Introduction
Many supplements have been introduced in the market with the purpose of enhancing athletes’ performance [
1]. Most of these supplements allegedly help athletes tolerate a higher degree of heavy training by helping athletes recover faster during intense sport training [
2]. Recently, supplements containing L-arginine have been introduced in the market claiming to promote vasodilatation by increasing nitric oxide (NO) production via nitric oxide synthase (NOS) activation. This vasodilatation would favor an increase perfusion as well as a higher nutrient and oxygen delivery to the active muscles during exercise, enhancing protein synthesis and muscle recovery [
2].
L-arginine is considered a semi-essential amino acid because the body normally produces it in sufficient amounts. However, supplementation may be needed in special conditions such as malnutrition, excessive ammonia production, burns, infections, peritoneal dialysis, rapid growth, urea synthesis disorders, and/or sepsis [
3].
Physiological concentrations of L-arginine in healthy individuals are enough to saturate endothelial NOS, which is ~ 3 μmol/L. Therefore, supplementary L-arginine should not promote increased enzyme activity; consequently, no further NO production should occur. However, there is evidence describing the NO-mediated biological effects associated with L-arginine supplementation despite the fact that nitric oxide synthase (NOS) is theoretically saturated with the physiological concentration of L-arginine—hence the condition known as the ‘L-arginine paradox’ [
4].
Early evidence suggests that L-arginine supplementation may help treat individuals with atherosclerosis risk factors, such as hypercholesterolemia, hypertension, diabetes mellitus, kidney failure, hyperhomocysteinemia, smoking, and aging—all of which are conditions that are associated with reduced NO biosynthesis [
5‐
9]. Böger R., [
10] had shown that plasma levels of asymmetric dimethylarginine (ADMA), an endogenous NOS inhibitor, are increased approximately 2–3 fold in the pathophysiological conditions associated with cardiovascular disease. For this reason, elevated ADMA concentration may be one possible explanation for endothelial dysfunction and decreased NO synthesis in this disease cluster. Therefore, it appears that only subjects with poor NO synthesis are likely to benefit from L-arginine supplementation.
Despite the theory regarding L-arginine supplementation improving vasodilatation from increased NO production, a recent review [
2] about the ergogenic effect of L-arginine supplementation in healthy subjects shows that there were only five studies that evaluated exercise performance after acute L-arginine supplementation, three of which reported significant improvements. Besides the improvements observed in physical performance, the authors of these studies did not measure the underlying mechanism that could explain how the results obtained may have been due to increased NO production.
Bailey et al., [
11] observed significant increases in the time to task failure with concomitant reductions in the O2 cost of moderate-intensity cycle exercise and slow oxygen uptake component amplitude on the group supplemented with 6 g of L-arginine, 1 h before a series of moderate- and severe-intensity exercise bouts for 3 days. Stevens et al., [
12] observed significant increase in peak torque, total work and fatigue index after supplementing with a product containing 6 g of L-arginine in three equal aliquots at 45, 30 and 10-min periods before isokinetic dynamometer exercise. Buford and Koch, [
13] observed significant improvement of average power during repeated sets of supra-maximal exercise during cycle ergometer on the group that consumed 6 g of L-arginine.
Based on the theory that physiological concentrations of L-arginine are enough to saturate endothelial NOS and no further NO production should occur in healthy individuals, it is our hypothesis that there should be no change inplasma concentration of NO2- and NO3- as a result of L-arginine supplementation when compared to placebo.
Due to the fact that other studies have demonstrated vascular and exercise performance benefits after L-arginine supplementation in healthy individuals [
2,
14], the question still remains as to whether this effect is NO-mediated. Therefore, in order to test the claim that L-arginine supplementation may increase NO synthesis, the present study was conducted to identify the acute effects of L-arginine supplementation on indirect markers of NO synthesis—NO
2- and NO
3-. It is to point out that, contrary to the present study, other studiesthat evaluated the effect of L-arginine supplementation on NO synthesis at rest have methodological limitations [
15] (e.g.: They did not control diet for food contain NO
2- and NO
3-), which may cause flaws in the results. The plasma levels of ADMA and SDMA at the beginning of the study have also been addressed.
Discussion
Dietary supplements containing the semi-essential amino acid L-arginine (the only substrate of NOS) have been introduced in the market, claiming to promote vasodilatation by increasing production of NO. In the present study, we found that in healthy subjects 6 g of oral L-arginine supplementation did not stimulate an increase in NO production when compared with the placebo group. Additionally, no significant increases were observed in plasma L-citrulline, which is the by-product of NO synthesis from L-arginine. Furthermore, no significant differences in plasma concentrations of ADMA and SDMA at onset of the study were observed between the L-arginine and placebo groups.
Several studies had showed no significant difference in NO production (measured by NO
2- and NO
3-) after L-arginine supplementation [
22‐
24]. Liu et al. [
22] did not observe any significant differences in plasma NO
2- and NO
3- concentrations after orally supplementing ten healthy male athletes with 6 g of L-arginine (as free form) or placebo for 3 days. Koppo et al. [
23] observed no significant difference in urinary NO
2- and NO
3- after 14 days of supplementing seven physically active healthy males with 7.2 g of L-arginine hydrochloride (3 × 3 capsules of 805 mg), and Tang et al. [
24] also did not observed any significant difference on NO synthesis (measured by plasma NO
2- and NO
3-) in eight healthy young men after an single dose of 10 g of L-arginine. Since all subjects of these studies were submitted to exercise, the results of these studies are not surprising due to the very nature of the underlying mechanism of NO synthesis: vascular shear stress is considered the main stimulus for endothelial NO production during exercise [
25]. Therefore, there should theoretically be no need for supplementary L-arginine to synthesize NO during exercise.
Regarding studies at rest condition, Blum et al. [
26] investigated the effects of oral L-arginine (9 g daily for one month) on NO bioactivity in 10 healthy postmenopausal women. After the supplementation period, the authors observed no significant difference in serum NO
2- and NO
3- concentrations. In another study, Evans et al. [
27] also did not find significant differences in serum NO
2- and NO
3- concentrations after submitting twelve healthy subjects to take L-arginine for 1-week periods at daily doses of 3, 9, 21, and 30 g. Schwedhelm et al., [
28] published a study with the purpose of investigating the pharmacokinetic and pharmacodynamics properties of oral L-arginine with regard to NO metabolism. Twenty healthy subjects were submitted to two doses of L-arginine supplementation (1 g of arginine sustained-release tree times per day or 1.6 g of immediate-release arginine two times per day) for 7 days. At baseline and on day 7, a single dose of L-arginine supplementation (half of the total daily dose, respectively) was administered and blood samples were drawn at 0, 0.5, 1, 2, 3, 4, 6, 8, 12, 16 and 24 h after supplementation. The authors observed no significant differences in urinary excretion of nitrate at any time point, despite increases in plasma L-arginine concentrations after the supplementation (T
max = 3.7 ± 1.3 h for arginine sustained-release and 0.7 ± 0.1 h for arginine immediate-release). In the present study, the plasma L-arginine concentrations increased significantly after 30 min of L-arginine supplementation when compared to placebo group, and maintained high throughout the study period. Furthermore, no significant differences were observed in plasma concentrations of L-ornithine and L-citrulline at any time point between the groups.
It is important to point out that, contrary to the present study, neither of the above mentioned studies controlled the intake of food rich in NO2- and NO3- from subject’s diet before or during the study. Most NO2- and NO3- comes from diet (vegetable products contain the highest levels of NO3-; meat and bean products contain the highest levels of NO2-), which may alter the results of the analysis. Thus, endogenous synthesis of NO may not be adequately measured by NO2- and NO3- in plasma and urine if the diet is not controlled.
Measurement of NO
2- and NO
3- in various biological fluids turned out to be the most suitable, practical and reliable non-invasive method to assess systemic NO synthesis in vivo [
19]. In the present study, NO synthesis was quantified by measuring plasma NO
2- + NO
3- (NOx) via high-performance liquid chromatography. Most of the studies [
22‐
24] had analyzed NO synthesis by using Griess reaction. The acidic conditions under which experiments using Griess reaction are conducted favor the formation of S-nitroso compounds from NO
2- and reduced thiols. Thus, the measurement of NO
2- and NO
3- in biological fluids by assays based on the Griess is subject to interference by many substances acting at different places in the Griess reaction and the following spectrophotometric measurement in the same wavelength (normal absorbance at 540 nm). Therefore, studies using Griess reaction to detect stable metabolites (NO
2- and NO
3-) to measure NO synthesis may have significant methodological limitations, as compared to fluorometric techniques associated with HPLC.
Bailey et al. [
11] observed significant increases in plasma NO
2- after supplementing nine healthy recreationally active men with a supplement that contained 6 g of L-arginine (dissolved in 500 mL of water) as compared to placebo. It is important to note that this study associated other amino acids besides L-arginine, including L-citrulline (quantities not expressed in the study), which have been shown to increase NO production, as measured by plasma concentrations of NO
2-[
29] and urinary excretion of NO
3- and cGMP [
28]. Interestingly, the authors did not measure plasma NO
2- at baseline; they had just done so 1 hour after supplementation, which is a major methodological limitation, since it is not known whether there were any differences in the samples prior to supplementation. Furthermore, taking into consideration that diet can influence nitrite plasma concentrations, no dietary control to limit the consumption of foods rich in NO
2- and NO
3- was conducted.
Others studies also have showed improvements in NO production by using L-arginine supplementation [
30‐
33]. However, all of these studies had administered L-arginine in subjects with some cardiovascular risk factors or cardiopathy. It appears that L-arginine is a limiting factor for NO synthesis in patients at risk for atherosclerosis, but not for healthy individuals [
34]. Therefore, L-arginine supplementation may be necessary only for individuals with atherosclerosis risk factors.
Among the possible explanations for this phenomenon is the presence of high levels of asymmetric dimethylarginine (ADMA), an endogenous NOS inhibitor. Higher concentrations of ADMA were encountered in individuals with atherosclerosis, as well as in individuals with atherosclerosis risk factors, such as hypercholesterolaemia, hypertension, diabetes mellitus, kidney failure, hyperhomocysteinaemia, smoking and aging [
10]. Physiological levels of L-arginine and the presence of normal concentrations of ADMA saturate the endothelial NOS enzyme, promoting NO production. In these conditions, L-arginine supplementation does not affect enzyme activity. In contrast, in the presence of elevated plasma concentrations of ADMA the endothelial NOS activity diminishes, resulting in lower physiological levels of NO production. SDMA has no effect on NOS activity but may compete with L-arginine for the y + transport system [
35]. Under these conditions, L-arginine supplementation may re-establish the L-arginine/ADMA ratio in order to activate endothelial NOS [
4]. Therefore, L-arginine supplementation may exert a beneficial effect on vascular function.
In the present study, we observed no significant difference in plasma concentrations of ADMA, SDMA and L-arginine/ADMA ratio at baseline between the groups. This finding may explain the absence of significant changes in NO production after L-arginine supplementation. The baseline plasma concentrations of ADMA observed in the present study are similar to previous studies in healthy subjects which reported ADMA concentrations ranging between 0.3 and 0.9 μmol/L [
36‐
38]. Therefore, it may be speculated that there should be no further increase in NO production after L-arginine supplementation in subjects with normal levels of ADMA.
Böger et al., [
34] observed that plasma ADMA levels were significantly higher in hypercholesterolemic subjects than in normocholesterolemic control subjects (2.17 ± 0.15 and 1.03 ± 0.09 μmol/L, respectively); and the higher levels of ADMA was associated with reduced NO synthesis. Therefore, it appears that high plasma ADMA concentrations may inhibit NOS enzyme, but low or normal plasma ADMA concentrations do not affect NO synthesis.
In conclusion, L-arginine supplementation does not increase NO production in healthy subjects with normal plasma ADMA concentrations. Therefore, it is not advisable to recommend dietary supplements containing L-arginine for the purposes of increasingacutely NO production in healthy subjects. This result does not discard the possible effect of L-arginine on NO production in individuals with pathophysiologicalconditions (e.g.: hypercholesterolaemia, hypertension, diabetes mellitus, kidney failure, hyperhomocysteinaemia, smoking and aging) and long-term studies are needed to identify whether L-arginine may provide some benefit.
Competing interests
Each author certifies that he or she has no commercial associations (e.g., consultancies, stock ownership, equity interest, patent/licensing arrangements, etc.) that might pose a conflict of interest in connection with the submitted article, except as disclosed on a separate attachment. All funding sources supporting the Work and all institutional or corporate affiliations of the authors are acknowledged in a footnote in the Work.
Authors’ contributions
TSA contributed substantially to data acquisition and chromatographic analysis, statistical analysis and data interpretation, and was the manuscript writer. CACJ contributed substantially to chromatographic analysis, interpretation of results, and reviewing the manuscript. JTS contributed to data interpretation and manuscript revision. VMFP contributed to data interpretation and manuscript revision. All authors read and approved the final manuscript.