European Heart Journal (1999) 20, 16761680 Article No. euhj.1999.1689, available online at http://www.idealibrary.

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Vitamin C improves endothelial function of epicardial coronary arteries in patients with hypercholesterolaemia or essential hypertension assessed by cold pressor testing
M. Jeserich, T. Schindler, M. Olschewski* M. Unmussig, H. Just and U. Solzbach
Medizinische Klinik III and *Medizinische Biometrie und Informatik, Universitat Freiburg, Freiburg, Germany

Aims There is evidence that formation of free radicals increases in patients with hypertension or hypercholesterolaemia, which may contribute to endothelial dysfunction of epicardial coronary arteries due to inactivation of the vasodilator NO. The present study was designed to test whether the abnormal constriction of epicardial coronary arteries due to sympathetic stimulation by the cold pressor test in patients with essential hypertension or hypercholesterolaemia could be reversed by administration of the antioxidant vitamin C. Methods and Results In 28 patients without relevant coronary artery stenosis the cold pressor test was performed before and after a 3 g infusion of vitamin C. In ve normal controls the cold pressor test led to a similar increase in luminal area before and after vitamin C (37 13% and 19 08%, ns vs before vitamin C). In nine hypercholesterolaemic patients the cold pressor test led to a 141 28% reduction in cross-sectional area before vitamin C. This constriction was signicantly improved after vitamin C to 76% 20, P=0027 vs before vitamin C. In nine hypertensive patients, the cold pressor test led to a

171 32% decrease in cross-sectional area before vitamin C, which was improved to 71 31 after vitamin C, P=0004 vs before vitamin C. This increase in luminal area was signicant in each group in comparison with normal controls (each P<005). Administration of saline (placebo group, ve patients) had no signicant eect on cold pressor test-induced constriction ( 69 39% before and 68 37% after saline). Conclusion The antioxidant vitamin C reverses cold pressor test-induced vasoconstriction of epicardial coronary arteries in patients with hypertension or hypercholesterolaemia. Our data suggest that enhanced oxidative stress contributes to impaired endothelial function in this patient population. (Eur Heart J 1999; 20: 16761680)  1999 The European Society of Cardiology Key Words: Cold pressor test, endothelium, antioxidant vitamin C. See page 1615 for the Editorial comment on this article

Introduction
Essential hypertension and hypercholesterolaemia are primary risk factors for coronary vascular disease. The endothelium plays a pivotal role in the regulation of vascular tone[1]. Impaired coronary endothelial vasodilator function is well established in patients with risk factors for coronary artery disease and is assumed to be an early step in the development of atherosclerosis[2,3]. Sympathetic stimulation by the cold pressor test in
Revision submitted 7 May 1999, and accepted 12 May 1999. Correspondence: Michael Jeserich, Praxis Dr Haggenmiller und Dr Jeserich, Konigstr. 39, 90402 Nurnberg, Germany. 0195-668X/99/221676+05 $18.00/0

humans leading to an increase in heart rate and mean arterial blood pressure has been shown to dilate normal coronary arteries, but to constrict epicardial coronary arteries in patients with risk factors for atherosclerosis[4,5]. The exact mechanism still remains to be claried, but there is increasing evidence that inactivation of the vasodilator NO by superoxide anions or oxidized LDLs might be involved[6,7]. Ascorbic acid, or vitamin C, is the main water-soluble antioxidant in human plasma[8]. It eectively scavenges superoxide and other reactive oxygen species[8]. Therefore, the present study was designed to test whether the antioxidant vitamin C reverses the abnormal constriction of epicardial coronary arteries due to sympathetic
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Table 1

Characteristics of the study population

Hypercholesterolaemia Hypertension 6/3 63 3* 70 3 115 5 196 18 124 12 15 1 Controls 1/4 53 2 74 4 102 5 183 15 108 8 14 1

Sex (male/female) Age (years) Heart rate (beats . min 1) Mean aortic pressure (mmHg) Cholesterol (mg . 100 ml 1) LDL cholesterol (mg . 100 ml 1) Haemoglobin (g . 100 ml 1) *P<005 vs controls; **P<001 vs controls.

5/4 59 1 72 4 102 4 279 7** 189 8** 15 1

stimulation in patients with essential hypertension of hypercholesterolaemia.

Methods Patient population

Twenty-eight patients were studied (Table 1). Nine patients had hypercholesterolaemia and another nine had a known history of essential hypertension and no other cardiovascular risk factors. In addition, ve controls with smooth epicardial coronary arteries and without known risk factors were examined. In ve patients (mean age 57 5 years) with hypertension (n=3) or hypercholesterolaemia (n=2), the cold pressor test was performed before and after infusion of 100 ml 09% saline infusion without vitamin C (placebo group). Patients with a history of unstable angina pectoris or myocardial infarction, valvular heart disease, diabetes mellitus, cigarette smoking and a history suggestive of variant angina or heart failure were excluded. Vasodilators and beta-blockers were discontinued at least 24 h before the study. All patients gave informed consent before the study. The study was approved by the local Ethics Committee of the University of Freiburg, Germany.

of 100 ml 09% saline containing 3 g of vitamin C over 10 min. Heart rate and aortic pressure were continuously measured and serial hand injections of non-ionic contrast material (Ultravist, Schering AG, Germany) were done. The maximal vasodilator response was tested by administration of nitroglycerin 02 mg i.c. Quantitative coronary angiography by automatic contour detection of the coronary artery was performed as previously described and evaluated[3,5,9].

Statistical analysis
For descriptive purposes, all data are presented as mean SEM or absolute frequencies where indicated. Dierences in baseline values between groups were tested by means of the KruskalWallis test for quantitative, or the Chi-square test for qualitative variables. Absolute changes of cold pressor test induced vascular responses before and after vitamin C infusion or placebo within each group were analysed with the Wilcoxon sign rank test. Comparison of these changes between groups was investigated by means of an analysis of covariance model (ANCOVA), with patient group and individual corresponding cold pressor test values at baseline as covariates. All test procedures were two-sided with a P value of less than 005 indicating statistical signicance.

Study protocol and quantitative coronary angiography

All patients underwent routine diagnostic cardiac catheterization for evaluation of chest pain. A prerequisite for inclusion in the study was the absence of angiographically signicant coronary artery disease. The left ventricular cineangiograms of all patients showed no evidence of segmental wall motion abnormalities. After routine coronary angiography, a control angiogram was performed. Special care was taken to avoid overlapping of coronary segments. A relatively straight left anterior descending coronary artery (in 13 patients) or left circumex coronary artery segment (in 15 patients) 48 mm long was pre-selected. This was followed by immersion of the patients right hand and forearm in ice water for 90 s before and after an intravenous infusion

Results
In ve normal controls, cold pressor test led to a similar increase in luminal area before and after vitamin C (Table 2). In patients with hypercholesterolaemia and in patients with essential hypertension, cold pressor testinduced constriction of epicardial arteries was signicantly improved after vitamin C vs before vitamin C and compared to normal controls, each P<005 (Table 2). Figures 1 and 2 depict the individual responses of the analysed coronary segments before and after vitamin C. In the placebo group, constriction of the epicardial arteries was similar before and after saline ( 69 39% before and 68 37% after saline). Individual responses are shown in Table 3. The maximal vasodilator response was tested by administration of nitroglycerin (Table 2). Vasodilator responses to nitroglycerin
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Table 2 Changes in cross-sectional area of the left anterior descending/left circumex coronary artery compared to baseline (%)
CPT before Vit C Hypercholesterolaemia Essential hypertension Controls 141% 171 +37 28 32 13 CPT after Vit C 76% 71 +19 20* 31 08 NTG +335 +395 +287 40 82 67

CPT=cold pressor test; LAD=left anterior descending coronary artery; LCX=left circumex coronary artery; Vit C=Vitamin C; NTG-nitroglycerin; *P=0027 vs cold pressor test before vitamin C, P=0004 vs cold pressor test before vitamin C; P<005 compared to corresponding changes in controls (ANCOVA).

10 5 0 5 (%) 10 15 20 25 30 35

* *

CPT before vit C

CPT after vit C

Figure 1 Individual responses of the analysed coronary segments to the cold pressor test (CPT) before and after 3 g of vitamin C (vit C) in hypertensive patients. were not signicantly dierent in patients who were hypercholesterolaemic, hypertensive or in controls. Vitamin C levels were measured before and after vitamin C infusion in ve patients with hypercholesterolaemia, four patients with hypertension and in four controls. The levels were between 055 and 138 mg/100 ml and showed no dierences between groups. In our laboratory the normal range is 06 to 20 mg/100 ml serum. After vitamin C there was a 20- to 30-fold increase in vitamin C serum levels. constriction of epicardial arteries due to cold pressor testing could be diminished by application of 3 g of vitamin C intravenously. Cold pressor testing has been shown to dilate normal coronary arteries, but to constrict epicardial coronary arteries in patients with risk factors for atherosclerosis[35]. This type of stimulation induces sympathetic release of norepinephrine and epinephrine, and leads to elevation in mean aortic pressure[10], an increase in coronary blood ow[4,5] and ow-mediated release of NO[11], resulting in dilation of epicardial coronary arteries in the normal setting[35]. Coronary vasomotion in response to sympathetic stimulation by cold pressor testing has been shown to mirror the eects of the endothelium-dependent vasodilator acetylcholine[3,5]. In a previous study[12] we have shown that vitamin C improves the acetylcholine-induced constriction of epicardial arteries in hypertensive patients. Compared to the application of acetylcholine by guiding catheter and intracoronary Doppler, measurements to test endothelial function cold pressor testing oer three main advantages: rstly, it tests sympathetic stimulation; secondly, compared to acetylcholine it is more easily

Discussion
The present study demonstrates that impaired coronary vasomotion following sympathetic stimulation in patients with essential hypertension or hypercholesterolaemia can be improved by administration of the antioxidant vitamin C. In a homogeneous group with either essential hypertension or hypercholesterolaemia and no further cardiovascular risk factors predisposing to endothelial dysfunction, we found that abnormal
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5 0 5 10 (%) 15 20 25

*
30 CPT before vit C CPT after vit C

Figure 2 Individual responses of the analysed coronary segments to cold pressor test (CPT) before and after 3 g of vitamin C (vit C) in patients with hypercholesterolaemia.

performed during routine coronary angiography; and thirdly, introduction of a guiding catheter is not necessary. Thereby it is less invasive and time consuming. Thus, cold pressor testing may be a promising tool to test the functional integrity of the endothelium during routine coronary angiography. The precise mechanism responsible for endotheliumderived dysfunction in patients with risk factors for coronary atherosclerosis is not known. In experimental models of hypertension, abnormal production of vasoactive substances such as endothelin or angiotensin II have been observed[13,14]. Interestingly, it has been suggested that angiotensin activates enzyme systems that promote superoxide generation in vascular smooth cells[15]. In addition, it has been shown that prolonged stretch, important in the pathophysiology of hypertension, increases superoxide production by activation of protein kinase[16]. Thus, there is increasing evidence that these pathological pathways might contribute to increased oxidative stress in hypertension. Table 3 Individual responses of the luminal area to cold pressor testing before and after placebo
Patient 1 2 3 4 5 Mean SEM Baseline 521 651 339 476 618 521 050 CPT before (%) Vit C (mm2) 521 660 288 383 610 493 062 004 139 1521 1939 132 691 386 CPT after (%) Vit C (mm2) 513 665 298 378 609 492 061 159 209 1208 2062 160 676 375

In hypercholesterolaemia, other mechanisms have been reported, including impairment of NO synthesis due to uncoupling of the receptor-Gi complex[17], or involvement of endogenous inhibitors of nitric oxide synthase[18]. However, similar to models of hypertension, reduced NO activity could be caused by enhanced catabolism[19]. Superoxide anions are known to react rapidly with NO[6]. Thus, increased catabolism of NO, through its reaction with superoxide, could be an important mechanism in hypercholesterolaemia and hypertension and might thus contribute to the observed endothelial dysfunction in this patient population. Ascorbic acid has been shown to be an ecient scavenger of many reactive oxygen species, including superoxide anion[8,20]. Indeed, previous studies in the forearm circulation[21] and in the coronary circulation[2224] have assumed that nitric oxide degradation by oxygenderived free radicals contributes to abnormal vascular reactivity in smokers or patients with hypertension, diabetes mellitus or coronary artery disease. The dose of vitamin C was chosen in view of previous studies by Heitzer et al.[21] and our group[12]. It is very likely that a lower dose of 1 or 2 g of vitamin C would have had similar results. Indeed, in an ongoing study, 1 g of vitamin C taken orally twice a day improves coronary perfusion in hypertensive patients with endothelial dysfunction after 3 months[25].

Clinical implications
Our results show that the antioxidant vitamin C improves endothelial dysfunction of coronary arteries in hypertensive and hypercholesterolaemic patients, assessed by cold pressor testing. The data suggest that enhanced oxidative stress contributes to impaired
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CPT=cold pressor test; Vit C=vitamin C.

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endothelial function in this patient population. Further studies investigating the long-term eects of vitamin C have to be performed before general administration of vitamin C in patients with hypertension or hypercholesterolaemia can be recommended.
We thank the nurses of the Cardiac Catheterization Laboratory at the University of Freiburg for invaluable assistance in the performance of the study. We thank Frau Baumann and Frau Dr Zurcher, MD for performing the vitamin C measurements and we thank Dr Koster, MD for providing the cholesterol measurements.

[12]

[13]

[14] [15]

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