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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:1128.)
© 2003 American Heart Association, Inc.

Letters to the Editor

A Peculiar Result and a Fanciful Hypothesis Regarding L-Arginine

John P. Cooke; Karsten Sydow

Program in Vascular Medicine and Biology, Stanford University School of Medicine, Calif

To the Editor:

In the January 2003 issue of this Journal, Chen et al¹ observed that L-arginine supplementation did not affect lesion formation in the hypercholesterolemic apolipoprotein E knockout mice, and it negated the protective effect that iNOS gene deficiency has in this model. Their findings are discordant with those observed in most other hypercholesterolemic models in which the administration of supplemental L-arginine improves vasodilation, increases endothelial synthesis of NO, reduces the generation of superoxide anion, suppresses the activation of oxidant sensitive transcriptional proteins, and reduces monocyte adhesion, infiltration, and lesion formation.^2–7 In most hypercholesterolemic models, L-arginine appears to be rate-limiting for the synthesis of NO. This may be due to increases in plasma asymmetric dimethylarginine (ADMA, an endogenous NOS inhibitor), expression of tissue arginase, or NO synthase uncoupling.^8–10

Attempting to explain the results of Chen et al, in his editorial Loscalzo¹¹ proposes the fanciful hypothesis that supplemental L-arginine could increase plasma levels of homocyst(e)ine, a substance known to induce endothelial dysfunction and atherosclerosis. Unfortunately, this hypothesis lacks any mechanistic basis or experimental support. Loscalzo notes correctly that L-arginine is a precursor for guanidinoacetate (GAA), a reaction catalyzed by L-arginine:glycine amidinotransferase (AGAT). The methylation of GAA by S-adenosylmethionine yields creatine and S-adenosylhomocysteine. Loscalzo speculates that supplemental arginine could increase homocysteine by this pathway. However, the article he cites to support his speculation revealed that GAA, not L-arginine, increased homocysteine levels in Sprague-Dawley rats.¹² Notably, GAA markedly suppressed the activity of AGAT. The negative regulation of AGAT activity by GAA would inhibit the conversion of L-arginine to GAA. Thus, the cited article does not provide evidence that L-arginine supplementation would increase homocysteine levels.

Not only is there absence of evidence, there is evidence of absence. L-arginine administration does not increase plasma homocysteine levels in humans. In patients with peripheral arterial disease and hyperhomocyst(e)inemia, oral L-arginine supplementation (24g/d for 8 weeks) did not affect homocyst(e)ine levels.¹³ In another study, intravenous infusion of L-arginine (3 g) in diabetic patients actually reduced plasma homocyst(e)ine.¹⁴ Notably, we have shown that homocysteine inhibits NO synthesis by increasing the elaboration of ADMA.¹⁵ Homocysteine impairs the activity of DDAH (dimethylarginine dimethylaminohydrolase), the enzyme that degrades ADMA. Because it has a critical sulfhydryl in its active site, DDAH is vulnerable to oxidative attack¹⁶ Homocysteine forms a mixed disulfide with DDAH, and thereby reduces the degradation of ADMA.¹⁵ This mechanism accounts for our recent observation that experimental hyperhomocysteinemia in humans induces an endothelial vasodilator dysfunction that is associated with an increase in plasma ADMA, and that is reversed by intravenous infusion of L-arginine (Stuhlinger MC, Oka RK, Graf EE, Schmolzer I, Upson BM, Kapoor O, Szuba A, Malinow MR, Wascher TC, Pachinger O, Cooke JP, unpublished observations, 2003).

To conclude, L-arginine does not increase plasma homocysteine levels. In most hypercholesterolemic animal models, and in hypercholesterolemic or hyperhomocysteinemic humans, the majority of investigators report that L-arginine supplementation improves vascular function. Supplemental L-arginine may exert this beneficial effect by opposing pathological increases in plasma levels of ADMA, tissue arginase, or NOS uncoupling.

References

1. Chen J, Kuhlencordt P, Urano F, Ichinose H, Astern J, Huang PL. Effects of chronic treatment with L-arginine on atherosclerosis in apoE knockout and apoE/inducible NO synthase double-knockout mice. Arterioscler Thromb Vasc Biol. 2003; 23: 97–103.[Abstract/Free Full Text]
2. Cooke JP, Singer AH, Tsao P, Zera P, Rowan RA, Billingham ME. Anti-atherogenic effects of L-arginine in the hypercholesterolemic rabbit. J Clin Invest. 1992; 90: 1168–1172.
3. Tsao P, McEvoy LM, Drexler H, Butcher EC, Cooke JP. Enhanced endothelial adhesiveness in hypercholesterolemia is attenuated by L-arginine. Circulation. 1994; 89: 2176–2182.[Abstract/Free Full Text]
4. Aji W, Ravalli S, Szabolcs M, Jiang XC, Sciacca RR, Michler RE, Cannon PJ. L-arginine prevents xanthoma development and inhibits atherosclerosis in LDL receptor knockout mice. Circulation. 1997; 95: 430–437.[Abstract/Free Full Text]
5. Boger RH, Bode-Boger SM, Brandes RP, Phivthong-ngam L, Bohme M, Nafe R, Mugge A, Frolich JC. Dietary L-arginine reduces the progression of atherosclerosis in cholesterol-fed rabbits: comparison with lovastatin. Circulation. 1997; 96: 1282–1290.[Abstract/Free Full Text]
6. De Nigris F, Lerman LO, Williams Ignarro S, Sica G, Lerman A, Palinski W, Ignarro LJ, Napoli C. Beneficial effects of antioxidants and L-arginine on oxidation-sensitive gene expression and endothelial NO synthase activity at sites of disturbed shear stress. Proc Natl Acad Sci U S A. 2003; 100: 1420–1425.[Abstract/Free Full Text]
7. Cooke JP. Does ADMA cause endothelial dysfunction? Arterioscler Thromb Vasc Biol. 2000; 20: 2032–2037.[Abstract/Free Full Text]
8. Ito A, Tsao PS, Adimoolam S, Kimoto M, Ogawa T, Cooke JP. Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase. Circulation. 1999; 99: 3092–3095.[Abstract/Free Full Text]
9. Wei LH, Wu G, Morris SM Jr, Ignarro LJ. Elevated arginase I expression in rat aortic smooth muscle cells increases cell proliferation. Proc Natl Acad Sci U S A. 2001; 98: 9260–9264.[Abstract/Free Full Text]
10. Pritchard KA Jr, Groszek L, Smalley DM, Sessa WC, Wu M, Villalon P, Wolin MS, Stemerman MB. Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion. Circ Res. 1995; 77: 510–518.[Abstract/Free Full Text]
11. Loscalzo J. Adverse effects of supplemental L-arginine in atherosclerosis: consequences of methylation stress in a complex catabolism. Arterioscler Thromb Vasc Biol. 2003; 23: 3–5.[Free Full Text]
12. Stead LM, Au KP, Jacobs RL, Brosnan ME, Brosnan JT. Methylation demand and homocysteine metabolism: effects of dietary provision of creatine and guanidinoacetate. Am J Physiol Endocrinol Metab. 2001; 281: E1095–E1100.[Abstract/Free Full Text]
13. Sydow K, Schwedhelm E, Arakawa N, Bode-Böger SM, Tsikas D, Hornig B, Frölich JC, Böger RH. ADMA and oxidative stress are responsible for endothelial dysfunction in hyperhomocyst(e)inemia: effects of L-arginine and B vitamins. Cardiovasc Res. 2003; 57: 244–252.[Abstract/Free Full Text]
14. Cassone Faldetta M, Laurenti O, Desideri G, Bravi MC, De Luca O, Marinucci MC, De Mattia G, Ferri C. L-arginine infusion decreases plasma total homocysteine concentrations through increased nitric oxide production and decreased oxidative status in type II diabetic patients. Diabetologia. 2002; 45: 1120–1127.[CrossRef][Medline] [Order article via Infotrieve]
15. Stuhlinger MC, Tsao PS, Her J-H, Kimoto M, Balint RF, Cooke JP. Homocysteine impairs the NO synthase pathway: role of asymmetric dimethylarginine. Circulation. 2001; 104: 2569–2575.[Abstract/Free Full Text]
16. Murray-Rust J, Leiper J, McAlister M, Phelan J, Tilley S, Santa Maria J, Vallance P, McDonald N. Structural insights into the hydrolysis of cellular nitric oxide synthase inhibitors by dimethylarginine dimethylaminohydrolase. Nat Struct Biol. 2001; 8: 679–683.[CrossRef][Medline] [Order article via Infotrieve]

Jiqiu Chen; Paul Huang

Cardiovascular Research Center, Charlestown, Mass

In Response:

Our original intent was to test the hypothesis that L-arginine supplementation would reduce atherosclerosis in apolipoprotein E (apoE) knockout mice by providing substrate for eNOS.¹ In light of recent data that the iNOS isoform is potentially proatherogenic,^2,3 we were concerned that L-arginine might also increase iNOS-derived NO, confounding our results. Thus, we studied both apoE knockout mice and apoE/iNOS double-knockout mice, reasoning that L-arginine could not serve as substrate for iNOS in the double knockout mice. We were surprised to find that L-arginine supplementation did not reduce atherosclerotic burden in the apoE knockout mice, a result in agreement with other reports of the apoE knockout mouse model of atherogenesis.⁴ Furthermore, we found that L-arginine supplementation eliminated the protective effect of iNOS deficiency in the apoE/iNOS double-knockout mice. To examine potential mechanisms for these effects, we measured oxidized and reduced biopterin levels, as well as MDA-TBA reactive material as markers of lipid oxidation.

In his accompanying editorial,⁵ Loscalzo pointed out another potential mechanism: that L-arginine may increase plasma levels of homocysteine, because creatine synthesis accounts for the major fraction of total body arginine utilization and results in a significant methylation demand. Loscalzo’s analysis of the literature and our unexpected results was, we believe, extremely logical and cogent. The hypothesis is an intriguing one. If true, the implications would be very broad indeed, as indiscriminate supplementation with L-arginine may be not be helpful and may in fact be contraindicated. Thus, the hypothesis certainly merits experimental testing.

We have three comments regarding Cooke’s and Sydow’s letter. First, they do not acknowledge that, while some studies suggest a beneficial effect of L-arginine on atherosclerosis and endothelial function, other studies fail to show such effects.^4,6–10 A review of the literature suggests that this issue is far from clear. Thus, it becomes all the more important to establish under what conditions L-arginine would be beneficial and restore endothelial function and under what conditions it may be harmful.

Second, their letter misstates that Loscalzo cites the article by Stead et al¹¹ to provide evidence that L-arginine supplementation increases homocysteine levels. Rather, if one reads the editorial carefully, Loscalzo cites numerous references^12–14 that show that creatine synthesis represents the major fraction of total body arginine utilization, while NO synthesis represents a much lower fraction (1.2%) of arginine flux. Loscalzo cites the article by Stead et al¹¹ to explain why creatine supplementation would be expected to suppress AGAT expression, thereby decreasing methylation stress. Creatine supplementation would therefore be one means to experimentally test the effects of L-arginine supplementation in vivo.

Third, we agree that hyperhomocysteinemia impairs endothelial function. However, these effects do not negate the possibility that L-arginine supplementation may, under certain conditions, increase homocysteine levels.

We believe the results of our study and the issues raised by Loscalzo in his editorial are best resolved by experimental study, so that we can determine when and if L-arginine supplementation is beneficial and, equally importantly, when it might not be.

Footnotes

This communication was supported by grants from the National Institutes of Health (RO1 HL63685 and PO1AI50153), and the Tobacco Related Disease Research Program (7RT-0128). Dr Sydow is a recipient of a research grant provided by the German Research Council (Sy 41/1-1).

References

1. Chen J, Kuhlencordt P, Urano F, Ichinose H, Astern J, and Huang PL. Effects of chronic treatment with L-arginine on atherosclerosis in apoE knockout and apoE/inducible NO synthase double-knockout mice. Arterioscler Thromb Vasc Biol. 2003; 23: 97–103.
2. Detmers PA, Hernandez M, Mudgett J, Hassing H, Burton C, Mundt S, Chun S, Fletcher D, Card DJ, Lisnock J, Weikel R, Bergstrom JD, Shevell DE, Hermanowski-Vosatka A, Sparrow CP, Chao YS, Rader DJ, Wright SD, Pure E. Deficiency in inducible nitric oxide synthase results in reduced atherosclerosis in apolipoprotein E–deficient mice. J Immunol. 2000; 165: 3430–3435.[Abstract/Free Full Text]
3. Kuhlencordt PJ, Chen J, Han F, Astern J, Huang PL. Genetic deficiency of inducible nitric oxide synthase reduces atherosclerosis and lowers plasma lipid peroxides in apolipoprotein E-knockout mice. Circulation. 2001; 103: 3099–3104.[Abstract/Free Full Text]
4. Kauser K, da Cunha V, Fitch R, Mallari C, Rubanyi GM. Role of endogenous nitric oxide in progression of atherosclerosis in apolipoprotein E-deficient mice. Am J Physiol Heart Circ Physiol. 2000; 278: H1679–H1685.[Abstract/Free Full Text]
5. Loscalzo J. Adverse effects of supplemental L-arginine in atherosclerosis: consequences of methylation stress in a complex catabolism? Arterioscler Thromb Vasc Biol. 2003; 23: 3–5.
6. Candipan RC, Wang BY, Buitrago R, Tsao PS, Cooke JP. Regression or progression: dependency on vascular nitric oxide. Arterioscler Thromb Vasc Biol. 1996; 16: 44–50.[Abstract/Free Full Text]
7. Jeremy RW, McCarron H, Sullivan D. Effects of dietary L-arginine on atherosclerosis and endothelium-dependent vasodilatation in the hypercholesterolemic rabbit response according to treatment duration, anatomic site, and sex. Circulation. 1996; 94: 498–506.[Abstract/Free Full Text]
8. Oomen CM, van Erk MJ, Feskens EJ, Kok FJ, Kromhout D. Arginine intake and risk of coronary heart disease mortality in elderly men. Arterioscler Thromb Vasc Biol. 2000; 20: 2134–2139.[Abstract/Free Full Text]
9. Blum A, Hathaway L, Mincemoyer R, Schenke WH, Kirby M, Csako G, Waclawiw MA, Panza JA, Cannon RO3rd. Oral L-arginine in patients with coronary artery disease on medical management. Circulation. 2000; 101: 2160–2164.[Abstract/Free Full Text]
10. Walker HA, McGing E, Fisher I, Boger RH, Bode-Boger SM, Jackson G, Ritter JM, Chowienczyk PJ. Endothelium-dependent vasodilation is independent of the plasma L-arginine/ADMA ratio in men with stable angina: lack of effect of oral L-arginine on endothelial function, oxidative stress and exercise performance. J Am Coll Cardiol. 2001; 38: 499–505.[Abstract/Free Full Text]
11. Stead LM, Au KP, Jacobs RL, Brosnan ME, Brosnan JT. Methylation demand and homocysteine metabolism: effects of dietary provision of creatine and guanidinoacetate. Am J Physiol Endocrinol Metab. 2001; 281: E1095–E1100.
12. Wu G, Morris SM, Jr. Arginine metabolism: nitric oxide and beyond. Biochem J. 1998; 336: 1–17.
13. Castillo L, Beaumier L, Ajami AM, Young VR. Whole body nitric oxide synthesis in healthy men determined from [15N] arginine-to-[15N]citrulline labeling. Proc Natl Acad Sci U S A. 1996; 93: 11460–11465.[Abstract/Free Full Text]
14. Minuskin ML, Lavine ME, Ulman EA, Fisher H. Nitrogen retention, muscle creatine and orotic acid excretion in traumatized rats fed arginine and glycine enriched diets. J Nutr. 1981; 111: 1265–1274.

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