European Heart Journal (1999) 20, Article No. euhj , available online at on

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1 European Heart Journal (1999) 20, Article No. euhj , available online at on 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. Unmüssig, H. Just and U. Solzbach Medizinische Klinik III and Medizinische Biometrie und Informatik, Universität 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 five normal controls the cold pressor test led to a similar increase in luminal area before and after vitamin C ( % and %, ns vs before vitamin C). In nine hypercholesterolaemic patients the cold pressor test led to a % reduction in cross-sectional area before vitamin C. This constriction was significantly improved after vitamin C to 7 6% 2 0, P=0 027 vs before vitamin C. In nine hypertensive patients, the cold pressor test led to a % decrease in cross-sectional area before vitamin C, which was improved to after vitamin C, P=0 004 vs before vitamin C. This increase in luminal area was significant in each group in comparison with normal controls (each P<0 05). Administration of saline (placebo group, five patients) had no significant effect on cold pressor test-induced constriction ( % before and % 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: ) 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 Correspondence: Michael Jeserich, Praxis Dr Haggenmiller und Dr Jeserich, Königstr. 39, Nürnberg, Germany X/99/ $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 clarified, 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].Iteffectively 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 1999 The European Society of Cardiology

2 Vitamin C improves endothelial function of epicardial coronary arteries 1677 Table 1 Characteristics of the study population Hypercholesterolaemia Hypertension Controls Sex (male/female) 5/4 6/3 1/4 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<0 05 vs controls; P<0 01 vs controls. 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, five controls with smooth epicardial coronary arteries and without known risk factors were examined. In five 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 0 9% 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 0 9% 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 0 2 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. Differences in baseline values between groups were tested by means of the Kruskal Wallis 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 0 05 indicating statistical significance. 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 significant 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 circumflex coronary artery segment (in 15 patients) 4 8 mm long was pre-selected. This was followed by immersion of the patient s right hand and forearm in ice water for 90 s before and after an intravenous infusion Results In five 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 significantly improved after vitamin C vs before vitamin C and compared to normal controls, each P<0 05 (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 ( % before and % 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

3 1678 M. Jeserich et al. Table 2 Changes in cross-sectional area of the left anterior descending/left circumflex coronary artery compared to baseline (%) CPT before Vit C CPT after Vit C NTG Hypercholesterolaemia 14 1% % Essential hypertension Controls CPT=cold pressor test; LAD=left anterior descending coronary artery; LCX=left circumflex coronary artery; Vit C=Vitamin C; NTG-nitroglycerin; P=0 027 vs cold pressor test before vitamin C, P=0 004 vs cold pressor test before vitamin C; P<0 05 compared to corresponding changes in controls (ANCOVA) (%) 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 significantly different in patients who were hypercholesterolaemic, hypertensive or in controls. Vitamin C levels were measured before and after vitamin C infusion in five patients with hypercholesterolaemia, four patients with hypertension and in four controls. The levels were between 0 55 and 1 38 mg/100 ml and showed no differences between groups. In our laboratory the normal range is 0 6 to 2 0 mg/100 ml serum. After vitamin C there was a 20- to 30-fold increase in vitamin C serum levels. 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 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 [3 5]. 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 flow [4,5] and flow-mediated release of NO [11], resulting in dilation of epicardial coronary arteries in the normal setting [3 5]. Coronary vasomotion in response to sympathetic stimulation by cold pressor testing has been shown to mirror the effects 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 offer three main advantages: firstly, it tests sympathetic stimulation; secondly, compared to acetylcholine it is more easily

4 Vitamin C improves endothelial function of epicardial coronary arteries (%) 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 Baseline CPT before (%) Vit C (mm 2 ) CPT after (%) Vit C (mm 2 ) Mean SEM CPT=cold pressor test; Vit C=vitamin C. In hypercholesterolaemia, other mechanisms have been reported, including impairment of NO synthesis due to uncoupling of the receptor-g i 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 efficient scavenger of many reactive oxygen species, including superoxide anion [8,20]. Indeed, previous studies in the forearm circulation [21] and in the coronary circulation [22 24] 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

5 1680 M. Jeserich et al. endothelial function in this patient population. Further studies investigating the long-term effects 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 Zürcher, MD for performing the vitamin C measurements and we thank Dr Köster, MD for providing the cholesterol measurements. References [1] Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980; 288: [2] Treasure CB, Manoukian SV, Klein JL et al. Epicardial coronary artery responses to acetylcholine are impaired in hypertensive patients. Circ Res 1992; 71: [3] Zeiher AM, Drexler H, Wollschläger H, Just H. 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