The Physiological E ﬀ ects of Amino Acids Arginine and Citrulline: Is There a Basis for Development of a Beverage to Promote Endurance Performance? A Narrative Review of Orally Administered Supplements

: Nutritional and ergogenic aid supplementation is prevalent within athletic or general ﬁtness populations, and is only continuing to gain momentum. Taken in isolation or as a combination, amino acid (AA) supplementation has the potential to increase endurance performance among other beneﬁts. L-Arginine (L-Arg) and L-Citrulline (L-Cit) are two AAs proposed to increase endothelial nitric oxide (NO) synthesis, with potential additional physiological beneﬁts, and therefore may contribute to enhanced performance outcomes such as increased power output, or time to exhaustion. However, the appropriate dose for promoting physiological and performance beneﬁts of these AAs, and their potential synergistic e ﬀ ects remains to be determined. Therefore, the aim of this review was to evaluate the varied concentrations used in the current literature, assess the e ﬀ ects of L-Arg and L-Cit in combination on physiological responses and endurance performance, and consider if there is a fundamental basis for providing these supplements in the form of a beverage. A total of six studies were considered eligible for the review which utilized a range of 3–8 g of the AA constituents. The ﬁndings support the notion that supplementing with a combination of L-Arg and L-Cit may increase NO production, enhance vasodilation, and therefore increase performance capacity in athletes. A beverage as a carrier for the two AAs is worth considering; however, there remains limited research assessing these outcomes across a consistent range of concentrations in order to see their full potential.


Introduction
Supplementation and athletic performance share a long history [1,2]. There is a continual search for the latest supplements for enhancing athletic performance, reducing fatigue and recovery time, and providing a 'competitive edge' [1]. However, due to the relatively easy addition of supplements to a diet or training regime, they are often misused or used in the absence of evidence supporting their claims [3,4]. Amino acids (AAs) are the basic building blocks of proteins, which play an essential role in the body for cellular signaling and transport [5]. These signaling and transport proteins affect basic and non-recreational individuals [13,26,27]. Generally, studies using either AAs or both in combination, have investigated predominantly physical and perceived parameters of exercise such as blood pressure (BP), sprinting time, jump height or Rating of Perceived Exhaustion (RPE) [9,12,28]. However, to date, an assessment of these outcomes against the diverse range of concentrations administered has not been performed. Therefore, the purpose of this review is to evaluate the range of L-Arg and L-Cit concentrations used in eligible studies, assess the effects of both AAs (individually or in combination) relating to performance outcomes, and to consider if there is a fundamental basis for providing these supplements in the form of a beverage.

Methods
Literature searches were performed in a non-systematic manner to assess the effectiveness of two commonly used AA, L-Arg and L-Cit as a combined supplement, on performance outcomes. Four electronic databases were searched (PubMed, Cochrane Library, Web of Science, and SCOPUS) using the terms "L-Arginine" OR "L-Citrulline" AND "athletes" AND "performance", (human and animal trials) in addition to manual searches of the identified article reference lists. Only peer-reviewed, randomized control trials published in the English language were of interest to the review. In particular, studies that used greater than 3 g of each supplement constituent in humans (or 0.05 g/kg of body mass) and at least 1.43 mmol·kg −1 of animal body mass were considered, based on inclusion criteria from previously published systematic reviews and meta-analyses [12,29]. Studies with a sample population considered 'healthy', placebo group as a comparator, and performance variables resulting from an exercise intervention in a trained population were of interest. All evidence was synthesized under the themes of AA bioavailability, vasodilation and NO biomarkers, physiological response, and performance outcomes as well as potential synergistic effects of both AAs. Results of the studies have been synthesized in Table 1.

Prevalence of Supplementation in Athletic and Recreational Populations
Supplementation for competitive or recreational use is widespread in today's society, and it has become increasingly popular in recent years [1]. Results of a survey in 2016, indicated around 50% of athletes are taking supplements regularly, although this does not capture the 'irregular' supplement users, or what a particular individual might define as a 'supplement' [30,31]. While the prevalence of supplementation has not necessarily increased over the period of a few years, a study by Shaw et al. (2016) observed a greater amount of supplements being used within the same populations [30]. In general, a number of different supplementation products are becoming increasingly available and predominately sold as food supplements such as gels, bars, protein powders, or beverages. These products also contain a plethora of caffeine, different sugars and other ingredients (e.g., herbal extracts) that might be more attributable to an increased performance outcome than the 'active' ingredient itself [2]. In addition, these beverages are also sources of an arbitrary combination of vitamins, minerals, and herbs; mixed with ergogenic aids such as nitrates (beetroot juice), and creatine, with the majority having limited or no scientific support behind their consumption [32]. Several institutions, such as the Australian Institute of Sport (AIS), provide a framework of supplement classifications, which includes an educational tool to rank sports foods and supplements (and their 'functional' ingredients) according to their scientific evidence and safety, allowing a practical contribution to an individual's performance goals [32]. In 2018, the International Olympic Committee (IOC) released a consensus statement on supplements and the high-performance athlete, indicating supplement use varied between different sports or activities [31]. This also increased with age, the level of training or performance, and was strongly influenced by perceived cultural and social norms [31]. Overall, an individual's motivation for supplement use is mainly attributable to the proposed health benefits, such as preventing nutrient deficiencies that may hinder performance or impair health, while supplementing for the purpose of a direct performance outcome was only secondary [30,31]. This further iterates the importance of using supplements that are evidence-based, and designed with the individual in mind. With supplements being increasingly consumed by active individuals, could the burden of handfuls of capsules, tablets or powders be alleviated through convenient and specifically developed beverages?

L-Arginine
One of the popular nutritional supplements is L-Arg, which is reported to have potentially beneficial effects on athletic performance and health in general [16]. It has been previously established that L-Arg's key roles in the health and upkeep of cardiac and skeletal muscles are important in the regulation of homeostasis and hemodynamics of the body [16,33], including its mediation of creatine synthesis and growth hormone release [34]. These parameters, in addition to the requirement of L-Arg for NO production, outline the integral role of L-Arg in vascular function, and skeletal muscle maintenance [34]. In endothelial cells, NO is synthesized from L-Arg by eNOS and induces smooth muscle relaxation through guanylate cyclase activation [35,36]. For this reason, it has been proposed circulating L-Arg in the blood produced by oral administration, may represent a possible therapeutic mechanism to increase the synthesis and bioavailability of NO [13].

L-Citrulline
The consumption of L-Cit, known for its vasodilatory and NO production properties, has been postulated to increase muscular repair and accelerate wound healing. In addition, many of the health-related applications of L-Cit supplementation are primarily centered on the capacity for L-Cit to increase the availability of L-Arg for NO production [22]. Unlike L-Arg, L-Cit is transported directly to the kidneys after ingestion, where it is then catabolized into L-Arg by arginosuccinate enzymes (synthase and lyase) [37]. Due to the suggested vasodilation activity, supplementation with L-Cit is thought to affect exercise performance positively, gaining increased interest in the sporting and nutrition fields [28]. However, currently, ambiguity surrounding the effects of acute vs. chronic L-Cit supplementation, the method of delivery, and particular considerations in terms of the bioavailability of the supplement, all remain to be confirmed [28].

The Bioavailability of L-Arg and L-Cit
A number of different factors are associated with affecting the bioavailability of AA, including their chirality (L-or D-forms), concentrations provided, site of absorption, gastrointestinal health, transit time through the gastrointestinal tract and food matrix in which they are consumed [38,39]. Therefore, the mode of delivery has a strong potential to influence AA absorption, potentially making them more readily available in the body, as it may interact with several different factors resulting in a difference in the speed of the absorption or in some cases, even impaired absorption [39]. As most of the essential AA are usually consumed as dietary sources in the form of protein-containing foods, after ingestion they are absorbed relatively slowly and undergo extensive systemic and pre-systemic elimination [21,39,40]. In contrast, non-essential AA can be readily absorbed (specifically the L-forms) relatively quickly, and can also further contribute to the synthesis of essential AA in most cases [21]. Orally available AA supplements are often consumed as a pressed tablet, capsule, or powder, and often other ingredients are added to lessen an unfavorable taste if provided in liquid form [41]. However, the physiologically relevant responses may exceed consumption of the several grams range, and consequently, providing capsules might not necessarily be the most effective and convenient way of supplementation [42]. The combination of AA in beverage supplements, other liquid forms and in some pre-prepared food products can also affect the bioavailability of the AA. Hence, subsequent effects cannot be solely attributed to the particular compound being marketed [42,43]. Interestingly, a pharmacokinetic study [21] assessing doses of both L-Arg and L-Cit ranging from 5 to 10 g, demonstrated L-Cit is more readily absorbed and bioavailable compared with L-Arg. Although from a performance perspective, Ermolao et al. (2017), observed no significant in sprint time when supplementing with 3 g of L-Arg in a 500 mL carbohydrate (CHO) beverage (Total CHO 26.7 g) [44]. However, the effect that this may have had on the binding of the AA with the CHO matrix was not considered [44]. Unfortunately, there is a lack of recent pharmacokinetic studies on L-Cit and L-Arg despite the need to determine an effective dose range or upper limit, nor how ingesting food before or alongside supplementation may interact with its metabolism in the body. This reiterates the need to explore the effects of these AA and their combination in potentially higher doses (6 g and greater) as they are reported to be relatively safe and are currently largely unexamined [40,44]. Moreover, there is growing interest to support the potential health properties of these AA surrounding their performance enhancing, and general beneficial health properties, including vasoactivity [40,44].

L-Arg and L-Cit on Vasodilation and NO Biomarkers
The endothelial-derived vasoactive factor, NO, is a potent signaling molecule that plays a major role in vasodilatory capacity and thereby increasing oxygen uptake in skeletal muscle [45]. L-Arg is a substrate for vascular NO (Figure 1), and endothelial NO synthase (eNOS)-dependent NO formation [14]. It has also been proposed that endogenous NO production is dependent on extracellular L-Arg concentration, and therefore, by increasing extracellular L-Arg levels, NO-dependent vasodilation will also be enhanced [27,46]. A study by Bailey et al. (2016), reported a significant increase (p < 0.05) in NO production when supplementing with standalone L-Arg (6 g) and tended to increase with standalone L-Cit (p = 0.08) at concentrations of 6 g when compared with placebo [46]. In an animal study by Morita et al. (2014), rats and New Zealand white rabbits received either 2.85 mmol/kg of L-Cit, 2.85 mmol·kg −1 of L-Arg or 1.43 mmol·kg −1 of each in combination, to assess the acute effects on NO and blood flow [13]. This L-Arg/L-Cit combination caused NO bioavailability and blood flow (measured in the central ear artery) to increase more rapidly when compared with the other treatment groups (p < 0.05) [13]. While promising, a caveat for future studies was that the dosage used was relevant to the weight of the animal; however, it is stated in the article that relatively large dosages (5-15 g daily) would be required to improve endothelial function in humans [13].

L-Arg and L-Cit on Physiological Response and Exercise Performance Outcomes
Relatively recent studies have investigated the effects of both L-Arg and L-Cit supplementation on physiological responses and improving exercise performance [9,10,26,27,35,44,[46][47][48]. Exercise intensity (i.e., velocity or power output) refers to the amount of work that can be accomplished to perform a given activity, often expressed as a percentage of an individual's VO 2max , or maximal power output, both typically measured using incremental exercise testing [49]. Exercise capacity refers to the ability of an individual to perform at a specific workload intensity [50].
Together with physiological responses associated with the autonomic nervous system (ANS) such as heart rate, heart rate variability and blood pressure, sub-maximal and maximal oxygen consumption (VO 2 ) and exercise economy, to name a few, are factors directly affecting the performance of an individual [51]. A study by Bailey et al. (2015), compared L-Arg and L-Cit individual supplementation on running economy and exercise performance in active males [46]. After acute supplementation with 6 g of L-Arg and 6 g of L-Cit, the authors reported no significant effects (p > 0.05) on either running economy or overall exercise performance after a moderate intensity cycle compared with placebo. This study, however, did not assess the potential effects of the AA in combination. In contrast, Chen et al. [10] assessed running economy in 12 athletes using L-Arg and L-Cit in combination using an oral tablet. The treatment group, who received 0.05 g/kg of body weight of L-Arg/L-Cit, improved performance over two consecutive days compared with PL (p = 0.002) [10]. Similarly, Hsueh et al.
(2018) reported male swimmers improved high-intensity interval training performance after a 50-m swim when supplementing with an average of 3.2 g for both L-Arg and L-Cit (average based on AA 0.05 g/kg body weight). [27]. While the studies assessing both L-Arg and L-Cit in combination had relatively small sample sizes (n = 10-24), overall, enhancing cardiorespiratory efficiency through NO modulation with a combination of L-Arg and L-Cit was seen to more efficiently enhance VO 2 kinetics (pulmonary O 2 uptake)-a valuable outcome in endurance athletes by positively affecting power output and metabolic responses. It is for these reasons that L-Arg and L-Cit require further investigation, specifically given their potential to enhance physiological responses, either as standalone supplements or in combination. Step 2. L-arginine is released systemically into blood vessels.
Step 3. L-arginine is transported into endothelial cells via CAT transporters and eNOS to produce nitric oxide (NO). Step 4. NO diffuses from endothelial cells into skeletal cells.
Step 5. NO presence and activation of GC and GTP produces cGMP causing cellular calcium efflux, leading to vasodilation. Abbreviations; ARGs = arginosuccinate synthase; CAT = cationic amino acid transporter; eNOS = extracellular nitric oxide synthase; NO = nitric oxide; GTP = guanosine triphosphate; GC = guanylyl cyclase; cGMP = cyclic guanosine monophosphate. Figure 1 was provided as a courtesy by Mr. Jackson Williams.

Potential Synergistic Effects of L-Arg and L-Cit
The effects of L-Arg are known to be hampered by its absorption due to intestinal arginase activity [39]. While L-Cit is made available rather quickly, it can also be metabolically converted into L-Arg, thus showing potential for a synergistic relationship between the two [14,52]. While only four known studies [10,13,26,27], to our knowledge, have assessed both AA in combination, it cannot be ruled out that their combined consumption has the potential to be more efficient in achieving improved performance outcomes. Three human trials [10,26,27] identified in this review, while having relatively small sample sizes (12-16 participants), still found enhanced responses for their collective outcome measures of fatigue modification and exercise performance. Previous human pharmacokinetic studies [21,40] have demonstrated the 'recycling' mechanism of L-Arg through the involvement of L-Cit in the urea cycle, and have focused on the suggestion that increasing L-Cit would result in increased plasma L-Arg [40]. From the studies assessed in this review, it can be speculated that due to their synergistic effects, supplementing with a combination of L-Arg and L-Cit may be more effective than supplementing with the individual AA, due to the potential for increased bioavailability and the reuptake of L-Arg [13,40]. However, there is no consensus on the appropriate concentration of each AA when used in combination, and if it is possible to achieve the expected results with a lesser dose but with chronic supplementation, compared with acute supplementation with a higher dose. Table 1. Summary of studies which tested L-Arginine and L-Citrulline in combination in humans and animal studies. To compare the effects of L-Cit and L-Arg supplementation on NO biomarkers,VO 2 kinetics, and exercise performance.
Plasma NO concentration was increased with L-Arg supplementation (p < 0.05) and tended to increase with L-Cit supplementation (p = 0.08) compared with PL.VO 2 during moderate-intensity cycle exercise was not significantly different (p > 0.05).

g (of each)
Due to the short study design, a familiarization and baseline testing session was conducted to discount a 'learning effect' in exercise testing.
Short study duration (seven days) and small sample size (n = 10). To examine the effect of co-ingestion of L-Arg and L-Cit on high-intensity interval swim performance in trained young swimmers.
Average swim time was shorter in treatment group when compared with PL. RPE was similar between the two groups. 0.05 g/kg of body weight (of each) Testing protocol began one hour after supplementation to allow for peak plasma levels.
Relatively small sample size (n = 16). Morita et al., 2014 [13] [animal] n = 24 To investigate the acute effects of a combination of oral L-Cit and L-Arg on plasma L-Arg and NO levels, as well as on blood circulation.
L-Arg and L-Cit combined caused a more rapid increase and enhancement of NO bioavailability compared with single individual AA.
2.85 mmol·kg −1 (of each) animal body weight, and 1.43 mmol/kg of combined L-Arg/L-Cit Strong study design, highlights future direction for human studies.
Animal trial-not directly transferrable? Silva et al., 2017 [8] [animal] n = 40 To evaluate the effects of supplementing with L-Arg and L-Cit on performance and oxidative stress in trained and untrained rats Supplementation improved physical performance in both control and trained groups.

mg/kg animal body weight
Strong study design, highlights future direction for human studies.
Animal trial-not directly transferrable? While there is relatively strong evidence related to improved sports performance, vasodilation and NO production after supplementation with L-Arg and L-Cit individually, trials assessing a combination of L-Arg and L-Cit are still relatively scarce. There is conflicting evidence relating to the most appropriate and effective concentration of both AA, and larger trials are needed in order to assess the true potential of L-Arg and L-Cit as a combined nutritional ergogenic aid.

Summary of Key Findings and Future Directions
Despite the consistent yet still emerging use of sporting supplements, there remains a lack of consensus underlying their effectiveness. Supplementation with L-Arg and L-Cit still requires further investigation using larger sample sizes, a consistent range of AA concentrations and timing of administration, to determine the optimal effect on physiological parameters. There also lies a general acceptance that the ratio at which L-Arg and L-Cit should be consumed in combination, typically a 2:1 L-Cit to L-Arg, as opposed to a 1:1 ratio, which may alter the effectiveness of functional beverages which contain both AAs. However, further research is needed to determine the optimal quantities of each AA required to be consumed in order to potentially exhibit beneficial effects in performance. Additionally, by providing supplementation in the form of a functional beverage, there is potential for more natural consumption of the product during various types of athletic events (i.e., running, weight lifting, and swimming), and whether the timing of supplementation could be spaced out and delivered at the most critical time points to promote improved performance.
Taking into account that sports beverages are primarily developed and formulated to be effective in improving performance, and to provide hydration and a cooling effect when required, traditionally, there are no universal agreements for the most efficacious formulations. The main reason for this could be seen in the versatility and convenience in the use of sports drinks. For example, regardless of formulation, a particular beverage could be used in several different sports or physical activities and more recently, their availability on the market has provided opportunities for use in general populations too. This requires a level of likeness or acceptability of a particular product in order to be selected by everyday individuals or recreational athletes-which in the case of L-Arg and L-Cit, in addition to the competitive nature of product development, can be a difficult obstacle. Although the flavor modulation in beverages is achieved with the use of sweeteners (carbohydrates, artificial and semi-artificial sweeteners), salts and encapsulation, it is important to select ingredients that will not interfere with the stability or in some cases the absorbability of the active compounds. Some of the potential avenues could include pH-sensitive encapsulation of the active ingredients targeting specific sites of absorption in the small intestine while masking the potential undesirable flavor of the active ingredients. Additionally, many of the commercially available L-Cit supplements currently on the market today are commonly prepared with citrulline malate, and future studies should look to investigate any differences in effectiveness between the salt and isolated compounds.

Conclusions
A beverage that provides the potential ergogenic benefits of L-Arg and L-Cit supplementation in the convenience of a sports drink with the tested stability of the active ingredients and flavor adaptation to make it more palatable is worth considering. Overall, there remains a relatively mixed consensus surrounding the quantities of L-Arg and L-Cit that need to be consumed to elicit the most beneficial effects in humans, while animal studies have highlighted a path for future human studies and potential mechanisms of action. Further research should assess these outcomes across a range of concentrations in order to see the full potential in the delivery of these AA on endurance performance for immediate use pre-, during-and post-event within the athletic, recreational sport, and general populations.