Nitrosation of Uric Acid by Peroxynitrite

Peroxynitrite (ONOO−), formed by the reaction between nitric oxide (·NO) and superoxide, has been implicated in the etiology of numerous disease processes. Low molecular weight antioxidants, including uric acid, may minimize ONOO−--mediated damage to tissues. The tissue-sparing effects of uric acid are typically attributed to oxidant scavenging; however, little attention has been paid to the biology of the reaction products. In this study, a previously unidentified uric acid derivative was detected in ONOO−-treated human plasma. The product of the uric acid/ONOO− reaction resulted in endothelium-independent vasorelaxation of rat thoracic aorta, with an EC50 value in the range of 0.03–0.3 μm. Oxyhemoglobin, a ·NO scavenger, completely attenuated detectable ·NO release and vascular relaxation. Uric acid plus decomposed ONOO− neither released ·NO nor altered vascular reactivity. Electrochemical quantification of ·NO confirmed that the uric acid/ONOO− reaction resulted in spontaneous (thiol-independent) and protracted (t½ ∼ 125 min) release of ·NO. Mass spectroscopic analysis indicated that the product was a nitrated uric acid derivative. The uric acid nitration/nitrosation product may play a pivotal role in human pathophysiology by releasing ·NO, which could decrease vascular tone, increase tissue blood flow, and thereby constitute a role for uric acid not previously described.

Reactive oxygen and nitrogen species are continuously generated in vivo and play an integral role in numerous physiologic and pathologic processes (1)(2)(3). To minimize the consequences of oxidant damage to biologic molecules, human plasma is endowed with an integrated antioxidant system of enzymatic and expendable soluble antioxidants. Uric acid is reportedly one of these physiologically important plasma antioxidants (4). Reactive species can oxidize uric acid to relatively stable products that can serve as quantitative estimates of oxidant stress in vivo (5). Uric acid is of special relevance to humans because it is the terminal oxidation product of purine metabolism due to the evolutionary loss of urate oxidase, which catabolizes uric acid to allantoin (6). Consequently, human plasma contains uric acid at concentrations approaching 500 M (7). Endogenous antioxidants constitute the first line of defense against oxidant-induced tissue injury. However, in a variety of pathologic conditions, antioxidant defenses may become overwhelmed, thus allowing reactive oxygen and nitrogen species to react with target molecules and to impair essential biochemical processes (8 -11).
Peroxynitrite is an oxidizing and nitrating agent that reacts with a variety of biomolecules, including lipids, proteins, carbohydrates, and deoxyribonucleic acid (14 -18). Potential pathophysiologic effects of ONOO Ϫ include its action as a bactericidal agent (19), inactivation of mitochondrial manganesesuperoxide dismutase (20) and glutamine synthetase (21), alteration of the lipid aggregatory properties of surfactant protein A (22,23), inactivation of sodium transport (24), inactivation of ␣ 1 -antiproteinase (17), and modification of tyrosine phosphorylation (25,26). Plasma antioxidants also serve as significant biologic targets that can be decreased by ONOO Ϫ-, leading to oxidative damage to tissues and compromised function (11,14,18,27,28). Nitration and nitrosation reactions are increasingly recognized as important mediators of damage in biologic systems, although the precise mechanism of interaction of the reactive nitrogen species with endogenous biomolecules remains unclear. Peroxynitrite, and possibly other reactive nitrogen species, can react readily with phenolic compounds, such as tyrosine, to form nitrated (3-nitrotyrosine) and dimerized (dityrosine) products (29,30). The stable 3-nitrotyrosine product has been detected in human atherosclerotic lesions (31), and tissues following acute injury (32,33), inflammatory disorders (34), and liver ischemia/reperfusion and preservation/ transplantation (35). It has also been reported that the reaction of ONOO Ϫ with proteins, carbohydrates, and thiols can result in the formation of products, which can act as ⅐NO donors (36 -38). Therefore, it was the purpose of this study to determine if the reaction between uric acid and ONOO Ϫ could result in the formation of recognized oxidation products as well as form novel uric acid nitration/nitrosation derivatives that could release ⅐NO and induce vasorelaxation. tima grade) was ordered from Fisher. Water used in solution preparation was purified by passage through a Milli-Q water purification system (Millipore Corp., Milford, MA). Prior to use, the purified water was polished using a Sep-Pak C 18 column (Waters) and then filtered with a 0.22-m membrane filter.
Incubation of Purines and Purine Oxidation Products with ONOO Ϫ -Hypoxanthine, xanthine, uric acid, allantoin, and parabanic acid (0.1-0.3 mM) were prepared in 100 mM potassium phosphate buffer containing 0.1 mM diethylenetriaminepentaacetic acid (DTPA) (pH 7.4) and standardized by spectrophotometry. In all experiments, ONOO Ϫ was kept on ice and shielded from light. Purine was added to the buffer; ONOO Ϫ (0, 0.25, 0.5, 0.75, or 1.0 mM) was added; and the samples were allowed to incubate at room temperature for 15 min. To control for nonspecific effects of ONOO Ϫ decomposition products, ONOO Ϫ was first incubated in the phosphate buffer for 15 min prior to the addition of purine or purine oxidation product (reverse order addition). Incubation with 1.2 N NaOH served as the vehicle control. Peroxynitrite reactivity is pH-dependent; therefore, the pH of all samples was determined and maintained at Յ7.8 under our experimental conditions. Peroxynitrite (0, 0.5, or 1.0 mM) was added to fresh plasma, and the mixture was allowed to incubate for 15 min at room temperature. Ice-cold acetonitrile (3:1, v/v) was added, and the samples were allowed to incubate for 5 min at room temperature. Samples were then centrifuged (15 min, 15,000 ϫ g, 4°C), and the supernatant was collected and concentrated (at ambient temperature) in a rotary evaporator (Speed-Vac, Savant Instruments, Inc., Holbrook, NY). The resulting residue was resuspended in 100 mM potassium phosphate buffer containing 0.1 mM DTPA (pH 7.4). To minimize the pH effects of addition of ONOO Ϫ to plasma in the reverse order addition experiments, ONOO Ϫ was first decomposed by incubation for 15 min in 500 mM potassium phosphate buffer (pH 7.0). An aliquot of the decomposed ONOO Ϫ (equivalent to 1.0 or 0.5 mM ONOO Ϫ ) was then added to the plasma. Again, incubation with 1.2 N NaOH served as the vehicle control, and the pH was maintained at Յ7.8 under our experimental conditions. For the vessel reactivity experiments, ONOO Ϫ (10 mM) or decomposed ONOO Ϫ was added to 2.5 mM uric acid in 100 mM potassium phosphate containing 0.1 mM DTPA (pH 7.4). The mixture was shielded from light and incubated for 20 min at room temperature. Serial dilutions of the uric acid stock solution were prepared in Krebs-Henseleit buffer (118 mM NaCl, 4.6 mM KCl, 27.2 mM NaHCO 3 , 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 1.75 mM CaCl 2 , 0.03 mM Na 2 EDTA, and 11.1 mM glucose). The nominal concentration of purine in the tissue bath (range ϭ 0.002-0.74 M) was significantly less than normal physiologic levels of the purines tested (41).
Absorbance Spectrum Analysis-Peroxynitrite (2.0 mM) was added to uric acid (0.3 mM) in 100 mM potassium phosphate containing 0.1 mM DTPA (pH 7.4) at 20°C. Evidence for the formation of a nitrated derivative was obtained by measuring the hypsochromic absorbance spectrum shift of the reaction at an acidic pH (4 -5) relative to the absorbance spectrum at pH 7.4 (42). The pH was adjusted using hydrochloric acid (5.0 N). Absorbance spectra were measured on a Beckman DU7500 UV-visible diode array spectrophotometer.
HPLC-Mass Spectrometric Analysis-Uric acid and ONOO Ϫ mixtures (as described above) were separated by reverse-phase HPLC on a 10 cm ϫ 2.1-mm (internal diameter) C 8 column, with 5% methanol and 10 mM ammonium acetate (pH 6.5) as the mobile phase at a flow rate of 0.2 ml/min. The eluate was split 1:1, and 100 l/min was introduced into the electrospray ionization interface of a PE-Sciex API III triple quadrupole mass spectrometer. Positive ion spectra were recorded with the needle and orifice potentials set at 5500 and 70 V, respectively.
Preparation of Oxyhemoglobin-Human hemoglobin was purified as described previously (43) and stored as carboxyhemoglobin. Oxyhemoglobin, a ⅐NO scavenger, was prepared by photolyzing the CO-Fe bond of carboxyhemoglobin while gently blowing oxygen over the solution at 4°C. Oxyhemoglobin concentrations were calculated using the extinction coefficient ⑀ 577 ϭ 14, 600 M Ϫ1 cm Ϫ1 per heme group.
⅐NO Microelectrode Experiments-Nitric oxide was detected electrochemically using a membrane-coated carbon fiber ⅐NO microelectrode (World Precision Instruments, Inc., Iso-NO 200) (32). For the ⅐NO detection experiments, ONOO Ϫ (1.0 mM) or decomposed ONOO Ϫ was added to uric acid (0.3 or 3.0 mM) or xanthine (0.3 mM) in 200 mM potassium phosphate (pH 7.4) with or without 0.1 mM DTPA. An aliquot of the purine/ONOO Ϫ mixture was then added to a temperature-controlled (maintained at 37°C) reaction chamber containing the microelectrode. The reaction chamber was extensively rinsed with distilled water between addition of aliquots of the purine/ONOO Ϫ mixture. The effect of thiols or metals on the reaction mixture was tested in a subset of experiments by adding 0.2 mM cysteine or glutathione followed by Cu 2ϩ (0.025-0.1 mM) to the reaction chamber. The thiol and metal were added to the reaction chamber either prior to or after the addition of the purine/ONOO Ϫ mixture. As an additional control, in a subset of experiments, oxyhemoglobin (0.01 mM) was added to the reaction chamber either prior to or following the addition of sample to the chamber. The mean half-life for each condition was calculated by fitting the data to a single exponential decay model (half-life ϭ y 0 ϩ A 1 e Ϫ(xϪx0 )/ t1 , where y 0 ϭ asymptotic value of the y variable, x 0 ϭ initial time, x ϭ time of decay, A 1 ϭ y value when x 0 ϭ 0, and 1/t 1 ϭ decay constant).
Vessel Reactivity Studies-Isometric tension was measured in isolated rat aortic ring segments. Animals were euthanized by an anesthetic overdose. The aorta was excised, cleansed of fat and adherent tissue, cut into ring segments (2-3 mm), and suspended from a forcedisplacement transducer in a tissue bath. Segments were bathed in a Krebs-Henseleit solution. A passive load of 2 g was applied and maintained throughout the experiment. Indomethacin (0.05 mM)-treated ring segments were depolarized with KCl (70 mM) to determine maximal vessel contractile capacity. In subsequent experiments, phenylephrine (ϳ3 ϫ 10 Ϫ8 M) was added to the indomethacin-treated ring segments to obtain a contraction equivalent to ϳ40% of the KCl response. When tension development reached a plateau, uric acid or xanthine reaction mixtures (obtained by treatment of purine with ONOO Ϫ or decomposed ONOO Ϫ ) were added to achieve a cumulative dose-response curve, expressed as the nominal concentration of purine to which the tissue was exposed.
In some experiments, endothelium was removed by rubbing the luminal surface of the vessel with a serrated forceps. Endothelial denudation was confirmed by the absence of vessel relaxation in response to acetylcholine (0.001 mM), an endothelium-dependent vasodilator. To determine whether vessel relaxation was due to the release of ⅐NO from the reaction products of ONOO Ϫ and purine, oxyhemoglobin (0.001 mM) was added to the tissue bath in a subset of experiments. Data are reported as weighted means representing two or three observations per animal (n ϭ eight animals). Real time data were acquired for all experiments using a computerized data acquisition system.
Statistical Analysis-Analyses for effects of ONOO Ϫ on purines and purine oxidation products were conducted by ANOVA with post hoc comparison of means by a Student-Newman-Keuls test. Comparison of the response of different purines to ONOO Ϫ was conducted by repeated measures ANOVA. Analysis of the effect of xanthine, uric acid, and ONOO Ϫ as well as the effects of light on aortic relaxation was conducted by repeated measures ANOVA for the various concentrations of purine. Comparison of two groups was done by repeated measures ANOVA with Bonferroni correction for multiple comparisons. For determination of the effects of temperature and light on the stability of ⅐NO release, the half-life was determined from each experiment and analyzed by ANOVA with a Student-Newman-Keuls post hoc test. All analyses were done using the SAS system for Microsoft Windows (Release 6.12).

RESULTS
Both uric acid and xanthine were significantly (p Ͻ 0.001) oxidized by 0.25, 0.5, 0.75, and 1.0 mM ONOO Ϫ (Fig. 1). The extent of oxidation of uric acid was markedly greater than that of xanthine at all concentrations of purine and ONOO Ϫ studied. Hypoxanthine was significantly (p Ͻ 0.05) oxidized only with 1.0 mM ONOO Ϫ . Parabanic acid and allantoin were not significantly oxidized at any concentration of ONOO Ϫ . Concomitant with uric acid oxidation was the formation of allantoin, parabanic acid, urazole, oxonic acid, and a novel peak not observed following addition of decomposed ONOO Ϫ . Spectral characteristics and retention times of authentic oxaluric acid, cyanuric acid, alloxan monohydrate, and uracil excluded these purine oxidation products as this novel peak. Xanthine oxidation also produced allantoin, parabanic acid, and a novel product, but this product exhibited a different retention time and different spectra than the ONOO Ϫ -treated nitrated uric acid product. The novel peak was not detected when xanthine was treated with decomposed ONOO Ϫ .
The hypsochromic shift of the uric acid/ONOO Ϫ product ( Fig.  2) is consistent with the pH-dependent absorbance changes of nitrated aromatic compounds (42) and suggests that uric acid may be nitrated (or nitrosated) upon reaction with ONOO Ϫ . Addition of decomposed ONOO Ϫ to uric acid resulted in an absorbance spectrum indistinguishable from uric acid alone and was pH-insensitive (data not shown).
In human plasma samples, the endogenous uric acid concentration was 0.20 Ϯ 0.01 mM (mean Ϯ S.E.) in pooled donor plasma. Upon addition of ONOO Ϫ , plasma uric acid decreased concurrently with the appearance of oxidation products (allantoin and parabanic acid) and the nitrated uric acid product, similar to the effect observed in buffer. 9 Ϯ 2% of the plasma uric acid was converted to the nitrated peak, whereas 13 Ϯ 4% was converted following exposure to 1.0 mM ONOO Ϫ , again similar to the results with uric acid in buffer (Fig. 3). Treatment with neither decomposed ONOO Ϫ nor vehicle resulted in significant oxidation of uric acid or formation of the nitrosation product.
Mass spectrophotometric analysis of the ONOO Ϫ -treated uric acid revealed that the uric acid concentration (m/z ϭ 169, (uric acid ϩ H) ϩ , retention time ϭ 5.4 min) was reduced 87% by addition of 1.0 mM ONOO Ϫ (Fig. 4A) compared with addition of an equivalent amount of decomposed ONOO Ϫ (Fig. 4B). Concomitant with the reduction in uric acid was the formation of a novel peak at 6.5 min. This product was predicted to have a M r of 146 based upon the mass-to-charge ratio (m/z) of 147 ((nitrated product ϩ H) ϩ ion) (Fig. 4C). Addition of decomposed ONOO Ϫ (equivalent to 1.0 mM active ONOO Ϫ ) did not result in a significant reduction in the uric acid concentration (Fig. 4B) or formation of the novel nitrated product at the retention time of 6.5 min or molecular ion at m/z 147 (Fig. 4D).
The ONOO Ϫ -treated uric acid product was scanned by electrospray ionization mass spectrum analysis. Scans acquired at 6.5 min (retention time of nitrated product) demonstrated that fragmentation of the nitrated product contained ions at 130, 104, 87, and 61 (Fig. 5A). The resulting spectrophotometry also   4)), and samples of fresh human plasma (uric acid concentration ϭ 0.20 Ϯ 0.01 mM) were also reacted with identical ONOO Ϫ concentrations. Both sets of samples were then allowed to incubate for 15 min at room temperature. Plasma samples were deproteinized with acetonitrile and vacuum-dried, and the resulting pellet was resuspended in potassium phosphate buffer. Both plasma and buffer samples were then analyzed by reverse-phase HPLC. A dose-dependent oxidation (hatched bars) and nitration (solid bars) of uric acid was demonstrated in plasma (left panel) and buffer (right panel). Treatment with neither decomposed ONOO Ϫ nor vehicle resulted in significant oxidation of uric acid, as represented by decomposed ONOO Ϫ -treated samples. The amount of uric acid nitration/nitrosation product is depicted relative to the uric acid standard or to the initial amount of plasma uric acid at base line ((uric acid area/nitrated peak area) ϫ 100). The nitrated peak was not formed with treatment with either decomposed ONOO Ϫ or vehicle. *, p Ͻ 0.05 versus inactive control.
Cumulative administration of the uric acid/ONOO Ϫ mixture induced a dose-dependent relaxation of rat aortic ring segments with EC 50 ϭ 0.27 Ϯ 0.19 M (Fig. 7A). The calculated EC 50 represents an underestimate due to the 10-fold dilution of the uric acid/ONOO Ϫ mixture in the tissue bath and the inability to directly quantify the nitrated product. The actual EC 50 is in the range of 0.03-0.3 M. The relaxation response was completely reversed by treatment with oxyhemoglobin. In contrast, uric acid alone or uric acid exposed to decomposed ONOO Ϫ had little effect on vessel tone, although a modest decrease in tension was observed at the highest cumulative concentration of uric acid treated with decomposed ONOO Ϫ . Treatment of xanthine with ONOO Ϫ or decomposed ONOO Ϫ did not yield reaction products with vasodilatory activity. Ring segments denuded of endothelium displayed similar dose-response characteristics for ONOO Ϫ -treated uric acid, as did intact vessels (Fig. 7B).
Direct electrochemical detection of ⅐NO, utilizing a selective microelectrode, demonstrated that the uric acid/ONOO Ϫ mixture spontaneously liberated ⅐NO (Fig. 8A). A similar extent of ⅐NO release was seen when either 0.3 or 3.0 mM uric acid was treated with 1.0 mM ONOO Ϫ . Therefore, in subsequent studies, 3.0 mM was used to ensure that excess substrate was present. Addition of oxyhemoglobin to the reaction chamber consumed the ⅐NO present and completely attenuated any further release of ⅐NO by subsequent additions of the uric acid/ONOO Ϫ mixture.
The reaction of 3.0 mM uric acid with 1.0 mM ONOO Ϫ resulted in the production of 13.7 Ϯ 1.1 M ⅐NO. There was no detectable ⅐NO produced from the reaction of ONOO Ϫ alone, uric acid alone, uric acid treated with decomposed ONOO Ϫ , or xanthine treated with ONOO Ϫ . The release of ⅐NO from the uric acid/ONOO Ϫ mixture was spontaneous and was not enhanced by the addition of thiols (cysteine or glutathione) or metal (Cu 2ϩ ). Addition of DTPA (0.1 mM), a metal chelator, did not alter ⅐NO release.
Finally, it was demonstrated that both temperature and light significantly (p Ͻ 0.01) alter the half-life of this ⅐NOdonating product. The half-life of ⅐NO release in the dark at 4°C was 123 Ϯ 20 min (Fig. 8B) and was significantly decreased at room temperature (20 Ϯ 3 min) and further decreased in the presence of light (13 Ϯ 1 min).

DISCUSSION
Peroxynitrite is both a nitrating and oxidizing agent that can compromise antioxidant defenses and that simultaneously results in oxidative damage to tissues (11). In this study, we demonstrated that uric acid is considerably more susceptible to oxidation by ONOO Ϫ than other purines (hypoxanthine and xanthine) or purine oxidation products (allantoin and parabanic acid). The interaction of ONOO Ϫ with both uric acid and xanthine resulted in the formation of putatively nitrated purine products. However, unlike the nitrated uric acid product, the xanthine product was not formed in our plasma experiments, nor did the reaction of xanthine with ONOO Ϫ result in release of ⅐NO or vascular relaxation.
Formation of the putatively nitrated uric acid product was dependent upon the concentration of ONOO Ϫ in both plasma and buffer systems. Over 50% of the endogenous uric acid was oxidized in human plasma, which was remarkable in view of the multitude of other potential target molecules present that could react with ONOO Ϫ . The pH-dependent hypsochromic absorbance shift of the uric acid product was suggestive of nitration, although a nitrosation product could not be dismissed solely on these data. A pathway for formation of the fragmentation products was developed from the liquid chromatography-mass spectrophotometric data. Electrospray ionization mass spectrometry indicated that uric acid (M r 168) was most likely oxidized to a product similar in structure to parabanic acid, a known uric acid oxidation product. This oxidation product could be subsequently nitrated or nitrosated to form a product(s) with a M r of 146. The structure of a nitrosated product would be consistent with a potential role as a ⅐NO donor, which was supported by ex vivo vessel relaxation data in this study. The ⅐NO donor produced by the reaction of uric acid with ONOO Ϫ resulted in a dose-dependent relaxation of rat aortic ring segments that was completely reversed by addition of oxyhemoglobin, a ⅐NO scavenger. Aortic ring segments denuded of endothelium displayed similar dose-response characteristics as intact vessels, indicating that the ⅐NO donating properties of the uric acid product were not mediated by the stimulation of endothelium-dependent ⅐NO production. The release of ⅐NO from the nitrated uric acid product was confirmed electrochemically and was not thiol-dependent, in marked contrast to organic nitrites that release ⅐NO (44). The treatment of uric acid with decomposed ONOO Ϫ did not result in production of a ⅐NO-donating metabolite. Based on these cumulative lines of evidence, we propose that the uric acid product is 2-nitrito-4-amino-5-hydroxyimidazoline (Fig. 6). Detection of the nitrosated uric acid product (in plasma) may also be a good index of ⅐NO-derived oxidant production and provide insight into the relative roles of reactive oxygen and nitrogen species in the etiology of oxidant-induced tissue injury.
Release of ⅐NO from the nitrosated uric acid derivative following insults such as ischemia/reperfusion could minimize tissue injury and thereby constitute a previously uncharacterized role for uric acid in physiology. Although ⅐NO-releasing drugs have been used clinically for Ͼ100 years in the treatment of cardiovascular dysfunction, the full potential for ⅐NO therapy is only now being recognized. Recently, it was demonstrated that ⅐NO could decrease lung injury following intestinal ischemia and that ⅐NO donors can prevent hydrogen peroxide-me- diated endothelial cell injury (45,46). This suggests that the cardiopulmonary complications associated with liver ischemia/ reperfusion, transplantation, and systemic shock states may be amenable to ⅐NO therapy. Nitric oxide is also currently being evaluated as a treatment for acute hypoxemia and/or pulmonary hypertension associated with persistent pulmonary hypertension of the newborn, acute respiratory distress syndrome, and numerous other disease states (47)(48)(49).
Vasodilator actions of organic nitrates and nitrites in vivo may be due to the formation of S-nitrosothiols. These stable sulfhydryl-containing compounds liberate ⅐NO and induce relaxation via activation of vascular smooth muscle cell guanylate cyclase (50,51). Conversion of numerous organic nitrates to their ⅐NO-donating forms has been attributed to the action of membrane-associated enzymes, whereas production of ⅐NO from organic nitrites appears to involve a distinct cytosolic enzyme (52,53). The nominal potency of 2-nitrito-4-amino-5hydroxyimidazoline as a ⅐NO donor (EC 50  Nitric oxide is also released following the reaction of ONOO Ϫ with glucose, glycerol, and other biologic molecules that contain an alcohol functional group (37, 55) by a thiol-and Cu 2ϩ -dependent mechanism. This is a property shared with organic nitrates and nitrites (44). Although the precise mechanisms of ⅐NO release remain somewhat controversial, it has been suggested that an S-nitrosothiol intermediate is involved and that the decomposition of the nitrosothiol is accelerated by Cu 2ϩ (56). In marked contrast, the release of ⅐NO from the nitrated uric acid product is spontaneous and not accelerated by addition of Cu 2ϩ or thiol.
In this study, efforts were made to minimize the potential contribution of bicarbonate/CO 2 to the nitration/nitrosation of uric acid. Bicarbonate is one of the most abundant constituents of the extracellular milieu (ϳ25 mM) and, because of the equilibrium with carbonic acid, results in ϳ1.3 mM being present in plasma as CO 2 . In plasma, it is possible that the reaction of ONOO Ϫ with bicarbonate/CO 2 may influence ONOO Ϫ -mediated oxidation and nitration reactions (57)(58)(59)(60). The reaction of ONOO Ϫ with bicarbonate/CO 2 is rapid (ϳ3 ϫ 10 4 M Ϫ1 s Ϫ1 ) (59) and reported to yield a nitrosoperoxycarbonate anion (ONO-OCO 2 Ϫ ) that can participate in oxidation and nitration reactions (57-59, 61). Bicarbonate enhances ONOO Ϫ -mediated nitration FIG. 7. Uric acid treated with ONOO ؊ acts as a vasodilator. Administration of uric acid treated with ONOO Ϫ (q) induced a dosedependent relaxation of rat aortic ring segments with an EC 50 in the range of 0.03-0.3 M (A). Addition of oxyhemoglobin (10 M), a scavenger of ⅐NO, completely ablated this response. Uric acid exposed to decomposed ONOO Ϫ (E) had little effect on vessel tone, although a slight decrease in tension was observed at the highest cumulative concentrations. Incubation with uric acid (Ⅺ), xanthine treated with ONOO Ϫ (OE), or xanthine treated with decomposed ONOO Ϫ (‚) did not exhibit significant vasodilatory activity. Ring segments denuded of endothelium (E) displayed similar dose-response characteristics for ONOO Ϫ -treated uric acid, as did intact vessels (q) (B). *, p Ͻ 0.05 versus inactive control.
FIG. 8. Release of ⅐NO and stability of product formed by treatment of uric acid with ONOO ؊ . Uric acid (UA; 3.0 mM) was reacted with ONOO Ϫ (1.0 mM). An aliquot of this reaction mixture was then added to a reaction chamber containing a ⅐NO microelectrode. It can be seen that with each repeated addition of the reaction mixture, ⅐NO was spontaneously released (A). Upon the addition of oxyhemoglobin to the reaction chamber, all of the ⅐NO was consumed, and further detection of ⅐NO by the reaction mixture was completely inhibited. Over a 5-h time period, aliquots of the uric acid (3.0 mM) and ONOO Ϫ (1.0 mM) mixture were added to the reaction chamber containing a ⅐NO microelectrode, and the ⅐NO released was detected. The reaction chamber was extensively rinsed with distilled water between the addition of aliquots of the purine/ONOO Ϫ mixture. In this manner, a curve was generated that depicts the stability of the nitrated uric acid product, as determined by the release of ⅐NO, with time (B). The reaction mixture was kept at 4°C in the dark throughout each experiment. The half-life of the ⅐NOdonating uric acid product was 123 Ϯ 20 min when fitted by a singleorder exponential decay equation (solid line).
of p-hydroxyphenyl acetate (59), albumin (58), and tyrosine (58,62). Uric acid effectively reduces ONOO Ϫ -induced nitration of albumin, whereas the addition of 0.2 mM uric acid decreases nitrotyrosine formation by ϳ40% (58). In this study, to ensure decomposition of ONOO Ϫ in the vessel relaxation studies, ONOO Ϫ was reacted with uric acid for 20 min in phosphate buffer (100 mM, pH 7.4) prior to addition to the vessel chamber containing Krebs-Henseleit buffer. The electrochemical ⅐NO detection experiments were also conducted in phosphate buffer at pH 7.4 prior to addition to the reaction vessel. It was unlikely that that the adventitious carbonate associated with absorbed CO 2 and contamination by the salts used in the buffer preparation was of sufficient concentration to catalyze nitration (59).
We have yet to demonstrate that this nitrated uric acid derivative is formed in vivo, and these studies are ongoing. If this derivative is formed in vivo, then the observation that the nitrated uric acid product releases ⅐NO in a continuous fashion suggests that the product could play a significant role in the pathophysiology of acute and chronic inflammation. In addition, the nitrated product may prove valuable to the rational design of a new class of mechanism-based, anti-ischemic drugs that mimic the properties of endogenous uric acid. It is possible that a mimic of the nitrated uric acid product could be utilized to alleviate myocardial ischemic syndromes following ischemia/ reperfusion, cardioplegic ischemic arrest, coronary artery thrombosis after thrombolysis, and restenosis after transluminal coronary angioplasty (63,64). The uric acid nitrosation product may play a pivotal role in human pathophysiology by releasing ⅐NO, which could decrease vascular tone and increase tissue blood flow (65,66). The nitrated uric acid product may also exhibit anti-inflammatory properties by inhibiting platelet aggregation/adherence (67) as well as by attenuating leukocyte adhesion to the endothelial cell surface (68). The combined vasodilator and anti-inflammatory properties of such a ⅐NO donor may make it ideally suited for minimizing tissue damage associated with organ transplantation, ischemic bowel disease, and multiple organ dysfunction associated with systemic shock states such as burns, hypovolemia, and trauma.