The Journal of Nutrition Vol. 129 No. 1 January 1999, pp. 109-112

Vitamins C and E Prolong Time to Arterial Thrombosis in Rats¹

Jason Mehta, Dayuan Li, and Jawahar L. Mehta²

Department of Medicine, University of Florida College of Medicine and the VA Medical Center, Gainesville, FL

	ABSTRACT

Abstract Introduction Methods Results Discussion References

To examine the modulation of arterial thrombosis by vitamins C and E, Sprague-Dawley rats were fed nonpurified diet, or diet mixed with vitamin C [100 mg/(kg body weight·d)], vitamin E [100 mg/(kg·d)] or both vitamins C and E [each 100 mg/(kg·d)], for a period of 9-19 d (mean 15 d). An occlusive aortic thrombus was created by application of a Whatman filter soaked in 1 mol/L FeCl₃. Both vitamins C and E and their combination decreased platelet aggregation and delayed time to occlusive thrombus formation (P < 0.05 vs. control). Vitamins C and E decreased arterial superoxide generation (P < 0.05 vs. control). Interestingly, vitamin E also increased endogenous superoxide dismutase activity (SOD) and protein expression in aortic tissues (P < 0.05 vs. control). The combination of vitamins C and E was not superior to each vitamin alone with regard to effect on time to thrombus formation, but it was more potent with regard to platelet inhibition. The increase in endogenous antioxidant activity by vitamin E is an intriguing observation. This study shows that the antioxidant vitamins C and E have important effects on platelet aggregation, SOD activity, superoxide generation and thrombus formation.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Free radicals, also known as oxidants, are molecules with an odd number of electrons. Because of this unpaired electron, free radicals are chemically reactive and lead to cell separation, vacuolization and nuclear fragmentation (Mehta et al. 1992). Free radicals can be injurious to vascular tissues for several reasons. They promote platelet aggregation, injure endothelium, promote monocyte-macrophage chemotaxis and enhance intravascular thrombosis (Lawson et al. 1990, Mehta et al. 1989, Salvemini and Botting 1993). These processes are also involved in the initiation and propagation of atherosclerosis and myocardial ischemia (Davies 1998). Accordingly, the use of antioxidants has increased substantially in the past decade in the prevention and treatment of complications of atherosclerosis (Hodis et al. 1995, Mehta 1998, Stephens et al. 1996).

Several experimental studies show release of free radicals during reperfusion of the coronary artery and extension of ischemic injury by free radicals, but limitation of reperfusion injury by the use of antioxidants (Mehta et al. 1990 and 1992, Werns et al. 1988). In an experimental model of coronary thrombosis, antioxidants have been shown to prevent coronary artery reocclusion after thrombolysis (Mehta et al. 1990). Freedman et al. (1996) showed that vitamin E inhibits ADP-induced platelet aggregation by a protein kinase C-dependent mechanism. It is likely that the reduction in the frequency of myocardial infarction in clinical trials using vitamin E (Stephens et al. 1996) is a result of a decrease in platelet aggregation and inhibition of intra-arterial thrombus formation. Although strong evidence exists for the salutary effect of vitamin E in patients with coronary artery disease, data on the beneficial effects of vitamin C are debatable (Enstrom et al. 1989, Mehta 1998, Nyysonen et al. 1997).

This study was designed to define differential effects of vitamins C and E and their combination on platelet aggregation and intra-arterial thrombosis.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Study protocol.  Male Sprague-Dawley rats (n = 35, weight 300-350 g) were used in this study. The rats were randomly fed nonpurified diet (Lab Rodent Chow #5001, Purina Mills, St. Louis) (n = 7), or diet mixed with vitamin C [100 mg/(kg body weight·d), n = 11], vitamin E [100 mg/(kg·d), n = 11], or a combination of both vitamins C and E [each 100 mg/(kg·d), n = 6]. Vitamin C (Nature's Bounty, Bohemia, NY) was dissolved in distilled water and allowed to soak into the food pellets. Vitamin E (Nature's Bounty) was also allowed to soak into food pellets. Feeding was continued for 9-19 d (mean 15 d). The treatment of rats and the procedures described below were approved by the University of Florida in accordance with the guidelines of the American Physiological Society.

An arterial thrombus model (Kurtz et al. 1990, Li et al. 1997) was used in this study. Briefly, the animals were anesthetized with sodium pentobarbital (30 mg/kg), the abdominal cavity opened and ~1.2 cm of the abdominal aorta isolated. Aortic blood flow was continuously recorded using an ultrasonic Doppler flow probe. Whatman paper soaked in 1 mol/L FeCl₃ was wrapped around the external surface of the aorta. After the thrombus was formed, the exposed aorta was removed and the thrombus weighed. Blood (~5 mL) was collected for platelet aggregation and measurement of superoxide dismutase (SOD)³ activity. The aortic segment proximal to the thrombus was saved for measurement of superoxide anion generation.

Platelet aggregation.  Blood was gently mixed with 130 mmol/L sodium citrate (9:1), centrifuged at 400 × g for 10 min at room temperature to obtain platelet-rich plasma (PRP) and centrifuged again at 1800 × g for 15 min to obtain platelet-poor plasma. Platelet count in PRP was determined and kept at ~2-3 × 10¹¹ cells/L. ADP (final concentration 20 µmol/L) was used as a stimulus for platelet aggregation (Wargovich et al. 1987).

Arterial superoxide anion generation.  Superoxide anion generation in aortic segments was determined by lucigenin (bis-N-methylacridiniun nitrate) chemiluminescence. HEPES buffer (1 mL), containing 0.25 mmol/L of lucigenin (pH 7.4), was placed in each glass scintillation vial and the aortic segment added. Chemiluminescence of lucigenin was then detected using a scintillation counter (LS 7000; Beckman Instruments, Fullerton, CA) in out-of-coincidence mode with a single active photomultiplier tube, for six times at interval of 3 min. Data on superoxide anion generation were expressed as Bq/mg aorta (Yang et al. 1998).

Determination of SOD activity and SOD protein.  Serum was assayed for SOD activity by tracking the inhibition of pyrogallol autoxidation and expressed as units/L (Chen et al. 1995). Abdominal aortas from control and vitamin E-fed rats were homogenized in ice-cold medium (20 mmol/L HEPES, 210 mmol/L mannitol and 70 mmol/L sucrose, pH 7.4) and the supernatant used for Western blot analyses. Protein (14 µg) was fractionated by 15% SDS-PAGE and electroblotted to nitrocellulose membranes (Sigma Chemical, St. Louis, MO). Nonspecific binding sites in the membranes were blocked by 40 g/L nonfat dry milk (Sigma) for 1 h at room temperature. The blots were then incubated with a specific primary antibody against manganese superoxide dismutase (MnSOD) (a gift of Harry Nick, University of Florida) at a dilution of 1:250 for 1 h at room temperature, followed by a 1-h incubation period with an anti-mouse alkaline phosphatase-conjugated second antibody at 1:3000 dilution. Relative intensities of the bands of interest were analyzed with use of a MSF-300G Scanner (Nikon, Japan) (Chen et al. 1996).

Statistical analysis.  All data are presented as means ± SEM. The significance of the differences was determined in multiple comparisons among independent groups of data in which ANOVA and the Scheffé F-test indicated the presence of significant differences. A P-value 0.05 was considered significant.

	RESULTS

Abstract Introduction Methods Results Discussion References

Time to thrombus formation and weight of thrombus.  Both vitamins C and E delayed time to occlusive thrombus formation compared with the control when the rats consumed vitamin C (for a mean of 14 d) or vitamin E (for a mean of 15 d) (both P < 0.05). The time to thrombus formation did not differ in vitamin C- and vitamin E-fed rats. Although significantly longer than that for the control (P < 0.05), time to thrombosis was not longer in rats given the combination (for a mean of 15 d) than in the rats fed vitamin C or vitamin E alone (Fig. 1). However, feeding of the antioxidant vitamins had no effect on the weight of the thrombus (data not shown).

View larger version (48K): [in this window] [in a new window]   Fig 1. Effects of vitamins C (given for a mean of 14 d) and E (given for a mean of 15 d) alone and their combination (given for a mean of 15 d) on time to formation of occlusive thrombus in the rat aorta (upper panel) and ADP-induced platelet aggregation (lower panel). Values are means ± SEM. Means with no letter in common differ, P < 0.05; n = 7 in control group; 11 in vitamin C group; 11 in vitamin E group; and 6 in vitamins C + E group.

Platelet aggregation.  Both vitamins C and E reduced platelet aggregation compared with control (P < 0.05). Platelet aggregation was not significantly different in vitamin C- and vitamin E-fed rats. Platelet aggregation was further reduced in rats given the combination of vitamins C and E (Fig. 1).

Superoxide anion generation.  Both vitamins C and E reduced arterial superoxide anion generation compared with control, and there was no difference between vitamin C- and vitamin E-fed rats. However, the superoxide anion generation in the vitamins C- and E-fed rats did not differ from that in the control group (Fig. 2).

View larger version (46K): [in this window] [in a new window]   Fig 2. Effect of vitamins C and E alone and their combination on superoxide anion formation in the rat aorta (upper panel) and superoxide dismutase (SOD) activity in the blood (lower panel). Duration of feeding was as in Figure 1. Values are means ± SEM. Means with no letter in common differ, P < 0.05; n = 7 in control group; 11 in vitamin C group; 11 in vitamin E group; and 6 in vitamins C + E group.

Serum SOD activity and arterial MnSOD protein expression.  SOD activity in vitamin C-fed rats did not differ from that in control rats, but there was markedly greater SOD activity in vitamin E-fed rats (P < 0.05 vs. control, Fig. 2). Serum SOD activity in the rats fed a combination of vitamins C and E was similar to that in the control rats (Fig. 2).

To study the effect of vitamin E on MnSOD protein expression, Western blots were performed on aortic segments from control and vitamin E-fed rats. MnSOD protein expression in vitamin E-fed rats was significantly higher than that in the control rats (P < 0.05, Fig. 3). Aortic MnSOD protein expression in vitamin C or vitamins C plus E fed rats was not examined because SOD activity was unaffected in these rats.

View larger version (46K): [in this window] [in a new window]   Fig 3. Identification by Western blot analysis of manganese superoxide dismutase (MnSOD) protein in aortic segments from rats fed vitamin E. The upper panel reflects the results of one representative experiment; the lower panel contains the densitometric data. The duration of feeding was as in Figure 1. Values are means ± SEM. Means with no letter in common differ, P < 0.05; n = 6 in both the control group and vitamin E group. MnSOD protein was not assessed in the other two groups because SOD activity was not different from control (see Fig. 2). The positive control was endothelial cell lysate.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

This study showed that both vitamins C and E significantly delayed time to thrombus formation, and that the effect of the two vitamins was similar whether fed individually or together. These vitamins also decreased platelet aggregation (whether fed individually or together) and superoxide anion generation (fed individually), but only vitamin E significantly increased endogenous antioxidant activity.

In the rat model used in this study, the thrombus formed rapidly (15-25 min) and was characterized by endothelial disruption, extensive platelet and red blood cell clumps and interspersed fibrin (Kurt et al. 1990, Li et al. 1997). This morphology is similar to that of thrombi in coronary arteries of patients with acute myocardial ischemia (Falk 1987). The low cost of the rats, ease of thrombus formation and similarity to human thrombus make this model appropriate for the study of the influence of different agents that can inhibit platelet aggregation.

Results of this study complement data from previous studies in animals and humans. For example, tissue-type plasminogen activator (t-PA) enhanced platelet adhesion, and co-administration of vitamin E with t-PA further decreased platelet adhesion in a rat model of mesenteric thrombosis (Jen et al. 1996). Nyyssonen et al. (1997) found an inverse relationship between the intake of vitamin C and risk of an acute myocardial infarction in 1605 middle-aged men. Recently, Losonczy et al. (1996) described fewer cardiac events in subjects taking vitamin C than in those not taking the vitamin in a study of 11,178 elderly subjects followed for 5 y. The Nurses Health Study (Stampfer et al. 1993), conducted in 87,245 female nurses followed for 8 y and the U.S. Male Health Professional Study (Rimm et al. 1993) involving 39,910 men followed for 4 y, found significant inverse associations between cardiac events and vitamin E intake. The beneficial effect of vitamin E persisted after adjustment of cardiac risk factors. Most importantly, the CHAOS trial (Stephens et al. 1996) found a significant 75% reduction in nonfatal myocardial infarction in cardiac patients taking vitamin E.

As expected, both vitamins C and E, which are chain-breaking antioxidants, significantly decreased vascular superoxide anion generation by 35-40%. Superoxide anion stimulates platelet aggregation (Salvemini and Botting 1993); thus, it was not surprising that administration of vitamin C or vitamin E decreased platelet aggregation. In keeping with the platelet-dependent nature of the arterial thrombus, rats fed vitamins C or E showed a significant delay in formation of occlusive thrombi. It is noteworthy that the weight of the thrombus was similar in all four groups of rats, implying that the thrombus can be formed in the presence of antioxidant vitamins; the antioxidants merely delay the formation of the thrombus. Actually, the morphologic features of the thrombi were not different in the four groups (data not shown).

One of the more interesting and intriguing observations was that vitamin E, but not vitamin C, enhanced the endogenous production of SOD. The MnSOD protein expression was also upregulated by feeding vitamin E to the rats. In two recent studies, one in rats fed a high fructose diet (Faure et al. 1997) and the other in rabbits given a high cholesterol diet (Chen et al. 1997), administration of vitamin E was associated with increases in SOD activity. A recent study (Parkes et al. 1998) indicated 40% longer life expectancy in fireflies that had high levels of SOD. Although the exact mechanism by which vitamin E increases endogenous SOD activity is unclear, the significance of this phenomenon is obvious. We measured MnSOD, rather than Cu-ZnSOD protein expression because of its powerful antioxidant effect.

The presence of both vitamins in the diet did cause different effects than C or E individually. Although both vitamins had a cumulative interaction relative to effects on platelet aggregation, the presence of vitamin C blocked the effect of vitamin E on SOD activity. The combination of vitamins also did not affect superoxide generation. Although the precise basis of the latter observation is unknown, antioxidants, especially -tocopherol, may act as prooxidants (Bowry et al. 1992), especially when given in large amounts (Keaney et al. 1994). Whether a similar mechanism underlies the reversal of the effects of vitamin E on superoxide anion generation and SOD activity when vitamin C was given concurrently is not clear.

The issues of the beneficial effect of antioxidants, their dosage, and appropriate combinations in combating coronary heart disease events are being debated at present. This is exemplified by the lack of consensus among cardiologists regarding prophylactic intake of antioxidants to prevent cardiac events (Mehta 1997). This study points to the beneficial effects of 100 mg/(kg body weight·d) of both vitamins C and E on arterial thrombosis.

	FOOTNOTES

Manuscript received 24 July 1998. Initial reviews completed 10 September 1998. Revision accepted 23 October 1998.

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