8-Nitroxanthine, a product of myeloperoxidase, peroxynitrite, and activated human neutrophils, enhances generation of superoxide by xanthine oxidase

ABB Archives of Biochemistry and Biophysics 418 (2003) 1–12 www.elsevier.com/locate/yabbi

8-Nitroxanthine, a product of myeloperoxidase, peroxynitrite, and activated human neutrophils, enhances generation of superoxide by xanthine oxidase George C. Yeh,a,1 Jeffrey P. Henderson,a Jaeman Byun,b D. Andre dÕAvignon,c and Jay W. Heineckeb,* a

Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA b Department of Medicine, University of Washington, Seattle, WA 98195, USA c Department of Chemistry, Washington University, St. Louis, MO 63110, USA Received 18 February 2003, and in revised form 30 April 2003

Abstract Reactive nitrogen and oxygen species are implicated in the damage of ischemic tissue that is reperfused. One important pathway may involve xanthine oxidase. Xanthine oxidase uses xanthine, a product of ATP degradation in ischemic tissue, to produce superoxide and hydrogen peroxide. Superoxide reacts rapidly with nitric oxide to form peroxynitrite, a powerful oxidant. Another potential source of reactive nitrogen species is the myeloperoxidase–hydrogen peroxide–nitrite system of activated phagocytes. We demonstrate that peroxynitrite and myeloperoxidase nitrate xanthine in vitro. Through 13 C NMR spectroscopy, UV/visible spectroscopy, and mass spectrometry, the major product was identified as 8-nitroxanthine. Xanthine nitration by peroxynitrite was optimal at neutral pH and was markedly stimulated by physiological concentrations of bicarbonate. Xanthine nitration by myeloperoxidase required hydrogen peroxide and nitrite. However, it was independent of chloride ion and little affected by scavengers of hypochlorous acid, suggesting that the reactive agent is a nitrogen dioxide-like species. 8-Nitroxanthine was generated by a low, steady flux of peroxynitrite, and also by the myeloperoxidase–hydrogen peroxide–nitrite system of activated human neutrophils, suggesting that the reactions may be physiologically relevant. 8-Nitroxanthine may exert biological effects because it markedly increased the production of superoxide by the xanthine oxidase–xanthine system. Our observations suggest a mechanism for the enhanced formation of superoxide in reperfused tissue, which might increase the production of peroxynitrite and 8-nitroxanthine. Generation of 8-nitroxanthine by peroxynitrite and myeloperoxidase could represent a positive feedback mechanism that enhances further the production of both reactive oxygen and nitrogen species in ischemic tissue that is reperfused. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Ischemia–reperfusion injury; Nitric oxide; Hydrogen peroxide; Nitrite

Reactive oxygen species and reactive nitrogen species may be of central importance in cellular injury in ischemic and reperfused tissue [1–3]. One important pathway for generating such species in heart and other tissues is likely to involve xanthine oxidase, an enzyme derived from xanthine dehydrogenase by proteolytic * Corresponding author. Present address: Box 356426, Division of Metabolism, Endocrinology and Nutrition, University of Washington, Seattle, WA 98195, USA. Fax: 1-2062685-3781. E-mail address: [email protected] (J.W. Heinecke). 1 Present address: Sigma–Aldrich, 3050 Spruce Street, St. Louis, MO 63103, USA.

cleavage or thiol oxidation [1–5]. Xanthine oxidase, a metalloflavoprotein, uses reducing agents to convert molecular oxygen to superoxide (O
2 ) and hydrogen peroxide (H2 O2 ) [6,7]. The ability of xanthine oxidase inhibitors such as allopurinol and oxypurinol to reduce post-ischemic tissue injury, together with the direct detection of free radicals in tissue by electron paramagnetic resonance [8–10], implicates reactive oxygen species in tissue damage. Hypoxanthine and xanthine are physiological substrates for xanthine oxidase. During ischemia, ATP is converted to ADP, AMP, and adenosine, which can be degraded further to inosine, hypoxanthine, and xanthine

0003-9861/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-9861(03)00256-X

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[1]. Thus, ischemia might provide xanthine oxidase with the reducing equivalents it needs to generate reactive oxygen species. Indeed, levels of hypoxanthine and xanthine rise 1000-fold in ischemic heart tissue [9]. Therefore, the supply of reducing equivalents by hypoxanthine and xanthine might be a critical regulator of oxidant production by xanthine oxidase. The observation that ischemic heart tissue exhibits a burst of free radical production by xanthine oxidase when it is reperfused [9] is consistent with this proposal. Moreover, the subsequent decline in radical production is associated with loss of xanthine and hypoxanthine from tissue rather than with a major change in xanthine oxidase activity. Nitrating species that might be important after ischemia and reperfusion include peroxynitrite (ONOO
) [11,12], which is generated when nitric oxide (NO) reacts with O
2 :
O
2 þ NO ! ONOO

ð1Þ O
2

Under normal conditions, concentrations may be limiting, resulting in little ONOO
formation. However, appreciable amounts of ONOO
could form if O
2 concentrations rise. In the early phase of reperfusion, production of both NO and O
2 increases [13], potentially boosting ONOO
production. Moreover, xanthine oxidase is inactivated when it is exposed to ONOO
[14] or NO [15] under certain conditions in vitro, suggesting that NO and its reactive products might alter the balance for the generation of reactive nitrogen species in reperfused ischemic tissue. Studies using HPLC analysis with electrochemical detection suggest that ONOO
nitrates xanthine, guanine, and adenine nucleosides [16]. Nitryl chloride (NO2 Cl) is another potential nitrating agent [17]. Spectroscopic and mass spectrometric studies have suggested that reagent NO2 Cl reacts with 20 -deoxyguanosine and calf thymus DNA to yield 8-nitroxanthine [18]. The relevance of these reactions to ischemia and reperfusion is not yet established. However, NO2 Cl can be produced by a pathway involving myeloperoxidase [19]. This heme enzyme is secreted by neutrophils, which accumulate at high levels in reperfused tissue [20–22]. Myeloperoxidase uses hydrogen peroxide (H2 O2 ) derived from the phagocyte NADPH oxidase or other sources to produce powerful oxidants [23–25]. The major product at plasma concentrations of chloride ion (Cl
) is generally thought to be hypochlorous acid (HOCl): Cl
þ H2 O2 þ Hþ ! HOCl þ H2 O

ð2Þ

HOCl can react with nitrite (NO
2 ), a degradation product of nitric oxide (NO) to produce NO2 Cl:
HOCl þ NO
2 ! NO2 Cl þ HO

ð3Þ

Myeloperoxidase can also produce nitrogen dioxide radical (NO2 ), another reactive nitrogen species

[17,26,27]. The reaction involves direct one-electron oxidation of NO
2 by compound I, a complex of myeloperoxidase and H2 O2 :  þ NO
2 þ compound I þ H ! NO2 þ H2 O þ compound II

ð4Þ In the current studies, we demonstrate that 8-nitroxanthine is generated by ONOO
in a model system that generates a low, steady flux of ONOO
, and also by the myeloperoxidase–H2 O2 –O
2 system of activated human neutrophils. The major pathway for the myeloperoxidase nitration reaction appears to involve the one-elec tron oxidation of NO
2 to NO2 (Eq. (4)). We also found that 8-nitroxanthine enhances O
2 production by xanthine oxidase. Our observations indicate that 8-nitroxanthine is a potential biomarker for oxidation by reactive nitrogen species. They also raise the possibility that generation of 8-nitroxanthine in reperfused ischemic tissue or other inflammatory conditions might modulate O
2 production by xanthine oxidase in vivo.

Experimental procedures Materials Unless otherwise indicated, all reagents were from Sigma (St. Louis, MO) or Fisher (Pittsburgh, PA). HPLC solvents were filtered through 0.22 lm nylon filters (MAGNA brand; Osmonics). Myeloperoxidase (donor: hydrogen peroxide, oxidoreductase, EC 1.11.1.7) was isolated by lectin affinity and size exclusion chromatographies from HL-60 cells [28,29] and stored at –20 °C. Purified enzyme had an A430 =A280 ratio of 0.8 and was apparently homogeneous on SDS–PAGE analysis; its concentration was determined spectrophotometrically (e430 ¼ 0:17 M
1 cm
1 ) [30]. Neutrophils were isolated from human blood anticoagulated with EDTA by density gradient centrifugation [31] and suspended in HanksÕ balanced salt solution (HBSS; GibcoBRL; Life Technology, Grand Island, NY). Reverse-phase HPLC analysis of xanthine oxidation products Xanthine oxidation products were analyzed on a Beckman System Gold 125 HPLC equipped with a Beckman Ultrasphere ODS reverse-phase column (5 lm resin, 4.6 mm  25 cm; Beckman Instruments, Berkeley, CA) coupled to a Waters 484 Tunable Absorbance Detector. Compounds were resolved by isocratic elution with 20 mM ammonium formate buffer (pH 6.3) [32] at a flow rate of 1.0 mL/min. Absorption spectra were acquired with a Beckman Gold 168 photodiode array detector.

G.C. Yeh et al. / Archives of Biochemistry and Biophysics 418 (2003) 1–12

Synthesis and purification of 8-nitroxanthine Xanthine (6–12 mM) was prepared in 100 mM NH4 OH with heating. Tetranitromethane (50 mM) was prepared in ethanol. 8-Nitroxanthine was synthesized by reacting xanthine with tetranitromethane (1/2, v/v) at room temperature for 1 h [33]. The reaction mixture was evaporated under vacuum and resuspended in MeOH. Silica (40 lm, Baker-Flex regular phase silica; J.T. Baker) was added and the solution was dried under vacuum. A mini-column was prepared with reaction products bound to silica and a Pasteur pipette. The mini-column was washed with 1-propanol and 8-nitroxanthine was eluted with MeOH. The dried eluant was resuspended in 20 mM ammonium formate and 8-nitroxanthine was isolated by reverse-phase HPLC as described above. Xanthine and 8-nitroxanthine were detected by monitoring absorbance at 269 and 377 nm, respectively. The concentration of 8-nitroxanthine was determined spectrophotometrically (e377 ¼ 11,750 M
1 cm
1 ) [33].

3

mode, methanol/water/acetic acid (50/49/1; v/v/v) was used for dissolving the samples and as a carrier solvent. In the negative ion mode, 10 mM ammonium acetate (pH 7.4)/acetonitrile (1/1; v/v) was used. Helium was used as a damping gas and collision activation partner. The flow of gas (1 mL/min) into the mass analyzer cavity was regulated by a pressure regulator and a capillary restrictor. The temperature of the heated capillary was 220 °C. For each full scan mass spectrum (m/z 120–230), 10 scans were signal-averaged and the background from 10 control scans was subtracted from the full scan mass spectrum. Preparation of xanthine solutions Stock solutions of xanthine (2 mM; pH 8–8.6) were prepared from concentrated xanthine stock solution (6–12 mM in 100 mM NH4 OH) by dilution into 200 mM ammonium acetate (ONOO
reactions) or 50 mM ammonium acetate (human neutrophils). The concentration of xanthine was determined spectrophotometrically (e269 ¼ 9120 M
1 cm
1 ) [36].

Synthesis of 8-aminoxanthine Xanthine oxidation by ONOO
8-Nitroxanthine was reduced to 8-aminoxanthine using a molar excess of sodium hydrosulfite at room temperature [27]. The disappearance of 8-nitroxanthine and the appearance of 8-aminoxanthine were assessed by reverse-phase HPLC, as described above. The concentration of 8-aminoxanthine was determined spectrophotometrically (e288 ¼ 15,780 M
1 cm
1 ) [34]. Sodium hydrosulfite (100 mM) was prepared in 50 mM NH4 OH. NMR 8-Nitroxanthine was prepared by suspending the dried compound in d6 -DMSO (Aldrich) and vortexing for 15 min. 13 C NMR spectra were recorded at 600 MHz at on a Varian Unity-600 NMR spectrometer (Varian; Palo Alto, CA) equipped with a broadband probe. The collection conditions were as follows: 40 °C, spectral width ¼ 35,000 Hz, 6000 transients, broadband H1 decoupling (WALTZ), and 8 ls pulse width (flip angle ¼ 50 °). A 2-Hz line broadening apodization was employed. 13 C chemical shifts were referenced to the internal solvent DMSO (39.5 ppm). Peak assignments were referenced to the 13 C NMR spectrum of xanthine [35]. Electrospray ionization mass spectrometry Full mass scanning, zoom scanning, and low-energy collisionally activated dissociation were performed on a Finnigan LCQ (ThermoFinnigan; San Jose, CA). Samples were introduced with an electrospray source and injected at a flow rate of 3 lL/min. In the positive ion

Xanthine (0.5 mM) was exposed to ONOO
or 3-morpholinosydnonimine hydrochloride (SIN-1; Alexis Biochemicals) at 25 °C in Cl
-free buffer A (50 mM sodium phosphate, pH 7.0) or Cl
-free buffer A supplemented with freshly prepared 10 mM NaHCO3 . ONOO
was synthesized from 0.6 M sodium nitrite, 0.6 M H2 O2 in 0.7 M HCl, and 1.2 M NaOH [12] and stored in 1.2 M NaOH at –20 °C. The concentration of ONOO
was determined spectrophotometrically (e302 ¼ 1670 M
1 cm
1 ) immediately before use [12]. ONOO
and SIN-1 solutions were kept on ice and shielded from light during the course of the experiments. Following the addition of an aliquot of ONOO
to the side of the reaction vessel, the reaction was initiated by rapid mixing of oxidant and reaction mixture by vortexing. Xanthine oxidation by myeloperoxidase Xanthine (0.5 mM) was incubated with myeloperoxidase in Cl
-free buffer A supplemented with 10 lM diethylenetriaminepentaacetic acid (DTPA2) at 37 °C. DTPA was included to inhibit metal catalyzed reactions [37]. NaNO2 and H2 O2 stock solutions were prepared daily. Reactions were initiated by the addition of H2 O2 2

Abbreviations used: DTPA, diethylenetriaminepentaacetic acid; HBSS, HanksÕ balanced salt solution; H2 O2 , hydrogen peroxide; ESI, electrospray ionization; MS, mass spectrometry; NO
2 , nitrite; NO2 Cl,
nitryl chloride; O
2 , superoxide; ONOO , peroxynitrite; SIN-1, 3-morpholinosydnonimine.

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and terminated by the addition of catalase (20 lg/mL; Boehringer Ingelheim). Formation of 8-nitroxanthine by human neutrophils The complete system consisted of 2  106 cells/mL in HBSS (final pH 7.0) supplemented with 0.05 mM xanthine, 5 mM phosphate, 50 lM NaNO2 , and 100 mM NaCl. Cells were activated by addition of 200 nM phorbol myristate acetate, incubated at 37 °C, and maintained in suspension by intermittent inversion. Reactions were terminated by pelleting the cells by centrifugation and removal of the supernatant. Uric acid production by xanthine oxidase in the presence of 8-nitroxanthine The conversion of xanthine to uric acid by xanthine oxidase (from bovine milk; Calbiochem) was monitored as the increase in absorbance of the reaction mixture at 295 nm [38]. Reactions were carried out at 25 °C in 50 mM sodium phosphate (pH 7.4) supplemented with 2.5–10 lM xanthine, 0–10 lM of 8-nitroxanthine, 0.4 nM xanthine oxidase, and 40 lM DTPA. The concentration of uric acid was calculated using an extinction coefficient of 11,000 M
1 cm
1 [39]. Superoxide production was monitored spectrophotometrically as the superoxide dismutase-inhibitable reduction of ferricytochrome c (e550 ¼21,000 M
1 cm
1 ) [40]. Reactions were carried out in sodium phosphate buffer (pH 7.4) supplemented with 2.5–15 lM xanthine, 0–15 lM of 8nitroxanthine, 1 nM xanthine oxidase, 100 lM cytochrome c, 40 lM DTPA, and 5 nM catalase. Reactions were performed in 96-well microplates and monitored on a Molecular Devices Thermomax microplate reader at 25 °C. Vo values for the production of uric acid and O
2 were determined from the initial rate of reaction using Microsoft Excel and SigmaPlot (SPSS). Under these experimental conditions, the initial rate of reaction was directly proportional to the concentration of xanthine oxidase.

Results Both ONOO
and the myeloperoxidase–H2 O2 –NO
2 system convert xanthine to 8-nitroxanthine To investigate potential mechanisms for oxidative modification of purines during ischemia–reperfusion injury, we exposed xanthine to two systems that might generate reactive nitrogen species in vivo. In the first experimental system, xanthine was incubated with reagent ONOO
(10:1, mol purine/mol oxidant) in buffer A (50 mM sodium phosphate, pH 7.0) for 60 min at 25 °C. In the second, it was incubated with the

Fig. 1. HPLC analysis of xanthine oxidized by ONOO
, of xanthine oxidized by the myeloperoxidase–H2 O2 –NO
2 system (MPO), and of authentic 8-nitroxanthine (8-NitroX). Xanthine (0.5 mM) was exposed to ONOO
in buffer A (50 mM sodium phosphate, pH 7.0) at 25 °C for 30 min. Xanthine (0.5 mM) was exposed to 5 nM myeloperoxidase in buffer A supplemented with 50 lM H2 O2 , 50 lM NO
2 , 100 mM NaCl, and 10 lM DTPA at 37 °C for 60 min. The reaction mixtures were then subjected to reverse-phase HPLC analysis with monitoring absorbance at 377 nm.

myeloperoxidase–H2 O2 –NO
2 system (10:1, mol purine/ mol H2 O2 ) in buffer A containing 100 mM NaCl and 10 lM DTPA for 120 min at 37 °C. Analysis of each reaction mixture by reverse-phase HPLC with monitoring absorbance at 377 nm (Fig. 1) revealed a major peak of new material that had the same retention time as 8-nitroxanthine prepared using tetranitromethane. This material was the only nitrated product that we detected as monitored by absorbance at 377 nm. In addition, the absorption spectrum of the oxidized reaction mixture was consistent with the presence of only the nitrated product and xanthine (see below). However, there could be other oxidation products that were not detected using these two approaches. Formation of the material required ONOO
or each component of the myeloper
oxidase–H2 O2 –NO
and 2 system. Thus, both ONOO
the myeloperoxidase–H2 O2 – NO2 system can convert xanthine to a major oxidation product that comigrates with authentic 8-nitroxanthine on reverse-phase HPLC. To identify this material, we first obtained an absorption spectrum, using reverse-phase HPLC and a diode array detector. The oxidation product (Fig. 1; retention time 16 min) produced by ONOO
or by the myeloperoxidase–H2 O2 –nitrate system absorbed

G.C. Yeh et al. / Archives of Biochemistry and Biophysics 418 (2003) 1–12

Fig. 2. Absorption spectra of the xanthine oxidation product produced by ONOO
and the myeloperoxidase–H2 O2 –NO
2 system. Samples were subjected to reverse-phase HPLC analysis. Spectra of the peaks of material eluting with a retention time of 16 min (Fig. 1) were acquired with an on-line diode array detector.

maximally at 377 nm (Fig. 2). The absorption spectra of both compounds were virtually identical to that of authentic 8-nitroxanthine. Absorbance in this region of the UV/visible spectrum is characteristic of aromatic compounds with a strongly electron-withdrawing nitroso (–NO), nitro (–NO2 ), or nitrosooxy (–ONO) group on the ring. These observations suggest that the aromatic ring stem of xanthine had remained intact during oxidation but had been modified with a nitro, nitroso, or nitrosooxy group. We used 13 C NMR to establish the location of the electron-withdrawing group on the purine ring. 13 C NMR should be more structurally informative than 1 H NMR, given the lack of non-exchangeable hydrogens on 8-nitroxanthine. To obtain the large amounts of material required for NMR analysis, we used HPLC to isolate xanthine that was oxidized by tetranitromethane. Fig. 3A shows the 13 C NMR spectrum of the purified xanthine oxidation product. Compared with the 13 C NMR spectrum of xanthine itself, the spectrum showed pronounced shifts in the resonances of two of the carbons, C-5 and C-8. The C-8 shift was 19 ppm downfield, consistent with enhanced electron-withdrawal. Furthermore, the signal intensity at this position in the purine oxidation product was lower than that of xanthine itself because the loss of the hydrogen atom on C-8 precludes dipole–dipole interactions between the C and H in the spin–lattice relaxation, lengthening relaxation times. Thus, the peak intensity of C-8 became similar to that of the other ring carbons, which lacked attached hydrogen atoms. In a long-range effect, the shift of C-5 in 8-nitroxanthine was about 11.5 ppm downfield.

5

Fig. 3. 13 C NMR spectrum of 8-nitroxanthine. Xanthine was oxidized with tetranitromethane and the reaction product was isolated by reverse-phase HPLC. The purified material was suspended in d6 -DMSO and subjected to high resolution 13 C NMR spectroscopy. Peak assignments are referenced to d6 -DMSO (39.5 ppm).

These observations indicate that a strong electronwithdrawing group had replaced the hydrogen on C-8 of the purine ring in the oxidation product. To establish the structure of the group that ONOO
or the myeloperoxidase–H2 O2 –NO
2 system adds at the C-8 position of xanthine, we determined the molecular mass of the oxidation product, using mass spectrometry. Modified xanthines were not detectable by ESI–MS in the positive ion mode (with methanol/H2 O/acetic acid as carrier solvent), presumably because the oxidation product lacked a group that could be protonated. Using the negative ion mode (with acetonitrile/ammonium formate as carrier solvent), a compound was readily detected with a mass-to-charge (m/z) ratio of 196, the anticipated m/z of deprotonated 8-nitroxanthine (Fig. 4A; [M + 46 ) H]
). Zoom scan analysis confirmed the m/z of this material and revealed an M + 1 peak consistent with the anticipated abundance (5%) of 13 C in a compound derived from xanthine. Tandem MS analysis with low energy collisional activation failed to fragment the precursor ion (data not shown). Collectively, these observations indicate that both ONOO
and the myeloperoxidase–H2 O2 –NO
2 system nitrate xanthine at the C-8 position. To confirm the identity of the oxidation product as 8-nitroxanthine, we reduced the material and analyzed it by ESI–MS. In contrast to 8-nitroxanthine, authentic 8-aminoxanthine is readily detectable in the positive ion

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analyses of the reduced and non-reduced xanthine oxidation products together with the 13 C NMR studies unequivocally identified the substituent at the C-8 position of the modified xanthine adduct as a nitro group. Production of 8-nitroxanthine by ONOO
is optimal at neutral pH and plasma concentrations of NaHCO3

Fig. 4. Negative ion ESI mass spectra of 8-nitroxanthine produced by ONOO
. Xanthine was oxidized with ONOO
as described in the legend to Fig. 1 and the reaction product was isolated by reverse-phase HPLC. (A) Full scan mass spectrum of the oxidation product. (B) Zoom scan mass spectrum of the oxidation product.

mode by ESI–MS analysis because the exocyclic amino group is readily protonated (data not shown). When the xanthine oxidation product generated by ONOO
or the myeloperoxidase–H2 O2 –NO
2 system was reduced with sodium hydrosulfite and subjected to positive-ion ESI– MS analysis, a major ion was detected at m/z 168 (Fig. 5A). We have previously demonstrated that 8-aminoguanine fragments upon subjection to tandem mass analysis, losing an amino group from the purine ring [27]. Tandem mass spectrometric analysis of the reduced xanthine oxidation product revealed a major product ion at m/z 151, which was consistent with loss of NH3 from the precursor ion (Fig. 5B). The MS and MS/MS

The pH dependence of the reaction of xanthine with ONOO
(Fig. 6A) indicated that a physiological pH (7.3) yielded the maximal amount of product. However, recent studies indicate that ONOO
reacts rapidly with carbon dioxide to generate ONO2 CO
2 [41,42]. Because the reactivity of ONO2 CO
2 differs from that of ONOO
, and bicarbonate/carbon dioxide is present at high concentrations in vivo, we determined whether adding NaHCO3 to the reaction buffer affected the yield of 8-nitroxanthine or the pH optimum of the reaction. Adding 10 mM NaHCO3 made the pH optimum slightly more acidic (pH 6.8 vs. 7.3). It also distinctly elevated the yield of 8-nitroxanthine, over a broad pH range. The absolute magnitude of the increase was similar over the pH range investigated, but the relative increase varied from about 3- to 4-fold at pH 5.5 to about 1.5-fold between pH 6.8 and 7.7. It is of interest that bicarbonate and carbon dioxide also enhance ONOO
-mediated formation of nitrotyrosine [41–44] and nitroguanine [45]. Production of 8-nitroxanthine showed an essentially linear dependence on [ONOO
], both in the presence and absence of added carbonate (Fig. 6B). Without exogenous NaHCO3 , the product yield was 5–10%. With 10 mM NaHCO3 , it was 8–14%. SIN-1 also converts xanthine to 8-nitroxanthine Most studies of ONOO
have used a concentrated bolus of the nitrating species. However, recent studies

Fig. 5. Positive ion ESI tandem mass spectrum of 8-aminoxanthine derived from 8-nitroxanthine produced by ONOO
. Xanthine was oxidized with ONOO
as described in the legend to Fig. 1 and the reaction product was isolated by reverse-phase HPLC. Following reduction with sodium hydrosulfite, the material was subjected to ESI–MS analysis. (A) Zoom scan mass spectrum. (B) Full scan low energy collisionally activated mass spectrum of the [M + H]þ ion of 8-aminoxanthine.

Fig. 6. Formation of 8-nitroxanthine by ONOO
. Xanthine (0.5 mM) was exposed to 0.05 mM ONOO
in Cl
-free buffer A at 25 °C for 30 min. 8-Nitroxanthine was quantified by reverse-phase HPLC. (A) Effect of pH (d, no added NaHCO3 ; s, plus 10 mM freshly prepared NaHCO3 ). (B) Effect of [ONOO
] (d, no added NaHCO3 , reactions performed at pH 7.30; s, plus 10 mM NaHCO3 , reactions performed at pH 6.95). Results represent means  SD of three independent experiments.

G.C. Yeh et al. / Archives of Biochemistry and Biophysics 418 (2003) 1–12

7

Fig. 7. Formation of 8-nitroxanthine by SIN-1. Xanthine (0.5 mM) was exposed to the indicated concentration of SIN-1 in 50 mM phosphate (pH 7.0) at 37 °C for 24 h. Where indicated, reactions included 10 mM NaHCO3 and/or 100 mM NaCl. d, no additions; s, plus NaCl; ., plus NaHCO3 ; and ,, plus NaHCO3 and NaCl. Results represent the average of two independent experiments.

suggest that L -tyrosine is a poor substrate for nitration when ONOO
is generated at a low, steady rate [46]. To investigate whether purine nitration occurs under these conditions, we incubated xanthine with SIN-1 (3-morpholinosydnonimine), which provides a constant supply of ONOO
by simultaneously generating NO and O
2 . The principal product was 8-nitroxanthine (Fig. 7), though the overall yield (1.2%, pH 7.0) was less than that obtained with a bolus of reagent ONOO
under the same conditions (8%, pH 7.0). As shown in Fig. 7, adding NaHCO3 (10 mM) to the reaction mixture enhanced 8-nitroxanthine formation, whereas adding NaCl (100 mM) was inhibitory. Adding both NaHCO3 and NaCl generated a larger yield of 8-nitroxanthine than adding NaCl alone, but the yield was less than with NaHCO3 alone. At physiological pH and plasma concentrations of NaCl and NaHCO3 , the product yield of 8-nitroxanthine derived from SIN-1 was 1.3%. These findings show that ONOO
generated at a low, steady rate will efficiently nitrate xanthine at plasma concentrations of NaHCO3 and Cl
. Myeloperoxidase generates 8-nitroxanthine at physiological levels of H2 O2 and NO
2 To determine whether myeloperoxidase can nitrate xanthine under conditions that mimic those in vivo, we exposed xanthine to the myeloperoxidase–H2 O2 –NO
2 system in buffer A (50 mM sodium phosphate) supplemented with 100 mM NaCl and 10 lM DTPA, and used HPLC to characterize the reaction (Fig. 8). Nitration was maximal at pH 5.8 (Fig. 8A). To examine the time course and [NO
2 ]- and [H2 O2 ]-dependence, we selected pH 6.25, which was close to both this pH optimum and to neutral conditions. Production of 8-nitroxanthine

Fig. 8. Reaction requirements for the formation of 8-nitroxanthine by the myeloperoxidase–H2 O2 –NO
2 system. Xanthine (0.5 mM) was exposed to 5 nM myeloperoxidase in Cl
-free buffer A (pH 6.25) supplemented with 100 lM H2 O2 , 100 lM NO
2 , and 10 lM DTPA at 37 °C for 60 min. Reactions were initiated by the addition of H2 O2 and terminated with 200 nM catalase. Where indicated, the pH (A), time of the incubation (B), [NO
2 ] (C) or [H2 O2 ] (D) was varied. Results represent means  SD of three independent experiments.

increased most rapidly over the first 10 min of the reaction. The rate diminished over the next 20 min and the reaction was essentially complete by 30 min. It occurred at physiologically relevant concentrations of [NO
2 ] (1– 100 lM) and [H2 O2 ] (1–35 lM). The product yield was sharply dependent on [H2 O2 ] (Fig. 8D), rising markedly between 0 and 35 lM H2 O2 and gradually decreasing at higher peroxide concentrations. At pH 6.25, the optimal ratio of [NO
2 ]/[H2 O2 ] for xanthine nitration was 2.5/ 1 mol/mol. Formation of 8-nitroxanthine required each component of the myeloperoxidase–H2 O2 –NO
2 system but was independent of Cl
(Table 1). Adding azide (a heme poison) or catalase (a scavenger of H2 O2 ) blocked the reaction. Including Cl
had only a small effect, suggesting that NO2 Cl [17] is not an intermediate in the myeloperoxidase-catalyzed nitration reaction. Likewise, neither taurine nor methionine (both potent scavengers of HOCl) inhibited the reaction in the absence of Cl
(Table 1). However, simultaneous addition of Cl
and taurine reduced 8-nitroxanthine formation by 20%, suggesting that NO2 Cl contributed to the formation of 8-nitroxanthine and/or that Cl
and NO
2 competed for oxidation by myeloperoxidase. Control experiments demonstrated that the concentrations of taurine and methionine used in these experiments completely

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Table 1 Requirements for conversion of xanthine to 8-nitroxanthine by the myeloperoxidase–NO
2 –H2 O2 system Reaction conditions


Complete system (Cl -free)* Complete system minus Myeloperoxidase H2 O2 NO
2 Complete system plus Cl
(100 mM) Cl
(100 mM)/taurine (1 mM) Cl
(100 mM)/HCO
3 (10 mM) Cl
(100 mM)/methionine (1 mM) Taurine (1 mM) HCO
3 (10 mM) Methionine (1 mM) Cysteine (1 mM) Azide (10 mM) Catalase (200 nM)

Table 2 Requirements for conversion of xanthine to 8-nitroxanthine by activated human neutrophils

8-Nitroxanthine (lM)

Reaction conditions

8-Nitroxanthine (nM)

1.28

Complete system Complete system minus Phorbol myristate acetate Neutrophils NO
2 Complete system plus Azide (10 mM) Cyanide (10 mM) Superoxide dismutase (480 nM) Taurine (1 mM) Catalase (200 nM)

19

0 0 0 1.19 1.05 0.95 0.98 1.21 0.95 1.26 0 0 0

Xanthine (0.5 mM) was exposed to 5 nM myeloperoxidase in Cl
free buffer A (50 mM sodium phosphate, pH 6.25) supplemented with 35 lM H2 O2 , 100 lM NO
2 , and 10 lM DTPA at 37 °C for 60 min (complete system). Reactions were initiated by the addition of H2 O2 and terminated with 200 nM catalase and 10 mM methionine. The concentration of 8-nitroxanthine was determined by reverse-phase HPLC. Results represent means of three independent experiments.

inhibited the nitration of xanthine by a combination of

HOCl and NO
2 . Unlike ONOO , HCO3 (1 mM) modestly inhibited (30%) the nitration of xanthine by myeloperoxidase. A high concentration of L -cysteine (1 mM; 10/1, mol/mol H2 O2 ), a thiol-containing amino acid, completely inhibited 8-nitroxanthine production. These observations suggest that under our experimental conditions the major pathway for the nitration of xanthine by myeloperoxidase at plasma concentrations of Cl
involves a NO2 -like species. Activated human neutrophils produce 8-nitroxanthine in the presence of NO
2 To determine whether human neutrophils can also generate 8-nitroxanthine, we incubated cells (2  106 per mL) at neutral pH in a physiological salt solution supplemented with 5 mM phosphate, 0.05 mM xanthine, and 50 lM NO
2 . The neutrophils were activated with phorbol ester and incubated for 60 min at 37 °C. 8Nitroxanthine production was assessed by reverse-phase HPLC. The activated neutrophils produced nanomolar concentrations of 8-nitroxanthine (Table 2). The nitration required neutrophils, NO
2 , and activation of the cells with phorbol ester. Adding azide, cyanide, or catalase inhibited 8-nitroxanthine formation, implicating myeloperoxidase in the reaction pathway. Superoxide dismutase increased the product yield, presumably by increasing the level of H2 O2 or protecting myeloperoxidase from inactivation [47]. Taurine failed to inhibit

0 0 0 0 0 65 26 0

Neutrophils (2  106 cells/mL) were incubated for 2 h at 37 °C in HBSS and 5 mM phosphate (pH 7.0) was supplemented with 0.05 mM
xanthine, 50 lM NO
2 , 100 mM Cl , and 200 nM phorbol ester (complete system). Cells were maintained in suspension by occasional inversion. At the end of the incubation, neutrophils were pelleted by centrifugation and the concentration of 8-nitroxanthine in the supernatant was determined by reverse-phase HPLC. Results represent means from three independent experiments.

8-nitroxanthine formation. These observations strongly support the hypothesis that activated human neutrophils use the myeloperoxidase–H2 O2 –NO
2 system to convert xanthine to 8-nitroxanthine by a reaction pathway independent of HOCl. 8-Nitroxanthine decreases the initial rate of uric acid production by the xanthine oxidase–xanthine system To determine how 8-nitroxanthine might interact with xanthine oxidase in situations such as reperfusion, we examined the rate at which xanthine oxidase converts xanthine to its usual product, uric acid, in the presence of 8-nitroxanthine. Increasing the concentration of 8-nitroxanthine decreased the initial rate of uric acid production (Fig. 9A), a disparity that became more pronounced with time. Both a double reciprocal plot and a Dixon plot indicated that 8-nitroxanthine was acting as a noncompetitive inhibitor (Figs. 9B and C) rather than as a competitive or uncompetitive inhibitor. Furthermore, 8-nitroxanthine did not appear to be a substrate for xanthine oxidase, as HPLC analysis of the spent reaction mixture indicated complete conversion of xanthine to uric acid and recovered all of the 8-nitroxanthine added (data not shown). These results indicate that 8-nitroxanthine inhibits the initial rate of conversion of xanthine to uric acid by xanthine oxidase. 8-Nitroxanthine increases production of O
by the 2 xanthine oxidase–xanthine system In vitro, electrons donated by xanthine to xanthine oxidase are subsequently transferred to oxygen, with production of H2 O2 and O
2 . A number of factors

G.C. Yeh et al. / Archives of Biochemistry and Biophysics 418 (2003) 1–12

9

Fig. 9. Effect of 8-nitroxanthine (8-NitroX) on the conversion of xanthine to uric acid by xanthine oxidase. (A) Reactions were carried out in 50 mM sodium phosphate buffer (pH 7.4) supplemented with 10 lM xanthine, 0.4 nM xanthine oxidase, and 40 lM DTPA. Reaction conditions were: d, no 8-NitroX; s, 2.5 lM 8-NitroX; ., 5 lM of 8-NitroX; and ,, and 15 lM of 8-NitroX. (B) Double-reciprocal plot for uric acid production at different concentrations of xanthine. Reaction conditions as in (A) with 2.5–10 lM xanthine and: d, no 8-NitroX; s, 5 lM of 8-NitroX; and ., 10 lM of 8NitroX. (C) Dixon plot for uric acid production at different concentrations of 8-NitroX. Reaction conditions as in (A) with 0–10 lM of 8-NitroX and d, 2.5 lM xanthine; s, 5 lM xanthine; and ., 10 lM xanthine.

influence the relative rates of the one and two electron transfer reactions [6,7]. To determine whether 8-nitroxanthine affects this transfer, we monitored changes in O
2 production by xanthine oxidase, quantifying the superoxide dismutase-inhibitable reduction of ferricytochrome c by xanthine [40]. Increasing concentrations of 8-nitroxanthine (1–15 lM) caused the reaction to proceed for progressively longer times, increasing the cumulative production of O
2 by the xanthine oxidase–xanthine system (Fig. 10A). At the highest concentration of 8-nitroxanthine examined (15 lM), total O
generation 2 nearly doubled. In striking contrast to the production of uric acid experiment, the initial rate of O
2 production was not affected by increasing the concentration of 8nitroxanthine at two different starting concentrations of xanthine (Fig. 10B). Moreover, there was no change in the cumulative production of uric acid under these conditions

Fig. 10. Effect of 8-nitroxanthine (8-NitroX) on O
2 production by the xanthine–xanthine oxidase system. (A) Progress curve of O
2 production. Reactions were carried out in 50 mM sodium phosphate buffer (pH 7.4) supplemented with 15 lM xanthine, 1 nM xanthine oxidase, 100 lM ferricytochrome c, 40 lM DTPA, and 5 nM catalase. Reaction conditions were: d, no 8-nitroxanthine; s, 2.5 lM of 8-NitroX; ., 7.5 lM of 8-NitroX; and ,, 15 lM of 8-NitroX. (B) Initial rate (Vo ) of O
2 production. Reaction conditions as in (A) with the indicated concentration of 8-nitroxanthine and 15 lM (d) or 25 lM (s) xanthine.

(Fig. 9A), indicating that alternations in the availability of xanthine did not account for the change in O
2 production. These observations demonstrate that 8-nitroxanthine prolongs O
2 production and increases the total amount of O
2 generated by the xanthine oxidase–xanthine system.

Discussion Our observations indicate that 8-nitroxanthine is the principal product when xanthine is exposed to either ONOO
or the myeloperoxidase–H2 O2 –NO
2 system, two potential sources of reactive nitrogen species in vivo. These observations may be physiologically relevant because xanthine was nitrated both by sustained, low concentration of ONOO
and by the myeloperoxidase system of activated human neutrophils. Production of 8-nitroxanthine by myeloperoxidase increased greatly when the concentration of NO
2 increased from 0 to 100 lM. Although plasma levels of NO
2 in normal humans range from 1 to 5 lM [48], levels as high as 100 lM have been reported in inflammatory conditions [49,50]. Indeed, recent studies provide strong evidence that myeloperoxidase generates reactive nitrogen species in vivo and that it operates in this fashion only when nitrite and nitrate become available [51]. The production of NO also increases during ischemia–reperfusion [3]. Thus, variations in NO
2 and NO concentration might modulate the rate of the nitrating reaction in vivo. Plasma and tissue levels of xanthine, xanthine oxidase, NO, and myeloperoxidase are elevated in a variety of inflammatory conditions, raising the possibility that the production of 8-nitroxanthine plays a role in the pathogenesis of vascular disease [52–57]. Indeed, production of O
2 by xanthine oxidase has been implicated in endothelial dysfunction during sickle cell disease and

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atherosclerosis [57,58]. Because xanthine occurs freely in plasma, it could be a more facile target for reactive nitrogen species and reactive oxygen species than intracellular purines, which are protected by much higher concentrations of antioxidants. The purine rings of both uric acid and 8-nitroxanthine are modified at the C-2, C4, and C-8 positions. Uric acid is not metabolized and is excreted freely in the urine, suggesting that 8-nitroxanthine might also be excreted unaltered, perhaps without resorption by the kidney. Such behavior would contrast with that of 3-nitrotyrosine, the most commonly studied biomarker of nitration. In vivo, 3-nitrotyrosine is metabolized to 3-nitro-4-hydroxyphenylacetic acid and 3-nitro-4-hydroxyphenyllactic acid [59]. Furthermore, recent studies have presented conflicting views on the levels of 3-nitrotyrosine that ONOO
generates under physiologically plausible conditions [46,60,61]. 3-Nitrotyrosine is also technically difficult to quantify in biological material because it is readily generated artifactually during sample workup [62–64]. Therefore, nitrated purines such as 8-nitroxanthine and 8-nitroguanine might have the potential to replace 3-nitrotyrosine as stable, noninvasive markers for in vivo formation of reactive nitrogen species. Myeloperoxidase generates reactive nitrogen intermediates from NO
2 by two distinct pathways. The reaction involving HOCl and NO2 Cl (Eqs. (2) and (3)) should be inhibited by sulfur- or amino-containing compounds that react with HOCl. The reaction involv ing the direct, one-electron oxidation of NO
2 to NO2 by compound I (Eq. (4)) should not require chloride. We found that under our experimental conditions nitration of xanthine by myeloperoxidase was only modestly inhibited by scavengers of HOCl and did not require Cl
. Formation of 8-nitroxanthine by activated human neutrophils was not affected by taurine, suggesting that nitration under these conditions was independent of HOCl. Moreover, the oxidation potentials of NO
2 and Cl
are )0.99 and )1.36 V, respectively, suggesting that myeloperoxidase might oxidize NO
2 even at plasma concentrations of Cl
[17]. These observations indicate that a NO2 -like species is likely to mediate the formation of 8-nitroxanthine by myeloperoxidase under physiological conditions. Alternatively, xanthine might be oxidized directly by compound I, a complex of myeloperoxidase and H2 O2 , to yield a radical species  that subsequently reacts with NO
2 or NO2 to generate the observed products. Indeed, certain oxidation reactions of myeloperoxidase occur at or near its sheltered active site [25,28,65]. Our observations suggest several mechanisms that potentially could regulate production of reactive intermediates during reperfusion and inflammatory conditions associated with elevated levels of xanthine and xanthine oxidase. We found that 8-nitroxanthine affects the activity of xanthine oxidase in at least two ways.

First, it inhibits the initial rate of xanthine oxidation. Second, it enhances the cummulative production of O
2 . These results are understandable in light of the fact that xanthine and oxygen interact with xanthine oxidase at different sites [7]. Xanthine binds near the enzymeÕs central molybdenum atom, whereas oxygen binds next to FAD. A previous study demonstrated that 8-bromoxanthine interacts with xanthine oxidase near the xanthine-binding domain [66]. It is likely that 8-nitroxanthine behaves similarly, interacting near the enzymeÕs molybdenum center to inhibit xanthine binding. Such an interaction would explain why 8-nitroxanthine acted as a noncompetitive inhibitor of xanthine oxidation by xanthine oxidase. In contrast, because only a fraction of the reducing equivalents derived from xanthine are used to convert oxygen to H2 O2 , the reduced rate of uric acid production that we observed in the presence of 8-nitroxanthine would not necessarily be expected to reduce the rate of O
2 production. Indeed, it is possible that 8-nitroxanthine favors a partially reduced form of xanthine oxidase that preferentially reduces oxygen by a one-electron transfer reaction [7,66]. By inhibiting the conversion of xanthine to uric acid, 8-nitroxanthine prolongs the activity of the enzyme when a fixed amount of substrate is available. Alternatively, xanthine oxidase, cytochrome P450 reductase, and inducible nitric oxide synthase can reduce nitro compounds to the radical anion intermediate, which subsequently donates an electron to oxygen to form O
2 [67–69]. Collectively, these factors may account in part for the increased cumulative production of O
2 that we observed. The ability of 8-nitroxanthine to increase O
2 production by xanthine oxidase suggests a potential mechanism for regulating the generation of free radicals during ischemia and reperfusion. In this scenario, reactive nitrogen species would convert xanthine to 8-nitroxanthine. This product would modulate the activity of xanthine oxidase, perhaps by inhibiting the oxidation of substrate while maintaining the enzymeÕs ability to produce O
2 . The net result would be to increase the production of O
2 , which in turn would react with NO to generate more ONOO
and therefore more 8-nitroxanthine. Nitration of xanthine by the myeloperoxidase system of inflammatory cells might contribute further to this process. Thus, conversion of xanthine to 8-nitroxanthine might represent a positive feedback mechanism that enhanced further the production of both reactive oxygen and nitrogen species in reperfused tissue. Our observations indicate that xanthine is nitrated by ONOO
and by the myeloperoxidase–H2 O2 –NO
2 system of neutrophils. To investigate the biological relevance of 8-nitroxanthine, it will be important to establish the presence of this nitrated purine in vivo. The mass spectrometric methods we have developed offer a useful starting point for identifying such compounds in

G.C. Yeh et al. / Archives of Biochemistry and Biophysics 418 (2003) 1–12

biological materials. Detecting 8-nitroxanthine in reperfused or inflamed tissue would raise the possibility that xanthine or other purines represent a physiologically important target for damage by reactive nitrogen species.

Acknowledgments We thank S.Y. Kassim for help with the xanthine oxidase studies. NMR experiments were performed at the High Resolution NMR Facility, Department of Chemistry, Washington University. Mass spectrometry experiments were performed at the Washington University School of Medicine Mass Spectrometry Resource. This work was supported by grants from the National Institutes of Health (AG19309, AG021191, HL64344, and RR00954). J.P.H. was supported by a Biophysics Training Grant from the National Institutes of Health.

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