jurnal stroke.pdf

1The 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 antioxi-dant capacity.1 Uric acid (UA) is an end-product of the metabo-lism of purines and a potent antioxidant compound.2 Its multiple effects include scavenging of hydroxyl radicals, hydrogen per-oxide, 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 out-come, and reduced the amount of brain damage in experimen-tal 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 endog-enous 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

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 (URICO-ICTUS) 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

2 Stroke August 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 bio-logical 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 thera-pies, although further validation is required to be used as sur-rogate 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 ther-apy. Moreover, we analyzed the effect of therapy on circulat-ing markers of oxidative stress and on infarct growth.

MethodsThe URICO-ICTUS methodology has been described in detail previ-ously.13,17 In short, the trial was approved by local institutional re-view 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 PopulationThe 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 6 and

Llull et al Uric Acid Therapy and Stroke in Women 3

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 uni-variate analysis, UA therapy was more effective than placebo 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 NIHSS score at study onset was the only additional independent factor associ-ated with excellent outcome at 90 days in women (odd ratio [95% confidence interval], 0.844 [0.7900.902]; P


findings at baseline when compared with the whole popula-tion 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 (Table 2), 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 (MannWhitney, P=0.605; Figure 3). Correspondingly, there was a significant interaction between treatment and UA levels on infarct growth in women (P

Llull et al Uric Acid Therapy and Stroke in Women 5

in relation with a higher AL/UA ratio indicative of a greater nonenzymatic degradation of UA. On the whole, these find-ings 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 URICO-ICTUS data13 and previous descriptive series.30 We now report that women also had lower serum allantoin levels, but interest-ingly 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 radicalmediated oxidation of UA. 911 Accordingly, we also argue that

women obtained greater clinical benefits after UA replenish-ment than men because they were in a greater need of antioxi-dants as the result of their lower constitutional levels of UA. We also believe that a lower UA-mediated antioxidant capac-ity 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 dimor-phic 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 apopto-sis is a typical consequence of low to moderate concentrations of peroxynitrite,4 whereas exposure of cells to higher concen-trations 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 antiox-idant 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 evalu-ate the course of the infarct, or serum samples collected for evaluation of the redox status using a larger battery of oxida-tive 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 random-ized, 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 confound-ers. 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 conclu-sion, 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 thromboly-sis within 4.5 hours of clinical onset. Given the major prac-tical 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 FundingThe 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).

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 (%) 0.238

No 5 (20) 2 (10)

Early 13 (52) 16 (76)

Late or undetermined 7 (28) 3 (14)

ASPECTS indicates Alberta Stroke Program Early CT score; NVT, nonviable tissue; and TAR, tissue at risk.

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.

by guest on July 13, 2015http://stroke.ahajournals.org/Downloaded from


DisclosuresNone.

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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% but

Anna Planas, Sergio Amaro and ngel ChamorroLaura Llull, Carlos Laredo, Arturo Ren, Beln Prez, Elisabet Vila, Vctor Obach, Xabier Urra,

Uric Acid Therapy Improves Clinical Outcome in Women With Acute Ischemic Stroke

Print ISSN: 0039-2499. Online ISSN: 1524-4628 Copyright 2015 American Heart Association, Inc. All rights reserved.

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