This Article Abstract Full Text (PDF) All Versions of this Article: 22/9/1402    most recent 01.ATV.0000027524.86752.02v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hink, H. U. Articles by Fukai, T. Search for Related Content PubMed PubMed Citation Articles by Hink, H. U. Articles by Fukai, T. Related Collections Pathophysiology Oxidant stress Mechanism of atherosclerosis/growth factors

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:1402.)
© 2002 American Heart Association, Inc.

Vascular Biology

Peroxidase Properties of Extracellular Superoxide Dismutase

Role of Uric Acid in Modulating In Vivo Activity

H. Ulrich Hink; Nalini Santanam; Sergey Dikalov; Louise McCann; Andrew D. Nguyen; Sampath Parthasarathy; David G. Harrison; Tohru Fukai

From the Division of Cardiology, Department of Medicine (H.U.H., S.D., L.M., A.D.N., D.G.H., T.F.), and the Department of Gynecology and Obstetrics (N.S., S.P.), Emory University School of Medicine, and the Atlanta Veterans Administration Hospital (L.M., D.G.H., T.F.), Atlanta, Ga.

Correspondence to Tohru Fukai, MD, PhD, Division of Cardiology, Emory University School of Medicine, 1639 Pierce Dr, WMB 319, Atlanta, GA 30322. E-mail tfukai{at}emory.edu

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Objective— The cytosolic form of Cu/Zn-containing superoxide dismutase (SOD1) has peroxidase activity, with H₂O₂ used as a substrate to oxidize other molecules. We examined peroxidase properties of the extracellular form of SOD (SOD3), a major isoform of SOD in the vessel wall, by using recombinant SOD3 and an in vivo model of atherosclerosis.

Methods and Results— In the presence of HCO₃^-, SOD3 reacted with H₂O₂ to produce a hydroxyl radical adduct of the spin trap 5-diethoxyphosphoryl-5methyl-1-pyrroline N-oxide (DEMPO). SOD1 and SOD3 were inactivated by H₂O₂ in a dose- and time-dependent fashion, and this was prevented by physiological levels of uric acid. To examine the in vivo role of uric acid on SOD1 and SOD3, control and apolipoprotein E-deficient (ApoE^-/-) mice were treated with oxonic acid, which inhibits urate metabolism. This treatment increased plasma levels of uric acid in control and ApoE^-/- mice by 3-fold. Although increasing uric acid levels did not alter aortic SOD1 and SOD3 protein expression, aortic SOD1 and SOD3 activities were increased by 2- to 3-fold in aortas from ApoE^-/- mice but not in aortas from control mice.

Conclusions— These studies show that SOD1 and SOD3 are partially inactivated in atherosclerotic vessels of ApoE^-/- mice and that levels of uric acid commonly encountered in vivo may regulate vascular redox state by preserving the activity of these enzymes.

Key Words: superoxide dismutase • peroxidase activity • uric acid • hydrogen peroxide • atherosclerosis

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Cytoplasmic Cu/Zn superoxide dismutase (SOD1) not only catalyzes dismutation of O₂^·- to H₂O₂ but also has peroxidase activity, with H₂O₂ used as a substrate, forming a copper-bound hydroxyl (^·OH) radical.¹ Several lines of evidence suggest that this bound ^·OH attacks 1 adjacent histidine residue that binds copper, leading to loss of copper and inactivation of the enzyme.² This inactivation of SOD1 by H₂O₂ may be prevented by coincubation with small anions or other reductants, such as formate, tocopherol, nitrite, and uric acid, resulting in the formation of their respective radicals.^1,3–5 Recently, it has been recognized that HCO₃^- is important in this reaction, either by formation of the bicarbonate radical⁴ or by facilitating the reaction of H₂O₂ with Cu²⁺ in the active site of the enzyme.⁶

See page 1367

Extracellular SOD (SOD3) is also a Cu/Zu-containing SOD that shares 50% sequence homology with SOD1 within its catalytic region.⁷ Compared with the x-ray crystallography structure data for SOD1,⁸ all ligands to the copper and zinc atoms can be identified in SOD3, as can the cysteines forming the intramolecular disulfide bond.⁷ Thus, these findings suggest that the conformation of the active copper-binding site of SOD3 may resemble the active site of SOD1. The rate constants for dismutation of O₂^·- by SOD1 and SOD3 are similar, and both enzymes are inhibited by cyanide, azide, and diethyldithiocarbamate.⁹ Because SOD3 constitutes 30% to 50% of the total SOD in vascular tissues¹⁰ and is localized in the extracellular space, it would be of considerable importance to determine whether SOD3 also has peroxidase activity and to determine which small molecule reductants can preserve its dismutase activity.

Although peroxidase properties of SOD1 have been studied extensively in vitro, evidence is lacking to prove that either SOD1 or SOD3 activities are affected by H₂O₂ in vivo. In the present study, we sought to determine whether SOD3 has peroxidase activities that are similar to those of SOD1 and to examine small molecules that might prevent the inactivation of SOD3 by using recombinant SOD3 expressed in Pichia pastoris. In these experiments, we found that physiological levels of uric acid completely prevented the inactivation of SOD1 and SOD3 by H₂O₂. In additional experiments, we provide evidence for peroxidase activity of SOD1 and SOD3 in vivo, by showing that these enzymes seem to be partially inactivated in the aortas of apolipoprotein E-deficient (ApoE^-/-) mice and that the activity of these enzymes can be restored by elevating the endogenous levels of uric acid.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials and Reagents
All reagents were purchased from Sigma Chemical Co, including bovine Cu/Zn SOD (SOD1, 3000 to 5000 U/mg), unless otherwise noted. Chelex-100 resin was purchased from Bio-Rad. The pPIC9K-vector was purchased from Invitrogen. Steri-Vac filters and Prep-Scale concentrators were obtained from Millipore. CNBr-activated sepharose was obtained from Amersham Pharmacia Biotech. Catalase was purchased from Roche. Silver staining was performed by using a commercially available kit (Pierce). The spin trap 5-diethyoxyphosphoryl-5methyl-1-pyrroline N-oxide (DEMPO) was obtained from Oxis International. Potassium phosphate buffers were prepared from KH₂PO₄ and K₂HPO₄ in the concentrations given. All buffers contained diethylenetriamine pentaacidic acid (DTPA, 100 µmol/L) and were treated with chelex-100 (5 mg/100 mL, with gentle stirring for 1 hour) before use.

Overexpression and Purification of Recombinant SOD3
The murine SOD3 was overexpressed in P pastoris. The cDNA encoding SOD3, minus its original signaling domain, was cloned into the EcoRI and NotI sites of the pPIC9K vector, and this vector was transfected into the GS115 P pastoris strain by electroporation. Large amounts of SOD3 were obtained by amplifying yeast in a basal fermentation media containing 160 µmol/L copper and zinc with the use of methanol induction. The medium containing SOD3 protein was filtered (Steri-Vac GP-10) and concentrated with the use of a flow concentrator (2.5 ft², Prep-Scale TFF [Millipore], molecular weight cutoff [MWCO] 10 000). After 2 washings in 50 mmol/L sodium phosphate buffer (pH 7.8) to eliminate low molecular impurities including loosely bound copper, SOD3 was affinity-purified by an anti-SOD3 antibody linked to a CNBr sepharose column according to manufacturer’s instructions. The concentrated protein fraction was further purified by use of a Resource Q FPLC column (Pharmacia). The pooled SOD3 fractions were dialyzed in 50 mmol/L Tris/HCl, pH 7.5. The resulting sample was applied to a Resource Q FPLC column. The column was washed with 50 mmol/L Tris/HCl, pH 7.5, and eluted with a linear gradient of NaCl (0 to 1 mol/L) at 0.5%/min with a flow rate of 1 mL/min. Fractions containing SOD3 were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and pooled. The resultant purified protein yielded 2 bands on silver staining with molecular masses of 33 and 66 kDa, corresponding with monomeric and dimeric forms of the enzyme. SOD activity was measured in 50 mmol/L phosphate buffer by inhibition of the reduction of cytochrome c (50 µmol/L) by superoxide generated by xanthine (0.1 mmol/L) and xanthine oxidase (0.01 U/mL) at pH 7.8, as described previously.¹¹ The specific activity of recombinant SOD3 was 2087 U/mg, similar to the previously purified SOD3.¹² This activity was completely blocked by KCN (1 mmol/L).

ESR Spectroscopy
Electron spin resonance (ESR) measurements were performed by using a Bruker EMX spectrometer at 9.78 GHz (ER 041 XG Microwave Bridge X-Band). DEPMPO was used as a spin trap. Spectrometer settings were as follows: microwave power 20.02 mW, time constant 163 ms, sweep time 83.89 seconds, center field 3480 G, sweep width 110 G, and modulation amplitude 1 G.

Animal Studies
ApoE^-/- mice, backcrossed 10 times to the C57BL/6 strain, were weaned at 4 weeks of age and maintained on regular chow. Experiments were performed when the mice were aged 6 to 8 months. C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, Me), fed regular chow, and studied at the same age. Diet and water were provided ad libitum. The Emory University Institutional Animal Care and Use Committee approved the protocol for animal use. Osmotic minipumps were inserted via a subscapular incision during anesthesia with 2.5% tribromoethanol (0.3 mL/25 g body wt IP), as previously described.¹³ Sham-operated animals underwent an identical surgical procedure, except that either no pump or an empty osmotic pump was implanted. Western analysis for SOD protein levels and analysis of aortic SOD activity were performed as described previously.^13–15

Determination of Serum Uric Acid Levels
Serum uric acid levels were measured by using a quantitative enzymatic assay (Sigma) according to the manufacturer’s protocol, and the results were standardized by using standard solutions of uric acid (Sigma).

Statistical Analysis
Data are presented as mean±SEM. Western blots were quantified by use of densitometry. Comparisons between groups of mice were performed by using ANOVA, followed by a Dunnett ad hoc test. Values of P<0.05 were considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of H₂O₂ on SOD Activity and Formation of DEPMPO-OH Adduct
Because the initial steps of the peroxidase reaction led to inactivation of SOD1 in the absence of cosubstrates,¹ we initially examined the ability of H₂O₂ to inactivate SOD3 compared with SOD1. The enzymes were incubated with H₂O₂ in 50 mmol/L phosphate buffer at pH 7.8. Both enzymes were inactivated to a similar extent by H₂O₂ in a time- and dose-dependent manner (Figure 1). The inactivation of these enzymes by H₂O₂ was not significantly affected by the addition of 23.5 mmol/L HCO₃^- (data not shown).

View larger version (14K): [in this window] [in a new window]   Figure 1. Inhibition of SOD1 and SOD3 activity by H2O2. A, SOD1 or SOD3 (1 U/mL) was incubated in the presence or absence of H2O2 (100 µmol/L) for up to 4 hours at 37°C. B, SOD1 or SOD3 (1 U/mL) was incubated in the presence or absence of various concentrations of H2O2 for 2 hours at 37°C. SOD activity was determined with the cytochrome c reduction assay at indicated time points in the presence of catalase (1000 U/mL) and DTPA (100 µmol/L). The data are expressed as the relative inhibition by H2O2 as mean±SEM from 3 independent experiments performed in duplicate.

To determine whether SOD3 could catalyze the formation of a hydroxyl-like species on reaction with H₂O₂, we performed additional studies with use of the spin trap DEPMPO. In the absence of HCO₃^-, incubation mixtures containing SOD3, DEPMPO, H₂O₂, and DTPA in 50 mmol/L phosphate buffer (pH 7.8) yielded no DEPMPO-OH (Figure 2). In the presence of HCO₃^-, however, the level of DEPMPO-OH adduct obtained on incubation of SOD3 with H₂O₂ was clearly increased. These data obtained with SOD3 are consistent with previous studies with SOD1, in which HCO₃^- was found to have minimal effects on the inhibition of SOD1 activity by H₂O₂ but dramatically increased the hydroxylation of DEPMPO by SOD1 in the presence of H₂O₂.^5,16

View larger version (18K): [in this window] [in a new window]   Figure 2. Formation of DEPMPO-OH adduct in the presence of SOD3 and H2O2. The spin adduct from DEPMPO (10 mmol/L) resulting from the reaction between SOD3 (20 U/mL each) and H2O2 (10 mmol/L) was measured by ESR spectroscopy. The pH level was measured immediately before and after the addition of 25 mmol/L HCO3- to ensure that the HCO3--mediated enhancement was not due to a change in the pH of the reaction mixture. A, Representative DEPMPO-·OH tracing. ESR spectrometer settings are as given in the text. The spectra shown are the average of 10 sequential scans. B, Quantification of the obtained ESR signals shown in arbitrary units as the mean±SEM of 5 independent experiments.

Cosubstrate Utilization by SOD1 and SOD3
Anionic radical scavengers, such as formate, urate, and azide, can prevent the inactivation of SOD1 by H₂O₂ because of the favorable electrostatic interaction.¹ To determine whether SOD3 shared this property, both SODs were exposed to various reductants during incubation with H₂O₂. Supraphysiological levels of nitrite (1 mmol/L) prevented the inactivation of SOD1 but only partially prevented the inactivation of SOD3 (Figure 3A). In contrast, physiological levels of urate (500 µmol/L) almost completely prevented the inactivation of SOD1 and SOD3 by H₂O₂ (Figure 3B). Other potential substrates, including formate, glutamate, tyrosine, lactate, -tocopherol, histidine, arginine, and uracil, were not as effective as either nitrite or urate, even when applied in high concentrations (1 mmol/L, Figure 3C).

View larger version (16K): [in this window] [in a new window]   Figure 3. Effect of various reductants on the H2O2-induced inhibition of SOD1 and SOD3. In the presence or absence of the indicated reductants (A, nitrite; B, uric acid; and C, various reductants as indicated), 2.5 U/mL of either enzyme was incubated with H2O2 (100 µmol/L) for 3.5 hours at 37°C. Thereafter, SOD activity was measured by cytochrome c reduction assay in the presence of catalase (1000 U/mL) and DTPA (100 µmol/L). Data are expressed as the mean±SEM from at least 3 independent experiments performed in duplicate.

In Vivo Evidence for Protection of SOD1 and SOD3 Activity by Uric Acid
Because uric acid is capable of preventing H₂O₂-induced inactivation of SOD1 and SOD3, we sought to determine whether increasing uric acid levels in vivo could increase the activity of these enzymes in the aortas of ApoE^-/- mice. When the animals were maintained and studied at 6 to 8 months of age and even when they were fed a normal chow diet, the aortas of these animals developed extensive atherosclerosis and exhibited increased production of vascular reactive oxygen species.¹⁷ We have previously shown that SOD3 protein levels are increased in their aortas.¹⁴ We reasoned that the increase in H₂O₂ levels resulting from increased O₂^·- production may partially inactivate SOD1 and SOD3 in this model. To increase endogenous levels of uric acid, oxonic acid was infused by osmotic minipump at a rate of 2.8 mg/kg per day. Oxonic acid is a potent inhibitor of the enzyme uricase, which in mice is responsible for the metabolism of uric acid to allantoin.¹⁸ A 7-day infusion of oxonic acid increased the plasma levels of uric acid by 3-fold in control and ApoE^-/- mice (Figure 4). The activities of SOD1 and SOD3 were not altered by oxonic acid infusion in vessels from control animals (Figure 5A). In ApoE^-/- mice, however, increased uric acid markedly increased the activities of SOD3 and SOD1 by 2.5±0.1- and 1.7±0.3-fold, respectively (Figure 5A). As previously shown,¹⁴ the protein level of SOD3 but not SOD1 was higher in aortic vessels from ApoE^-/- mice than in aortic vessels from control mice, but changing uric acid levels had no effect on either protein (Figure 5B).

View larger version (10K): [in this window] [in a new window]   Figure 4. Effect of oxonic acid (OXO) treatment on plasma uric acid levels. Plasma uric acid levels (mg/dL) in control and ApoE-/- mice before and after OXO infusion with osmotic minipumps for 7 days (n =4). *P<0.05 between 2 groups.

View larger version (27K): [in this window] [in a new window]   Figure 5. Effect of oxonic acid on SOD1 and SOD3 activity and protein expression. Oxonic acid was infused in control (C57BL/6) and ApoE-/- mice for 7 days by osmotic minipumps. A, Aortas were homogenized, and SOD activity was assayed by examining the inhibition of cytochrome c reduction by xanthine/xanthine oxidase at pH 7.4. The activity of SOD3 was determined after separation with concanavalin A-sepharose. Experiments were performed on 4 pooled aortas on 3 separate occasions. The results were presented as mean±SEM, and the values were expressed as units per milligram of total protein. *P<0.05 vs control. B, Protein levels of SOD3 and SOD1 were determined by Western analysis (20 µg protein of homogenate from aortic tissue per lane). Representative blots are from 4 individual experiments.

We considered the possibility that oxonic acid might directly prevent SOD3 inactivation by H₂O₂ or might inhibit the production of H₂O₂ by enzyme systems in the vessel walls of ApoE^-/- mice. In additional studies, we found that oxonic acid in concentrations up to 1 mmol/L had no effect on the inactivation of SOD3 by H₂O₂ (data not shown). Furthermore, we examined the effect of oxonic acid on the production of O₂^·- by cultured mouse aortic vascular smooth muscle cells. These cells were exposed to oxonic acid (100 µmol/L) for 24 hours, and the production of O₂^·- was determined by examining oxidation of the spin trap CP-H to CP^·by using electron spin resonance. Cells treated or untreated with oxonic acid produced identical levels of O₂^·- (data not shown). These data indicate that oxonic acid has no effect on the production of reactive oxygen species by vascular cells.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we found that SOD3 has peroxidase properties that are very similar to those of SOD1. SOD3 is inactivated by H₂O₂ in a fashion similar to that of SOD1. In the presence of HCO₃^-, a DEPMPO-OH adduct was formed when SOD3 was exposed to H₂O₂, as has been shown previously for SOD1. In physiologically relevant concentrations, uric acid prevented this inactivation of SOD1 and SOD3, whereas other small molecules were much less effective, or they affected SOD activities only in supraphysiological concentrations. Importantly, we found that increased levels of uric acid in vivo rather dramatically increased the activity of SOD1 and SOD3 in the aortas of ApoE^-/- mice but not in those of control mice. These findings provide the first evidence that peroxidase properties of SOD1 and SOD3 modulate the activity of these enzymes in vivo and provide a new mechanism whereby H₂O₂, by inactivating SOD, can worsen oxidative stress in the vessel wall.

Figure 6 illustrates the peroxidase activity of SOD3 as demonstrated in the present studies. Inactivation of SOD3 by H₂O₂ likely involves the formation of either SOD-Cu-^·OH or another similar species in a fashion similar to that reported for SOD1.¹⁹ Using the spin trap DEPMPO, we could readily observe the formation of a DEPMPO-OH adduct when SOD3 was exposed to H₂O₂. As has been reported for SOD1, formation of this adduct occurred only in the presence of the HCO₃^- anion. Several studies have shown that the HCO₃^- anion greatly enhances the peroxidase activity of SOD1.^4–6,16 Although there is some debate about the mechanisms involved, recent data support the concept that HCO₃^-, because of its negative charge and small size, readily enters the active site of SOD1, where it is oxidized to the bicarbonate radical (CO₃^·-) by Cu²⁺-·OH, which was previously formed by the reaction of H₂O₂ with SOD1.^4,5 The bicarbonate radical can readily diffuse out of the active site of SOD1 to react with other small molecules. In the case of nitron spin traps, such as 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and DEPMPO, the reaction with CO₃^·leads to formation of their respective nitron radical cations, which then undergo hydrolysis to form DMPO-OH and DEPMPO-OH.⁴ Our current findings suggest that the HCO₃^- anion and its radical have a similar role in the peroxidase activity of SOD3.

View larger version (46K): [in this window] [in a new window]   Figure 6. Peroxidase activity of SOD 1 and SOD3. The active SOD3 reacts with H2O2 to form an intermediate (SOD3-OH radical) that can interact with the bicarbonate to ultimately lead to the formation of a DEPMPO-OH adduct. In the absence of a cosubstrate, SOD3 (SOD1) is inactivated by H2O2. In the presence of physiological levels of uric acid, however, SOD3 (SOD1) inactivation is prevented. The urate radical formed has low oxidative potential and may react with ascorbic acid to regenerate uric acid.

In the original characterization of SOD3, Marklund⁹ showed that this enzyme was inactivated by H₂O₂. Our present study extends these observations by showing that this involves a peroxidase-like activity, with formation of a hydroxyl-like radical. In addition, a major goal of the present study was to determine which small molecules were effective in preventing H₂O₂ inactivation of SOD3. Although it is impossible to study all potential reductants, we focused on molecules previously reported to prevent the inactivation of SOD1 and, in particular, those present in vivo. Of note, in the present study, the HCO₃^- anion did not prevent H₂O₂ inactivation of either enzyme. This result is in keeping with the recent finding of Liochev and Fridovich,¹⁶ who showed that HCO₃^- had no effect on the inactivation of SOD1. Likewise, Gross et al⁵ have recently shown that HCO₃^- minimally protects against the inactivation of SOD1 by H₂O₂. Several other potential substrates, including formate, glutamate, tyrosine, lactate, -tocopherol, histidine, arginine, and uracil, in concentrations exceeding those encountered in vivo, had minimal impact on the inactivation of either enzyme by H₂O₂. Of note, as reported previously, nitrite (1 mmol/L) prevented the inactivation of SOD1. This concentration of nitrite only partially prevented the inactivation of SOD3 by H₂O₂. It should be noted that the plasma concentrations of nitrite are markedly lower than this, ranging from 4.2 to 6.1 µmol/L.²⁰

Further experiments demonstrated that uric acid, in concentrations present in human plasma, was able to almost completely prevent the inactivation of SOD3 by H₂O₂ in a fashion similar to that demonstrated for SOD1 by Hodgson and Fridovich.¹ Importantly, the resulting urate radical likely formed in this process is relatively stable (redox potential 0.59 V at pH 7) and does not react with oxygen,²¹ a property that contributes to the radical chain-breaking potential of urate. In human plasma, the urate radical can react readily with the weaker oxidant ascorbate, thereby regenerating urate and producing the even more innocuous ascorbate radical (Asc^·) with a redox potential of only 0.28 V.²² Because normal human plasma concentrations of urate (125 to 500 µmol/L) are 60-fold higher than those of nitrite,²⁰ the most likely product of the peroxidase reaction catalyzed by SOD3 in vivo is the relatively unreactive urate radical, which will be regenerated to urate by ascorbic acid. These findings indicate a new and potentially important antioxidant role of urate, aside from its known ability to react with potent oxidants such as peroxynitrite.²³

In a previous study,¹⁴ we found that the protein expression of SOD3 is increased by as much as 3-fold in the aortas of ApoE^-/- mice, seemingly from lipid-laden macrophages. In contrast to this 3-fold increase in protein expression, the activity of SOD3 was increased by only 2-fold. Our present findings provide some insight into this apparent discrepancy and suggest that SOD1 and SOD3 are partially inactivated in the aortas of ApoE^-/- mice. By infusing oxonic acid, an inhibitor of uricase, we were able to increase uric acid levels by 3-fold. In ApoE^-/- mice, this increase in uric acid rather dramatically increased the vascular activity of SOD1 and SOD3, while having no effect on the amounts of protein of either of these enzymes as determined by Western analysis. Importantly, oxonic acid infusion had no effect on SOD1 or SOD3 activity in aortas from control mice, although it increased the plasma levels of uric acid to the same extent in control mice and in ApoE^-/- mice. These findings suggest that in the presence of a pro-oxidant state such as atherosclerosis, these enzymes are partially inactivated. This phenomenon may contribute to the increased levels of O₂^·- observed in atherosclerotic vessels, because inactivation of these superoxide dismutases would reduce clearance of this radical.

Of interest, oxonic acid infusion in ApoE^-/- mice caused a rather dramatic increase in SOD1 activity above that observed in control mice, without a change in SOD1 protein expression (Figure 5). The mechanisms responsible for this augmentation in SOD1 activity remain undefined but clearly reflect a posttranslational change in enzyme function. Recently, SOD1 activity in myeloid cells has been shown to be inhibited by phosphorylation in response to granulocyte colony-stimulating factor.²⁴ Whether phosphorylation of SOD1 is occurring in the setting of atherosclerosis remains unclear.

Uric acid is a potent scavenger of peroxynitrite; however, it is unlikely that its effect on SOD 1 and SOD3 activities observed in the present study was due to the scavenging of peroxynitrite by uric acid. Previous studies from other laboratories²⁵ and our preliminary studies indicate that peroxynitrite does not alter the activity of either SOD1 or SOD3.

The role of uric acid in cardiovascular disease remains equivocal. Although some epidemiological studies have shown an independent association between elevated serum levels of uric acid and increased cardiovascular disease,^26–28 others have suggested that this association is confounded by the coexistence of conditions such as hypertension, obesity, hyperlipidemia, and diabetes mellitus, all known to be independent risk factors for atherosclerosis.^29–31 Interestingly, in most mammals, uric acid is further metabolized to allantoin by the enzyme uricase. In humans and higher primates, however, uric acid is the final product of purine metabolism.³² It has been hypothesized that uric acid may be an evolutionary antioxidant substitute for the loss of the ability to synthesize ascorbate in higher primates.³³ Indeed, in vitro experiments have demonstrated that uric acid is an effective scavenger of free radicals³³ and can chelate transition metal ions.³⁴ Paradoxically, uric acid has also been reported to increase oxidative damage,³⁵ to enhance platelet adhesiveness,³⁶ and to stimulate vascular smooth muscle cell growth.³⁷ Hence, the overall functional importance of urate in vivo remains unknown. Our present data suggest that one potential beneficial effect of uric acid may relate to its ability to prevent the inactivation of SOD1 and SOD3 by H₂O₂. In humans, the levels of uric acid normally encountered in the Western countries are generally high enough to prevent H₂O₂ inactivation of either SOD1 or SOD3. Of note, levels of uric acid may be decreased in Fanconi’s syndrome,³⁸ in the acquired immunodeficiency syndrome,³⁹ during space flight,⁴⁰ in type II diabetes, ⁴¹ and by allopurinol treatment. In these conditions, it is conceivable that H₂O₂ inactivation of SOD1 and SOD3 may occur and contribute to oxidant stress.

Because other H₂O₂-metabolizing enzymes, such as glutathione peroxidase and catalase, are located intracellularly, it is possible that SOD3 has a predominant role in the disposal of H₂O₂ in the extracellular space. This may have particular importance in vascular homeostasis. We have previously shown that vascular expression of SOD3 seems to be highly regulated in pathophysiological and physiological conditions, including atherosclerosis,^14,42 angiotensin II-induced hypertension,¹³ and exercise training.¹⁵ It is interesting to speculate that under these conditions, increased SOD3 expression not only reduces interstitial O₂^·- but also minimizes interstitial H₂O₂ levels.

In summary, these studies demonstrate that SOD3 has peroxidase properties that are similar to those of SOD1. We further show that physiological levels of uric acid importantly modulate the activity of SOD1 and SOD3 by preventing the inactivation of these enzymes by H₂O₂. In the setting of increased vascular oxidant stress, as encountered in atherosclerotic vessels, SOD1 and SOD3 are partially inactivated, and this can be prevented by increasing the circulating levels of uric acid within the physiological range. These data show that the inactivation of SOD by H₂O₂ may represent a new mechanism contributing to oxidant stress in vivo, and they also show an important role of uric acid in modulating this phenomenon.

	Acknowledgments

 
This research was supported by National Institutes of Health grants RO-1 HL-39006, RO-1 HL-59248, RO1 HL-70187, and Program Project Grant HL-5800; by a Veterans Administration Merit Grant; by Deutsche Forschungs Gemeinschaft HI 712/1-1; and by an American Heart Association National Scientist Development Grant (No. 0030180N).

Received April 4, 2002; accepted June 11, 2002.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Hodgson EK, Fridovich I. The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide: inactivation of the enzyme. Biochemistry. 1975; 14: 5294–5299.[CrossRef][Medline] [Order article via Infotrieve]

2. Jewett SL, Rocklin AM, Ghanevati M, Abel JM, Marach JA. A new look at a time-worn system: oxidation of CuZn-SOD by H2O2. Free Radic Biol Med. 1999; 26: 905–918.[CrossRef][Medline] [Order article via Infotrieve]

3. Singh RJ, Goss SP, Joseph J, Kalyanaraman B. Nitration of gamma-tocopherol and oxidation of alpha-tocopherol by copper-zinc superoxide dismutase/H₂O₂/NO₂-: role of nitrogen dioxide free radical. Proc Natl Acad Sci U S A. 1998; 95: 12912–12917.[Abstract/Free Full Text]

4. Zhang H, Joseph J, Felix C, Kalyanaraman B. Bicarbonate enhances the hydroxylation, nitration, and peroxidation reactions catalyzed by copper, zinc superoxide dismutase: intermediacy of carbonate anion radical. J Biol Chem. 2000; 275: 14038–14045.[Abstract/Free Full Text]

5. Goss SP, Singh RJ, Kalyanaraman B. Bicarbonate enhances the peroxidase activity of Cu,Zn-superoxide dismutase: role of carbonate anion radical. J Biol Chem. 1999; 274: 28233–28239.[Abstract/Free Full Text]

6. Sankarapandi S, Zweier JL. Bicarbonate is required for the peroxidase function of Cu,Zn-superoxide dismutase at physiological pH. J Biol Chem. 1999; 274: 1226–1232.[Abstract/Free Full Text]

7. Hjalmarsson K, Marklund SL, Engstrom A, Edlund T. Isolation and sequence of complementary DNA encoding human extracellular superoxide dismutase. Proc Natl Acad Sci U S A. 1987; 84: 6340–6344.[Abstract/Free Full Text]

8. Tainer JA, Getzoff ED, Richardson JS, Richardson DC. Structure and mechanism of copper, zinc superoxide dismutase. Nature. 1983; 306: 284–287.[CrossRef][Medline] [Order article via Infotrieve]

9. Marklund SL. Properties of extracellular superoxide dismutase from human lung. Biochem J. 1984; 220: 269–272.[Medline] [Order article via Infotrieve]

10. Strålin P, Karlsson K, Johansson BO, Markland SL. The interstitium of the human arterial wall contains very large amounts of extracellular superoxide dismutase. Arterioscler Thromb Vasc Biol. 1995; 15: 2032–2036.[Abstract/Free Full Text]

11. Crapo JD, McCord JM, Fridovich I. Preparation and assay of superoxide dismutases. Methods Enzymol. 1978; 53: 382–393.[Medline] [Order article via Infotrieve]

12. Tibell L, Hjalmarsson K, Edlund T, Skogman G, Engstrom A, Marklund SL. Expression of human extracellular superoxide dismutase in Chinese hamster ovary cells and characterization of the product. Proc Natl Acad Sci U S A. 1987; 84: 6634–6638.[Abstract/Free Full Text]

13. Fukai T, Siegfried MR, Ushio-Fukai M, Griendling KK, Harrison DG. Modulation of extracellular superoxide dismutase expression by angiotensin II and hypertension. Circ Res. 1999; 85: 23–28.[Abstract/Free Full Text]

14. Fukai T, Galis ZS, Meng XP, Parthasarathy S, Harrison DG. Vascular expression of extracellular superoxide dismutase in atherosclerosis. J Clin Invest. 1998; 101: 2101–2111.[Medline] [Order article via Infotrieve]

15. Fukai T, Siegfried MR, Ushio-Fukai M, Cheng Y, Kojda G, Harrison DG. Regulation of the vascular extracellular superoxide dismutase by nitric oxide and exercise training. J Clin Invest. 2000; 105: 1631–1639.[Medline] [Order article via Infotrieve]

16. Liochev SI, Fridovich I. On the role of bicarbonate in peroxidations catalyzed by Cu,Zn superoxide dismutase. Free Radic Biol Med. 1999; 27: 1444–1447.[CrossRef][Medline] [Order article via Infotrieve]

17. Laursen JB, Somers M, Kurz S, McCann L, Warnholtz A, Freeman BA, Tarpey M, Fukai T, Harrison DG. Endothelial regulation of vasomotion in apoE-deficient mice: implications for interactions between peroxynitrite and tetrahydrobiopterin. Circulation. 2001; 103: 1282–1288.[Abstract/Free Full Text]

18. Fridovich I. The competitive inhibition of uricase by oxonate and related derivatives of s-triazines. J Biol Chem. 1965; 240: 2491–2494.[Free Full Text]

19. Yim MB, Chock PB, Stadtman ER. Copper, zinc superoxide dismutase catalyzes hydroxyl radical production from hydrogen peroxide. Proc Natl Acad Sci U S A. 1990; 87: 5006–5010.[Abstract/Free Full Text]

20. Moshage H, Kok B, Huizenga JR, Jansen PL. Nitrite and nitrate determinations in plasma: a critical evaluation. Clin Chem. 1995; 41: 892–896.[Abstract/Free Full Text]

21. Simic MG, Jovanovic SV. Antioxidation mechanisms of uric acid. J Am Chem Soc. 1989; 111: 5778–5782.[CrossRef]

22. Maples KR, Mason RP. Free radical metabolite of uric acid. J Biol Chem. 1988; 263: 1709–1712.[Abstract/Free Full Text]

23. Whiteman M, Halliwell B. Protection against peroxynitrite-dependent tyrosine nitration and alpha 1-antiproteinase inactivation by ascorbic acid: a comparison with other biological antioxidants. Free Radic Res. 1996; 25: 275–283.[Medline] [Order article via Infotrieve]

24. Csar XF, Wilson NJ, Strike P, Sparrow L, McMahon KA, Ward AC, Hamilton JA. Copper/zinc superoxide dismutase is phosphorylated and modulated specifically by granulocyte-colony stimulating factor in myeloid cells. Proteomics. 2001; 1: 435–443.[CrossRef][Medline] [Order article via Infotrieve]

25. Ischiropoulos H, Zhu L, Chen J, Tsai M, Martin JC, Smith CD, Beckman JS. Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase. Arch Biochem Biophys. 1992; 298: 431–437.[CrossRef][Medline] [Order article via Infotrieve]

26. Freedman DS, Williamson DF, Gunter EW, Byers T. Relation of serum uric acid to mortality and ischemic heart disease: the NHANES I Epidemiologic Follow-Up Study. Am J Epidemiol. 1995; 141: 637–644.[Abstract/Free Full Text]

27. Liese AD, Hense HW, Lowel H, Doring A, Tietze M, Keil U. Association of serum uric acid with all-cause and cardiovascular disease mortality and incident myocardial infarction in the MONICA Augsburg cohort: World Health Organization Monitoring Trends and Determinants in Cardiovascular Diseases. Epidemiology. 1999; 10: 391–397.[CrossRef][Medline] [Order article via Infotrieve]

28. Alderman MH, Cohen H, Madhavan S, Kivlighn S. Serum uric acid and cardiovascular events in successfully treated hypertensive patients. Hypertension. 1999; 34: 144–150.[Abstract/Free Full Text]

29. Wannamethee SG, Shaper AG, Whincup PH. Serum urate and the risk of major coronary heart disease events. Heart. 1997; 78: 147–153.[Abstract/Free Full Text]

30. Culleton BF, Larson MG, Kannel WB, Levy D. Serum uric acid and risk for cardiovascular disease and death: the Framingham Heart Study. Ann Intern Med. 1999; 131: 7–13.[Abstract/Free Full Text]

31. Moriarity JT, Folsom AR, Iribarren C, Nieto FJ, Rosamond WD. Serum uric acid and risk of coronary heart disease: Atherosclerosis Risk in Communities (ARIC) Study. Ann Epidemiol. 2000; 10: 136–143.[CrossRef][Medline] [Order article via Infotrieve]

32. Wu XW, Lee CC, Muzny DM, Caskey CT. Urate oxidase: primary structure and evolutionary implications. Proc Natl Acad Sci U S A. 1989; 86: 9412–9416.[Abstract/Free Full Text]

33. Ames BN, Cathcart R, Schwiers E, Hochstein P. Uric acid provides an antioxidant defense in humans against oxidant- and radical-caused aging and cancer: a hypothesis. Proc Natl Acad Sci U S A. 1981; 78: 6858–6862.[Abstract/Free Full Text]

34. Davies KJ, Sevanian A, Muakkassah-Kelly SF, Hochstein P. Uric acid-iron ion complexes: a new aspect of the antioxidant functions of uric acid. Biochem J. 1986; 235: 747–754.[Medline] [Order article via Infotrieve]

35. Vasquez-Vivar J, Santos AM, Junqueira VB, Augusto O. Peroxynitrite-mediated formation of free radicals in human plasma: EPR detection of ascorbyl, albumin-thiyl and uric acid-derived free radicals. Biochem J. 1996; 314: 869–876.

36. Ginsberg MH, Kozin F, O’Malley M, McCarty DJ. Release of platelet constituents by monosodium urate crystals. J Clin Invest. 1977; 60: 999–1007.

37. Rao GN, Corson MA, Berk BC. Uric acid stimulates vascular smooth muscle cell proliferation by increasing platelet-derived growth factor A-chain expression. J Biol Chem. 1991; 266: 8604–8608.[Abstract/Free Full Text]

38. Gaspar GA, Puig JG, Mateos FA, Oria CR, Gomez ME, Gil AA. Hypouricemia due to renal urate wasting: different types of tubular transport defects. Adv Exp Med Biol. 1986; 195: 357–363.

39. Maesaka JK, Cusano AJ, Thies HL, Siegal FP, Dreisbach AW. Hypouricemia in acquired immunodeficiency syndrome. Am J Kidney Dis. 1990; 15: 252–257.[Medline] [Order article via Infotrieve]

40. Leach CS. Biochemical and hematologic changes after short-term space flight. Microgravity Q. 1992; 2: 69–75.[Medline] [Order article via Infotrieve]

41. Gonzalez-Sicilia L, Garcia-Estan J, Martinez-Blazquez A, Fernandez-Pardo J, Quiles JL, Hernandez J. Renal metabolism of uric acid in type I insulin-dependent diabetic patients: relation to metabolic compensation. Horm Metab Res. 1997; 29: 520–523.[Medline] [Order article via Infotrieve]

42. Luoma JS, Stralin P, Marklund SL, Hiltunen TP, Sarkioja T, Yla-Herttuala S. Expression of extracellular SOD and iNOS in macrophages and smooth muscle cells in human and rabbit atherosclerotic lesions: colocalization with epitopes characteristic of oxidized LDL and peroxynitrite-modified proteins. Arterioscler Thromb Vasc Biol. 1998; 18: 157–167.[Abstract/Free Full Text]

This article has been cited by other articles:

				D. W. Trott, F. Gunduz, M. H. Laughlin, and C. R. Woodman Exercise training reverses age-related decrements in endothelium-dependent dilation in skeletal muscle feed arteries J Appl Physiol, June 1, 2009; 106(6): 1925 - 1934. [Abstract] [Full Text] [PDF]

				H.-Y. Zhang, J.-C. Lu, Y.-F. Huang, and N.-Q. Lu Standardization and Quality Control for the Determination of Uric Acid Level in Seminal Plasma Lab Med, January 1, 2009; 40(1): 23 - 26. [Abstract] [Full Text] [PDF]

				M. C. Gongora, H. E. Lob, U. Landmesser, T. J. Guzik, W. D. Martin, K. Ozumi, S. M. Wall, D. S. Wilson, N. Murthy, M. Gravanis, et al. Loss of Extracellular Superoxide Dismutase Leads to Acute Lung Damage in the Presence of Ambient Air: A Potential Mechanism Underlying Adult Respiratory Distress Syndrome Am. J. Pathol., October 1, 2008; 173(4): 915 - 926. [Abstract] [Full Text] [PDF]

				H. Alcaino, D. Greig, M. Chiong, H. Verdejo, R. Miranda, R. Concepcion, J. L. Vukasovic, G. Diaz-Araya, R. Mellado, L. Garcia, et al. Serum uric acid correlates with extracellular superoxide dismutase activity in patients with chronic heart failure Eur J Heart Fail, July 1, 2008; 10(7): 646 - 651. [Abstract] [Full Text] [PDF]

				J. F. Baker, H. R. Schumacher, and E. Krishnan Serum Uric Acid Level and Risk for Peripheral Arterial Disease: Analysis of Data From the Multiple Risk Factor Intervention Trial Angiology, September 1, 2007; 58(4): 450 - 457. [Abstract] [PDF]

				D. Patschan, S. Patschan, G. G. Gobe, S. Chintala, and M. S. Goligorsky Uric Acid Heralds Ischemic Tissue Injury to Mobilize Endothelial Progenitor Cells J. Am. Soc. Nephrol., May 1, 2007; 18(5): 1516 - 1524. [Abstract] [Full Text] [PDF]

				T. Fukai Extracellular SOD Inactivation in High-Volume Hypertension: Role of Hydrogen Peroxide Arterioscler Thromb Vasc Biol, March 1, 2007; 27(3): 442 - 444. [Full Text] [PDF]

				O. Jung, S. L. Marklund, N. Xia, R. Busse, and R. P. Brandes Inactivation of Extracellular Superoxide Dismutase Contributes to the Development of High-Volume Hypertension Arterioscler Thromb Vasc Biol, March 1, 2007; 27(3): 470 - 477. [Abstract] [Full Text] [PDF]

				A. A. Ejaz, W. Mu, D.-H. Kang, C. Roncal, Y. Y. Sautin, G. Henderson, I. Tabah-Fisch, B. Keller, T. M. Beaver, T. Nakagawa, et al. Could Uric Acid Have a Role in Acute Renal Failure? Clin. J. Am. Soc. Nephrol., January 1, 2007; 2(1): 16 - 21. [Abstract] [Full Text] [PDF]

				M. C. Gongora, Z. Qin, K. Laude, H. W. Kim, L. McCann, J. R. Folz, S. Dikalov, T. Fukai, and D. G. Harrison Role of Extracellular Superoxide Dismutase in Hypertension Hypertension, September 1, 2006; 48(3): 473 - 481. [Abstract] [Full Text] [PDF]

				D. D. Heistad Oxidative Stress and Vascular Disease: 2005 Duff Lecture Arterioscler Thromb Vasc Biol, April 1, 2006; 26(4): 689 - 695. [Abstract] [Full Text] [PDF]

				V. Jeney, S. Itoh, M. Wendt, Q. Gradek, M. Ushio-Fukai, D. G. Harrison, and T. Fukai Role of Antioxidant-1 in Extracellular Superoxide Dismutase Function and Expression Circ. Res., April 15, 2005; 96(7): 723 - 729. [Abstract] [Full Text] [PDF]

				S. P. Wilkinson and A. Grove HucR, a Novel Uric Acid-responsive Member of the MarR Family of Transcriptional Regulators from Deinococcus radiodurans J. Biol. Chem., December 3, 2004; 279(49): 51442 - 51450. [Abstract] [Full Text] [PDF]

				T. Ohtsubo, I. I. Rovira, M. F. Starost, C. Liu, and T. Finkel Xanthine Oxidoreductase Is an Endogenous Regulator of Cyclooxygenase-2 Circ. Res., November 26, 2004; 95(11): 1118 - 1124. [Abstract] [Full Text] [PDF]

				R. Stocker and J. F. Keaney Jr. Role of Oxidative Modifications in Atherosclerosis Physiol Rev, October 1, 2004; 84(4): 1381 - 1478. [Abstract] [Full Text] [PDF]

				D. Seo, T. Wang, H. Dressman, E. E. Herderick, E. S. Iversen, C. Dong, K. Vata, C. A. Milano, F. Rigat, J. Pittman, et al. Gene Expression Phenotypes of Atherosclerosis Arterioscler Thromb Vasc Biol, October 1, 2004; 24(10): 1922 - 1927. [Abstract] [Full Text] [PDF]

				P. C. Schulze, J. Yoshioka, T. Takahashi, Z. He, G. L. King, and R. T. Lee Hyperglycemia Promotes Oxidative Stress through Inhibition of Thioredoxin Function by Thioredoxin-interacting Protein J. Biol. Chem., July 16, 2004; 279(29): 30369 - 30374. [Abstract] [Full Text] [PDF]

				H. B. Suliman, M. Ali, and C. A. Piantadosi Superoxide dismutase-3 promotes full expression of the EPO response to hypoxia Blood, July 1, 2004; 104(1): 43 - 50. [Abstract] [Full Text] [PDF]

				S.-P. Hsu, M.-F. Pai, Y.-S. Peng, C.-K. Chiang, T.-I Ho, and K.-Y. Hung Serum uric acid levels show a 'J-shaped' association with all-cause mortality in haemodialysis patients Nephrol. Dial. Transplant., February 1, 2004; 19(2): 457 - 462. [Abstract] [Full Text] [PDF]

				P. F. Leite, A. Danilovic, P. Moriel, K. Dantas, S. Marklund, A. P. V. Dantas, and F. R.M. Laurindo Sustained Decrease in Superoxide Dismutase Activity Underlies Constrictive Remodeling After Balloon Injury in Rabbits Arterioscler Thromb Vasc Biol, December 1, 2003; 23(12): 2197 - 2202. [Abstract] [Full Text] [PDF]

				J. Kanellis and R. J. Johnson Editorial Comment--Elevated Uric Acid and Ischemic Stroke: Accumulating Evidence That It Is Injurious and Not Neuroprotective Stroke, August 1, 2003; 34(8): 1956 - 1957. [Full Text] [PDF]

				H. Zhang, C. Andrekopoulos, J. Joseph, K. Chandran, H. Karoui, J. P. Crow, and B. Kalyanaraman Bicarbonate-dependent Peroxidase Activity of Human Cu,Zn-Superoxide Dismutase Induces Covalent Aggregation of Protein: INTERMEDIACY OF TRYPTOPHAN-DERIVED OXIDATION PRODUCTS J. Biol. Chem., June 20, 2003; 278(26): 24078 - 24089. [Abstract] [Full Text] [PDF]

				R. J. Johnson, D.-H. Kang, D. Feig, S. Kivlighn, J. Kanellis, S. Watanabe, K. R. Tuttle, B. Rodriguez-Iturbe, J. Herrera-Acosta, and M. Mazzali Is There a Pathogenetic Role for Uric Acid in Hypertension and Cardiovascular and Renal Disease? Hypertension, June 1, 2003; 41(6): 1183 - 1190. [Abstract] [Full Text] [PDF]

				J. M. Hare and R. J. Johnson Uric Acid Predicts Clinical Outcomes in Heart Failure: Insights Regarding the Role of Xanthine Oxidase and Uric Acid in Disease Pathophysiology Circulation, April 22, 2003; 107(15): 1951 - 1953. [Full Text] [PDF]

				U. Landmesser and H. Drexler Toward Understanding of Extracellular Superoxide Dismutase Regulation in Atherosclerosis: A Novel Role of Uric Acid? Arterioscler Thromb Vasc Biol, September 1, 2002; 22(9): 1367 - 1368. [Full Text] [PDF]

				H.U. HINK and T. FUKAI Extracellular Superoxide Dismutase, Uric Acid, and Atherosclerosis Cold Spring Harb Symp Quant Biol, January 1, 2002; 67(0): 483 - 490. [Abstract] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 22/9/1402    most recent 01.ATV.0000027524.86752.02v1 Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hink, H. U. Articles by Fukai, T. Search for Related Content PubMed PubMed Citation Articles by Hink, H. U. Articles by Fukai, T. Related Collections Pathophysiology Oxidant stress Mechanism of atherosclerosis/growth factors