Consumption of vitamin E in coronary circulation in patients with variant angina

Abstract

Objectives: The plasma status of vitamin E has been suggested to be linked to the activity of coronary artery spasm. This study was designed to determine whether vitamin E is actually consumed in the coronary circulation in patients with active variant angina having repetitive spasm-induced transient myocardial ischemia and reperfusion. Methods: Blood samples were obtained simultaneously from the aortic root, coronary sinus and right atrium in 12 patients with variant angina due to spasm of the left coronary artery, nine patients with stable effort angina and nine control subjects. Plasma vitamin E (α- and γ-tocopherol) concentrations were determined by use of high-performance liquid chromatography and plasma lipid peroxides were measured as thiobarbituric acid-reactive substances (TBARS). Results: At baseline, both plasma α- (p<0.01) and γ- (p<0.05) tocopherol levels were significantly lower in the coronary sinus (5.50±0.50 and 0.55±0.07 mg/l, mean±SEM) than in the aortic root (6.63±0.57 and 0.63±0.08 mg/l) and also in the right atrium (6.44±0.61 and 0.63±0.09 mg/l) in the variant angina group. The TBARS level was significantly (p<0.05) higher in the coronary sinus than in the aortic root in this group. In contrast, these levels were not significantly different between the samples from the coronary sinus and the aortic root or the right atrium in the control group and also in the stable effort angina group. The coronary sinus–aortic difference in plasma vitamin E levels in the variant angina group was not significantly altered after left coronary artery spasm induced by intracoronary injection of acetylcholine. Also, the plasma vitamin E levels in the aortic root, coronary sinus and right atrium all remained unchanged in the stable effort angina group after pacing-induced angina and in the control group after intracoronary administration of acetylcholine. Conclusions: Transcardiac reduction in plasma vitamin E concentrations concomitant with lipid peroxide formation was demonstrated in patients with active variant angina, suggesting actual consumption of this major endogenous antioxidant. Oxidative stress and vitamin E exhaustion may be involved in the pathogenesis of coronary artery spasm.

Vitamin E, Variant angina, Coronary spasm, Myocardial ischemia, Reperfusion

Time for primary review 21 days.

1 Introduction

It is generally believed that the most important biological defence against lipid peroxidation is vitamin E [1, 2]. The incorporation of this lipophilic antioxidant vitamin into the lipoprotein particles protects low-density lipoprotein (LDL) from the pro-oxidant environment of the arterial wall by scavenging both oxygen and lipid free radicals [3]. Its level in blood has been reported to be inversely related to the risk of coronary heart disease [4, 5]. Recent studies also indicated that antioxidant therapy with vitamin E supplementation can be an effective treatment for both primary and secondary prevention against coronary heart disease [6–8].

Variant angina is a form of unstable angina characterized by coronary spasm-induced severe transient ischemic episodes of short duration, which may be frequently repeated, although the precise underlying mechanisms by which coronary spasm is triggered remain to be elucidated [9, 10]. We have recently reported that LDL from patients with active variant angina was highly susceptible to peroxidative modification induced by cupric ion, and vitamin E levels were significantly lower both in plasma and LDL fractions from these patients than from subjects without coronary spasm, suggesting an association between vitamin E deficiency and coronary artery spasm [11, 12]. However, the exact causal relationship between them remains unknown. It is possible that increased lipid oxidation stress, such as free radical production induced by frequently repeated alteration between severe transient regional myocardial ischemia and reperfusion, may cause exhaustion of vitamin E, resulting in higher susceptibility of LDL to oxidation [13]. However, free radical formation and/or antioxidant vitamin E consumption in the coronary circulation during ischemia/reperfusion by coronary spasm has not been demonstrated.

In the present study, to determine whether vitamin E is actually consumed in the coronary circulation in the case of coronary spasm, plasma vitamin E concentrations and products of lipid peroxidation were determined simultaneously in the aortic root, coronary sinus and right atrium in patients with variant angina before and after left coronary artery spasm induced by intracoronary injection of acetylcholine.

2 Methods

2.1 Study patients

Patients included in this study were divided into three groups. The variant angina group consisted of 12 patients (11 men and a woman; mean age, 57±3 years) who had attacks of chest pain associated with ST segment elevation in the precordial leads on the ECG, occurring at rest, and in whom spasm of the left coronary artery was induced by injection of acetylcholine into the left coronary artery. In the present study, coronary spasm was defined as total or subtotal occlusion of the coronary artery associated with ischemic ST segment changes on the ECG with or without chest pain. The stable effort angina group consisted of nine patients (seven men and two women; mean age, 59±4 years) with a significant (>75% narrowing of luminal diameter) organic left coronary artery stenosis having exertional angina and positive treadmill exercise stress test but no rest angina. The control group consisted of nine patients (seven men and two women; mean age, 56±5 years) who were admitted due to suspected coronary artery disease but finally diagnosed to be free from coronary artery disease. In these patients, coronary arteriographic findings revealed neither fixed coronary artery stenosis (>25% of luminal diameter) nor coronary artery spasm induced by the intracoronary injection of acetylcholine. None of them had positive treadmill exercise stress test results. Patients receiving any drugs containing vitamin E were excluded.

2.2 Procedures for catheterization and blood sampling

All drugs were withdrawn >24 h before cardiac catheterization except for sublingual nitroglycerin. The study was performed in the morning while the patients were in the fasting state. For blood sampling, a 6F Judkins catheter was advanced from the femoral artery and placed in the ascending aorta and a 7.5F Opticathe® (Model P7110; Oximetrix) was positioned in the coronary sinus through the internal jugular vein. The position of the catheter was confirmed by occasional injection of the contrast medium. Coronary arteriography was performed using the Judkins technique. All patients were given 5000 U of heparin at the insertion of the Judkins catheter. A blood sample was obtained from the right atrium via the Opticathe® before it was advanced into the coronary sinus. Blood samples were then collected from the aortic root and the coronary sinus simultaneously. To avoid artificial hemolysis, blood samples were drawn carefully and slowly through the catheter. The time interval between the blood sampling from the right atrium and the coronary sinus through the same catheter was a few minutes. Written informed consent was obtained from all the study patients, and the study protocol was approved by the ethical committee of our institution.

2.3 Intracoronary acetylcholine provocative test

Before coronary arteriography, a temporary pacing catheter was inserted into the right ventricle via a femoral vein and connected to a pulse generator with a demand-driven rate of 50 beats/min, both in the variant angina and control groups. After control blood sampling, 20 to 100 μg of acetylcholine was injected into the left coronary artery to induce coronary spasm both in the variant angina and the control groups [14]. A coronary angiogram was obtained when ST segment changes or chest pain appeared or 2 min after each injection. The angina was relieved promptly by intracoronary injection of nitroglycerin (0.2–0.5 mg). After confirming the resolution of the spasm in the repeated coronary arteriogram, blood samples were taken, again simultaneously from the coronary sinus and the aortic root.

2.4 Pacing stress test

After control blood sampling, atrial pacing was commenced in the stable effort angina group. The pacing rate was begun at 90 beats/min and was increased until the patients experienced angina that was sustained for 3 or 5 min. Then, sublingual nitroglycerin was administered to relieve angina. Blood samples then were collected simultaneously from both the coronary sinus and the aortic root after pacing-induced angina.

In all of the study patients, blood samples were also obtained from the right atrium through the catheter, which was pulled back to the right atrium from the coronary sinus immediately after sampling blood from the coronary sinus.

2.5 Assay of plasma vitamin E concentration

All of the blood samples were collected into vacutainer tubes containing ethylenediaminetetraacetic acid. Plasma was immediately separated by centrifugation and stored at −20°C until use. The vitamin E concentration in plasma was estimated by the high-performance liquid chromatography method, with vitamin E acetate as internal standard, added before lipid extraction [15]. The plasma vitamin E concentration was expressed as mg/l.

2.6 Assay of lipid peroxidation products

Plasma lipid peroxidation products were assayed as thiobarbituric acid-reactive substances (TBARS), as described previously [11]and results were expressed as nmol of malondialdehyde equivalents.

2.7 Statistical analysis

All data are reported as mean±SEM. Intergroup comparisons concerning serum lipid levels, plasma tocopherol levels and plasma TBARS levels were made with a Bonferroni multiple comparisons test after analysis with one-way ANOVA. Student's paired t-test was used to make a comparison between the tocopherol or TBARS levels in the coronary sinus and in the aortic root or in the right atrium in the same subjects. To compare the prevalence between the groups, a chi-square test was performed with Yates’ correction if one of the frequency tables was smaller than five. Differences were considered to be statistically significant at p<0.05.

Table 1

Clinical characteristics and angiographic findings of the study groups

	Control subjects	Variant angina	Stable effort angina
Number of patients	9	12	9
Age, years
Mean±SEM	56±5	57±3	59±4
Range	28–72	26–66	38–72
Male/female	7/2	11/1	7/2
Old myocardial infarction, n	0	3	3
Blood pressure >150/90 mmHg, n	1	3	3
Smokers, n	5	9	5
Diabetes mellitus, n	2	1	4
Obesity, n	2	4	2
Serum cholesterol, mg/dl	197±11	193±10	183±12
Serum triglycerides, mg/dl	126±21	146±26	137±8
HDL–cholesterol, mg/dl	50±6	40±4a	36±4a
Extent of coronary vessel
organic stenosis
75% stenosis, n	0	2	9
Coronary artery spasm, n
Single-vessel spasm	0	4	0
Multivessel spasm	0	8	0
Ejection fraction <50%, n	0	0	1

a

p<0.05 vs. control subjects.

3 Results

Table 1 shows clinical data and plasma lipid profiles in the study groups. There were no significant differences among the three groups in any of the following variables: age, sex, hypertension, smoking, diabetes mellitus, obesity, serum cholesterol and serum triglycerides. The high-density lipoprotein (HDL)–cholesterol level was significantly (p<0.05) lower both in the variant angina and the stable effort angina groups compared with the control group.

3.1 Vitamin E and TBARS levels in coronary circulation at baseline

In the variant angina group, five patients had spontaneously occurring anginal attacks with ST-segment elevation on ECG at night or in the morning prior to coronary arteriography. All of these patients had anginal attacks also during coronary arteriographic examination. The plasma vitamin E concentrations in the aortic root, coronary sinus and also right atrium at baseline in the study patients are shown in Table 2 and Fig. 1. Plasma α-tocopherol levels in the aortic root, in the coronary sinus and in the right atrium were significantly (p<0.01 for each) lower in the variant angina group than those in the stable effort angina group and also in the control group, respectively. Plasma γ-tocopherol levels in the aortic root, in the coronary sinus and in the right atrium were significantly (p<0.01 for each) lower in the variant angina group and also in the stable effort angina group compared with those in the control group, respectively. As shown in Table 2, the variant angina group showed significantly (p<0.05 for each) higher TBARS when compared to the stable effort angina group and the control group, irrespective of the sampling location, i.e. in the aortic root, in the coronary sinus or in the right atrium. In the variant angina group, both plasma α- and γ-tocopherol levels were significantly (p<0.01 for each) lower in the coronary sinus than in the aortic root and in the right atrium, respectively. The plasma TBARS level was significantly (p<0.05) higher in the coronary sinus than in the aortic root. In contrast, these levels were not significantly different between the samples from the coronary sinus and the aortic root or the right atrium in the stable effort angina group or the control group.

Table 2

Plasma vitamin E and TBARS levels in coronary circulation in study patients

Variant angina		Baseline	After ACh	Units
AO	α-Tocopherol	6.63±0.57	6.58±0.43	(mg/l)
	γ-Tocopherol	0.63±0.08	0.56±0.07	(mg/l)
	TBARS	2.3±0.3	2.4±0.5	(μmol/l)
CS	α-Tocopherol	5.50±0.50a	5.97±0.42b	(mg/l)
	γ-Tocopherol	0.55±0.07a	0.57±0.07	(mg/l)
	TBARS	3.2±0.5 c	2.9±0.8	(μmol/l)
RA	α-Tocopherol	6.44±0.61	6.58±0.52	(mg/l)
	γ-Tocopherol	0.63±0.09	0.61±0.07	(mg/l)
	TBARS	2.6±0.5	2.7±0.7	(μmol/l)
CS−AO	α-Tocopherol	−1.13±0.27	−0.61±0.22	(mg/l)
	γ-Tocopherol	−0.08±0.02	0.01±0.02	(mg/l)
	TBARS	0.9±0.4	0.5±0.6	(μmol/l)

Stable effort angina		Baseline	After pacing
AO	α-Tocopherol	8.81±0.37	8.94±0.30	(mg/l)
	γ-Tocopherol	0.52±0.06	0.54±0.06	(mg/l)
	TBARS	1.5±0.2	1.5±0.2	(μmol/l)
CS	α-Tocopherol	8.77±0.36	8.83±0.35	(mg/l)
	γ-Tocopherol	0.53±0.06	0.53±0.06	(mg/l)
	TBARS	1.6±0.1	1.5±0.1	(μmol/l)
RA	α-Tocopherol	9.09±0.43	9.23±0.36	(mg/l)
	γ-Tocopherol	0.56±0.06	0.56±0.06	(mg/l)
	TBARS	1.6±0.2	1.5±0.2	(μmol/l)
CS−AO	α-Tocopherol	−0.04±0.14	−0.11±0.10	(mg/l)
	γ-Tocopherol	0.01±0.02	−0.01±0.01	(mg/l)
	TBARS	0.1±0.1	0.0±0.1	(μmol/l)

Control		Baseline	After ACh
AO	α-Tocopherol	9.88±1.33	9.23±1.28	(mg/l)
	γ-Tocopherol	0.83±0.08	0.78±0.09	(mg/l)
	TBARS	1.5±0.2	1.7±0.3	(μmol/l)
CS	α-Tocopherol	9.69±1.36	9.50±1.14	(mg/l)
	γ-Tocopherol	0.80±0.09	0.82±0.09	(mg/l)
	TBARS	1.6±0.2	1.6±0.3	(μmol/l)
RA	α-Tocopherol	9.68±1.46	9.72±1.19	(mg/l)
	γ-Tocopherol	0.85±0.10	0.83±0.09	(mg/l)
	TBARS	1.6±0.2	1.5±0.2	(μmol/l)
CS−AO	α-Tocopherol	−0.19±0.11	0.27±0.33	(mg/l)
	γ-Tocopherol	−0.03±0.02	0.04±0.03	(mg/l)
	TBARS	0.1±0.1	−0.1±0.1	(μmol/l)

a

p<0.01 vs. AO and RA.

^bp<0.05 vs. AO and RA.

^cp<0.05 vs. AO.

Abbreviations: AO, aortic root; CS, coronary sinus; RA, right atrium.

CS−AO, coronary sinus−aortic difference.

After ACh, after intracoronary injection of acetylcholine in the left coronary artery.

After pacing, after pacing-induced angina.

TBARS, thiobarbituric acid-reactive substances (malondialdehyde equivalents).

Fig. 1

Comparison of the α-tocopherol level between the plasma samples simultaneously obtained from the aortic root and the coronary sinus and also the coronary sinus and the right atrium at baseline (Baseline) and after intracoronary injection of acetylcholine in the left coronary artery (ACh) or after rapid atrial pacing-induced angina (Pacing) in the study patients. Control: control subjects (a); Variant angina: patients with variant angina (b); Stable effort angina: patients with stable effort angina (c).

3.2 Vitamin E and TBARS levels in coronary circulation after spasm

The injection of acetylcholine into the left coronary artery induced left coronary artery spasm in all of the patients of the variant angina group. Total occlusion of the left anterior descending branch was induced in nine, and subtotal or severe diffuse vasoconstriction was induced in the remaining three. In the three patients, spasm of both the left anterior descending branch and the left circumflex branch was demonstrated angiographically. All of the episodes of induced spasm were associated with chest pain and electrocardiographic ischemic ST-segment changes. Ten patients had attacks associated with ST-segment elevation in the precordial leads and the other two had ST-segment depression. In contrast, coronary artery spasm was not induced in the left coronary artery after the intracoronary injection of acetylcholine in the control group. In all those in the stable effort angina group, anginal attacks were induced during rapid atrial pacing. The plasma vitamin E concentrations in the aortic root and coronary sinus after injection of acetylcholine or after rapid atrial pacing are shown in Table 2 and Fig. 1. In the variant angina group, the coronary sinus–aortic difference in the plasma α-tocopherol levels failed to be altered significantly after the left coronary artery spasm induced by intracoronary injection of acetylcholine. The plasma α-tocopherol level was still significantly (p<0.05) lower in the coronary sinus than in the aortic root and in the right atrium but the γ-tocopherol level was not significantly different after the spasm. Both α- and γ-tocopherol levels remained unchanged and were not significantly different between the samples from the coronary sinus and the aortic root or the right atrium in the stable effort angina group after rapid atrial pacing or in the control group after intracoronary injection of acetylcholine. Just as demonstrated at baseline, plasma α-tocopherol levels in the aortic root, in the coronary sinus and in the right atrium were significantly (p<0.01 for each) lower in the variant angina group than those in the stable effort angina group and also in the control group, respectively. Plasma γ-tocopherol levels in the aortic root, in the coronary sinus and in the right atrium were significantly (p<0.01 for each) lower in the variant angina group and also in the stable effort angina group compared with those in the control group, respectively. The coronary sinus–aortic difference in the plasma TBARS levels failed to be altered significantly after the spasm induced by intracoronary injection of acetylcholine in each group (Table 2).

4 Discussion

In our recent reports [11, 13], plasma low density lipoprotein in patients with variant angina has been shown to be vitamin E-deficient and highly susceptible to peroxidative modification compared with that in patients without a coronary spastic tendency. Also, circulating plasma vitamin E levels were demonstrated to be significantly lower in patients with active variant angina than in patients without coronary spasm [12]. In contrast, a low level of antioxidant vitamin E was not demonstrated in patients with an inactive stage of variant angina, suggesting that the plasma status of vitamin E is linked to the activity of coronary artery spasm [11, 12]. The precise mechanisms for low plasma levels of vitamin E in patients with active variant angina and the relation between the level of vitamin E and coronary artery spasm have to be elucidated. In the present study, both plasma α- and γ-tocopherol levels were significantly lower in patients with variant angina compared with patients without spasm, irrespective of the sampling location, i.e. in the aortic root, in the coronary sinus or in the right atrium. In addition, the plasma γ-tocopherol level was significantly lower in patients with stable effort angina compared with control subjects, results that are similar to those of our previous report [12].

4.1 Vitamin E uptake in coronary circulation in variant angina

In the present study, the plasma vitamin E concentrations (α- and γ-tocopherol) were demonstrated to be significantly lower in the coronary sinus compared with the aortic root in patients with variant angina with spasm in the left coronary artery at baseline, suggesting a significant vitamin E consumption in the coronary circulation in these patients. The TBARS level was significantly higher in the coronary sinus than in the aortic root. As malondialdehyde, as estimated by this method, is a measure of lipid peroxidation, which occurs when polyunsaturated fatty acids are attacked by free radicals of oxygen, free-radical activity or free radical-induced oxidant stress appears to be increased in coronary circulation in these patients. In contrast, no significant difference in vitamin E concentrations between the plasma samples from the aortic root and coronary sinus was noted in the control subjects or in the patients with an organic coronary stenosis and stable effort angina but without coronary spasm, suggesting that there was no apparent uptake of vitamin E in the coronary circulation in them. The apparent uptake of vitamin E at baseline in patients with variant angina may be due to either an ongoing vitamin E consumption for scavenging free radicals released from activated neutrophils staying in coronary circulation and/or possible supplementation for exhausted vitamin E in cellular membranes and lipoproteins in coronary circulation after spasm and reperfusion. It is possible that vitamin E-deficient plasma in these patients may contain intermediate oxidation products of tocopherols that cannot be detected as an active form of vitamin E and some of which may be regenerated to active form by vitamin C elsewhere, such as liver.

4.2 Vitamin E levels in coronary circulation after spasm

The coronary sinus–aortic difference in plasma vitamin E levels appeared to be unchanged or rather reduced after left coronary spasm and reperfusion compared with baseline in the patients with variant angina, suggesting no detectable increase of vitamin E uptake in the coronary circulation after a single spasm of short duration and reperfusion. Although the uptake of vitamin E in the coronary circulation was not quantitatively determined, as the coronary blood flow was not estimated in the present study, and it cannot be excluded that the vitamin E consumption might increase immediately after spasm and reperfusion, the ischemia/reperfusion itself appeared to cause no significant amount of vitamin E consumption in coronary circulation. Similarly, the apparent uptake of vitamin E in coronary circulation failed to increase after the pacing-induced ischemia related to the increased oxygen demand in patients with stable effort angina. These results suggest that lower plasma levels of vitamin E in the coronary sinus at baseline in the patients with active variant angina may be the result of exhaustion of this major endogenous antioxidant by increased lipid peroxidation stress, such as free radical production from a variety of cells activated during the inflammatory process triggered by frequently repeated alterations between severe transient regional myocardial ischemia and reperfusion.

4.3 Oxidative stress in coronary circulation

Recently, Mazzone et al. [16]reported that neutrophil and monocyte CD11b/CD18 adhesion molecules show a higher expression in the coronary sinus blood of patients with unstable angina, indicating that an inflammatory reaction takes place within their coronary tree. These leucocytes, once activated, may release a variety of potentially toxic and vasoactive substances, in particular, the lipoxygenase-derived metabolites of arachidonic acid, leucotrienes [17]. These substances have been shown to induce coronary vasoconstriction and decrease coronary flow in a variety of preparations. In addition, the respiratory burst results in the formation of oxygen-derived free radicals. In their subsequent report [18], among clinical and angiographic findings in patients with unstable angina, only the occurrence of chest pain within 48 h of blood sampling was related to a higher expression of the adhesion molecules in the coronary sinus blood. Many of our patients with variant angina had spontaneous anginal attacks during the 12 h prior to blood sampling. When Lafont et al. [19]studied plasma α-tocopherol concentration before and after reperfusion by coronary angioplasty in patients with acute myocardial infarction, reduction of α-tocopherol started immediately after abrupt reperfusion and continued for 3 h after angioplasty. The underlying hypothesis is that reperfusion is associated with increased free radical production [20–23], which may be indirectly reflected by the acute reductions in a major endogenous free radical scavenger, vitamin E.

4.4 Vitamin E deficiency and oxidative stress in the genesis of coronary artery spasm

Although vitamin E consumption in the coronary circulation was demonstrated in patients with variant angina, the exact causal relationship between vitamin E deficiency and coronary spasm still remains to be elucidated. Vitamin E deficiency as a possible cause of coronary artery spasm cannot be excluded by the evidence of vitamin E consumption. It is also possible that vitamin E deficiency could be directly related to the pathogenesis of coronary spasm. Consumption of vitamin E results in a reduction in the resistance of low-density lipoprotein to oxidation. The formation of oxidized low-density lipoprotein has been shown to enhance agonist-induced coronary vascular contractions and reduce endothelium-dependent vasorelaxations, causing increased coronary vasoreactivity to agonists [24–26]. Vitamin E has other beneficial actions on vascular reactivity, such as antiplatelet properties [27], inhibition of vascular smooth muscle cell proliferation [28]and also inhibition of protein kinase C stimulation [29]. Oxygen-derived free radicals from activated neutrophils and monocytes within the coronary vasculature may initiate a vicious circle by vitamin E consumption, which, in turn, may inhibit endothelium-dependent vasodilation and also potentiate agonist-induced vasoconstriction, leading to coronary spasm and transient regional myocardial ischemia as well as endothelial damage [13]. The cellular sources of reactive oxygen species may also include endothelial cells, smooth muscle cells and fibroblasts within the vessel wall [30]. Vitamin E deficiency may be involved in this vicious circle or chain-reaction in the pathogenesis of coronary artery spasm.

5 Conclusion

In conclusion, an apparent consumption of vitamin E concomitant with lipid peroxide generation in the coronary circulation was demonstrated at baseline in patients with variant angina, suggesting free radical formation and increased lipid oxidation stress in association with coronary spasm. In contrast, no apparent consumption of vitamin E in the coronary circulation was observed in patients without coronary artery disease or in patients with an organic coronary artery stenosis but without spasm. Increased oxidative stress and depletion of plasma vitamin E, including α- and γ-tocopherol, may be involved in the pathogenesis of coronary artery spasm and unstable angina.

Acknowledgements

We are grateful to our catheterization team for blood sampling. We are greatly indebted to Eisai Co., Ltd., Japan, for technical guidance in the assay of vitamin E.