Development and validation of an analytical assay for electrochemical detection and quantification of protein‐bound 3‐nitrotyrosine in biological samples and comparison with classical, antibody‐based methods

Abstract

BackgroundReactive oxygen species (ROS) cause oxidative damage which is associated with endothelial dysfunction and cardiovascular disease. Majority of ROS are formed by mitochondria and NADPH oxidase. Overproduction of ROS leads to oxidative damage and disease progression, therefore, accurate determination of ROS and reactive nitrogen species (like nitric oxide, superoxide and peroxynitrite) is of great importance but also represents a great challenge, since these species are short‐lived and methods that are usually used relay on indirect detection. Here we compared 3 different methods for the detection of 3‐nitrotyrosine, a footprint of peroxynitrite formation in vivo, in biological samples.Methods and ResultsNitrated proteins were generated by incubation with peroxynitrite or 3‐morpholino sydnonimine (SIN‐1) and digested using pronase, a mixture of different proteases. The free 3‐nitrotyrosine was then separated by HPLC and quantified by a coulometric method using the CoulArray system. Concentration‐response‐curves of 3‐nitrotyrosine standards were highly linear (detection limit 50 nM). Concentration‐response‐curves of free 3‐nitrotyrosine obtained from digested nitrated bovine serum albumin standards were also highly linear before reaching a plateau, probably representing nitration of all available tyrosine residues. The presence of 3‐nitrotyrosine in nitrated bovine serum albumin standards was validated by other methods (ELISA and dot blot analysis using specific 3‐nitrotyrosine antibodies), showing a similar pattern as compared to HPLC/ECD detection. Ex vivo nitration of isolated mitochondrial or membranous fractions as well as serum/plasma yielded a concentration‐dependent 3‐nitrotyrosine signal using the HPLC/ECD method. Ongoing studies are dedicated to the quantification of 3‐nitrotyrosine in tissue samples of nitrate‐tolerant, diabetic, hypertensive and septic mice and rats. A major draw‐back seems to be the separation of the 3‐nitrotyrosine peak in complex biological samples, which is currently our main focus.ConclusionsBased on our ex vivo data, the CoulArray quantification method for 3‐nitrotyrosine seems to have some advantages regarding sensitivity and selectivity. In the future, we hope to be able to establish a reliable automated HPLC assay for the routine quantification of 3‐nitrotyrosine in biological samples of cell culture, animal and human origin. Our most recent data show that protein‐bound 3‐nitrotyrosine not only represents a footprint of peroxynitrite formation and marker of oxidative stress but also correlates with increased mitochondrial superoxide formation rates pointing towards self‐propagating oxidative stress vicious circles.Support or Funding InformationVascular biology research grant from the Boehringer Ingelheim Foundation for the collaborative research group „Novel and neglected cardiovascular risk factors: molecular mechanisms and therapeutic implications”