmed/32384768
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/3672
AB  - 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.
T2  - Antioxidants (Basel, Switzerland)
T2  - Antioxidants (Basel, Switzerland)
T1  - 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.
IS  - 5
VL  - 9
DO  - 10.3390/antiox9050388
SP  - 388
ER  - 

@article{
author = "Vujačić-Mirski, Ksenija and Bruns, Kai and Kalinović, Sanela and Oelze, Matthias and Kröller-Schön, Swenja and Steven, Sebastian and Mojović, Miloš and Korać, Bato and Münzel, Thomas and Daiber, Andreas",
year = "2020",
abstract = "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.",
journal = "Antioxidants (Basel, Switzerland), Antioxidants (Basel, Switzerland)",
title = "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.",
number = "5",
volume = "9",
doi = "10.3390/antiox9050388",
pages = "388"
}

Vujačić-Mirski, K., Bruns, K., Kalinović, S., Oelze, M., Kröller-Schön, S., Steven, S., Mojović, M., Korać, B., Münzel, T.,& Daiber, A.. (2020). 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.. in Antioxidants (Basel, Switzerland), 9(5), 388.
https://doi.org/10.3390/antiox9050388

Vujačić-Mirski K, Bruns K, Kalinović S, Oelze M, Kröller-Schön S, Steven S, Mojović M, Korać 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.. in Antioxidants (Basel, Switzerland). 2020;9(5):388.
doi:10.3390/antiox9050388 .

Vujačić-Mirski, Ksenija, Bruns, Kai, Kalinović, Sanela, Oelze, Matthias, Kröller-Schön, Swenja, Steven, Sebastian, Mojović, Miloš, Korać, Bato, Münzel, Thomas, Daiber, Andreas, "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." in Antioxidants (Basel, Switzerland), 9, no. 5 (2020):388,
https://doi.org/10.3390/antiox9050388 . .