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Effect of oral l -arginine supplementation on blood pressure: A meta-analysis of randomized, double-blind, placebo-controlled trials
Curriculum in Cardiology
Effect of oral L-arginine supplementation on blood pressure: A meta-analysis of randomized, double-blind, placebo-controlled trials Jia-Yi Dong, BSc, a Li-Qiang Qin, MD, PhD, a Zengli Zhang, MD, PhD, b Youyou Zhao, PhD, c Junkuan Wang, PhD, c Fabrizio Arigoni, PhD, d,e and Weiguo Zhang, MD, PhD c Suzhou, and Beijing, China; and Tokyo, Japan
Background
Previous studies suggest that L-arginine, an amino acid and a substrate of nitric oxide synthase, may have blood pressure (BP)-lowering effect. Because some studies were performed with limited number of patients with hypertension and therefore limited statistical power with sometimes inconsistent results, we aimed to examine the effect of oral L-arginine supplementation on BP by conducting a meta-analysis of randomized, double-blind, placebo-controlled trials.
Methods
PubMed, Cochrane Central Register of Controlled Trials, and the ClinicalTrials.gov databases were searched through June 2011 to identify randomized, double-blind, placebo-controlled trials of oral L-arginine supplementation on BP in humans. We also reviewed reference lists of obtained articles. Either a fixed-effects or, in the presence of heterogeneity, a random-effects model was used to calculate the combined treatment effect.
Results
We included 11 randomized, double-blind, placebo-controlled trials involving 387 participants with oral L-arginine intervention ranging from 4 to 24 g/d. Compared with placebo, L-arginine intervention significantly lowered systolic BP by 5.39 mm Hg (95% CI −8.54 to −2.25, P = .001) and diastolic BP by 2.66 mm Hg (95% CI −3.77 to −1.54, P b .001). Sensitivity analyses restricted to trials with a duration of 4 weeks or longer and to trials in which participants did not use antihypertensive medications yielded similar results. Meta-regression analysis suggested an inverse, though insignificant (P = .13), relation between baseline systolic BP and net change in systolic BP.
Conclusions
This meta-analysis provides further evidence that oral L-arginine supplementation significantly lowers both systolic and diastolic BP. (Am Heart J 2011;162:959-65.)
Hypertension is a huge public health burden, affecting approximately one billion individuals worldwide. 1 It has been widely believed that lifestyle and dietary factors play an important role in the development of hypertension. This view has been reinforced by studies such as the DASH which demonstrates that a diet rich in vegetables, fruits, and low-fat dairy foods and low in saturated and total fat can substantially lower blood pressure (BP). 2 Moreover, the DASH diet contains more protein than the
From the aDepartment of Nutrition and Food Hygiene, School of Public Health, Soochow University, Suzhou, China, bDepartment of Toxicology, School of Public Health, Soochow University, Suzhou, China, cNestle Research Center Beijing, Nestle R&D Center Beijing, Beijing, China, dNestle Research Center, Tokyo, Japan, and eGraduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan. Submitted June 27, 2011; accepted September 13, 2011. Reprint requests: Weiguo Zhang, MD, PhD, Nestle Research Center Beijing, Nestle R&D Center Beijing, Beijing 100095, China. E-mail: [email protected] 0002-8703/$ - see front matter © 2011, Mosby, Inc. All rights reserved. doi:10.1016/j.ahj.2011.09.012
control diet, 2 and arginine-rich protein has been hypothesized to contribute to the BP-lowering effect of this diet. L-Arginine, a semi-essential amino acid, is the natural substrate for nitric oxide (NO) synthase and responsible for the production of the endothelium-derived relaxing factor NO, which is involved in a wide variety of regulatory mechanisms of the cardiovascular system. 3 This property led to the hypothesis that L-arginine may have BP-lowering effect. It is attractive to lower BP and prevent hypertension through effective lifestyle modification, particularly dietary supplements in persons with prehypertension, given the lack of evidence that antihypertensive drugs reduce cardiovascular morbidity and mortality in this population. 4 During the past decades, a number of clinical trials have been carried out to evaluate the role of L-arginine in BP regulation. However, the sample size of these trials was small, the quality varied from low to high, and the results were inconsistent. We therefore aimed to examine the effect of oral L-arginine supplementation on BP by conducting a meta-analysis of randomized, double-blind, placebo-controlled trials.
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Methods
Figure 1
Search strategy We attempted to follow the Preferred Reporting Items for Systematic Reviews and Meta-Analyses 5 guidelines in the report of this meta-analysis. We searched PubMed, Cochrane Central Register of Controlled Trials, and the ClinicalTrials.gov databases through June 2011 for relevant studies, using terms of “arginine” and “L-arginine” in combination with “blood pressure” and “hypertension.” Our search was limited to randomized controlled trials of oral L-arginine supplementation in humans. In addition, we systemically searched the reference lists of obtained articles. No attempt was made to identify unpublished studies.
Study selection Studies were included if they: (1) were randomized, doubleblind, placebo-controlled trials; (2) used oral L-arginine supplementation as intervention; and (3) reported the net changes of BP and the associated standard deviations (or data to calculate them). Studies were excluded if they: (1) had a short duration of intervention (b1 week); (2) had L-arginine administered by infusion; (3) were single-blind or open-label; (4) used L-arginine as part of intervention; (5) or lacked a concurrent placebocontrolled group.
Data extraction We recorded study characteristics as follows: first author's last name, publication year; design details, including whether parallel or crossover; study duration; number of participants; antihypertensive medication use; daily dose of L-arginine treatment; and adverse effects. Participant characteristics including health status, mean age, and baseline BP were also recorded. Further, we assess the methodological quality of each included trial using the Jadad scale, which assigned scores for reported randomization, blinding, and withdrawals. 6 Two of the authors independently performed the literature search, data extraction, and bias assessment, with disagreements resolved by discussion.
Statistical analysis For parallel trials, the net changes were calculated by the difference (intervention minus control) of the changes (final values minus baseline values) of the mean values. For cross-over trials, the net changes were calculated as the difference of mean values at the end of the intervention and control periods. Where necessary, standard errors, CIs, and P values were converted to standard deviations for the analysis. Standard deviations for changes from baseline in each group were obtained. If not specified, we computed the missing standard deviations using the method proposed by Follmann et al 7 in which a correlation coefficient of 0.5 was assumed. The homogeneity of the effect size among studies was tested using the Q test at the P b .10 level of significance. We also calculated the I 2 statistic, 8,9 a quantitative measure of inconsistency across studies. An I 2 value N 50% was considered to indicate substantial heterogeneity between trials. Either a fixedeffects or, in the presence of heterogeneity, a random-effects model was used to calculate the combined effect size. We did not conduct subgroup analysis because of the small number of
Flow chart of study selection.
included studies. Rather, we performed sensitivity analyses to explore potential sources of heterogeneity across studies and to test the robustness of the results based on various criteria regarding the magnitude of BP reduction, the duration of intervention, and the use of antihypertensive medication. We also investigated the influence of a single study on the overall effect estimate by omitting one study in each turn. Furthermore, we conducted meta-regression analyses to assess whether BP reductions were related to study or subject characteristics, including L-arginine dose, intervention duration, and baseline BP levels. Potential publication bias was assessed by Begg's test 10 and Egger's test 11 at the P b .10 level of significance. All analyses were performed using STATA version 11.0 (StataCorp, College Station, TX). P b .05 was considered statistically significant, except where otherwise specified. The study was sponsored by Nestec Ltd, Vevey, Switzerland. No other extramural funding was used to support this work.
Results Characteristics of the studies We identified 11 studies 12-22 that fully met the inclusion criteria for this meta-analysis. A flow chart of literature search and study selection is presented in Figure 1. The characteristics of the selected trials are presented in Table I. The included trials were published between 1996 and 2010. The sample size varied from 12 to 79, reaching a total of 387. All trials were randomized,
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Table I. Characteristics of the trials included in the meta-analysis
Study, year
Therapy l-arginine duration Sex Mean dose Design (wks) Status (M/F) age (y) (g/day)
Chin-Dusting et al, 1996 12 Clarkson et al, 1996 13 Piatti et al, 2001 14 Sydow et al, 2003 15 West et al, 2005 16 Lucotti et al, 2006 17 Facchinetti et al, 2007 18 Lucotti et al, 2009 19 Ast et al, 2010 20 Battaglia et al, 2010 21 Neri et al, 2010 22
R, P, DB, PC R, X, DB, PC R, P, DB, PC R, P, DB, PC R, X, DB, PC R, P, DB, PC R, P, DB, PC R, P, DB, PC R, P, DB, PC R, P, DB, PC R, P, DB, PC
Baseline BP (mm Hg)
Antihypertensive Adverse Jadad drug use effects score
4
Healthy
16/0
21
20
114/56 vs 118/57
0% vs 0%
3
0% vs 0%
Not observed Diarrhea
4
HC
18/9
29
21
119/76 vs 117/77
4
T2DM
8/4
58
9
128/70 vs 120/71
0% vs 0%
–
4
8
PAOD
15/3
66.4
24
162/86 vs 164/83
–
–
4
3
HC
16/0
45
12
134/87 vs 134/87
0% vs 0%
–
4
3
T2DM
8/25
56.4
8.3
151/90 vs 149/89
–
4
32.7
4
144/91 vs 147/93
15% vs 20%
Not observed Diarrhea
2
GH
0/74
24
CAD
28/2
64
6.4
129/79 vs 122/73
0% vs 0%
4
0% vs 0%
3
0% vs 0%
Not observed Not observed –
24% vs 45%
–
5
24
PCOS
0/28
24.8
8
138/85 vs 134/82⁎, 119/71 vs 115/69 † 108/67 vs 112/68
12
GH
0/79
34.1
4
127/79 vs 128/77
4
Healthy 30/24
38.7
12
4
5
5
(–), No information available; CAD, Coronary artery disease; DB, double-blind; GH, gestational hypertension; HC, hypercholesterolemia; P, Parallel; PAOD, peripheral arterial occlusive disease; PC, placebo-controlled; PCOS, polycystic ovary syndrome; R, randomized; T2DM, type 2 diabetes mellitus; X, crossover. ⁎ Hypertensive group. † Normotensive group.
double-blind, and placebo-controlled, of which 9 trials were parallel-designed and 2 had a crossover design. Five trials 16,18 , 20-22 had BP as a primary outcome. The duration of intervention lasted from 2 to 24 weeks, with a median of 4 weeks. Dose of L-arginine varied from 4 to 24 g/day, with a median of 9 g/day. Information on adverse effects of L-arginine intervention was available in 6 trials. Characteristics of participants enrolled in these trials varied across studies. Of note, two trials 18,22 were conducted in pregnant women with hypertension, and another trial 20 presented results separately by hypertension status. Few of the remaining trials specified hypertension status, and most included participants were normotensive as indicated by mean BP levels at baseline.
Effect of L-arginine on BP The net changes and corresponding 95% CIs for systolic and diastolic BP in each trial, and overall, are presented in Figure 2 and Figure 3. Compared with placebo, oral L-arginine intervention was associated with an average net change ranging from −23.0 to 2.8 mm Hg for systolic BP and from −11.0 to 1.0 mm Hg for diastolic BP. Most trials showed an intervention-related trend toward BP reductions, but only a few reached statistical significance. The combined effect size of L-arginine on systolic BP was −5.39 mm Hg (95% CI −8.54 to -2.25, P = .001), and
substantial heterogeneity was observed (P b .001, I 2 = 73.3%). For diastolic BP, the combined effect size was -2.66 mm Hg (95% CI −3.77 to −1.54, P b .001), with little evidence of heterogeneity (P = .12, I 2 = 34.4%). Neither Begg's test nor Egger's test provided evidence of publication bias regarding effect of L-arginine on systolic or diastolic BP (all P N .30).
Sensitivity analyses To explore potential sources of heterogeneity across studies of oral L-arginine supplementation on systolic BP and to test robustness of the results, we performed sensitivity analyses. After excluding two trials 14,17 that showed large systolic BP reductions in response to L-arginine intervention, there was no heterogeneity (P = .59, I 2 = 0%), and the combined effect size was −3.34 mm Hg (95% CI −4.93 to −1.86, P b .001). Restricting analysis to 8 trials with a duration of 4 weeks or longer did not change the overall BP estimates (systolic BP −3.96 mm Hg, 95% CI −5.68 to −2.24; diastolic BP -2.62 mm Hg, 95% CI −4.11 to −1.14). Restricting analysis to 7 trials in which participants did not use antihypertensive medications yielded similar results (systolic BP −3.92 mm Hg, 95% CI −6.47 to −1.37; diastolic BP −2.50 mm Hg, 95% CI −3.75 to −1.25). Additional analyses examining the influence of an individual trial on the combined effect size by omitting one trial in each turn yielded a range from −3.66
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Figure 2
Meta-analysis of the effect of oral L-arginine supplementation on systolic blood pressure as compared with placebo. WMD, weighted mean difference.
Figure 3
Meta-analysis of the effect of oral L-arginine supplementation on diastolic blood pressure as compared with placebo. WMD, weighted mean difference.
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Table II. Characteristics associated with net change in blood pressure (BP): univariate meta-regression analysis Systolic BP
Dose Duration Baseline BP
Diastolic BP
Coefficient
95% CI
P
Coefficient
95% CI
P
0.36 0.05 −0.22
−0.39 to 1.12 −0.56 to 0.67 −0.52 to 0.08
0.31 0.85 0.13
0.21 −0.15 0.06
−0.09 to 0.51 −0.38 to 0.09 −0.29 to 0.17
0.15 0.19 0.58
(95% CI −5.54 to −1.78) to −5.92 mm Hg (95% CI −9.33 to −2.51) for systolic BP and a range from −2.40 (95% CI −3.55 to −1.26) to −3.20 mm Hg (95% CI −4.68 to −1.73) for diastolic BP. None of the individual studies appeared to have appreciable impacts on the overall combined effect sizes.
Figure 4
Meta-regression analyses We next performed meta-regression analyses to assess whether BP reductions were related to L-arginine dose, intervention duration, or baseline BP levels. None of these covariates had significant impacts on the combined effect sizes (Table II). However, there was a trend toward greater reductions in systolic BP among subjects with higher systolic BP at baseline (r = −0.22, P = .13, Figure 4).
Discussion This meta-analysis of randomized, double-blind, placebo-controlled trials brought evidence that oral L-arginine supplementation, compared with placebo, significantly lowered systolic BP by 5.39 mm Hg (95% CI −8.54 to −2.25) and diastolic BP by 2.66 mm Hg (95% CI −3.77 to −1.54). The magnitudes of the BP reductions in response to Larginine supplementation in this meta-analysis are moderate but detectable. It should be noted that most participants included in these studies were normotensive, a category in which there may be less room for improving. In fact, one trial 20 that performed separate analysis according to hypertension status showed greater BP reductions in hypertensive participants than in normotensive ones (systolic BP: −5.6 vs −1.8 mm Hg, diastolic BP: −3.8 vs −1.8 mm Hg). It would be useful to perform stratified analysis by hypertension status, but the small number of trials conducted in hypertensive subjects 18,20,22 precluded such analysis. Yet our metaregression analysis suggested an inverse, although not significant (P = .13), relation between baseline systolic BP and net change in systolic BP. It is therefore possible that L-arginine supplementation could exert larger BP-lowering effect in those with high BP. Given the inherent limitations of meta-regression analysis, this finding should be regarded as hypothesis-generating and need to be verified in future studies.
Net change in systolic blood pressure (SBP) according to baseline SBP (r = -0.22, P = .13).
The observed heterogeneity among trials of L-arginine on systolic BP appeared to be due to 2 trials 14,17 that showed large BP reductions. After exclusion of these two trials, heterogeneity disappeared, and the combined effect size did not substantially change and remained significant. In one trial, 17 both treatment and control groups were submitted to a low-caloric diet and exercise training program, and L-arginine supplementation combined with lifestyle modification and dietary therapy may have led to the pronounced BP reductions. For another trial, 14 the disparate results were likely due to chance as the sample size was rather small (n = 12). In addition, all the participants enrolled in these 2 trials 14,17 were type 2 diabetic patients. Several mechanisms may be responsible for the beneficial effect of L-arginine on BP. As a substrate for NO synthase, L-arginine may exhibit antihypertensive activities by augmenting the production of NO in endothelium and improving its bioavailability in vascular smooth muscle cells, which are essential to maintain vascular homeostasis. 23,24 A recent meta-analysis 25 suggests that oral L-arginine supplementation is effective at improving endothelial cell function in individuals
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with endothelial dysfunction. In addition, L-arginine has been shown to improve insulin resistance, 14,26 which plays an important role in the etiology of hypertension associated with metabolic syndrome. 27,28 It is also worth mentioning the “L-arginine paradox” that exogenous L-arginine supplementation improves NO-mediated biological effects despite high endogenous concentration of L-arginine. 29 One possible explanation may be related to asymmetric dimethylarginine (ADMA). ADMA is an endogenous inhibitor of NO synthase and has been shown to reduce the sensitivity of NO synthase to L-arginine. 23,24,30 There is also considerable evidence that ADMA modulates endothelial NO synthase activity within the concentration range found in patients with vascular disease. 23,24,29 Overcoming the inhibition of NO synthase by ADMA may therefore underlie the beneficial effects of L-arginine supplementation on BP. 31 Our study had stringent inclusion and exclusion criteria. All included studies were randomized, doubleblind, placebo-controlled trials, which minimized biases and suggested a high internal validity. We excluded studies 32,33 using L-arginine supplementation as part of intervention, and hence the BP-lowering effect was mainly attributable to L-arginine supplementation. We were able to detect the potential sources of heterogeneity among studies with the use of sensitivity analyses. In addition, results of sensitivity analyses supported the robustness of the findings. However, the results of this meta-analysis should be interpreted with caution because of several limitations. First, the sample sizes of individual trials were relatively small, which limited the capacity of randomization to minimize the potential influences of confounding factors. For example, in one trial 22 more participants in the placebo group used antihypertensive medication than those in the L-arginine group (45% vs 24%), and this imbalance may have obscured the effect of L-arginine on BP. Nevertheless, restricting analysis to trials in absence of antihypertensive medication use did not change the overall BP estimates. Second, the validity of our metaanalysis depended upon the quality of the individual studies. Although all studies were randomized and double-blind, allocation concealment, quality of randomization, and details of withdrawals were not always reported. In addition, information on the adverse effects of L-arginine supplementation was available in a few studies. Third, the included studies had a short duration, with the majority shorter than 3 months. Therefore, the effect of L-arginine supplementation on BP as well as its safety in long term is uncertain. Fourth, only 11 studies were eligible for this meta-analysis. Most of them were conducted in patients with specific diseases and disorders, such as gestational hypertension, type 2 diabetes, and hypercholesterolemia, which may have limited the
generalization of the findings. Finally, as with any metaanalyses, publication bias may affect the results. Although formal statistical tests 10,11 did not detect evidence of this bias in our meta-analysis, the power of these tests were limited due to the small number of studies. In conclusion, this meta-analysis of randomized, double-blind, placebo-controlled trials provides evidence that oral L-arginine supplementation significantly lowers both systolic and diastolic BP. Large-scale, long-term randomized controlled trials are warranted to confirm the BP-lowering effect of L-arginine supplementation, in particular among the hypertensive populations. While it is premature to recommend L-arginine supplementation to treat and control hypertension, adopting a healthy diet that contains L-arginine–rich foods such as fish, soy, whole grains, beans, and nuts may contribute to hypertension prevention.
Disclosures The authors have no conflicts of interest. The authors are solely responsible for the design and conduct of this study, all study analyses and drafting and editing of the manuscript and its final contents.
References 1. Kearney PM, Whelton M, Reynolds K, et al. Global burden of hypertension: analysis of worldwide data. Lancet 2005;365:217-23. 2. Appel LJ, Moore TJ, Obarzanek E, et al. A clinical trial of the effects of dietary patterns on blood pressure. DASH Collaborative Research Group. N Engl J Med 1997;336:1117-24. 3. Boger RH, Ron ES. L-Arginine improves vascular function by overcoming deleterious effects of ADMA, a novel cardiovascular risk factor. Altern Med Rev 2005;10:14-23. 4. Pimenta E, Oparil S. Prehypertension: epidemiology, consequences and treatment. Nat Rev Nephrol 2010;6:21-30. 5. Moher D, Liberati A, Tetzlaff J, et al. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. Ann Intern Med 2009;151:264-9, W64. 6. Jadad AR, Moore RA, Carroll D, et al. Assessing the quality of reports of randomized clinical trials: is blinding necessary? Control Clin Trials 1996;17:1-12. 7. Follmann D, Elliott P, Suh I, et al. Variance imputation for overviews of clinical trials with continuous response. J Clin Epidemiol 1992;45: 769-73. 8. Higgins JP, Thompson SG. Quantifying heterogeneity in a metaanalysis. Stat Med 2002;21:1539-58. 9. Higgins JP, Thompson SG, Deeks JJ, et al. Measuring inconsistency in meta-analyses. BMJ 2003;327:557-60. 10. Begg CB, Mazumdar M. Operating characteristics of a rank correlation test for publication bias. Biometrics 1994;50:1088-101. 11. Egger M, Davey Smith G, Schneider M, et al. Bias in meta-analysis detected by a simple, graphical test. BMJ 1997;315:629-34. 12. Chin-Dusting JP, Alexander CT, Arnold PJ, et al. Effects of in vivo and in vitro L-arginine supplementation on healthy human vessels. J Cardiovasc Pharmacol 1996;28:158-66.
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13. Clarkson P, Adams MR, Powe AJ, et al. Oral L-arginine improves endothelium-dependent dilation in hypercholesterolemic young adults. J Clin Invest 1996;97:1989-94. 14. Piatti PM, Monti LD, Valsecchi G, et al. Long-term oral L-arginine administration improves peripheral and hepatic insulin sensitivity in type 2 diabetic patients. Diabetes Care 2001;24:875-80. 15. Sydow K, Schwedhelm E, Arakawa N, et al. ADMA and oxidative stress are responsible for endothelial dysfunction in hyperhomocyst(e) inemia: effects of L-arginine and B vitamins. Cardiovasc Res 2003;57: 244-52. 16. West SG, Likos-Krick A, Brown P, et al. Oral L-arginine improves hemodynamic responses to stress and reduces plasma homocysteine in hypercholesterolemic men. J Nutr 2005;135:212-7. 17. Lucotti P, Setola E, Monti LD, et al. Beneficial effects of a long-term oral L-arginine treatment added to a hypocaloric diet and exercise training program in obese, insulin-resistant type 2 diabetic patients. Am J Physiol Endocrinol Metab 2006;291:E906-12. 18. Facchinetti F, Saade GR, Neri I, et al. L-arginine supplementation in patients with gestational hypertension: a pilot study. Hypertens Pregnancy 2007;26:121-30. 19. Lucotti P, Monti L, Setola E, et al. Oral L-arginine supplementation improves endothelial function and ameliorates insulin sensitivity and inflammation in cardiopathic nondiabetic patients after an aortocoronary bypass. Metabolism 2009;58:1270-6. 20. Ast J, Jablecka A, Bogdanski P, et al. Evaluation of the antihypertensive effect of L-arginine supplementation in patients with mild hypertension assessed with ambulatory blood pressure monitoring. Med Sci Monit 2010;16:CR266-71. 21. Battaglia C, Mancini F, Battaglia B, et al. L-arginine plus drospirenone-ethinyl estradiol in the treatment of patients with PCOS: a prospective, placebo controlled, randomised, pilot study. Gynecol Endocrinol 2010;26:861-8. 22. Neri I, Monari F, Sgarbi L, et al. L-arginine supplementation in women with chronic hypertension: impact on blood pressure and maternal and neonatal complications. J Matern Fetal Neonatal Med 2010;23:1456-60.
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23. Thomas GD, Zhang W, Victor RG. Nitric oxide deficiency as a cause of clinical hypertension: promising new drug targets for refractory hypertension. JAMA 2001;285:2055-7. 24. Augustyniak RA, Thomas GD, Victor RG, et al. Nitric oxide pathway as new drug targets for refractory hypertension. Curr Pharm Des 2005;11:3307-15. 25. Bai Y, Sun L, Yang T, et al. Increase in fasting vascular endothelial function after short-term oral L-arginine is effective when baseline flow-mediated dilation is low: a meta-analysis of randomized controlled trials. Am J Clin Nutr 2009;89:77-84. 26. Wascher TC, Graier WF, Dittrich P, et al. Effects of low-dose L-arginine on insulin-mediated vasodilatation and insulin sensitivity. Eur J Clin Invest 1997;27:690-5. 27. Ferrannini E, Buzzigoli G, Bonadonna R, et al. Insulin resistance in essential hypertension. N Engl J Med 1987;317:350-7. 28. Reaven GM. Insulin resistance, hyperinsulinemia, and hypertriglyceridemia in the etiology and clinical course of hypertension. Am J Med 1991;90:7S-12S. 29. Boger RH. Asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, explains the q L-arginine paradoxq and acts as a novel cardiovascular risk factor. J Nutr 2004;134:2842S-7S [discussion 2853S]. 30. Vallance P, Leone A, Calver A, et al. Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 1992; 339:572-5. 31. Rajapakse NW, Mattson DL. Role of L-arginine in nitric oxide production in health and hypertension. Clin Exp Pharmacol Physiol 2009;36:249-55. 32. Martina V, Masha A, Gigliardi VR, et al. Long-term N-acetylcysteine and L-arginine administration reduces endothelial activation and systolic blood pressure in hypertensive patients with type 2 diabetes. Diabetes Care 2008;31:940-4. 33. Figueroa A, Sanchez-Gonzalez MA, Perkins-Veazie PM, et al. Effects of watermelon supplementation on aortic blood pressure and wave reflection in individuals with prehypertension: a pilot study. Am J Hypertens 2011;24:40-4.
Effect of oral L-arginine supplementation on blood pressure: A meta-analysis of randomized, double-blind, placebo-controlled trials Jia-Yi Dong, BSc, a Li-Qiang Qin, MD, PhD, a Zengli Zhang, MD, PhD, b Youyou Zhao, PhD, c Junkuan Wang, PhD, c Fabrizio Arigoni, PhD, d,e and Weiguo Zhang, MD, PhD c Suzhou, and Beijing, China; and Tokyo, Japan
Background
Previous studies suggest that L-arginine, an amino acid and a substrate of nitric oxide synthase, may have blood pressure (BP)-lowering effect. Because some studies were performed with limited number of patients with hypertension and therefore limited statistical power with sometimes inconsistent results, we aimed to examine the effect of oral L-arginine supplementation on BP by conducting a meta-analysis of randomized, double-blind, placebo-controlled trials.
Methods
PubMed, Cochrane Central Register of Controlled Trials, and the ClinicalTrials.gov databases were searched through June 2011 to identify randomized, double-blind, placebo-controlled trials of oral L-arginine supplementation on BP in humans. We also reviewed reference lists of obtained articles. Either a fixed-effects or, in the presence of heterogeneity, a random-effects model was used to calculate the combined treatment effect.
Results
We included 11 randomized, double-blind, placebo-controlled trials involving 387 participants with oral L-arginine intervention ranging from 4 to 24 g/d. Compared with placebo, L-arginine intervention significantly lowered systolic BP by 5.39 mm Hg (95% CI −8.54 to −2.25, P = .001) and diastolic BP by 2.66 mm Hg (95% CI −3.77 to −1.54, P b .001). Sensitivity analyses restricted to trials with a duration of 4 weeks or longer and to trials in which participants did not use antihypertensive medications yielded similar results. Meta-regression analysis suggested an inverse, though insignificant (P = .13), relation between baseline systolic BP and net change in systolic BP.
Conclusions
This meta-analysis provides further evidence that oral L-arginine supplementation significantly lowers both systolic and diastolic BP. (Am Heart J 2011;162:959-65.)
Hypertension is a huge public health burden, affecting approximately one billion individuals worldwide. 1 It has been widely believed that lifestyle and dietary factors play an important role in the development of hypertension. This view has been reinforced by studies such as the DASH which demonstrates that a diet rich in vegetables, fruits, and low-fat dairy foods and low in saturated and total fat can substantially lower blood pressure (BP). 2 Moreover, the DASH diet contains more protein than the
From the aDepartment of Nutrition and Food Hygiene, School of Public Health, Soochow University, Suzhou, China, bDepartment of Toxicology, School of Public Health, Soochow University, Suzhou, China, cNestle Research Center Beijing, Nestle R&D Center Beijing, Beijing, China, dNestle Research Center, Tokyo, Japan, and eGraduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan. Submitted June 27, 2011; accepted September 13, 2011. Reprint requests: Weiguo Zhang, MD, PhD, Nestle Research Center Beijing, Nestle R&D Center Beijing, Beijing 100095, China. E-mail: [email protected] 0002-8703/$ - see front matter © 2011, Mosby, Inc. All rights reserved. doi:10.1016/j.ahj.2011.09.012
control diet, 2 and arginine-rich protein has been hypothesized to contribute to the BP-lowering effect of this diet. L-Arginine, a semi-essential amino acid, is the natural substrate for nitric oxide (NO) synthase and responsible for the production of the endothelium-derived relaxing factor NO, which is involved in a wide variety of regulatory mechanisms of the cardiovascular system. 3 This property led to the hypothesis that L-arginine may have BP-lowering effect. It is attractive to lower BP and prevent hypertension through effective lifestyle modification, particularly dietary supplements in persons with prehypertension, given the lack of evidence that antihypertensive drugs reduce cardiovascular morbidity and mortality in this population. 4 During the past decades, a number of clinical trials have been carried out to evaluate the role of L-arginine in BP regulation. However, the sample size of these trials was small, the quality varied from low to high, and the results were inconsistent. We therefore aimed to examine the effect of oral L-arginine supplementation on BP by conducting a meta-analysis of randomized, double-blind, placebo-controlled trials.
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Methods
Figure 1
Search strategy We attempted to follow the Preferred Reporting Items for Systematic Reviews and Meta-Analyses 5 guidelines in the report of this meta-analysis. We searched PubMed, Cochrane Central Register of Controlled Trials, and the ClinicalTrials.gov databases through June 2011 for relevant studies, using terms of “arginine” and “L-arginine” in combination with “blood pressure” and “hypertension.” Our search was limited to randomized controlled trials of oral L-arginine supplementation in humans. In addition, we systemically searched the reference lists of obtained articles. No attempt was made to identify unpublished studies.
Study selection Studies were included if they: (1) were randomized, doubleblind, placebo-controlled trials; (2) used oral L-arginine supplementation as intervention; and (3) reported the net changes of BP and the associated standard deviations (or data to calculate them). Studies were excluded if they: (1) had a short duration of intervention (b1 week); (2) had L-arginine administered by infusion; (3) were single-blind or open-label; (4) used L-arginine as part of intervention; (5) or lacked a concurrent placebocontrolled group.
Data extraction We recorded study characteristics as follows: first author's last name, publication year; design details, including whether parallel or crossover; study duration; number of participants; antihypertensive medication use; daily dose of L-arginine treatment; and adverse effects. Participant characteristics including health status, mean age, and baseline BP were also recorded. Further, we assess the methodological quality of each included trial using the Jadad scale, which assigned scores for reported randomization, blinding, and withdrawals. 6 Two of the authors independently performed the literature search, data extraction, and bias assessment, with disagreements resolved by discussion.
Statistical analysis For parallel trials, the net changes were calculated by the difference (intervention minus control) of the changes (final values minus baseline values) of the mean values. For cross-over trials, the net changes were calculated as the difference of mean values at the end of the intervention and control periods. Where necessary, standard errors, CIs, and P values were converted to standard deviations for the analysis. Standard deviations for changes from baseline in each group were obtained. If not specified, we computed the missing standard deviations using the method proposed by Follmann et al 7 in which a correlation coefficient of 0.5 was assumed. The homogeneity of the effect size among studies was tested using the Q test at the P b .10 level of significance. We also calculated the I 2 statistic, 8,9 a quantitative measure of inconsistency across studies. An I 2 value N 50% was considered to indicate substantial heterogeneity between trials. Either a fixedeffects or, in the presence of heterogeneity, a random-effects model was used to calculate the combined effect size. We did not conduct subgroup analysis because of the small number of
Flow chart of study selection.
included studies. Rather, we performed sensitivity analyses to explore potential sources of heterogeneity across studies and to test the robustness of the results based on various criteria regarding the magnitude of BP reduction, the duration of intervention, and the use of antihypertensive medication. We also investigated the influence of a single study on the overall effect estimate by omitting one study in each turn. Furthermore, we conducted meta-regression analyses to assess whether BP reductions were related to study or subject characteristics, including L-arginine dose, intervention duration, and baseline BP levels. Potential publication bias was assessed by Begg's test 10 and Egger's test 11 at the P b .10 level of significance. All analyses were performed using STATA version 11.0 (StataCorp, College Station, TX). P b .05 was considered statistically significant, except where otherwise specified. The study was sponsored by Nestec Ltd, Vevey, Switzerland. No other extramural funding was used to support this work.
Results Characteristics of the studies We identified 11 studies 12-22 that fully met the inclusion criteria for this meta-analysis. A flow chart of literature search and study selection is presented in Figure 1. The characteristics of the selected trials are presented in Table I. The included trials were published between 1996 and 2010. The sample size varied from 12 to 79, reaching a total of 387. All trials were randomized,
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Table I. Characteristics of the trials included in the meta-analysis
Study, year
Therapy l-arginine duration Sex Mean dose Design (wks) Status (M/F) age (y) (g/day)
Chin-Dusting et al, 1996 12 Clarkson et al, 1996 13 Piatti et al, 2001 14 Sydow et al, 2003 15 West et al, 2005 16 Lucotti et al, 2006 17 Facchinetti et al, 2007 18 Lucotti et al, 2009 19 Ast et al, 2010 20 Battaglia et al, 2010 21 Neri et al, 2010 22
R, P, DB, PC R, X, DB, PC R, P, DB, PC R, P, DB, PC R, X, DB, PC R, P, DB, PC R, P, DB, PC R, P, DB, PC R, P, DB, PC R, P, DB, PC R, P, DB, PC
Baseline BP (mm Hg)
Antihypertensive Adverse Jadad drug use effects score
4
Healthy
16/0
21
20
114/56 vs 118/57
0% vs 0%
3
0% vs 0%
Not observed Diarrhea
4
HC
18/9
29
21
119/76 vs 117/77
4
T2DM
8/4
58
9
128/70 vs 120/71
0% vs 0%
–
4
8
PAOD
15/3
66.4
24
162/86 vs 164/83
–
–
4
3
HC
16/0
45
12
134/87 vs 134/87
0% vs 0%
–
4
3
T2DM
8/25
56.4
8.3
151/90 vs 149/89
–
4
32.7
4
144/91 vs 147/93
15% vs 20%
Not observed Diarrhea
2
GH
0/74
24
CAD
28/2
64
6.4
129/79 vs 122/73
0% vs 0%
4
0% vs 0%
3
0% vs 0%
Not observed Not observed –
24% vs 45%
–
5
24
PCOS
0/28
24.8
8
138/85 vs 134/82⁎, 119/71 vs 115/69 † 108/67 vs 112/68
12
GH
0/79
34.1
4
127/79 vs 128/77
4
Healthy 30/24
38.7
12
4
5
5
(–), No information available; CAD, Coronary artery disease; DB, double-blind; GH, gestational hypertension; HC, hypercholesterolemia; P, Parallel; PAOD, peripheral arterial occlusive disease; PC, placebo-controlled; PCOS, polycystic ovary syndrome; R, randomized; T2DM, type 2 diabetes mellitus; X, crossover. ⁎ Hypertensive group. † Normotensive group.
double-blind, and placebo-controlled, of which 9 trials were parallel-designed and 2 had a crossover design. Five trials 16,18 , 20-22 had BP as a primary outcome. The duration of intervention lasted from 2 to 24 weeks, with a median of 4 weeks. Dose of L-arginine varied from 4 to 24 g/day, with a median of 9 g/day. Information on adverse effects of L-arginine intervention was available in 6 trials. Characteristics of participants enrolled in these trials varied across studies. Of note, two trials 18,22 were conducted in pregnant women with hypertension, and another trial 20 presented results separately by hypertension status. Few of the remaining trials specified hypertension status, and most included participants were normotensive as indicated by mean BP levels at baseline.
Effect of L-arginine on BP The net changes and corresponding 95% CIs for systolic and diastolic BP in each trial, and overall, are presented in Figure 2 and Figure 3. Compared with placebo, oral L-arginine intervention was associated with an average net change ranging from −23.0 to 2.8 mm Hg for systolic BP and from −11.0 to 1.0 mm Hg for diastolic BP. Most trials showed an intervention-related trend toward BP reductions, but only a few reached statistical significance. The combined effect size of L-arginine on systolic BP was −5.39 mm Hg (95% CI −8.54 to -2.25, P = .001), and
substantial heterogeneity was observed (P b .001, I 2 = 73.3%). For diastolic BP, the combined effect size was -2.66 mm Hg (95% CI −3.77 to −1.54, P b .001), with little evidence of heterogeneity (P = .12, I 2 = 34.4%). Neither Begg's test nor Egger's test provided evidence of publication bias regarding effect of L-arginine on systolic or diastolic BP (all P N .30).
Sensitivity analyses To explore potential sources of heterogeneity across studies of oral L-arginine supplementation on systolic BP and to test robustness of the results, we performed sensitivity analyses. After excluding two trials 14,17 that showed large systolic BP reductions in response to L-arginine intervention, there was no heterogeneity (P = .59, I 2 = 0%), and the combined effect size was −3.34 mm Hg (95% CI −4.93 to −1.86, P b .001). Restricting analysis to 8 trials with a duration of 4 weeks or longer did not change the overall BP estimates (systolic BP −3.96 mm Hg, 95% CI −5.68 to −2.24; diastolic BP -2.62 mm Hg, 95% CI −4.11 to −1.14). Restricting analysis to 7 trials in which participants did not use antihypertensive medications yielded similar results (systolic BP −3.92 mm Hg, 95% CI −6.47 to −1.37; diastolic BP −2.50 mm Hg, 95% CI −3.75 to −1.25). Additional analyses examining the influence of an individual trial on the combined effect size by omitting one trial in each turn yielded a range from −3.66
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Figure 2
Meta-analysis of the effect of oral L-arginine supplementation on systolic blood pressure as compared with placebo. WMD, weighted mean difference.
Figure 3
Meta-analysis of the effect of oral L-arginine supplementation on diastolic blood pressure as compared with placebo. WMD, weighted mean difference.
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Table II. Characteristics associated with net change in blood pressure (BP): univariate meta-regression analysis Systolic BP
Dose Duration Baseline BP
Diastolic BP
Coefficient
95% CI
P
Coefficient
95% CI
P
0.36 0.05 −0.22
−0.39 to 1.12 −0.56 to 0.67 −0.52 to 0.08
0.31 0.85 0.13
0.21 −0.15 0.06
−0.09 to 0.51 −0.38 to 0.09 −0.29 to 0.17
0.15 0.19 0.58
(95% CI −5.54 to −1.78) to −5.92 mm Hg (95% CI −9.33 to −2.51) for systolic BP and a range from −2.40 (95% CI −3.55 to −1.26) to −3.20 mm Hg (95% CI −4.68 to −1.73) for diastolic BP. None of the individual studies appeared to have appreciable impacts on the overall combined effect sizes.
Figure 4
Meta-regression analyses We next performed meta-regression analyses to assess whether BP reductions were related to L-arginine dose, intervention duration, or baseline BP levels. None of these covariates had significant impacts on the combined effect sizes (Table II). However, there was a trend toward greater reductions in systolic BP among subjects with higher systolic BP at baseline (r = −0.22, P = .13, Figure 4).
Discussion This meta-analysis of randomized, double-blind, placebo-controlled trials brought evidence that oral L-arginine supplementation, compared with placebo, significantly lowered systolic BP by 5.39 mm Hg (95% CI −8.54 to −2.25) and diastolic BP by 2.66 mm Hg (95% CI −3.77 to −1.54). The magnitudes of the BP reductions in response to Larginine supplementation in this meta-analysis are moderate but detectable. It should be noted that most participants included in these studies were normotensive, a category in which there may be less room for improving. In fact, one trial 20 that performed separate analysis according to hypertension status showed greater BP reductions in hypertensive participants than in normotensive ones (systolic BP: −5.6 vs −1.8 mm Hg, diastolic BP: −3.8 vs −1.8 mm Hg). It would be useful to perform stratified analysis by hypertension status, but the small number of trials conducted in hypertensive subjects 18,20,22 precluded such analysis. Yet our metaregression analysis suggested an inverse, although not significant (P = .13), relation between baseline systolic BP and net change in systolic BP. It is therefore possible that L-arginine supplementation could exert larger BP-lowering effect in those with high BP. Given the inherent limitations of meta-regression analysis, this finding should be regarded as hypothesis-generating and need to be verified in future studies.
Net change in systolic blood pressure (SBP) according to baseline SBP (r = -0.22, P = .13).
The observed heterogeneity among trials of L-arginine on systolic BP appeared to be due to 2 trials 14,17 that showed large BP reductions. After exclusion of these two trials, heterogeneity disappeared, and the combined effect size did not substantially change and remained significant. In one trial, 17 both treatment and control groups were submitted to a low-caloric diet and exercise training program, and L-arginine supplementation combined with lifestyle modification and dietary therapy may have led to the pronounced BP reductions. For another trial, 14 the disparate results were likely due to chance as the sample size was rather small (n = 12). In addition, all the participants enrolled in these 2 trials 14,17 were type 2 diabetic patients. Several mechanisms may be responsible for the beneficial effect of L-arginine on BP. As a substrate for NO synthase, L-arginine may exhibit antihypertensive activities by augmenting the production of NO in endothelium and improving its bioavailability in vascular smooth muscle cells, which are essential to maintain vascular homeostasis. 23,24 A recent meta-analysis 25 suggests that oral L-arginine supplementation is effective at improving endothelial cell function in individuals
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with endothelial dysfunction. In addition, L-arginine has been shown to improve insulin resistance, 14,26 which plays an important role in the etiology of hypertension associated with metabolic syndrome. 27,28 It is also worth mentioning the “L-arginine paradox” that exogenous L-arginine supplementation improves NO-mediated biological effects despite high endogenous concentration of L-arginine. 29 One possible explanation may be related to asymmetric dimethylarginine (ADMA). ADMA is an endogenous inhibitor of NO synthase and has been shown to reduce the sensitivity of NO synthase to L-arginine. 23,24,30 There is also considerable evidence that ADMA modulates endothelial NO synthase activity within the concentration range found in patients with vascular disease. 23,24,29 Overcoming the inhibition of NO synthase by ADMA may therefore underlie the beneficial effects of L-arginine supplementation on BP. 31 Our study had stringent inclusion and exclusion criteria. All included studies were randomized, doubleblind, placebo-controlled trials, which minimized biases and suggested a high internal validity. We excluded studies 32,33 using L-arginine supplementation as part of intervention, and hence the BP-lowering effect was mainly attributable to L-arginine supplementation. We were able to detect the potential sources of heterogeneity among studies with the use of sensitivity analyses. In addition, results of sensitivity analyses supported the robustness of the findings. However, the results of this meta-analysis should be interpreted with caution because of several limitations. First, the sample sizes of individual trials were relatively small, which limited the capacity of randomization to minimize the potential influences of confounding factors. For example, in one trial 22 more participants in the placebo group used antihypertensive medication than those in the L-arginine group (45% vs 24%), and this imbalance may have obscured the effect of L-arginine on BP. Nevertheless, restricting analysis to trials in absence of antihypertensive medication use did not change the overall BP estimates. Second, the validity of our metaanalysis depended upon the quality of the individual studies. Although all studies were randomized and double-blind, allocation concealment, quality of randomization, and details of withdrawals were not always reported. In addition, information on the adverse effects of L-arginine supplementation was available in a few studies. Third, the included studies had a short duration, with the majority shorter than 3 months. Therefore, the effect of L-arginine supplementation on BP as well as its safety in long term is uncertain. Fourth, only 11 studies were eligible for this meta-analysis. Most of them were conducted in patients with specific diseases and disorders, such as gestational hypertension, type 2 diabetes, and hypercholesterolemia, which may have limited the
generalization of the findings. Finally, as with any metaanalyses, publication bias may affect the results. Although formal statistical tests 10,11 did not detect evidence of this bias in our meta-analysis, the power of these tests were limited due to the small number of studies. In conclusion, this meta-analysis of randomized, double-blind, placebo-controlled trials provides evidence that oral L-arginine supplementation significantly lowers both systolic and diastolic BP. Large-scale, long-term randomized controlled trials are warranted to confirm the BP-lowering effect of L-arginine supplementation, in particular among the hypertensive populations. While it is premature to recommend L-arginine supplementation to treat and control hypertension, adopting a healthy diet that contains L-arginine–rich foods such as fish, soy, whole grains, beans, and nuts may contribute to hypertension prevention.
Disclosures The authors have no conflicts of interest. The authors are solely responsible for the design and conduct of this study, all study analyses and drafting and editing of the manuscript and its final contents.
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