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Original Contribution

Uric Acid Therapy Improves Clinical Outcome in Women


With Acute Ischemic Stroke
Laura Llull, MD; Carlos Laredo, MSc; Arturo Ren, MD; Beln Prez, PhD; Elisabet Vila, PhD;
Vctor Obach, MD; Xabier Urra, MD, PhD; Anna Planas, PhD; Sergio Amaro, MD, PhD;
ngel Chamorro, MD, PhD
Background and PurposeIt is unknown whether women and men with acute ischemic stroke respond similar to an
antioxidant regimen administered in combination with thrombolysis. Here, we investigated the independent effect of sex
on the response to uric acid (UA) therapy in patients with acute stroke treated with alteplase.
MethodsIn the Efficacy Study of Combined Treatment With Uric Acid and rtPA in Acute Ischemic Stroke (URICOICTUS) trial, 206 women and 205 men were randomized to UA 1000 mg or placebo. In this reanalysis of the trial, the
primary outcome was the rate of excellent outcome at 90 days (modified Rankin Scale, 01, or 2, if premorbid score of
2) in women and men using regression models adjusted for confounders associated with sex. The interaction of UA levels
by treatment on infarct growth was assessed in selected patients.
ResultsExcellent outcome occurred in 47 of 111 (42%) women treated with UA, and 28 of 95 (29%) treated with placebo,
and in 36 of 100 (36%) men treated with UA and 38 of 105 (34%) treated with placebo. Treatment and sex interacted
significantly with excellent outcome (P=0.045). Thus, UA therapy doubled the effect of placebo to attain an excellent
outcome in women (odd ratio [95% confidence interval], 2.088 [1.0504.150]; P=0.036), but not in men (odd ratio [95%
confidence interval], 0.999 [0.5161.934]; P=0.997). The interactions between treatment and serum UA levels (P<0.001)
or allantoin/UA ratio (P<0.001) on infarct growth were significant only in women.
ConclusionsIn women with acute ischemic stroke treated with alteplase, the administration of UA reduced infarct growth
in selected patients and was better than placebo to reach excellent outcome.
Clinical Trial RegistrationURL: https://clinicaltrials.gov/ct2/show/NCT00860366. Unique identifier: NCT00860366.
(Stroke. 2015;46:00-00. DOI: 10.1161/STROKEAHA.115.009960.)
Key Words: allantoin oxidative stress sex stroke uric acid

he ischemic brain is particularly vulnerable to oxidative


stress as the result of its high consumption of oxygen, its
rich content of iron and unsaturated lipids, and its low antioxidant capacity.1 Uric acid (UA) is an end-product of the metabolism of purines and a potent antioxidant compound.2 Its multiple
effects include scavenging of hydroxyl radicals, hydrogen peroxide, and peroxynitrite, suppression of the Fenton reaction, and
limitation of lipid peroxidation and free radical induced damage
to DNA.3,4 Previous studies showed that UA protected cultured
neurons against excitotoxic death,5 improved the functional outcome, and reduced the amount of brain damage in experimental models of intraluminal,5 or thromboembolic transient brain
ischemia in male rodents.6 Of note, UA formation in humans
is accompanied by the emergence of free radicals,7,8 which
may degrade the molecule to allantoin through nonenzymatic

mechanisms.9 Thus, the resulting allantoin/UA (AL/UA) ratio


is considered a reliable marker of the oxidative stress burden
in patients with various conditions.10,11
Recently, a systematic review and meta-analysis of 8131
patients with acute ischemic stroke showed that the endogenous serum UA levels had a protective effect on neurological
outcome as patients with good outcomes had a higher serum
UA levels than those with poor outcome at follow-up.12 At the
bed side, the Efficacy Study of Combined Treatment With Uric
Acid and rtPA in Acute Ischemic Stroke (URICO-ICTUS) trial
recently suggested that the addition of UA to thrombolytic
therapy in patients with acute ischemic stroke improved the
rate of excellent outcome compared with placebo in women,
but not in men.13 However, the URICO-ICTUS trial did not
evaluate whether the greater efficacy of UA therapy observed

Received May 2, 2015; final revision received May 19, 2015; accepted May 21, 2015.
From the Department of Neuroscience, Comprehensive Stroke Center, Hospital Clinic, University of Barcelona, August Pi i Sunyer Biomedical
Research Institute (Institut dInvestigacions Biomdiques Agust Pi i Sunyer), Barcelona, Spain (L.L., C.L., A.R., V.O., X.U., S.A., .C.); Departament
de Farmacologia, Teraputica i Toxicologia, Universitat Autnoma de Barcelona, Bellaterra, Spain (B.P., E.V.); Department of Brain Ischemia and
Neurodegeneration, Institute for Biomedical Research of Barcelona, Spanish Research Council, Barcelona, Spain (A.P.); and Institut dInvestigacions
Biomdiques Agust Pi i Sunyer, Barcelona, Spain (A.P.).
Guest Editor for this article was Donna M. Ferriero, MD.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.
115.009960/-/DC1.
Correspondence to ngel Chamorro, MD, PhD, Hospital Clinic, Villarroel 170, 08036 Barcelona, Spain. E-mail achamorro@clinic.ub.es
2015 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org

DOI: 10.1161/STROKEAHA.115.009960

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2StrokeAugust 2015
in women could have resulted from unreported confounders
including the sex effect on stroke pathophysiology.14
In the early stages of the evaluation of neuroprotective
therapies it is recommended to obtain evidence of the biological effect of the treatment under investigation.15 These
biomarkers may include the effect of the drug in circulating
levels of molecules, such as oxidative stress biomarkers, or in
the longitudinal change in infarct volume between treatment
groups.15 Infarct growth has been related to relevant clinical
outcomes and may capture the effect of neuroprotective therapies, although further validation is required to be used as surrogate measure of clinical end points.16
Here, we reanalyzed the URICO-ICTUS data to test the
independent effect of sex in the clinical response to UA therapy. Moreover, we analyzed the effect of therapy on circulating markers of oxidative stress and on infarct growth.

Methods
The URICO-ICTUS methodology has been described in detail previously.13,17 In short, the trial was approved by local institutional review boards, and written informed consent was obtained from the
patients or from legally acceptable surrogates. URICO-ICTUS was
overseen by a data safety monitoring board, and it was registered with
ClinicalTrials.gov (NCT00860366).

URICO-ICTUS Primary Study Hypothesis and


Patient Population
The primary hypothesis of the URICO-ICTUS trial was that treatment
with UA 1000 mg would increase the rate of excellent outcome at 90
days compared with placebo in patients with acute ischemic stroke
treated with alteplase within 4.5 hours of symptom onset. Excellent
outcome was defined as a modified Rankin Scale (mRS) of 0 to 1, in
patients with a premorbid mRS score <2, or a mRS of 2, in patients
with a premorbid mRS score equal to 2. Additional eligibility criteria were age 18 years, baseline National Institutes of Health Stroke
Scale (NIHSS) >6 and <25, and premorbid mRS of <2. Exclusion
criteria were history of gouty arthritis or nephropathy, recent intake of
allopurinol or lithium, or serum creatinine levels >1.5 mg/dL.

Study Design and Conduct


URICO-ICTUS was a multicenter, double-blind, phase 2b/3 trial,
where 421 patients were randomly allocated (1:1) to receive UA or
placebo infused intravenously in 90 minutes during the infusion of alteplase. Of those, 411 were finally included in the target population of
the study. Six patients with stroke mimics and 4 patients who did not
start the experimental medication were excluded adhering to recommendations of the International Conference on Harmonisation Topic
E9 (CPMP/ICH/363/96), International Conference on Harmonisation
Topic E9 guidelines.18 The study patients had a noncontrast computed tomography at baseline and at 36 hours and were admitted into
stroke dedicated units. The vital signs and blood pressure were monitored at least for 24 hours, the neurological status was assessed with
the NIHSS every 4 to 8 hours during the days of hospitalization, and
the functional outcome was assessed with the mRS at 90 days by certified stroke neurologists. The qualifying stroke subtype was defined
following the Trial of Org 10 172 in Acute Stroke Treatment criteria
as large-artery atherosclerosis, cardioembolism, small-vessel occlusion, stroke of other determined cause, and stroke of undetermined
cause.19 Vascular risk factors were defined according to usual definitions, as previously reported.13

Efficacy Outcomes
The primary outcome measure of the current reanalysis of the
URICO-ICTUS data was the rate of excellent outcome at 90 days

in separate analyses for women and men. Secondary analyses included the correlations in women and men between serum UA and
AL/UA ratio with infarct growth as measured using multimodal brain
imaging.

Advanced Brain Imaging and Serum Biomarkers


Multimodal brain imaging was performed in 46 participants admitted
into 1 prespecified study site, and serial blood samples were collected
in 43 of these patients (samples were not available in 3 subjects for
technical reasons). The advanced brain imaging protocol included
the assessment of the following parameters: (1) the Alberta Stroke
Program Early CT Score (ASPECTS) on baseline noncontrast computed tomography20; (2) the nonviable tissue volume on computed
tomographic perfusion at the end of alteplase21; (3) the infarct volume
on diffusion-weighted imaging brain magnetic resonance imaging at
72 hours22; (4) the arterial patency on computed tomographic angiography, MR angiography, or digital subtraction angiography at the end
of thrombolysis, and at 72 hours; and (5) the collateral score on computed tomographic angiography (Methods in the online-only Data
Supplement).23 Infarct growth was defined on brain imaging as the
difference between 72-hour diffusion-weighted imaging infarct volume and baseline nonviable tissue volume on computed tomographic
perfusion. Serum UA and allantoin were measured in blood samples
collected before treatment onset, at 6 to 12 hours, at 48 hours, and
at 90 days.24 The AL/UA ratio and the percent change of serum UA
from baseline values were also calculated (Methods in the onlineonly Data Supplement). The ancillary imaging and biomarker data
were analyzed by investigators blinded to clinical information.

Statistical Methods
The URICO-ICTUS target population was divided into women and
men. Baseline demographic features, risk factors, baseline biological
findings, stroke subtypes, neurological course, and radiological findings were compared between the 2 sex groups. Continuous variables
were reported as meanSD or median with interquartile ranges and
were compared with the Student t test, 1-way ANOVA, ANCOVA,
MannWhitney, or KruskalWallis tests as appropriate. Correlations
were assessed with Pearson or Spearman coefficients, and categorical variables were compared with the 2 and Fisher exact tests. A
treatmentsex interaction on the primary outcome was assessed in a
model adjusted for treatment delay and the trial stratification factors
(study site and baseline NIHSS score). Afterward, excellent outcome
was estimated individually in women and men using separated binary
logistic regression models adjusted for variables associated with sex
and outcome in univariate analysis with a P value of <0.10. These
variables were age, premorbid mRS, NIHSS score at randomization,
stroke subtype, history of hypertension, dyslipidemia, coronary artery
disease, smoking, alcohol intake, and creatinine on admission. The
analyses were performed using SPSS Version 19.0 and the level of
significance was established at the 0.05 level (2-sided).

Results
Main Characteristics of Women and Men in the
URICO-ICTUS Trial
In this study, 206 women and 205 men had significant differences
in demographics, risk factors, stroke subtypes, and clinical severity at baseline (Table1). At 90 days, 75 (36.4%) women and
74 (36.1%) men achieved an excellent outcome (P=0.301).

UA Therapy Was More Effective in Women


Excellent outcome at 90 days was increased in women treated
with UA compared with men treated with UA (Table1).
The interaction between treatment (UA or placebo) and
sex had a significant effect on the rate of excellent outcome
(P=0.045), indicating that the effect of therapy on outcome

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Llull et al Uric Acid Therapy and Stroke in Women 3


Table 1. Baseline Data, Clinical Course, and Primary Outcome
in the URICO-ICTUS Target Population According to Sex (n=411
Patients)
Females
(n=206)

Males
(n=205)

P Value

79 (7283)

72 (6379)

0.000

0 (01)

0 (00)

0.047

20 (10)

51 (25)

0.000

2 (1)

24 (12)

0.000

Hypertension (treated
or >140/90), n (%)

158 (77)

122 (60)

0.000

Diabetes mellitus (treated or


glucose >110 mg/dL), n (%)

60 (29)

58 (28)

0.852

Dyslipidemia (treated
or cholesterol >220 mg/
dL), n (%)

74 (36)

94 (46)

0.045

Atrial fibrillation, n (%)

60 (29)

30 (15)

0.000

Ischemic heart disease


(MI or angina), n (%)

20 (10)

39 (19)

0.007

6 (3)

9 (4)

0.424

20 (10)

29 (14)

0.171

129 (18)

126 (17)

0.105

Temperature, C, median (IQR)

36 (3637)

36 (3637)

0.521

Glucose, mg/dL, median (IQR)

128 (111161)

Age, y, median (IQR)


Preadmission mRS, median (IQR)
Medical history
Smoking (>5 d cigarettes
per last 6 mo), n (%)
Alcohol intake (>60 g/d [male]
n(%) >40 g/d [female]), n (%)

Peripheral vascular
disease, n (%)
Previous stroke, n (%)
Baseline biological findings
Mean blood pressure,
mmHg, mean (SD)

Creatinine, mg/dL,
median (IQR)

126 (108150) 0.098

0.8 (0.71.0)

1.0 (0.81.1)

0.000

Pre-rtPA NIHSS score,


median (IQR)

15 (919)

12 (917)

0.027

Screening NIHSS score,


median (IQR)

15 (918)

12 (817)

0.026

Time to rtPA infusion,


min, median (IQR)

145 (100180)

145 (105182) 0.717

Time to uric acid or placebo,


min, median (IQR)

180 (135230)

180 (145220) 0.997

Rescue endovascular
treatment, n (%)

18 (9)

27 (13)

TOAST classification

0.028

Atherothrombotic, n (%)

23 (11)

31 (15)

Cardioembolic, n (%)

98 (48)

84 (41)

Lacunar, n (%)

0.150

6 (3)

13 (6)

77 (37)

67 (33)

2 (1)

19 (5)

NIHSS at end of rtPA infusion,


median (IQR)

11 (618)

9 (415)

0.025

NIHSS at 24 h, median (IQR)

7 (216)

7 (214)

0.610

NIHSS at 72 h, median (IQR)

4 (114)

5 (112)

0.869

NIHSS at day 5 or discharge,


median (IQR)

3 (012)

4 (012)

0.679

Undetermined cause, n (%)


Other cause, n (%)
Neurological course

(Continued)

Table 1. Continued

NIHSS at day 5 or discharge,


median (IQR)

Females
(n=206)

Males
(n=205)

P Value

2 (013)

2 (010)

0.978

Primary outcome
Excellent outcome, n (%)
Uric acid group

47/111 (42)

36/110 (36)

0.056


Placebo group

28/95 (29)

38/105 (34)

0.977

IQR indicates interquartile range; MI, myocardial infarction; mRS, modified


Rankin Scale; NIHSS, National Institutes of Health Stroke Scale; r-tPA,
recombinant tissue-type plasminogen activator; TOAST, Trial of Org 10 172
in Acute Stroke Treatment; and URICO-ICTUS, Efficacy Study of Combined
Treatment With Uric Acid and rtPA in Acute Ischemic Stroke.

differed according to sex. Therefore, multivariate predictor


models were built to assess the effect of therapy in men and
women separately. Thus, in discrete logistic regression models
adjusted for variables associated with sex and outcome in univariate analysis, UA therapy was more effective than placebo
in women (odd ratio [95% confidence interval], 2.088 [1.050
4.150]; P=0.036), but not in men (odd ratio [95% confidence
interval], 0.999 [0.5161.934]; P=0.997). The NIHSS score at
study onset was the only additional independent factor associated with excellent outcome at 90 days in women (odd ratio
[95% confidence interval], 0.844 [0.7900.902]; P<0.001),
and men (odd ratio [95% confidence interval], 0.821 [0.762
0.886]; P<0.001], in the multivariate models.

Serum Biomarkers and Sex


UA and allantoin were highly correlated in serum at baseline
(r=0.619; P<0.001), at 6 to 12 hours (r=0.769; P<0.001), at 48
hours (r=0.422; P=0.014), and at 90 days (r=0.681; P<0.001).
Treatment and sex had a significant interaction with UA levels
at 6 to 12 hours (P=0.026), serum allantoin at 6 to 12 hours
(P=0.029) and at 48 hours (P=0.023), and the AL/UA ratio at
6 to 12 hours (P=0.034) and at 48 hours (P=0.028), respectively. The interactions remained significant after adjustment
for the creatinine levels.
In untreated patients, serum UA declined at 6 to 12 hours
(P=0.298), 48 hours (P=0.010), and 90 days (P=0.030) after
stroke onset, and the serum UA levels were lower in women
than in men (Figure1A). After UA therapy both sex groups
had increased serum UA levels at 6 to 12 hours (P<0.001;
Figure1B). Serum allantoin did not change over time in the placebo group and women had lower levels than men at 6 to 12 hours
(P<0.040), and 48 hours (P=0.006), but not at 90 days (P=0.163;
Figure1C). Women and men showed a significant rise in allantoin levels at 6 to 12 hours (P<0.001), 48 hours (P<0.001), and
at 90 days (P=0.012) of UA therapy (Figure1D). The AL/UA
ratio did not differ significantly between women and men in the
placebo group, but women had a greater AL/UA ratio than men
at 6 to 12 hours of UA therapy (P=0.021; Figure2).

Infarct Growth and Sex


Advanced brain imaging was performed in a cohort of 46
patients (25 women and 21 men) that showed similar clinical

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4StrokeAugust 2015

Figure 1. Serum biomarkers and sex.


Uric acid (UA) levels declined in patients
treated with placebo but lower levels were
observed in women at baseline (P=0.112),
at 6 to 12 hours (P=0.109), at 48 hours
(P=0.129), and at 90 days (P=0.017; A).
Serum UA levels increased transiently after
UA therapy in both sex groups (B). Serum
allantoin did not change significantly over
time in women and men treated with placebo (C), and after UA therapy increased
similarly in both sex groups (D). Data are
mean (95% confidence interval), milligram
per deciliter of UA, or microgram per milliliter of allantoin, respectively.

findings at baseline when compared with the whole population included in the URICO-ICTUS trial (data not shown). In
this cohort, women and men also had similar brain imaging
parameters at baseline and at follow-up (Table2), and there
was no significant interaction between age and sex with regard
to infarct growth (P=0.177).
The interaction between treatment and sex with regard to
infarct growth was not significant (P=0.973). However, UA
therapy was better than placebo to reduce infarct growth in
women (MannWhitney, P=0.012), and not in men (Mann
Whitney, P=0.605; Figure3). Correspondingly, there was a
significant interaction between treatment and UA levels on
infarct growth in women (P<0.001), but not in men (P=0.184).
Furthermore, infarct growth decreased in women with higher
AL/UA ratio at 6 to 12 hours after UA treatment (Pearson

Figure 2. Allantoin (AL) to uric acid (UA) ratio and sex. Women
and men treated with placebo had similar AL/UA ratios but UA
therapy resulted in a greater early AL/UA ratio in women than in
men. Data are mean (95% confidence interval), milligram allantoin/milligram UA.

r=0.849; P=0.004), but this correlation was not significant in


men (Pearson r=0.268; P=0.426).

Discussion
The primary end point of this reanalysis of the URICO-ICTUS
data showed that compared with placebo UA therapy doubled
the rate of excellent outcome after acute ischemic stroke in
women but not in men. Importantly, we identified a significant
interaction between treatment (UA or placebo) and sex on the
rate of excellent outcome and confirmed that this finding was
also significant in multivariate models that accounted for the
differences observed between women and men at study onset
in demographics, risk factors, initial severity of stroke, and
creatinine levels.
In the study, we did not find different rates of vessel
recanalization after thrombolysis between women and men
as previously suggested in some2527 but not all cohorts.28,29
Furthermore, the effect of UA therapy on functional outcome
was assessed in models adjusted for stroke subtype, age, initial severity of stroke, and for the variable effects of risk factors. Therefore, it is sensible to think that the clinical response
to UA therapy was heightened in women compared with that
in men and that the effect of the experimental drug was not
biased by a sex-dependent susceptibility to thrombolytic therapy or by unbalanced effects of clinical variables.
The interaction between treatment and sex on infarct
growth did not have sufficient statistical power but nevertheless the study found that infarct growth was decreased in
women treated with UA, but not in men. In addition, UA therapy and the actual serum UA levels in treated patients had a
significant interaction with infarct growth in women but not in
men. Women, but not men, also showed reduced infarct growth

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Llull et al Uric Acid Therapy and Stroke in Women 5


Table 2. Main Baseline Traits on Multimodal Imaging
According to Sex (n=46)
Women
(n=25)

Men
(n=21)

P
Value

Advanced imaging
Volume 72 h, mL, median (IQR)

10.1 (3.223.2) 12.0 (7.643.1) 0.447

Collaterals score, median (IQR)

2 (13)

2 (23)

0.248

ASPECTS score, median (IQR)

9 (810)

9 (810)

0.743

Hypoperfusion, mL, median (IQR)

116 (55192)

119 (47194)

0.718

TAR, mL, median (IQR)

91 (54125)

73 (29132)

0.732

NVT, mL, median (IQR)

27 (550)

22 (755)

0.909

Recanalization, n (%)

No

Early
Late or undetermined

0.238
5 (20)

2 (10)

13 (52)

16 (76)

7 (28)

3 (14)

ASPECTS indicates Alberta Stroke Program Early CT score; NVT, nonviable


tissue; and TAR, tissue at risk.

in relation with a higher AL/UA ratio indicative of a greater


nonenzymatic degradation of UA. On the whole, these findings supported that UA therapy reduced infarct growth more
effectively in women than in men, although further study will
be required to confirm these results in larger imaging cohorts.
The time course of UA and allantoin in serum revealed sex
differences that gave additional clues to understand the relative
efficacy of UA therapy in women and men. Women had lower
serum UA levels than men, in accord with the original URICOICTUS data13 and previous descriptive series.30 We now report
that women also had lower serum allantoin levels, but interestingly women also had a greater AL/UA ratio than men during
a time course that was consistent with the elimination half-life
of UA therapy (44 hours).24Although these findings did not
actually prove that free radical formation in vivo was increased
in women compared with that in men, the results showed that
women were more exposed to the effects of free radicals, as
allantoin production in humans only results from free radical
mediated oxidation of UA. 911 Accordingly, we also argue that

women obtained greater clinical benefits after UA replenishment than men because they were in a greater need of antioxidants as the result of their lower constitutional levels of UA.
We also believe that a lower UA-mediated antioxidant capacity in women could be a mechanism that might contribute to
explain the more ominous course of stroke described in women
than in men.14,31
It is increasingly recognized that stroke is a sexually dimorphic disease both experimentally and clinically, although these
sex differences are still poorly understood.32 Experimental
data suggest the existence of sex-dependent pathways in cell
death after ischemic stroke.33 Namely, in males, cell death is
triggered by poly (ADP-ribose) polymerase activation and
nuclear translocation of apoptosis-inducing factor,34 whereas
caspase activation is the major pathway involved in women
after experimental brain ischemia.33 UA is a fundamental
scavenger of peroxynitrite and it is well described that apoptosis is a typical consequence of low to moderate concentrations
of peroxynitrite,4 whereas exposure of cells to higher concentrations of the oxidant has been associated with necrosis.35 In
light of our findings, it can be speculated that sex-dependent
mechanisms of cell death and their interaction with the antioxidant capacity were involved in the beneficial clinical response
to UA therapy observed in women compared with men, but
this possibility requires further study.
Limitations of the current study were the smaller cohort
of patients who had multimodal brain imaging data to evaluate the course of the infarct, or serum samples collected for
evaluation of the redox status using a larger battery of oxidative or antioxidant compounds other than UA and allantoin.
However, it must be emphasized that UA is the most powerful
extracellular antioxidant in human plasma,36 and the AL/UA
ratio is considered a useful oxidative marker.911 Alternatively,
other oxidative stress biomarkers have shown a more limited
value.37 Strong points of the current study were its randomized, double blind design, and that careful measures were
taken to minimize the risk of bias including the assessment
of sex in multivariate models adjusted for relevant confounders. Although the ancillary tests were performed in a smaller
cohort of individuals, the study showed that these patients
were representative of the whole study cohort. In conclusion, this exploratory reanalysis of the URICO-ICTUS trial
highlighted the clinical value of the administration of UA in
women with acute ischemic stroke who received thrombolysis within 4.5 hours of clinical onset. Given the major practical implications that could be derived in the field of acute
ischemic stroke, larger confirmatory studies will be urgently
required to confirm these encouraging results.

Sources of Funding

Figure 3. Infarct growth and sex. Boxes represent 25% to 75%


of interquartile range (IQR); central horizontal bar the median, and
outer horizontal bars 10% to 90% (IQR) to illustrate that women
but not men had significantly less infarct growth after uric acid
therapy.

The URICO-ICTUS trial was funded by the Institute of Health


Carlos III (ISCIII) of the Spanish Ministry of Health (Grant No.
EC07/90276), by the Spanish Ministry of Economy and Competence
(RETICS-INVICTUS R012/0014), and by a private grant from
Fundacin Doctor Melchor Colet. The medication in the study was
supplied by Grifols, Barcelona, Spain. We also thank the support of
the Spanish Ministry of Economy and Competitiveness for grant to
Dr Amaro (PI13/01268, funded as part of the Plan Nacional R+D+I
and cofinanced by ISCIII-Subdireccin General de Evaluacin and
by the Fondo Europeo de Desarrollo Regional).

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6StrokeAugust 2015

Disclosures
None.

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SUPPLEMENTARY MATERIAL

Supplementary Methods
Liquid chromatography
For allantoin measurement samples were centrifuged (5 min; 12.000 g) and from the
supernatant, 10 l were injected into the HPLC system. Separation of allantoin was
performed on a Synergi Hydro-RP C-18 reversed-phase column (250mm 4.6 mm I.D.,
5 m particle size) from Phenomenex (Torrance,CA, USA). Allantoin elution was at 4
minutes performed with potassium dihydrogen phosphate (pH 2.7; 10 mM): acetonitrile
(85:15) and ultraviolet detection (235nm).
Advanced Brain imaging methods
The advanced brain imaging protocol was obtained at one predefined study site that
reported the availability of the required imaging techniques during working hours.
CTPerfusion maps were calculated by commercial software MIStar (Apollo Medical
Imaging Technology, Melbourne, Australia). An absolute threshold of 2s was selected
on the Delay Time map to obtain the Hypoperfusion Tissue volume on CT-Perfusion
and, inside this area, a relative threshold of 30% of the mean Cerebral Blood Flow value
in the unaffected/contralateral hemisphere was used to obtain Non-Viable Tissue
volumes. Tissue-At-Risk was obtained by subtracting the Non-Viable Tissue volume to
the Hypoperfused Tissue volume. DWI lesion volumes were calculated by means of a
semi-automated thresholding method to identify regions of interests with high DWI
signal intensity (exceeding at least three standard deviations the intensity of the values
in the contralateral hemisphere). Each 24 to 48 hours DWI was then co-registered to the
corresponding CT-Perfusion maps. Using Statistical Parametric Mapping (SPM8,
Functional Imaging Laboratory, University College London, London, UK) the DWI
image was automatically subjected to a sequence of two 3D registration procedures to
match CT-Perfusion maps using Time of Flight MRI as an intermediary. Once
CTPerfusion and DWI were placed in register, images were analyzed pixel-wise to
obtain the infarct volume change over time.
Arterial recanalization and assessment of collaterals
Full arterial recanalization indicated a TIMI 2 or 3 score on CT- angiography or
MRangiography, and further classified as early onset if confirmed at the end of
thrombolytic therapy, or late onset, if first confirmed at 72 hours. Patients with a brain
infarct on MRI without arterial occlusion at baseline were also classified as early onset
recanalization. Full arterial recanalization confirmed at seventy-two hours but not
assessed at baseline was classified as uncertain onset and grouped with the late onset
patients. Absence of recanalization defined a TIMI score of 0 or1 confirmed at
seventytwo hours.
Collaterals were scored in CT- angiography according a validated grading system that
ranged from 0 to 3 (0, absent collateral supply; 1, collateral supply filling <50%; 2,
collateral supply filling >50% but <100% of the occluded arterial territory; and 3, 100%
collateral supply).

Uric Acid Therapy Improves Clinical Outcome in Women With Acute Ischemic Stroke
Laura Llull, Carlos Laredo, Arturo Ren, Beln Prez, Elisabet Vila, Vctor Obach, Xabier Urra,
Anna Planas, Sergio Amaro and ngel Chamorro
Stroke. published online July 9, 2015;
Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright 2015 American Heart Association, Inc. All rights reserved.
Print ISSN: 0039-2499. Online ISSN: 1524-4628

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