l-Arginine is one of the most metabolically versatile amino acids. In addition to its role in the synthesis of nitric oxide, l-arginine serves as a precursor for the synthesis of polyamines, proline, glutamate, creatine, agmatine and urea. Several human and experimental animal studies have indicated that exogenous l-arginine intake has multiple beneficial pharmacological effects when taken in doses larger than normal dietary consumption.
Such effects include:
L-arginine is a basic natural amino acid. Its occurrence in mammalian protein was discovered by Hedin in 1895.
Fig. 1. Overview of mammalian arginine metabolism. Only enzymes that directly use or produce arginine, ornithine, or citrulline are identified, and not all reactants and products are shown. Inhibition of specific enzymes is indicated by dashed lines and the dash within a circle. Amino acid residues within proteins are identified by brackets. Key to abbreviations: ADC, arginine decarboxylase; AGAT, arginine: glycine amidinotransferase; ARG, arginase; ASL, argininosuccinate lyase; ASS, argininosuccinate synthetase; DDAH, dimethylarginine dimethylaminohydrolase; Me2, dimethyl; OAT, ornithine aminotransferase; ODC, ornithine decarboxylase; OTC, ornithine transcarbamylase; P5C, l-D1-pyrroline-5-carboxylate; PRMT, protein–arginine methyltransferase. It is worth mentioning that the processes described in Fig. 1 do not all occur within each cell; instead, they are differentially expressed according to cell type, age and developmental stage, diet, and state of health or disease. In fact, Fig. 1 is somewhat misleading in that it summarises the metabolism of arginine at a whole body level; it does not represent arginine metabolism in any particular cell type, nor does it indicate which enzymes are expressed under different conditions, which enzymes are regulated, the presence of various inter- and intracellular transport systems or how substrates are divided into the different pathways.
L-arginine and the gastrointestinal tract
NO donors have been repeatedly shown to protect gastric mucosa against damage induced by various agents. In addition, reports from different laboratories have demonstrated the importance of endogenous NO in the protection of gastric mucosa.
Two studies from Pique’s laboratory have shown that NO plays a vasodilatory role in gastric microcirculation during acid secretion. Other studies have accredited the role of NO as an endogenous modulator of leukocyte adhesion. In support, Calatayud et al. have shown that transdermal nitroglycerine protected against indomethacin-induced gastric ulceration through maintenance of mucosal blood flow and reduction of leukocyte–endothelial cell rolling and adherence. Moreover, Wallace has stated that reduction of gastric blood flow is the main predisposing factor in the induction of non-steroidal anti-inflammatory drugs (NSAID) gastropathy. Other than the role of NO in maintenance of blood flow, NO may protect against NSAID damage by promotion of prostaglandin synthesis. A mutual interaction has been observed between NOS and cyclooxygenase (COX) enzymes. NO donors were shown to enhance COX activity whereas NOS inhibitors blocked prostaglandin E2 (PGE2) production.
In a study from our lab, we demonstrated the role of NO in protecting against indomethacin-induced gastric ulceration. Intraperitoneal (i.p.) injection of l-arginine (300 mg/kg) 30 min before i.p. injection of 30 mg/kg indomethacin to rats almost completely protected the rats against indomethacin-induced gastric ulceration by a mechanism independent of modulation of acid secretion, mucin content or pepsin activity, but via maintenance of mucosal NO. On the other hand, pre-treatment of rats with the NOS inhibitors l-NAME (50 mg/kg), a non-selective constitutive nitric oxide synthase/inducible nitric oxide synthase (cNOS/iNOS) inhibitor, or the selective iNOS inhibitor aminoguanidine (AMG) (50 mg/kg) worsens the ulcer index (the sum of the length (mm) of all lesions in the fundic region) Fig. 3). In support to the anti-ulcerogenic effect of l-arginine, reports by Lazaratos et al. and Jimenez et al. have indicated the protective role of l-arginine against the ulcerogenic action of endothelin-1 and ibuprofen, respectively.
Fig. 3. Ulcer index (mm) of normal, indomethacin, l-NAME, aminoguanidine, and l-arginine treated rats. Results are mean ± SEM of 6–10 animals. **Significantly different from indomethacin at p < 0:01.
Reports have not restricted the role of NO to gastric protection, but also discussed the acceleration of ulcer healing. Konturek et al. have shown that glyceryl trinitrate is capable of ulcer healing and that suppression of NO synthesis resulted in impaired ulcer healing. It is possible that NO directly accelerates ulcer repair by promoting the growth of smooth muscles, as suggested by Hogaboam et al.
In a recent study (in press), we have tested the effect of NO modulation on peptic ulcer healing using the NO precursor; l-arginine, a competitive inhibitor of NOS, l-NAME and the NO donor; nitroglycerine (NTG). Rats were injected with a single oral dose of indomethacin (30 mg/kg) and then treated with l-arginine, NTG or l-NAME, once daily for 7 days starting 4 h after the indomethacin injection. Gross lesion examination and histological assessment were done. Gastric tissue content of NO, PGE2 and mucin were detected. In addition, oxidative stress markers including glutathione (GSH) and lipid peroxides were measured. l-Arginine and NTG were found to accelerate the healing of indomethacin-induced ulcers, as evident in macroscopic and histological examination, to restore normal levels of NO and GSH and to significantly attenuate the increase in PGE2 and lipid peroxides induced by indomethacin. On the other hand, l-NAME was found to exacerbate the mucosal damage.
Table 3. Gross examination of the effect of treatment with l-arginine, NTG or l-NAME on gastric ulcer induced by indomethacin in rats.
|Groups||No. of dead rats||Ulcer No||Ulcer index (mm)||Ulcer score|
|Indomethacin||3||13.25 ± 0.75||19.0 ± 1.45||3.62 ± 0.26|
|Indomethacin + l-arginine||1||0||–||–|
|Indomethacin + NTG||2||0||–||–|
|Indomethacin + l-NAME||5||17.11 ± 0.65||23.2 ± 1.15||4.55 ± 0.17|
Gastric ulcer was induced by a single oral injection of indomethacin (30 mg/kg), and then 4 h later, treatment schedule was given daily for 1 week as follows: l-arginine (200 mg/kg), NTG (1 mg/kg) and l-NAME (15 mg/kg). Measurements were done 7 days later. Values given are means of 10–15 observations ± SEM. Ulcer index = sum of lengths of all lesions in each stomach; ulcer score indicates severity of gastric lesion, where 1 (ulcerated area 1–6 mm2), 2 (ulcerated area 7–12 mm2), 3 (ulcerated area 13–18 mm2), 4 (ulcerated area 19–24 mm2) and 5 (ulcerated area > 24 mm2) In parallel, Brzozowski et al. have shown that intragastric administration of l-arginine (32.5–300 mg/kg/day) enhanced the healing rate of acetic acid-induced ulcers in a dose-dependent manner, while d-arginine was not effective.
Very few articles have investigated the effects of l-arginine supplementation on CNS function. However, accumulating evidence is beginning to indicate that NO plays a part in the formation of memory. In vitro, after specific receptor stimulation, NO is released from a postsynaptic source to act pre-synaptically on one or more neurons. This leads to a further increase in the release of glutamate and, as a result, to a stable increase in synaptic transmission, a phenomenon known as long-term potentiation. This is thought to be linked to memory function.
Experiments in animals also suggest that NO is involved in memory, because inhibiting NO synthesis in vivo impairs learning behaviour.
Fig. 5. The role of nitric oxide in the long-term potentiation of neuronal activity. Glutamate released from the presynaptic nerve terminal activates different types of receptors on the dendrites of the postsynaptic neuron. Under normal conditions the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors mediate most of the effects of glutamate. During high-frequency synaptic transmission, however, the activation of N-methyl-d-aspartate (NMDA) receptors results in an increase in intracellular calcium, which stimulates the constitutive nitric oxide synthase (NOS). The nitric oxide (NO) that is produced diffuses back to the presynaptic neuron, where it enhances the release of glutamate. The increased glutamate release leads to greater activation of postsynaptic glutamate receptors, thereby increasing the effectiveness of that synapse. Plus signs indicate stimulation, and l-arg denotes l-arginine.
l-arginine (1.6 g/day) in 16 elderly patients with senile dementia has been found to be effective in reducing lipid peroxidation and increasing cognitive function. In their recent report, Jing et al. explored the possible role of l-arginine in Alzheimer’s disease (AD), taking into consideration known functions for l-arginine in atherosclerosis, redox stress and the inflammatory process, regulation of synaptic plasticity and neurogenesis, and modulation of glucose metabolism and insulin activity. They provided evidence that l-arginine may play a prominent role in protection from age-related degenerative diseases such as AD. Further investigation is still needed to cover this virgin area of research.
In an open-label randomised limited study conducted by the author,
5 g/day l-arginine base was administered orally once at night for 28 days in 21 subjects with age ranging between 41 and 75 years old (14 between 41 and 49 years, 4 between 50 and 59 years, 2 between 60 and 69 years, and 1 between 70 and 79 years), 16 were males and 5 females, 17 were non-smokers and 4 smokers, and 18 of the 21 subjects were taking other medications to control either hypertension, myocardial ischemia, diabetes, gastro-oesophageal reflux disease (GERD) and hyperacidity, hypothyroidism, neuritis, or rheumatoid. All recruited subjects gave written informed consent that complied with the principles of the Helsinki declaration.
A questionnaire was given to the subjects to be completed weekly for 4 weeks. The subjects were advised to write their health status before and after taking l-arginine. The questionnaire included 30 points regarding their mental, muscular, sexual, circulatory, GIT, and other functions during the 4-week administration. Scoring was recorded from 1 to 5; 1 was a remarkable improvement, 2 was a mild improvement, 3 no difference, 4 was worse than before, and 5 was not applicable. The subjects were also advised to report any adverse reactions developed during the administration of the supplement. In addition, they were asked if they wanted to continue taking the supplement after termination of the study. Tables 4 and 5 summarise the most noteworthy information of this pilot study
|Feature||% of cases (total = 21 cases)|
|Mental capability||Remarkable improvement||Mild improvement||No change|
|Ability to concentrate||55||35||10|
|Delay in mental exhaustion||75||15||10|
|Reduction in severity of anxiety and stress||60||20||20|
|Reduction in nervousness||72||21||60|
|Deepness of sleep||80||10||10|
|Muscular activity||Remarkable improvement||Mild improvement||No change|
|Delay in muscular exhaustion||60||15||25|
|Sexual performance in males||54||33||13|
|Overall feeling of well being||65||20||15|
Table 5. Additional observations at the end of the 4-week study reported by some subjects.
At the end of the study, none of the 21 cases experienced any side effects or aggravation of health problems from l-arginine administration. All the 21 cases wanted to continue taking the supplement after termination of the study.
l-arginine is traditionally classified as a semi-essential or conditionally essential amino acid; it is essential in children and non-essential in adults. Homeostasis of plasma l-arginine concentrations is regulated by dietary arginine intake, protein turnover, arginine synthesis, and metabolism. This may explain why, under certain conditions, l-arginine may become an essential dietary component. The main tissue in which endogenous l-arginine synthesis occurs is the kidney, where l-arginine is formed from citrulline, which is released mainly by the small intestine. The liver is also capable of synthesising considerable amounts of l-arginine; however, this is completely reutilised in the urea cycle so that the liver contributes little or not at all to plasma arginine flux.
l-arginine normally constitutes approximately 5–7% of the amino acid content of a typical healthy adult diet. This accounts to an average intake of 2.5–5 g/day, which only meets the body’s minimal requirements for tissue repair, protein synthesis and immune cell maintenance.
L-arginine delivered via the gastrointestinal tract (GIT) is absorbed in the jejunum and ileum of the small intestine. A specific amino acid transport system (the y+ transporter) facilitates this process; this transport system is also responsible for assisting the transport of other basic amino acids l-lysine and l-histidine.
About 60% of the absorbed l-arginine is metabolised by the GIT, and only 40% reaches the systemic circulation intact. Most dietary proteins have a relatively balanced mixture of amino acids, and thus the only way to selectively deliver more l-arginine to an individual would be to supplement with the individual amino acid itself.
There is little evidence to support an absolute dietary deficiency as a cause of vascular dysfunction in humans. However, evidence that supports the importance of an exogenous supply of l-arginine for a healthy vascular system has been provided by Kamada et al. In this study, vascular endothelial function was examined in a lysinuric protein intolerant (LPI) patient that had a genetic defect of dibasic amino acid transport caused by mutations in the SLC7A7 gene. The transporter is normally expressed in intestinal and renal epithelial cells, and deficient expression leads to impaired dietary uptake of exogenous l-arginine and impaired renal tubular reabsorption of filtered l-arginine. As a result, plasma l-arginine concentration in the patient was considerably lower than normal (reduced by 79%).
Assessment of NO-dependent endothelial function in this patient revealed serum levels of nitrogen oxides (NOx) and flow-mediated brachial artery vasodilator response approximately 70% lower than in controls. The patient also suffered from reduced circulating platelet count, increased plasma levels of the thrombin–antithrombin III complex, and elevated plasma fibrin (ogen) degradation products.
Intravenous infusion of l-arginine reversed all these effects. The conclusion that can be derived from these results is that the extracellular supply of l arginine is essential for proper endothelial nitric oxide synthase (eNOS) activity, despite the fact that intracellular l-arginine may far exceed the Km for eNOS, a phenomenon termed in literature ‘arginine paradox’.
Intravenous infusion of l-arginine reversed all these effects. The conclusion that can be derived from these results is that the extracellular supply of l-arginine is essential for proper endothelial nitric oxide synthase (eNOS) activity, despite the fact that intracellular l-arginine may far exceed the Km for eNOS, a phenomenon termed in literature ‘arginine paradox’.
Most investigators believe that this phenomenon is due to the colocalisation of cation arginine transporter (CAT-1) with membrane-bound eNOS in plasmalemmal caveoli. The importance of the external supply of l-arginine suggests the definition of l-arginine as a ‘semi-essential’ amino acid in adults.
Wound healing involves platelets, inflammatory cells, fibroblasts and epithelial cells. All of these cell types are capable of producing NO either constitutively or in response to inflammatory cytokines. NO produced by both iNOS and eNOS plays many important roles in wound healing, from the inflammatory phase through to scar re-modeling. NO has cytostatic, chemotactic and vasodilatory effects during early wound repair, regulates proliferation and differentiation of several cell types, modulates collagen deposition and angiogenesis, and affects wound contraction.
Fig. 4. Schematic of the hypothesised roles of in wound healing. Production of NO from eNOS or iNOS leads to modulation of cytokines (e.g., MCP-1, RANTES, VEGF, and TGFb1), which in turn modulates the various facets of wound healing (e.g., chemoattraction, proliferation, collagen deposition, and angiogenesis).
L-arginine was first noted to enhance wound healing in 1978. Since then dietary l-arginine has been shown to improve collagen deposition and wound strength in both humans and animals. This effect may be due in part to the subsequent increase in production of ornithine by the action of arginase enzyme, a precursor of l-proline during collagen synthesis. The direct role of NO as a cofactor in the promotion of wound healing by l-arginine has also been reported.
L-arginine might improve wound immune cell function by decreasing the inflammatory response at the wound site.The healing effect of L-arginine is also extended to cover burn injuries. Oral dietary l-arginine supplementation of 100–400 mg/kg/day shortened re-epithelisation times, increased amounts of hydroxyproline, and accelerated the synthesis of reparative collagen in burned rats. Burn injuries significantly increase arginine oxidation and fluctuations in arginine reserves. Total parenteral nutrition (TPN) increases conversion of arginine to ornithine and proportionally increases irreversible arginine oxidation. These make arginine conditionally essential in severely burned patients receiving TPN.
Diabetes is associated with reduced plasma levels of arginine and elevated levels of the NOS inhibitor ADMA. Evidence suggests that arginine supplementation may be an effective way to improve endothelial function in individuals with diabetes mellitus (DM). As well, low dose IV arginine has been shown to improve insulin sensitivity in obese, type 2 DM, and healthy subjects. Arginine may also counteract lipid peroxidation and thereby reduce microangiopathic long-term complications of DM.A double-blind trial found oral arginine supplementation (3 g three time/day, 1 month) significantly improved, but did not completely normalise, peripheral and hepatic insulin sensitivity in patients with type 2 DM. Moreover, l-arginine regulates insulin release by NO-dependent and NO-independent pathways
L-Arginine has been purported to have ergogenic potential. Athletes have taken arginine for three main reasons:
In a double-blind study, the effect of a 4-week treatment with arginine aspartate on 21 athletes was assessed. The treated group showed enhanced maximal oxygen consumption as well as a significantly decreased plasma lactate concentration at work intensity of 200, 300 and 400 W (running workout) on the treadmill as compared to the control group. In another study, 8 weeks of oral l-arginine administration (3 g) to 20 male subjects on an exercise program with weights caused a significant increase in muscle strength and mass as compared to the non-treated group.
Duchenne muscular dystrophy (DMD) is a lethal, X-linked disorder associated with dystrophin deficiency that results in chronic inflammation, sarcolemma damage, and severe skeletal muscle degeneration. Recently, the use of l-arginine, the substrate of neuronal nitric oxide synthase (nNOS), has been proposed as a pharmacological treatment to attenuate the dystrophic pattern of DMD. Hnia et al. were able to demonstrate that l-arginine decreases inflammation and enhances muscle regeneration in mdx mice (an animal model of Duchenne myopathy). The inhibitory effect of l-arginine on the NF-kappaB/Metalloproteinase cascade reduces beta-dystroglycan cleavage and translocates utrophin and nNOS throughout the sarcolemma. Evidence suggests that l-arginine up-regulates utrophin in muscles, which could compensate for the lack of dystrophin in DMD. Utrophin has over 80% homology with dystrophin.