Analysis of Kinetics of Dihydroethidium Fluorescence with Superoxide Using Xanthine Oxidase and Hypoxanthine Assay

Abstract

Superoxide (O₂⁻) is an important reactive oxygen species (ROS), and has an essential role in physiology and pathophysiology. An accurate detection of O₂⁻ is needed to better understand numerous vascular pathologies. In this study, we performed a mechanistic study by using the xanthine oxidase (XOD)/hypoxanthine (HX) assay for O₂⁻ generation and a O₂⁻ sensitive fluorescent dye dihydroethidium (DHE) for O₂⁻ measurement. To quantify O₂⁻ and DHE interactions, we measured fluorescence using a microplate reader. We conducted a detailed reaction kinetic analysis for DHE–O₂⁻ interaction to understand the effect of O₂⁻ self-dismutation and to quantify DHE–O₂⁻ reaction rate. Fluorescence of DHE and 2-hydroethidium (EOH), a product of DHE and O₂⁻ interaction, were dependent on reaction conditions. Kinetic analysis resulted in a reaction rate constant of 2.169±0.059×10³ M⁻¹s⁻¹ for DHE-O₂⁻ reaction that is ~ 100× slower than the reported value of 2.6±0.6×10⁵ M⁻¹s⁻¹. In addition, the O₂⁻ self-dismutation has significant effect on DHE-O₂⁻ interaction. A slower reaction rate of DHE with O₂⁻ is more reasonable for O₂⁻ measurements. In this manner, the DHE is not competing with superoxide dismutase and NO for O₂⁻. Results suggest that an accurate measurement of O₂⁻ production rate may be difficult due to competitive interference for many factors; however O₂⁻ concentration may be quantified.

Introduction

Superoxide (O₂⁻), which is one of the major reactive oxygen species (ROS), is one-electron reduction product of molecular oxygen. O₂⁻ participates in signaling pathways^{4, 9, 12} and is a common marker for oxidative stress. O₂⁻ leads to the formation of other ROS including hydrogen peroxide (H₂O₂), and hydroxyl radical (^•OH), and reactive nitrogen species (RNS) ^{7, 22, 25}. The accurate detection of O₂⁻ levels is critical for understanding numerous physiological phenomenon and pathologies such as cell proliferation, apoptosis, cardiovascular disease, diabetes and cancer^{8, 15, 26, 30}.

In the previous literature, several methods have been described to detect O₂⁻. These methods include lucigenin and luminol assays, electron paramagnetic resonance, high performance liquid chromatography (HPLC) coupled with mass spectrometry (MS), and fluorescence^{7, 8, 30, 32}. Most of these methods are not specific for O₂⁻ detection. For example, lucigenin and dichlorodihydrofluorescein-diacetate (H₂DCF-DA) can generate O₂⁻ and react with other oxidative stress. In addition, some methods can be very harmful to the human biological system. For example, lucigenin can stimulate oxidative stress formation to inhibit endothelium-dependent relaxation ²⁹, and hydroethidine (45 μM) has cytotoxic effect and there is an inverse correlation between hydroethidine uptake and cell survival ³⁵.

Since the early 1990’s, fluorescence resulting from the oxidation of dihydroethidium (DHE) has been used as an O₂⁻ probe with success²⁷. DHE has been established to detect O₂⁻ with end-product fluorescence measurement by many researchers^{5, 6, 24, 33, 34, 37}. DHE fluorescence is inhibited by O₂⁻ scavengers such as superoxide dismutase (SOD) and nitric oxide (NO)²⁰. Products for the oxidation of DHE by O₂⁻ are well established. In the presence of O₂⁻, DHE is oxidized to EOH and intermediate products. These intermediate products can react with ^•OH or H₂O₂ to form ethidium (E⁺) to a much lesser extent. Whereas E⁺ fluorescence is measured at excitation of 500–530nm and emission of 590–620nm, the EOH fluorescence is measured at an excitation and emission wavelength of 480nm and 567nm, respectively^{8, 33}. EOH is stable within the cell, allowing for precise measurement of DHE fluorescence without risk of intra-conversion variability.

Fluorescence measurement of DHE yields O₂⁻ levels specific for either intracellular or extracellular locations²². However, it is difficult to detect and quantify O₂⁻ because of its short life-time (a few seconds), low flux rate, fast self-dismutation rate, rapid reaction with multiple intra- and extracellular scavengers such as SOD, vitamin C and E, glutathione (GSH) and NO ^{3, 8}, and fluorescent dye reacting with various species to form other products or intermediate products^{10, 21, 35}. For the detection of O₂⁻ using DHE fluorescence, HPLC, microplate reader analysis, and fluorometry have been proven effective for qualitative and quantitative measurement of O₂⁻ concentrations ¹⁶. However, the quantitative understanding of O₂⁻ interaction with DHE in experiments is needed. Without this, only qualitative measurement of O₂⁻ is possible. A quantitative measurement of O₂⁻ interaction will help us to further study the impact and mechanism of O₂⁻ resulting damage.

Several studies have focused on quantitative interaction of DHE with O₂^{− 2, 6, 13, 18, 33, 36}. Laurindo et al. ¹³ described the possible way to separate using HPLC and to quantify EOH, which is the main product of DHE and O₂⁻ interaction, and E⁺, which is the main product of DHE and H₂O₂ interaction. Fink Bruno et al. ⁶ reported that EOH formation is inhibited in the presence of SOD, which catalyzes O₂⁻ dismutation. After the addition of H₂O₂, peroxynitrite (ONOO⁻) or hypochlorous acid to O₂⁻ generation system of potassium superoxide and XOD, they reported no additional change in EOH formation. Benov et al. ² showed that the ratio of E⁺ formation/O₂⁻ decreased as the flux of O₂⁻ increased. In addition, they showed that the DHE can catalyze the dismutation of O₂⁻. Fernandes et al. ⁵ showed that EOH increased after treatment of smooth muscle cells with AngII by detecting with HPLC and fluorescence micro-plate reader.

Zhao et al. ³³ reported that the addition of DEA/NO (an NO donor) inhibits the EOH fluorescence in xanthine/XOD system. In addition, using competition kinetic analysis with SOD, they estimated a reaction rate constant for DHE and O₂⁻ interaction of 2.6 ± 0.6 × 10⁵ M⁻¹s⁻¹. Later, Zielonka et al. ³⁶ used pulse radiolysis technique to calculate the rate constant between O₂⁻ and DHE is 2.0 × 10⁶ M⁻¹s⁻¹. They also reported that DHE interaction with only O₂⁻ results in EOH formation³⁴. Previous measurements of DHE interactions with O₂⁻ ignored the self-dismutation of O₂⁻, which can lead to the overestimation of this reaction rate constant.

In this study, we systematically investigated interactions of O₂⁻ with DHE by measuring DHE and oxidized product EOH using XOD catalysis of HX oxidation to form O₂⁻ using a microplate reader. We varied enzyme (XOD), substrate (HX), and reactant (DHE) concentration over a wide range to understand how these can affect the DHE and O₂⁻ interaction. We also developed a biochemical kinetic model to quantify DHE and O₂⁻ interactions and to understand the effect of self-dismutation of O₂⁻. As a result of this study, we provide a new reaction rate constant for the reaction between DHE and O₂⁻.

Materials and Methods

Materials

XOD, HX, catalase, ferricytochrome-c and phosphate buffer solution (PBS) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). DHE was purchased from Invitrogen Corp. (Carlsbad, CA, USA).

O₂⁻ and DHE reaction

Reactions were conducted in a 96-well Microtest^™ Optilux^™ Plate with a transparent bottom holding a total reagent volume of 250 μL in PBS (10 mM, pH 7.4). O₂⁻ was produced using HX and XOD enzyme system ¹¹. Catalase was used to remove excess H₂O₂, which is generated from the dismutation of O₂⁻ and the XOD/HX system. The reaction volume of 250 ml contained DHE, HX, catalase (100 U/ml) with or without XOD.

To understand the kinetics of the DHE and O₂⁻ reaction, we modified the kinetic analysis from an earlier study by Zhao et al. ³³ that used the competition kinetic analysis to provide a rate constant of 2.6 ± 0.6 × 10⁵ M⁻¹s⁻¹ for the O₂⁻ and DHE reaction. Three sets of experiments were conducted. First, we varied XOD concentration 1, 1.5, 2.5, 5 mU/ml, and kept HX and DHE concentration constant at 0.25 mM and 5 μM, respectively. Second, we varied DHE concentration 2, 5, 10, 20, and 50 μM and kept HX and XOD concentration constant at 0.25 mM and 1.5 mU/ml, respectively. Finally, the HX concentration was varied to 0.0625, 0.125, and 0.25 mM and XOD and DHE concentration was kept at 1.5 mU/ml and 5 μM, respectively.

Fluorescence measurement

In this study, EOH fluorescence was monitored by a Synergy 2 multi-detection microplate reader using a Gen5 microplate data collection & analysis software (BioTek Instruments Inc., VT, USA). As described by Zhao ³³, Robinson ²⁴ and Laurindo ¹³, the excitation/emission wavelength was chosen as 420/590 nm with a 50nm or 35nm slit width, respectively. The DHE fluorescence was measured at excitation/emission wavelength of 360/460 nm with a 40nm slit width. The plate was read from 50% top, which was suitable for all these filters, and the sensitivity was set to be 70. The kinetic measurements were performed for 120 min and fluorescence was recorded every 3 min. The fluorescence values were obtained by subtracting the background fluorescence values obtained in PBS with HX, catalase and DHE in the absence of XOD.

To obtain the DHE concentration, we generated a standard curve of DHE (concentration vs. fluorescence) by measuring DHE fluorescence at DHE concentration of 1, 2, 5, 10, 20, and 50 M in the same solution as experiment group without XOD. We observed that the DHE fluorescence without XOD in the solution (i.e. without superoxide) does not change significantly over 120 min.

O₂⁻ production

O₂⁻ production was measured using a widely used rapid reduction of ferri-cytochrome c reduction assay ^{14, 16}. Absorbance readings at 550 nm were collected every min over 30 min. A molar extinction coefficient 21,000 M⁻¹cm^{−1 33} and a path length 0.975 cm were used to calculate O₂⁻ production of different HX and XOD concentration experiments. The absorbance assay volume was 250 μL in PBS with reagents including XOD (1, 1.5, 2.5, and 5 mU/ml), HX (0.0625, 0.125, and 0.25 mM), catalase (100 U/mL), and ferri-cytochrome c (81 μM). We determined the rate of O₂⁻ generation by multiplying the slope of ferro-cytochrome c generation with the reciprocal of extinction coefficient of ferro-cytochrome c and path length.

DHE and O₂⁻ reaction kinetic analysis

We consider the following reactions occurring in the system to quantify the DHE and O₂⁻ interactions.

1. Reaction between DHE and O₂⁻ yields a fluorescent product at a rate constant of k_DHE as follows:

   $$\text{DHE}+{\text{O}}_2^{-}\stackrel{{\text{k}}_{\text{DHE}}}{\to }\text{Fluorescent}\text{Product}(\text{F})$$ (1)
2. We included the self-dismutation of O₂⁻ in the analysis. Earlier studies neglected this reaction ^{31, 33}. We also analyzed the effect of this reaction on the results of our study. In addition, we added large amount of catalase to scavenge H₂O₂. The rate constant for self-dismutation of O₂⁻ (k_D) ^{1, 23} is 8 × 10⁷ M⁻¹s⁻¹ and the reaction occurs as follows:

   $${\text{HO}}_{\text{2}}^{·}+{\text{O}}_2^{-}+{\text{H}}^{+}\stackrel{{\text{k}}_{\text{D}}}{\to }{\text{O}}_2+{\text{H}}_2{\text{O}}_2$$ (2)

Thus, the mass balance equation in the reaction system for the species of interests, O₂⁻, and DHE, can be written as:

$$\frac{{dC}_{o_2^{-}}}{dt}=S_{o_2^{·-}}-k_{\mathit{DHE}}{C}_{\mathit{DHE}}{C}_{o_2^{·-}}-k_D{C}_{{HO}_2^{·}}{C}_{o_2^{-}},$$ (3)

$$\frac{{dC}_{\mathit{DHE}}}{dt}=-k_{\mathit{DHE}}{C}_{\mathit{DHE}}{C}_{o_2^{-}}$$ (4)

Note that i) $S_{o_2^{-}}$ is the rate of O₂⁻ production in the system and was obtained from ferri-cytochrome c reduction assay as described in the previous subsection, and ii) the rate of O₂⁻ dismutation can be simplified assuming a rapid equilibrium of ${\text{HO}}_2^{·}/{{\text{O}}_2}^{-}$ with ${\text{C}}_{{\text{HO}}_2^{·}}/{\text{C}}_{{\text{o}}_2^{-}}=0.0025$, which is based on pK_a=4.8 and the pH value of 7.4 and the relationship for the mole fraction of ${\text{HO}}_2^{·}(\text{f}=\frac{1}{1+{10}^{({pk}_{\mathit{per}-pH})}})$. We assumed initial concentration of O₂⁻ is zero.

For a given experimental condition, Equations 3 and 4 were solved using the MatLab^® with initial concentrations of DHE (varied) and O₂⁻. We fitted the experimental data of DHE concentration (measured at ex/em of 360/460 nm) with the model predictions by varying k_DHE. As shown in Equation 5, we used least square estimation and minimized the sum of squares of error to obtain the rate constant, k_DHE.

$$Q={\sum }_{j=1}^k{\sum }_{i=1}^n{(y_{\mathit{exp}}-y_{\mathit{model}})}^2$$ (5)

Where y_exp and y_model are the average measured and model predicted DHE concentration values, respectively for one experiment condition. For each of the three sets of experiments mentioned earlier, k represents the number of varying conditions in one set of experiments and n represents the number of time data points in one experimental conditions. We fit the data only for first 18 min of experiments, therefore n is equal to 7. For each set of experiments, a new value of Q was calculated. The reported rate constant in this manuscript corresponds to the minimum Q.

Statistical analysis

Three runs were performed for each experimental condition and all values are reported as mean ± st. err. We used the Matlab function ode15s to solve the simultaneous differential equations and used absolute error and relative error of 1 × 10⁻¹³. For optimization of k_DHE, we used fminsearch function with a toleration error for Q of 1 × 10 ⁻¹³.

Results

DHE concentration related change in DHE and EOH fluorescence

Figure 1A and B show the reduction of DHE fluorescence and the accumulation of EOH fluorescence for DHE concentration of 2, 5, 10, 20, and 50 μM over 120 minutes. The initial XOD and HX concentration was 1.5 mU/ml and 0.25 mM, respectively. Both DHE and EOH fluorescence had two phases of fluorescence change. The fluorescence changed at an initial high slope followed by a small slope. The DHE fluorescence decreased linearly for the first 20 min for all DHE concentrations. In addition, an initial faster decrease in DHE fluorescence and increase in EOH fluorescence was observed at higher DHE concentrations (≥ 10 μM). After 40 min, DHE reacted completely and the formation of EOH reached a plateau except for the DHE concentration of 20 and 50 μM.

Figure 1. Measured DHE and EOH fluorescence as a function of DHE concentration.

Figure 1A and B shows the DHE and EOH fluorescence with respect to time for DHE concentration of 2, 5, 10, 20, and 50 M with XOD concentration of 1.5 mU/ml and HX concentration of 0.25 mM for 120 min.

Enzyme XOD concentration related change in DHE and EOH fluorescence

Figure 2A and B show DHE and EOH fluorescence for XOD concentration of 1, 1.5, 2.5, and 5 mU/ml over 120 min. The initial DHE and HX concentration was 5 μM and 0.25 mM, respectively. The DHE and EOH fluorescence again show the two phases for changes of fluorescence density; an initial steep change for the first 20 min followed by a slow change. In addition, Figure 2A and B show that the higher the XOD concentration, the faster the initial decrease in DHE fluorescence and the increase in EOH fluorescence. At XOD concentration of 1 mU/ml, Figure 2B shows that the DHE did not completely transform into EOH as demonstrated by non-convergence of RFU at 120 min with other XOD concentrations. This may be possible because of auto-oxidation of DHE resulting in other products and not EOH or sufficient superoxide formation to react with DHE.

Figure 2. Measured DHE and EOH fluorescence as a function of XOD concentration.

Figure 2A and B shows the DHE and EOH fluorescence for XOD concentration of 1.0, 1.5, 2.5 and 5 mU/ml for HX (=0.25 mM) and DHE (=5 mM) for 120 min.

Substrate HX concentration related change in DHE and EOH fluorescence

Figure 3A and B show the effect of HX concentration of 0.0625, 0.125, and 0.25 mM on DHE and EOH fluorescence, respectively. The initial DHE concentration was 5 μM and 1.5 mU/ml for XOD. The same two phases in DHE and EOH fluorescence were also present in this set of experiment. However, the initial DHE and EOH fluorescence changes were higher at increased HX concentrations. Figure 3B also shows that DHE did not completely convert into EOH at HX concentration of 0.0625 and 0.125 mM as compared with HX concentration of 0.25 mM.

Figure 3. Measured DHE and EOH fluorescence as a function of HX concentration.

Figure 3A and B shows the DHE and EOH fluorescence for HX concentration of 0.0625, 0.125 and 0.25 mM with XOD (=1.5 mU/ml) and DHE (=5 M) for 120 min.

Absorbance measurements of O₂⁻ generation (product of XOD and HX)

Figure 4A and B describe ferrocytochrome c absorbance for the measurement of O₂⁻ formation rate as a function of XOD concentration and HX concentration, respectively. These data is required for the kinetic analysis. As shown, the ferrocytochrome c absorbance increased linearly with varying slope for the first 20 min for all conditions. It implies that O₂⁻ formation rate is constant for a given condition over the first 20 min. For a given HX and XOD concentration, the O₂⁻ formation rate was calculated by multiplying the slope of increase in ferrocytochrome c absorbance with the reciprocal of EC value (21 mM⁻¹ cm⁻¹) and path length (0.975 cm). At 1, 1.5, 2.5, and 5 mU/ml XOD concentration, the O₂⁻ formation rate was 0.943, 1.465, 1.636, and 2.623 μM/min, respectively. The O₂⁻ formation rate was 0.866, 1.189, and 1.473 μM/min at 0.0625, 0.125, and 0.25 mM HX concentration. This data indicated the higher substrate and enzyme concentration leads to the higher O₂⁻ formation rate.

Figure 4. O₂⁻ generation rate by HX-XOD system determined by measuring ferrocytochrome c at 550 nm (EC=21 mM⁻¹cm⁻¹).

Figure 4A shows the absorbance of ferrocytochrome c for XOD concentration of 1.0, 1.5, 2.5 and 5 mU/ml for HX (=0.25 mM). Figure 4B shows the absorbance of ferrocytochrome c for HX concentration of 0.0625, 0.125 and 0.25 mM with XOD (=1.5 mU/ml).

Standard curve of DHE fluorescence vs. concentration

As shown in Figure 5, we generated the standard curve of DHE fluorescence vs. DHE concentration by fitting DHE concentrations (1, 2, 5, 10, 20, and 50 μM) with their corresponding initial fluorescence. As we used a very wide range of DHE concentration, the optimal fit was not linear. However, the nonlinear relationship (R² = 0.9965) between DHE concentration and RFU is [DHE], M= 1 × 10⁻⁷ × (DHE RFU)² + 0.0014 × (DHE RFU).

Figure 5. Standard curve of DHE concentration vs. DHE fluorescence.

Figure 5 shows DHE relative fluorescence unit (RFU) vs. DHE concentration for standard curve.

Kinetic analysis of DHE and O₂⁻ reaction

In order to obtain the kinetic rate constant, we fitted measured experimental data with model data by varying k_DHE. We used non-linear relationships from previous section to convert DHE RFU into DHE concentration.

First, we fitted experimental data from Figure 1A with varying DHE concentration and the constant O₂⁻ formation rate of 1.469 μM/min, which is the average value of 1.465 and 1.473 μM/min based on the ferrocytochrome c absorbance data at XOD=1.5 mU/ml and HX=0.25 mM. The minimum sum of squares of error between experimental data and predicted model data (Q) was 4.045 × 10³ M⁻¹s⁻¹. The minimum Q was obtained with a reaction rate constant of DHE and O₂⁻ of 2.116 × 10³ M⁻¹s⁻¹. The resulting fit with experimental data is shown in Figure 6A. As seen, the model data closely predicted each point of experimental data for 18 min for all DHE concentrations.

Figure 6. Measured and Predicted DHE concentration profiles using kinetic analysis.

Figure 6A-C shows the kinetic model results (solid lines) for experimental data (discrete symbols) of varying DHE concentration, XOD concentration and HX concentrations, respectively.

Next, we fitted experimental data from Figure 2A and 3A for varying XOD concentration and HX concentration, respectively. These conditions resulted in varying O₂⁻ formation rates as described in pervious sections for ferrocytochrome c absorbance data. Using different O₂⁻ formation rates, we obtained k_DHE for each of the XOD and HX concentration variation. The resulting fit with experimental data is shown in Figure 6B and 6C. At 1, 1.5, 2.5, and 5 mU/ml XOD concentration, the k_DHE is 1.933 × 10³, 1.827 × 10³, 2.626 × 10³, and 2.372 × 10³ M⁻¹s⁻¹, and at the HX concentration of 0.0625, 0.125, and 0.25 mM, the k_DHE reaction is 2.191 × 10³, 2.208 × 10³, and 2.295 × 10³ M⁻¹s⁻¹ resulted in the best fit between experimental and predicted data. The respective Q value was 5.436 × 10⁻¹⁵, 2.415 × 10⁻¹⁴, 1.447 × 10⁻¹³, 7.072 × 10⁻¹⁵, 7.327 × 10⁻¹⁵, 4.122 × 10⁻¹⁵, and 2.628 × 10⁻¹⁵. Based on all these reaction rate constant of DHE and O₂⁻, the mean rate of reaction of DHE with O₂⁻, k_DHE, is 2.169 ± 0.059 × 10³ M⁻¹s⁻¹.

Discussion

O₂⁻ is the precursor of many derivative ROS, and can rapidly react with NO to inactivate NO bioavailability and augment production of RNS. Therefore, the quantitative measurement of O₂⁻ measurement is critical. Traditionally, the fluorescence product of DHE was measured at the excitation/emission wavelength of 510/595 nm, which is the wavelength for detecting E⁺. The reaction of O₂⁻ and DHE also generates EOH that is confirmed to be the specific products formed. Researchers have started using excitation/emission wavelength to detect EOH fluorescence at either sensitive or specific wavelengths of 480/580 nm and 396/579 nm, respectively ^{5, 13, 24, 33}. In this study, we have confirmed that DHE fluorescence can be used to quantify O₂⁻ by HX-XOD system using microplate readers like other methods of O₂⁻ measurement ^{8, 13, 30, 32, 33}. The development of mathematical model for DHE interaction with O₂⁻ is novel and can be extended further for understanding many aspects of oxidative stress interactions including NO at molecular level. In addition, we provide a calibration curve for DHE concentration using RFU for the first time. We propose a DHE and O₂⁻ reaction rate constant of 2.169 ± 0.059 × 10³ M⁻¹s⁻¹.

O₂⁻ measurements

A predictable cell-free O₂⁻ generating system was utilized to achieve measurements of O₂⁻ concentration using a common fluorescence detection method and microplate reader analysis. The formation of EOH and depletion of DHE was specific to O₂⁻ as seen in Figure 1 with varying DHE concentration. The change in the rate of DHE fluorescence was dependent on O₂⁻ formation from the HX-XOD system as seen in Figures 2 and 3. However, in the absence of O₂⁻, DHE depletion was also influenced by autooxidation of DHE at later time points (>40 min). Both the increase in EOH and the decrease in DHE occurred in two linear phases: a steep initial change followed by a slower, steadier change. As the DHE concentration or the rate of O₂⁻ formation increased through the increase in the concentration of HX and XOD, both the decrease in DHE fluorescence and increase in EOH fluorescence would be accelerated. From these profiles, we showed that the higher the concentration of reactant, the faster the reaction between DHE and O₂⁻. The biphasic curve in Figures 1–3 was because of the change in the superoxide production rate at later time points that were seen after 30 min in most cases (data not shown) during absorbance assay (Figure 4). We showed that using a wide ranging superoxide production rate (varying HX and XOD concentrations for the first 18 min), we were able to fit the data with the k_DHE.

Currently, much research begins to pay attention to the accuracy of fluorescence detection of DHE-O₂⁻ reaction ^{13, 35}. These studies suggest that HPLC analysis is a reliable method for measurement of DHE detection of O₂. Due to the extremely short O₂⁻ half-life and rapid dismutation reaction rate, DHE fluorescence detection is prone to underestimation of actual O₂⁻ production^{34, 37}. Additionally, studies have shown that DHE can react with various oxidants found within the cell, including mitochondrial cytochrome hemes, hypochlorous acid, myeloperoxidase, horseradish peroxidase ONOO⁻, ^•OH, H₂O₂, compounds I and II, Fenton reagent, and so on^{10, 19, 21, 35}. Shao et al. ²⁸ also demonstrated that certain potent diet antioxidants can greatly decrease O₂⁻ concentration even in extreme acute oxidative stress. The total O₂⁻ production measurements are impossible as reported ⁸. At best, it will be an estimate due to the varying amount of O₂⁻ consumption among various interactions with other species.

However, Munzel ¹⁷ has suggested that it is wise to use two or more techniques to detect O₂⁻. Reliable conclusions could be drawn from similar results by using different measurements. In this study, we used cytochrome c absorbance and DHE fluorescence to detect O₂⁻ generation. Our results show that the formed products between superoxide and DHE or Cyt c respectively increase linearly. Moreover, by fitting our experimental data with computational model, we confirmed that DHE fluorescence measurement and microplate reader analysis can describe as a quantitative measurement of localized O₂⁻ concentration in vivo, which will provide us an effective method to detect O₂⁻.

DHE-O₂⁻ reaction rate constant of 2.169 ± 0.059 × 10³ M⁻¹s⁻¹

Based on experimental measurements and computational modeling in this study, we report a reaction rate constant of ~ 2.169 ± 0.059 × 10³ M⁻¹s⁻¹, which is ~ 100 × lower than the previously reported value of 2.6 ± 0.6 × 10⁵ M⁻¹s^{−1 33}. There are two possible reasons for this discrepancy. Firstly, the kinetic analysis in our study is more detailed with respect to the reactions occurring in the system. Secondly, the self-dismutation of O₂⁻ was considered in present study that increased O₂⁻ consumption and was not considered in previous study. The rate constant of O₂⁻ self-dismutation (8.0 × 10⁷ M⁻¹s⁻¹) is lower than that of O₂⁻ and SOD reaction (1.6 × 10⁹ M⁻¹s⁻¹)²³, but higher than that of DHE and O₂⁻ reaction (2.169 ± 0.059 × 10³ M⁻¹s⁻¹). Therefore, it is necessary to include O₂⁻ self-dismutation in DHE and O₂⁻ reaction system as done in this study.

We analyzed reasons behind a lower reaction rate constant for DHE reaction with O₂⁻. We simulated the experiment performed by Zhao et al. ³³ for k_DHE of 2.17 × 10³ and 2.69 × 10⁵ M⁻¹s^{−1 33} with and without the inclusion of self-dismutation of O₂⁻. We used a 50 μM initial DHE and 8 μM/min O₂⁻ generation rate ³³, and the results are plotted in Figure 7. Figures 7A and 7B show results for k_DHE of 2.69 × 10⁵ M⁻¹s⁻¹ without and with the inclusion of self-dismutation of O₂⁻, respectively, while Figure 7C and 7D show results for k_DHE of 2.17 × 10³ M⁻¹s⁻¹ without and with the inclusion of self-dismutation of O₂⁻, respectively. Figures 7 A–C are similar to each other and show that DHE is completely consumed and transformed to EOH at 6, 7, and 8 min respectively. However, as Zhao et al. ³³ described that the intensity of product fluorescence linear increased (Figure 10 in reference³³) and kept the same trend for first 20 minutes until they added DEA/NO into this reaction buffer. The model simulation results are not in agreement with experimental results. Therefore, we can conclude that DHE would fast consume for both k_DHE when self-dismutation of O₂⁻ is neglected.

Figure 7. Predicted O₂⁻, EOH, and DHE concentration profiles using kinetic analysis.

Figure 7 shows the predicted concentration profiles from the kinetic model for DHE and O₂⁻ reaction for 120 min. DHE and EOH concentration are shown on left axis. O₂⁻ concentration is shown on right axis. Figure A–D shows DHE and O₂⁻ reaction under O₂⁻ formation of 8 μM/min and DHE of 50μM, and Figure E–F shows DHE and O₂⁻ reaction under O₂⁻ formation of 1.465μM/min and DHE of 5μM. Figure A and B shows the profile for without and with O₂⁻ self-dismutation, respectively for 2.6 × 10⁵ M⁻¹s⁻¹ reaction rate constant of DHE and O₂⁻ reaction. Figure C and E shows the profile for without O₂⁻ self-dismutation for 2.17 × 10³ M⁻¹s⁻¹ reaction rate constant of DHE and O₂⁻ reaction. Figure D and F shows the profile for with O₂⁻ self-dismutation for 2.17 × 10³ M⁻¹s⁻¹ reaction rate constant of DHE and O₂⁻ reaction

However, when we include self-dismutation with the lower k_DHE, Figure 7D shows that there is an initial linear increase in EOH and decrease in DHE for the first 20 min followed by a slow change in EOH and DHE. In addition, we also simulated the experimental results of the current study without (Figure 7E) or with (Figure 7F) self-dismutation reaction at 5 μM DHE and 1.465 μM/min superoxide production rate (HX (0.25 mM) and XOD (1.5 mU/ml). As shown in Figure 7E, the depletion of DHE occurred in the first 10 minutes without self-dismutation reaction. Figure 7F shows that the model predictions with self dismuation reaction are similar to the experiment result i.e. DHE can last much longer. This indicated that O₂⁻ self-dismutation cannot be neglected and model results are in agreement with experimental results.

Implications of slower reaction rate of DHE with O₂⁻

A slower reaction rate of DHE with O₂⁻ is more reasonable for O₂⁻ measurements. In addition to superoxide production rate, the most important factors for the determination of O₂⁻ concentration in a given system are SOD and NO. SOD can fast dismutate O₂⁻ into H₂O₂ and NO can quickly react with O₂⁻ to generate RNS. The slower reaction rate of DHE-O₂⁻ implies that the presence of DHE does not affect O₂⁻ concentration, whereas the faster reaction rate implies that DHE competes with SOD and NO for O₂⁻ and reduces O₂⁻ concentration. Therefore, the respective changes in DHE fluorescence indicate change in O₂⁻ concentration in the system for the slower reaction rate. Many other chemical reactions simultaneously occur in the physiological system, the results from this study can be extrapolated to explain the effect of these reactions on oxidative stress. The change in DHE fluorescence will be in the same trend as these other reactions affect the O₂⁻ concentration. Thus, the fluorescence probes designed to exhibit a relatively low rate constant with the analyte detected will be ideal in vivo detection of O₂⁻. In ROS and RNS mathematical modeling, SOD can fast dismutate O₂⁻ into H₂O₂ and NO can quickly react with O₂⁻ to generate RNS. We conclude that DHE can be used to detect the O₂⁻ concentration without the interference of pathways in the cell system. It will be useful to determine the accumulated O₂⁻ concentrations and to predict damages in cell systems.

In conclusion, an accurate method for O₂⁻ measurement would be highly beneficial to the potential diagnosis and treatment of a number of different biological issues. This current study describes some necessary considerations for experimental measurement and analysis of O₂⁻ concentrations. DHE measurement of O₂⁻ may provide a more accurate description of oxidative stress, especially for O₂⁻. This detailed kinetic analysis study provided a DHE and O₂⁻ reaction rate constant of ~ 2.169 ± 0.059 × 10³ M⁻¹s⁻¹ that is ~ 100 × lower than the previously reported value of 2.6 ± 0.6 × 10⁵ M⁻¹s⁻¹, and is more appropriate for DHE O₂⁻ measurement.

Abbreviations

DHE

Dihydroethidium

E⁺

Ethidium

EOH

2-Hydroethidium

GSH

Glutathione

H₂DCF-DA

Dichlorodihydrofluorescein-diacetate

H₂O₂

Hydrogen peroxide

HPLC

High performance liquid chromatography

HX

Hypoxanthine

MS

Mass spectrometry

NO

Nitric oxide

O₂⁻

Superoxide

^•OH

Hydroxyl radical

ONOO⁻

Peroxynitrite

PBS

Phosphate buffer solution

ROS

Reactive oxygen species

RNS

Reactive nitrogen species

SOD

Superoxide dismutase

XOD

Xanthine oxidase