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(Circulation. 1997;95:2617-2622.)
© 1997 American Heart Association, Inc.

Articles

Vitamin C Improves Endothelium-Dependent Vasodilation in Forearm Resistance Vessels of Humans With Hypercholesterolemia

Henry H. Ting, MD; Farris K. Timimi, MD; Elizabeth A. Haley, BA; Mary-Anne Roddy, BSN; Peter Ganz, MD; Mark A. Creager, MD

the Vascular Medicine and Atherosclerosis Unit, Cardiovascular Division, Brigham and Women's Hospital and Harvard Medical School, Boston, Mass.

Correspondence to Mark A. Creager, MD, Cardiovascular Division, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115.

	Abstract

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Background Endothelium-dependent vasodilation is impaired in humans with hypercholesterolemia. Oxidative degradation of endothelium-derived nitric oxide plays a major role in endothelial dysfunction in animal models of hypercholesterolemia. To assess whether this mechanism is relevant to humans, we studied the effect of vitamin C, an antioxidant, on vasodilator function in forearm resistance vessels of patients with hypercholesterolemia.

Methods and Results We studied 11 hypercholesterolemic and 12 healthy control subjects. Forearm blood flow was determined by venous occlusion plethysmography. Endothelium-dependent vasodilation was assessed by intra-arterial infusion of methacholine (0.3 to 10 µg/min). Endothelium-independent vasodilation was measured by intra-arterial infusion of nitroprusside (0.3 to 10 µg/min) and verapamil (10 to 300 µg/min). Forearm blood flow dose-response curves were determined for each drug before and during coadministration of vitamin C (24 mg/min). In hypercholesterolemic subjects, endothelium-dependent vasodilation to methacholine was augmented by coinfusion of vitamin C (P=.001); in contrast, endothelium-independent vasodilation to nitroprusside and verapamil were not affected by coinfusion of vitamin C (P=.8 and P=.3, respectively). In control subjects, vitamin C administration did not alter endothelium-dependent vasodilation (P=.2).

Conclusions We conclude that vitamin C improves endothelium-dependent vasodilation in the forearm resistance vessels of patients with hypercholesterolemia. These findings suggest that nitric oxide degradation by oxygen-derived free radicals contributes to abnormal vascular reactivity in hypercholesterolemic humans.

Key Words: antioxidants • hypercholesterolemia • endothelium • free radicals

	Introduction

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Hypercholesterolemia is causally associated with increased morbidity and mortality from cardiovascular disease.^{1 2 3} Elevated serum cholesterol accelerates atherogenesis, leading to the development of fatty streaks and atherosclerotic plaques.^{4 5} Impairment of normal endothelial function occurs early in atherogenesis and can be detected before the development of atherosclerotic lesions.

The endothelium plays an important role in maintaining vascular integrity, in part by the synthesis and release of vasoactive substances such as NO.^{6 7 8 9} Hypercholesterolemia impairs endothelium-dependent vasodilation in human coronary and peripheral blood vessels.^{10 11 12 13} The mechanisms accounting for the observed endothelial dysfunction have not been completely elucidated, but increased vascular oxidative stress has been implicated as a possible cause. Indeed, in cholesterol-fed animals, endothelium-dependent vascular relaxation can be normalized by treatment with antioxidants.^{14 15 16 17 18 19 20}

The purpose of this study was to determine whether observations made in animal models of experimental hypercholesterolemia can be extended to humans. Vitamin C is the primary water-soluble antioxidant in human plasma, capable of scavenging oxygen-derived free radicals and sparing other endogenous antioxidants from consumption.^{21 22 23} Accordingly, we sought to test the hypothesis that vitamin C can restore endothelium-dependent vasodilation in patients with hypercholesterolemia.

	Methods

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Subjects
The study population included 11 hypercholesterolemic subjects (mean age, 49±2 years; range, 42 to 60 years) and 12 healthy control subjects (mean age, 46±2 years; range, 36 to 63 years). Hypercholesterolemic subjects were eligible if they had a total cholesterol 240 mg/dL and LDL cholesterol 160 mg/dL measured after an overnight fast. The duration of hypercholesterolemia averaged 10±1 years (5 to 15 years). Hypercholesterolemic subjects received treatment with diet alone (n=8) or diet plus HMG-CoA reductase inhibitor (n=3). Hypercholesterolemic subjects discontinued HMG-CoA reductase inhibitors 12 weeks before participation in this study. All subjects were recruited from the Boston area via advertisements in local newspapers. Subjects were screened by a medical history, physical examination, and laboratory analysis and were excluded if any of the following were present: hypertension (blood pressure >140/90 mm Hg); diabetes mellitus; tobacco use within the past year; clinical or laboratory evidence of cardiac, pulmonary, renal, hepatic, or hematological abnormalities; and current use of cardiac or other vasoactive medications, antioxidant vitamins, or hormone replacement therapy. No subject had clinical evidence of atherosclerosis, as documented by the absence of symptoms including angina, claudication, and cerebrovascular ischemia and the absence of physical findings including diminished pulses, asymmetrical blood pressure, and bruits. This study was approved by the Human Research Committee of Brigham and Women's Hospital, and each subject gave written informed consent.

Drug Infusion Protocol
Methacholine chloride (Roche Laboratories), a congener of acetylcholine, was administered via the brachial artery to assess vasodilation resulting from endothelium-derived NO. FBF was measured during infusion of increasing concentrations of methacholine at doses of 0.3, 1.0, 3.0, and 10.0 µg/min. Sodium nitroprusside (Elkins-Sinn Inc) was administered via the brachial artery to assess the vasodilator response to an exogenous NO donor. FBF was determined during infusion of increasing concentrations of nitroprusside at doses of 0.3, 1.0, 3.0, and 10.0 µg/min. Verapamil (American Reagent Laboratory Inc), a calcium channel blocker, was administered via the brachial artery to assess vascular smooth muscle relaxation not dependent on endothelium-derived or exogenous NO. FBF was measured during infusion of increasing concentrations of verapamil at doses of 10, 30, 100, and 300 µg/min. The doses of each drug were chosen to achieve a significant change in FBF and FVR without causing systemic effects. Hemodynamic measurements were performed after infusion of methacholine, nitroprusside, or verapamil for 3 minutes at each dose, administered at 0.4 mL/min.

Vitamin C (sodium ascorbate, Abbott Laboratories) was administered via the brachial artery to assess whether this antioxidant vitamin modified the vasodilator responses to methacholine, nitroprusside, or verapamil. Vitamin C was infused at a constant dose of 24 mg/min and rate of 0.4 mL/min to approximate a local forearm concentration of 1 to 10 mmol/L. This vitamin C concentration has been shown to protect human plasma from free radical–mediated lipid peroxidation²² and to improve endothelium-dependent vasodilation in patients with diabetes mellitus²⁴ and in chronic smokers.²⁵

Experimental Protocol
Each subject was studied in the postabsorptive state in the vascular research laboratory, a quiet, dim, and temperature-controlled (23°C) room. Alcohol, caffeine, and all medications were withheld for 12 hours before the study. Aspirin and nonsteroidal anti-inflammatory medications were withheld 7 days before the study. Under local anesthesia and sterile conditions, a 20-gauge polyethylene catheter was inserted into a brachial artery of each subject for determination of blood pressure and infusion of drugs. All subjects rested for at least 30 minutes after catheter placement to achieve a stable baseline before data collection.

At the beginning of each protocol, normal saline (0.9% sodium chloride) was infused intra-arterially at a rate of 0.4 mL/min, and baseline measurements of FBF and blood pressure were obtained every 10 minutes until stable. All subjects participated in the following protocol: (1) an initial FBF dose-response curve to increasing concentrations of methacholine, (2) a 60-minute rest period to reestablish baseline measurements, (3) intra-arterial administration of vitamin C for 10 minutes, and (4) a second FBF dose-response curve during concomitant infusion of methacholine and vitamin C. Using a similar experimental design, hypercholesterolemic subjects also participated in two additional protocols on separate dates to assess FBF dose-response curves to nitroprusside and verapamil before and during coinfusion of vitamin C.

To ensure that any observed vascular effects were due to vitamin C and not secondary to repeated administration of methacholine, five subjects with hypercholesterolemia participated in an identical protocol as described above with coinfusion of vehicle (0.9% sodium chloride) instead of vitamin C.

Hemodynamic Measurements
Bilateral FBF was determined by venous occlusion strain-gauge plethysmography with calibrated mercury-in-Silastic strain gauges (D.E. Hokanson) and expressed as mL·100 mL tissue^-1·min^-1. Each arm was supported above the level of the heart. Venous occlusion pressure averaged 36±1 mm Hg. Circulation to the hand was prevented by a wrist cuff inflated to suprasystolic pressures before each FBF determination. Each FBF determination comprised at least five separate measurements at 10- to 15-second intervals. The vascular response to each drug was determined by measurement of FBF in the drug infusion arm, and potential systemic effects were assessed by measurement of FBF in the contralateral arm. FVR was calculated as the ratio of mean blood pressure to FBF and expressed as units reflecting mm Hg·mL·100 mL tissue^-1·min^-1.

Blood pressure was measured via the arterial cannula, which was attached to a Statham P23 pressure transducer aligned to an amplifier on a Gould physiological recorder. Heart rate was determined from a simultaneously obtained ECG signal and calculated from the RR interval.

Statistical Analysis
All data are presented as mean±SEM. Group comparisons with respect to clinical characteristics were made with unpaired and two-tailed t tests. FBF dose-response curves for each drug before and during coadministration of vitamin C were analyzed with two-way repeated-measures ANOVA followed by post hoc two-tailed t tests adjusted with a Bonferroni correction for multiple comparisons. Statistical significance was accepted at the 95% confidence level (P<.05).

	Results

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The clinical characteristics of the study population are provided in the Table. Hypercholesterolemic subjects had higher total cholesterol, LDL cholesterol, and triglyceride levels than control subjects. The age, body mass index, mean blood pressure, HDL cholesterol, glucose, blood urea nitrogen, and creatinine were similar in both groups. No subject had evidence of hypertension, diabetes mellitus, tobacco use, or cardiovascular disease.

View this table: [in this window] [in a new window]   Table 1.

Effect of Vitamin C on Basal FBF
The baseline FBF was similar between hypercholesterolemic and control subjects, 2.2±0.4 and 2.6±0.3 mL·100 mL tissue^-1·min^-1, respectively (P=NS). The baseline FVR was 46.6±5.6 U in hypercholesterolemic subjects and 34.8±3.6 U in control subjects (P=NS). Intra-arterial administration of vitamin C did not change baseline FBF in hypercholesterolemic subjects, 2.2±0.4 to 2.3±0.2 mL·100 mL tissue^-1·min^-1 (P=NS), or control subjects, 2.6±0.3 to 2.4±0.2 mL·100 mL tissue^-1·min^-1 (P=NS).

Effect of Vitamin C on Endothelium-Dependent Vasodilation
The vasodilator response to methacholine was attenuated in hypercholesterolemic subjects compared with control subjects (P=.001 by ANOVA, Fig 1). At the highest dose of methacholine (10 µg/min), the FBF increased to 12.2±1.3 mL·100 mL tissue^-1·min^-1 in hypercholesterolemic subjects compared with 21.1±1.6 mL·100 mL tissue^-1·min^-1 in control subjects (P<.01). At this dose, the FVR decreased to 7.8±1.1 U in hypercholesterolemic subjects compared with 3.9±0.5 U in control subjects (P<.05).

View larger version (16K): [in this window] [in a new window]   Figure 1. FBF dose-response curves to methacholine in hypercholesterolemic () and control () subjects. Endothelium-dependent vasodilation to methacholine was significantly attenuated in hypercholesterolemic subjects compared with control subjects (P=.001 by ANOVA). FBFs at each methacholine dose between hypercholesterolemic and control subjects were compared by unpaired t tests adjusted with a Bonferroni correction for multiple comparisons (*P<.05, **P<.01).

In hypercholesterolemic subjects, the vasodilator response to methacholine was augmented during coadministration of vitamin C (P=.001 by ANOVA, Fig 2). At the highest dose of methacholine (10 µg/min), the FBF increased from 12.2±1.3 to 16.4±1.5 mL·100 mL tissue^-1 ·min^-1 during coinfusion of vitamin C (P<.01). At this dose, the FVR decreased from 7.8±1.1 to 5.9±0.7 U during coinfusion of vitamin C (P<.05).

View larger version (15K): [in this window] [in a new window]   Figure 2. FBF dose-response curves to methacholine () and methacholine plus vitamin C () in hypercholesterolemic subjects. Endothelium-dependent vasodilation to methacholine was augmented during concomitant infusion of methacholine and vitamin C (P=.001 by ANOVA). FBFs at each methacholine dose before and during vitamin C administration were compared by paired t tests adjusted with a Bonferroni correction for multiple comparisons (**P<.01).

In contrast, the vasodilator response to methacholine in control subjects was not altered by coadministration of vitamin C (P=.2 by ANOVA, Fig 3). At the highest dose of methacholine (10 µg/min), the FBF was 21.1±1.6 mL·100 mL tissue^-1·min^-1 before and 21.2±1.8 mL·100 mL tissue^-1·min^-1 during coinfusion of vitamin C (P=NS). At this dose, the FVR was unaffected by coinfusion of vitamin C, 3.9±0.5 versus 4.1±0.4 U (P=NS).

View larger version (15K): [in this window] [in a new window]   Figure 3. FBF dose-response curves to methacholine () and methacholine plus vitamin C () in control subjects. Endothelium-dependent vasodilation to methacholine was not altered during concomitant infusion of methacholine and vitamin C (P=.2 by ANOVA).

FBF and FVR in the contralateral arm did not change during methacholine infusion in either group of subjects. Blood pressure and heart rate also were not altered during methacholine infusion.

Effect of Vitamin C on Endothelium-Independent Vasodilation
In hypercholesterolemic subjects, the vasodilator response to nitroprusside was not altered during coadministration of vitamin C (P=.8 by ANOVA, Fig 4). At the maximal dose of nitroprusside (10 µg/min), the FBF was 14.1±2.8 mL·100 mL tissue^-1·min^-1 before and 13.0±2.7 mL·100 mL tissue^-1·min^-1 during coinfusion of vitamin C (P=NS). At this dose, the FVR was similar before and during coinfusion of vitamin C, 7.7±1.6 and 8.4±1.4 U, respectively (P=NS). Nitroprusside infusion did not change FBF or FVR in the contralateral arm and did not affect blood pressure or heart rate.

View larger version (14K): [in this window] [in a new window]   Figure 4. FBF dose-response curves to nitroprusside () and nitroprusside plus vitamin C () in hypercholesterolemic subjects. Endothelium-independent vasodilation to nitroprusside was not altered during concomitant infusion of nitroprusside and vitamin C (P=.8 by ANOVA).

Also, the vasodilator response to verapamil was not altered during coadministration of vitamin C in hypercholesterolemic subjects (P=.3 by ANOVA, Fig 5). At the maximal dose of verapamil (300 µg/min), the FBF was similar before and during coinfusion of vitamin C, 16.0±2.0 and 16.2±2.1 mL·100 mL tissue^-1·min^-1, respectively (P=NS). At this dose, the FVR was 5.6±1.1 U before and 6.0±0.8 U during coinfusion of vitamin C (P=NS). Verapamil infusion did not affect FBF or FVR in the contralateral arm and did not alter blood pressure or heart rate.

View larger version (15K): [in this window] [in a new window]   Figure 5. FBF dose-response curves to verapamil () and verapamil plus vitamin C () in hypercholesterolemic subjects. Endothelium-independent vasodilation to verapamil was not altered during concomitant infusion of verapamil and vitamin C (P=.3 by ANOVA).

Effect of Vehicle on Endothelium-Dependent Vasodilation
In five subjects with hypercholesterolemia, forearm vascular responses to methacholine were assessed before and during coinfusion of vehicle (0.9% sodium chloride). The vasodilator response to methacholine in hypercholesterolemic subjects was not altered by coadministration of vehicle (P=.1 by ANOVA). At the highest dose of methacholine (10 µg/min), the FBF was 14.2±1.9 mL·100 mL tissue^-1·min^-1 before and 14.0±2.2 mL·100 mL tissue^-1·min^-1 during coinfusion of vehicle (P=NS). At this dose, the FVR was unaffected by coinfusion of vehicle, 6.6±1.2 versus 6.9±1.4 U (P=NS). Methacholine infusion did not change FBF or FVR in the contralateral arm and did not affect blood pressure or heart rate.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The salient finding of this study is that administration of vitamin C, an antioxidant, improves endothelium-dependent vasodilation in humans with hypercholesterolemia. Vitamin C did not alter endothelium-independent vasodilation in hypercholesterolemic subjects and did not modify endothelium-dependent vasodilation in healthy control subjects. Taken together, these findings suggest that increased vascular oxidative stress may play a role in the abnormal endothelium-dependent vasodilation observed in the forearm resistance vessels of hypercholesterolemic humans.

Effect of Hypercholesterolemia on Endothelium-Dependent Vasodilation
The endothelium is an important modulator of vascular tone and function, in part by the synthesis and release of NO.^{6 7 8 9} The vascular response to muscarinic agonists such as acetylcholine and methacholine depends on the health and integrity of the endothelium and the endothelium-derived NO pathway. Endothelium-dependent vasodilation is impaired in blood vessels of animals with experimental hypercholesterolemia^{26 27 28 29 30 31} and in the coronary^{10 11} and forearm^{12 13} vasculature of humans with hypercholesterolemia. The mechanisms responsible for endothelial dysfunction in hypercholesterolemia are not completely understood but may be explained by decreased bioavailability of NO due to either decreased production by endothelial cells or increased degradation by oxygen-derived free radicals.

Oxidative Stress in Experimental Models of Hypercholesterolemia
Enhanced oxidative degradation of endothelium-derived NO contributes to endothelial dysfunction in animal models of hypercholesterolemia. In cholesterol-fed rabbits, Minor and coworkers³² demonstrated that endothelium-dependent vascular relaxation was diminished despite increased endothelial NO synthase activity and NO production. This finding suggests that the decreased NO bioavailability is more likely to be secondary to increased degradation by oxygen-derived free radicals rather than decreased production by endothelial cells. NO is readily inactivated by superoxide anion,^{33 34 35} generating peroxynitrite anion.³⁶ Peroxynitrite anion spontaneously yields hydroxyl and peroxynitrite radicals, which are capable of causing further oxidative stress and damage.³⁶ Indeed, Ohara and colleagues^{16 17} have shown that blood vessels from cholesterol-fed rabbits produced threefold to fivefold more superoxide anion than control rabbits. Several studies in animal models of hypercholesterolemia have evaluated whether antioxidant treatment can reverse endothelial dysfunction and normalize vascular superoxide production. In cholesterol-fed rabbits, treatment with SOD,^{14 15} oxypurinol,¹⁶ -tocopherol,^{18 19} or probucol²⁰ restores endothelium-dependent vasodilation. Moreover, treatment with oxypurinol¹⁶ or probucol²⁰ has been shown to normalize vascular superoxide production.

Effect of Vitamin C on Endothelium-Dependent Vasodilation
The present study extends observations made in these experimental models to humans with hypercholesterolemia. In this study, vitamin C administration selectively improved endothelium-dependent vasodilation in response to methacholine in hypercholesterolemic subjects. It is possible that administration of a higher dose or longer duration of vitamin C could have scavenged more superoxide radicals and achieved a larger benefit.

Vitamin C is the main water-soluble antioxidant in human plasma^{21 22 23} and is capable of scavenging superoxide anion.^{37 38 39 40 41} The reaction rate constants are estimated to be 3x10⁵ (mol/L) ^-1·s^-1 between vitamin C and superoxide anion^{37 39 40} and 2x10⁹ (mol/L) ^-1·s^-1 between SOD and superoxide anion.^{38 40 42} Despite a 10⁴ times slower reaction rate observed with vitamin C, tissue vitamin C concentrations are 10⁴ times higher than that of SOD,^{37 38 43} enabling vitamin C to competitively scavenge superoxide anion. Vitamin C may also improve endothelial function by sparing intracellular glutathione from oxidative degradation. Glutathione is an important source of intracellular reduced thiols and can be degraded by oxidation to glutathione disulfide.^{44 45} Depletion of intracellular reduced thiols impairs NO synthase activity in cultured endothelial cells.^{46 47} Furthermore, reduced thiols can react with NO to form S-nitrosothiol species, which may result in stabilization and increased bioavailability of NO.^{48 49}

Previous Studies
Three previous studies have demonstrated that antioxidants can improve endothelium-dependent vasodilation in humans with atherosclerosis documented by coronary angiography. Meredith and colleagues⁵⁰ showed that in patients with atherosclerosis, acute administration of SOD attenuated the abnormal vasoconstrictor response of the coronary arteries to acetylcholine. Anderson and coworkers¹¹ demonstrated that treatment with a combination of lovastatin and probucol for 12 months improved coronary artery endothelium-dependent vasodilator response to acetylcholine compared with dietary treatment alone. Recently, Levine and colleagues⁵¹ demonstrated that a single oral dose of vitamin C (2 g) acutely improved endothelium-dependent vasodilation of the brachial artery in patients with coronary atherosclerosis. The present study differs from the above reports in that we studied the effect of an antioxidant, vitamin C, on endothelial function in humans with hypercholesterolemia in the absence of other cardiovascular risk factors and clinical evidence of atherosclerosis.

Two previous studies have shown no beneficial effect of antioxidants on endothelium-dependent vasodilation in humans with hypercholesterolemia in the absence of atherosclerosis. Garcia and coworkers⁵² demonstrated that SOD did not modify endothelium-dependent vasodilation in the forearm resistance vessels of hypercholesterolemic humans. However, this negative finding may be explained by the inability of copper-zinc SOD to cross the cell membrane and scavenge intracellular sources of superoxide radicals. Gilligan et al⁵³ treated hypercholesterolemic patients with a combination of vitamin C, vitamin E, and ß-carotene for 1 month and found no improvement in endothelium-dependent vasodilation of forearm resistance vessels. The lack of a beneficial effect may be explained by the relatively small doses and insufficient intracellular concentrations achieved. Furthermore, this report differs from the present study in the route (oral versus intra-arterial) of vitamin C administration.

Conclusions
In summary, this study demonstrates that the antioxidant vitamin C selectively improves endothelium-dependent vasodilation in forearm resistance vessels of humans with hypercholesterolemia. This finding supports the hypothesis that oxygen-derived free radicals may contribute to abnormal vascular reactivity in hypercholesterolemic humans.

	Selected Abbreviations and Acronyms

 

FBF = forearm blood flow FVR = forearm vascular resistance HMG-CoA = hydroxy-3-methylglutaryl coenzyme A NO = nitric oxide SOD = superoxide dismutase

	Acknowledgments

 
This research was supported by a National Institutes of Health Program Project Grant in Vascular Biology and Medicine (HL-48743). Dr Ting is a recipient of a grant from the Clinical Investigator Training Program: Harvard/MIT Health Sciences and Technology–Beth Israel Deaconess Medical Center, in collaboration with Pfizer Inc. Dr Creager is a recipient of a National Heart, Lung, and Blood Institute Academic Award in Systemic and Pulmonary Vascular Medicine (HL-02663).

Received September 17, 1996; revision received December 12, 1996; accepted January 4, 1997.

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