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Proc Natl Acad Sci U S A. Aug 10, 2010; 107(32): 14351–14356.
Published online Jul 26, 2010. doi:  10.1073/pnas.1001072107
PMCID: PMC2922538
Medical Sciences

Treating gout with pegloticase, a PEGylated urate oxidase, provides insight into the importance of uric acid as an antioxidant in vivo


A high plasma urate concentration (PUA), related to loss of urate oxidase in evolution, is postulated to protect humans from oxidative injury. This hypothesis has broad clinical relevance, but support rests largely on in vitro data and epidemiologic associations. Pegloticase therapy generates H2O2 while depleting urate, offering an in vivo test of the antioxidant hypothesis. We show that erythrocytes can efficiently eliminate H2O2 derived from urate oxidation to prevent cell injury in vitro; during therapy, disulfide-linked peroxiredoxin 2 dimer did not accumulate in red blood cells, indicating that their peroxidase capacity was not exceeded. To assess oxidative stress, we monitored F2-Isoprostanes (F2-IsoPs) and protein carbonyls (PC), products of arachidonic acid and protein oxidation, in plasma of 26 refractory gout patients receiving up to five infusions of pegloticase at 3-wk intervals. At baseline, PUA was markedly elevated in all patients, and plasma F2-IsoP concentration was elevated in most. Pegloticase infusion rapidly lowered mean PUA to ≤1 mg/dL in all patients, and PUA remained low in 16 of 21 patients who completed treatment. F2-IsoP levels did not correlate with PUA and did not increase during 15 wk of sustained urate depletion. There also was no significant change in the levels of plasma PC. Because refractory gout is associated with high oxidative stress in spite of high PUA, and profoundly depleting uric acid did not increase lipid or protein oxidation, we conclude that urate is not a major factor controlling oxidative stress in vivo.

Keywords: F2-Isoprostanes, hyperuricemia, hypouricemia, oxidative stress, Peroxiredoxin 2

Urate oxidase (uricase) converts uric acid to 5-hydroxy isourate and H2O2, leading to the formation of allantoin (1). Mutation of the uricase gene during human evolution eliminated this source of H2O2 and resulted in an average plasma urate concentration (PUA) of ~0.3 mM (5 mg/dL), 5- to >20-fold higher than in most other mammals (2, 3). Ames et al. (4) proposed that, by scavenging free radicals, a higher PUA protects humans from cancer and other life-shortening disorders. In support, they showed that urate inhibits lipid peroxidation by products of the reaction of H2O2 with hemoglobin. In plasma, ascorbate is much more effective than urate in scavenging peroxyl radicals (5), but because of its higher concentration, urate often is cited as the major water-soluble antioxidant in human plasma (6). The antioxidant hypothesis has broad clinical relevance; for example, it underlies speculation that low PUA is a cause of neurodegenerative diseases and that raising PUA may be an effective therapy for these disorders (7, 8).

Ascribing benefit to high PUA must contend with health risks associated with hyperuricemia, usually defined as PUA >7 mg/dL (0.42 mM), which is about the limit of sodium urate solubility. Chronic hyperuricemia leads to the deposition in tissues of monosodium urate (MSU) crystals, which are proinflammatory and cause gout, a form of arthritis affecting 3–5 million people in the United States (911). Both hyperuricemia itself and gout are strongly associated with conditions thought to result from or to cause oxidative stress, including obesity, hypertension, the metabolic syndrome, and cardiovascular disease (CVD) (1218).The role of urate in these conditions remains uncertain.

The relationship of uricase expression and PUA to oxidative stress is directly relevant to pegloticase, a PEGylated recombinant porcine uricase under investigation as an orphan drug for treating refractory gout when other therapies have failed to maintain PUA below 6 mg/dL (0.36 mM), the usual therapeutic target (19, 20). In such patients, i.v. pegloticase can lower PUA rapidly to <2 mg/dL (0.12 mM), considered the lower limit of normal. Markedly decreasing PUA can facilitate the dissolution of MSU deposits to control the debilitating effects of advanced gout (21, 22). However, because oxidative injury may exacerbate CVD and other gout-associated disorders, it is important to know whether inducing hypouricemia while simultaneously generating H2O2 with pegloticase increases oxidant stress.

We have addressed two questions relevant to this issue: During pegloticase therapy, (i) does H2O2 production exceed the physiologic capacity to eliminate H2O2, and (ii) does oxidative stress status increase? Regarding the first question, we consider the role of erythrocytes in preventing H2O2 from accumulating and causing injury. To address the second question, we have monitored plasma levels of F2-isoprostanes (F2-IsoPs) and protein carbonyls (PCs), biomarkers of lipid and protein oxidation (23, 24), during a phase II clinical trial of pegloticase for refractory gout. Because of the extreme range of PUA encountered in each patient, our findings offer valuable insight into the in vivo role of urate as an antioxidant.


The Role of Erythrocytes in Eliminating H2O2 Generated by Pegloticase.

Speculation that pegloticase therapy may increase H2O2 to toxic levels (25) implies a greater rate of production than elimination. Before clinical testing we considered this possibility unlikely for these reasons: (i) because of PEGylation, infused pegloticase would be restricted to plasma (20); (ii) H2O2 freely crosses cell membranes; and (iii) erythrocytes are known to have a prodigious capacity to decompose H2O2 generated within the vascular compartment (26, 27). We estimate that erythrocytes can eliminate H2O2 2–3 orders of magnitude faster than it can be produced by pegloticase therapy (Table 1).

Table 1.
H2O2 production and degradation during pegloticase therapy

The importance of this rate disparity is evident in studies performed with the CEM human T-cell leukemia line (Fig. 1), in which culture medium was supplemented with 0.3 or 0.5 mM urate and 40 mU/mL of pegloticase, close to the maximal plasma activity during therapy (20), and able to oxidize the urate in a few minutes. The combination of urate plus pegloticase, but neither alone, was highly toxic, decreasing CEM viability (Fig. 1A) and arresting growth (Fig. 1B). This cytotoxicity was prevented by adding either excess catalase (Fig. 1A), showing that the cytotoxicity was caused by H2O2, or 4 × 107/mL human RBC (Fig. 1B), ~1% of the RBC count in normal blood. Human RBC previously have been shown to protect murine leukemia cells from reactive oxygen species (ROS) generated by the combination of hypoxanthine and xanthine oxidase (26).

Fig. 1.
Catalase and erythrocytes protect CEM T-lymphoblastoid cells from H2O2 generated by urate plus pegloticase. CEM cell viability determined by trypan blue exclusion (A) and fold increase in CEM cell number (B) was followed during culture for 72 h in medium ...

Effect of Urate Oxidation on Peroxiredoxin 2 in Erythrocytes.

We have explored a potential way to assess exposure of RBC to excess H2O2 during pegloticase therapy. Peroxiredoxin 2 (Prx2), a homodimeric peroxidase with high reactivity for H2O2 (Km ~0.7 μM), is abundant in RBC and is thought to protect them from H2O2 generated within the vascular compartment (2830). During catalysis, an active Cys site on each Prx2 subunit forms a disulfide bond with another Cys on the opposing subunit, resulting in a dimer on nonreducing immunoblots. The disulfides then are reduced by thioredoxin to regenerate active enzyme. Normally, reduced Prx2 predominates, but the dimer accumulates when H2O2 concentration increases sufficiently (e.g., during oxidative stress).

Preliminary experiments confirmed that the state of Prx2 depends on both H2O2 and RBC concentration (28). Thus, exposing 5 × 106/mL RBC to 5 μM H2O2 caused the quantitative, but reversible, conversion of Prx2 to dimer (Fig. 2A). By contrast, under conditions mimicking the blood of a markedly hyperuricemic, moderately anemic patient, rapidly generating ~160-fold more H2O2 with pegloticase caused only a trace of Prx2 dimer to appear transiently in RBC (Fig. 2B). Over a 70-day period, we observed no Prx2 dimer in erythrocytes of a patient receiving pegloticase (Fig. 2C). In this patient PUA fell from 9.0 to 4.9 mg/dL within 2 h after the first infusion (the earliest posttreatment sample) and was <2 mg/dL thereafter. The absence of Prx2 dimer indicates that the capacity of RBC to eliminate H2O2 was never exceeded, consistent with the rate estimates in Table 1.

Fig. 2.
Effects of H2O2 and urate oxidation on Prx2 in erythrocytes. (A) Reversible effect of H2O2 on Prx2 in a dilute suspension of erythrocytes. RBC (5 × 106/mL in PBS, 5 mM glucose) were incubated at 37 °C with 5 μM H2O2 for 5 min; ...

Characteristics of Refractory Gout Patients and Response to Pegloticase.

The 26 patients studied had clinical features (Table 2) similar to those of refractory gout patients treated in other trials of pegloticase (19, 20, 31). Notably, 92% had tophaceous MSU deposits, indicating advanced disease, and the prevalence of hypertension, diabetes, and CVD was very high.

Table 2.
Patient characteristics

Plasma levels of urate and F2-IsoP are plotted in Fig. 3 A and B, respectively. At baseline (Fig. 3A, “pre”), all patients were markedly hyperuricemic, with a mean PUA of 10.8 ± 1.3 mg/dL (range 9.0–13.7 mg/dL). PUA fell rapidly in all patients after the first infusion of pegloticase, to a mean of 7 mg/dL within 2 h and to ≤1 mg/dL at 48 h and 168 h. The response to pegloticase subsequently diverged, as is evident from the two distinct clusters of PUA values measured after the last infusion (the red and blue symbols in the last two columns of Fig. 3A). The wide range of PUA measured at a follow-up visit reflects variability in the terminal clearance of pegloticase from plasma.

Fig. 3.
PUA (A) and F2-IsoP (B). In each panel, the data in the first four columns from the left were obtained from all 26 patients before (“pre”) and at the indicated times after the first infusion of pegloticase. Data in the last two columns ...

Among the 21 patients who received all five infusions, 16 had persistently low PUA, averaging 0.9 ± 0.5 mg/dL in samples obtained weekly over a 15-wk period from day 2 after the first dose of pegloticase through 3 wk after the last dose (the calculation is given in Materials and Methods). PUA is a measure of the soluble pool of uric acid, but tophaceous MSU deposits were depleted also, as illustrated in Fig. 4 for one of these patients. The remaining five patients who completed the trial had a transient response to pegloticase, with PUA returning to baseline levels by the second to fourth infusion and averaging 6.6 ± 2.1 mg/dL over this 15-wk period. At the follow-up visit 7 wk after the last dose of pegloticase, the “persistent” and “transient” responders had mean PUAs of 5.7 ± 3.0 mg/dL and 11.2 ± 1.7 mg/dL, respectively. As observed in previous trials, the early loss of PUA control was caused by accelerated clearance of pegloticase associated with the appearance of antibodies to PEG (20, 31).

Fig. 4.
Resolution of prominent digital tophi (s.c. MSU deposits) in a persistently hypouricemic patient. (A) Before treatment with pegloticase. (B) After five infusions of pegloticase, 8 mg every 3 wk.

Oxidative Stress Status and Relationship with PUA During Pegloticase Therapy.

F2-IsoPs are produced solely by free radical attack on cell membrane-associated arachidonic acid (32); their measurement by GC-MS has been validated as a reliable index of oxidative stress status (23, 33). The mean plasma concentration of F2-IsoPs at baseline (Fig. 3B, “pre”) was 69 ± 38 pg/mL (range, 30–182 pg/mL). In previous studies using the same GC-MS methodology, a level of 35 ± 6 pg/mL was found for normal human plasma (34), and a value >50 pg/mL was considered elevated in a study involving 396 nonsmoking healthy adults (35). Of the gout patients in the present study, 17 (65%) exceeded the normal mean by 2 SD, and 15 (58%) exceeded the normal mean by 3 SD; the baseline plasma F2-IsoP concentration in these 15 patients ranged from 53–182 pg/mL.

Consistent with the reported association between obesity and increased oxidative stress (35, 36), body mass index correlated with baseline plasma F2-IsoP concentration (R2 = 0.22, P = 0.016); interactions with neither age nor a diagnosis of CVD reached statistical significance. We did not collect information on ascorbate levels or smoking status, which also might have influenced plasma F2-IsoP levels (35, 37).

If urate is important in limiting lipid peroxidation in vivo, plasma F2-IsoP levels might be expected to correlate with PUA or to increase when urate is markedly depleted, particularly in patients under oxidative stress. However, there was no significant change in mean plasma F2-IsoP concentration during treatment with pegloticase (Fig. 3B). Regression analysis of the data from Fig. 3 indicated no relationship between overall plasma urate and F2-IsoP levels (R2 = 0.01; P = 0.21). We also analyzed the relative change from baseline (Fig. 5). At no sampling time was the mean change from baseline in F2-IsoP concentration statistically significant, although there was a slight downward trend. One week after the first infusion of pegloticase (Fig. 5, Left), the mean decline for all 26 patients was 7.5%. One week after the fifth infusion (Fig. 5, Right), the 16 still-hypouricemic patients showed a mean 12% decrease from their baseline F2-IsoP levels, whereas the five patients who had again become hyperuricemic showed a 13% increase. At follow-up, F2-IsoP concentration had returned to baseline in the “persistent responders” and was 28% above baseline in the hyperuricemic “transient responders,”

Fig. 5.
Relative change in plasma F2-IsoP concentration from baseline during pegloticase therapy. For each patient, the F2-IsoP concentration at each sampling point was expressed as a percent of baseline (“pre”) value; the mean percent of baseline ...

The level of carbonyl groups in plasma proteins has been used to assess oxidative stress in various disease states (24, 3840). We used a standardized ELISA kit to measure PC levels in plasma obtained before treatment, and 1 wk after the first and fifth doses of pegloticase (Table 3). There was no statistically significant difference in mean plasma PC levels at these times. At the last sampling time, mean PC levels were about 9% above baseline in the hypouricemic “persistent responders” and 20% above baseline in the hyperuricemic “transient responders,” suggesting that this modest increase was not related to urate depletion.

Table 3.
Concentration of protein carbonyls in plasma


Although urate contributes significantly to plasma radical-scavenging antioxidant capacity, the in vivo significance of this in vitro measurement is unclear. The association of low-normal serum urate levels with Parkinson's disease and multiple sclerosis has led to proposals that higher PUA protects neurons from toxic effects of peroxynitrite (7, 4143). However, urate concentration in brain is much lower than in plasma (7, 44, 45); urate is a poor scavenger of peroxynitrite (4648); and reactions of urate with free radicals have pro- as well as antioxidant consequences (46, 47, 49, 50). Moreover, significantly raising serum urate concentration for 2 years was of no benefit in patients with relapsing-remitting multiple sclerosis (51). A positive correlation between plasma urate and PC levels has suggested that urate is ineffective in controlling oxidative stress in some other clinical settings (39, 40). Although rare, inherited deficiencies of xanthine oxidase and the Urat1 transporter both cause lifelong hypouricemia, but neither has been associated with neurodegenerative diseases or other chronic illnesses linked to oxidative injury (52, 53).

Pegloticase therapy for refractory gout offers a unique advantage for assessing the antioxidant role of urate in vivo. PUA encountered in epidemiologic studies is limited at the low range, and the converse situation exists in experimental animals. Conventional therapy for gout rarely causes hypouricemia, and the uricase inhibitor oxonic acid increases PUA in rats only to the low-normal range for humans. Uricase knockout mice have high PUA, but, rather than conferring an advantage, this high PUA causes renal failure soon after birth (54, 55). By contrast, PUA ranged from >10 to <0.5 mg/dL in almost every patient in the present study. Hypouricemia was induced rapidly by pegloticase and in most cases was sustained, permitting evaluation of acute and chronic effects, with each patient serving as his or her own control. The production of H2O2 accompanying urate oxidation, along with comorbidities associated with gout, provides an oxidative challenge, increasing the ability to detect a meaningful loss of antioxidant protection caused by hypouricemia and urate depletion.

I.v. pegloticase can be viewed as ectopic uricase replacement, acting in plasma rather than in hepatic peroxisomes, where uricase is expressed in most mammals, and with RBC replacing the organelle as a rich source of peroxidases to eliminate H2O2 derived from urate oxidation. In vitro, erythrocytes at low density completely protected CEM lymphoblasts from H2O2 generated by concentrations of urate and pegloticase mimicking those at the start of therapy. The absence of Prx2 dimer in the RBC of a patient during treatment, including at 2 h after the first infusion when the soluble urate pool is being depleted and H2O2 production is maximal, confirms that erythrocytes have more than sufficient capacity to prevent H2O2 from achieving a level capable of causing cell injury.

As noted (Table 1), H2O2 production declines by about 10-fold beyond the initial 24–48 h of treatment, whereas hypouricemia is maintained as long as plasma uricase activity exceeds the rate of urate production. During this period, the impact of low PUA itself on oxidative stress status can be assessed. Three of our findings suggest that plasma urate provides little protection from oxidants in vivo. First, in spite of marked hyperuricemia, pretreatment plasma F2-IsoP concentrations were significantly elevated in most patients, indicating a high level of oxidative stress. Second, over the wide range of PUA observed during treatment with pegloticase, there was no correlation between PUA and plasma F2-IsoP concentrations. Third, inducing hypouricemia did not cause F2-IsoP levels to increase; on the contrary, maintaining PUA at <1 mg/dL for 15 wk was associated with a trend toward a decline in F2-IsoP levels from baseline.

To our knowledge, systemic oxidative stress status has not previously been examined in gout. However, it is plausible that chronic gout is at least partly responsible for the increased oxidative stress. At hyperuricemic levels, prooxidant effects of soluble urate may predominate. MSU crystals also may play a role by activating the NALP-3 inflammasome in phagocytic cells, resulting in IL-1β secretion, recruitment of neutrophils, and generation of inflammatory mediators, including ROS (56, 57). At early stages of gout, attacks of arthritis are isolated, but in refractory gout extensive MSU deposits cause chronic, widespread inflammation. Eliminating proinflammatory MSU deposits, as illustrated in Fig. 4 and as reported previously (58), might reduce systemic oxidative stress. This possibility deserves further investigation during longer periods of therapy, as does the possibility that this effect may reduce morbidity from gout-associated diseases.

In judging the antioxidant advantage resulting from loss of uricase, it is useful to consider the contribution of urate to the antioxidant capacity of whole blood, rather than plasma. Whereas plasma urate reacts stoichiometrically with oxidants, catalase, superoxide dismutase, glutathione peroxidase, and Prx2 in RBC react catalytically and far more rapidly, conferring an immense capacity to consume H2O2 generated by the auto-oxidation of hemoglobin, as well as H2O2 and other oxidants, including peroxynitrite, produced within the vasculature. For example, the apparent second-order rate constant for the reaction of urate with peroxynitrite is ~5 × 102 M−1·s−1 (46), compared with ≥107 M−1·s−1 for the reactions of Prx2 with H2O2 and peroxynitrite (30). The concentration of erythrocyte Prx2 in blood is ~240 μM, close to that of plasma urate and high enough to scavenge H2O2 noncatalytically (2830). As a means of protection from systemic oxidant damage, localizing high levels of peroxidases within RBC seems far superior to elevating PUA, and less hazardous. The effect of losing urate oxidase on human evolution will continue to be a subject of interest and speculation.

Materials and Methods


Mouse monoclonal anti-2-CysPrx (LF-MA0073) was obtained from Lab Frontier, and goat anti-mouse IgG, peroxidase conjugate was obtained from Calbiochem.

Cell Culture.

Mycoplasma-free CCRF-CEM human T-lymphoblastoid cells (American Type Culture Collection) were obtained from the Duke University Cell Culture Facility. They were maintained in RPMI 1640 medium (Invitrogen) with 2 mM L-glutamine and 10% dialyzed FBS. Cell counts were performed with a Cellometer Auto T4 (Nexcelom). When CEM cells were cocultivated with human erythrocytes, the latter were lysed before counting by dilution into 10 mM KHCO3, 150 mM NH4Cl, 0.1 mM EDTA, pH 8.0.

Concentrations of Urate, F2-IsoPs, and PCs in Plasma.

Fresh heparinized blood was placed on ice and centrifuged at 4 °C within 15 min. An aliquot of the plasma was acidified for measuring PUA by HPLC (20, 31). Additional aliquots of plasma were frozen at −80 °C until they were sent on dry ice to Vanderbilt University for measurement of F2-IsoPs by GC negative ion chemical ionization mass spectrometry (34). PCs in plasma were determined according to the manufacturer’s instructions using an enzyme immunoassay kit (Biocell PC Test; Biocell Corp, Ltd.). PUA is expressed as mg/dL (multiplying by 0.06 gives mmol/L); if urate was undetectable, a value of 0.25 mg/dL (i.e., half the lower quantifiable limit) was assigned. F2-IsoP levels are expressed as pg/mL and PC levels as nmol/mg protein.

In Vitro Effects of H2O2 and Urate Oxidation on Peroxiredoxin 2 in Erythrocytes.

Erythrocytes prepared from fresh heparinized blood were washed three times in PBS, then resuspended in PBS containing 5 mM glucose or in plasma, followed by incubation with H2O2, or urate plus pegloticase, as indicated in figure legends. After incubation, RBC were centrifuged at 4 °C and washed three times with ice-cold 100 mM NEM in PBS as described (28). The RBC then were resuspended in 0.1 mL of nonreducing SDS sample buffer (65.8 mM Tris-HCL pH 6.8, 10.5% glycerol, 2.1% SDS) containing 100 mM N-ethylmaleimide (NEM) and stored at −80 °C. For immunoblot analysis of Prx2, aliquots containing 10 μg protein (determined with BSA as standard) were electrophoresed on 12% SDS PAGE gels, transferred to a nitrocellulose membrane, blocked with 2% BSA, and probed with anti-2-CysPrx, followed by goat anti-mouse IgG, peroxidase conjugate. Prx2 bands were identified after incubation with diaminobenzidine and H2O2.

Erythrocyte Peroxiredoxin 2 During Treatment with Pegloticase.

Heparinized blood obtained from patients at various times during treatment with pegloticase (8 mg every 3 wk) was processed within 15 min at 4 °C. After plasma was removed, 10 μL of packed RBC were washed three times with freshly prepared ice-cold 100 mM NEM in PBS, essentially as described (28). The RBC pellets then were resuspended in 0.1 mL of nonreducing SDS sample buffer containing 100 mM NEM and were stored at −80 °C before immunoblot analysis of Prx2, as described above.

Patients and Clinical Trial.

Patients participated, with informed consent, in an open-label, phase II trial of i.v. pegloticase (provided by Savient Pharmaceuticals) for treatment of refractory gout (ClinicalTrials.gov ID NCT00111657). The Duke University Institutional Review Board approved the trial protocol and an addendum to analyze F2-IsoP in stored plasma samples. Participants were ≥18 y old, with symptomatic gout and PUA >7 mg/dL Use of other urate-lowering medications during the trial was prohibited. Except for inclusion of organ transplant recipients, exclusion criteria were similar to those in other trials of pegloticase (19, 20, 31).

The trial protocol called for five 8-mg infusions of pegloticase administered at 3-wk intervals. As prophylaxis against gout flares, patients received colchicine or a nonsteroidal anti-inflammatory drug (unless contraindicated). As prophylaxis against infusion reactions, patients received prednisone (20 mg) and fexofenadine (60 mg) orally the evening before each pegloticase infusion. Fexofenadine was repeated on the morning of the infusion, along with hydrocortisone (200 mg i.v.).

Analysis of the Relationship Between Plasma Urate and F2-IsoP Concentrations.

Stored samples suitable for analysis were available from 26 subjects, including 21 who had received all five infusions of pegloticase and five who had discontinued treatment after one to three infusions because of adverse events or loss of ability to maintain PUA below 6 mg/dL. PUA was measured before and 2 h after each pegloticase infusion and weekly until 21 days after the last dose; in addition, PUA was determined at 48 h after the first and fifth infusions and at a follow-up visit 7 wk after the last infusion. The average PUA between day 2 after infusion 1 and day 21 after infusion 5 was determined by dividing the area under the concentration curve for PUA by the number of days in the monitoring period (nominally 103).

To assess F2-IsoP levels at both extremes as well as intermediate values of PUA, a set of six frozen plasma samples was analyzed: samples 1–4 were, respectively, the baseline (pretreatment) and specimens obtained 2, 48, and 168 h after infusion during the first week of treatment; samples 5 and 6 were obtained 1 wk and 7 wk after the final (fifth) infusion of pegloticase. Samples 1–4 were available from all 26 patients; samples 5 and 6 were available from the 21 patients who completed the study. From four patients who received only two or three infusions, a fifth sample was obtained 1 or 2 wk after their last infusion, and in two cases, a sixth sample was obtained at a follow-up visit 4 or 11 wk after the last (second) infusion. The samples for F2-IsoP determination were coded and sent in two batches to Vanderbilt University on dry ice, without patient identifiers or information about PUA, number of infusions completed, or response to therapy. After the analysis was completed, the F2-IsoP and PUA data for individual samples were matched to assess the correlation between these parameters.


Except as indicated, data are expressed as mean ± SD. Statistical analyses were performed using JMP software, version 8.0 (SAS Institute). The overall correlation between plasma F2-IsoP and urate concentrations was assessed by linear regression. The changes from baseline in PUA and levels of plasma F2-IsoP and PC were assessed by paired t test and also by multivariate ANOVA for repeated measurements. Means for patient subgroups were compared using ANOVA. Nonparametric methods were tested also but did not result in any change in assessment of statistical significance. P < 0.05 was considered significant.


We thank Theresa Rosario-Jansen and Zebulon Horowitz of Savient Pharmaceuticals for their support and David Pisetsky for comments on the manuscript. This research was supported by US Food and Drug Administration Office of Orphan Product Development Grant RO1 FD 002537; National Institutes of Health Grant R37 GM42056; and Savient Pharmaceuticals, Inc. Pegloticase supplied by Savient Pharmaceuticals was used under Investigator IND no. 11274 held by J.S.S., and research was performed in the Clinical Research Unit at Duke University Medical Center with support from National Institutes of Health Grant M01-RR-30.


*This Direct Submission article had a prearranged editor.

Conflict of interest statement: M.S.H., J.S.S., and L.J.R. have acted as paid consultants to Savient Pharmaceuticals. Duke University, M.S.H. and S.J.K., and Mountain View Pharmaceuticals hold patent rights in pegloticase and its use, which have been licensed to Savient Pharmaceuticals. Duke University, M.S.H., and S.J.K. will receive royalties from sales if pegloticase receives Food and Drug Administration approval.


1. Kahn K, Tipton PA. Spectroscopic characterization of intermediates in the urate oxidase reaction. Biochemistry. 1998;37:11651–11659. [PubMed]
2. Wu XW, Muzny DM, Lee CC, Caskey CT. Two independent mutational events in the loss of urate oxidase during hominoid evolution. J Mol Evol. 1992;34:78–84. [PubMed]
3. Roch-Ramel F, Peters G. Urinary excretion of uric acid in nonhuman mammalian species. Handb Exp Pharmacol. 1978;51:211–255.
4. 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 USA. 1981;78:6858–6862. [PMC free article] [PubMed]
5. Frei B, England L, Ames BN. Ascorbate is an outstanding antioxidant in human blood plasma. Proc Natl Acad Sci USA. 1989;86:6377–6381. [PMC free article] [PubMed]
6. Ghiselli A, Serafini M, Natella F, Scaccini C. Total antioxidant capacity as a tool to assess redox status: Critical view and experimental data. Free Radic Biol Med. 2000;29:1106–1114. [PubMed]
7. Ascherio A, et al. Parkinson Study Group DATATOP Investigators. Urate as a predictor of the rate of clinical decline in Parkinson disease. Arch Neurol. 2009;66:1460–1468. [PMC free article] [PubMed]
8. Koch M, De Keyser J. Uric acid in multiple sclerosis. Neurol Res. 2006;28:316–319. [PubMed]
9. Becker MA. Hyperuricemia and gout. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. New York: McGraw-Hill; 2001. pp. 2513–2535.
10. Saag KG, Mikuls TR. Recent advances in the epidemiology of gout. Curr Rheumatol Rep. 2005;7:235–241. [PubMed]
11. Wallace KL, Riedel AA, Joseph-Ridge N, Wortmann R. Increasing prevalence of gout and hyperuricemia over 10 years among older adults in a managed care population. J Rheumatol. 2004;31:1582–1587. [PubMed]
12. Choi HK, Curhan G. Independent impact of gout on mortality and risk for coronary heart disease. Circulation. 2007;116:894–900. [PubMed]
13. Choi HK, Ford ES, Li C, Curhan G. Prevalence of the metabolic syndrome in patients with gout: The Third National Health and Nutrition Examination Survey. Arthritis Rheum. 2007;57:109–115. [PubMed]
14. Cirillo P, et al. Uric acid, the metabolic syndrome, and renal disease. J Am Soc Nephrol. 2006;17(12, Suppl 3):S165–S168. [PubMed]
15. Feig DI, Soletsky B, Johnson RJ. Effect of allopurinol on blood pressure of adolescents with newly diagnosed essential hypertension: A randomized trial. JAMA. 2008;300:924–932. [PMC free article] [PubMed]
16. Ioachimescu AG, Brennan DM, Hoar BM, Hazen SL, Hoogwerf BJ. Serum uric acid is an independent predictor of all-cause mortality in patients at high risk of cardiovascular disease: A preventive cardiology information system (PreCIS) database cohort study. Arthritis Rheum. 2008;58:623–630. [PubMed]
17. Krishnan E, Svendsen K, Neaton JD, Grandits G, Kuller LH. MRFIT Research Group. Long-term cardiovascular mortality among middle-aged men with gout. Arch Intern Med. 2008;168:1104–1110. [PubMed]
18. Strasak A, et al. VHM&PP Study Group. Serum uric acid and risk of cardiovascular mortality: A prospective long-term study of 83,683 Austrian men. Clin Chem. 2008;54:273–284. [PubMed]
19. Sundy JS, et al. Pegloticase Phase 2 Study Investigators. Reduction of plasma urate levels following treatment with multiple doses of pegloticase (polyethylene glycol-conjugated uricase) in patients with treatment-failure gout: Results of a phase II randomized study. Arthritis Rheum. 2008;58:2882–2891. [PubMed]
20. Sundy JS, et al. Pharmacokinetics and pharmacodynamics of intravenous PEGylated recombinant mammalian urate oxidase in patients with refractory gout. Arthritis Rheum. 2007;56:1021–1028. [PubMed]
21. Hershfield MS. Reassessing serum urate targets in the management of refractory gout: Can you go too low? Curr Opin Rheumatol. 2009;21:138–142. [PMC free article] [PubMed]
22. Perez-Ruiz F, Lioté F. Lowering serum uric acid levels: What is the optimal target for improving clinical outcomes in gout? Arthritis Rheum. 2007;57:1324–1328. [PubMed]
23. Basu S. F2-isoprostanes in human health and diseases: From molecular mechanisms to clinical implications. Antioxid Redox Signal. 2008;10:1405–1434. [PubMed]
24. Levine RL. Carbonyl modified proteins in cellular regulation, aging, and disease. Free Radic Biol Med. 2002;32:790–796. [PubMed]
25. Terkeltaub R. Gout. Novel therapies for treatment of gout and hyperuricemia. Arthritis Res Ther. 2009;11:236. [PMC free article] [PubMed]
26. Agar NS, Sadrzadeh SMH, Hallaway PE, Eaton JW. Erythrocyte catalase. A somatic oxidant defense? J Clin Invest. 1986;77:319–321. [PMC free article] [PubMed]
27. Winterbourn CC, Stern A. Human red cells scavenge extracellular hydrogen peroxide and inhibit formation of hypochlorous acid and hydroxyl radical. J Clin Invest. 1987;80:1486–1491. [PMC free article] [PubMed]
28. Low FM, Hampton MB, Peskin AV, Winterbourn CC. Peroxiredoxin 2 functions as a noncatalytic scavenger of low-level hydrogen peroxide in the erythrocyte. Blood. 2007;109:2611–2617. [PubMed]
29. Low FM, Hampton MB, Winterbourn CC. Peroxiredoxin 2 and peroxide metabolism in the erythrocyte. Antioxid Redox Signal. 2008;10:1621–1630. [PubMed]
30. Manta B, et al. The peroxidase and peroxynitrite reductase activity of human erythrocyte peroxiredoxin 2. Arch Biochem Biophys. 2009;484:146–154. [PubMed]
31. Ganson NJ, Kelly SJ, Scarlett E, Sundy JS, Hershfield MS. Control of hyperuricemia in subjects with refractory gout, and induction of antibody against poly(ethylene glycol) (PEG), in a phase I trial of subcutaneous PEGylated urate oxidase. Arthritis Res Ther. 2006;8:R12. [PMC free article] [PubMed]
32. Morrow JD, Awad JA, Boss HJ, Blair IA, Roberts LJ., 2nd Non-cyclooxygenase-derived prostanoids (F2-isoprostanes) are formed in situ on phospholipids. Proc Natl Acad Sci USA. 1992;89:10721–10725. [PMC free article] [PubMed]
33. Kadiiska MB, et al. Biomarkers of oxidative stress study II: Are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning? Free Radic Biol Med. 2005;38:698–710. [PubMed]
34. Morrow JD, Roberts LJ., 2nd Mass spectrometric quantification of F2-isoprostanes in biological fluids and tissues as measure of oxidant stress. Methods Enzymol. 1999;300:3–12. [PubMed]
35. Block G, et al. The effect of vitamins C and E on biomarkers of oxidative stress depends on baseline level. Free Radic Biol Med. 2008;45:377–384. [PMC free article] [PubMed]
36. Keaney JF, Jr, et al. Framingham Study. Obesity and systemic oxidative stress: Clinical correlates of oxidative stress in the Framingham Study. Arterioscler Thromb Vasc Biol. 2003;23:434–439. [PubMed]
37. Morrow JD, et al. Increase in circulating products of lipid peroxidation (F2-isoprostanes) in smokers. Smoking as a cause of oxidative damage. N Engl J Med. 1995;332:1198–1203. [PubMed]
38. Paton LN, Mocatta TJ, Richards AM, Winterbourn CC. Increased thrombin-induced polymerization of fibrinogen associated with high protein carbonyl levels in plasma from patients post myocardial infarction. Free Radic Biol Med. 2010;48:223–229. [PubMed]
39. Sinha S, Singh SN, Ray US. Total antioxidant status at high altitude in lowlanders and native highlanders: Role of uric acid. High Alt Med Biol. 2009;10:269–274. [PubMed]
40. Tsukimori K, Yoshitomi T, Morokuma S, Fukushima K, Wake N. Serum uric acid levels correlate with plasma hydrogen peroxide and protein carbonyl levels in preeclampsia. Am J Hypertens. 2008;21:1343–1346. [PubMed]
41. Andreadou E, et al. Serum uric acid levels in patients with Parkinson's disease: Their relationship to treatment and disease duration. Clin Neurol Neurosurg. 2009;111(9):724–728. [PubMed]
42. Chen H, Mosley TH, Alonso A, Huang X. Plasma urate and Parkinson's disease in the Atherosclerosis Risk in Communities (ARIC) study. Am J Epidemiol. 2009;169:1064–1069. [PMC free article] [PubMed]
43. Koprowski H, Spitsin SV, Hooper DC. Prospects for the treatment of multiple sclerosis by raising serum levels of uric acid, a scavenger of peroxynitrite. Ann Neurol. 2001;49:139. [PubMed]
44. Langemann H, Feuerstein T, Mendelowitsch A, Gratzl O. Microdialytical monitoring of uric and ascorbic acids in the brains of patients after severe brain injury and during neurovascular surgery. J Neurol Neurosurg Psychiatry. 2001;71:169–174. [PMC free article] [PubMed]
45. Tohgi H, Abe T, Takahashi S, Kikuchi T. The urate and xanthine concentrations in the cerebrospinal fluid in patients with vascular dementia of the Binswanger type, Alzheimer type dementia, and Parkinson's disease. J Neural Transm Park Dis Dement Sect. 1993;6:119–126. [PubMed]
46. Santos CX, Anjos EI, Augusto O. Uric acid oxidation by peroxynitrite: Multiple reactions, free radical formation, and amplification of lipid oxidation. Arch Biochem Biophys. 1999;372:285–294. [PubMed]
47. Squadrito GL, et al. Reaction of uric acid with peroxynitrite and implications for the mechanism of neuroprotection by uric acid. Arch Biochem Biophys. 2000;376:333–337. [PubMed]
48. Whiteman M, Ketsawatsakul U, Halliwell B. A reassessment of the peroxynitrite scavenging activity of uric acid. Ann N Y Acad Sci. 2002;962:242–259. [PubMed]
49. Gersch C, et al. Reactions of peroxynitrite with uric acid: Formation of reactive intermediates, alkylated products and triuret, and in vivo production of triuret under conditions of oxidative stress. Nucleosides Nucleotides Nucleic Acids. 2009;28:118–149. [PMC free article] [PubMed]
50. Patterson RA, Horsley ET, Leake DS. Prooxidant and antioxidant properties of human serum ultrafiltrates toward LDL: Important role of uric acid. J Lipid Res. 2003;44:512–521. [PubMed]
51. Gonsette RE, et al. ASIIMS study group Boosting endogenous neuroprotection in multiple sclerosis: The Association of Inosine and Interferon beta in relapsing- remitting Multiple Sclerosis (ASIIMS) trial. Mult Scler. 2010;16:455–462. [PubMed]
52. Ichida K, et al. Clinical and molecular analysis of patients with renal hypouricemia in Japan-influence of URAT1 gene on urinary urate excretion. J Am Soc Nephrol. 2004;15:164–173. [PubMed]
53. Raivio KO, Saksela M, Lapatto R. Xanthine oxidoreductase - role in human pathophysiology and in hereditary xanthinuria. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. New York: McGraw-Hill; 2001. pp. 2639–2652.
54. Kelly SJ, et al. Diabetes insipidus in uricase-deficient mice: A model for evaluating therapy with poly(ethylene glycol)-modified uricase. J Am Soc Nephrol. 2001;12:1001–1009. [PubMed]
55. Wu X, et al. Hyperuricemia and urate nephropathy in urate oxidase-deficient mice. Proc Natl Acad Sci USA. 1994;91:742–746. [PMC free article] [PubMed]
56. Martinon F, Pétrilli V, Mayor A, Tardivel A, Tschopp J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature. 2006;440:237–241. [PubMed]
57. So A. Developments in the scientific and clinical understanding of gout. Arthritis Res Ther. 2008;10:221. [PMC free article] [PubMed]
58. Baraf HS, Matsumoto AK, Maroli AN, Waltrip RW., 2nd Resolution of gouty tophi after twelve weeks of pegloticase treatment. Arthritis Rheum. 2008;58:3632–3634. [PubMed]

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