Protocol

Introduction

Superoxide radical anion (O₂^- ), the one-electron reduction product of molecular oxygen, has been implicated in various physiological and pathological intracellular signaling processes^{1, 2}. Increased production of O₂^- , its dismutation to hydrogen peroxide and molecular oxygen, as well as its rapid reaction with nitric oxide (NO) cause cellular oxidative stress resulting in the modification of lipids, proteins and DNA. However, because of its short lifetime, low fluxes and rapid reaction with cellular components including superoxide dismutase (SOD) or NO, the detection and quantification of O₂^- in cellular systems has remained a challenging task³. In most chemical and biochemical systems, O₂^- can be detected and quantified by way of its reaction with probes that produce easily detectable and relatively stable compounds (e.g., reduction of ferricytochrome c, oxidation of epinephrine to adrenochrome, spin trapping with cyclic nitrones). The specificity of each of these approaches can be validated by SOD, that is, an SOD-inhibitable signal would indicate an O₂^- -dependent reaction of the probe. In cellular systems, however, the fluorogenic probe hydroethidine (HE, also known as dihydroethidium, DHE, Fig. 1) remains the probe of choice³. Recently, the derivative of HE bearing triphenylphosphonium moiety, called MitoSOX Red or Mito-HE (Fig. 1), has been synthesized. This probe was reported to accumulate in mitochondria and trap mitochondria-derived O₂^- (ref. 4).

Figure 1: Structures of hydroethidine (HE), Mito-HE and their oxidation products.

2-OH-E⁺, 2-hydroxyethidium.

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Recent studies categorically proved that the product of the reaction of HE with O₂^- is 2-hydroxyethidium (2-OH-E⁺), and that no ethidium (E⁺) is produced from this reaction (Figs. 1, 2)^{5, 6}. Reports also show that no other biologically relevant oxidant reacts with HE to form 2-OH-E⁺. Thus, 2-OH-E⁺ is a specific marker for O₂^- (refs. 3–6). Rather conveniently, it has proven possible to prepare the authentic standard 2-OH-E⁺ by an independent chemical synthetic route (Fig. 3)⁷. Reaction between HE and nitrosodisulfonate radical anion (Fremy's salt) yields 2-hydroxyethidium as the sole product⁷. Please note that an analogous reactivity also leads to the production of 2-OH-Mito-E⁺ from Mito-HE; see Box 1.

Figure 2: Proposed mechanism of 2-hydroxyethidium (2-OH-E⁺) formation from the reaction between hydroethidine (HE) and O₂^- .

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Figure 3: Mechanism of the reaction of hydroethidine (HE) with nitrosodisulfonate radical dianion (NDS) adapted from ref. 7.

2-OH-E⁺, 2-hydroxyethidium.

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With regard to the mechanism of the reaction of O₂^- with HE, it has been postulated that the radical formed in the first step of the reaction between HE and O₂^- reacts quickly with another O₂^- to give hydroperoxide, which, upon water elimination, would form the imino-quinone derivative of HE, which rearranges to form 2-OH-E⁺ (Figs. 2, 3)^{7, 8}. A rate constant of 2 10⁶ M^{- 1} s^{- 1} has been estimated for the first stage of this reaction, the formation of the HE-derived radical from HE and O₂^- (ref. 8). The proposed mechanism of the reaction of HE with superoxide has the following implications for the intracellular detection and quantification of O₂^- using HE as the detection probe:

1. The HE-radical intermediate formed in the assay does not react with oxygen (aromatic aminyl radicals are not reactive toward molecular oxygen). Therefore, unlike assays with other probes (lucigenin, 2',7'-dichlorofluorescin, luminol)⁹, the use of HE should not lead to the artifactual formation of superoxide.
2. As the rate constant of the reaction of O₂^- with HE is 1,000 times lower than that of the reaction of O₂^- with SOD, the relative concentrations of HE and SOD are expected to affect substantially the yield of 2-OH-E⁺.
3. With a constant flux of O₂^- , the yield of 2-OH-E⁺ may be increased by the presence of enzymes or other oxidizing species capable of oxidizing HE to its radical cation (HE⁺) or the neutral radical (HE(NH)). This radical intermediate may, in fact, subsequently react with O₂^- to form the product, 2-OH-E⁺. This reactive pathway would, however, result in an overall stoichiometric ratio of 1:1 between O₂^- and 2-OH-E⁺ formed, as opposed to the expected 2:1.
4. In oxidizing environments, other products of HE-derived radicals reactions (e.g., dimerization and/or disproportionation) will be formed which, in turn, will affect the effective HE concentration for superoxide trapping.

Although the measured amount of 2-OH-E⁺ is a good index of superoxide formation it cannot be directly equated to the value of intracellular flux of superoxide radical anion because of the following reasons:

1. Owing to the competition with SOD and/or other intracellular superoxide scavengers, only a fraction of superoxide is scavenged by HE.
2. Despite the theoretical stoichiometry of two superoxide molecules per molecule of 2-OH-E⁺ formed, it has been postulated that, similar to SOD, HE could also catalyze the dismutation of superoxide, potentially leading to an underestimation of superoxide levels⁹.
3. Intracellular availability of HE may vary with time, and the fraction of superoxide scavenged by HE may, therefore, not be constant throughout the incubation period.

Despite these limitations, one can compare the amount of 2-OH-E⁺ produced in different conditions and obtain semi-quantitative data regarding the effect of various treatments or pathophysiological conditions on intracellular steady-state levels (or flux) of superoxide.

Methods of 2-hydroxyethidium quantification

UV-visible absorption spectroscopy. A major advantage of the spectrophotometric detection of 2-OH-E⁺ (and 2-OH-Mito-E⁺) is the ready availability of spectrophotometers in most laboratories, short time for analysis and the possibility of real-time monitoring of 2-OH-E⁺ formation in chemical and biochemical systems. As shown in Figure 4, 2-OH-E⁺ spectrum exhibits absorption maxima at 266 and 470 nm at pH 7.4. As the spectral overlap with other solutes and with HE in the UV range is prominent, the absorption maximum at 470 nm is preferred for 2-OH-E⁺ quantification. However, even at this wavelength the quantification may be ambiguous owing to interference from other oxidation products of HE absorbing at this wavelength (e.g., E⁺, Fig. 4a). Therefore, the spectrophotometric detection should be accompanied by HPLC analysis of the products. In some cases, 2-OH-E⁺ can be separated from other solutes absorbing light at 470 nm by extracting 2-OH-E⁺ with n-BuOH. After the subsequent evaporation of n-BuOH, the dry residue should be redissolved in the appropriate solvent¹⁴. It should be noted that it is much easier to dissolve the dry residue containing 2-OH-E⁺ in acidic solution [0.1 M hydrochloric acid (HCl)] than in pure water owing to protonation of the compound (see below), which leads to higher solubility in the aqueous solution. In this case, however, the analysis needs to be adjusted to the experimental conditions. As shown in Figure 4b,c, the UV-visible spectral properties of 2-OH-E⁺ and E⁺ change with the pH of the solution. Comparison of the pK_a values of E⁺ (pK_as of 0.4 and 2.1, see inset in Fig. 4c) and those of 2-OH-E⁺ (pK_as of 0.5, 2.2 and 7.3, see inset in Fig. 4b) clearly indicates the involvement of the aromatic hydroxyl group in the acid–base equilibria at neutral pH in the case of 2-OH-E⁺. As the extinction coefficient of 2-OH-E⁺ is sensitive to changes in pH, a rigorous control of pH is required for the spectrophotometric assay of 2-OH-E⁺. Please note that, as Mito-E⁺ and 2-OH-Mito-E⁺ have almost the same acid–base properties as E⁺ and 2-OH-E⁺ (pK_a = 0.2 and 2.0 for Mito-E⁺ and 0.4, 1.9 and 7.2 for mito-2-OH-E⁺), the pH should be rigorously controlled also when working with the mitochondria-targeted analog. The absorption spectrum of 2-OH-E⁺ is also affected by environmental factors (binding to DNA or polarity of the solvent) as shown in Figure 4d. Therefore, a calibration curve should be prepared and the extinction coefficient of 2-OH-E⁺ determined under the same experimental conditions.

Figure 4: UV-visible absorption spectra.

(a) Hydroethidine (HE), 2-hydroxyethidium (2-OH-E⁺) and E⁺ (10 M each) in 10 mM Tris buffer (pH 7.4) containing 1 mM EDTA. (b,c) The dependence of UV-visible absorption spectra of 2-OH-E⁺ (10 M, panel b) and E⁺ (10 M, panel c) on pH of the solution. Insets: pH titration curve of the absorbance detected at 430 nm (for E⁺ and 2-OH-E⁺). Red solid line shows the fitted curve based on the determined pK_a values. (d) 10 M 2-OH-E⁺ in the absence (black line) and presence (red line) of DNA (1 mg ml^{- 1}) in 10 mM Tris buffer (pH 7.4) containing 1 mM EDTA. The spectrum of the compound in n-octanol saturated with PBS is shown as a green line.

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Fluorescence spectroscopy. Fluorescence spectroscopy, while still allowing the 'real time' monitoring of oxidation/hydroxylation of HE to 2-OH-E⁺, is more selective than spectrophotometry. The former approach, in fact, enables quantitative analysis in the presence of multiple components absorbing at 470 nm. Fluorescence-based techniques (including fluorescence microscopy and flow cytometry) have been used for the HE-based detection of superoxide radical anion in a variety of biological systems for over a decade^{15, 16}. The fluorescence spectra of HE, E⁺ and 2-OH-E⁺ are shown in Figure 5. Whereas HE will not interfere with the fluorescence detection of 2-OH-E⁺, there is significant spectral overlap between 2-OH-E⁺ and E⁺. Therefore, the detection and quantification of 2-OH-E⁺ by fluorescence spectroscopy also requires HPLC analysis to ensure that 2-OH-E⁺ is the only fluorescent product formed following HE oxidation. Note that, in virtually all the biological systems investigated, we detected both 2-OH-E⁺ and E⁺ in different ratios and hence the use of the fluorescent microscopy methods alone can be misleading. The fluorescence intensity of 2-OH-E⁺ and E⁺ (as well as their mitochondria-targeted analogs) increases upon binding of these compounds to DNA (Fig. 5b,c). The fluorescence intensities of both E⁺ and 2-OH-E⁺ increase when excitation light in the range of 450–500 nm is used. 2-OH-E⁺ displays an additional excitation band with a maximum between 350 and 400 nm (as indicated by an arrow in Fig. 5b,d). This particular spectral feature has been used in the selective detection of 2-OH-E⁺ (and 2-OH-Mito-E⁺)⁴. However, E⁺ also is fluorescent when excited at those wavelengths. Thus, depending on its concentration, E⁺ can significantly contribute to the total florescence intensity measured, thereby confounding the analysis. Moreover, that new excitation band between 350 and 400 nm increases in intensity when n-octanol is used as a solvent (Fig. 5d) or following decrease of the pH of the solution. We attribute this excitation band to the protonated form of 2-OH-E⁺, and conclude that, depending on the intracellular distribution of 2-hydroxyethidium, different fluorescence intensity can be detected. These results further strengthen the case for using HPLC analysis to validate the formation of 2-OH-E⁺ and 2-OH-Mito-E⁺.

Figure 5: Fluorescence properties of hydroethidine (HE) and its oxidation products.

In all panels the blue lines represent the excitation spectra and red lines the emission spectra. (a) Fluorescence spectra of HE (1 M, dashed lines), 2-hydroxyethidium (2-OH-E⁺) (10 M, solid lines) and E⁺ (10 M, dotted lines) in 10 mM Tris buffer (pH 7.4) containing 1 mM EDTA. (b,c) The fluorescence spectra of 2-OH-E⁺ (panel b) and E⁺ (panel c) in the absence (dotted lines, compounds' concentration: 10 M) and the presence (solid lines, compounds' concentration: 1 M) of DNA (1 mg ml^{- 1}) in 10 mM Tris buffer (pH 7.4) containing 1 mM EDTA. (d) Fluorescence spectra of 2-OH-E⁺ (10 M) in Dulbecco's PBS (DPBS) (dotted lines) and in n-octanol saturated with DPBS (solid lines). The arrows indicate an additional excitation band present in the case of 2-OH-E⁺.

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Separation of the oxidation products. As UV-visible absorption and fluorescence spectroscopy alone cannot be used to accurately quantify 2-OH-E⁺ in the presence of ethidium, the separation of these products is essential^{5, 6}. HPLC enables the analysis of complex mixtures and the quantification of both HE and its oxidation products. Several methods of extraction of these products from cells and tissues have been reported^{6, 11, 17, 18, 19}, and, depending on either the cell type or the tissue type, different extraction protocols may be required to obtain satisfactory extraction efficiency.

As a consequence of the fluorescent properties of 2-OH-E⁺ and E⁺, HPLC has been used in conjunction with fluorescence detection^{4, 5, 6, 7, 8, 11, 17, 20, 21}. Recently, we have shown that electrochemical detection is approximately ten times more sensitive than fluorescence detection^{8, 10, 13}. The selective detection of 2-OH-E⁺ in pure solution and in extracts from cells (in the presence of E⁺) has been also achieved by HPLC-mass spectrometry (HPLC-MS)^{5, 7, 19}. This technique has the advantage that the compounds can be resolved based not only on their different retention time but also on their different m/z values (314 and 330 for E⁺ and 2-OH-E⁺, respectively), thereby increasing selectivity compared with regular HPLC.

Other methods for the selective detection and quantification of 2-OH-E⁺ extracted from biological systems include micellar electrokinetic chromatography coupled with laser-induced fluorescence detection^{22, 23} and purification of the cell (or tissue) extract by cation-exchange and hydrophobic micro-column chromatography followed by detection of the fluorescence intensity of the mixture of 2-OH-E⁺ and E⁺ in the presence of DNA before and after consumption of 2-OH-E⁺ by an HRP/H₂O₂ system¹⁸. It is noteworthy that the micellar electrokinetic chromatography coupled with laser-induced fluorescence detection is the most sensitive method available for detection of 2-OH-E⁺ reported to date (limit of detection: 0.15 amol)²³.

Materials

Reagents

• Acetonitrile, CH₃CN, MeCN, gradient grade, for HPLC (Sigma-Aldrich, cat. no. 34851)
  Caution Highly flammable and harmful.
• BSA (Sigma, cat. no. A8806)
• Argon gas (4.8 grade; Praxair)
• Bradford reagent (Sigma, cat. no. B6916)
  Caution Corrosive.
• 1-Butanol, n-BuOH (Sigma-Aldrich, cat. no. 34867)
  Caution Harmful.
• DTPA (Fluka, cat. no. 32319)
  Caution Irritant, dangerous for the environment.
• DMSO, ACS spectrophotometric grade (Sigma-Aldrich, cat. no. 15,493-8)
• Dulbecco's PBS (DPBS; Sigma, cat. no. D8537)
• EtOH (200 proof; Aldrich, cat. no. E7023)
  Caution Highly flammable.
• Ethidium bromide, E⁺Br^- —5-ethyl-6-phenyl-3,8-diaminophenanthridinium bromide (Fluka, cat. no. 46065)
  Caution Very toxic.
• Formic acid (HCOOH, 98.0–100%; Riedel-de Haën, cat. no. 27001)
  Caution Corrosive.
• HCl (fuming, 37%; Fluka, cat. no. 84415)
  Caution Corrosive.
• HE or DHE—5-ethyl-5,6-dihydro-6-phenyl-3,8-diaminophenanthridine (Invitrogen, cat. no. D-1186 or Fluka, cat. no. 37291).
  Caution Potentially harmful—undergoes oxidation to a very toxic compound, ethidium.
• MeOH (for HPLC; Sigma-Aldrich, cat. no. 34860)
  Caution Highly flammable and toxic.
• Mito-HE—5-(triphenylphosphonium)hexyl-5,6-dihydro-6-phenyl-3,8-diaminophenanthridine, MitoSOX Red (Invitrogen, cat. no. M36008)
  Caution Potentially harmful—undergoes oxidation to an analog of ethidium.
• Perchloric acid (HClO₄, 70%; Fluka, cat. no. 77227)
  Caution Corrosive and oxidizing.
• Phosphoric acid (H₃PO₄, 85 weight % in H₂O; Sigma-Aldrich, cat. no. 215104)
  Caution Corrosive.
• Potassium nitrosodisulfonate, NDS, Fremy's salt (Aldrich, cat. no. 220930)
  Caution Harmful.
• Potassium phosphate dibasic (K₂HPO₄; Fluka, cat. no. 60356)
• Potassium phosphate monobasic (KH₂PO₄; Sigma-Aldrich, cat. no. P0662)
• Tetrachloro-p-benzoquinone, chloranil (optional, see Box 1; Fluka, cat. no. 23280)
  Caution Irritant, dangerous for the environment.
• Trifluoroacetic acid (TFA, 100%; Pierce, cat. no. 28903)
  Caution Corrosive.
• Triton X-100 (Sigma-Aldrich, cat. no. T9284)
  Caution Harmful, dangerous for the environment.
• Water for the solution preparation and for HPLC-EC mobile phase should be purified by Milli-Q system (Millipore) and passed through a Prevail C18 SPE cartridge (Alltech) to remove traces of organic contaminants.

Equipment

• HPLC system with electrochemical detection system (optional, see Step 53, ESA Biosciences Inc.) consisting of Model 582 Pump, with RS-232 Control (ESA Biosciences Inc., cat. no. 70-4050); Gradient Upgrade for CoulArray System (ESA Biosciences Inc., cat. no. 70-4051); Model 5600A CoulArray Detector with computer, 8 Channel (ESA Biosciences Inc., cat. no. 70-4325); CoulArray Organizer with a Temperature Control (ESA Biosciences Inc., cat. no. 70-4340T); Model 542 Autosampler with Sample Tray Cooling, Biocompatible Fluid Path and Standard vial tray (84 positions; ESA Biosciences Inc., cat. no. 70-4152); Model 542 Autosampler Start-up Kit for CoulArray and Coulchem Systems (ESA Biosciences Inc., cat. no. 70-4139)
• HPLC system with absorption and fluorescence detectors and gradient mixer (optional, see Step 53, Agilent 1100 system; Agilent)
• HPLC system with MS detector (optional, see Step 53, LC-MSD SL system based on a HPLC Agilent 1100; Agilent)
• CO₂ incubator (Fisher)
• Refrigerated centrifuge (Eppendorf, model 5417R)
• Anaerobic chamber (model 855-AC, Plas-Labs Controlled Atmosphere (Anaerobic) Chamber)
• Safelight lamp (Kodak 5.5" round adjustable safelight lamp, model B; Kodak, cat. no. 141 2212, equipped with Kodak OC safelight filter; Kodak, cat. no. 152 1483)
• Rotary evaporator (Büchi Rotavapor R-205 equipped with heating bath B-490, vacuum controller V-800 and vacuum pump Vac V-500, Büchi)
• Low temperature (- 80 °C) freezer (model 8581, Thermo Forma Ultra Low Temperature Upright Freezer; Thermo Forma)
• C₁₈ column, optional—for methods A and B (see Step 53), Kromasil C₁₈ HPLC column, 250 mm 4.6 mm, 5 m, 100 Å (Alltech, cat. no. 62000); guard cartridge (Kromasil C₁₈ guard cartridges; Alltech, cat. no. 96163); cartridge holder (All-Guard cartridge holder; Alltech, cat. no. 80101)
• Ether-linked phenyl column, optional—for method C (see Step 53), Synergi Polar RP HPLC column, 250 mm 4.6 mm, 4 m, 80 Å (Phenomenex, cat. no. 00G-4336-E0); guard cartridge (Synergi Polar RP Securityguard cartridges; Phenomenex, cat. no. AJ0-6076); guard cartridge holder (Phenomenex, cat. no. KJ0-4282)
• Ether-linked phenyl column, optional—for method D (see Step 53), Synergi Polar RP HPLC column, 250 mm 2.0 mm, 4 m, 80 Å (Phenomenex, cat. no. 00G-4336-B0); guard cartridge (Synergi Polar RP Securityguard cartridges; Phenomenex, cat. no. AJ0-6075); guard cartridge holder (Phenomenex, cat. no. KJ0-4282)
• Semi-preparative HPLC column (Beckman Ultrasphere ODS, 250 mm 10 mm; Beckman, cat. no. 235328)
• SPE cartridges—Extract Clean SPE Prevail C18 1000 (Alltech, cat. no. 605430)
• Adjustable-volume pipettes 2.5 l, 10 l, 20 l, 100 l, 200 l, 1 ml + tips (adjustable-volume pipettes; Eppendorf Research)
• HPLC amber vials (wide opening screw top amber vials with write-on spot; Agilent, cat. no. 5182-0716)
• Inserts for HPLC vials (100-l deactivated glass inserts with polymer feet and mandrel interior; Agilent, cat. no. 5181-8872)
• Screw caps with septa for HPLC vials (polypropylene screw caps with septa for wide opening screw top vials, PTFE/red silicone rubber septa; Agilent, cat. no. 5182-0717)
• 1.5-ml microcentrifuge clear tubes (VWR, cat. no. 89000-028)
• 1.5-ml microcentrifuge amber tubes (VWR, cat. no. 89000-030)
• 15-ml centrifuge tubes (ISC BioExpress, cat. no. C-3394-1)
• Insulin syringes for cell lysis (Becton Dickinson, cat. no. 329461)
• 0.45-m membrane filter (Versapor —450 membrane; PALL, cat. no. 66408)

Reagent setup

• 0.5 M phosphate buffer pH 7.4 Mix KH₂PO₄ (final concentration: 0.11 M) with K₂HPO₄ (final concentration: 0.39 M) and dissolve these solids in an amount of water for HPLC-EC that is up to 90% of the desired final volume of the solution. To check the pH of the buffer, take an aliquot, dilute it with nine volumes of water and measure the pH of this 1:10-diluted buffer solution. If the pH value is not in the range 7.3–7.5, adjust the pH of the original buffer by addition of concentrated HCl or NaOH and check the buffer's pH again as above. After obtaining the desired pH of the buffer solution, add water to obtain the final solution volume as planned. Finally, filter the buffer solution by passing it through 0.45-m membrane filter and store in the 4 °C refrigerator for up to 1 month.
• 1 M phosphate buffer pH 2.6 Prepare as described above, but by mixing H₃PO₄ (final concentration: 0.3 M) with KH₂PO₄ (final concentration: 0.7 M). The final pH of the buffer, after 1:10 dilution with water should be in the range 2.5–2.7.
• 0.2 M HClO₄ in MeOH Add 85.5 l of 70% HClO₄ per 4.915 ml of ice-cold MeOH. Keep the resulting solution in the 4 °C refrigerator.

Procedure

1. Points from here (point 1) up to and including point 19 are related to Preparation of 20 mM HE stock solution in a darkroom (under amber light)Timing: 3 hPlace in an anaerobic chamber 50 plastic 1.5-ml amber or black Eppendorf tubes, 100-l and 1-ml pipettes, appropriate pipette tips, 5 ml DMSO in a 15-ml tube. Please note that, alternatively, instead of DMSO the solvent used at this point may be an aqueous solution of H₃PO₄ (0.05 M) or HClO₄ (0.1 M). For most experiments it is most convenient to use the stock solution of HE in DMSO. However, when DMSO could interfere with the assay (e.g., in the case of reaction of HE with Fenton's reagent or for spectroscopic measurements <300 nm) DMSO should be avoided. To prepare a 5 mM HE solution in 0.05 M H₃PO₄ (or in 0.1 M HClO₄), add 634 l of 0.05 M aqueous solution of H₃PO₄ (or of 0.1 M HClO₄) per 1 mg of HE in amber glass vial, vortex for 1 min and keep on ice. Check then the concentration of HE by the procedure analogous to the one described below for HE stock solution in DMSO (Steps 11–19).
2. Purge the chamber with argon gas.
3. Remove oxygen from DMSO by bubbling with argon inside the anaerobic chamber for 30 min.
4. Turn off the room light. Use darkroom amber light.
5. Place the HE vial in the anaerobic chamber.
6. Weigh 5–6 mg of HE in 1.5-ml Eppendorf tube. Write down the exact mass and calculate the volume of DMSO needed to prepare a 20 mM solution according to the equation:

   

   where m–is the mass of HE (in g), 315.42 g mol^{- 1}—molecular weight of HE, 0.02 mol dm^{- 3}—concentration of HE in the stock solution (for 0.00631 g HE, one should add 1.0 ml DMSO)

7. Add the calculated volume of deoxygenated DMSO and shake the tube to make sure all HE has been dissolved.
8. Take 20 l aliquots of the HE stock solution and transfer into the prepared amber (or black) Eppendorf tubes. Take the tubes out of the anaerobic chamber.
9. Place the tubes containing HE stock solution in a - 80 °C freezer.Pause Point The stock solution of HE in DMSO thus prepared is stable at - 80 °C for at least 6 months.
10. Before each experiment (see below), thaw the HE stock solution at room temperature for 5 min.
11. Determine the concentration of the HE stock solution by spectrophotometry: place 0.998 ml of aqueous solution of 50 mM phosphate buffer pH 7.4 containing 100 M DTPA in a quartz cuvette.
12. Add 2.5 l 20 mM HE stock solution in DMSO.
13. Mix the solution quickly and collect the UV-visible spectrum in the range 200–800 nm.
14. Scan the appropriate blank by the same procedure but using pure DMSO instead of HE stock solution.
15. Subtract the spectrum of the blank solution from the spectrum of HE solution.
16. Read the absorbance values at 265 and 345 nm.
17. Calculate the concentration of HE in the cuvette using the absorbance values at 265 and 345 nm using the equation:

    

18. Plug the relevant extinction coefficients listed in Table 1 into the equation in Step 17.

    Table 1: The extinction coefficient values for the standards of HE and Mito-HE and their oxidation products at pH 7.4.

    

    
    Full table
    
    
19. Calculate the concentration of HE in stock solution (c_stock) using the average value of the concentration determined at 265 and 345 nm (c_cuvette).

    

20. Points from here (point 20) up to and including point 26 are related to Synthesis of 2-hydroxyethidiumTiming: 3 hAdd 10 ml of 50 mM aqueous solution of phosphate buffer pH 7.4 containing 100 M DTPA to 3.6 mg NDS (Fremy's salt). Note that the commercially available NDS powder contains 75% of the compound (based on spectrophotometric analysis). It is normal that dissolution of the yellow powder of Fremy's salt in aqueous solution colors the solution blue. This is due to the dissociation of the (yellow) dimeric nitrosodisulfonate (Fremy's salt powder) into the (blue) monomeric form of nitroxide radical.
    Critical step Owing to its instability, especially in acidic solution, the NDS solution should be prepared fresh, stored at 4 °C and used within a couple of hours of preparation.
21. Record the UV-visible absorption spectrum of the NDS solution prepared in Step 20 after adjusting the baseline reading with 50 mM aqueous solution of phosphate buffer pH 7.4 containing 100 M DTPA (blank solution).
22. Record the absorbance values at 248 and 545 nm.
23. Calculate the concentration of NDS in the cuvette using the absorbance values (A) at 248 and 545 nm and the optical path length (l = 1.0 cm) according to the equation:

    

24. Use the extinction coefficient values of 1.69 10³ and 20.8 dm³ mol^{- 1} cm^{- 1} at 248 and 545 nm, respectively, to calculate an average NDS concentration.
25. To 24 ml of water in a 100-ml amber glass bottle, add 4 ml of 0.5 M aqueous phosphate buffer pH 7.4, 4 ml of 1 mM aqueous solution of DTPA and 200 l of 20 mM solution of HE in DMSO (see above). After mixing, add slowly 8.0 ml of 1.0 mM NDS while slowly stirring the solution. If the determined concentration of NDS (calculated in Step 24) is different from 1 mM, the volume should be adjusted accordingly. Leave the solution at room temperature for 2 h. The solution should turn yellow after mixing. After incubation analyze the obtained mixture by HPLC to determine the retention time of 2-OH-E⁺ (major peak).
    Critical step Excess of NDS should be avoided as it will react with the product, 2-OH-E⁺, to form undefined product(s) which will lower the yield of 2-OH-E⁺.
26. Along with the reduction products of NDS, the final reaction mixture may contain small amounts (<10%) of E⁺. The 2-OH-E⁺ thus synthesized can be purified by three alternative approaches: method A, HPLC-based purification; method B, purification on silica gel column⁷; and method C, purification on Alltech Prevail SPE C₁₈ cartridge¹⁰. Method A is recommended for small quantities (<1 mg) of 2-OH-E⁺. It requires the semi-preparative HPLC column and yields the compound as a trifluoroacetate salt. Method B is recommended for large quantities (>50 mg) of 2-OH-E⁺. Method C is recommended for intermediate amounts of 2-OH-E⁺ (0.5–50 mg).
    1. HPLC-based purificationTiming: 75 min per injection
       1. Follow the method described in Box 1 for the synthesis of 2-OH-Mito-E⁺, but use the gradient starting at 10% MeCN and change it to 70% MeCN over a time of 46 min.
    2. Purification on silica gel columnTiming: 6 h
       1. Follow the procedure described elsewhere⁷.
    3. Purification on Alltech Prevail SPE C₁₈ cartridgeTiming: 6 h
       1. Condition the cartridge by passing 6 ml of water, followed by 3 ml of water/MeOH (50/50) mixture, 3 ml of pure MeOH and again 6 ml of pure water.
       2. Load the reaction mixture (obtained after Step 25) onto the cartridge.
          Critical step All the fractions obtained in Steps 26C(ii)–(vii) should be collected and the volumes written down to allow subsequent estimation of the amount of 2-OH-E⁺ purified.
       3. Wash the cartridge with 4 3 ml of water.
       4. Wash the cartridge with 2 3 ml of water/MeOH (50/50) mixture.
       5. Wash the cartridge with 3 3 ml of water/MeOH (20/80) mixture. 2-OH-E⁺ (the orange band) should elute in this step. Use more water/MeOH (20/80) mixture if the eluate is still colorful, which indicates incomplete elution.
       6. Wash the cartridge with 2 3 ml of pure MeOH.
       7. Continue washing the cartridge until the second band (E⁺, the pink band) is eluted.
       8. Analyze by HPLC 10 l aliquots of each fraction after mixing with 90 l of 0.1 M aqueous phosphate buffer pH 2.6 solution containing 25% MeOH.
       9. Based on HPLC analysis take the fraction containing pure 2-OH-E⁺ and lyophilize it or evaporate the solvent under air flux. The other fractions containing 2-OH-E⁺ and E⁺ should be kept for repurification.
       10. Based on the volume of the 2-OH-E⁺ fraction collected and the concentration of 2-OH-E⁺ (the concentration in the eluate was 10 higher than in the solution analyzed by HPLC) calculate the amount of 2-OH-E⁺ and the volume of 0.1 M HCl needed to prepare a 10 mM 2-OH-E⁺ solution. Follow the directions reported below for final dilution and determination of the concentration of 2-OH-E⁺.
27. Points from here (point 27) up to and including point 35 are related to Preparation of the stock solutions of the standards for oxidation products of HE (and Mito-HE)Timing: 1 hTo prepare each solution, first add 0.1 M HCl to the solid and vortex until the compound is completely dissolved to form a 10 mM solution and then dilute the solution 1:100 with water to obtain 0.1 mM solution of the standard in 1 mM HCl.Pause Point The solutions of the standards in 1 mM aqueous solution of HCl should be stable for at least 6 months when stored at 4 °C.
28. To determine the concentration of the standards by UV-visible spectrophotometry, place 100 l of aqueous solution of 100 mM phosphate buffer pH 7.4 containing 200 M DTPA in an amber glass vial.
29. Add 100 l of the 100 M standard stock solution prepared in Step 27.
30. Mix the solution quickly, transfer it into a microcuvette (designed for 100 l of the solution) and collect the UV-visible spectrum in the range 200–800 nm.
31. Scan the appropriate blank by the same procedure but using 100 l of 1 mM HCl instead of standard stock solution.
32. Subtract the spectrum of the blank solution from the spectrum of standard solution.
33. Read the absorbance values at the relevant _max values for each standard solution (see Table 1).
34. Calculate the concentration of standard in the cuvette (c_cuvette) using the measured absorbance values (A) and the optical path length (l = 1.0 cm) according to the equation:

    

35. Calculate the concentration of the standard in stock solution (c_stock) using the determined concentration c_cuvette.

    

36. Points from here (point 36) up to and including point 53 are related to Detection of 2-OH-E⁺ in cellsTiming: 7 hPrepare three sets of 1.5-ml tubes: the first set (empty tubes) will be used to store aliquots of cell lysate for protein determination; the second set (filled with 100 l of 0.2 M HClO₄ in MeOH per each tube) will be used to perform protein precipitation from the lysate; and the third set (filled with 100 l of 1 M phosphate buffer pH 2.6) will be used to precipitate perchlorate anions and adjust pH of the cell extract. Place all three sets of tubes on ice.
    Critical step Steps 37–53 should be carried out under subdued light and the samples should always be kept on ice during sample processing. The procedure described herein can be used for 60-mm diameter dishes. The reagents' volumes should be appropriately scaled, depending on the size of the cell dish.
37. Incubate the cells of choice with cell culture medium containing 10 M HE (5 l of 20 mM HE in DMSO per 10 ml of medium) for 30 min.
38. To stop the incubation, remove the medium and wash the cells with ice-cold DPBS buffer.
39. Scrape the cells immediately in 1 ml of ice-cold DPBS, transfer the cell suspension into a 1.5-ml Eppendorf tube and place the tube on ice.
40. Pellet the cells by centrifugation: 5 min 1,000g at 4 °C.
41. Remove the supernatant carefully by aspiration.Pause Point Although it is recommended that the samples be analyzed immediately, the cell pellets can be frozen and stored at - 80 °C, typically for up to 1 month, until the day of the HPLC analysis. Please note that all subsequent steps should be carried out on the day of the HPLC analysis.
42. To the cell pellets add 150 l of ice-cold DPBS containing 0.1% (by vol) of Triton X-100 (lysis buffer).
43. Lyse the cells by drawing the mixture in and out of the insulin syringe. Repeat ten times.
44. Spin down the unlysed cells by centrifugation: 5 min 1,000g at 4 °C.
45. Transfer 100 l of the lysate supernatant into the tube containing 100 l of 0.2 M HClO₄ in MeOH (see Step 36), vortex the tube for 10 s and place it back on ice to allow protein precipitation. Leave it on ice for 1–2 h.
46. Transfer 2 l of the lysate supernatant into an empty 1.5-ml tube placed on ice.Pause Point The cell lysate mixed with acidified MeOH may be stored on ice for 2 h. During that time one can analyze the protein levels in the lysates as well as take a break.
47. Determine the protein concentration in the lysates using the Bradford reagent as follows: Prepare the blank solution (no BSA) and BSA standard solutions (0.5, 1, 1.5, 2, 3, 4 and 5 mg ml^{- 1}) in DPBS containing 0.1% Triton X-100. Transfer 2 l aliquots of the BSA standard solutions into empty 1.5-ml tubes. Add 998 l (per sample) of Bradford reagent to the vials containing the BSA standard solutions and to those containing 2 l each of the cell lysates from Step 46. Vortex the tubes and place them in the area shielded from light. Incubate the mixtures for 20 min at room temperature. Before absorption measurement vortex each tube once more (2 s) and transfer the mixture into a disposable plastic cuvette. Place the cuvette in the spectrophotometer. Measure immediately the absorbance at 595 nm. Based on a calibration curve obtained using the BSA standard solutions, calculate the protein concentration in the cell lysates.
48. Pellet the protein precipitate (obtained in Step 45) by centrifugation: 30 min 20,000g at 4 °C.
49. Transfer 100 l of the supernatant to the tube containing 100 l of 1 M phosphate buffer pH 2.6, vortex for 5 s.
50. Pellet the excess buffer and KClO₄ precipitate by centrifugation: 15 min 20,000g at 4 °C.
51. Transfer 150 l of the supernatant into the HPLC vial equipped with 200-l conical glass insert, seal the vial and place it on ice.
52. When all samples are in HPLC vials, place them in the autosampler with the tray cooled down to 4 °C.
53. Analyze the standards (HE, 2-OH-E⁺ and E⁺) and the samples from the cell lysis experiment by HPLC. For compound detection, one can use fluorescence and UV-visible absorption (HPLC-Fl)⁷, method A; electrochemistry (HPLC-EC)^{8, 10}, methods B and C; or MS^{5, 7, 19} (HPLC-MS), method D. To make an informed decision as to which analytical approach to implement, please note that the HPLC-electrochemistry (HPLC-EC) system affords greater sensitivity than HPLC-Fl in the analysis of HE and its oxidation products. Moreover, the electrochemical properties of HE, 2-OH-E⁺ and E⁺ are distinctly different to one another^{8, 10}, thus the analytes can be resolved not only based on their different retention times but also based on their different oxidation potentials. HPLC-MS is the most selective approach for the detection of the analytes, but the cost of such a system is much higher than either of the other systems mentioned above. Please note that, although when applying HPLC-Fl using a C₁₈ column (with the mobile phase containing TFA) we obtain good separation between the peaks of 2-OH-E⁺ and E⁺, in the case of HPLC-EC, when acidic phosphate buffer is used instead of TFA, the separation is not always satisfactory. Therefore, the additional option (method C) is described, in which the C₁₈ column is replaced by an ether-linked phenyl column, which affords a much better peak separation. The same type of column is also used for HPLC-MS, for which the mobile phase contains HCOOH instead of TFA. Also in case of unsatisfactory resolution on C₁₈ column in the case of TFA-modified mobile phase (HPLC-Fl) the ether-linked phenyl column can be used. Please note that the schematic HPLC conditions that we recommend for the analogous analyses of Mito-HE, 2-OH-Mito-E⁺ and Mito-E⁺ are reported in Box 2 and Tables 2, 3, 4.

    Table 2: Elution conditions for the analysis of Mito-HE and its oxidation products by HPLC with fluorescence detection (Box 2, method A).

    

    
    Full table
    
    

    Table 3: Elution conditions for the analysis of Mito-HE and its oxidation products by HPLC with electrochemical detection (Box 2, method B).

    

    
    Full table
    
    

    Table 4: Elution conditions for the analysis of Mito-HE and its oxidation products by HPLC with electrochemical detection (Box 2, method C).

    

    
    Full table
    
    
    1. HPLC with fluorescence and UV-visible absorption detectionTiming: 75 min per sample
       1. To separate HE, E⁺ and 2-OH-E⁺, inject 50 l of sample (the actual sample to be tested or the 2-OH-E⁺ standard prepared in Steps 21–28 above) into the HPLC system with a Kromasil C₁₈ column equilibrated with 10% CH₃CN in water containing 0.1% (vol/vol) TFA.
       2. Use a gradient elution method with two mobile phases: A—water containing 0.1% (vol/vol) TFA and B—99.9% acetonitrile, 0.1% (vol/vol) TFA. Increase linearly the concentration of B phase from 10 to 70% in 46 min at a flow rate of 0.5 ml min^{- 1}.
       3. During the next 1 min (46–47 min after injection) increase the concentration of B to 100% and keep this concentration fixed for the next 8 min (55 min after injection).
       4. Over the next 5 min (from 55 to 60 min after injection) gradually decrease B concentration to the initial value of 10% then re-equilibrate the column for 15 min.
       5. Use fluorescence detection at 356 and 510 nm (excitation) and 595 nm (emission) as well as the absorbance at 220, 250, 290, 370 and 500 nm to monitor the reaction products in the test sample.
       6. Measure the areas of the peak of 2-OH-E⁺ obtained following HPLC of the 2-OH-E⁺ standards prepared in Steps 20–26, and use this values to infer a correlation between those areas and the known concentrations of the standards. Identify the peak due to 2-OH-E⁺ in the test sample through the fluorescence trace recorded using an excitation at 510 nm and emission at 595 nm, and measure the area of the peak. Use the area/concentration correlation obtained through the 2-OH-E⁺ standards to calculate the concentration in the test sample.
    2. HPLC with electrochemical detection (HPLC-EC) using a Kromasil C₁₈ columnTiming: 60 min per sample
       1. Use a gradient elution method with two mobile phases: A—50 mM phosphate buffer (pH 2.6), 10% acetonitrile, 90% water; and B—50 mM phosphate buffer (pH 2.6), 60% acetonitrile, 40% water. Deoxygenate the mobile phase by bubbling with argon gas. Start mobile phase deoxygenation at least 30 min before beginning HPLC analysis and continue bubbling argon through the mobile phase during analysis of the samples.
       2. Separate HE, 2-OH-E⁺ and E⁺ on a Kromasil C₁₈ column using a gradient elution with ratios of A and B mobile phases changing from 7:3 to 3:7 over a period of 20 min (ref. 10).
       3. Over the next 5 min (20–25 min after injection) increase the fraction of the mobile phase B to 100% and maintain this setting for the next 15 min (25–40 min after injection).
       4. During the next 5 min (40–45 min after injection) decrease the concentration of CH₃CN gradually in the mobile phase to the initial values, then re-equilibrate the column for 15 min.
       5. Throughout the experiment, set the detector channels to the potentials 0, 200, 280, 365, 400, 450, 500 and 600 mV versus the palladium reference electrode.
       6. The quantification of 2-OH-E⁺ is performed by adding the areas of the peaks observed at 200, 280 and 365 mV. While some 2-OH-E⁺ will undergo oxidation at 200 mV, most of the compound should undergo oxidation at 280 mV and the remaining compound will be oxidized at 365 mV. Other potentials can be used for quantification of HE (0 and 200 mV), E⁺ (365–500 mV) and other products.
       7. Quantify the amount of 2-OH-E⁺ in the test sample in an analogous way as described in Step 53A(vi).
    3. HPLC with electrochemical detection (HPLC-EC) using an ether-linked phenyl columnTiming: 60 min per sample
       1. To obtain a better separation between 2-OH-E⁺ and E⁺ peaks than in method B, use an ether-linked phenyl column (250 mm 4.6 mm) instead of a C₁₈ column⁸.
       2. The elution method uses the same two mobile phases as described in method B above. Separate HE, 2-OH-E⁺ and E⁺ using a gradient elution with ratios of A and B mobile phases changing from 3:2 to pure B phase over a period of 30 min using a flow rate 0.5 ml min^{- 1}.
       3. Pump the pure mobile phase B through the column for the next 10 min (30–40 min after injection).
       4. Over the next 5 min (40–45 min after injection) restore the initial composition of the mobile phase then re-equilibrate the column for 15 min.
       5. Calculate the peak areas in the same way as described in Step 53B(vi).
       6. Quantify the amount of 2-OH-E⁺ in the test sample in an analogous way as described in Step 53A(vi).
    4. HPLC with MS detectionTiming: 90 min per sample
       1. For the separation and detection of HE, 2-OH-E⁺ and E⁺, use an HPLC system with an MS detector (LC-MSD SL, see EQUIPMENT), an electrospray ionization source and a single quadrupole mass analyzer. Inject 20 l of sample into the HPLC system with an ether-linked phenyl column (250 mm 2.0 mm) equilibrated with 20% CH₃CN in water containing 0.1% (vol/vol) HCOOH.
          Critical step As MS detectors are typically not compatible with phosphate buffer, the HPLC-MS system should be equipped with a valve allowing online buffer removal. Alternatively, cell pellets (obtained in Step 41) should be extracted directly with organic solvent (EtOH) and the supernatant diluted 1:1 with water before injection.
       2. Use a gradient elution method with two mobile phases: A—water containing 0.1% (vol/vol) HCOOH and B—99.9% acetonitrile, 0.1% (vol/vol) HCOOH. Separate HE and its oxidation products through a linear increase in B phase concentration from 20 to 100% in 60 min at a flow rate of 0.1 ml min^{- 1}.
       3. Over the next 10 min (60–70 min after injection), pump the B mobile phase through the column.
       4. Decrease the concentration of B back to 20% over the next 5 min (70–75 min after injection).
       5. Re-equilibrate the column by pumping the mobile phase containing 20% B through the column over the next 15 min.
       6. For the 2-OH-E⁺ quantification use the peak area detected at m/z = 330. Quantify the amount of 2-OH-E⁺ in the test sample in an analogous way as described in Step 53A(vi).Troubleshooting

Timing

Steps 1–9, preparation of the solutions of HE and standards of oxidation products: 3 h; Steps 10–19: 30 min; Steps 20–25: 3 h; Step 26: 6 h; Steps 27–35: 1 h; Step 36, detection of 2-OH-E+ in cells: 30 min; Steps 37–39, depending on the actual experiment, but typically <2 h; Steps 40 and 41: 15 min; Steps 42–44: 25 min; Steps 45 and 46: 15 min; Step 47: up to 2 h 35 min; Step 48: 45 min; Steps 49 and 50: 30 min; Steps 51 and 52: 15 min; Step 53, HPLC analysis: 75 min (method A), 60 min (methods B and C) or 90 min (method D) per sample. Include at least four more samples for running the blank and standards (HE, 2-OH-E+ and E+) samples.
Note: As for the Step 53 the samples are placed in the refrigerated autosampler, there is no need for the person carrying out the analysis to be present during the whole time the HPLC analysis is being done. The HPLC sequence can be started at the beginning of the break after Step 46, so that at least one standard will be done before the end of cell samples preparation, giving the person a chance to control whether the HPLC system is working properly.

Troubleshooting

Troubleshooting advice can be found in Table 5.

Table 5: Troubleshooting table.

Full table

Anticipated results

The typical chromatograms that are obtained by analysis of the extracts from unstimulated RAW 264.7 cells incubated for 20 min with HE (10 M in the medium) are shown in Figure 6 (HPLC with absorption and fluorescence detection), Figure 7 (HPLC with electrochemical detection) and Figure 8 (HPLC with MS detection). Note that HPLC enables the detection of several products formed from HE reactions: HE alone, 2-OH-E⁺, E⁺ and other derivatives, some of them we attribute to HE radical dimerization products²⁴. As shown in Figure 6, at least under the chosen HPLC elution conditions, only 2-OH-E⁺ and E⁺ contribute significantly to the total fluorescence intensity. The calculation of the amount of 2-OH-E⁺ based on the HPLC peak area gives the value of 10 pmol of 2-OH-E⁺ per mg protein. From the same chromatogram the amount of E⁺ is calculated as 100 pmol of E⁺ per mg protein, clearly indicating that E⁺ should be considered as a significant contributor to the total fluorescence intensity observed in fluorescence microscopy and flow cytometry measurements in cells.

Figure 6: HPLC of the extract from RAW 264.7 cells.

Cells were treated with hydroethidine (HE; 10 M) in DMEM containing 2% FBS for 20 min at 37 °C and processed as described in the PROCEDURE. An HPLC system with absorption and fluorescence detectors equipped with a Kromasil C₁₈ column was used (method A). 2-OH-E⁺, 2-hydroxyethidium.

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Figure 7: HPLC-EC chromatogram of the extract from RAW 264.7 cells.

Sample was prepared and processed as described in the Figure 6 caption. The HPLC-EC system equipped with a Synergi Polar RP HPLC column was used (method C). 2-OH-E⁺, 2-hydroxyethidium; EC, electrochemistry; HE, hydroethidine.

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Figure 8: HPLC-MS chromatogram of the cell extract from RAW 264.7 cells after incubation with HE as described in the Figure 6 caption.

The cell pellet was extracted with pure ethanol, centrifuged (15 min 20,000g, 4 °C) and the supernatant mixed with water (1:1) before the injection on column. An HPLC-MS system equipped with a Synergi Polar RP HPLC column was used (method D). Total ionic current (TIC) represents the mass range m/z from 100 to 1,000. The calculated (and detected) m/z values are as follows: HE: M+H⁺: 316.2, M+2H⁺: 158.6; 2-OH-E⁺: M: 330.2; E⁺: M: 314.2; dimer E⁺-E⁺: M: 313.2; dimer HE-HE: M+H⁺: 629.3, M+2H⁺: 315.2, M+3H⁺: 210.5; dimer HE-E⁺: M: 627.3, M+H⁺: 314.2, M+2H⁺: 209.8 where M denotes the parent compound. 2-OH-E⁺, 2-hydroxyethidium; HE, hydroethidine; MS, mass spectrometry.

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