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(Circulation. 1997;96:2545-2550.)
© 1997 American Heart Association, Inc.

Articles

Randomized, Double-Blind, Placebo-Controlled Study of Supplemental Vitamin E on Attenuation of the Development of Nitrate Tolerance

Hideki Watanabe, MD; Masaaki Kakihana, MD; Sadanori Ohtsuka, MD; ; Yasuro Sugishita, MD

From the Department of Cardiology, KINU Medical Association Hospital, Mitsukaido (H.W.); Ibaraki Prefectural University of Health Science, Ami (M.K.); and the Cardiovascular Division, Department of Internal Medicine, University of Tsukuba (S.O., Y.S.), Ibaraki, Japan.

Correspondence to Hideki Watanabe, MD, Department of Cardiology, KINU Medical Association Hospital, 13-3 Araigi-cho, Mitsukaido City, Ibaraki 303, Japan. E-mail wata-h{at}xa2.so-net.or.jp

	Abstract

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Background The attenuation of intracellular production of cGMP has been known to be a mechanism of nitrate tolerance. A recent in vitro study showed an increase in superoxide levels and a reduced activation of guanylate cyclase in tolerant vessels. We investigated the preventive effect of an antioxidant, vitamin E, on the development of nitrate tolerance.

Methods and Results In this double-blind, placebo-controlled study, 24 normal volunteers and 24 patients with ischemic heart disease (IHD patients) were randomized to receive either vitamin E (200 mg TID vitamin E group) or placebo (placebo group). Vasodilator response to nitroglycerin was assessed with forearm plethysmography by measurement of the change in the forearm blood flow before and 5 minutes after sublingual administration of 0.3 mg nitroglycerin, and at the same time, blood samples were taken from veins to measure the platelet cGMP level. Measurements of the forearm blood flow and blood sampling were obtained serially at baseline (day 0), 3 days after vitamin E or placebo alone was taken (day 3), and 3 days after application of a 10-mg/24-h nitroglycerin tape concomitantly with oral vitamin E or placebo (day 6). The responses of forearm blood flow (%FBF) and cGMP (%cGMP) after sublingual nitroglycerin on day 0 (%FBF: normal volunteers, 32±12 versus 31±11; IHD patients, 35±15 versus 34±15; %cGMP: normal volunteers, 38±10 versus 35±11; IHD patients, 37±11 versus 38±12, vitamin E group versus placebo group) and day 3 (%FBF: normal volunteers, 33±9 versus 32±12; IHD patients, 35±12 versus 33±13; %cGMP: normal volunteers, 38±10 versus 37±11; IHD patients, 36±14 versus 37±10, vitamin E group versus placebo group) were not different between the two groups. On day 6, %FBF and %cGMP in the placebo group were significantly lower compared with day 0, and there were significant differences in them between the two groups (%FBF: normal volunteers, 30±12 versus 17±9, P<.01; IHD patients, 28±14 versus 17±8, P<.01; %cGMP: normal volunteers, 35±11 versus 8±5, P<.01; IHD patients, 38±10 versus 12±4, P<.01, vitamin E group versus placebo group).

Conclusions These results indicate that the combination therapy with vitamin E is potentially a useful method to prevent the development of nitrate tolerance.

Key Words: antioxidants • nitroglycerin • platelets

	Introduction

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Organic nitrates have been widely used in cardiovascular medicine. However, continuous administration of organic nitrates has been shown to result in rapid development of tolerance.^{1 2 3} The underlying mechanisms responsible for nitrate tolerance probably are multifactorial⁴ and may include neurohormonal counterregulatory mechanisms,⁵ intravascular volume expansion,⁶ or intrinsic abnormalities such as desensitization of the target enzyme guanylate cyclase⁷ or a decrease in nitroglycerin biotransformation.⁸ Recent experimental data demonstrated that nitrate tolerance is associated with increased vascular superoxide anion production.⁹ Probably these radicals inactivate the enzymes involved in the release of nitric oxide from nitroglycerin, leading to impaired cGMP production and tolerance. More recent experimental data demonstrated that tolerance is associated with enhanced propensity for vasoconstriction secondary to increased endothelin expression within the vascular smooth muscle.¹⁰ Vitamin E is the most important endogenous lipid-soluble antioxidant, and the resistance of LDL to oxidation has been shown to be increased by oral intake of vitamin E.¹¹ On the basis of these considerations, this study was designed to investigate the effect of vitamin E, an antioxidant, on nitrate tolerance during continuous administration of nitroglycerin in normal subjects and patients with ischemic heart disease (IHD patients).

	Methods

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Patient Population
The study population was composed of 24 normal volunteers (18 men and 6 women) 19 to 38 years old and 24 IHD patients (19 men and 5 women) 45 to 74 years old. Normal volunteers had no history of renal or cardiac disease, and none had been exposed to any nitrate compounds or any cardiac medications. Among the IHD patients, 18 had received long-acting nitrate (ISDN-Retard, isosorbide dinitrate retard, 20 mg BID) and 16 had received calcium channel blockers. Long-acting nitrates were discontinued 72 hours before this study. Other medications were continued during this study. Their characteristics appear in the Table.

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

Study Protocol
The study design consisted of a series of measurements of plethysmographic vasodilator response and platelet cGMP response to sublingual nitroglycerin in the following three periods: (1) at baseline (day 0), (2) after 3 days of administration of vitamin E or placebo (day 3), and (3) after 3 days after the application of a 10-mg/24-h nitroglycerin tape concomitantly with oral vitamin E or placebo (day 6). An 18-gauge heparin lock was placed in the contralateral forearm of each subject to allow venous blood sampling for measurements of platelet cGMP.

Baseline plethysmographically measured venous volume was recorded before and 5 minutes after 0.3 mg sublingual nitroglycerin on day 0. Subjects were then randomized by a double-blind parallel-group design to receive either vitamin E 200 mg TID (Juvela, Eisai [vitamin E group, n=12]) or placebo TID (placebo group, n=12). Subjects returned after 3 days for measurements of forearm blood flow and platelet cGMP before and after sublingual nitroglycerin on vitamin E or placebo alone. A 5-mg nitroglycerin tape (Millisrol tape, Nippon Kayaku) was then applied twice a day. The final phase of the study was performed 3 days after treatment with combined continuous transdermal nitroglycerin and vitamin E or placebo (Fig 1).

View larger version (23K): [in this window] [in a new window]   Figure 1. Study protocol. NTG indicates nitroglycerin.

The study protocol was approved by the Ethics Committee of Tsukuba University, Tsukuba, Japan, and written informed consent for participation in this study was obtained from all subjects.

Assessment of Vasodilator Response to Nitroglycerin
To evaluate the vasodilator response to nitroglycerin, the forearm blood flow was measured by use of a mercury-in-Silastic strain-gauge plethysmograph and the venous occlusion technique. The strain gauge was placed 5 cm below the antecubital crease and connected to a calibrated plethysmograph to measure forearm blood flow as the rate of change of forearm volume (mL · min^-1 · 100 mL forearm^-1). The pressure in the venous occlusion or congesting cuff was 40 mm Hg. Circulation to the hand was arrested during determination of forearm blood flow by a cuff around the wrist inflated to a suprasystolic pressure. Forearm blood flow was taken as the average of three measurements made at 15-second intervals.

Preparation of Platelet cGMP
Blood samples were drawn into syringes containing 5 mmol/L EDTA and a cGMP phosphodiesterase inhibitor (10^-3 mol/L 2-O-propoxyphenyl-8-azapurin-6-one dissolved in 1% triethanolamine). Immediately after blood sampling, platelet-rich plasma and platelet-poor plasma were prepared by centrifugation at 200g for 20 minutes. Platelet-rich plasma was further centrifuged at 2500g for 10 minutes, and the supernatant was discarded. The pellet was suspended in modified Tyrode's solution (containing 0.35% BSA and 5 mmol/L HEPES, pH 7.35) to obtain a final platelet count of 2x10⁶ to 3x10⁶ platelets/µL, and the samples were kept frozen at -70°C until analysis.¹²

Platelet cGMP Assay
To 1 mL of platelet samples, 0.5 mL of trichloroacetic acid (final concentration, 6%) was added. After centrifugation at 2500g for 20 minutes, trichloroacetic acid was extracted four times from the supernatant with water-saturated ether. The aqueous phase was then assayed for cGMP with a commercially available radioimmunoassay kit (Yamasa Shoyu),¹³ and the results were expressed as picomoles per 10⁹ platelets. The coefficients of variation averaged 3.4% for intra-assay error and 11.9% for interassay error.

Statistical Analysis
Results are expressed as mean±SD in the forearm blood flow and as mean±SEM in the platelet cGMP level. Differences among 3 testing days were analyzed by means of a repeated-measures ANOVA with Bonferroni test, and those between before and after sublingual nitroglycerin by Student's t test. Differences between the normal volunteers and IHD patients were also analyzed by Student's t test. A significance level was defined for values of P<.05.

	Results

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Forearm Blood Flow
Fig 2 shows the change of the forearm blood flow before and after sublingual nitroglycerin in the normal volunteers and IHD patients.

View larger version (25K): [in this window] [in a new window]   Figure 2. Forearm blood flow before and after sublingual nitroglycerin. Data are mean±SD. *P<.05 vs day 0 and day 3. IHD indicates ischemic heart disease.

Forearm blood flow (mL · min^-1 · 100 mL arm^-1) after sublingual nitroglycerin was increased both on day 0 (vitamin E group: normal volunteers, 2.60±0.58 to 3.43±0.68; IHD patients, 2.40±0.48 to 3.24±0.56; placebo group: normal volunteers, 2.64±0.67 to 3.46±0.62; IHD patients, 2.46±0.64 to 3.29±0.69) and on day 3 (vitamin E group: normal volunteers, 2.61±0.54 to 3.47±0.72; IHD patients, 2.51±0.48 to 3.38±0.51; placebo group: normal volunteers, 2.60±0.66 to 3.43±0.74; IHD patients, 2.54±0.52 to 3.38±0.61). There was no significant difference in the forearm blood flow before and after nitroglycerin between the two groups in the normal volunteers and IHD patients.

On day 6, the forearm blood flow (mL · min^-1 · 100 mL arm^-1) in the placebo group was increased from 2.58±0.64 to 3.02±0.75 in the normal volunteers and from 2.46±0.72 to 2.89±0.73 in the IHD patients after sublingual administration of nitroglycerin. But the flow after sublingual nitroglycerin was significantly lower than on days 0 and 3. Conversely, in the vitamin E group, the change of the forearm blood flow after sublingual administration of nitroglycerin was similar to those on days 0 and 3 (normal volunteers, 2.61±0.70 to 3.39±0.84; IHD patients, 2.54±0.72 to 3.26±0.79). The forearm blood flow after sublingual nitroglycerin in the vitamin E group was significantly greater than that of the placebo group in the normal volunteers (P<.01) and the IHD patients (P<.01).

The percentage of the increase of forearm blood flow after sublingual administration of nitroglycerin is shown in Fig 3.

View larger version (34K): [in this window] [in a new window]   Figure 3. Percent increase of forearm blood flow after sublingual nitroglycerin. Data are mean±SD. *P<.05 vs day 0 and day 3. IHD indicates ischemic heart disease.

There was no significant difference on days 0 and 3 between the placebo group and the vitamin E group (day 0: vitamin E group, normal volunteers, 32±12%; IHD patients, 35±15%; placebo group, normal volunteers, 31±11%; IHD patients, 34±15%; day 3: vitamin E group, normal volunteers, 33±9%; IHD patients, 35±12%; placebo group, normal volunteers, 32±12%; IHD patients, 33±13%).

On day 6, the percentage of the increase in the placebo group (normal volunteers,17±9%; IHD patients, 17±8%) was significantly reduced compared with that on days 0 and 3 (P<.01). Conversely, in the vitamin E group, the percentage of the increase of forearm blood flow after nitroglycerin (normal volunteers, 30±12%; IHD patients, 28±14%) was maintained on day 6, and it was significantly greater than in the placebo group (P<.01).

Platelet cGMP
The levels of platelet cGMP before and after sublingual administration of nitroglycerin are shown in Fig 4.

View larger version (24K): [in this window] [in a new window]   Figure 4. Platelet cGMP level before and after sublingual nitroglycerin. Data are mean±SEM. *P<.05 vs day 0 and day 3. IHD indicates ischemic heart disease.

The level of platelet cGMP in the normal volunteers was significantly increased after sublingual administration of nitroglycerin on day 0 (vitamin E group, 0.58±0.04 to 0.80±0.05; placebo group, 0.63±0.05 to 0.85±0.06) and on day 3 (vitamin E group, 0.59±0.05 to 0.81±0.06; placebo group, 0.61±0.04 to 0.84±0.06). There was no significant difference between the two groups on days 0 and 3. The level of platelet cGMP in the IHD patients was also increased after sublingual administration of nitroglycerin on day 0 (vitamin E group, 0.35±0.03 to 0.48±0.04; placebo group, 0.39±0.05 to 0.54±0.04) and on day 3 (vitamin E group, 0.38±0.06 to 0.52±0.04; placebo group, 0.38±0.05 to 0.52±0.06). There was no significant difference between the two groups on days 0 and 3.

On day 6, the platelet cGMP level before nitroglycerin was significantly lower in the placebo group than that on days 0 and 3 (normal volunteers, 0.49±0.04, P<.05 versus days 0 and 3; IHD patients, 0.22±0.03, P<.05 versus days 0 and 3). The level after sublingual nitroglycerin was also significantly lower (normal volunteers, 0.53±0.05, P<.05 versus days 0 and 3; IHD patients, 0.25±0.04, P<.05 versus days 0 and 3), whereas in the vitamin E group, the platelet cGMP level on day 6 was increased from 0.62±0.04 to 0.84±0.05 in the normal volunteers and from 0.37±0.04 to 0.51±0.06 in the IHD patients after sublingual nitroglycerin. There was no significant difference in the platelet cGMP level in the vitamin E group among the testing days, and it was significantly higher than that of the placebo group (P<.05).

In the present study, the level of platelet cGMP in the IHD patients was significantly lower than in the normal volunteers in the two groups on all testing days (P<.05).

The percentage of the increase of platelet cGMP after sublingual administration of nitroglycerin is shown in Fig 5.

View larger version (38K): [in this window] [in a new window]   Figure 5. Percent increase of platelet cGMP level after sublingual nitroglycerin. Data are mean±SD. *P<.05 vs day 0 and day 3. IHD indicates ischemic heart disease.

On days 0 and 3, there was no difference between the placebo group and the vitamin E group (day 0: vitamin E group, normal volunteers, 38±10%; IHD patients, 37±11%; placebo group, normal volunteers, 35±11%; IHD patients, 38±12%; day 3: vitamin E group, normal volunteers, 38±10%; IHD patients, 36±14%; placebo group, normal volunteers, 37±11%; IHD patients, 37±10%). On day 6, the percentage of the increase of cGMP in the placebo group (normal volunteers, 8±5%; IHD patients, 12±4%) was significantly lower than on days 0 and 3 (P<.01). Conversely, in the vitamin E group, the percentage of the increase of platelet cGMP was maintained on day 6 (normal volunteers, 35±11%; IHD patients, 38±10%), and it was significantly greater than that in the placebo group (P<.01).

	Discussion

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The present study demonstrated that the response in forearm blood flow and the production of platelet cGMP after the sublingual administration of nitroglycerin during transdermal application of nitroglycerin was decreased in the placebo group but not in the vitamin E group. These findings suggest that concomitant administration of an antioxidant, vitamin E, prevents the development of nitrate tolerance during continuous nitrate therapy in the normal volunteers and IHD patients.

Mechanisms of Nitrate Tolerance
Although the phenomenon of nitrate tolerance was first described during the early part of this century,¹⁴ clinicians did not consider it clinically important.¹⁵ However, intense research in the past decade has clearly indicated the clinical importance of nitrate tolerance as a limitation for the efficacy of these drugs in the treatment of patients with ischemic heart disease as well as congestive heart failure.^{16 17 18} The mechanism of nitrate tolerance is multifactorial.^{4 17 19} Nitrate tolerance is thought to be due to an inability of the vascular tissue to respond to nitroglycerin.²⁰ Münzel and coworkers¹⁸ hypothesized four mechanisms to be responsible for attenuation of nitroglycerin action after chronic exposure: (1) a desensitization of the target enzyme guanylate cyclase⁷ ; (2) an increase in phosphodiesterase activity²¹ ; (3) intracellular sulfhydryl group depletion²² ; and (4) impaired nitroglycerin biotransformation.²³ Recently, they demonstrated that enhanced activity of angiotensin II results in an increased production of oxygen-derived radicals, which inhibit the dilator action of nitroglycerin-derived nitric oxide.⁹ These radicals inactivate the enzymes involved in the release of nitric oxide from nitroglycerin, leading to impaired cGMP production and nitrate tolerance. In our study, an antioxidant, vitamin E, prevented the attenuation of the response in vasodilation and the intracellular production of cGMP to sublingual administration of nitroglycerin during continuous transdermal application of nitroglycerin. Thus, this observation strongly supports the concept that increased production and activity of oxygen-derived free radicals contribute to the development of nitrate tolerance in long-term therapy with organic nitrate.

Effect of Vitamin E on Nitrate Tolerance
Vitamin E has been reported to be the most important endogenous lipid-soluble antioxidant.¹¹ Recently, the effect of dietary vitamin E in the secondary prevention (nonfatal myocardial infarction) of coronary heart disease was reported in the Cambridge Heart Antioxidant Study (CHAOS).²⁴ Miwa and coworkers²⁵ demonstrated that plasma vitamin E levels were significantly lower in patients with variant angina. Moreover, vitamin E has been shown to protect myocardial cells from free radical–induced structural injury in regional ischemia in vivo²⁶ and to reduce the adhesion and aggregation of platelets.²⁷ To the best of our knowledge, our findings constitute the first report that the development of nitrate tolerance may be prevented by supplementation with an antioxidant, vitamin E, in clinical investigation. The present study demonstrates that supplementation of vitamin E can be an effective treatment in patients with coronary artery disease not only in the secondary prevention of coronary artery disease but also in the prevention of nitrate tolerance in patients with coronary artery disease receiving continuous nitrate therapy.

Previous Studies on the Prevention of Nitrate Tolerance
Because of the potential role of nitrate tolerance in limiting the therapeutic effects of these drugs, an extensive effort has been made in recent years by numerous investigators to develop effective strategies for the prevention of this phenomenon. Several groups reported that concomitant administration of ACE inhibitors with nitroglycerin reversed tolerance or prevented the development of nitrate tolerance.^{28 29 30 31} However, other groups failed to prevent tolerance.^{32 33} Although it is difficult to explain the different efficacies of ACE inhibitors in these studies, we may need higher doses of ACE inhibitors to inhibit angiotensin II formation. A more recent study demonstrated that high-dose enalapril reversed nitrate tolerance in vivo.³⁴ An increase in blood volume has been reported in patients receiving nitrate therapy and has been suggested as a mechanism for early attenuation of the hemodynamic effects.^{5 6 33} But the concomitant use of diuretic drugs in normal volunteers³⁵ and in patients with ischemic heart disease and heart failure^{36 37} failed to prevent the development of nitrate tolerance. More importantly, in these previous studies, there is no investigation evaluating the intracellular production of cGMP. Therefore, more information is needed to determine the clinical role of ACE inhibitors or diuretics in the prevention of nitrate tolerance.

Study Limitations
There are some limitations in this study. First, we investigated the effects of vitamin E on the prevention of nitrate tolerance in the normal volunteers and IHD patients. In the present study, the level of platelet cGMP in the IHD patients was lower than that in normal volunteers. However, we could not evaluate the mechanisms in this study, because characteristics of the IHD patients were not matched to those of the normal volunteers. Second, we measured platelet cGMP levels to evaluate the intracellular production of cGMP in this study. It is not possible to evaluate the effects of nitroglycerin on the intracellular production of cGMP in the vascular smooth muscle cells in vivo except by biopsy. Nitroglycerin also activates soluble guanylate cyclase in platelets, and the increased level of platelet cGMP inhibits platelet adhesion.^{38 39} Platelets are known to contain predominantly the soluble form of guanylate cyclase.^{40 41} Therefore, platelets are a convenient material for the clinical measurement of intracellular cGMP. In our previous study, we demonstrated that platelet cGMP can be used as an indicator for in situ evaluation of the effects of nitroglycerin and the development of nitrate tolerance.¹² Third, we cannot measure superoxide levels and vitamin E levels in vessels or platelets. However, on the basis of the previous study by Münzel and coworkers,⁹ we believe that inhibition of enhanced superoxide by vitamin E is its main effect. Fourth, we did not investigate effects of vitamin E on cross-tolerance, and the effects of other antioxidants were not evaluated in our study.

Conclusions
Our findings demonstrate that combination therapy with an antioxidant, vitamin E, is a potentially useful method to prevent the development of nitrate tolerance during continuous nitrate therapy in IHD patients. Further studies are required to clarify the possible beneficial effects of antioxidant supplementation on the development of nitrate tolerance.

	Footnotes

 
Presented in part at the 69th Scientific Sessions of the American Heart Association, New Orleans, La, November 10-13, 1996, and published in abstract form (Circulation. 1996;94[suppl I]:I-503-I-504).

Received April 10, 1997; revision received May 16, 1997; accepted May 19, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Abram J. Tolerance to organic nitrates. Circulation. 1986;74:1181-1185.[Free Full Text]

2. Packer M, Lee W, Kessler P, Gottlieb S, Medina N, Yushak M. Prevention and reversal of nitrate tolerance in patients with congestive heart failure. N Engl J Med. 1987;317:799-804.[Abstract]

3. Zimrin D, Reichek N, Bogin K, Aurigemma G, Douglas P, Berko B, Fung H-L. Antianginal effects of intravenous nitroglycerin over 24 hours. Circulation. 1988;77:1376-1384.[Abstract/Free Full Text]

4. Fung H. Solving the mystery of nitrate tolerance: a new scent on the trail? Circulation. 1993;88:322-324.[Free Full Text]

5. Parker J, Farrel B, Fenton T, Cohanim M, Parker J. Counterregulatory responses to continuous and intermittent therapy with nitroglycerin. Circulation. 1991;84:2336-2345.[Abstract/Free Full Text]

6. Dupis J, Lalonde G, Lemieux R, Rouleau J. Tolerance to intravenous nitroglycerin in patients with congestive heart failure: role of increased intravascular volume, neurohumoral activation and lack of prevention with N-acetylcysteine. J Am Coll Cardiol. 1990;16:922-931.

7. Waldman S, Rapoport R, Ginsburg R, Murad F. Desensitization to nitroglycerin in vascular smooth muscle from rat and human. Biochem Pharmacol. 1986;35:3525-3531.[Medline] [Order article via Infotrieve]

8. Schroder H, Leitman D, Bennett B, Waldman S, Murad F. Glyceryl trinitrate-induced desensitization of guanylate cyclase in cultured rat lung fibroblasts. J Pharmacol Exp Ther. 1988;245:413-418.[Abstract/Free Full Text]

9. Münzel T, Sayegh H, Freeman B, Tarpey M, Harrison D. Evidence for enhanced vascular superoxide anion production in nitrate tolerance. J Clin Invest. 1995;95:187-194.

10. Münzel T, Giaid A, Kurz S, Stewart D, Harrison D. Evidence for a role of endothelin-1 and protein kinase C in nitrate tolerance. Proc Natl Acad Sci U S A. 1995;92:5244-5248.[Abstract/Free Full Text]

11. Esterbauer H, Dieber-Rotheneder M, Striegl G, Waeg G. Role of vitamin E in preventing the oxidation of low-density lipoprotein. Am J Clin Nutr. 1991;53(suppl):314S-321S.

12. Watanabe H, Kakihana M, Ohtsuka S, Enomoto T, Yasui K, Sugishita Y. Platelet cyclic GMP: a potentially useful indicator to evaluate the effects of nitroglycerin and nitrate tolerance. Circulation. 1993;88:29-36.[Abstract/Free Full Text]

13. Honma M, Satoh T, Takezawa J, Ui M. An ultrasensitive method for the simultaneous determination of cyclic AMP and cyclic GMP in small volume samples from blood and tissue. Biochem Med. 1977;18:257-273.[Medline] [Order article via Infotrieve]

14. Crandall L, Leake C, Loevenhart A, Muehlberger C. Acquired tolerance to and cross-tolerance between the nitrous and nitric acid esters and sodium nitrite in man. J Pharmacol Exp Ther. 1931;41:103.[Abstract/Free Full Text]

15. Elkayam U, Aronow W. Glyceryl trinitrate (nitroglycerin) ointment and isosorbide dinitrate: a review of their pharmacological properties and therapeutic use. Drugs. 1982;23:165-194.[Medline] [Order article via Infotrieve]

16. Elkayam U. Tolerance to organic nitrates: evidence, mechanisms, clinical relevance, and strategies for prevention. Ann Intern Med. 1991;114:667-677.

17. Mangione N, Glasser S. Phenomenon of nitrate tolerance. Am Heart J. 1994;128:137-146.[Medline] [Order article via Infotrieve]

18. Münzel T, Kurz S, Heitzer T, Harrison D. New insights into mechanisms underlying nitrate tolerance. Am J Cardiol. 1996;77:24C-30C.[Medline] [Order article via Infotrieve]

19. Packer M. What causes tolerance to nitroglycerin? The 100 year old mystery continues. J Am Coll Cardiol. 1990;16:932-935.[Medline] [Order article via Infotrieve]

20. Slack C, McLaughlin B, Brien J, Marks G, Nakatsu K. Biotransformation of glyceryl trinitrate and isosorbide dinitrate in vascular smooth muscle made tolerant to organic nitrates. Can J Physiol Pharmacol. 1989;67:1381-1385.[Medline] [Order article via Infotrieve]

21. Axelsson K, Andersson R. Tolerance towards nitroglycerin, induced in vivo, is correlated to a reduced cGMP response and an alteration in cGMP turnover. Eur J Pharmacol. 1983;88:71-79.[Medline] [Order article via Infotrieve]

22. Needleman P, Johnson EMJ. Mechanism of tolerance development to organic nitrates. J Pharmacol Exp Ther. 1973;184:709-715.[Abstract/Free Full Text]

23. Feelisch M, Kelm M. Biotransformation of organic nitrates to nitric oxide during degradation of organic nitrates and activation of guanylate cyclase. Eur J Pharmacol. 1987;139:19-30.[Medline] [Order article via Infotrieve]

24. Stephens N, Parsons A, Schofield P, Kelly F, Cheeseman K, Mitchinson M, Brown M. Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS). Lancet. 1996;347:781-786.[Medline] [Order article via Infotrieve]

25. Miwa K, Miyagi Y, Igawa A, Nakagawa K, Inoue H. Vitamin E deficiency in variant angina. Circulation. 1996;94:14-18.[Abstract/Free Full Text]

26. Klein H, Pich S, Lindert S, Nebendahl K, Niedmann P, Kreuzer H. Combined treatment with vitamin E and C in experimental myocardial infarction in pigs. Am Heart J. 1989;118:667-673.[Medline] [Order article via Infotrieve]

27. Steiner M. Influence of vitamin E on platelet function in humans. J Am Coll Nutr. 1991;10:466-473.[Abstract]

28. Katz R, Levy W, Buff L, Wasserman A. Prevention of nitrate tolerance with angiotensin converting enzyme inhibitors. Circulation. 1991;83:1271-1277.[Abstract/Free Full Text]

29. Muiesan M, Boni E, Castellano M, Beschi M, Cefis G, Cerri B, Verdecchia P, Porcellati C, Pollavini G, Agabiti-Rosei E. Effects of transdermal nitroglycerin in combination with an ACE-inhibitor in patients with chronic stable angina pectoris. Eur Heart J. 1993;14:1701-1708.[Abstract/Free Full Text]

30. Pizzulli L, Hagendorff A, Zirbes M, Fehske W, Ewig S, Jung W, Lüderitz B. Influence of captopril on nitroglycerin-mediated vasodilation and development of nitrate tolerance in arterial and venous circulation. Am Heart J. 1996;131:342-349.[Medline] [Order article via Infotrieve]

31. Lawson D, Nichols W, Mehta P, Mehta J. Captopril-induced reversal of nitroglycerin tolerance: role of sulfhydryl group vs. ACE inhibitory activity. J Cardiovasc Pharmacol. 1991;17:411-418.[Medline] [Order article via Infotrieve]

32. Dadak N, Makhoul N, Flugelman M, Merdler A, Shehadeh H, Schneeweiss A, Halon D, Lewis B. Failure of captopril to prevent nitrate tolerance in congestive heart failure secondary to coronary artery disease. Am J Cardiol. 1990;66:608-613.[Medline] [Order article via Infotrieve]

33. Parker JD, Parker JO. Effect of therapy with an angiotensin-converting enzyme inhibitor on hemodynamic and counterregulatory response during continuous therapy with nitroglycerin. J Am Coll Cardiol. 1993;21:1445-1453.[Abstract]

34. Münzel T, Bassenge E. Long-term angiotensin-converting enzyme inhibition with high-dose enalapril retards nitrate tolerance in large epicardial arteries and prevents rebound coronary vasoconstriction in vivo. Circulation. 1996;93:2052-2058.[Abstract/Free Full Text]

35. Parker JD, Farrell B, Fenton T, Parker JO. Effects of diuretic therapy on the development of tolerance during continuous therapy with nitroglycerin. J Am Coll Cardiol. 1992;20:616-622.[Abstract]

36. Elkayam U, Kulick D, McIntosh N, Roth A, Hseuh W. Incidence of early tolerance to hemodynamic effects of continuous infusion of nitroglycerin in patients with coronary artery disease and heart failure. Circulation. 1987;76:577-584.[Abstract/Free Full Text]

37. Parker JD, Parker AB, Farrell B, Parker JO. Effects of diuretic therapy on the development of tolerance to nitroglycerin and exercise capacity in patients with chronic stable angina. Circulation. 1996;93:691-696.[Abstract/Free Full Text]

38. Bowen R, Haslam R. Effects of nitrovasodilators on platelet cyclic nucleotide levels in rabbit blood: role for cyclic AMP in synergistic inhibition of platelet function by SIN-1 and prostaglandin E1. J Cardiovasc Pharmacol. 1991;17:424-433.[Medline] [Order article via Infotrieve]

39. Pohl U, Busse R. EDRF increases cyclic GMP in platelets during passage through the coronary vascular bed. Circ Res. 1989;65:1798-1803.[Abstract/Free Full Text]

40. Rapoport R, Murad F. Endothelium-dependent and nitrovasodilator-induced relaxation of vascular smooth muscle: role of cyclic GMP. J Cyclic Nucleotide Pro Phosphoryl Res. 1983;9:281-296.

41. Glass D, Frey W, Carr D, Goldberg N. Stimulation of human platelet guanylate cyclase by fatty acid. J Biol Chem. 1977;252:1279-1285.[Abstract/Free Full Text]

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