Abstract
Objectives. Endothelial dysfunction is a well known risk factor for atherosclerosis. Uric acid levels are associated with endothelial dysfunction and atherosclerosis even if in physiological range. Xanthine oxidase inhibition with allopurinol decreases uric acid levels and oxidative stress and improves endothelial function. We have investigated the effect of high-dose and long-term allopurinol therapy on endothelial function in diabetic normotensive patients. Methods. This study is a randomized, single-blind, placebo-controlled trial. Both treatment and placebo groups consisted of 50 patients. In the treatment group, daily oral 900 mg allopurinol was started after randomization and maintained for 12 weeks. Brachial artery flow-mediated dilatation (FMD) and nitrate-induced dilatation (NID) were measured at baseline and after the allopurinol therapy to evaluate endothelial function. Results. HbA1c and uric acid levels decreased after allopurinol therapy (6.1 ± 2.1 vs 5.5 ± 1.0%, 5.0 ± 0.8 vs 3.3 ± 0.5 mg/dl, respectively, p = 0.01) but no change was observed in the placebo group (7.7 ± 1.9% vs 7.6 ± 2.0%, 5.3±2.1 vs 5.6 ± 0.8 mg/dl, respectively, p > 0.05). FMD and NID increased significantly in the treatment group (5.6 ± 2.1% vs 8.5 ± 1.2%, 10 ± 7.4% vs 14 ± 4.0%, 10 ± 7.4% vs 14 ± 4.0%, respectively, p = 0.01), whereas no change was observed in the placebo group (5.8 ± 1.8% vs 6.1 ± 0.8%, 12 ± 9.5 vs 10 ± 3.8%, respectively, p > 0.05). Conclusion. Long-term and high-dose allopurinol therapy significantly improved endothelial function in diabetic normotensive patients. In addition, allopurinol therapy contributes to the lower HbA1c levels.
Introduction
Uric acid (UA) is the end oxidation product of purine metabolism and it is associated with obesity, hypertension insulin resistance and endothelial dysfunction. It has been demonstrated that there is a prominent relationship between UA levels and cardiovascular risk and mortality even if in physiological ranges (Citation1,Citation2). It is well known that diabetic patients have increased oxidative stress with different degrees of endothelial dysfunction and increased cardiovascular risk (Citation3).
Endothelium-dependent flow-mediated dilatation (FMD) of the brachial artery is a method of detecting endothelial dysfunction. This method evaluates the capacity of endothelial cells, which respond to the pathological vasoconstrictive stimuli via enhanced production and bioavailability of nitric oxide and a consequent vasodilatation. The response of a peripheral artery to a shear stress reflects the endothelial function and correlates with the atherosclerotic involvement of coronary arteries (Citation4).
Allopurinol is a structural analogue of hypoxanthine and it decreases serum UA levels by inhibiting the xanthine oxidase (XO) enzyme, which also produces superoxide radicals (Citation5). Butler et al. (Citation6) demonstrated the ability of 300 mg allopurinol treatment to improve endothelial function in diabetic patients with systolic hypertension. However, the role of allopurinol therapy on endothelial function in normotensive diabetic patients is not clearly known.
In this study, we have investigated the effect of high-dose and long-term allopurinol therapy on endothelial function in diabetic normotensive patients.
Methods
Study design
This study was a randomized, single-blind, placebo-controlled trial. In present study, patients with diagnosed diabetes mellitus were included in both treatment and control groups. Diabetes mellitus was defined as a fasting blood glucose level of 127 mg/dl or greater, or a clinical diagnosis of diabetes with using oral antidiabetic agents or on insulin treatment. The exclusion criteria were hypertension, smoking, known heart disease, presence of congestive heart failure, history of coronary artery disease and other comorbid situations.
One hundred diabetic normotensive patients (39 women and 51 men, mean age 46 ± 2 years) were enrolled into the study among 362 screened diabetic patients. Age, gender, body mass index, duration of diabetes mellitus and blood pressures were recorded. After the baseline evaluation, patients were randomly selected to treatment or placebo groups. Both treatment and control groups consisted of 50 patients. In the treatment group, daily oral 900 mg allopurinol divided into three individual doses was started after randomization and maintained for 12 weeks (Urikoliz® 300 mg, Sandoz Ilaç San. ve Tic. A.S, Kocaeli, TR). Ultrasonographic and electrocardiographic examinations and biochemical analyses were performed in baseline term and at the end of the 12 weeks. The differences between the post-treatment and baseline UA levels were described as ΔUA.
This study was approved by the ethics committee and the institutional review board of Erciyes University Faculty of Medicine, and informed consent was obtained from each subject.
Biochemical analyses
Blood samples were taken at the beginning and at the end of the study (12 weeks later) Serum UA levels, high-sensitivity C-reactive protein, fasting blood glucose, hemoglobin A1c (HbA1c), total cholesterol, high-density lipoprotein (HDL)-cholesterol, low-density lipoprotein (LDL)-cholesterol and triglyceride levels were measured. Blood samples were drawn and centrifuged at 4°C. Measurements were performed Olympus Chemistry analyzer AU640-type 643-03 device (Manufactory Mishima Olympus Co. Ltd, Shizuoka, Japan).
Ultrasonographic examination
B mode and Doppler USG were used to assess brachial artery response to antiuricemic therapy as previously described (Citation7). Ultrasonographic measurements were performed at the onset of the study and at the end of the treatment (12 weeks later) by specialists of cardiology and radiology using commercially available machine (Vivid 7® GE Medical System, Horten, Norway) with a 7.5-MHz linear array transducer. All echocardiographic examinations were performed in a quiet and temperature-controlled room (22°C) before 12 a.m.
None of the patients had taken vasoactive agents such as angiotensin-converting enzyme (ACE) inhibitors, calcium-channel blockers or beta-blocker. The brachial artery was longitudinally scanned in the antecubital fossa after 10 min of rest in the supine position. When a proper position was found, the skin was marked for reference for later examinations. Measurements were taken at the end of diastole and the mean diameters were calculated from at least seven consecutive cardiac cycles. After measuring the baseline diameters, the blood pressure cuff was placed around the forearm and inflated with a pressure of 200 mmHg for 5 min. Therefore, ischemia was generated on the forearm and then the cuff was released. The second measurements of brachial artery were performed continuously from 30 s before to the 90 s after cuff deflation.
To evaluate endothelium-independent vasodilatation, 0.4 mg of sublingual nitroglycerine spray was administered after 10 min of rest. The last measurements were performed at 4–5 min after nitroglycerine. FMD was defined as a relative percentage change from the baseline diameter of brachial artery. Flow-mediated vasodilatation (FMD, %)=(reactive hyperemia diameter–baseline diameter) / baseline diameters × 100). The intra-observer and inter-observer coefficients (CV) of variations for overall FMD and nitrate-induced dilatation (NID) measurements were 3.2%, 2.8%, 4.1% and 3.3%, respectively. The differences between the post-treatment and baseline FMD and NID values were described as ΔFMD and ΔNID, respectively.
Statistically analyses
The Kolmogorov–Smirnov test was used to determine whether the distributions of continuous variables exist. We report continuous data as mean and standard deviation or median and interquartile range. Categorical data were compared with the chi-square test. Continuous variables were compared using unpaired t-tests or the Wilcoxon two-sample rank sum test between groups. ANOVA and paired t-test were used to evaluate repeated measures in groups. Pearson correlation coefficients were used to examine the degree of relationship between examined variables. p < 0.05 was accepted to be statistically significant. The SPSS 13 software program was used for statistical analysis.
Results
All patients continued to use initial medical therapy including same anti-diabetic treatment during 12 weeks observation. There were no statistically significant differences between study groups, regarding demographic characteristics and baseline laboratory findings, which have been summarized in . Systolic and diastolic blood pressures were in normal range in all patients. Most of patients were taking insulin, statin and aspirin therapy in both groups. Baseline serum UA levels were in normal range and similar between the groups (5.0 ± 0.8 vs 4.8 ± 1.1 mg/dl, p > 0.05).
Table I. Demographic characteristics of patients in treatment and control groups.
There were no statistically significant differences between the study groups, regarding the baseline diameters and the flow volumes of brachial artery, which is shown in . Impaired FMD and NID were determined in both groups.
Table II. Baseline diameter and blood flow of brachial artery in both groups.
Blood pressures and laboratory parameters of treatment group before and after allopurinol therapy are shown in . Systolic and diastolic blood pressures and lipid profiles of patients did not change. Statistically significant decrements were obtained in HbA1c and UA levels with allopurinol therapy (6.1 ± 2.1% vs 5.5 ± 1.0%, 5.0 ± 0.8 vs 2.3 ± 0.5 mg/dl, respectively, p = 0.01). However, there were no statistically significant differences in the placebo group, regarding laboratory parameters including HbA1c and UA levels (7.7 ± 1.9% vs 7.6 ± 2.0%, 5.3 ± 2.1 vs 5.6 ± 0.8 mg/dl, respectively, p > 0.05), which have been summarized in . Systolic and diastolic blood pressures and lipid profiles of patients also did not change in the placebo group.
Table III. Blood pressures and laboratory parameters in the treatment group.
Table IV. Blood pressures and laboratory parameters in the placebo group.
There was significant improvement in endothelial function after 12 weeks of allopurinol therapy. As shown in , FMD and NID increased significantly in the treatment group (5.6 ± 2.1% vs 9.5 ± 1.2%, 10 ± 7.4% vs 14 ± 4.0%, 10 ± 7.4% vs 14 ± 4.0%, respectively, p = 0.01). However, no change was observed at FMD and NID in the placebo group (5.8 ± 1.8% vs 6.1 ± 0.8%, 12 ± 9.5% vs 10 ± 3.8%, respectively, p > 0.05) as shown in .
Table V. The diameter and blood flow of brachial artery in the treatment group
Table VI. The diameter and blood flow of brachial artery in the placebo group.
Pearson correlation analysis was performed to evaluate the correlation between hypouricemic action of allopurinol and changes in FMD and NID in the treatment group. For this, the difference between the post-treatment and baseline FMD, NID and UA levels were described as ΔFMD, ΔNID and ΔUA, respectively, and all of this data were calculated. It has noted that ΔUA was significantly correlated with ΔFMD (r = 0.62, p = 0.001) and ΔNID (r = 0.58, p = 0.001) in the treatment group.
Discussion
In this study, we have firstly demonstrated that high-dose and long-term allopurinol therapy was well tolerated and has a favorable effect on endothelial function in normotensive patients with type 2 diabetes mellitus. Moreover, it has shown that allopurinol therapy may contribute to the decreased HbA1c levels.
Endothelial dysfunction has been accepted as an early stage of atherosclerosis and plays an important role in the development, progression and clinical complications of atherosclerosis (Citation8,Citation9). It has been accepted as a marker of subclinical cardiovascular disease. FMD is widely used in clinic practice for non-invasive evaluation of endothelial function. Impaired brachial FMD is related to the prevalence and extent of coronary atherosclerosis and predicts cardiovascular events (Citation10,Citation11). Previous studies suggested that improvement in endothelium-dependent vasodilatation with treatment was associated with decrement in cardiovascular risk (Citation12). Several studies demonstrated that FMD was lower in diabetic subjects when compared with control subjects (Citation13–15). Endothelial dysfunction is still a serious clinical issue in diabetic patients, although treatment with many different therapeutic agents such as statins and ACE inhibitors is useful.
In our study, we have determined that adding 900 mg allopurinol therapy to the standard treatment increased FMD levels from 5.6% to 9.5% in normotensive patients with type 2 diabetes mellitus after 12 weeks. Allopurinol therapy was well tolerated in all patients and no serious adverse effects occurred. Previously, several studies showed that XO inhibition with allopurinol improved endothelial function. In these studies, allopurinol improved endothelial function in patients with coronary artery disease and chronic heart failure and smokers (Citation16,Citation17). Similarly to our study, Butler et al. (Citation6) found that the allopurinol improved endothelial dysfunction in patients with type 2 diabetes mellitus with systolic hypertension (11 patients with type 2 diabetes and 12 healthy age-matched control subjects). However, in that study, factors including the presence of hypertension, smoking status and use of any medical therapy, which could affect endothelial function, were not excluded. Moreover, a 300-mg/day dose of allopurinol was used for 1 month and endothelial function was evaluated using only venous occlusion plethysmography. In another study, it was shown that endothelial function improved by 52% by increasing the allopurinol dose from 300 to 600 mg (Citation18). As a difference from this study, our patients used a 900-mg allopurinol dose for 3 months. We also excluded some conditions such as hypertension, smoking and ACE inhibitor use, which may affect endothelial function. In another study, Mercuro et al. (Citation19) investigated the effect of 300-mg allopurinol therapy for 3 months on endothelial function in hyperuricemic patients. Although this study provided useful information in patients with hyperuricemic and high cardiovascular risk, there is no information about normouricemic and diabetic patients. In our study, we investigated the effects of 900-mg allopurinol therapy in normouricemic diabetic patients. It was shown that the overall prevalence of the hyperuricemia was infrequent in different patient groups (approximate; 8–10%) (Citation20). Therefore, we think that our findings have more importance in clinical practice.
The antioxidant effect of allopurinol may be one of the underlying mechanisms. Previously, the presence of oxidative stress has been shown in diabetic patients (Citation3). Increased oxidative stress is one of the causes of developing early and severe atherosclerosis in diabetic patients. It was shown that XO increased in plasma and liver of diabetic rats and arterial rings produced superoxide in the presence of XO, and treatment with allopurinol decreased oxidative stress by preventing hemoglobin glycation, glutathione oxidation and lipid peroxidation (Citation21). XO may play an important role in the generation of oxidative stress and endothelial dysfunction (Citation22,Citation23). In a previous study, George et al. investigated the effects of allopurinol therapy on endothelial function in heart failure and authors state that the mechanism of improvement, in endothelial dysfunction, had occurred by through reducing vascular oxidative stress and not by urate reduction (Citation18). These observations suggested that prevention of oxidative stress and the inhibition of XO contribute to recovery from endothelial dysfunction.
The UA lowering effect of allopurinol may be other possible mechanism. It was shown that allopurinol improved endothelial function in hyperuricemic subjects but did not improve in normouricemic subjects (Citation19). In this study, 300 mg/day allopurinol therapy was used over 3 months. Some factors that could affect endothelial function such as hypertension and smoking had not been excluded in this study. Because of any change in normouricemic subjects, the authors suggested that the beneficial effect of allopurinol could have been a direct consequence of the reduced UA levels rather than of superoxide anions mediated by XO inhibition. In our study, although baseline UA levels were normal, it has been determined that UA levels decreased from 5.0±0.8 mg/dl to 2.3±0.5 mg/dl with 900-mg allopurinol therapy after 12 weeks. Therefore, we suggested that the favorable effects of allopurinol therapy on endothelial function may be dependent on both of antioxidant and UA lowering effects of allopurinol.
One of the important findings of our study is that allopurinol therapy may contribute to the decreased HbA1c levels. Borges et al. (Citation24) found that hyperuricemia was related with insulin resistance and development of metabolic syndrome, indicating potential benefits of lowering serum UA levels. Facchini et al. (Citation25) showed that urinary UA clearance appeared to decrease in proportion to increases in insulin resistance in normal volunteers, leading to an increase in serum UA concentration. Urukawa et al. (Citation26) showed that oxidative stress was associated with insulin resistance in men. Allopurinol therapy may have been decreased HbA1c levels by providing lower serum UA levels and reducing oxidative stress.
There are some limitations to our study. We did not measure oxidative stress markers. These measurements could provide important pathophysiological information. Although we have shown that FMD improved with allopurinol therapy, further double-blinded studies, with larger sample size, are needed to evaluate the effects of allopurinol therapy on both endothelial function and cardiovascular risk.
Conclusion
In this study, we demonstrated that long-term and high-dose allopurinol therapy may have favorable effects on endothelial function in normotensive diabetic patients. In addition, allopurinol therapy may contribute to the decreased HbA1c levels. However, further clinical studies are needed to confirm the clinical significance of long-term and high-dose allopurinol therapy.
Conflict of interest
None for all authors.
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