Introduction
Also known as endothelium-derived relaxing factor (EDRF), nitric oxide (NO) is biosynthesized endogenously from L-arginine and oxygen, by various nitric oxide synthase (NOS) enzymes and by reduction of inorganic nitrate [
1]. Cell types containing NOS have been demonstrated to be able to reutilize L-citrulline, the byproduct of NO synthesis, to L-arginine by the arginine-citrulline cycle [
2]. Nitric oxide is a gaseous signaling molecule which activates soluble guanylate cyclase (sGC) in smooth muscle cells, thereby catalyzing cyclic guanosine monophosphate (cGMP) synthesis. Intracellular cGMP serves as a cellular messenger and plays a role in a variety of biological processes, and in human blood vessels, results in vasodilation [
3]. Cell types containing NOS have been demonstrated to be able to reutilize L-citrulline, the byproduct of NO synthesis, to L-arginine by the arginine-citrulline cycle [
2]. An elevation in plasma L-arginine has been shown to improve endothelial function because the vascular endothelium uses NO to signal the surrounding smooth muscle to relax, thus resulting in vasodilation and increasing blood flow [
4]. During exercise, vasodilation occurs as a result of various intracellular events, including the production and release of NO. However, it has recently been shown that seven days of oral L-arginine supplementation at 12 g/day, while effective in elevating plasma L-arginine and NO metabolites nitrite and nitrate (NOx) after exercise, was ineffective at increasing blood flow during exercise [
5].
L-citrulline has been indicated to be a second NO donor in the NOS-dependent pathway, since it can be converted to L-arginine [
6]. Dietary L-citrulline supplementation has shown conflicting results regarding its effectiveness at improving exercise performance [
7,
8]. Moreover, results showing favorable effects in exercise performance [
8] did not assess NO status; therefore, this response cannot be related to an improvement in exercise performance. The importance of L-citrulline towards ergogenic support is based on the premise that L-citrulline is not subject to pre-systemic elimination and, consequently, could be a more efficient way to elevate extracellular levels of L-arginine. L-Citrulline can perhaps improve the effects on nitrate elimination during the course of recovery from exhaustive muscular exercise, and also serves as an effective precursor of L-arginine. It has been shown that three grams daily of oral L-citrulline supplementation for seven days elevated plasma L-arginine concentration and augmented NO-dependent signaling [
9].
Glutathione is a low molecular weight, water-soluble tripeptide composed of the amino acids cysteine, glutamic acid, and glycine. Glutathione is an important antioxidant and plays a major role in the detoxification of endogenous metabolic products, including lipid peroxides. Intracellular glutathione exists in both the oxidized disulfide form (GSSG) or in reduced (GSH) state; the ratio between GSH and GSSG is held in dynamic balance depending on many factors including the tissue of interest, intracellular demand for conjugation reactions, intracellular demand for reducing power, and extracellular demand for reducing potential. In some cell types, GSH appears to be necessary for NO synthesis and NO has been shown to be correlated with intracellular GSH [
10]. GSH stimulates total L-arginine turnover and, in the presence of GSH, NOS activity is increased [
11]. This suggests that GSH may play an important role in protection against oxidative reaction of NO, thus contributing to the sustained release of NO. Therefore, combining L-citrulline with GSH may augment the production of NO. However, the effectiveness for oral GSH supplementation in humans, particularly in combination with L-citrulline has not been clearly delineated.
Using in vitro (cell culture) and in vivo approaches in rodents and humans, the overall purpose of this study was to determine the efficacy of L-citrulline and/or GSH supplementation towards increasing the levels of cGMP, nitrite, and NOx. We hypothesized that the combination of L-citrulline and GSH would preferentially increase the concentrations of cGMP, nitrite, and NOx levels when compared to control conditions.
Discussion
In the present study, we sought to determine the effectiveness of L-citrulline and/or GSH in increasing NO synthesis during in vivo conditions with rodents and humans and also in an in vitro condition using HUVEC. Collectively, in phase one and three of the study we observed combining L-citrulline with GSH to be more effective at increasing the concentrations of nitrite and/or NOx than with control/placebo in HUVEC and humans, respectively. In phase two, we observed L-citrulline combined with GSH to be more effective at increasing plasma NOx.
L-citrulline is a ubiquitous amino acid in mammals [
13], and in the kidneys, vascular endothelium, and other tissues can be readily converted to L-arginine thus raising plasma and tissue levels of L-arginine which increases NOS synthesis and subsequent NO production [
14]. Additionally, L-citrulline has been indicated to be a secondary NO donor in the NOS-dependent pathway, since it can be converted to L-arginine. Nitrate and nitrite are the main substrates to produce NO via the NOS-independent pathway. These anions can be reduced
in vivo to NO and other bioactive nitrogen oxides.
Previous studies have reported that L-citrulline could increase plasma L-arginine concentration by the L-citrulline-NO cycle [
15]. Fu et al. [
16] showed that pre-treatment with L-citrulline in rodents for seven days at doses of 300, 600, and 900 mg/kg increased the NO content. Since L-citrulline can be readily converted to L-arginine, it provides a recycling pathway for the conversion of L-citrulline to NO via L-arginine [
14,
17]. In phase three of the present study, we observed seven days of L-citrulline supplementation, with and without GSH, to result in significant increases in the levels of plasma citrulline and arginine. Our present data support previous results [
18] showing that a 10-g oral bolus of L-citrulline significantly enhanced plasma citrulline and arginine levels compared with placebo. Therefore, our present observations indicate that L-citrulline is indeed a precursor to L-arginine formation which subsequently increases circulating levels of NOx, and that recycling of L-citrulline to L-arginine may maintain substrate concentration in favor of NO synthesis [
19].
It has been shown in some mammalian cell types, that GSH and NO activity are linked [
20]. Furthermore, results suggest that GSH is necessary in HUVEC for NO synthesis rather than for the NO-related effect on guanylate cyclase, because when cells were depleted of GSH, citrulline synthesis and cGMP production were inhibited in a concentration-dependent manner [
21]. This may be explained based on the premise that the synthesis of NO, detected as L-citrulline production, in HUVEC and murine endothelial cells has been shown to be correlated with intracellular GSH [
10]. A previous study suggested that in some cell types, the activity of NO is influenced by the endogenous antioxidant GSH [
22]. It is conceivable that GSH activity may be augmented by L-citrulline as it has been shown that pre-treatment with L-citrulline in rodents for seven consecutive days lead to an elevation in the level of GSH [
23].
Furthermore, in phase one of the present study, we showed that combining L-citrulline and GSH effectively increased nitrite concentration in HUVEC cells compared to control; although, both L-citrulline and GSH alone had no effect on nitrite. However, in phase two, the combined L-citrulline and GSH provided to rodents resulted in a significant increase in plasma NOx one hr following ingestion compared to control and L-citrulline. Moreover, we observed a similar response in phase three compared to phase one, where combining L-citrulline and GSH effectively increased plasma nitrite and NOx concentration in humans compared to placebo.
Oral supplementation with L-arginine can increase plasma L-arginine levels; although, oral supplementation with L-citrulline, a precursor for arginine biosynthesis, has been shown to be more efficient than oral L-arginine in increasing plasma L-arginine [
9], due to splanchnic catabolism of ingested L-Arginine [
24]. NO synthesis is primarily dependent upon intracellular arginine availability and is affected by: 1) the transport of extracellular arginine; 2) intracellular synthesis of arginine from citrulline, which is dependent on citrulline availability; and 3) the activity of arginase [
17]. Moreover, this latter point can be further supported based on the data demonstrating increased arginine availability in cultured cell model or by supplementation
in vivo was able to overcome the effects of arginase and to enhance NO synthesis [
25]. Based on results from all three phases of the present study, it is evident that L-citrulline supplementation impacted extracellular arginine concentration and the subsequent intracellular arginine synthesis based on the responses we observed in nitrite and NOx concentrations.
In phase 3 of the present study, we were also interested to determine if increased plasma arginine availability and subsequent NO synthesis due to oral L-citrulline and/or GSH supplementation was effected by resistance exercise. Interestingly, we observed increases in plasma NOx in all four groups immediately following resistance exercise, which indicates this response in plasma NOx to be particularly due to the stimulus of resistance exercise. These results are similar to our previous study where resistance exercise increased plasma NOx, independent of increased plasma arginine, due to seven days of L-arginine supplementation [
5]. In the same way as NOx, plasma cGMP levels were increased by the combination of L-citrulline and GSH; however, this increase was not significantly different. Nevertheless, this suggests a possible synergistic effect from GSH that may be partially mediated by the formation of the NO-GSH complex. However, in the present study, significantly different increases in NOx occurred 30 min following resistance exercise, and only for the L-citrulline + GSH group. This suggests that a resistance exercise-related mechanism of inducing plasma NO, perhaps due to increased shear stress that triggered an up-regulation in NO-cGMP signaling, is a conceivable candidate for this response.
Consequently, there are possible physiological benefits of having high NO levels at 30 min post-exercise relative to its impact on muscle protein metabolism and possible muscle performance in response to resistance exercise training. It has been shown that NOS activity is necessary for calcium-induced activation of the Akt pathway (involved in translation initiation and thus muscle protein synthesis), and that NO is sufficient to elevate Akt activity in primary myotubes. Nitric oxide appears to influence Akt signaling though a cGMP/PI3K-dependent pathway [
26], which is the primary pathway for up-regulating translation initiation and protein synthesis in skeletal muscle. Additionally, nitrite has been shown to enhance the proliferation and mTOR activity of myoblasts [
27]. Similarly, NO seems to influence skeletal muscle function through effects on excitation-contraction coupling, myofibrillar function, perfusion, and metabolism. Another study showed that by using an agent to inhibit phosphodiesterase-5, that the augmentation of NO-cGMP signaling increased protein synthesis and reduced fatigue in human skeletal muscle [
28]. In the present study, L-citrulline + GSH showed an improvement in cGMP activity suggesting that this outcome could likely play a role in muscle protein synthesis and muscle performance when combined with resistance training.
Our present data suggest that the oral supplementation of L-citrulline combined with GSH provides an augmenting effect on plasma NOx. Based on results from recent studies, this may be explained based on the premise that in some cell types, the activity of NO is influenced by the endogenous antioxidant, GSH [
10]. Therefore, GSH may play an important role in protection against oxidative reaction of NO, thus contributing to the sustained release of NO.
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Competing interests
Masahiko Morita is an employee of KYOWA HAKKO BIO CO., LTD. The other co-authors declare no conflicts of interest.
Authors’ contributions
SM served as the study coordinator and was involved in participant recruitment, testing, laboratory analyses, and assisted in manuscript preparation. TA was involved in testing and laboratory analyses. MM was involved in conducting the phase 1 and 2 portions of this study, in performing the plasma L-citrulline and L-arginine analyses in phase 3, and was involved in manuscript preparation. DSW was the principal investigator and, was responsible for securing grant funding and developing the experimental design. He was also involved in training and mentoring for laboratory analyses, provided primary oversight during the course of the study, and supervised manuscript preparation. All authors read and approved the final manuscript.