Rapid and sensitive gradient liquid chromatography method for the quantitation of 2-hydroxyethidium ion from neutrophils a a a a b a Pablo J. Lebed , Jaiver Osorio Grisales , Sonia Keunchkarian , Javier Gotta , Miriam Giambelluca and Cecilia Castells a Division Química Analítica y CIDEPINT, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina. b Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, La Plata, Argentina E-mail: castells@isis.unlp.edu.ar ABSTRACT EXPERIMENTAL 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 1-5 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 6-9 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 + + 10 ethidium (E ), as had been previously thought, and that 2-OH-E did not arise from the action of other intracellular oxidants. 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. Synthesis and purification 2-hydroxyethidium The synthesis of 2-OH-E+ was performed by incubation of HE (50 M) with xanthine (1 mM) and xanthine oxidase (0.05 U/ml) in a phosphate buffer (pH 7.4, 50 mM) containing ethylenediaminetetraacetate (100 M). The volume of the mixture was 25 mL. The mixture was stirred several times. After 60 min at room temperature, the reaction product was purified in a two-step procedure. First, the reaction mixture was solid-phase extracted with a Phenomenex Strata C18-E cartridge preconditioned with water, followed by a (50/50) water/methanol mixture, and finally by water. The clean-up was performed with water followed by a (50/50) water/methanol mixture, and pure methanol (1mL) was then used to elute the 2-OH-E+. The second step consisted in a semipreparative-HPLC separation based on the optimized gradient elution using the phenyl column as described in HPLC analysis. The solvent from the collected fraction was evaporated and the solid resuspended in 0.1 M HCl until the compound was completely dissolved to form a 10 mM stock solution. This solution was kept at 4°C, and aliquots were diluted with water to obtain 0.1 mM standard solutions. Spectral measurements The ultraviolet- (UV-) visible measurements were recorded by means of a Thermo Scientific UV/Visible spectrophotometer equipped with 1.0-cm-path-length quartz cells. The concentrations of the 2-OH-E+ standards were calculated by the UV-absorbance measurements at an extinction coefficient of 1.2 x 104 dm3mol-1cm-1 at 470 nm. The fluorescence-emission (with excitation fixed at 470 nm) and -excitation (emission measured at 595 nm) spectra of the standard were measured in a LS50B Perkin Elmer Luminiscence spectrometer. Excitation and emission slit widths were set at 10 nm. The liquid chromatography mass spectrometer used was an Agilent 1100 LC system (Agilent Technologies Inc., USA) equipped with a binary pump and a diode-array detector and coupled with a MSD VL quadrupole (Agilent Technologies, USA) with an electrospray ionization (ESI) interface. The data acquisition and analyses were performed through the use of the LC/MSD Agilent ChemStation. The electrospray ionization was performed with nitrogen to assist nebulization at a flow rate of 7 l/min. The capillary temperature and voltage were set at of 330°C and 3,000 V, respectively. The negative and positive scan-ion modes were used for identifying the base- peak m/z values for each analyte. The ESI-MS was used to prove the quality of the standard compound. HPLC analysis Fluorescent compounds were separated in an Agilent HP1100 liquid chromatograph (Agilent Technologies Inc., USA) equipped with an auto injector and fluorescence detector. The columns used were a Zorbax SB-C18 column (150 mm x 4.6 mm, 5 m) or a Zorbax SB-Phenyl column (250 mm x 4.6 mm, 5 m). Both columns were equilibrated with a mobile phase of 35% CH3CN and 65% water, both containing a 0.1% (v/v) of TFA and these conditions were used for the separation of the peaks of E+ and 2-OH-E+ by isocratic elution at a flow rate of 1ml/min with both columns. Gradient elutions were optimized for both columns. A gradient from 35% to 65% in 15 min at a flow of 1ml/min was used with the C18 column or, alternatively, from 35% to 55% in 5 min at a flow rate of 2 ml/min was used with the phenyl column. The injection volume was 10 l. The fluorescence excitation was set at 470 nm and the emission was measured at 595 nm. Cell treatment Cell collection and pretreatment Human neutrophils, obtained from fresh peripheral blood drawn by venipuncture, were isolated by Histopaque double-gradient centrifugation as described by the manufacturer (Sigma-Aldrich, Procedure Nr. 1119). Stated in brief, neutrophils recovered from the interface between solutions of Histopaque 1119 and 1077 were washed once in 10 mL of HEPES-buffered solution and then suspended in the same solution to a concentration of 2 x 107 cells/ml. The viability was greater than 95%, as assessed by trypan-blue dye exclusion and homogeneity greater than 98%, as determined by differential cell counting. The cells (106/ml) were preincubated with HE (20 M) for 15 min at 37°C with continuous shaking and thereafter the suspensions divided into three aliquots for separate treatments. Accordingly, the tubes were incubated with no addition or with one effector, either PMA (150 nM) or fMLP (100 nM), for 15 min at 37°C. After activation, the polymorphonuclear leukocytes were placed on ice to stop the reactions. Sample preparation for HPLC analysis The reaction medium was removed and the cells washed twice by centrifuging with ice-cold HEPES solution. After a final centrifugation and removal of the supernatant, the resulting cell pellet was treated with lysis buffer (iced-cold HEPES solution with 0.1% (v/v) Triton X-100). The unlysed cells were then separated by centrifugation (5 min at 1,000 g and 4°C). The next steps were done following the methodology proposed by Zielonka et al. [23]. Stated in brief, the lysate supernatant was transferred to a tube containing 0.2 M HClO4 in MeOH and the sample placed on ice to precipitate the proteins. The precipitates were pelleted by centrifuging for 30 min at 20,000 g and the supernatants transferred to a tube containing 1 M phosphate buffer pH 2.6. The excess buffer and KClO4 were precipitated (15 min at 20,000 g) and the supernatant transferred into a HPLC vial. RESULTS Fig. 1. RPLC isocratic separation of ethidium and 2-hydroxyethidium. A. Zorbax + + SB-C18 column. B. Zorbax SB-Phenyl. Peaks of E and 2-OH-E were separated with (35/65) CH3CN/water containing 0.1% (v/v) TFA at a flow rate of 1ml/min. Injection volume: 10 ml. Fluorescence detection: excitation at 510 nm and emission at 595 nm. Fig. 2. RPLC gradient elution of ethidium and 2-hydroxyethidium. A. Zorbax SB-C18 column with mobile phase: gradient from 35% to 65% CH3CN in 15 min at a flow rate of 1ml/min. B. Zorbax SB-Phenyl, gradient from 35% to 55% in 5 min at a flow rate of 2 ml/min. 0 1 2 3 4 5 6 Time (min) Basal fMLP PMA E+ 2-OH-E+ 2-OH-E+ 2-OH-E+ E+ E+ Basal fMLP PMA 0.0 0.5 1.0 1.5 + Detector response / 2-OH-E Fig. 3. Stimulation of neutrophils with fMLP and PMA. (A) Chromatograms obtained from extracts of neutrophils incubated with HE. Comparison of the chromatogram of the control with those obtained after a 15-min exposure to fMLP or to PMA. The chromatographic conditions were as in + Fig. 4B. (B) Mean ± SD of three replicates (2-OH-E peak height). ACKNOWLEDGMENTS REFERENCES [1] V.J. Thannickal, B.L. Fanburg, Reactive oxygen species in cell signaling, Am. J. Physiol. Lung Cell Mol. Physiol. 279 (2000) 1005-1028. [2] C.C. Winterbourn, Reconciling the chemistry and biology of reactive oxygen species, Nat. Chem. Biol. 4 (2008) 278-286. [3] J.F. Turrens, Mitochondrial formation of reactive oxygen species, J. Physiol. 552 (2003) 335-344. [4] M.P. Murphy, How mitochondria produce reactive oxygen species, Biochem. J. 417 (2009) 1-13. [5] G. Bartosz, Reactive oxygen species: destroyers or messengers?, Biochem. Pharm. 77 (2009) 1303-1315. [6] B. Fink, K. Laude, L. McCann, A. Doughan, D.G. Harrison, S. Dikalov, Detection of intracellular superoxide formation in endothelial cells and intact tissues using dihydroethidium and an HPLC-based assay, Am. J. Physiol. Cell Physiol. 287 (2004) 895-902. [7] C.D. Georgiou, I. Papapostolou, N. Patsoukis, T. Tsegenidis, T. Sideris, An ultrasensitive fluorescent assay for the in vivo quantification of superoxide radical in organisms, Anal. Biochem. 347 (2005) 144-151. [8] E. Emregül, Development of a new biosensor for superoxide radicals, Anal. Bioanal. Chem. 383 (2005) 947-954. [9] J. Zielonka, M. Hardy, B. Kalyanaraman, HPLC study of oxidation products of hydroethidine in chemical and biological systems: ramifications in superoxide measurements, Free Radical Bio. Med. 46 (2009) 329-338. [10] H. Zhao, Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide, Free Radical Bio. Med. 34 (2003) 1359-1368. CONCLUSION 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. -1 Table 1. Least-squares regression parameters of peak area (mAU.s ) and peak + height (mAU) versus 2-OH-E concentration (mM), detection and quantification limits, and recoveries. Slope Intercept 2 R a LOD (M) a LOQ (M) Analyte Recovery, n:3 3.6 M 12.6 M Peak Area Peak Height 2.72 (±0.01) 0.350 (±0.001) - 0.9 (±0.2) 0.004 (±0.01) 0.998 0.999 0.37 1.15 0.24 0.75 81 (±4) % 100 (±7) % 0 1 2 3 4 5 6 Time (min) Basal fMLP PMA E+ 2-OH-E+ 2-OH-E+ 2-OH-E+ E+ E+ PMA fMLP Intracellular Signal PLC G i   fMLPR 2+ Ca NADPH + + NADP + H O 2 HE + 2-OH-E phox gp22 phox gp91 phox p40 phox p47 phox p67 P P P P phox p40 phox p47 phox p67 phox gp91 phox gp22 DAG IP 3 PIP 2 PKC MAPK 2+ [Ca ] i . - O 2 PMA and fMLP are commonly used as agonists that, in different cell zones, trigger a signaling cascade that finally lead to the activation of NADPH oxidase with the subsequent production of an oxidative burst in neutrophiles. ע" LOD= 3.28s 0 and LOQ= 10s 0 ; фф 2 x / y 0 Q x m 1 3 1 slope s s    1 where s y/x is the standard deviation of the regression, m is the number of calibration 2 solutions, ? x average concentration of calibration standards, Qxx = sum of squares of x. 3 HPLC optimization Calibration Synthesis of 2-hydroxyethidium Application of the method to human neutrophils