Rapid and Sensitive Gradient Liquid Chromatography Method for the Quantitation of 2-Hydroxyethidium Ion from Neutrophils

LEBED PABLO; OSORIO GRISALES JAIVER; KEUNCHKARIAN SONIA; GOTTA JAVIER; GIAMBELLUCA MIRIAM; CASTELLS CECILIA

Budapest

Congreso; 36th International Symposium on High Performance Liquid Phase Separations and Related Techniques; 2011

Hungarian Society for Separation Sciences

Reactive oxygen species (ROS) are physiologically generated in small amounts during mitochondrial oxidative phosphorylation as well as during the oxidative burst of polymorphonuclear leukocytes. Studies in molecular and cell biology indicates that these oxidants are specifically associated with a variety of biological processes, such as cellular signaling and respiratory burst. Because these species have very short half-lives, their direct analysis is not feasible, so the use of indirect techniques becomes mandatory.Among these approaches, fluorescent probes as hydroethidine (HE) are used for the specific detection of the intracellular superoxide radical by means of fluorescence microscopy and flow cytometry in different types of cells and tissues. Recently, Zhao et al. discovered that the reaction product between HE and the superoxide radical was 2-hydroxyethidium (2-OH-E+) and not ethidium (E+), as had been previously thought, and that 2-OH-E+ did not arise from the action of other intracellular oxidants [1]. We developed an improved reversed-phase high-performance liquid chromatography (RPLC) assay for the rapid separation and determination of the 2-hydroxyethidium ion. The 2-hydroxyethidium ion is the specific product of the redox reaction between hydroethidine with superoxide radical. High resolution between the chromatographic bands corresponding to ethidium and 2-hydroxyethidium ions was achieved within a practicable analysis time. The RPLC-fluorescence method can reliably detect 2-hydroxyethidium ion concentrations down to 0.12 μM (or 1.2 pmol) and the signal is linear with concentration beyond 50 μM. An application of the method to neutrophil samples demonstrated that intracellular quantification of 2-hydroxyethidium was reproducible, as evidenced by low values of the relative standard deviations: 0.016 for non-stimulated cells, and 0.056 and 0.0125 for neutrophils incubated with agonists phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (fMLP), respectively. The resulting analytical method combines a rapid separation of the relevant peaks with the degree of sensitivity required for use in routine biological analyses.