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(Circulation. 1999;99:1272-1276.)
© 1999 American Heart Association, Inc.

Correspondence

Vitamin C and Endothelial Dysfunction: What Is New?

Antonio Ceriello, MD

Chair of Internal Medicine University of Udine, Udine, Italy

To the Editor:

Several papers recently published in Circulation,^{1 2 3} and 1 in the Journal of Clinical Investigation,⁴ report that vitamin C is able to improve endothelial dysfunction. In each of these studies, the hypothesis is formulated that vitamin C may cause such an effect through its antioxidant activity, protecting NO from inactivation by superoxide anion.

Regarding all these studies, 3 questions should be taken into consideration:

1. The reported experiments do not constitute a strong support for the attribution of the effects of vitamin C to its antioxidant action. A demonstration that different antioxidants reproduce the same effect would have been useful.

2. Clinical trials based on the administration of vitamin C should be suggested with caution. Apart from the possibly increased risk of the formation of urinary oxalate stones during therapy with megadoses of ascorbic acid,⁵ vitamin C has been shown to increase the production of advanced glycation end products in diabetic patients.⁶ Moreover, several studies have demonstrated that vitamin C contributes only about 10% to the total antioxidant capacity of plasma.^{7 8}

3. The bibliographies of all the above-mentioned studies do not adequately correspond with the existing literature. Indeed, my studies on the protective effects of several antioxidants, among with vitamin C, against endothelial dysfunction in diabetic patients⁹ and the antihypertensive effects of the same substances¹⁰ were published in 1990 and 1991, respectively. If my publications had been cited, the authors would have reported much of their works as refined and more-detailed confirmations of previously known data. Moreover, they would have found extensive evidence to support the hypothesis that vitamin C exerts certain effects because it is an antioxidant, as illustrated in my review on the possible role of oxidative stress in hypertension, published in 1993.¹¹ In fact, my studies showed that the same effect observed with vitamin C can be reproduced with 2 other antioxidants, thiopronine and glutathione, respectively.^{9 10}

The question of missing citations is surely an important one, and particularly so when the paternity of a finding or concept of some value is to be attributed.

References

1. Levine GN, Frei B, Koulouris SN, Gerhard MD, Keaney JF, Vita JA. Ascorbic acid reverses endothelial vasomotor dysfunction in patients with coronary artery disease. Circulation. 1996;93:1107–1113.
   
2. Heitzer T, Just H, Münzel T. Antioxidant vitamin C improves endothelial dysfunction in chronic smokers. Circulation. 1996;94:6–9.
   
3. Solzbach U, Hornig B, Jeserich M, Just H. Vitamin C improves endothelial dysfunction of epicardial coronary arteries in hypertensive patients. Circulation. 1997;96:1513–1519.
   
4. Ting HH, Timimi FK, Boles KS, Creager SJ, Ganz P, Creager MA. Vitamin C improves endothelium-dependent vasodilation in patients with non–insulin-dependent diabetes mellitus. J Clin Invest. 1996;97:22–28.
   
5. Sutton RAL, Walker VR. Enteric and mild hyperoxaluria. Miner Electrolyte Metab. 1994;20:352–360.
   
6. Ceriello A, Quatraro A, Giugliano D. New insights on non-enzymatic glycosylation may lead to therapeutic approaches for the prevention of diabetic complications. Diabet Med. 1992;9:297–299.
   
7. Wayner DDM, Burton GW, Ingold KU, Barclay LRC, Locke SJ. The relative contributions of vitamin E, urate, ascorbate and proteins to the total peroxyl radical-trapping antioxidant activity of human blood plasma. Biochim Biophys Acta. 1987;924:408–419.
   
8. Ghiselli A, Serafini M, Maiani G, Azzini E, Ferro-Luzzi A. A fluorescence-based method for measuring total plasma antioxidant capability. Free Radic Biol Med. 1995;18:29–36.
   
9. Ceriello A, Quatraro A, Caretta F, Varano R, Giugliano D. Evidence for a possible role of oxygen free radicals in the abnormal functional arterial vasomotion in insulin-dependent diabetes. Diabetes Metab. 1990;16:318–322.
   
10. Ceriello A, Giugliano D, Quatraro A, Lefebvre PJ. Anti-oxidants show an anti-hypertensive effect in diabetic and hypertensive subjects. Clin Sci (Colch). 1991;81:739–742.
    
11. Ceriello A, Quatraro A, Giugliano D. Diabetes mellitus and hypertension: the possible role of hyperglycaemia through oxidative stress. Diabetologia. 1993;36:265–266.
    

Response

John F. Keaney, Jr, MD; Joseph A. Vita, MD

Boston University School of Medicine, Boston, Mass

We are grateful for the opportunity to respond to Dr Ceriello's comments concerning our recent publication in Circulation.¹ In response to his comments, we offer the following points.

We agree that our study, as well as others, did not directly examine the mechanism responsible for improved endothelial vasomotor function with ascorbic acid in patients with or at risk for vascular disease. However, we disagree with Dr Ceriello's characterization that our study concluded "vitamin C may cause such an effect through its antioxidant activity, protecting NO from inactivation by superoxide anion." In our study, ascorbic acid treatment produced a plasma concentration of 114±11 µmol/L. Although ascorbic acid has the capacity to scavenge superoxide anion, the rate constant for that reaction (3.3x10⁵ mol · L^-1 · s^-1)² is 10⁵-fold less than the rate constant for the reaction between superoxide anion and NO (1.9x10¹⁰ mol · L^-1 · s^-1).³ On the basis of an estimated local NO concentration of 0.1 to 1 µmol/L,⁴ one would predict that an ascorbic acid concentration of 10 to 100 mmol/L would be required to effectively compete for the reaction between NO and superoxide anion. This prediction was recently confirmed in our laboratory using an in vitro model of superoxide-mediated endothelial dysfunction.^4A As mentioned in our initial article¹ and in a more recent study,⁵ an effect of ascorbic acid on cellular redox state or glutathione metabolism should be considered. It remains possible that an effect of ascorbic acid to scavenge superoxide anion may contribute to the findings of other recent studies that used intra-arterial infusions that produced ascorbic acid concentrations in the 1 to 10 mmol/L range.⁶

We also disagree with Dr Ceriello's implication that his prior studies using ascorbic acid, glutathione, and an analogue of N-acetylcysteine (thiopronine) provide additional support that scavenging of superoxide accounts for the effect of ascorbic acid.^{7 8} Although glutathione is known to interact with superoxide, the rate of this reaction (10² to 10³ mol · L^-1 · s^-1)⁹ dictates that glutathione competition with NO for superoxide would require extremely high glutathione concentrations (>100 mmol/L). A more plausible explanation for a concerted action of ascorbic acid, glutathione, and thiopronine would involve some effect on intracellular redox state, as suggested by our recent publication.⁵

Finally, Dr Ceriello was very concerned that we had not cited his papers published in the journals Diabetes and Metabolism (Paris) and Clinical Science in 1990 and 1991, respectively.^{7 8} Although we thank Dr Ceriello for bringing his work to our attention, we could not have cited those papers as previous demonstrations that ascorbic acid improves endothelial function because neither paper specifically examined endothelial function. The Clinical Science paper demonstrated that a rapid intravenous infusion of ascorbic acid (1 g) acutely lowers systemic blood pressure in patients with diabetes and/or hypertension.⁸ In the Diabetes and Metabolism paper, Dr Ceriello reported that the same dose of ascorbic acid increased the extent of postischemic reactive hyperemia in diabetic subjects.⁷ Subsequent studies have demonstrated that peak reactive hyperemia is largely an endothelium-independent response.¹⁰ Furthermore, the latter study was flawed because ascorbic acid and the other compounds used in the study induced large changes in baseline forearm blood flow (and presumably blood pressure), making it hard to interpret the findings. On the basis of those publications, Dr Ceriello claims "paternity" for the idea that "oxidative stress" plays a role in vascular dysfunction in atherosclerosis and related disease states. In response, we can only refer Dr Ceriello to earlier and concurrent work on this subject.^{11 12 13 14}

References

1. Levine GN, Frei B, Koulouris SN, Gerhard MD, Keaney JF Jr, Vita JA. Ascorbic acid reverses endothelial vasomotor dysfunction in patients with coronary artery disease. Circulation. 1996;96:1107–1113.
   
2. Gotoh N, Niki E. Rates of interactions of superoxide with vitamin E, vitamin C, and related compounds as measured by chemiluminescence. Biochim Biophys Acta. 1992;1115:201–207.
   
3. Kissner R, Nauser T, Bugnon P, Lye PG, Koppenol WH. Formation and properties of peroxynitrite as studied by laser flash photolysis, high pressure stopped-flow technique, and pulsed radiolysis. Chem Res Toxicol. 1997;10:1285–1292.
   
4. Malinski T, Taha Z, Grunfeld S, Patton S, Kapturczak M, Tomboulian P. Diffusion of nitric oxide in the aorta wall monitored in situ by porphyrinic microsensors. Biochem Biophys Res Commun. 1993;193:1076–1082.
   
4. Jackson TS, Xu A, Vita JA, Kearney JF Jr. Ascorbate prevents the interaction of superoxide and nitric oxide only at high physiologic concentrations. Circ Res. 1998;83:916–922.
   
5. Vita JA, Frei B, Holbrook M, Gokce N, Leaf C, Keaney JF Jr. L-2-oxothiazolidine-4-carboxylic acid reverses endothelial dysfunction in patients with coronary artery disease. J Clin Invest. 1998;101:1408–1414.
   
6. Ting HH, Timimi FK, Boles KS, Creager SJ, Ganz P, Creager MA. Vitamin C improves endothelium-dependent vasodilation in patients with non–insulin-dependent diabetes mellitus. J Clin Invest. 1996;97:22–28.
   
7. Ceriello A, Quatraro A, Caretta F, Giugliano D. Evidence for a possible role of oxygen free radicals in the abnormal functional arterial vasomotion in insulin dependent diabetes. Diabete Metab. 1990;16:318–322.
   
8. Ceriello A, Giugliano D, Quatraro A, Lefebvre P. Anti-oxidants show an anti-hypertensive effect in diabetic and hypertensive subjects. Clin Sci (Colch). 1991;81:739–742.
   
9. Winterborn CC, Metodiewa D. The reaction of superoxide with reduced glutathione. Arch Biochem Biophys. 1994;314:284–290.
   
10. Tagawa T, Imaizumi T, Endo T, Shiramoto M, Harasawa Y, Takeshita A. Role of nitric oxide in reactive hyperemia in human forearm vessels. Circulation. 1994;90:2285–2290.
    
11. Wei EP, Kontos JA, Christman CW, DeWitt DS, Povlishock JT. Superoxide generation and reversal of acetylcholine-induced cerebral arteriolar dilation after acute hypertension. Circ Res. 1985;57:781–787.
    
12. Gryglewski RJ, Palmer RM, Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature. 1986;320:454–456.
    
13. Rubanyi GM, Vanhoutte PM. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am J Physiol. 1986;250:H822–H827.
    
14. Mugge A, Elwell JK, Peterson TE, Harrison DG. Release of intact endothelium-derived relaxing factor depends on endothelial superoxide dismutase activity. Am J Physiol. 1991;260:C219–C225.
    

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