Hydrogen Peroxide is the Major Oxidant Product of Xanthine Oxidase

Abstract

Xanthine oxidase (XO) is a critical source of reactive oxygen species (ROS) in inflammatory disease. Focus, however, has centered almost exclusively on XO-derived superoxide (O₂^•−) while direct H₂O₂ production from XO has been less well-investigated. Therefore, we examined the relative quantities of O₂^•− and H₂O₂ produced by XO under a range (1–21%) of O₂ tensions. At O₂ concentrations between 10 and 21 %, H₂O₂ accounted for ~ 75% of ROS production. As O₂ concentrations were lowered, there was a concentration-dependent increase in H₂O₂ formation, accounting for 90% of ROS production at 1% O₂. Alterations in pH between 5.5 and 7.4 did not affect the relative proportions of H₂O₂ and O₂^•− formation. Immobilization of XO, by binding to heparin-Sepharose, further enhanced relative H₂O₂ production by ~30%, under both normoxic and hypoxic conditions. Furthermore, XO bound to glycosaminoglycans (GAGs) on the apical surface of bovine aortic endothelial cells demonstrated a similar ROS production profile. These data establish H₂O₂ as the dominant (70–95%) reactive product produced by XO under clinically relevant conditions and emphasize the importance of H₂O₂ as a critical factor when examining the contributory roles of XO-catalyzed ROS in inflammatory processes as well as cellular signaling.

Introduction

The molybdoflavin enzyme, xanthine oxidoreductase (XOR) catalyzes the terminal two steps of purine degradation (hypoxanthine → xanthine → uric acid) in humans. XOR is transcribed as a single gene product, xanthine dehydrogenase (XDH). Substrate-derived electrons at the Mo-cofactor of XDH are transferred via Fe/S centers to a FAD moiety where NAD⁺ is reduced to NADH. During inflammatory conditions, post-translational modification by oxidation of critical cysteine residues or limited proteolysis converts XDH to xanthine oxidase (XO) (1, 2). The key difference between XDH and XO is the structural conformation and electrostatic microenvironment surrounding the FAD resulting in a decreased affinity for NAD⁺ and enhancement of affinity for O₂ (3). Substrate-derived electrons at the Mo-cofactor of XO reduce O₂ at the FAD-cofactor both univalently, generating superoxide (O₂^•−), and divalently, forming hydrogen peroxide (H₂O₂). However, conversion to XO is not requisite for ROS production, as XDH displays partial oxidase activity under conditions in which NAD⁺ levels are diminished such as the ischemic/hypoxic microenvironment encountered in vascular inflammation (4). This same inflammatory milieu leads to enhanced XO levels and thus increased XO-derived ROS formation resulting in activation of redox-dependent cell signaling reactions and alterations in vascular function. Evidence of this role for XO is exemplified by numerous studies in which XO inhibition attenuates symptoms of vascular disease including congestive heart failure, sickle cell anemia and diabetes (5–8).

Reports of XO-derived ROS production frequently address XO as the O₂^•−-producing form of XDH and H₂O₂ is produced as a secondary byproduct of spontaneous or enzymatic dismutation of O₂^•−. A crucial concept is often overlooked, specifically, that under relatively physiologic conditions (21% O₂ and pH 7.0) XO catalyzes the reduction of O₂ to H₂O₂ and O₂^•− at a ratio of 4:1 (H₂O₂:O₂^•−) or ~80% H₂O₂ and ~20% O₂^•− , whereas production of 100% O₂^•− requires an environment of 100% O₂ at pH 10 (9). While some studies have acknowledged this characteristic of XO (10–15), it is vastly underappreciated in the literature where focus remains fixed on O₂^•− as the key reactive product derived from XO. In addition, a limited number of biochemical studies addressing XO-mediated H₂O₂ production have centered on hyperoxia and/or alkaline conditions, which are less reflective of pathophysiologic conditions under which XOR most likely exerts significant influences (9, 16–18). With renewed attention being focused on XO-derived ROS in numerous inflammatory processes, the relationship between O₂ concentration and XO-catalyzed H₂O₂/O₂^•− formation is crucial for the evaluation of contributory roles of XO and subsequent design of pharmacological approaches for treatment. In toto, these issues affirm the need for the examination of XO-derived ROS under clinically relevant conditions as performed herein.

Materials and Methods

Materials

Xanthine, allopurinol, diphenyleneiodonium chloride (DPI), Chelex resin, catalase and uric acid were from Sigma (St. Louis, MO). Medium 199 and fetal bovine serum (FBS) were from Invitrogen (Carlsbad, CA). Superoxide dismutase (CuZnSOD) was from OXIS International Inc. (Portland, OR).

Buffer Treatment

Buffers for all experiments were prepared from MilliQ H₂O and treated with Chelex resin to remove adventitious metals and thus minimize loss of ROS by metal-catalyzed reactions.

XO Activity

Enzyme was purified from fresh bovine cream by the method of Rajagopalan and stored in ammonium sulfate at 4°C until immediately before use (19). Enzymatic activity was determined either spectrophotometrically by the rate of uric acid formation monitored at 292 nm in 50 mM potassium phosphate (KPi), pH 7.4 (ε = 11 mM⁻¹ cm⁻¹) or electrochemically via reverse phase HPLC analysis of uric acid production (ESA CoulArray System, Chelmsford, MA), (1 Unit = 1 µmole urate/min) as previously (20). XDH activity was distinguished from XO activity by incubation with NAD⁺ as previously described (21). Formation of O₂^•− was assessed by the SOD-inhibitable reduction of cytochrome c (550 nm) (9).

Univalent/Divalent Flux

Univalent flux was determined as previously reported (9). Briefly, under saturating xanthine concentrations, the total electron flux through the enzyme to O₂ was calculated as the rate of uric acid formation. Under saturating cytochrome c concentrations, the rate of SOD-inhibitable cytochrome c reduction represents a measure of electron flux via the univalent reduction of O₂ to O₂^•−. Dividing the cytochrome c reduction rate (1 e⁻) by the uric acid formation rate (2 e⁻) gives the % univalent flux (cyto c / 2(uric acid) ×100). As O₂ is the sole oxidizing substrate in the system, the divalent flux is derived by subtracting % univalent flux from 100.

XO Binding to GAGs

Xanthine oxidase was bound to heparin Sepharose 6B (HS6B) as previously (20). Briefly, XO (2 mg/ml) was added to a fixed amount of gel (0.05 g dry weight) and the mixture gently stirred in 5 mM KPi, pH 7.4 (2 ml final volume) at 25°C for 30 min. The suspension was centrifuged at 10,000 × g for 5 min, washed and the pellet resuspended in 5 mM KPi, pH 7.4. A quantity of HS6B-XO, equaling 5 mU/ml of XO activity, was added to PBS pH 7.4 in a 3 ml cuvette containing a small stir bar. Continuous gentle stirring was maintained with a Helma electronic stirrer placed inside the spectrophotometer cavity.

Oxygen Tension Experiments

Experiments at specific oxygen tensions were performed in a table-top glove box (Coy Instruments, Grass Lake, MI, USA) purged with N₂. All buffers were equilibrated >18 h before use. Glove box atmospheric O₂ conditions were followed with an O₂ monitor (Maxtec, Salt Lake City, UT) and O₂ concentrations verified with a clinical blood gas analyzer. Spectrophotometric determinations were carried out in gas tight cuvettes. Real time concentrations of molecular O₂ were determined polarographically using an Apollo 4000 Free Radical Analyzer (World Precision Instruments, Sarasota, FL, USA). Experiments were performed at standard temperature (25°C) and pressure (1 Atm).

Cellular Studies

Bovine aortic endothelial cells (BAEC) were isolated as previously (22). Primary cell culture, routine passage and experimental manipulations were all conducted in the absence of proteases. Cells were propagated by sub-culturing (1:4 ratios) in Medium 199 containing 5% FBS and thymidine (10 µM). Cells were utilized between passages 4 and 8 and were monitored visually for typical cobblestone morphology indicative of endothelial cells and by staining for von Willebrand factor expression. For O₂ consumption studies, confluent BAEC were exposed to XO (5 mU/ml) for 20 min at 25°C, harvested by mechanical dissociation, washed thoroughly (3 times with ice-cold PBS, pH 7.4), resuspended as a single-cell suspension (2 × 10⁶ cells /ml) and placed on ice (for less than 1 h) until warmed to 25°C immediately before evaluation. This method minimizes cellular internalization of the enzyme as we have previously demonstrated (23). Cell viability was 93% following 1 h on ice as determined by Trypan blue dye exclusion. Oxygen consumption studies were performed under various O₂ tensions, as above, in the presence and absence of 50 U/ml CAT and/or SOD.

Statistics

Data were analyzed using one way analysis of variance followed by Tukey’s range test for multiple pair-wise comparisons. Significance was determined as p < 0.05.

Results

Univalent/Divalent Flux

At 21% O₂, oxidation of xanthine (100 µM) to uric acid by XO (5 mU/ml) resulted in 28.1 ± 1.4% univalent flux (O₂^•− formation) in accordance with our previous reports, Table 1 (20, 24). Between 21 and 5% O₂, univalent flux remained relatively constant at ~70 %. As O₂ concentration was lowered below 5%, there was an O₂-dependent decrease in univalent flux so that O₂^•− formation accounted for only 10% of electron flow through the enzyme at 1% O₂. Likewise, XO-dependent divalent reduction of O₂ to H₂O₂ increased from 72 % (21% O₂) to 90% (1% O₂). As O₂ tensions dropped below the K_m for O₂ (46 µM) an O₂-dependent decrease in the total electron flux (as determined by the formation of uric acid) through the enzyme was observed (1% = 13 µmoles/min, 2.5% = 18 µmoles/min and 5% = 2.3 µmoles/min) (1, 2.5 and 5% O₂ = ~13, 29 and 59 µM O₂, respectively) (25). This was reflected by a decrease in the overall rates of both uric acid formation and cytochrome c reduction. Control experiments conducted in the presence of catalase (CAT) demonstrated no H₂O₂-mediated reoxidation of cytochrome c during the initial 60 s of the reaction (the time frame for all univalent flux studies herein) that could account for the observed reduction in O₂^•− formation. Additional control experiments demonstrated that XO preparations were not contaminated with SOD as determined by both western-blot and activity assays. When O₂ tension was elevated to 95%, the percent univalent flux increased to 47.2 ± 2.3 (data not shown).

Table 1. Oxygen dependence of univalent vs. divalent flux for XO.

Purified XO (5 mU/ml) was exposed to xanthine (100 µM) and superoxide was measured by the SOD-inhibitable reduction of cytochrome c (λ = 550 nm). Divalent flux was derived by subtracting the percent univalent flux values from 100. Experiments were performed at standard temperature (25°C) and pressure (1 Atm).

[O2] %	O2•− % Univalent Flux	H2O2 % Divalent Flux
1	10.4 ± 0.6	90
2.5	14.3 ± 0.9	86
5	24.6 ± 2.3	76
10	28.5 ± 0.7	71
15	27.8 ± 0.5	72
21	28.1 ± 1.4	72

Values for univalent flux were calculated by monitoring the SOD-inhibitible reduction of cytochrome c (550 nm) as described in the methods. Values for divalent flux were derived by subtracting the percent univalent flux from 100.

Oxygen Consumption

Initiation of XO turnover by addition of xanthine (100 µM), at 1% O₂, resulted in a rapid rate of O₂ consumption, Fig 1A. Increasing the O₂ concentration from 1–5% resulted in an O₂-dependent increase in the rate of O₂ consumption similar to the effects observed for univalent flux studies. At 1% O₂, the addition of CAT (50 U/ml) after 4.7 µM of O₂ was consumed, produced an immediate and pronounced evolution of O₂ (2.1 µM) from Reaction 1, Fig. 1B. Similar experiments carried out at different O₂ concentrations revealed an O₂-dependent diminution of CAT-induced O₂ evolution between 1–5% O₂ with no further decrease in O₂ evolved above 5% O₂, Fig. 1C and 1D. It is important to note that the quantity of H₂O₂ consumed by CAT is represented by 2 × O₂ evolved from Reaction 1. For example, at 1% O₂, CAT addition resulted in 2.1 µM O₂ evolution representing the enzymatic catalysis of 4.2 µM H₂O₂ and thus 89% of the O₂ consumed was due to H₂O₂ formation. Similar calculations for % H₂O₂ formation from O₂ consumption at the various O₂ tensions yielded: (89% (1% O₂), 77% (2.5% O₂), 72% (5% O₂), 64% (10% O₂), 68% (15% O₂) and 65% (21% O₂)).

$$2{\mathrm{H}}_2{\mathrm{O}}_2\to 2{\mathrm{H}}_2\mathrm{O}+{\mathbf{O}}_{\mathbf{2}}$$ Reaction 1

$${{\mathrm{O}}_2}^{•-}+{{\mathrm{O}}_2}^{•-}\to {\mathrm{H}}_2{\mathrm{O}}_2+{\mathbf{O}}_{\mathbf{2}}$$ Reaction 2

Fig. 1. H₂O₂ is the major ROS produced by XO at low O₂ tensions.

(A) XO (20 mU/ml, PBS pH 7.2) was exposed to xanthine (100 µM) in a 1% O₂ atmosphere and O₂ concentration monitored polargraphically over time. (B) During the linear phase of O₂ consumption 50 U/ml catalase (CAT) (2H₂O₂ → 2H₂O + O₂) was added to the reaction. (C) Scheme showing total O₂ evolved (2.1 µM) by the addition of 50 U/ml CAT at 1% O₂ and the effect of increasing O₂ tension (1–5%) on the quantity of CAT-dependent O₂ evolution. (D) Using the quantity of O₂ evolved at 1% O₂ as maximum (100%), CAT-dependent O₂ evolution values are plotted as (% Maximum) for O₂ concentrations (1–21%). For each O₂ tension, CAT was added to the samples immediately after the same XO-dependent depletion of O₂ (4.7 µM). Values represent the mean of at least 4 independent determinations.

Addition of SOD (50 mU/ml) during enzyme turnover did not alter the rate of O₂ consumption from Reaction 2, Fig. 2A. At 1% O₂, when SOD or CAT was added to the sample before xanthine, CAT significantly decreased the rate of O₂ consumption while SOD did not, Fig. 2B. However, the addition of both SOD and CAT resulted in an additional decrease in the O₂ consumption rate, compared to that produced by CAT alone, as diagrammed in Fig. 2C. Plotting the O₂ consumption rates in the presence of (SOD + CAT) as a percentage of the CAT only rates for each O₂ tension revealed an O₂-dependent decrease in the SOD + CAT rates from 1–5% O₂, Fig. 2D. For all O₂ consumption studies, addition of boiled CAT or SOD did not affect O₂ evolution or rates of O₂ consumption, not shown.

Fig. 2. Oxygen dependence of O₂^•− formation from XO.

(A) XO (20 mU/ml, PBS pH 7.2) was exposed to xanthine (100 µM) at 1% O₂ as in Fig. 1. During the linear phase of O₂ consumption 50 U/ml SOD (O₂^•− + O₂^•− → H₂O₂ + O₂) was added to the reaction. (B) Either SOD or CAT were added to the sample before the initiation of enzyme turnover with xanthine (100 µM) and compared to control in the absence antioxidant. (C) Shown is a representative diagram of experiments conducted similar to those in B, but with SOD and CAT added together before xanthine. (D) The (SOD + CAT)-dependent decreases in the rate of O₂ consumption are plotted as percentage of the CAT only values (CAT only = 100%) at each O₂ concentration (1–21%). These (SOD + CAT)-dependent decreases in the rate of O₂ depletion result from evolution of O₂ from the following reactions: SOD (O₂^•− + O₂^•− → H₂O₂ + O₂) and CAT (2H₂O₂ → 2H₂O + O₂). Shown are representative experiments of at least 4 independent determinations.

Effects of pH

The effect of pH (5.5–7.4) on the relative proportions of O₂^•− and H₂O₂ formation by XO were determined under various O₂ concentrations (1–21%) at saturating xanthine concentrations (100 µM). There was no effect of pH on univalent/divalent flux at any of the O₂ concentrations examined, data not shown.

XO-Immobilization

At 21% O₂, immobilization of XO by binding to heparin-Sepharose 6B (HS6B-XO) reduced the rates of urate formation and cytochrome c reduction as we reported previously (20), Table 2 and online supplement. In addition to an immobilization-induced reduction of total electron flow through the enzyme, the relative proportion of O₂^•− and H₂O₂ was also altered, reducing univalent electron transfer to O₂ by 30% compared to XO in solution. This immobilization-induced reduction in univalent flux was consistent for all O₂ tensions examined, such that at 1% O₂, univalent flux was only 6.9 % for bound XO compared to 10.4% univalent flux for free XO.

Table 2. Oxygen dependence of univalent vs. divalent flux for GAG-imobilized XO.

XO was immobilized on heparin-Sepharose 6B (HS6B-XO), exposed to xanthine (100 µM) and univalent flux (O₂^•− formation) determined by the SOD-inhibitable reduction of cytochrome c (λ = 550 nm) under the indicated O₂ tensions. Divalent flux (H₂O₂ formation) was derived by subtracting the percent univalent flux values from 100. Experiments were performed at standard temperature (25°C) and pressure (1 Atm).

[O2] %	% O2•−		% H2O2
	free	bound	free	bound
1	10.4 ± 0.6	6.9 ± 1.2	90	93
5	24.6 ± 2.3	17.4 ± 4.2	76	83
10	28.5 ± 0.7	18.9 ± 2.1	71	81
21	28.1 ± 1.4	19.8 ± 1.1	72	80

Values for univalent flux were calculated by monitoring the SOD-inhibitible reduction of cytochrome c (550 nm) as described in the methods. Values for divalent flux were derived by subtracting the percent univalent flux from 100.

Endothelial cell-bound XO

Xanthine oxidase was bound to extracellular GAGs on the apical surface of BAEC as described in the methods. Cellular O₂ consumption studies were performed at various O₂ tensions (1–21%) in which CAT (200 U/ml), SOD (200 U/ml) or both were present before addition of xanthine. The effect of the presence of the antioxidants on the rate of XO-dependent O₂ consumption was determined and plotted as percent decrease when compared to control rates (no SOD or CAT) at each O₂ tension, Fig. 3. The presence of CAT significantly decreased the rate of O₂ consumption at all O₂ tensions resulting in a 27% decrease at 1% O₂ while SOD produced small, but significant, decreases (>5%) at O₂ levels above 2.5%. The presence of both SOD and CAT did not significantly alter rates of O₂ consumption compared to samples containing only CAT. Rates of O₂ consumption in the absence of xanthine were determined and subtracted from all values reported. When XO-treated cells were washed and then exposed to trypsin (0.25%) for 3 min at 37°C to remove/inactivate GAG-associated enzyme, no O₂ consumption was observed upon addition of xanthine demonstrating that ROS formation was from cell-associated XO. Furthermore, treatment with the XO-specific inhibitor, Febuxostat (25 µM), before addition of xanthine, abolished XO-dependent O₂ consumption, not shown.

Fig. 3. H₂O₂ is the major ROS produced by endothelial cell-associated XO.

BAEC were exposed to XO (5 mU/ml) as described in the methods. Cell suspensions (2 × 10⁶ cells /ml) were monitored for O₂ consumption upon the addition of xanthine. Either SOD (200 U/ml), CAT (200 U/ml) or both were added to the samples before xanthine as indicated. The effects of the presence of (CAT, SOD, CAT+SOD) are represented as % decrease (inset) in the rate of O₂ consumption from control samples without antioxidant and are plotted for the indicated O₂ tensions. Values represent the mean of 3 independent determinations. (*) indicates p < 0.05 compared to CAT only and/or CAT+SOD.

Discussion

Studies on reactive species derived from XO have confirmed the biological production of O₂^•− (26). However, under normoxic conditions at neutral pH, XO produces significantly greater quantities of H₂O₂ (9). Perhaps of more significance, is that under the lower O₂ tensions and pH encountered during inflammation or ischemia, H₂O₂ accounts for 90–95% of XO-derived ROS formation, Table 1 and Table 2. These data confirm an O₂ dependence for ROS formation where lower O₂ concentrations lead to even greater H₂O₂ formation by XO. Conceptually, this can be illustrated by consideration of the overall oxidation/reduction state of the enzyme, Fig. 4. This scheme, albeit a very simplified representation of a complex series of electronic interactions between cofactors, summarizes the concept that a more reduced XO favors H₂O₂ production while a more oxidized XO favors O₂^•− formation. This effect of the redox state of XO on H₂O₂ vs. O₂^•− production has been previous reported with purified enzyme at pH 8.5 and confirms that high concentrations of xanthine and low O₂ tensions result in greater H₂O₂ production while low concentrations of xanthine and high O₂ tensions result in greater O₂^•− formation (18, 27–29). This is critical to note as hypoxia/inflammation leads to lower O₂ tensions, increased XO expression as well as increased hypoxanthine/xanthine levels from ATP catabolism and thus sets the stage for enhance vascular H₂O₂ production. Therefore, recognizing that both reducing substrate concentration and O₂ tension are the key factors determining the identity XO-mediated ROS formation, we examined the effects of O₂ tension in the presence of a saturating xanthine concentration (100 µM). We chose this xanthine level as it reflects the concentrations encountered in vivo under ischemic/hypoxic conditions where ATP breakdown results in elevation of hypoxanthine/xanthine levels from ~2 µM to 50–100 µM, well above its K_m (6.5 µM) (20). In aggregate, our studies were designed to confirm and expand the observations of previous studies under clinically-relevant conditions and to refocus attention on XO-derived H₂O₂.

Fig. 4. Effects of O₂ on XO-derived ROS formation.

Purine-derived electrons enter XO via the Mo-cofactor and egress through the FAD-cofactor by reaction with molecular O₂. Therefore, at physiological pH, the oxidation/reduction status of XO is dependent upon both the concentration of purine (xanthine/hypoxanthine) and O₂. Under saturating purine concentrations, O₂ tension is the prime determinant of this redox state of XO. Shown are 2 cartons depicting the extreme redox states (either fully oxidized or reduced) of the various co-factors in XO in the presence of saturating xanthine concentrations. On the left, under anoxia, the enzyme is fully reduced. Upon oxygenation, the reduced FAD-cofactor reacts with O₂ diavalently to produce H₂O₂. As O₂ levels increase so does O₂-mediated electron egress via the FAD-cofactor until it begins to out compete electron entry at the Mo-cofactor resulting in an increase in the overall oxidation of the enzyme. This increase in the oxidation of the enzyme shifts FAD-cofactor-O₂ reactions from divalent to univalent producing more O₂^•− and less H₂O₂.

Electron flux studies utilize uric acid formation and cytochrome c reduction to determine univalent flux; however, they do not directly measure divalent flux and thus it is a derived value. In a simple system in which O₂ is the sole electron acceptor, this is a valid calculation; however, an alternative approach which measures XO-derived H₂O₂ formation would confirm conclusions based on this derivation. Oxygen consumption was chosen to examine XO-dependent H₂O₂ formation in the presence and absence of CAT and SOD, the enzymes most often used to substantiate contributions of H₂O₂ and O₂^•− in biological systems, Fig. 1. This avoids possible direct interactions with XOR, ROS specificity issues and the potential of redox cycling which limit the specificity and sensitivity of several dye-based H₂O₂ assays (30). At 1% O₂, CAT addition resulted in significant evolution of O₂, indicating that 89% of the O₂ consumed by XO was reduced to H₂O₂. Values obtained at other O₂ tensions were also similar to, yet slightly lower than, the divalent fluxes listed in Table 1 supporting the validity of this approach. Lower yields for the O₂ evolution experiments may be due to the extended time (~2 min) of enzyme turnover and thus potential loss of H₂O₂ before the addition of CAT and/or subtle variations in temperature and barometric pressure.

Since CAT addition experiments confirmed the proportion of H₂O₂ formed during XO-catalyzed O₂ consumption, then the remainder of O₂ consumption should be attributable to O₂^•− formation. However, at 1% O₂, the presence of SOD before or after (Fig. 2) the addition of xanthine resulted in neither O₂ evolution nor alteration of the O₂ consumption rate. It is hypothesized that loss of O₂^•− from spontaneous dismutation (~2 × 10⁵ M⁻¹ s⁻¹), reaction with protein in the sample and degree of assay sensitivity all contributed to the absence of observable effects. To further address this issue, experiments were performed in which both SOD and CAT were present before initiation of enzyme turnover. At all O₂ tensions, addition of SOD + CAT before xanthine decreased the rate of O₂ consumption compared to samples containing only CAT, Fig. 2C and 2D. It is assumed that both O₂ evolution and the formation of additional CAT substrate (H₂O₂) by SOD (Reaction 2) served to augment total O₂ evolution. The CAT + SOD-induced decreases in O₂ consumption rates were not O₂-dependent above 5% O₂ (Fig. 2D) which is consistent with data from univalent flux studies showing no significant increase in O₂^•− formation from 5–21% O₂. This result is also consistent with the argument that as O₂ levels exceed the K_m of O₂ with the FAD (46 µM), a constant rate of O₂^•− formation would ensue where the presence of SOD offers no additional contribution to O₂ evolution and subsequent reduction in the rate of O₂ consumption in this O₂ concentration range (46–235 µM or 5–21%).

Inflammation is often characterized by both hypoxia and lowered pH; thus, it is important to consider the effects of H⁺ ion concentration on ROS production by XO. The oxidation of xanthine at the Mo-cofactor is base-catalyzed and thus sensitive to changes of pH in the physiological range as well as substrate concentration (27). However, under saturating xanthine concentrations, the relative proportion of H₂O₂ to O₂^•− at each O₂ tension examined was not altered by pH (5.5–7.4).

During inflammatory conditions XDH is released into the circulation, rapidly converted to XO and avidly binds (K_d = 6 nM) to negatively charged glycosaminoglycans (GAGs) on the surface of vascular endothelial cells (24, 31–33). This sequestration of XO by GAGs substantially amplifies local enzyme concentration, diminishes its rotational and translational mobility, thus, altering kinetic properties and conferring resistance to both product-induced and pharmacological inhibition (20, 24). GAG association of XO decreases substrate binding affinity and thus increases the K_m for xanthine (6.5 µM free vs. 21.2 µM bound) and K_i for oxypurinol (85 nM free vs. 451 nM bound) when compared to XO in solution (24, 34). These characteristics indicate that XO bound to endothelial cell GAGs could serve as a long-lived source of ROS in this microenvironment. It is in this setting that XO-generated ROS can critically impact vessel function, emphasizing the need to more clearly define the effects of O₂ tension on ROS production by GAG-bound XO. Our previous studies demonstrate immobilization of XO on heparin-Sepharose reduces univalent flux by 30% in room air (20). At every O₂ concentration examined, GAG immobilization of XO produced a similar ~30% reduction in univalent flux, so that at 1% O₂, 93% of the ROS formed was H₂O₂. These data suggest XO-derived ROS production is almost exclusively H₂O₂ when XO is bound to endothelial GAGs in a hypoxic vascular milieu. To test this hypothesis in a cellular model, BAEC were exposed to purified XO and XO-dependent O₂ consumption was monitored over time in the presence of SOD, CAT or CAT + SOD, Fig. 3. The presence of SOD slightly altered O₂ consumption rates only when O₂ tensions exceeded 5%. In contrast, CAT significantly decreased rates of O₂ consumption as XO-produced H₂O₂ was converted to H₂O and O₂. These CAT-dependent decrements in O₂ consumption rates were seen at all O₂ tensions, with the greatest effects observed below 5% where H₂O₂ production would be expected to account for a preponderance of total ROS formation. The presence of both CAT and SOD produced O₂ consumption rates that were not statistically different from rates produced by samples containing only CAT. While differences between the CAT + SOD and CAT only values did not demonstrate statistical significance, the individual mean values for CAT + SOD from 5–21% O₂ were all greater than the corresponding CAT only values, possibly suggesting contributions from SOD near the limit of detection. Combined, these data demonstrate that quantification and characterization of ROS from these cellular studies is problematic due to loss of both H₂O₂ and O₂^•− before reaction with CAT and SOD from many mechanisms including reactions with cellular constituents such as GSH. However, they do reveal similar trends to those observed in the biochemical studies and qualitatively demonstrate that H₂O₂ is the major ROS product of XO, especially at O₂ tensions relevant to vascular pathology.

Over the past several years, there is renewed interest in the role of XO in vascular inflammation as the number of studies demonstrating salutary effects of XO inhibition accumulate. In order to successfully evaluate the contribution of XO in these and future studies, it is crucial to understand the identity and relative proportions of the ROS produced by this complex enzyme under conditions reflective of the microenvironment in which they are formed. Herein, we revisit the underappreciated detail that H₂O₂ is the major (~75%) ROS produced by XO under normal aerobic conditions and expand this observation to demonstrate that under pathophysiologic conditions XO-derived H₂O₂ formation approaches 95%. These results affirm the need to more critically evaluate the role of H₂O₂ in studies where XO-derived ROS have been proposed to play a contributory role. For example, many of the vessel studies in which XO-derived O₂^•− reduces NO-mediated vasodilatation were conducted using 95% O₂. This O₂ tension is significantly higher than O₂ levels experienced in vivo, and also serves to greatly enhance XO-derived O₂^•− formation and possibly lead to misconceptions regarding the role XO in this model. Furthermore, assigning a causative role for XO-derived O₂^•− based upon the beneficial actions of allopurinol administration, in the absence of controls for XO-derived H₂O₂, may result in neglecting pivotal signaling events mediated by H₂O₂, a molecule with an expanding number of targets in various cell signaling pathways (35–37).

Combined, the results from this report refocus attention on previous and commonly overlooked studies reporting H₂O₂ as the major ROS product of XO under normoxia. Furthermore, these data demonstrate that under pathophysiologic conditions XO-derived H₂O₂ formation approaches 95% and thus underscores the danger of failing to appreciate this attribute when investigating contributory roles for XO in pathology.

Abbreviations

BAEC

bovine aortic endothelial cells

CAT

catalase

GAGs

glycosaminoglycans

ROS

reactive oxygen species

SOD

superoxide dismutase

XDH

xanthine dehydrogenase

XO

xanthine oxidase

XOR

xanthine oxidoreductase