Effect of vitamin E on resistance vessel endothelial dysfunction induced by methionine

Effect of Vitamin E on Resistance Vessel Endothelial Dysfunction Induced by Methionine Geetha Raghuveer, MD, Christine A. Sinkey, RN, Catherine Chenard, Phyllis Stumbo, PhD, and William G. Haynes, FRCPE

MS,

We tested if vitamin E, a fat-soluble antioxidant, prevents resistance vessel endothelial dysfunction caused by methionine-induced hyperhomocysteinemia in humans. Moderate elevations in plasma homocysteine concentrations are associated with atherosclerosis and hypertension. Homocysteine causes endothelial dysfunction possibly through several mechanisms. No previous study has tested if a fat-soluble antioxidant can prevent endothelial dysfunction caused by experimental hyperhomocysteinemia. Ten healthy subjects participated in a 2 ⴛ 2 factorial, double-blind crossover study, receiving L-methionine (100 mg/kg at – 6 hours) or vehicle, with and without vitamin E (1,200 IU at –13 hours). Endothelial function of forearm resistance vessels was assessed using forearm blood flow responses to brachial artery administration of endothelium-dependent and endothelium-independent agents. Forearm resistance vessel dilatation to acetylcholine was significantly impaired 7

hours after methionine (placebo, 583 ⴞ 87% vs methionine 30 ⴞ 68%; p <0.05). Dilatation to bradykinin was also impaired (placebo, 509 ⴞ 54% vs methionine 289 ⴞ 48%; p <0.05). Methionine did not alter vasodilatation to the endothelium-independent vasodilators, nitroprusside, and verapamil. Methionine-induced impairment of resistance vessel dilatation to acetylcholine and bradykinin (p <0.05 vs placebo) was prevented by administration of vitamin E (acetylcholine, p ⴝ 0.004; bradykinin, p ⴝ 0.004; both vs methionine alone). Experimentally increasing plasma homocysteine concentrations by oral methionine rapidly impairs resistance vessel endothelial function in healthy humans and this effect is reversed with administration of the fat-soluble antioxidant, vitamin E. 䊚2001 by Excerpta Medica, Inc. (Am J Cardiol 2001;88:285–290)

M

with enhanced lipid peroxidation in vivo.11 However, other studies have not observed changes in lipid peroxidation products in hyperhomocysteinemic humans.12 Lipid peroxidation and oxidation of low-density lipoprotein may contribute to endothelial dysfunction and development of atherosclerosis. Vitamin E is known to prevent lipid peroxidation. The effect of a fat-soluble antioxidant on methionine-induced endothelial dysfunction in humans has not been tested. Therefore, we studied the hypothesis that vitamin E, a fat-soluble antioxidant, prevents resistance vessel endothelial dysfunction caused by experimental hyperhomocysteinemia.

oderate elevation in plasma homocysteine is associated with atherosclerosis and hypertension.1,2 High concentrations of homocysteine have deleterious effects on vascular endothelium in vitro3 and in vivo,4 which may underlie its effects on atherosclerosis. Patients with hyperhomocysteinemia exhibit endothelial dysfunction.5,6 Experimental hyperhomocysteinemia induced by oral methionine produces endothelial dysfunction in conduit and resistance vessels.7,8 Homocysteine increases oxidant stress in vitro.9 We have previously shown that endothelial dysfunction caused by methionine administration can be reversed by oral administration of the water-soluble antioxidant vitamin C.10 High fasting plasma homocysteine concentrations are associated From the Departments of Pediatrics and Internal Medicine, and the General Clinical Research Center, University of Iowa College of Medicine, Iowa City, Iowa. This study was supported in part by the National Institutes of Health (HL58972; NCRR General Clinical Research Centers program: RR00059) and the Department of Veterans Affairs, Bethesda, Maryland. Dr Raghuveer received training Grant HL 07413 in Pediatric Cardiology from the National Institutes of Health, Bethesda, Maryland. Dr Haynes is supported by a Faculty Development Grant from the Pharmaceutical Research Manufacturers of America Foundation, Washington, DC. Manuscript received January 9, 2001; revised manuscript received and accepted February 21, 2001. Address for reprints: William G. Haynes, FRCPE, Department of Internal Medicine, University of Iowa College of Medicine, 200 Hawkins Drive, Iowa City, IA 52242. E-mail: william-g-haynes@ uiowa.edu. ©2001 by Excerpta Medica, Inc. All rights reserved. The American Journal of Cardiology Vol. 88 August 1, 2001

METHODS

Subjects: Ten healthy subjects without risk factors for or clinical evidence of atherosclerosis were recruited by advertisement. The studies were conducted after receiving written informed consent from each subject, and with the approval of the institutional review board. No subject received vasoactive drugs in the week before the study and all abstained from alcohol for 24 hours and from caffeine for at least 12 hours before any measurements were obtained. Studies were performed in a quiet room maintained at a constant temperature between 22°C and 25°C. Measurements: Forearm blood flow was measured in both arms before and after intra-arterial infusion of vasoactive agents using venous occlusion plethysmography with mercury-in-silastic strain gauges (model 0002-9149/01/$–see front matter PII S0002-9149(01)01642-3

285

9.8 11.8 12.3 6.1 13.1 11.6 6.2 17.9 7.8 5.2 10.2 ⫾ 1.2 277 338 551 522 261 306 499 265 565 863 436 ⫾ 59 8 17 20 16 17 16 20 6 15 20 15 ⫾ 2 426 374 486 498 263 387 420 226 593 455 400 ⫾ 35 80 92 79 75 85 90 84 69 78 77 81 ⫾ 2 91 115 40 64 78 99 83 73 151 65 88 ⫾ 9 51 48 55 49 73 39 65 44 38 68 53 ⫾ 4 131 101 40 80 70 106 52 69 128 53 91 ⫾ 13 168 183 103 129 165 159 158 131 215 144 159 ⫾ 10 1.76 1.90 1.74 1.62 1.89 1.78 1.72 1.74 1.80 1.79 1.80 ⫾ 3.20 75 79 63 43 94 70 75 77 95 72 76 ⫾ 5 20 M 23 M 24 F 19 F 22 M 24 M 27 M 28 F 19 M 42 F 25 ⫾ 2 6/4 1 2 3 4 5 6 7 8 9 10 Mean ⫾ SE

Fasting Homocysteine (␮mol/L) Erythrocyte Folate (7,260 ng/ml; range) Folate (3.2–20.1 ng/ml) Vitamin B12 (250–1,100 pg/ml) Glucose (mg/dl) LDL (mg/dl) HDL (mg/dl) Triglycerides (mg/dl) Cholesterol (mg/dl) Height (m) Weight (kg) Age (yrs) & Sex Subject Number

TABLE 1 Demographic Data

EC-4, Hokanson Inc., Bellevue, Washington). The left brachial artery was cannulated under local anesthesia with a 27-gauge steel needle attached to an 18-gauge epidural catheter. Baseline forearm blood flows were obtained during infusion of 0.9% saline (1 ml/min) for 30 minutes. Acetylcholine (3 to 30 ␮g/min, Iolab, Claremont, California), bradykinin (0.1 to 0.4 mg/ min, Clinalfa, AG, Switzerland), nitroprusside (1 to 10 ␮g/min, Elkins-Sinn, Cherry Hill, New Jersey), and verapamil (10 to 100 ␮g/min, Solopak Labs, Elk Grove Village, Illinois) were separately administered, with each dose infused for 6 minutes. The order of intra-arterial drugs was randomized (except for verapamil) with individual subjects receiving drugs in the same order on the 4 study days. Verapamil was always administered last because of its long duration of action. Forearm blood flow was measured in the last 3 minutes of each dose. Saline was infused for at least 12 minutes between drugs to allow blood flow to return to basal levels, with at least 90% reversal of vasodilatation. Arterial pressure was measured twice at baseline and after each dose. Laboratory assays: Plasma homocysteine assays were performed in the University of Iowa General Clinical Research Center Core Lab, by high performance liquid chromatography as previously described.10 Plasma vitamin E, B12, plasma and red cell folate, cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol were measured using standard methods. Study design: This was a 4-phase, randomized, double-blind, placebo-controlled, 2 ⫻ 2 factorial crossover study comparing the effects of oral L-methionine and placebo, with and without vitamin E, on endothelial function with a washout period of at least 1 week between the study days. The 4 phases were: (1) placebo vehicle at 7 A.M.; (2) L-methionine 100 mg/kg at 7 A.M.; (3) vitamin E 1,200 IU at midnight followed by placebo at 7 A.M., and (4) vitamin E 1,200 IU at midnight followed by L-methionine at 7 A.M. Vitamin E, at concentrations similar to those achieved in humans after 1,200 IU orally, improves endothelial function in hypercholesterolemic animals.13,14 Vitamin E was administered 14 hours before assessment of endothelial function, because this is the time of maximal plasma concentrations after single-dose oral administration.15,16 The L-methionine dose was chosen based on previous studies in which plasma homocysteine concentrations increased fourfold after 6 to 8 hours.10 Subjects were admitted to the University of Iowa General Clinical Research Center the night before each study. An antecubital vein of the noninfused arm was cannulated with a 22-gauge catheter for blood sampling. Vitamin E was given orally at midnight at phases 3 and 4. Subjects received oral L-methionine 100 mg/kg dissolved in cranberry juice, or cranberry juice alone, at about 7 A.M. on phases 2 and 4. A standard breakfast containing 70 mg of L-methionine was served 2 hours after methionine or placebo administration at all phases. Venous blood for assay of plasma homocysteine was obtained before and 6 and 8 hours after L-methionine/placebo administration.

286 THE AMERICAN JOURNAL OF CARDIOLOGY姞

VOL. 88

AUGUST 1, 2001

TABLE 2 Arterial Pressure and Heart Rate on Different Study Days Placebo/Placebo

Placebo/Meth

Vit E/Placebo

Vit E/Meth

Mean Arterial Pressure (mm Hg) Baseline Acetylcholine Bradykinin Nitroprusside Verapamil

89 89 88 88 92

⫾ ⫾ ⫾ ⫾ ⫾

2 2 2 2 3

86 86 86 85 90

⫾ ⫾ ⫾ ⫾ ⫾

2 2 3 2 2

89 88 89 87 93

⫾ ⫾ ⫾ ⫾ ⫾

2 2 2 2 2

Heart Rate (beats/min) Baseline Acetylcholine Bradykinin Nitroprusside Verapamil

58 58 60 59 59

⫾ ⫾ ⫾ ⫾ ⫾

3 3 3 2 2

58 57 57 57 61

⫾ ⫾ ⫾ ⫾ ⫾

2 2 2 2 2

60 60 89 61 60

⫾ ⫾ ⫾ ⫾ ⫾

Meth ⫽ methionine; Vit ⫽ vitamin.

Plasma vitamin E levels were estimated at midnight, 7 A.M., 1 and 3 P.M. (i.e., before and 7, 13, and 15 hours after vitamin E administration). Resistance vessel vasomotor function was assessed from 6 to 8 hours after administration of L-methionine/placebo. Data analysis and statistics: All analyses were performed by observers blinded to treatment assignment. Basal blood flow and blood pressure were obtained as the average of the 3 baseline recording periods during saline infusion. In addition to absolute blood flows, we calculated percent change from baseline in the ratio of blood flow between infused and noninfused arms, because this halves variability in blood flow responses to infused agents.17 Two-way repeated measure analysis of variance was used to compare the effects of methionine and placebo on resistance vessel dilatation to vasoactive agents. All doses of drugs were used in the analysis of variance. Tukey’s test was used for post hoc analysis if the analysis of variance was positive. Data are expressed as mean ⫾ SE. A p value ⬍0.05 was considered statistically significant. Data were analyzed using StatView software (Brainpower Inc., Calabasas, California).

RESULTS

Demographics: The study group included 10 subjects (6 men) aged between 19 and 42 years, with normal blood pressure, lipids, glucose, and B-vitamin levels (Tables 1 and 2). Effect of experimental hyperhomocysteinemia on endothelial function: Homocysteine levels were normal at

baseline (Table 1). Methionine loading slowly increased plasma homocysteine concentrations fourfold to a maximum of 44.8 ⫾ 0.8 ␮mol/L at 8 hours (Figure 1). Baseline blood pressure, heart rate, and forearm blood flow did not differ between study days (Tables 2 and 3). Brachial artery infusion of each vasodilator agent significantly increased forearm blood flow in the infused arm on each study day (p ⬍0.05), but did not alter blood pressure or blood flow in the noninfused arm, confirming that drug effects were confined to the infused arm (Tables 2 and 3).

2 2 2 3 2

88 90 90 88 93

⫾ ⫾ ⫾ ⫾ ⫾

1 2 2 1 2

However, methionine significantly impaired dilatation to acetylcholine (p ⫽ 0.0034) and bradykinin (p ⬍0.001 vs placebo; Figures 2 [left] and 3 [left]). Nitroprusside and verapamil responses did not differ between the methionine and placebo days (Table 3). Effect of vitamin E on homocysteine-induced endothelial dysfunction:

The increase in plasma homocysteine produced by methionine ad57 ⫾ 2 ministration was not altered by vita58 ⫾ 3 min E (Figure 1). Plasma ␣-tocoph58 ⫾ 3 59 ⫾ 2 erol increased approximately 62 ⫾ 3 twofold 13 and 15 hours after administration of vitamin E (i.e., 6 to 8 hours after methionine or placebo) (Figure 4). Endothelium-dependent dilatation tended to be lower after vitamin E administration, but this did not achieve statistical significance (Table 3, acetylcholine, p ⫽ 0.365; bradykinin, p ⫽ 0.293). Methionine-induced impairment of resistance vessel dilatation to acetylcholine and bradykinin (p ⬍0.05 vs placebo) was prevented by administration of vitamin E (acetylcholine, p ⫽ 0.004; bradykinin, p ⫽ 0.004; both vs methionine alone) (Figures 2 [right] and 3 [right]). Vitamin E and methionine, when administered alone or in combination, did not alter forearm blood flow response to intra-arterial administration of the endothelium-independent vasodilators nitroprusside and verapamil (Table 3).

DISCUSSION In this study we have described for the first time the effects of vitamin E administration on resistance vessel endothelial dysfunction caused by acute methionine loading in humans. We confirm our previous observations that experimental elevation of plasma homocysteine concentrations rapidly impairs resistance vessel endothelium-dependent dilatation to acetylcholine in human subjects.10 We have now demonstrated that endothelial dysfunction also occurs with a different endothelium-dependent dilator, bradykinin. Endothelial dysfunction could be reversed by administration of the lipid soluble antioxidant, vitamin E. These data support the hypothesis that reversal of methionine induced endothelial dysfunction by vitamin C in previous studies was due to the antioxidant effects of vitamin C. Oxidant effects of homocysteine: Homocysteine increases generation of free radical oxidant species in vitro through a number of mechanisms including autooxidation, inhibition of glutathione peroxidase, and oxidation of low-density lipoprotein.9,11,18 Several studies have shown increased lipid oxidation caused by homocysteine. Experimentally induced hyperhomocysteinemia in pigs is associated with increased cardiac malondialdehyde and unsaturated fatty acid concentrations.19 In a human study using methionine loading, there was a linear correlation between plasma

CARDIOVASCULAR PHARMACOLOGY/VITAMIN E PREVENTS ENDOTHELIAL DYSFUNCTION

287

FIGURE 1. Effect of oral L-methionine (Meth) (100 mg/kg) or placebo with and without vitamin E on plasma homocysteine concentrations (open square, placebo; open circle, L-methionine; solid square, placebo and vitamin E; solid circle, L-methionine and vitamin E). *p <0.05 versus baseline.

TABLE 3 Forearm Blood Flow Data Placebo/Placebo

Placebo/Meth

Vit E/Placebo

Vit E/Meth

Forearm Blood Flow Infused (ml/100 ml/min) Baseline Pre-Acetylcholine Acetylcholine Pre-Bradykinin Bradykinin Pre-Nitroprusside Nitroprusside Pre-Verapamil Verapamil

2.6 ⫾ 0.2 4.0 ⫾ 0.8 16.9 ⫾ 2.9 2.90 ⫾ 0.7 14.8 ⫾ 2.2 3.6 ⫾ 0.6 13.9 ⫾ 1 3.5 ⫾ 0.3 16 ⫾ 3.3

2.8 ⫾ 0.2 3.4 ⫾ 0.5 12.3 ⫾ 2.1* 3.8 ⫾ 0.3 12 ⫾ 1.8* 3.4 ⫾ 0.5 15.6 ⫾ 0.9 4.0 ⫾ 0.4 15.5 ⫾ 1.5

3.1 ⫾ 0.3 3.9 ⫾ 0.5 19.0 ⫾ 2.6 4.0 ⫾ 0.5 15.7 ⫾ 1.9 3.7 ⫾ 0.4 16.9 ⫾ 2.2 3.9 ⫾ 0.4 12.9 ⫾ 1.6

3.5 ⫾ 0.4 4.5 ⫾ 0.6 19.7 ⫾ 1.6 4.2 ⫾ 0.4 16.8 ⫾ 1.5 4.0 ⫾ 0.3 17.7 ⫾ 1.9 4.3 ⫾ 0.3 14.2 ⫾ 1.6

Forearm Blood Flow Noninfused (ml/100 ml/min) Baseline Pre-Acetylcholine Acetylcholine Pre-Bradykinin Bradykinin Pre-Nitroprusside Nitroprusside Pre-Verapamil Verapamil

2.0 1.9 1.9 2.0 1.9 2.0 1.7 2.2 2.3

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.2 0.2 0.2 0.7 0.3 0.6 0.3 0.3 0.4

2.5 2.5 2.6 2.7 2.8 2.5 2.5 2.8 2.9

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.2 0.2 0.2 0.3 0.2 0.5 0.2 0.4 0.2

2.3 2.2 2.3 2.2 2.0 2.2 2.0 2.4 2.5

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.3 0.3 0.3 0.5 0.3 0.4 .3 0.4 0.3

2.7 2.7 2.5 2.6 2.5 2.6 2.4 2.8 2.6

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.3 0.4 0.3 0.4 0.3 0.3 0.3 0.3 0.3

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

12 81 14 67 8 73 13 83

— 45 626 41 481 27 562 39 401

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

19 132 20 78 12 83 18 78

% ⌬ in Ratio (infused/noninfused) Baseline Pre-Acetylcholine Acetylcholine Pre-Bradykinin Bradykinin Pre-Nitroprusside Nitroprusside Pre-Verapamil Verapamil

— 57 583 58 509 31 594 41 526

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

20 87 15 54 12 36 13 114

— 26 330 33 289 27 499 34 436

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

13 68* 10 48* 14 30 11 64

— 24 483 28 475 18 508 29 355

* p ⬍0.05 versus placebo only, vitamin E only, vitamin E and methionine. Data are shown for highest dose of each drug. Abbreviations as in Table 2.

288 THE AMERICAN JOURNAL OF CARDIOLOGY姞

VOL. 88

AUGUST 1, 2001

homocysteine and malondialdehyde and thiobarbituric acid–reactive substance levels, which are indicators of lipid peroxidation.20 Elevated total plasma homocysteine levels in human subjects are linearly correlated with plasma F2–isoprostane levels, a marker of lipid peroxidation.11 Thus, there are limited and contradictory data on whether homocysteine causes lipid peroxidation in humans. Vitamin E, oxidant stress, and endothelial dysfunction: Vitamin E is a

potent scavenger of reactive oxygen species. Vitamin E deficiency alone induces hepatic lipid peroxidation in rats, and maintenance of adequate or higher levels of vitamin E acts as a protective factor against free radical generation.21 Endothelium-dependent relaxation and low-density lipoprotein oxidation in hypercholesterolemic rabbits normalizes after vitamin E therapy.22 Human studies with vitamin E have been less clear. There was a linear correlation between plasma vitamin E levels and endothelium-dependent dilatation of coronary vessels.23 However, vitamin E (1,000 IU/day) for 10 weeks did not improve flow-mediated vasodilatation in conduit vessels in older patients with age-related endothelial dysfunction.24 In contrast, vitamin E caused improvement in basal and simulated

FIGURE 2. Left and right, forearm vasodilatation to intra-arterial administration of acetylcholine after placebo or oral L-methionine with and without vitamin E. Left, dilatation to acetylcholine after placebo (open boxes) and methionine (closed boxes) (n ⴝ 10). Right, dilatation to acetylcholine after placebo (open circles) and methionine (solid circles) when pretreated with vitamin E (n ⴝ 10). *p <0.05 versus methionine without vitamin E.

FIGURE 3. Left and right, forearm vasodilation to intra-arterial administration of bradykinin after placebo or oral L-methionine with and without vitamin E. Left, dilatation to bradykinin after placebo (open boxes) and methionine (closed boxes) (n ⴝ 10). Right, dilatation to bradykinin after placebo (open circles) and methionine (solid circles) when pretreated with vitamin E (n ⴝ 10). *p <0.05 versus methionine without vitamin E.

nitric oxide–related resistance vessel endothelial function in subjects with hypercholesterolemia.25 Our previous study and other studies using vitamin C have shown that a water-soluble antioxidant can completely reverse methionine-induced endothelial dysfunction in humans.10,26 However, it is possible that reversal of endothelial dysfunction by vitamin C is not due to its antioxidant effects, but due to other actions of ascorbic acid, such as changes in protein synthesis. The improvement in endothelial dysfunction by vitamin E alone in this study suggests that reversal of endothelial dysfunction by vitamin C may be due to its antioxidant effects. The beneficial effect of vitamin E may have been due to a mechanism distinct from its antioxidant effects, such as inhibition of platelet aggregation by a protein kinase C-dependent mechanism.27 However, given the protective effect of 2 distinct antioxidants, it appears likely that alterations in oxidant stress underlie methionine-induced endothelial dysfunction. During hyperhomocysteinemic states, soluble oxidant radicals may be produced initially, and these then may cause lipid peroxidation and endothelial dysfunction. Thus, vitamins C and E may block different stages of the pathophysiologic process by which homocysteine causes vascular injury. Further studies are needed to confirm this mechanism of action of vitamin E in hyperhomocysteinemic states. Potential limitations: Our model for inducing experimental hyperhomocysteinemia uses oral methionine loading. A nonspecific effect of amino acids on endothelial function appears unlikely, because high concentrations of amino acids such as L-arginine and N-acetylcysteine do not impair endotheliumdependent relaxation in humans.28,29 However it is possible that endothelial dysfunction in this model is caused by increased methionine rather than homocysteine concentrations. However, a recent report has shown similar endothelial dysfunction after oral homocysteine administration as occurs after oral methionine.30 Although our results suggest that homocysteine may act to increase lipid peroxidation, this specific mechanism was not explored in this study and is thus speculative. The differences in forearm blood flow responses are unlikely to be due to the

CARDIOVASCULAR PHARMACOLOGY/VITAMIN E PREVENTS ENDOTHELIAL DYSFUNCTION

289

FIGURE 4. Plasma vitamin (Vit) E levels on the 4 study days (open boxes, placebo; open circle, L-methionine; solid box, placebo and vitamin E; solid circle, L-methionine and vitamin E. *p <0.05 versus placebo only and methionine only.

antioxidant effect of cranberry juice, because this was used as a vehicle during all the 4 sessions. In addition, it is possible that antioxidant therapy with vitamin C or E will not prevent endothelial dysfunction in patients with hyperhomocysteinemia due to causes other than high methionine intake (i.e., folate or vitamin B6 deficiency). The protective effect of vitamin E was demonstrated in healthy subjects in this study. We cannot extrapolate these findings in situations in which there may be other confounding risk factors for endothelial dysfunction such as smoking or hypercholesterolemia. Our study tested only the acute effects of elevated homocysteine and vitamin E. It is possible that any vascular injury caused by more chronic elevation in homocysteine will not be reversible with vitamin E, or that chronic vitamin E therapy will not exhibit the benefits of acute treatment. Clinical intervention trials may provide answers to this question. 1. Sutton-Tyrell K, Bostom A, Selhub J, Zeigler-Johnson C. High homocysteine

levels are independently related to isolated systolic hypertension in older adults. Circulation 1997;96:1745–1749. 2. Nygard O, Nordrehaug JE, Refsum H, Ueland PM, Farstad M, Vollset SE. Plasma homocysteine levels and mortality in patients with coronary artery disease. N Engl J Med 1997;337:230 –236. 3. Stamler JS, Osborne JA, Jaraki O, Rabbani LE, Mullins M, Singel D, Loscalzo J. Adverse vascular effects of homocysteine are modulated by endotheliumderived relaxing factor and related oxides of nitrogen. J Clin Invest 1993;91: 308 –318. 4. Lentz SR, Sobey CG, Piegors DJ, Bhopatkar MY, Faraci FM, Malinow MR, Heistad DD. Vascular dysfunction in monkeys with diet-induced hyperhomocyst(e)inemia. J Clin Invest 1996;98:24 –29. 5. Celermajer DS, Sorensen K, Ryalls M, Robinson J, Thomas O, Leonard JV, Deanfield JE. Impaired endothelial function occurs in the systemic arteries of children with homozygous homocystinuria but not in their heterozygous parents. J Am Coll Cardiol 1993;22:854 – 858.

290 THE AMERICAN JOURNAL OF CARDIOLOGY姞

VOL. 88

6. Tawakol A, Omland T, Gerhard M, Wu JT, Creager MA. Hyperhomocyst(e)inemia is associated with impaired endothelium-dependent vasodilation in humans. Circulation 1997;95:1119 –1121. 7. Chambers JC, McGregor A, Jean-Marie J, Kooner JS. Acute hyperhomocysteinaemia and endothelial dysfunction. Lancet 1998;351:36 –37. 8. Bellamy MF, McDowell IFW, Ramsey MW, Brownlee M, Bones C, Newcombe RH, Lewis MJ. Hyperhomocysteinemia after an oral methionine load acutely impairs endothelial function in healthy adults. Circulation 1998;98:1848 – 1852. 9. Starkebaum G, Harlan JM. Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine. J Clin Invest 1993;77:1370 – 1376. 10. Kanani PM, Sinkey CA, Browning RL, Allaman M, Knapp HR, Haynes WG. Role of oxidant stress in endothelial dysfunction produced by experimental hyperhomocysteinemia in humans. Circulation 1999;100:1161–1168. 11. Voutilainen S, Morrow JD, Roberts LJ, Alfthan G, Alho H, Nyyssonen K, Salonen JT. Enhanced in vivo lipid peroxidation at elevated plasma total homocysteine levels. Arterioscler Thromb Vasc Biol 1999;19:1263–1266. 12. Chao CL, Kuo TL, Lee YT. Effects of methionine-induced hyperhomocysteinemia on endothelium-dependent vasodilation and oxidative status in healthy adults. Circulation 2000;485– 490. 13. Keaney JF Jr, Gaziano JM, Xu A, Frei B, Curran-Celentano J, Shwaery GT, Loscalzo J, Vita JA. Low-dose alpha-tocopherol improves and high-dose alphatocopherol worsens endothelial vasodilator function in cholesterol-fed rabbits. J Clin Invest 1994;93:844 –951. 14. Dieber-Rotheneder M, Puhl H, Waeg H, Streigl G, Esterbauer H. Effect of oral supplementation with D-alpha-tocopherol on the vitamin E content of human low density lipoproteins and resistance to oxidation. J Lipid Res 1991;32:1325– 1332. 15. Ferslew KE, Acuff RV, Daigneault EA, Woolley TW, Stanton PE Jr. Pharmacokinetics and bioavailability of the RRR and all racemic stereoisomers of alpha-tocopherol in humans after single oral administration. J Clin Pharmacol 1993;33:84 – 88. 16. Cheeseman KH, Holley AE, Kelly FJ, Wasil M, Hughes L, Burton G. Biokinetics in humans of RRR-alpha-tocopherol: the free phenol, acetate ester, and succinate ester forms of vitamin E. Free Radic Biol Med 1995;19:591–598. 17. Petrie JR, Perry C, Cleland SJ, Murray LS, Elliott HL, Connell JMC. Forearm plethysmography: does the right arm know what the left is doing? Clin Sci 2000;98:209 –210. 18. Upchurch GR, Welch GN, Fabian AJ, Freedman JE, Johnson JL, Keaney JF, Loscalzo J. Homocyst(e)ine decreases bioavailable nitric oxide by a mechanism involving glutathione peroxidase. J Biol Chem 1997;272:17,012–17,017. 19. Young PB, Kennedy S, Molloy AM, Scott JM, Weir DG, Kennedy DG. Lipid peroxidation induced in vivo by hyperhomocysteinemia in pigs. Atherosclerosis 1997;129:67–71. 20. Domagala TB, Libura M, Szczeklik A. Hyperhomocysteinemia following oral methionine load is associated with increased lipid peroxidation. Thromb Res 1997;4:411– 416. 21. Vannuchi H, Junior AAJ, Iglesias AC, Morandi MV, Chiarello PG. Effect of different dietary levels of vitamin E on lipid peroxidation in rats. Arch Latino Am Nutr 1997;471:34 –37. 22. Boger RH, Bode-Boger SM, Phivthong-Ngam L, Brandes RP, Schwedhelm E, Mugge A, Bohme M, Tsikas D, Frolich JC. Dietary L-arginine and alphatocopherol reduce vascular oxidative stress and preserve endothelial function in hypercholesterolemic rabbits via different mechanisms. Atherosclerosis 1998; 141:31– 43. 23. Kinlay S, Fang JC, Hikita H, Ho I, Delagrange DM, Frei B, Suh JH, Gerhard M, Creager MA, Selwyn AP, Ganz P. Plasma alpha-tocopherol and coronary endothelium-dependent vasodilator function. Circulation 1999;100:219 –221. 24. Simons LA, Von Konigsmark M, Simons J, Stocker R, Celermajer DS. Vitamin E ingestion does not improve arterial endothelial dysfunction in older adults. Atherosclerosis 1999;143:193–199. 25. Green D, O’Driscoll G, Rankin JM, Maiorana AJ, Taylor RR. Beneficial effect of vitamin E administration on nitric oxide function in subjects with hypercholesterolemia. Clin Sci 1998;95:361–367. 26. Chambers JC, McGregor A, Jean-Marie J, Obeid OA, Kooner JS. Demonstration of rapid onset vascular endothelial dysfunction after hyperhomocysteinemia: an effect reversible with vitamin C therapy. Circulation 1999;99:1156 – 1160. 27. Freedman JE, Farhat JH, Loscalzo J, Keaney JK. ␣-Tocopherol inhibits aggregation of human platelets by a protein kinase C-dependent mechanism. Circulation 1996;94:2434 –2440. 28. Adams MR, Forsyth CJ, Jessup W, Robinson J, Celermajer DS. Oral L-arginine inhibits platelet aggregation but does not enhance endothelium-dependent dilatation in healthy young adults. J Am Coll Cardiol 1995;26:1054 –1061. 29. Creager MA, Roddy MA, Boles K, Stamler JS. N-acetylcysteine does not influence the activity of endothelium-derived relaxing factor in vivo. Hypertension 1996;29:668 – 672. 30. Hanratty CG, McGrath LT, McAuley DF, Young IS, Johnston GD. The effects of oral methionine and homocysteine on endothelial function (abstr). J Am Coll Cardiol 2000;35;288A.

AUGUST 1, 2001