Alvares et al. Nutrition & Metabolism 2012, 9:54 http://www.nutritionandmetabolism.com/content/9/1/54 RESEARCH Open Access Acute L-Arginine supplementation does not increase nitric oxide production in healthy subjects Thiago Silveira Alvares1,2*, Carlos Adam Conte-Junior1, Joab Trajano Silva1 and Vânia Margaret Flosi Paschoalin1 Abstract Dietary supplements containing L-arginine have been marketed with the purpose of increasing vasodilatation, and thus, blood and oxygen supply to the exercising muscle. The present study evaluated the acute effect of L-arginine - - supplementation on indicators of NO production, nitrite (NO2) + nitrate (NO3) (NOx), in healthy subjects. Plasma concentrations of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) have also been addressed. Seventeen healthy males participated in a randomized, double-blind, placebo-controlled study. Blood samples were drawn from a left antecubital vein at baseline (T0). Afterwards, subjects were randomly submittedto 6 g of oral L-arginine supplementation (as L-arginine hydrochloride) or placebo (as corn starch); afterwards, the subjects remained at rest in supine position and blood samples were drawn again at 30 (T1), 60 (T2), 90 (T3) and - - 120 minutes (T4) after supplementation. To analyze NO production, NO3 was converted to NO2 by nitrate reductase, - followed by the derivatization of NO2 with 2,3-diaminonaphthalene. NOx, ADMA and SDMA were analyzed using a high-performance liquid chromatography system and monitored with a fluorescence detector. Two-way ANOVA with repeated measures showed no significant changes in NOx concentrations on the L-arginine group as compared to placebo group at any of the fivetime points (T0: 17.6 ± 3.9 vs 14.6 ± 2.3 μmol/L; T1: 15.8 ± 2.4 vs 14.3 ± 1.7 μmol/L; T2: 16.8 ± 4.9 vs 13.7 ± 2.7 μmol/L; T3: 16.7 ± 3.9 vs 14.6 ± 2.1 μmol/L; T4: 15.1 ± 2.8 vs 13.5 ± 3.5 μmol/L). Furthermore, plasma levels of ADMA and SDMA were not statistically significant between the L-arginine and placebo groups at T0 (0.43 ± 0.19 vs 0.39 ± 0.15 μmol/L and 1.83 ± 1.13 vs 1.70 ± 0.62 μmol/L), respectively. In conclusion, acute L-arginine supplementation does not increase plasma concentration of NOx in healthy individuals with normal plasma concentrations of ADMA. Keywords: Amino acids, Nitric oxide, Asymmetric dimethylarginine, Symmetric dimethylarginine, Nitrite, Nitrate, HPLC Introduction in the market claiming to promote vasodilatation by in- Many supplements have been introduced in the market creasing nitric oxide (NO) production via nitric oxide syn- with the purpose of enhancing athletes’ performance [1]. thase (NOS) activation. This vasodilatation would favor an Most of these supplements allegedly help athletes tolerate increase perfusion as well as a higher nutrient and oxygen a higher degree of heavy training by helping athletes re- delivery to the active muscles during exercise, enhancing cover faster during intense sport training [2]. Recently, protein synthesis and muscle recovery [2]. supplements containing L-arginine have been introduced L-arginine is considered a semi-essential amino acid because the body normally produces it in sufficient * Correspondence: [email protected] amounts. However, supplementation may be needed in 1Laboratory of Advanced Analysis in Biochemistry and Molecular Biology, special conditions such as malnutrition, excessive am- Department of Biochemistry, Chemistry Institute, Federal University of Rio de Janeiro, Brazil monia production, burns, infections, peritoneal dialysis, 2Present address: Laboratório de Análises Avançadas em Bioquímica e rapid growth, urea synthesis disorders, and/or sepsis [3]. Biologia Molecular, Departamento de Bioquímica – Instituto de Química, Physiological concentrations of L-arginine in healthy Universidade Federal do Rio de Janeiro, Avenida Athos da Silveira Ramos, 149 Centro de Tecnologia, Bloco A, Sala 545, Ilha do Fundão, Rio de Janeiro, individuals are enough to saturate endothelial NOS, RJ 21941-909, Brazil which is ~ 3 μmol/L. Therefore, supplementary L- © 2012 Alvares et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Alvares et al. Nutrition & Metabolism 2012, 9:54 Page 2 of 8 http://www.nutritionandmetabolism.com/content/9/1/54 arginine should not promote increased enzyme activity; individuals, it is our hypothesis that there should be no - - consequently, no further NO production should occur. change inplasma concentration of NO2 and NO3 as a re- However, there is evidence describing the NO-mediated sult of L-arginine supplementation when compared to biological effects associated with L-arginine supplemen- placebo. tation despite the fact that nitric oxide synthase (NOS) Due to the fact that other studies have demonstrated is theoretically saturated with the physiological concen- vascular and exercise performance benefits after L- tration of L-arginine—hence the condition known as the arginine supplementation in healthy individuals [2,14], ‘L-arginine paradox’ [4]. the question still remains as to whether this effect is Early evidence suggests that L-arginine supplementa- NO-mediated. Therefore, in order to test the claim that tion may help treat individuals with atherosclerosis risk L-arginine supplementation may increase NO synthesis, factors, such as hypercholesterolemia, hypertension, dia- the present study was conducted to identify the acute betes mellitus, kidney failure, hyperhomocysteinemia, effects of L-arginine supplementation on indirect mar- - - smoking, and aging—all of which are conditions that are kers of NO synthesis—NO2 and NO3. It is to point out associated with reduced NO biosynthesis [5-9]. Böger R., that, contrary to the present study, other studiesthat [10] had shown that plasma levels of asymmetric evaluated the effect of L-arginine supplementation on dimethylarginine (ADMA), an endogenous NOS inhibi- NO synthesis at rest have methodological limitations tor, are increased approximately 2–3 fold in the patho- [15] (e.g.: They did not control diet for food contain - - physiological conditions associated with cardiovascular NO2 and NO3), which may cause flaws in the results. disease. For this reason, elevated ADMA concentration The plasma levels of ADMA and SDMA at the begin- may be one possible explanation for endothelial dysfunc- ning of the study have also been addressed. tion and decreased NO synthesis in this disease cluster. Therefore, it appears that only subjects with poor NO Methods synthesis are likely to benefit from L-arginine Subjects supplementation. Seventeen healthy males (25.5 ± 3.5 yr, 78.7 ± 10.5 kg; Despite the theory regarding L-arginine supplementa- 176.1 ± 7.5 cm and 25.3 ± 2.3 kg.m-2 BMI) were recruited tion improving vasodilatation from increased NO pro- to participate in the study. All subjects were fully duction, a recent review [2] about the ergogenic effect of informed of the nature and purpose of the investigation L-arginine supplementation in healthy subjects shows and gave their written consent to participate. The exclu- that there were only five studies that evaluated exercise sion criteria for participation in the study were any performance after acute L-arginine supplementation, known cardiovascular, pulmonary or metabolic diseases three of which reported significant improvements. Be- (asthma, diabetes, hypertension, dyslipidemia etc.), and sides the improvements observed in physical perform- the use of nutritional and pharmacological ergogenics. ance, the authors of these studies did not measure the All experimental procedures were performed in accord- underlying mechanism that could explain how the ance with the ethical standards of the Helsinki Declar- results obtained may have been due to increased NO ation and were approved by the Institutional Ethics production. Committee of the Hospital Universitário Clementino Bailey et al., [11] observed significant increases in the Fraga Filho (protocol # 0118.0.197.000-10) from Rio de time to task failure with concomitant reductions in the Janeiro, Brazil. O2 cost of moderate-intensity cycle exercise and slow oxygen uptake component amplitude on the group sup- Experimental design plemented with 6 g of L-arginine, 1 h before a series of In a randomized, double-blind and placebo-controlled moderate- and severe-intensity exercise bouts for 3 days. study, subjects reported to the laboratory. Blood samples Stevens et al., [12] observed significant increase in peak were drawn from an antecubital forearm vein at baseline torque, total work and fatigue index after supplement- after a ten-minute period of quiet rest in supine position. ing with a product containing 6 g of L-arginine in Afterwards, subjects were randomly divided into either a three equal aliquots at 45, 30 and 10-min periods be- placebo or an L-arginine group and rested again in su- fore isokinetic dynamometer exercise. Buford and Koch, pine position in a quiet room. Blood samples were [13] observed significant improvement of average drawn again at 30 (T0), 60 (T1), 90 (T2) and 120 min- power during repeated sets of supra-maximal exercise utes (T4) after supplementation (see Figure 1). During during cycle ergometer on the group that consumed this period, the subjects consumed no food and drink. 6 g of L-arginine. Based on the theory that physiological