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(Circulation. 1997;96:3287-3293.)
© 1997 American Heart Association, Inc.

Articles

Endothelial Dysfunction Is Associated With Cholesterol Levels in the High Normal Range in Humans

Helmut O. Steinberg, MD; Basel Bayazeed, MD; Ginger Hook, RN; Ann Johnson, RN; Jessica Cronin, RN; ; Alain D. Baron, MD

From the Indiana University Medical Center and the Richard L. Roudebush Veterans Affairs Medical Center, Indianapolis.

Correspondence to Alain D. Baron, MD, Division of Endocrinology and Metabolism, Indiana University Medical Center, 541 N Clinical Dr, Clinical Bldg 459, Indianapolis, IN 46202-5111. E-mail abaron{at}mdep.iupui.edu

	Abstract

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Background The purpose of this study was to test the hypothesis that cholesterol levels in the high normal range are associated with impaired endothelium-dependent vasodilation.

Methods and Results We studied leg blood flow (LBF) responses to graded intrafemoral artery infusions of the endothelium-dependent vasodilator methacholine chloride (MCh) or the endothelium-independent vasodilator sodium nitroprusside (SNP) in normal volunteers exhibiting a wide range of total cholesterol levels within the normal range (<75th percentile). LBF increased in a dose-dependent fashion in response to the femoral artery infusions of MCh and SNP (P<.001). LBF responses to MCh were significantly blunted (P<.001) in subjects with high normal cholesterol (195±6 mg/dL, n=13) compared with subjects with low normal cholesterol (146±5 mg/dL, n=20). Maximal endothelium-dependent vasodilation in the high normal group was decreased by nearly 50% compared with the low normal group (146±13% versus 268±34%, P<.01). There was a negative correlation between total cholesterol levels and maximal endothelium-dependent vasodilation (total cholestero,l r=-.41, P<.02; LDL cholesterol, r=-.42, P<.02). On the other hand, LBF responses to the endothelium-independent vasodilator SNP did not differ between groups.

Conclusions These data suggest that an inverse and continuous relationship exists between the prevailing cholesterol level and endothelium-dependent vasodilation. Moreover, cholesterol levels even in the normal range may be associated with endothelial dysfunction, thus potentially contributing to the increased risk of macrovascular disease conferred by cholesterol elevations.

Key Words: cholesterol • endothelium • vasodilation • methacholine • sodium nitroprusside

	Introduction

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Cholesterol is one of the most well-established risk factors for premature CAD^{1 2 3} and peripheral vascular disease. Cholesterol levels and CAD risk show a strong and linear relationship.¹ Hypercholesterolemia with total cholesterol levels of 260 mg/dL and LDL cholesterol levels of 200 mg/dL are associated with impaired endothelium-dependent vasodilation in the forearm^{4 5 6 7} and in the coronary vasculature,⁸ indicating impaired endothelial function. Endothelium-dependent vasodilation is mediated at least in part through the release of EDNO.⁹ EDNO, the most powerful endogenous vasodilator, participates in the regulation of systemic blood pressure and local vascular tone regulation.¹⁰ Furthermore, EDNO inhibits proliferation and migration of vascular smooth muscle cells^{11 12} as well as lipid oxidation,^{13 14} which all are involved in the development and progression of atherosclerotic vascular disease. Thus, impaired production/release of EDNO under conditions of elevated cholesterol levels may be an important mechanistic link between hypercholesterolemia and increased rates of CAD.

Given the similar effect of hypercholesterolemia on endothelial function in both the coronary and the peripheral vascular beds and given the continuous relationship between cholesterol levels and coronary artery risk, we hypothesized that cholesterol levels are continuously related to endothelial function. In other words, cholesterol levels even in the normal range would have a negative effect on endothelial function. To this end, we studied the LBF responses to graded intrafemoral artery infusions of the endothelium-dependent vasodilator MCh or the endothelium-independent vasodilator SNP in two groups of healthy volunteers exhibiting low and high serum cholesterol concentrations in the normal range.

	Methods

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Subjects
Table 1 gives the demographic characteristics of the subject groups in each. All study subjects were healthy and had total and LDL cholesterol levels <75 percentile according to the NHANES III data.¹⁵ All subjects were on no medications and had normal cuff blood pressure determinations and normal 75-g oral glucose tolerance tests.¹⁶ Studies were approved by the Indiana University Human Subjects Internal Review Board, and all volunteers gave informed consent.

View this table: [in this window] [in a new window]   Table 1. Characteristics of the Study Groups

Diet
All subjects were admitted to the Indiana University General Clinical Research Center 2 days before study and were fed a weight-maintaining diet the caloric content of which was distributed as 50% carbohydrate, 30% fat, and 20% protein.

Drugs
All infusates were prepared under sterile conditions on the morning of the study. MCh (Roche Labs) was dissolved in normal saline to a concentration of 25 µg/mL, and SNP (Roche Labs) was dissolved in normal saline to a concentration of 7 µg/mL. MCh or SNP was infused directly into the femoral artery with a Harvard programmable pump (model 44, Harvard Apparatus).

Protocol
Separate groups of subjects were studied under two distinct study protocols designed to examine the effect of normal range cholesterol concentrations on endothelium-dependent (MCh study) and endothelium-independent vasodilation (SNP study). Aspects of the protocol that are common to both studies are described below.

At 7 AM, after an overnight 14-hour fast, a catheter was inserted into the antecubital vein. Subsequently, the right femoral artery and vein were cannulated. A 5F sheath (Cordis Corp) was placed in the right femoral vein to allow insertion of a custom-designed 5F double-lumen thermodilution catheter (Baxter Scientific, Edwards Division) to measure LBF as previously described.¹⁷ The right femoral artery was cannulated with a 5.5F double-lumen catheter (Arrow International) to allow simultaneous infusion of substances through the proximal port (most caudad) and invasive blood pressure monitoring through the distal port (most cephalad). Heart rate and MAP were monitored continuously via precordial leads and a pressure transducer connected to a vital signs monitor (VSM 1, Physiocontrol).

Hemodynamic Measurements
All hemodynamic measurements were obtained with subjects in the supine position in a quiet, temperature-controlled room and after the subject had emptied his or her bladder. Baseline measurements of LBF, MAP, and heart rate were obtained after allowing 30 minutes of rest after insertion of the catheters. Rates of LBF were determined by injecting 1 mL of iced normal saline into the femoral vein via the thermodilution catheter. The thermodilution curves were recorded on a chart recorder and visually inspected for integrity. LBF was calculated by a cardiac output computer (model 9520A, American Edwards Laboratories) that integrates the area under the thermodilution curve and displays the flow rate in liters per minute. During graded intrafemoral artery infusion of drugs (MCh or SNP), LBF measurements were begun 2 minutes after the onset of each dose. LBF measurements were performed every 30 seconds for a total of 10 determinations at each drug dose. Invasively determined MAP and heart rate were recorded with every other LBF determination.

Endothelium-Dependent Vasodilation (MCh Study)
To study the effect of normal range cholesterol on endothelium-dependent vasodilation, we administered graded intrafemoral artery infusions of MCh at sequential doses of 2.5, 5.0, 7.5, 10.0, and 12.5 µg/min. In this fashion, dose-response curves for MCh to cause endothelium-dependent vasodilation were obtained. Each MCh dose was administered for 8 minutes. The volume of MCh delivered ranged from 0.1 to 0.5 mL/min.

Endothelium-Independent Vasodilation (SNP Study)
To study the effect of normal range cholesterol levels on endothelium-independent vasodilation, graded intrafemoral artery infusions of SNP were administered at rates of 1.75, 3.5, and 7.0 µg/min. In this fashion, dose-response curves for the effect of SNP to cause endothelium-independent vasodilation were obtained. Each SNP dose was administered for 8 minutes. The volume of SNP delivered ranged from 0.25 to 1.0 mL/min.

Analytical Methods
Serum total cholesterol and triglyceride levels were measured on an Ektachem 702 analyzer with an enzymatic method. HDL cholesterol was measured with the Magnetic HDL kit (Reference Diagnostics, Inc), and LDL cholesterol was calculated according to the formula of Friedewald et al.¹⁸ Free fatty acids were measured according to the method described by Novak.¹⁹ Body fat content was determined by dual energy x-ray absorptiometry (DXA, Lunar DPX-L, with system software 1.2).

Statistical Analysis
Results are shown as the mean±SEM. MAP is expressed in millimeters of mercury, and LBF is expressed in liters per minute. Changes in blood flow are expressed as percent change (%) to adjust for differences at baseline. LVR was calculated as MAP divided by LBF and is presented in arbitrary units. Total cholesterol levels of <25th percentile (NHANES III¹⁵ ) were used to define the group with low normal range cholesterol (n=20), and total cholesterol levels of >25th but <75th percentile (NHANES III¹⁵ ) were used to define the group with high normal range cholesterol (n=13). Therefore, both groups were made up of subjects exhibiting serum cholesterol levels <75th percentile according to criteria established by NHANES III. Therefore, the terms "low cholesterol" and "high cholesterol" group refer to stratification according to total cholesterol levels. To analyze the effect of normal range LDL cholesterol levels on endothelium-dependent vasodilation, NHANES III percentiles¹⁵ for LDL cholesterol were applied in exactly the same fashion to define groups with LDL cholesterol levels in the low (<25th percentile) and high (>25th but <75th percentile) normal ranges. Two-way ANOVA was used to compare the changes in LBF in response to the graded drug infusions between the groups with low and high normal range cholesterol levels. When significant differences between groups were found by ANOVA, this was followed by post hoc testing with Fisher's protected least significant difference.

Simple linear regression analysis was performed to assess the relationship between the maximal increase in LBF in response to the intrafemoral artery infusions of MCh or SNP and serum total cholesterol, HDL cholesterol, LDL cholesterol, and triglyceride and free fatty acid levels.

Statistical significance was accepted at a level of P<.05. Statistics were performed on a Power Macintosh computer with StatView IV (Abacus Concepts, Inc).

	Results

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MCh Study
Lipids
Whole group. Mean total cholesterol levels were 165.2±5.7 mg/dL, and LDL and HDL cholesterol levels were 101.1±5.1 and 42.4±2.5 mg/dL, respectively. Triglyceride levels averaged 97.9±8.0 mg/dL and were in the normal range.

High and low cholesterol groups. Grouping the subjects according to their percentiles of total cholesterol levels of <25th percentile (low) or >25th percentile (high) resulted as expected in two groups with significantly different total and LDL cholesterol levels. HDL cholesterol levels were similar in both groups. Triglyceride levels were slightly higher in the high cholesterol group but were still in the normal range. Table 2 shows the lipid levels of the low and high cholesterol groups. Table 2 also reveals the subject characteristics of the two groups that were comparable throughout.

View this table: [in this window] [in a new window]   Table 2. Characteristics and Lipid Levels of the Study Groups Stratified According to Their Total Cholesterol Levels of 25th (Low) or >25th (High) Percentile

Hemodynamic Measurements
Whole group. Basal MAP was 89.6±1.5 mm Hg, and basal LBF was 0.234±0.020 L/min. In response to the graded intrafemoral artery infusions of MCh, LBF increased in a dose-dependent fashion (P<.0001) with no changes in MAP.

High and low cholesterol groups. Both groups had comparable basal rates of LBF and levels of MAP (Table 2). However, the LBF response to the graded intrafemoral artery infusions of MCh was much more pronounced (P<.0001) in the low compared with the high cholesterol group. This difference in LBF response between the groups was clearly evident when comparing the rates of absolute LBF, as seen in Fig 1A, or the percent increase above baseline, as shown in Fig 1B. The differences in LBF response to Mch were equally impressive when the LBF response was assessed in the groups according LDL cholesterol levels of lesser or greater than the 25th percentile, as seen in Fig 2A and 2B. Furthermore, percent maximal endothelium-dependent vasodilation in the high cholesterol group (146±13%) was nearly half that (268±34%) observed in the low cholesterol group (P<.01, high versus low). Changes in LVR in response to graded intrafemoral artery infusions of Mch mirrored the changes in LBF. Basal LVR was 448±53 and 494±43 units in the low and high cholesterol groups, respectively (P=NS). LVR decreased in a dose-dependent fashion in both groups, but the decrease in LVR was more pronounced in the low cholesterol group. In response to the graded intrafemoral artery infusion of Mch, LVR declined to 324±35, 227±27, 195±23, 175±23, and 145±19 units in the low cholesterol group and to 381±35, 313±40, 266±23, 231±16, and 212±16 units in the high cholesterol (P<.0005 low versus high). The data indicate that both LDL and total cholesterol levels in the upper normal range are associated with decreased endothelium-dependent vasodilation.

View larger version (21K): [in this window] [in a new window]   Figure 1. A, LBF under basal conditions and in response to graded intrafemoral artery infusions of MCh in the low () and high () normal cholesterol groups (P<.0001, ANOVA, low vs high). B, Percent increments (%) in LBF above baseline in response to graded intrafemoral artery infusions of MCh in the low and high normal cholesterol groups (P<.0001, ANOVA, low vs high).

View larger version (20K): [in this window] [in a new window]   Figure 2. A, LBF under basal conditions and in response to graded intrafemoral artery infusions of MCh in subjects with LDL cholesterol levels <25th percentile () and >25th but <75th percentile () (P<.0001, ANOVA). B, Percent increments (%) in LBF above baseline in response to graded intrafemoral artery infusions of MCh in subjects with LDL cholesterol levels <25th percentile () and >25th but <75th percentile () (P<.0001, ANOVA).

Nitroprusside Study
Lipids
Whole group. Mean total cholesterol levels were 167.1±5.4 mg/dL, and LDL and HDL cholesterol levels were 107.1±4.9 and 36.4±2.5 mg/dL, respectively. Triglyceride levels were 126.9±10.0 mg/dL and also in the normal range.

High and low cholesterol groups. Grouping the subjects into low and high cholesterol groups resulted as expected in two groups with significantly different total and LDL cholesterol levels. HDL cholesterol levels were similar in both groups. Triglyceride levels were higher in the high cholesterol group but were still in the normal range. Table 2 gives the lipid levels of the low and high cholesterol. Table 2 also reveals that the subject characteristics of the two groups were comparable throughout.

Hemodynamic Measurements
Whole group. Basal MAP was 89.8±1.6 mm Hg, and basal LBF was 0.243±0.028 L/min. In response to the graded intrafemoral artery infusions of SNP, LBF increased in a dose-dependent fashion (P<.0001) with no changes in MAP.

High and low cholesterol groups. Both the high and low cholesterol groups had comparable basal rates of LBF and levels of MAP (Table 2). LBF in response to the graded intrafemoral artery infusions of SNP was somewhat higher in the low compared with the high cholesterol group (P<.05 by ANOVA; Fig 3A). However, this difference in LBF response between the groups completely disappeared when comparing the percent increase above baseline (Fig 3B). Assessing the LBF response to SNP in the groups according LDL cholesterol levels of lesser or greater than the 25th percentile (NHANES III)¹⁵ yielded the same result as for total cholesterol. Changes in LVR in response to graded intrafemoral artery infusions of SNP mirrored the changes in LBF. Basal LVR was 405±52 and 510±26 units in the low and the high cholesterol groups, respectively (P=NS). LVR decreased in a dose-dependent fashion in both groups. In response to the graded intrafemoral artery infusion of SNP, LVR declined to 297±54, 253±47, and 212±42 units in the low cholesterol group and to 465±51, 391±45, and 239±24 units in the high cholesterol group (P=NS). These results demonstrate that both total and LDL cholesterol in the normal range have no effect on modulating endothelium-independent vasodilation.

View larger version (19K): [in this window] [in a new window]   Figure 3. A, LBF under basal conditions and in response to graded intrafemoral artery infusions of SNP in the low () and high () normal cholesterol groups. B, Percent increments (%) in LBF above baseline in response to graded intrafemoral artery infusions of SNP in the low and high cholesterol groups.

Correlational Analyses
Simple linear regression analyses examining the relationship between maximum increment in LBF in response to MCh or SNP and lipid parameters are shown in Table 3. Total cholesterol levels correlated inversely and significantly with maximal endothelial-dependent vasodilation as determined by the response to the intrafemoral artery infusions of Mch (Fig 4A) but had no impact on endothelium-independent vasodilation achieved by intrafemoral infusion of SNP. LDL cholesterol levels were also inversely and significantly correlated (Fig 4B) with maximal endothelium-dependent vasodilation. However, LDL cholesterol levels had no effect on endothelium-independent vasodilation. In contrast to the inverse relationship between total and LDL cholesterol and maximal endothelium-dependent vasodilation, HDL cholesterol, triglycerides, and free fatty acids did not have any relation to the maximal endothelium-dependent or -independent vasodilation. Thus, these results suggest that endothelium-dependent vasodilation is inversely and continuously related to prevailing total and LDL cholesterol levels.

View this table: [in this window] [in a new window]   Table 3. Correlations Between Maximal Percent Change in LBF and Lipid Levels in Response MCh or SNP

View larger version (19K): [in this window] [in a new window]   Figure 4. A, Relationship between the maximum percent increments (%) in LBF above baseline in response to graded intrafemoral artery infusions of MCh and total cholesterol levels. B, Relationship between the maximum percent increments in LBF above baseline in response to graded intrafemoral artery infusions of MCh and LDL cholesterol levels.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this study, we tested the hypothesis that cholesterol levels in the normal range may be associated with impaired endothelium-dependent vasodilation. The results of our study demonstrate that (1) total cholesterol and LDL cholesterol levels at the upper end of the normal range are associated with significant decreases in endothelium-dependent vasodilation, (2) a significant inverse continuous relationship exists between maximum endothelium-mediated vasodilation and both total and LDL cholesterol levels, and (3) there is no effect of total or LDL cholesterol on endothelium-independent vasodilation. These findings suggest that cholesterol even at levels within the normal range may cause endothelial dysfunction.

Cholesterol is now recognized as a major cause of macrovascular disease.^{1 2 3} The mechanism by which serum cholesterol elevations cause cardiovascular disease is not completely understood. Impaired endothelium-dependent vasodilation associated with elevated cholesterol levels could represent one such mechanism. Endothelium-dependent vasodilation, which depends at least in part on the production/release of nitric oxide,²⁰ has been demonstrated to be impaired in subjects with moderately elevated cholesterol levels (260 mg/dL).^{4 5 6 7} Because the relationship between cholesterol levels and cardiovascular disease is linear and apparently without a threshold,^{1 2} it is reasonable to ask whether cholesterol levels even in the normal range might be associated with impaired endothelium-dependent vasodilation. It is important to note that the levels of total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides in our subjects were nearly identical to those reported in the control subjects of the studies that found decreased endothelium-dependent vasodilation in hypercholesterolemic subjects.^{4 5 6 7} Although our study group had cholesterol levels that were within the normal range, there were impressive differences in the LBF response to the endothelium-dependent vasodilator MCh between subjects on the upper versus the lower range of normal cholesterol levels. The subjects in the upper range exhibited only half the LBF increase in response to MCh. Although this difference could have been artificially generated by the selection of the percentiles for the definition of high versus low normal cholesterol groups, this is not likely because nearly identical differences were found when we compared endothelium-dependent vasodilation between groups with <50th and >50th but <75th percentile total cholesterol levels (NHANES III¹⁵ ). Moreover, regression analysis showed a significant albeit modest continuous linear inverse relationship between cholesterol levels and maximum achieved endothelium-dependent increases in LBF. It should be noted that the relationship between total cholesterol or LDL cholesterol levels and maximum endothelium-dependent vasodilation was quite variable. However, this variability is not unexpected given the number of factors that modulate endothelium-dependent vasodilation, eg, blood pressure,^{21 22} age,²³ insulin sensitivity,^{24 25} and body fat content.²⁵ Therefore, it is clear that cholesterol levels alone cannot serve as a predictor of endothelial function. Nevertheless, it is important to realize that cholesterol levels even in the normal range may explain 20% of the variance in endothelium-dependent vasodilation.

The blunted response to MCh associated with cholesterol levels in the high normal range are likely the result of alterations in endothelial release of vasoactive substances, altered metabolism of these substances, or impaired action of these substances at the level of the vascular smooth muscle cell. MCh causes vasodilation via the production/release of EDNO⁹ but also through the release of other vasoactive factors like endothelium-dependent hyperpolarizing factor^{26 27 28} and prostaglandins.^{29 30} In subjects with high normal cholesterol levels, production/release of EDNO or endothelium-dependent hyperpolarizing factor could be reduced in response to MCh, leading to lower increases in LBF. Currently, no in vivo data are available regarding rates of nitric oxide production in response to MCh or regarding the contribution of endothelium-dependent hyperpolarizing factor to the vasodilatory response to MCh in subjects with normal or elevated cholesterol levels. However, hypercholesterolemic animals have been shown to exhibit increased basal rates of EDNO production.³¹ Furthermore, basal EDNO-dependent blood flow and blood flow responses to bradykinin that are largely EDNO dependent have been reported to be normal in frankly hypercholesterolemic humans,^{5 7} suggesting that production/release of EDNO in hypercholesterolemic subjects is normal. It is therefore possible that higher cholesterol levels may cause rapid degradation of NO perhaps via formation of oxygen radicals as suggested by others.^{32 33} Alternatively, cholesterol elevations could either increase the release of vasoconstrictor prostaglandins like thromboxane or reduce the release of vasodilator prostaglandins like prostacyclin. Changes in production of prostaglandins, which have been shown to be released in response to acetylcholine, could thus theoretically explain the blunted response to MCh in subjects with higher cholesterol levels. However, this seems unlikely because inhibition of prostaglandin production in humans does not appear to change the blood flow response to acetylcholine.³⁴ Unfortunately, no studies have assessed the effect of hypercholesterolemia on prostaglandin production/release. If endothelial function and EDNO production/release are not impaired by higher cholesterol levels, it follows that the response to EDNO at the level of the vascular smooth muscle cell must be diminished. This explanation does not appear likely because the response to SNP that gauges nitric oxide action at the level of the vascular smooth muscle cell was comparable between groups with low and high normal cholesterol levels. Furthermore, the absence of any correlation between the maximum LBF response to SNP and cholesterol or other lipid levels indicates no effect of lipids on endothelium-independent vasodilation. Clearly, more research is needed to elucidate the mechanism(s) underlying the diminished endothelium-dependent vasodilation in the subjects with high cholesterol levels.

Before one can conclude that the large difference in blood flow response to MCh was due mainly to cholesterol, one has to exclude other possible factors that have been associated with impaired endothelium-dependent vasodilation. Besides the difference in cholesterol levels, triglyceride levels were significantly higher in the high normal group. Although we cannot completely rule out the possibility that elevated triglyceride levels are associated with impaired endothelium-dependent vasodilation, this seems to be unlikely. Indeed, there was no relationship between triglyceride levels and maximum endothelium-dependent vasodilation. Furthermore, increases in triglyceride concentrations in humans have not been reported to blunt endothelium-dependent vasodilation. On the other hand, obesity/insulin resistance, diabetes mellitus, hypertension, age, and smoking have been shown to be associated with decreased responses to endothelium-dependent vasodilators. Our groups were well matched for body weight, body fat content, fasting glucose and insulin levels, blood pressure, age, sex, and smoking history, suggesting that these factors are not likely to explain the differences in endothelium-dependent vasodilation between the low and high normal cholesterol groups. We did not determine insulin sensitivity in our subjects and thus cannot dismiss the possibility that differences in insulin resistance could explain part of the decreased response to Mch.²⁵ This possibility is unlikely because the major determinants of insulin sensitivity, namely body fat content and blood pressure, were comparable between the groups. Furthermore, one would expect higher fasting insulin levels in insulin-resistant subjects, but fasting insulin levels were comparable between groups. Finally, hypercholesterolemia per se is not associated with increased insulin resistance.^{35 36}

Our finding that cholesterol levels even in the normal range may be inversely related to endothelium-dependent vasodilation has important clinical implications. It suggests that lowering cholesterol levels even within in the normal range may improve the production/release of EDNO. This idea is supported by recent reports that lowering cholesterol levels enhances endothelium-dependent vasodilation not only in subjects with massively elevated cholesterol levels³⁷ but also in subjects with normal cholesterol levels.³⁸ It is important to note that lowering of average cholesterol levels in patients with documented CAD³⁹ leads to decreased rates of myocardial infarction. Improvement of endothelium-dependent vasodilation in the coronary vasculature was also achieved in hypercholesterolemic patients with documented CAD by lowering cholesterol levels,⁴⁰ which demonstrates a qualitatively comparable effect of cholesterol to modulate endothelium-dependent vasodilation at the level of the heart and skeletal muscle. Finally, our findings provide a physiological rationale for the decision of the National Cholesterol Education Program and the American Heart Association⁴¹ to abandon normal cholesterol levels and to recommend "desirable" lower cholesterol levels in the population that, if achieved, would be expected to result in greatly reduced rates of CAD and cardiovascular mortality.

	Selected Abbreviations and Acronyms

 

CAD = coronary artery disease EDNO = endothelium-derived nitric oxide LBF = leg blood flow LVR = leg vascular resistance MAP = mean arterial pressure MCh = methacholine chloride SNP = sodium nitroprusside

	Acknowledgments

 
This work was supported by grants DK42469, MO1-RR750-19, and DK20452 from the NIH, a Veterans Affairs Merit Review Award, and Grant-in-Aid A3392 from the American Heart Association. We wish to thank Joyce Ballard for her expert and invaluable help in preparing the manuscript and Dr Naomi Fineberg from the Division of Biostatistics at the Indiana University Medical Center for her expertise and support in performing the statistical analysis.

Received May 27, 1997; revision received July 10, 1997; accepted July 21, 1997.

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