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Keywords:

  • Arterial wave reflection;
  • oxidative stress;
  • stroke;
  • uric acid;
  • xanthine oxidase

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of Interest
  9. References

The importance of xanthine oxidase and its products is being increasingly recognized in cardiovascular medicine. Patients who have had a stroke are at high risk of future cardiovascular events and this risk is higher in those with high urate levels. The aim of this pilot study was to see if inhibiting xanthine oxidase altered arterial wave reflection, determined from the augmentation index (AIx). In a double-blind study, 30 patients with high urate (≥0.38 mmol/L) were randomized to 300 mg allopurinol or placebo for 8 weeks. AIx measurements were made before and after treatment using the validated SphygmoCor pulse waveform analysis system. For patients treated with allopurinol, there was a reduction in AIx from 26.08 ± 3.31% to 20.15 ± 2.23% compared with an increase in the placebo group from 23.57 ± 3.13% to 27.64 ± 3.44% (P= 0.031, ANOVA). The vascular benefits of allopurinol are rapidly emerging. We have demonstrated that allopurinol has beneficial effects on AIx, a validated measure of vascular function. A further larger study is warranted to look at whether a therapeutic intervention with allopurinol will impact positively on mortality and morbidity in stroke survivors.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of Interest
  9. References

The importance of xanthine oxidase and its products is being increasingly recognized in cardiovascular medicine. There are, however, two separate aspects to xanthine oxidase and its products. First, xanthine oxidase forms uric acid, which has been shown to be a marker of cardiovascular risk [1–3]. In most cases, this is independent of conventional risk factors for atherosclerosis [4]. However, it is unclear whether uric acid itself is a culprit or whether elevated uric acid level is a marker identifying asymptomatic vascular dysfunction.

The second and different aspect relates to the vascular effects of blocking xanthine oxidase. Allopurinol inhibits xanthine oxidase and therefore also reduces not only uric acid but also the formation of superoxide anions, which are formed in the same reaction as uric acid. Several studies have shown that allopurinol improves endothelial function [5,6], but its effect on measures of arterial stiffness, such as augmentation index (AIx), is not known and determining this was our aim. The impact of oxidative stress on cardiovascular function is well recognized and it is worth noting that the beneficial effects of xanthine oxidase inhibition on endothelial function appear to relate more to changes in oxidative stress, rather than to uric acid levels per se[7]. However, the vascular effects of allopurinol have never been studied before in stroke survivors despite their being known to suffer greatly from both cardiac events and recurrent stroke [8]. Thus, there are two novel aspects of our study: we studied stroke survivors, a high-risk group, for the first time and measured AIx also for the first time.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of Interest
  9. References

Patients

Thirty stroke survivors with high serum urate (≥0.38 mmol/L—the local upper limit of normal) were randomly selected from the stroke clinic at Ninewells Hospital and Medical School, Dundee, Scotland. Ethical approval for the study was obtained from the local ethics committee and all subjects gave written, informed consent. All study-related procedures were conducted according to institutional guidelines and the Declaration of Helsinki. Visits were conducted mid-afternoon. Subjects arrived fasted for 6-h, and refrained from smoking and drinking alcohol or caffeinated drinks for at least 12-h prior to study measurements. Following 15 min of rest, blood was withdrawn from the ante-cubital fossa for measurements of urate, urea and electrolytes, liver function tests, and plasma lipids. All urate measurements were available within 1 h to determine if the subject was eligible (i.e., urate (≥0.38 mmol/L) to take part in the study. A 12-lead ECG was recorded on a Burdick Eclipse 850 (Spacelabs, Hertford, UK). Blood pressure was recorded using a validated automated device (Omron705CPII; Omron Electronics Ltd, Milton Keynes, UK), and a full clinical history was taken including measurements of height and weight. The presence or absence of coronary heart disease and peripheral vascular disease was determined from a review of the medical notes. Presence of coronary heart disease included a documented history of angina/myocardial infarction requiring a cardiology review. Presence of peripheral vascular disease (PVD) was determined by a vascular surgeon documenting claudication pain and an ankle brachial pressure index (ABPI) <0.90.

Patients were randomly allocated to receive either allopurinol 300 mg once daily or matched placebo using a randomization sequence determined by the local hospital pharmacy. Measurements were taken at baseline and after 8 weeks of treatment. Patients attended nonfasted for one additional visit at 2 weeks for measurements of urea and electrolytes, liver function testing, and for monitoring of any adverse reactions.

Pulse Wave Analysis

Subjects were rested in a supine position for at least 10 min after which blood pressure was measured in triplicate using an automated blood pressure monitor (Omron705 CPII). An index of arterial stiffness was assessed noninvasively, using the validated SphygmoCor pulse waveform analysis system (AtCor Medical, Gloucestershire, UK) [9–11], by measuring the AIx, which is an estimate of systemic arterial and muscular stiffness. Peripheral pressure waveforms were recorded at the radial artery by applanantion tonometry using a high fidelity micromanometer. At least 15 high quality pressure waveform recordings were obtained from which the central aortic pressure waveform was calculated using a validated generalized transfer function. From the averaged aortic pulse wave, the AIx, defined as the augmented pressure divided by the pulse pressure and expressed as a percentage, was calculated. In addition, the AIx normalized for a heart rate of 75 beats per minute (AIx@75) was calculated to take into account the effect of heart rate on AIx [10,11]. The time to return of the reflected wave (Tr) was determined and used as an indirect marker of pulse wave velocity [10]. Assessments and data analyses were carried out by individuals who were blinded to the urate levels and also to whether the patients were receiving placebo or allopurinol.

Statistical Analysis

Values are expressed as means ± 1SD, unless stated otherwise. Baseline differences in variables between groups were examined using t-tests or χ2-tests. Before and after differences in AIx between the allopurinol and placebo treated groups were tested using ANOVA. Based on repeat measurements in 12 subjects on two separate occasions with a mean difference (SD) in AIx of 1.13 (5.08), eight subjects would be required to detect a before and after difference of 1SD (paired comparison) with 80% power at a 5% significance level. A P-value of <0.05 was considered significant. All analyses were performed using SPSS statistical package (version 13; SPSS Inc., Chicago, IL, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of Interest
  9. References

Clinical details and characteristics of the participants are given in Table 1. In the allopurinol treated group, six patients were diagnosed as having had an ischemic stroke, one a hemorrhagic event, and seven had a transient ischemic attack (TIA) where all symptoms had resolved within 24-h. Additionally, three patients in the allopurinol group had concomitant coronary artery disease and two had peripheral vascular disease. For the placebo group, eight patients had an ischemic stroke, one a hemorrhagic event, and five had a TIA. The diagnosis in all patients was confirmed by CT scan, which was carried out within 24-h of admission to the hospital. In the placebo group, four patients had concomitant coronary artery disease and two had peripheral vascular disease. No subject had type 1 diabetes and one subject in the allopurinol group had type 2 diabetes. The average time since the cerebrovascular accident was 138 ± 94 weeks and 145 ± 101 weeks in the allopurinol and placebo group, respectively. There was no change in drug therapy in any of the patients during the study period.

Table 1.  Baseline characteristics (mean ± SD) of patients in the placebo (n= 14) and allopurinol groups (n= 14)
ParameterPlacebo (n= 14)Allopurinol (n= 14)P-value
  1. Actual numbers are given in parentheses.

  2. SBP = systolic blood pressure; DBP = diastolic blood pressure; ACE = angiotensin coverting enzyme; HDL = high density lipoprotein.

Age (years)68.7 ± 10.368.2 ± 5.40.676
Height (m)1.71 ± 0.091.70 ± 0.090.641
Weight (kg)85.6 ± 17.480.0 ± 9.500.382
Body mass index (m2/kg)29.8 ± 8.227.8 ± 3.640.500
Males (%)79 (11)79 (11)1.000
Peripheral SBP (mmHg)129.0 ± 12.0137.6 ± 11.10.258
Peripheral DBP (mmHg)73.3 ± 7.179.7 ± 8.10.581
Pulse pressure (mmHg)56.1 ± 10.759.2 ± 14.50.198
Central SBP (mmHg122.0 ± 14.4120.7 ± 17.50.676
Central DBP (mmHg)74.4 ± 9.580.1 ± 10.10.450
Heart rate (beats/min)64.9 ± 12.465.1 ± 14.70.712
Smoking%
 Never42.9 (6)35.7 (5)0.499
 Former57.1 (8)57.1 (8)1.000
 Current0 (0)7.1 (1) 
Alcohol intake%
 None28.6 (4)14.3 (2)0.479
 ≤ 14 unit female or 21 unit male57.1 (8)78.6 (11)0.648
 >14 unit female or 21 unit male14.3 (2)7.1 (1)0.349
Drug treatment (%)
 ACE inhibitors50 (7)78 (11)0.373
 Beta blockers21 (3)28 (4)0.788
 Statins93 (13)87 (12)0.622
 Antiplatelet93 (13)93 (13)1.000
 Diuretics42 (6)50 (7)0.809
Creatinine (μmol/L)106.5 ±12.592.9 ± 25.40.185
Glucose (mmol/L)5.24 ±0.444.99 ± 0.330.289
Total chol (mmol/L)4.52 ±1.134.23 ± 0.730.434
HDL (mmol/L)1.46 ±0.341.41 ± 0.320.695
Triglycerides (mmol/L)1.44 ±0.921.37 ± 0.360.813
Urate (mmol/L)0.46 ±0.060.47 ± 0.070.933

Two patients were withdrawn from the study—one in the placebo arm due to suffering a further stroke during the study period, and another, on allopurinol, was treated for hyponatraemia. Data from 28 patients were therefore analyzed. Table 1 shows that the systolic and diastolic blood pressure was higher in the allopurinol group than in the placebo group, although this difference was not statistically significant. There were no other significant differences in baseline characteristics of the patients allocated to the placebo or allopurinol group (Table 1).

Placebo treatment had no significant effect on urate levels (0.46 ± 0.06 mmol/L vs. 0.47 ± 0.07 mmol/L), whereas allopurinol treatment significantly lowered urate levels from 0.47 ± 0.07 mmol/L to 0.24 ± 0.04 mmol/L (P < 0.001).

There were no significant changes in peripheral blood pressure before and after allopurinol (systolic 137.6 ± 11.1 mmHg to 133.3 ± 17.6 mmHg, diastolic 79.7 ± 8.1 mmHg to 78.8 ± 9.0 mmHg, respectively) or in central blood pressure (systolic 120.7 ± 17.5 mmHg to 113.6 ± 17.1 mmHg, diastolic 80.1 ± 10.1 mmHg to 77.2 ± 9.4, respectively). In the placebo group, peripheral systolic blood pressure did not change significantly (129.0 ± 12.0 mmHg to 130.3 ± 19.9 mmHg) but there was a significant reduction in diastolic blood pressure (73.3 ± 7.1 mmHg to 68.0 ± 7.4 mmHg, P= 0.008). There were no significant changes in central blood pressure (systolic 122.0 ± 14.4 mmHg to 119.7 ± 19.7 mmHg, diastolic 74.4 ± 9.5 mmHg to 72.6 ± 11.7 mmHg, respectively). Heart rate did not change significantly before and after allopurinol (64.9 ± 12.4 beats/min to 65.7 ± 15.4 beats/min, respectively) or placebo treatment (65.1 ± 14.7 beats/min to 60.0 ± 8.5 beats/min, respectively).

Comparing the differences in AIx before and after treatment showed that there was a significant reduction in the allopurinol group compared with changes in the placebo group (P= 0.031, ANOVA) (Table 2). Adjusting for changes in systolic and diastolic blood pressure in the ANOVA model did not alter the significant difference in AIx changes between the allopurinol and placebo group (P= 0.033). A significant difference between the allopurinol and placebo treated groups was also found for AIx@75 (P= 0.020) (Table 2), and adjustment for blood pressure did not alter the difference (P= 0.022). The allopurinol group showed a reduction in AIx of 7.00 ± 3.04% compared with an increase of 5.28 ± 3.89% in the placebo group (P= 0.021 comparing the absolute change in AIx). For AIx@75, the reduction was 5.15 ± 2.67% in the allopurinol group compared with an increase of 5.54 ± 3.74% in the placebo group (P= 0.029). In the allopurinol treated group, there was no correlation between the change in AIx@75 and the change in urate levels (r=−0.114, P= 0.711).

Table 2.  Changes in measures of arterial stiffness before and after treatment with placebo (n= 14) and allopurinol (n= 14)
 PlaceboAllopurinol
  1. The change in AIx and AIx@75 was significantly different between the allopurinol and placebo treated groups (P= 0.031 and P= 0.020, ANOVA, respectively). Values are means ± SEM. AIx = augmentation index; AIx@75 = augmentation index normalized for a heart rate of 75 beats per minute;

  2. Tr = time to return of the reflected wave.

AIx (%)
Before23.57 ± 3.1326.08 ± 3.31
After27.64 ± 3.4420.15 ± 2.23
AIx@75 (%)
Before19.65 ± 2.2521.47 ± 2.59
After23.76 ± 2.8616.32 ± 1.83
Tr (msec)
Before138.4 ± 2.6147.1 ± 7.5
After137.9 ± 44.7155.8 ± 10.6

Tr showed a small increase from 147.1 ± 7.5 msec to 155.8 ± 10.6 msec, suggestive of a decrease in pulse wave velocity, compared with reduction in the placebo group from 138.4 ± 2.6 msec to 137.9 ± 44.7 msec, but the difference between the groups did not reach statistical significance (P= 0.354).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of Interest
  9. References

Arterial stiffness is an established marker of cardiovascular risk and this association has been well validated in the literature [12–16]. In the present study in stroke survivors, we found that inhibition of xanthine oxidase with allopurinol, significantly improved a marker of arterial stiffness as shown by a 23% reduction in the AIx. The degree of reduction in AIx that we found following treatment with allopurinol compares favorably with other therapeutic intervention studies using arterial stiffness as a measure of vascular dysfunction [17]. Patients receiving the active and placebo treatment were well matched for secondary preventative treatments, with the exception of a slightly greater angiotensin-converting enzyme inhibitor use in the allopurinol group, and therefore concurrent therapy is unlikely to have a confounding effect of arterial stiffness. In spite of concurrent therapy known to improve vascular function, such as angiotensin-converting enzyme and HMG CoA reductase inhibitors, allopurinol was still able to show an improvement in arterial stiffness, suggesting that inhibition of xanthine oxidase may provide an additive mechanism for improving vascular function.

Inhibition of xanthine oxidase has two effects—to lower levels of uric acid and to reduce the formation of superoxide anions which are formed in the same reaction. The precise role that uric acid plays is not yet clear and there is much controversy about the effect of serum urate on vascular function. Elevated serum uric acid can increase the incidence of ischemic stroke [18,19] and it is also an independent predictor of poor vascular outcome in stroke survivors [20,21].

No previous study has examined the relationship between xanthine oxidase inhibition and arterial wave reflection in stroke patients. Our current data shows that allopurinol treatment significantly improves AIx, although the degree of improvement in AIx was not correlated with the amount of lowering of uric acid. Our finding is at odds with those from Mercuro et al. [22] who showed that the improvement in endothelial function correlated with the amount of urate lowering in patients at increased cardiovascular risk. However, similar to our results, Baldus et al. found that inhibition of xanthine oxidase with oxypurinol improved endothelial function in patients with coronary artery disease, independently of any change in uric acid levels [23]. Thus, it is possible that there may be an alternative mechanism by which allopurinol improves arterial wave reflection. One possible mechanism for the action of allopurinol on arterial wave reflection is inhibition of superoxide anion formation and therefore, a reduction in oxidative stress damage to the vascular wall [24]. Recent studies have confirmed that AIx and pulse pressure amplification correlate with superoxide anion production [12]. Our previous work on endothelial function has also shown that allopurinol is effective in patients with chronic heart failure and that allopurinol can completely abolish the oxidative stress that would have otherwise been sensitive to high dose vitamin C infusions [7]. Thus, our current results are entirely consistent with the known pharmacological actions of allopurinol in reducing superoxide anion-induced vascular stiffness. Uric acid may even be produced by the body as a counter-mechanism to the free radicals that are generated by xanthine oxidase action. This may explain why acute infusions of uric acid improve endothelial function [25]. When the oxidative stress burden is increased in various conditions (fe.g., by up-regulation of other systems such as NADPH oxidase as well as xanthine oxidase) and begins to overwhelm compensatory mechanisms, uric acid is no longer able to fully deal with the oxidative stress burden and becomes more useful as a marker of xanthine oxidase activity.

Recently, treatment with 300-mg allopurinol for 3 months in patients with hyperuricemia and normal renal function has been shown to lower C-reactive protein levels and blood pressure [26], and because elevated C-reactive protein levels are associated with increased AIx [27], it is possible that allopurinol might have affected the AIx through an antiinflammatory effect. Unfortunately, we did not obtain measures of C-reactive protein, although we found no significant changes in blood pressure with allopurinol treatment.

A limitation of our study is that we did not obtain measures of oxidative stress and therefore are unable to comment with certainty whether the induced changes in arterial function with lowering urate are a direct result of changes in oxidative stress, although this is a likely mechanism. Large-scale, controlled trials of serum urate lowering in high-risk individuals are required to determine causality. Another limitation is that blood pressure was higher in the allopurinol group than in the placebo group, although this difference was not statistically different. However, adjusting for systolic and diastolic blood pressure in the ANOVA model did not alter the significant difference in AIx changes between the allopurinol and placebo group.

The changes in AIx with allopurinol were modest, which might be a reflection of the relatively small sample size in this pilot study, and submaximal inhibition of xanthine oxidase. Nevertheless, we do not believe that our results have been obtained by chance. Based on our sample size calculations, eight subjects was sufficient to detect a change in AIx of 5.08 (we detected a change in AIx of 5.93) with 80% power at a significance level of P < 0.05. Improvements in vascular function with xanthine oxidase inhibition have been shown in previous studies using similar sample sizes [23]. The number of females in each group was small (n= 3), however analysis of data for males only did not change the overall outcome. Although our changes in AIx with allopurinol were independent of changes in urate levels, our study numbers were probably too small to carry out an accurate regression analysis to determine the independent effect of allopurinol on AIx. With respect to the sensitivity of AIx to reflect subtle changes in arterial stiffness, a recent study showed significant changes in AIx following inhibition of nitric oxide synthase using a smaller sample size than in the present study [28].

We appreciate that the gold standard measure of arterial stiffness is pulse wave velocity. AIx is mainly a function of the speed of wave travel, the amplitude of the reflected waves, and the elastic properties of the aorta, in addition to the duration and pattern of ventricular ejection. Nevertheless, AIx has also been shown to be an important determinant of cardiovascular outcome. It is an independent predictor of all-cause mortality in patients with end stage renal disease [29] and an independent marker for severity of coronary obstruction [30]. The time for return of the reflected wave, which is an indirect measure of pulse wave velocity, was increased in the allopurinol treated group compared with the placebo group, suggestive of a decrease in pulse wave transmission. However, this difference did not reach statistical significance and the study was not powered adequately to measure such a difference.

In conclusion, we have demonstrated in this pilot study a further possible benefit of allopurinol, that is, on the AIx, a validated measure of vascular function. Since the FDA approved allopurinol in 1966, it has primarily been used as an agent to treat gout. Studies showing its vascular benefits are emerging rapidly and the current study further highlights the potential of allopurinol as a low-cost and effective option in patients with high cardiovascular risk. A further larger study is warranted to look at whether a therapeutic intervention with allopurinol will impact positively on mortality and morbidity in patients who have survived a stroke.

What Is Known about the Topic

  • • 
    Patients who have had a stroke are at high risk of future cardiovascular events.
  • • 
    Uric acid has been shown to be a marker of cardiovascular risk.
  • • 
    Xanthine oxidase inhibition improves endothelial function, but its effect on arterial wave reflection is unknown.

What This Study Adds

  • • 
    Inhibition of xanthine oxidase with allopurinol significantly improves AIx, a marker of arterial stiffness.
  • • 
    The beneficial effect of allopurinol might be mediated through its effect on oxidative stress.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of Interest
  9. References

Chest, Heart and Stroke Scotland (Grant Ref: Res 02/ A60), Heart Research UK (Grant Ref 2464/02/05) provided funds to cover salary and consumable costs. They had no input in study design, data collection, data analysis, manuscript preparation, and/or publication decisions. This trial has been registered with the ISRCTN: Number 98638368.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Conflict of Interest
  9. References