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Clin Chem Lab Med 2010;48(3):383390 2010 by Walter de Gruyter Berlin New York. DOI 10.1515/CCLM.2010.065

Decrease in uric acid in acute ischemic stroke correlates with stroke severity, evolution and outcome

Raf Brouns13, Annick Wauters4, Gerda Van De Vijver3, Didier De Surgeloose5, Rishi Sheorajpanday2,3 and Peter P. De Deyn2,3,*
1

Department of Neurology, University Hospital Brussels, Vrije Universiteit Brussel, Brussels, Belgium 2 Department of Neurology and Memory Clinic, ZNA Middelheim Hospital, Antwerp, Belgium 3 Laboratory for Neurochemistry and Behavior, Institute of Born-Bunge, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium 4 Department of Clinical Biology, ZNA Middelheim Hospital, Antwerp, Belgium 5 Department of Radiology, ZNA Middelheim Hospital, Antwerp, Belgium

not change significantly in patients with TIA. Changes in UA concentrations from admission to day 7 (DUAday 7) correlated with the NIHSS score (rs0.32; p-0.001), stroke progression (rs0.29; ps0.001), infarct volume (rs0.37; p-0.001), mRS score (rs0.28; ps0.001) and mortality (ps0.010). Conclusions: Decreases in UA during the first week after onset of stroke correlates with more severe stroke, unfavorable stroke evolution, and poor long-term stroke outcome. Clin Chem Lab Med 2010;48:38390. Keywords: acute cerebral infarction; stroke evolution; stroke outcome; stroke severity; uric acid.

Abstract
Background: Although uric acid (UA) is one of the most important antioxidants in plasma and appears to be neuroprotective in animal models, results from human studies are controversial. In this study, we investigated the kinetics of serum UA concentrations in the acute, subacute and chronic phase of ischemic stroke and its relation with initial stroke severity, stroke evolution in the subacute phase and longterm stroke outcome. Methods: Serum concentrations of UA were measured in 199 stroke patients at admission (median, 2.8 h after stroke onset), at 24 h, 72 h, day 7, month 1 and month 3 after onset of stroke. We evaluated the relationship between changes in UA concentrations and (a) stroke severity wpatients with transient ischemic attack (TIA) vs. stroke patients, National Institutes of Health Stroke Scale (NIHSS) score at admissionx, (b) stroke evolution (stroke progression, infarct volume at 72 h), and (c) stroke outcome wmodified Rankin scale (mRS) score at month 3, mortalityx. Results: UA concentrations decreased significantly during the first 7 days after stroke onset before returning to baseline (p-0.001). Mean plasma UA concentrations decreased from 336.66"113.01 mmol/L at admission to 300.37"110.04 mmol/L at day 7 (p-0.001) in patients with stroke, but did
*Corresponding author: Prof. Dr. Peter P. De Deyn, Laboratory of Neurochemistry and Behaviour, Institute of Born-Bunge, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium Phone: q32 3 265 26 20, Fax: q32 3 265 26 18, E-mail: peter.dedeyn@ua.ac.be Received August 1, 2009; accepted October 19, 2009; previously published online December 18, 2009

Introduction
Oxidative stress occurs when the physiological balance between oxidants and antioxidants is disrupted in favor of the former, with potential damage to the organism (1). An increased production of free radicals and other chemical species has been demonstrated in ischemic stroke, and oxidative stress is proposed as a fundamental mechanism of brain damage in this condition (2). With a concentration that exceeds other antioxidants by almost ten-fold, uric acid (UA) is one of the most important antioxidants in plasma, and contributes as much as two-thirds of all free radical scavenging capacity in plasma (3). It is particularly effective in quenching hydroxyl, superoxide and peroxynitrite radicals, and may play a protective physiological role by preventing lipid peroxidation (4, 5). In animal models, UA was shown to be neuroprotective (6, 7), but results from human studies have been controversial (8). In some reports, increased plasma UA concentrations in patients with ischemic stroke were associated with poor outcome (911), while other studies found an inverse relationship between UA concentration and stroke severity (12, 13). Additionally, administration of UA reduced oxidative stress in healthy volunteers and in stroke patients (1416). Currently, a multicenter phase III clinical trial is assessing whether the administration of UA increases the clinical benefits of recombinant tissue plasminogen activator (17). In this study, we investigated the kinetics of serum UA concentrations in the acute, subacute and chronic phases of ischemic stroke and the relationship to initial stroke severity, stroke evolution in the subacute phase and long-term outcome following stroke.

2010/434

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Materials and methods

Study population
This study is part of the Middelheim Interdisciplinary Stroke Study, which is a project studying the clinical, biochemical, neuroimaging, neuropsychological and electrophysiological evaluation of patients with ischemic stroke or transient ischemic attack (TIA) at ZNA Middelheim Hospital, Antwerp. Other biochemical analyses from this project have been reported elsewhere (1822). In this study, we focused on the 199 patients with ischemic stroke (ns156) or TIA (ns43), in whom analysis of UA was available within 24 h after onset of stroke symptoms. Treatment with intravenous or intra-arterial thrombolytics was administered to 30 patients (15%). The study was performed in accordance with the revised Declaration of Helsinki (1998) and the guidelines of the Ethics Committees of ZNA Antwerp and the University of Antwerp. Characteristics of the study population and the occurrence of possible confounding factors for UA concentrations are shown in Table 1.

after onset of stroke. Venous blood samples were collected in 4.9 mL S-Monovette Serum-Gel tubes (Sarstedt Monovette , Numbrecht, Germany) and sent immediately to the hospital laboratory. Serum UA concentrations were measured using a colorimetric uricase method with the Vitros 950AT and Vitros 5.1FS (Vitros URIC slides, Ortho-Clinical Diagnostics, Beerse, Belgium). Imprecision ranges from 1.4% at a concentration of 249.84 mmol/L to 1.2% at 636.44 mmol/L. The reference interval is 208.18505.58 mmol/L for men and 148.7368.78 mmol/L for women.

Evaluation of stroke severity, evolution and outcome

The neurological deficit was quantified using the National Institutes of Health Stroke Scale (NIHSS) by trained stroke physicians at admission, 24 h, 72 h and at day 7. Thirty-two patients developed progressing stroke as defined by the European Progressing Stroke Study criteria (23). In addition to neuroimaging at admission, all patients underwent magnetic resonance imaging (MRI) of the brain at an average of 3.1 days after onset of stroke, except 10 patients who died before repeat neuroimaging was performed, and 13 patients who had contraindications for MRI. These patients were evaluated by computed tomography of the brain at an average of 3.0 days following onset of stroke. Infarct volume was assessed by two independent observers as described previously (1822). The median infarct volume was 13.9 mL (interquartile range, 1.864.7 mL). Outcome was assessed at 3 months after stroke by means of the modified Rankin scale (mRS) and all-cause mortality. In agreement with the literature, poor outcome was defined as a mRS score )3 (24).

Blood sampling and measurement of uric acid

Blood collection and measurement of UA was performed at admission wmedian, interquartile range; 2.8 h (1.46.9 h) after onset of stroke symptomsx and at 24 h, 72 h, 7 days, 1 month and 3 months Table 1 Patient demographics and stroke characteristics in the 199 patients with acute ischemic stroke or TIAa. Characteristics Value

Patient characteristics Age, years 71.9"12.4 Male gender 102, 51.3 Caucasian race 194, 97.5 Time to blood sampling at admission, h 2.8, 1.46.9 Stroke characteristics TIA/ischemic stroke 43, 21.6/156, 78.4 NIHSS score at admission 5, 215 Stroke progressionb 42, 21.1 Infarct volume, mL 13.9, 1.864.7 mRS at month 3 mRS 03 144, 72.4 mRS 46 55, 27.6 Mortality at month 3 32, 16.1 Possible confounding factors for uric acid Sodium serum concentration, mEq/L 139"8 75"28 eGFR, MDRD, mL/min/1.73 m2 Uric acid fractional excretion rate, 8.4"3.9 %, ns128 Body mass index 26"5 Diuretics 69, 34.7 Cytotoxic agents 1, 0.5 Malignancy 18, 9.0 Psoriasis 3, 1.5 Alcohol abuse 11, 5.5 History of gout 8, 4.0 a Data given as mean"SD, as number, percentage or as median, interquartile range. bStroke progression defined by the European Progressing Stroke Study criteria. TIA, transient ischemic attack; NIHSS, National Institutes of Health Stroke Scale; mRS, modified Rankin scale; eGFR, estimated glomerular filtration rate; MDRD, Modification of Diet in Renal Diseases formula.

Evaluation of possible confounding factors for serum uric acid levels

Changes in serum UA concentrations may be the result of alterations in hydration, purine metabolism, purine dietary intake, renal function or UA oxidation. In order to evaluate a possible influence of hydration status or renal function on UA concentrations, for all time points we measured the urea:creatinine ratio and calculated the estimated glomerular filtration rate (eGFR) using the Modification of Diet in Renal Diseases (MDRD) formula. In addition, the fractional excretion rate of UA was assessed at 72 h after stroke onset in a subset of patients (ns128). It cannot be excluded that ischemiainduced breakdown of cerebral tissue leads to increased purine concentrations and increased UA production. The extent of cerebral tissue damage was estimated by infarct volumetry. Three out of eight patients with a history of gout were treated with allopurinol at admission.

Statistical analyses
Statistical computations were performed with the SPSS software package version 15.0 (SPSS Inc., Chicago, IL, USA). Normally distributed data (Kolmogorov-Smirnov test) is presented as mean" standard deviation (SD), data not normally distributed data is shown as median and interquartile range. We used the t-test for independent samples to assess differences in UA between groups. To evaluate differences in UA at various time points within groups, we used the t-test for paired samples. The relationship between changes in UA concentrations from admission to day 7 after stroke onset (DUAday 7) and parameters for stroke severity, evolution and outcome was assessed using bivariate correlations (Spearmans r). Logistic regression analysis was performed to determine factors that could be considered independent predictors for stroke outcome.

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Finally, we analyzed long-term mortality using the Kaplan-Meier method. The statistical significance for differences between the groups was assessed with the log rank test for survival.

Results
Uric acid concentrations

Serum UA concentrations for the total stroke population are shown in Figure 1. The mean UA concentrations were 342.61"110.01 mmol/L at admission, 332.49"115.99 mmol/L at 24 h, 314.65"114.20 mmol/L at 72 h, 308.11" 108.85 mmol/L at day 7, 331.90"100.52 mmol/L at month 1 and 328.92"93.38 mmol/L at month 3. UA concentrations decreased significantly between admission and day 7 (p-0.001), and then returning to basal concentrations after one month (p-0.001).
Uric acid concentrations in relation to initial stroke severity

Based on the NIHSS score at admission, patients were categorized as having mild stroke (NIHSS -7, ns113) and moderate to severe stroke (NIHSS )7, ns86) (25). In patients with moderate to severe stroke, UA concentrations decreased significantly between admission and day 7, which was followed by an increase until month 3 (mean"SD; 330.11"97.55 mmol/L at admission to 267.66"110.04 mmol/L at day 7, p-0.001; 317.03"100.52 mmol/L at 3 months, ps0.004). This is in contrast to patients with mild stroke who displayed a non-significant decrease in UA concentrations. Thus, the change in UA concentrations from admission to day 7 was significantly greater in patients with moderate to severe stroke than in those with mild stroke (mean DUAday 7"SD; 58.29"82.68 mmol/L vs. 14.87" 73.16 mmol/L; ps0.001). In addition, the DUAday 7 correlated positively with the NIHSS score at admission (rs0.32; p-0.001). A line graph displaying the kinetics of UA in relation to stroke severity is shown in Figure 2.
Uric acid concentrations in relation to evolution of subacute stroke

In patients with stroke, UA concentrations decreased significantly from admission to day 7 (mean"SD; 336.66" 113.01 mmol/L at admission to 300.37"110.04 mmol/L at day 7; p-0.001), and then increased significantly from day 7 to the chronic phase at month 3 (328.92"90.41 mmol/L at month 3; p-0.001). This is in contrast to patients with TIA who showed no significant changes in UA concentration.

In the first 72 h after stroke onset, 42 patients (21%) developed progressing stroke as defined by the European Progressing Stroke Study criteria (23). The decrease in UA concentrations in the first 7 days was significant, both in patients with progressing stroke (mean"SD; 322.98" 107.66 mmol/L at admission to 257.55"133.24 mmol/L at day 7; ps0.004) and in those without stroke progression

Figure 1 Bar graphs demonstrating mean UA concentrations in the total study population at admission, 24 h, 72 h, day 7, month 1 and at month 3. The whiskers in each bar represent the 95% confidence interval. Significant differences between various time points are indicated with *(p-0.001).

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Figure 2 Line graph demonstrating the kinetics of mean UA concentrations in relation to initial stroke severity (NIHSS score dichotomized at 7). The whiskers represent the standard deviation.

(mean"SD; 347.36"110.63 mmol/L at admission to 317.62"103.50 mmol/L at day 7; ps0.001). When assessing UA kinetics, it is obvious that the decrease in UA concentration during the first 7 days is more pronounced in patients suffering from progressing stroke compared to those without progression (mean DUAday 7"SD; 72.57"73.16 mmol/L vs. 25.58"79.11 mmol/L; ps0.012). Also, the DUAday 7 and the occurrence of stroke progression showed positive correlation (rs0.29; ps0.001). Infarct volume on neuroimaging at 72 h after stroke onset is the result of initial stroke severity and stroke evolution in the subacute phase. Patients were classified based on their infarct volume as having small infarction (-5 mL, ns107) and moderate to large infarctions ()5 mL, ns82). UA concentrations in patients with small infarctions did not decrease significantly from admission through day 7 (mean"SD; 342.01"116.58 mmol/L at admission to 327.73"104.09 mmol/L at day 7; ps0.053). However, patients with infarct volumes )5 mL showed a more pronounced consumption profile of UA (mean"SD; 343.20"101.12 mmol/L at admission to 277.77"109.44 mmol/L at day 7; p-0.001) (Figure 3). The change in plasma UA concentrations between admission and day 7 was larger in patients with moderate to large infarctions compared to patients with small infarctions (DUAday 7"SD; 59.48"70.78 mmol/L vs. 13.09"80.89 mmol/L; p-0.001). In line with these findings, a positive correlation between DUAday 7 and infarct volume was present (rs0.37; p-0.001).

Uric acid concentrations in relation to long-term stroke outcome

The outcome at 3 months after stroke was poor (mRS 46) in 55 patients (27.6%) and good to moderate (mRS 03) in 144 patients (72.4%). The decrease in UA concentrations during the first 7 days was statistically significant in patients with favorable outcome (mean"SD; 352.12"110.63 mmol/L at admission, 328.92"95.17 mmol/L at day 7; ps0.003) and in patients with poor outcome (mean"SD; 314.65"104.69 mmol/L at admission, 243.87"124.31 mmol/L at day 7; p-0.001) (Figure 4). However, a larger decline in serum UA concentrations during the first week was found in patients with poor outcome when compared to patients with favorable outcome (DUAday 7"SD; 67.21" 73.76 mmol/L vs. 23.20"79.11 mmol/L; ps0.006). Also, mRS scores at month 3 were positively correlated with DUAday 7 (rs0.28; ps0.001). Using receiver operating characteristic curve analysis, the optimal cut-point for DUAday 7 was calculated to differentiate between poor and favorable outcomes. The area under the curve was 0.71 w95% confidence interval (CI) 0.600.83x and the cut-point was set at 57.10 mmol/L, showing a sensitivity of 0.77 (95% CI, 0.730.80) and specificity of 0.70 (95% CI, 0.550.82). Using this cut-point, the odds ratio for poor outcome was 7.8 (95% CI, 3.219.0) and the relative risk 1.7 (95% CI, 1.32.0). Stepwise logistic regression analysis (using inclusion criteria of p-0.01) that included patient characteristics (age, gender), stroke characteristics (NIHSS score at admis-

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Figure 3 Line graph demonstrating the kinetics of mean UA concentrations in relation to evolution of stroke in the subacute phase (infarct volume at 72 h, dichotomized at 5 mL). The whiskers represent the standard deviation.

sion, infarct volume) and possible confounding factors for UA concentrations (eGFR, serum sodium concentration, body mass index, history of malignancy, gout, alcohol abuse,

psoriasis and the use of diuretics, thrombolytics or cytotoxic drugs) as covariates, identified the DUAday 7, the NIHSS score and the infarct volume as predictors for stroke outcome

Figure 4 Line graph demonstrating the kinetics of mean UA concentrations in relation to long-term outcome from stroke (mRS score dichotomized at 3). The whiskers represent the standard deviation.

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at month 3. This result was to be expected since we found highly significant correlation between the DUAday 7, NIHSS score and infarct volume. Thirty-two patients (16%) died within 3 months after stroke. Mortality was higher in patients with the DUAday 7 )57.10 mmol/L (ps0.01). The Kaplan-Meier plot for survival illustrates higher mortality in patients with more pronounced decreases in serum UA concentrations during the first 7 days after onset of stroke (ps0.010) (Figure 5).
Uric acid levels in relation to possible confounding factors

Discussion
The association between hyperuricemia and increased cardiovascular risk has been recognized for more than 50 years (26). Furthermore, increased serum UA concentrations are associated with increased risk of stroke in high-risk groups (27, 28). However, given the absence of compelling biological evidence of a causal link, the significance of this relationship remains subject to considerable debate (8). It also remains unclear whether UA is advantageous or injurious in acute stroke. In animal models, UA was shown to be neuroprotective (6, 7), but results from human studies have been controversial (8). In some reports, increased plasma UA concentrations in ischemic stroke patients were associated with poor outcome (911), while others found an inverse relationship between UA concentrations and severity of stroke (12, 13). Additionally, administration of UA reduced oxidative stress in healthy volunteers and patients with stroke (1416). We found six studies in the literature that evaluated serum UA concentrations in acute stroke patients. UA concentrations in our study are similar to those in these other reports. In three studies, single serum UA measurements were obtained ;24 h after onset of stroke. Higher UA concentrations were associated with poor outcome in two studies (9, 11), but appeared to be a strong predictor of favorable outcome in the third study (12). Kinetics of UA in plasma after acute ischemic stroke were assessed in three small studies (10, 16, 29). However, these studies may have been hampered by small study populations (ranging from 8 to 38 patients), the application of exhaustive exclusion criteria and the use of CT-based diagnosis of stroke. In agreement with the findings of Gariballa et al., we found a gradual decrease in UA concentrations between admission and day 7 (29).

The urea:creatinine ratio and eGFR were similar at admission and day 7 after onset of stroke (ps0.060 and 0.058, respectively). This indicated that the observed changes in UA concentrations may not be attributed to changes in hydration status or renal function. The mean fractional excretion rate of UA at 72 h was within normal limits (8.4%"3.9%) and did not correlate with initial stroke severity (TIA vs. stroke, NIHSS score at admission), stroke evolution (stroke progression, infarct volume at 72 h), stroke outcome (mRS score at month 3, mortality), or with the DUAday 7. A contribution of cerebral ischemia-induced necrosis to plasma UA concentrations may not be present, given the absence of a correlation between infarct volume and UA concentration at admission or at 24 h (ps0.833 and 0.409), and an inverse correlation at 72 h (rs0.20, ps0.014) and day 7 after onset of stroke (rs0.26, ps0.002). Plasma UA kinetics (DUAday 7) and key stroke characteristics, including initial stroke severity, stroke evolution and stroke outcome were similar in patients receiving treatment with allopurinol and those not receiving allopurinol.

Figure 5 Kaplan-Meier plot showing 3-month survival in relation to evolution of UA concentration in the first 7 days after stroke (DUAday 7 dichotomized at 57.10 mmol/L).

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Amaro et al. reported a maximal drop in serum UA concentration as soon as 6 h after onset of stroke in a population of eight stroke patients receiving thrombolytic therapy (16), a finding we could not reproduce in our subpopulation of 30 patients treated with thrombolytics (data not shown). Cherubini et al. described a gradual increase in UA concentration toward day 7 (10). To the best of our knowledge, this is the first study to evaluate the kinetics of serum UA concentrations in the acute, subacute and chronic phases of ischemic stroke in a large population. The present study shows that UA serum concentration displays a pronounced decrease in the first 7 days after onset of stroke, followed by a return to baseline after 1 month. In addition, this biphasic kinetic with its nadir at day 7 was more pronounced in patients with severe stroke, unfavorable stroke evolution and poor longterm outcome. Also, the correlation between the DUAday 7 and the NIHSS score at admission, the occurrence of stroke progression, the infarct volume at 72 h, and the mRS score at month 3 further illustrates the relationship between UA and stroke characteristics. Several mechanisms could explain our observations: serum UA concentrations may change due to alterations in hydration, renal function, purine metabolism, purine dietary intake or UA oxidation. However, hydration status and renal function remained stable, the UA excretion rate did not correlate with key stroke characteristics or with the DUAday 7 and UA concentrations were not positively correlated with infarct volume, indicating that cerebral ischemia-induced tissue breakdown does not contribute to serum UA concentrations. We hypothesize that the progressive decrease in UA concentrations reflects ongoing consumption of UA in the first week after stroke, followed by a return to baseline. This is consistent with previous reports on oxidative stress, showing a marked decrease in concentrations of other non-enzymatic antioxidants including vitamins A, C, E and carotenoids (30, 31). In addition, clinical and experimental studies have shown a gradual consumption of antioxidant capacity after transient cerebral ischemia; the magnitude of antioxidant consumption seemed to be correlated with the extent and severity of brain ischemia, growth of infarction and longterm functional outcome (17). Consequently, it is plausible that consumption of antioxidants, including UA, is more pronounced in patients with more severe stroke, unfavorable stroke evolution and poor outcome. As demonstrated by the logistic regression model and data from the literature, initial stroke severity and subsequent evolution strongly influence long-term outcome following stroke (32). Therefore, it is likely that the relationship between the consumption profile of UA and stroke outcome is secondary to initial stroke severity and stroke evolution. Some limitations of this study should be acknowledged. Despite the presence of highly significant changes in UA concentrations in the total study population, the lack of data regarding UA concentrations before onset of stroke or in controls may limit the generalization of the findings. In addi-

tion, the inter-individual variations in UA concentrations is important, and as we deliberately chose not to exclude patients with conditions known to influence UA concentrations, confounding effects from comorbidity or concomitant medication cannot be ruled out, despite the results of the logistic regression model. Since we did not assess markers of UA oxidation, such as allantoin or other markers for oxidative stress, this study provides no definite proof that the observed decreases in serum UA concentrations is secondary to scavenging of free radicals. However, this hypothesis is plausible, especially since the changes in UA concentrations cannot be explained by changes in UA generation, UA excretion or changes in plasma volume. This study improves insight into the role of oxidative stress in acute ischemic stroke and secondary ischemic injury. This may contribute to the development of more adequate therapeutics. Administration of UA as an adjuvant therapy with recombinant tissue plasminogen activator has shown to be safe (16) and is currently being tested in a multicenter phase III clinical trial (17). The results presented indicate that ongoing oxidative stress may be an important source of secondary injury after the initial ischemic event, a phenomenon that can be tackled by enhancing antioxidant capacity by means of administration of UA.

Conflict of interest statement

Authors conflict of interest disclosure: The authors stated that there are no conflicts of interest regarding the publication of this article. Research funding played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication. Research funding: This research was also supported by the Institute Born-Bunge; the agreement between the Institute Born-Bunge and the University of Antwerp; the Interuniversity Attraction Poles (IAP) program P6/43 of the Belgian Federal Science Policy Office, Belgium; and the Medical Research Foundation Antwerp. Employment or leadership: R.B. was a research assistant of the Fund for Scientific Research Flanders (FWO-Vlaanderen). Honorarium: None declared.

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