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Xanthine Oxidase Inhibition With Oxypurinol Improves Endothelial Vasodilator Function in Hypercholesterolemic but Not in Hypertensive Patients

Originally publishedhttps://doi.org/10.1161/01.HYP.30.1.57Hypertension. 1997;30:57–63

    Abstract

    Abstract Hypercholesterolemic and hypertensive patients have impaired endothelium-dependent vasorelaxation because of decreased nitric oxide activity, but the mechanism underlying this abnormality is unknown. This study sought to determine whether an increased breakdown of nitric oxide by xanthine oxidase–generated superoxide anions could participate in these forms of endothelial dysfunction. We studied vascular responses to intrabrachial infusion of acetylcholine (an endothelium-dependent vasodilator, 7.5 to 30 μg/min) and sodium nitroprusside (a direct smooth muscle dilator, 0.8 to 3.2 μg/min) by strain-gauge plethysmography before and during the combined administration of oxypurinol (300 μg/min), a xanthine oxidase inhibitor, in 20 hypercholesterolemic patients, 20 essential hypertensive patients, and 20 normal subjects. The vasodilator response to acetylcholine was blunted in hypercholesterolemic (highest flow, 8.2±8 mL·min−1·dL−1) and hypertensive (8.5±4 mL·min−1·dL−1) patients compared with control subjects (13.8±6.6 mL·min−1·dL−1) (both P<.001); however, no differences were observed in the response to sodium nitroprusside. Oxypurinol did not change the response to acetylcholine in control subjects (P=.26) and improved, but did not normalize, its vasodilator effect in hypercholesterolemic patients (P<.01). Oxypurinol did not affect the response to acetylcholine in hypertensive patients (P=.34) and did not modify the response to sodium nitroprusside in any group. These results suggest that xanthine oxidase–generated superoxide anions are partly responsible for the impaired endothelial vasodilator function of hypercholesterolemic patients. In contrast, this mechanism does not appear to play a significant role in essential hypertension.

    The synthesis of nitric oxide (NO) by the vascular endothelium importantly contributes to the maintenance of vasodilator tone. Moreover, endothelium-derived NO inhibits the proliferation of smooth muscle cells, reduces platelet aggregability, and prevents leukocyte adhesion to the vascular surface.1 These relevant physiological actions in the regulation of cardiovascular homeostasis suggest that an impaired bioavailability of endothelium-derived NO may contribute to the development of the atherosclerotic process. Endothelial dysfunction related to decreased NO activity has previously been described in patients with known risk factors for atherosclerosis, such as hypercholesterolemia234 and arterial hypertension.567 However, the pathophysiological mechanisms underlying the NO abnormalities in these conditions remain unclear.

    Recent studies in animal models of hypercholesterolemia and hypertension have linked the pathogenesis of endothelial dysfunction with an increased degradation of NO. Principal among the substances involved in this breakdown process is superoxide anion, an avid scavenger of endothelium-derived NO.89 An increased production of superoxide radical has been directly assessed by chemiluminescence in the aortic wall of cholesterol-fed rabbits,1011 and chronic treatment with superoxide dismutase has been shown to be effective in restoring endothelium-dependent vasodilator function in this model of hypercholesterolemia.12 Previous studies have also shown that the administration of superoxide dismutase, a scavenger of superoxide anion,13 decreases blood pressure in hypertensive but not in normotensive rats,14 supporting the concept that superoxide anion might be increased in the arterial wall of spontaneously hypertensive rats and might trigger the development of hypertension, probably by inactivating the vasodilator effect of NO.

    Superoxide anions may be generated by different enzymatic and nonenzymatic sources. In the vascular endothelium, the xanthine oxidase system is one of the main sources of superoxide anion within and around endothelial cells, both directly and through the activation of circulating neutrophils.1516 Inhibition of xanthine oxidase can be achieved by oxypurinol, which has a molecular structure similar to that of xanthine and binds to xanthine oxidase, preventing the formation of uric acid and superoxide radicals.17

    In the present study, we tested the hypothesis that an increased activity of superoxide anions formed by the xanthine oxidase system could be involved in the decreased bioavailability of NO in patients with hypercholesterolemia and patients with essential hypertension. To this purpose, we assessed endothelium-dependent vascular relaxation to acetylcholine before and during the infusion of oxypurinol in control subjects and in hypercholesterolemic and hypertensive patients.

    Methods

    Study Population

    The clinical characteristics and lipid profiles of the 60 subjects who took part in the study are reported in Table 1.

    The hypercholesterolemic group included 20 patients selected for the study because their plasma cholesterol levels after a 12-hour fasting period were greater than 250 mg/dL. Eighteen of the 20 patients had low-density lipoprotein (LDL) plasma levels greater than 160 mg/dL. Their plasma high-density lipoprotein and triglyceride levels (Table 1) were not significantly different from those of the control group (P=.32 and P=.10, respectively). Patients had not taken any cholesterol-lowering agent within the previous 2 months or any antioxidant vitamin supplement in the preceding 6 months. No effort was made to change the patients’ diet before studies were performed. All hypercholesterolemic patients had normal blood pressure values.

    The hypertensive group included 20 patients with a history of chronically elevated blood pressure (≥145/95 mm Hg) without any apparent underlying cause who were followed at the outpatient clinic of the National Heart, Lung, and Blood Institute. Each patient had been previously treated with one or more antihypertensive agents for more than 3 years. Patients were asked to discontinue their current antihypertensive therapy 2 weeks before the study day; during that period, they were closely monitored for any evidence of accelerated or malignant hypertension. Patients in whom withdrawal of antihypertensive therapy was considered hazardous, mostly because of severely elevated blood pressure despite medications, were excluded from the study. All hypertensive patients had normal plasma cholesterol levels (<200 mg/dL).

    A population of 20 normal volunteers with no evidence of present or past hypertension and hypercholesterolemia (plasma cholesterol ≤200 mg/dL) was selected as a control group. They were matched with the patients of both experimental groups for approximate age and sex.

    Before admission, participants were screened by clinical history, physical examination, routine chemical analyses, electrocardiography, and chest radiography. Exclusion criteria were history or evidence of present or past diabetes mellitus, cardiac disease, peripheral vascular disease, coagulopathy, or any other disease predisposing them to vasculitis or Raynaud’s phenomenon. One normal volunteer, 1 hypercholesterolemic patient, and 3 hypertensive patients were smokers. Six normal volunteers, 4 hypercholesterolemic patients, and 5 hypertensive patients were postmenopausal women; none of them were on estrogen replacement therapy at the time of the study.

    All participants gave written informed consent for all procedures; the study protocol was approved by the National Heart, Lung, and Blood Institute Investigational Review Board.

    Protocol

    All studies were performed in the morning in a quiet room with a temperature of approximately 22°C. Participants were asked to refrain from drinking alcohol or beverages containing caffeine and from smoking for at least 24 hours before studies.

    Each study consisted of infusion of drugs into the brachial artery and measurement of the response of the forearm vasculature by means of strain-gauge venous-occlusion plethysmography. All drugs used in this study were approved for human use by the Food and Drug Administration in the form of Investigational New Drug (IND) and were prepared by the Pharmaceutical Development Service of the National Institutes of Health following specific procedures to ensure accurate bioavailability and sterility of the solutions.

    While the participants were supine, a 20-gauge polytetrafluoroethylene catheter (Arrow Inc) was inserted into the brachial artery of the nondominant arm (left in most cases). This arm was slightly elevated above the level of the right atrium, and a mercury-filled silicone elastomer strain gauge was placed on the widest part of the forearm.18 The strain gauge was connected to a plethysmograph (model EC-4, DE Hokanson) calibrated to measure the percent change in volume and connected in turn to a chart recorder to record flow measurements. For each measurement, a cuff placed around the upper arm was inflated to 40 mm Hg with a rapid cuff inflator (model E-10, Hokanson) to occlude venous outflow from the extremity. A wrist cuff was inflated to suprasystolic pressures 1 minute before each measurement to exclude hand circulation.19 Flow measurements were recorded for approximately 7 seconds every 15 seconds; seven readings were obtained for each mean value.

    Basal measurements were obtained after a 3-minute infusion of 5% dextrose solution at 1 mL/min. Forearm blood flow was measured after infusion of sodium nitroprusside and acetylcholine. Sodium nitroprusside was used as an endothelium-independent vasodilator because its vasodilator effect is largely due to its direct action on smooth muscle cells.20 Acetylcholine, in contrast, induces vasodilation by stimulating the release of relaxing factors from the vascular endothelium.21

    Sodium nitroprusside was infused at 0.8, 1.6, and 3.2 μg/min and acetylcholine chloride (Sigma Chemical Co) at 7.5, 15, and 30 μg/min (the infusion rates were 0.25, 0.5, and 1 mL/min, respectively, for each drug). Each dose was infused for 5 minutes, and forearm blood flow was measured during the last 2 minutes. A 30-minute rest period was allowed and another basal measurement obtained between infusions of the two drugs.

    Oxypurinol (Sigma), dissolved in 5% dextrose solution, was infused at 300 μg/min (infusion rate, 1 mL/min) for 30 minutes, and baseline flow measurements were obtained. The oxypurinol dose was chosen to achieve, at baseline flow conditions, an intravascular concentration of 10 μg/mL, which has been shown to achieve more than 90% inhibition of xanthine oxidase activity in the forearm blood vessels.22

    Subsequently, cumulative dose-response curves for acetylcholine and sodium nitroprusside were repeated during the concomitant infusion of oxypurinol using the same doses, infusion rates, and resting interval reported above. Oxypurinol infusion was continued during the resting period.

    The sequence of acetylcholine and sodium nitroprusside infusions, both before and after oxypurinol infusion, was randomized to avoid any bias related to the order of drug infusion.

    During the studies, participants were unaware of the drug being infused. All blood pressures were recorded directly from the intra-arterial catheter after each flow measurement. Forearm vascular resistance was calculated as the mean arterial pressure divided by the forearm blood flow.

    Statistical Analysis

    Differences among means of the three groups were analyzed by ANOVA followed by Dunnett’s test for post hoc comparisons between patients and control subjects when the global test showed statistical significance. Absolute values of hemodynamic variables were used for these comparisons when basal values were similar in patients and control subjects; however, because basal forearm vascular resistance was higher in the hypertensive group, changes in vascular resistance were expressed as percentage of baseline values. Oxypurinol effects on baseline hemodynamic variables were analyzed by paired Student’s t test. The responses to acetylcholine and sodium nitroprusside before and after oxypurinol were compared by ANOVA for repeated measures. All calculated probability values are two-tailed, and a value of P<.05 was considered statistically significant. All group data are reported as mean±SD, except in the figures, where values represent mean±SEM.

    Results

    Basal Blood Flow and Vascular Resistance

    Basal forearm blood flow, measured at the beginning of the study, was similar in control subjects and hypercholesterolemic and hypertensive patients (P=.43) (Table 2). As expected, basal forearm vascular resistance was significantly higher in hypertensive patients than control subjects (P<.01); basal vascular resistance did not differ significantly between control subjects and hypercholesterolemic patients (P=NS).

    Blood Pressure and Heart Rate

    Group differences in baseline mean arterial pressure and heart rate values are reported in Table 1. Throughout the study, during the infusion of the different substances, no significant change from baseline in mean arterial pressure and heart rate was observed in each of the three groups.

    Vascular Response to Acetylcholine and Sodium Nitroprusside

    As shown in Fig 1, the increase in blood flow and decrease in vascular resistance induced by acetylcholine infusion were significantly reduced in hypercholesterolemic and hypertensive patients compared with control subjects. Forearm blood flow measured at the highest dose of acetylcholine was 13.8±6.6 mL·min−1·dL−1 in control subjects compared with 8.2±8 in hypercholesterolemic patients and 8.5±4 in hypertensive patients (both P<.001). In contrast, no significant difference was observed among the three groups in the forearm blood flow and vascular resistance responses to sodium nitroprusside (P=NS) (Table 3).

    Effects of Oxypurinol on Basal Blood Flow

    As reported in Table 2, oxypurinol infusion did not produce any significant change in blood flow or vascular resistance in either control subjects (P=.77 and P=.80, respectively, compared with baseline values) or hypercholesterolemic (P=.93 and P=.65, respectively, compared with baseline values) and hypertensive (P=.96 and P=.89, respectively, compared with baseline values) patients.

    Effect of Oxypurinol on Vascular Responses to Acetylcholine and Sodium Nitroprusside

    In control subjects, the vasodilator response to acetylcholine was not significantly modified after oxypurinol infusion (forearm blood flow at the highest dose of acetylcholine was 15.2±7.6 mL·min−1·dL−1; P=.26 compared with values obtained without oxypurinol) (Fig 2). Although a trend was observed toward higher blood flow during oxypurinol, no such trend was found in vascular resistance, indicating that the slight changes in forearm blood flow were not related to changes in vascular tone induced by oxypurinol.

    In contrast, in hypercholesterolemic patients, both the increase in forearm blood flow and the decrease in vascular resistance were significantly greater after oxypurinol infusion (highest forearm blood flow, 9.8±7.3 mL·min−1·dL−1; lowest vascular resistance, 14±10.1 mm Hg·mL−1·min−1 · dL−1) (P<.01 and P<.05, respectively, compared with values obtained without oxypurinol) (Fig 3).

    In hypertensive patients, the response to acetylcholine was not significantly modified during concomitant oxypurinol infusion (Fig 4). Forearm blood flow responses to acetylcholine before and during oxypurinol administration were not significantly different (P=.34), although similar to findings in control subjects, there was a trend toward greater forearm blood flow during oxypurinol infusion (highest forearm blood flow, 9.9±5.2 mL·min−1·dL−1). However, no such trend was observed in the response to acetylcholine in terms of vascular resistance (P=.64); in fact, at the highest acetylcholine dose, vascular resistance without oxypurinol was 16.2±7.2 mm Hg·mL−1·min−1·dL−1 compared with 18.5±18.5 with oxypurinol.

    Oxypurinol infusion did not modify the vasodilator response to sodium nitroprusside in normal subjects or in hypercholesterolemic or hypertensive patients (Table 3).

    Discussion

    We designed the present study to investigate the possibility that an enhanced breakdown of NO by xanthine oxidase–generated superoxide anions may contribute to the impaired endothelium-dependent vasodilation in patients with hypercholesterolemia and in those with essential hypertension. We hypothesized that if the activity of superoxide anions produced by xanthine oxidase were increased in these conditions, then oxypurinol, by virtue of its antagonistic effect on xanthine oxidase, would allow a greater delivery of endothelium-derived NO to the underlying smooth muscle. This, in turn, would improve the abnormal response to acetylcholine previously reported in these patients.

    The main finding of our study was that oxypurinol infusion improved acetylcholine-induced vasodilation in hypercholesterolemic patients, an effect not observed in control subjects. This beneficial effect of oxypurinol was observed at all doses of acetylcholine infused, whereas no difference in basal vascular tone was found with oxypurinol administration. Because many regulatory mechanisms independent of endothelial release of NO contribute to the determination of basal vascular tone, these findings may indicate that increased NO breakdown by xanthine oxidase–generated superoxide anions affects vascular tone only in situations of stimulated NO release. The results of this study are in keeping with those of studies in animal models of hypercholesterolemia which have shown that exposure to oxypurinol for 30 minutes normalizes the increased production of superoxide anions and improves acetylcholine-induced relaxation only in aortic preparations from hypercholesterolemic rabbits, without any effect in control animals.10 These results have recently been confirmed in the same animal model after dietary correction of hypercholesterolemia for 1 month.23 The overproduction of superoxide anions in hypercholesterolemic animals appears to occur predominantly in endothelial cells, since endothelium removal normalizes superoxide anion generation.1012 Thus, the inactivation of NO within endothelial cells, or shortly after its release from the endothelium, is one possible mechanism by which superoxide anion excess leads to endothelial dysfunction. Moreover, endothelium-derived superoxide anions also participate in the oxidation of LDL.24 Oxidized LDL, in turn, inhibits endothelium-dependent relaxation by affecting either membrane receptors25 or intracellular signal transduction pathways26 and by degrading NO directly,27 further promoting endothelial dysfunction and atherosclerosis. It has recently been reported that lipoprotein(a), another atherogenic plasma lipoprotein, could also influence vascular tone in a fashion similar to that of oxidized LDL. In fact, exposure to oxidized lipoprotein(a) increased superoxide anion production and suppressed acetylcholine-induced dilator response in rabbit renal artery,28 suggesting that enhanced NO inactivation by reactive oxygen species could be the underlying mechanism for the impaired endothelium-dependent relaxation. Evidence that abnormal vascular oxidative stress plays an important role in the endothelial dysfunction of hypercholesterolemic animals stems from studies demonstrating that dietary antioxidants such as probucol increase LDL resistance to oxidative modification, prevent the increase in vascular superoxide generation, and normalize relaxation to acetylcholine even without lowering plasma cholesterol levels.29 Human studies seem to be in agreement with these findings, since combined LDL-lowering and antioxidant therapy has been shown to provide a greater improvement in the vasoconstrictor response to acetylcholine in patients with hypercholesterolemia and atherosclerosis than other LDL-lowering treatments.30

    When taken together, these observations emphasize the role of free radicals (especially superoxide anions) and oxidative metabolism in the endothelial dysfunction characteristic of dyslipidemia. We have previously investigated the possible involvement of extracellular superoxide anion generation in the pathogenesis of endothelial dysfunction in patients with hypercholesterolemia using copper-zinc superoxide dismutase (CuZn SOD) as a scavenger of superoxide anions.31 The infusion of CuZn SOD did not have any demonstrable effect on acetylcholine-induced vasorelaxation. Because CuZn SOD has poor intracellular penetrance because of its negative charge,32 those findings suggested that extracellular breakdown of NO is not responsible for the impaired endothelial responses. In contrast, the findings of the present investigation demonstrate that inhibition of xanthine oxidase–mediated formation of superoxide anions by oxypurinol improves endothelium-dependent vasodilator response in hypercholesterolemic humans. Thus, the present findings support the concept that excess vascular oxidative stress derived from the xanthine oxidase system is present within the endothelial cells in the microcirculation of these patients.

    It must be noted, however, that oxypurinol infusion improved but did not completely restore the abnormal endothelial vasodilator response to acetylcholine in hypercholesterolemic patients. In fact, oxypurinol restored only about 30% of the difference in the mean forearm blood flow response to the three acetylcholine doses between normal subjects and hypercholesterolemic patients. This suggests that the pathophysiology of endothelial dysfunction in hypercholesterolemic patients is probably multifactorial and only partly related to the generation of superoxide anions via the xanthine–xanthine oxidase pathway. Other sources of free radicals, such as the NADPH oxidase system33 or even a decreased bioavailability of l-arginine,3435 may also contribute to this abnormality. It is also possible that a greater potentiation of the response to acetylcholine could be observed with higher doses of oxypurinol. However, this is unlikely given the results of previous studies demonstrating that inhibition of xanthine oxidase is maximal with intravascular concentrations of oxypurinol similar to those achieved with the doses used in the present study participants.22

    In contrast to the findings in hypercholesterolemic patients, oxypurinol did not significantly modify the endothelium-dependent response to acetylcholine in patients with essential hypertension. Previous studies in animal models of hypertension have demonstrated that oxygen free radicals generated by xanthine oxidase cause greater vasoconstriction in arteries from spontaneously hypertensive rats than in those from normotensive animals36 and that xanthine oxidase inhibition by oxypurinol is effective in reducing blood pressure in spontaneously hypertensive rats but not in normotensive controls.14 These results are consistent with the possibility of xanthine oxidase–mediated increased production of oxygen free radicals that destroy endothelium-derived NO and contribute to the pathogenesis of high blood pressure in hypertensive animals. However, whether reactive oxygen species scavengers effectively improve the abnormal endothelial vasodilator function observed in different hypertensive rat models has not been reported. An alternative possibility is that the enhanced pressor effect of oxygen free radicals in hypertensive animals could be independent of endothelial production of NO. This explanation is supported by the observation that the vasoconstrictor effect of free radicals in hypertensive arteries is enhanced in both the presence and absence of endothelium.36

    Using CuZn SOD, we have previously tested the possibility that an increased extracellular oxidative stress in the arterial wall could be involved in the impairment of endothelial vasodilator function in hypertensive humans.37 Similar to the results of the current study, we observed that CuZn SOD administration did not result in improvement of the abnormal endothelium-mediated vasodilator function of these patients. The use of scavengers of superoxide anions that reach intracellular space may be helpful to further characterize the role of these oxygen free radicals in this condition. It must also be pointed out that the present observations are consistent with the concept that the endothelial dysfunction of hypertension is due to either reduced NO synthesis or to a non–NO-related mechanism, such as an increased production of vasoconstrictor prostanoids.3839

    In the present study, the vascular responses to sodium nitroprusside were similar in hypercholesterolemic and hypertensive patients compared with control subjects, confirming that the abnormality in endothelial vasodilator function of hypercholesterolemic and hypertensive patients is not related to an abnormal responsiveness of arterial smooth muscle cells to vasodilator stimuli. Oxypurinol infusion did not modify the response to sodium nitroprusside in any of the three subject groups. This observation further emphasizes the specificity of the beneficial effect of oxypurinol on the response to acetylcholine in hypercholesterolemic patients.

    In conclusion, the results of the present study demonstrate that oxypurinol improves the impaired endothelial vasodilator function of patients with hypercholesterolemia. This suggests that increased NO breakdown by superoxide anion generated via xanthine oxidase participates in the pathophysiology of this abnormality. In contrast, this mechanism does not appear to be involved in the abnormal endothelial function of patients with essential hypertension.

    Table 1. Clinical Characteristics of the Study Population

    CharacteristicNormal SubjectsHypercholesterolemic PatientsHypertensive Patients
    Sex, M/F10/1012/810/10
    Age, y53±652±750±7
    MAP, mm Hg81±1084±10113±101
    HR, bpm67±1065 ±1067±10
    Total cholesterol, mg/dL176±28287±431179±27
    LDL cholesterol, mg/dL113±26205±491112±26
    HDL cholesterol, mg/dL45±1448±1541±13
    Triglycerides, mg/dL97±66170±142133 ±79

    MAP indicates mean arterial pressure; HR, heart rate; LDL, low-density lipoprotein; and HDL, high-density lipoprotein.

    1P<.001 vs normal subjects. All other comparisons between each patient group and normal subjects were not statistically significant.

    Table 2. Baseline Forearm Blood Flow and Vascular Resistance Before and During Oxypurinol Infusion (300 μg/min)

    Normal SubjectsHypercholesterolemic PatientsHypertensive Patients
    ParameterBeforeDuringBeforeDuringBeforeDuring
    FBF, mL·min−1·dL−13.3±1.33.3±0.92.9±0.92.9±0.92.9±1.42.9±1.3
    FVR, mm Hg·mL−1·min−1·dL−129.3±13.928.9±10.231.3±13.431.9±11.546.5±18.9146.1±21.12

    FBF indicates forearm blood flow; FVR, forearm vascular resistance.

    1P<.01,

    2P<.01 vs normal subjects during oxypurinol.

    Table 3. Forearm Blood Flow and Vascular Resistance Responses to Sodium Nitroprusside Before and During Oxypurinol Infusion (300 μg/min)

    Normal SubjectsHypercholesterolemic PatientsHypertensive Patients
    ParameterBeforeDuringBeforeDuringBeforeDuring
    FBF, mL·min−1·dL−1
    SNP 0.8 μg/min6.8±2.36.3 ±2.46.4±1.86.1±1.66.1±2.65.7±2.4
    SNP 1.6 μg/min8.8±3.19±3.38.2±2.38.3±2.47.4±2.67.5±2.8
    SNP 3.2 μg/min10.7±411.3±410.7±2.610.8±3.59.4±3.49.8±3.5
    FVR, mm Hg·mL−1·min−1·dL−1
    SNP 0.8 μg/min13.4±4.315.6 ±6.114±4.515.8±7.722±9.224.3±11.3
    SNP 1.6 μg/min10.4±3.310.8±4.211.2±4.512.4±9.117.2±6.417.6±6.8
    SNP 3.2 μg/min8.7±38.6±3.48.4±2.79.9±8.213.7±5.213.3±4.4

    FBF indicates forearm blood flow; FVR, forearm vascular resistance; and SNP, sodium nitroprusside. All within-group comparisons of the effect of sodium nitroprusside before and during oxypurinol infusion were not statistically significant.

    
          Figure 1.

    Figure 1. Forearm blood flow and vascular resistance responses to acetylcholine in 20 normal control subjects (□), 20 hypercholesterolemic patients (▴), and 20 hypertensive patients (•). Values represent mean and SEM. P values refer to comparison of the curves obtained in hypercholesterolemic and hypertensive patients vs that of control subjects by ANOVA.

    
          Figure 2.

    Figure 2. Forearm blood flow and vascular resistance responses to acetylcholine in 20 normal control subjects before (○) and after (•) oxypurinol infusion. Values represent mean and SEM. P values refer to comparison of the two curves by ANOVA for repeated measures.

    
          Figure 3.

    Figure 3. Forearm blood flow and vascular resistance responses to acetylcholine in 20 hypercholesterolemic patients before (○) and after (•) oxypurinol infusion. Values represent mean and SEM. P values refer to comparison of the two curves by ANOVA for repeated measures.

    
          Figure 4.

    Figure 4. Forearm blood flow and vascular resistance responses to acetylcholine in 20 hypertensive patients before (○) and after (•) oxypurinol infusion. Values represent mean and SEM. Probability values refer to comparison of the two curves by ANOVA for repeated measures.

    Footnotes

    Correspondence to Julio A. Panza, MD, Cardiology Branch, National Institutes of Health, Building 10, Room 7B-15, Bethesda, MD 20892-1650. E-mail

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