Abstract
Red blood cells are constantly exposed to reactive species under physiological or pathological conditions or during administration of xenobiotics. Regardless of the source, its accurate quantification is paramount in the area of theragnostics, which had been elusive up until now. Even if there are a lot of approaches to evaluate the oxidative stress, very sensitive methods are missing for the blood system. We therefore sought to apply a highly sensitive approach, by liquid chromatography coupled to mass spectrometry (UPLC–MS), for the quantification of reactive species such as superoxide radical and hydrogen peroxide using dihydroethidium (DHE) and coumarin boronic acid (CBA) probes respectively through the detection of 2-hydroxyethidium (2OH-E+) and 7-hydroxycoumarin (COH). The use of the high-resolution mass spectrometry associated to UPLC ensured a selective detection of superoxide and hydrogen peroxide in the blood system under diverse conditions such as oxidized red blood cells (RBCs), untreated and treated parasitized RBCs. Moreover, this technique allowed the determination of reactive species in human plasma. This protocol provides a huge opportunity for in-depth study of several pathological conditions vis-a-vis their treatment in modern medicine.
Highlights
To unravel the biological roles of reactive oxygen species (ROS), the ability to detect, identify and quantify the reactive species involved at the cellular level is paramount
We report how the liquid chromatography (LC)–MS method can be successfully applied to erythrocytes and human plasma for quantifying superoxide radicals and its reduced form, hydrogen erythrocytes and human plasma for quantifying superoxide radicals and its reduced form, hydrogen peroxide in the erythrocyte system under diverse conditions
A significant increase of both species was observed for PHZ treated red blood cells (RBCs) in comparison with untreated ones
Summary
To unravel the biological roles of reactive oxygen species (ROS), the ability to detect, identify and quantify the reactive species involved at the cellular level is paramount. Oxidative stress results from an imbalance between the antioxidant system and generation of reactive species that normally takes place in healthy organism. Oxidative stress can be measured directly by the quantification of reactive oxygen, nitrogen species or indirectly by measuring the level of oxidative markers such as lipid peroxidation or antioxidant enzymes or glutathione [3]. The direct and indirect methods are usually complementary to each other in stating the oxidative state of a cell, but the direct approach is often more challenging. Several direct approaches exist for eukaryotic cells among them luminescent assays (fluorescent, chemiluminescent and bioluminescent) [4] and electron paramagnetic resonance (EPR) [5]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
