Introduction

The initial description in 1980 by Furchgott and Zawadzki [33] of endothelium-derived relaxing factor (EDRF) has stimulated more than two decades of intense research into the elucidation of its basic biology and importance in the clinical setting, such as in human coronary atherosclerosis. The identification of the endothelium-derived vasodilator as nitric oxide (NO) with smooth-muscle relaxing effects led to the recognition that nitrovasodilators act by providing an exogenous source of NO to the diseased blood vessel. NO is formed in the endothelium from the amino acid L-arginine by endothelial isoform of NO synthase (eNOS), which is the product of the NOS 3 gene. In addition to producing NO constitutively, the enzyme may be stimulated to increase NO synthesis by a variety of physiological agonists, shear stress, and pharmacological agents including acetylcholine. NO mediates a number of protective functions of the endothelium: It inhibits platelet aggregation and adhesion, it inhibits smooth muscle proliferation, and it limits vascular recruitment of leukocytes by inhibiting the expression of proinflammatory cytokines, chemokines, and leukocyte adhesion molecules. In the presence of known atherogenic risk factors, the normal endothelium can become dysfunctional. In the setting of many vascular disorders, including essential hypertension, several forms of dyslipidemia, diabetes mellitus, cigarette smoking, aging, and hyperhomocysteinemia, endothelium-dependent dilation occurs. The endothelium is a direct, sensitive target for the damaging effects of atherogenic risk factors, as evidenced from the experimental introduction of risk factors into healthy subjects; for example, eating a high-fat meal leads to endothelial vasodilator dysfunction in a time span of just a few hours [32].

Central to the development of endothelial dysfunction, regardless of its cause, is a loss of bioactive endothelial NO. There are two fundamental mechanisms for the loss of NO bioactivity: reduced synthesis and increased oxidative inactivation by reactive oxygen intermediates. Reactive oxygen species (ROS) are produced in abundance in the dysfunctional endothelium, and limitation of ROS generation increases the availability of NO. For this reason, antioxidative therapy with vitamin C [36] and cholesterol-lowering therapy with HMG-CoA reductase inhibitors improves endothelial function [75]. An alternative approach to increase levels of bioactive NO and to improve endothelial function is to increase the synthesis of NO. Enhanced synthesis of NO can be achieved by increased availability of agonists that stimulate release of NO from the endothelial cells, such as bradykinin.

Another straightforward approach to increase NO synthesis is to provide additional substrate to the endothelial cell. The semi-essential amino acid L-arginine serves as the substrate for the enzyme eNOS. First isolated from lupin seedlings in 1886 by Schulze and Steiger [64], L-arginine was shown to be a product of protein hydrolysis by Hedin 9 years later [40], but its structure was not proven until Sorensen in 1910 [65]. While much is known about the intermediary metabolism of L-arginine in several metabolic pathways, such as the urea cycle, the importance of this amino acid was heightened by the recognition in the 1980s that L-arginine is a precursor of NO synthesis: NO synthase catalyzes the 5-electron oxidation of L-arginine to L-citrulline and produces stoichiometric amounts of NO in the process. Providing supplementation substrate to individuals with inadequate NO, therefore, has been suggested as a rational approach to increase NO production by the NO synthase, and this therapeutic paradigm has been met with some success in recent years.

The first evidence for the effect of L-arginine came from animal studies showing that acute or chronic administration of L-arginine in vivo improves vascular responsiveness, probably via enhanced NO elaboration [27]. Long-term oral administration of L-arginine has been associated with a significant improvement in NO-dependent vasodilation in cholesterol-fed rabbits and in slowing the development of intimal plaques in the carotid arteries [14]. In 1991, Drexler and colleagues [31] demonstrated that local intracoronary infusion of L-arginine normalized coronary vasomotor responses to acetylcholine in hypercholesterolemic humans. A similar observation was also made upon intravenous infusion of L-arginine in hypercholesterolemic subjects in whom endothelium-dependent forearm vasodilation was improved [28]. Since that time, numerous studies have confirmed that both the acute administration of L-arginine and its chronic administration improve vascular function in hypercholesterolemia [26], in exercising patients with stable angina pectoris [22], in patients with heart failure [62], at sites of coronary artery stenosis [70], and in small-vessel disease [48]. In hypertensive patients, however, the majority of studies on L-arginine revealed a lack of effect of this amino acid on endothelial function [59]. Long-term effects of L-arginine in patients with hypertension require further investigation.

So, the use of L-arginine as a tool to examine the NO pathway grew into a potential role for L-arginine as a therapeutic tool in cardiovascular disease. In a series of clinical studies, we investigated the clinical pharmacology of L-arginine. First, we showed that an intravenous infusion of 30 g of L-arginine significantly increased arterial blood flow in the femoral artery of healthy subjects [8]. Plasma L-arginine concentration increased to 6.0±0.4 μmol/l. A lower dose of L-arginine (6 g) administered by either the intravenous or the oral route increased plasma levels of L-arginine (822±59 μmol/l and 310±152 μmol/l, respectively) but failed to produce acute vasodilation. In a study in patients suffering from severe peripheral arterial disease, we demonstrated that an acute intravenous infusion of 30 g of L-arginine increased femoral arterial blood flow in the more severely affected leg. The vasodilator effect of L-arginine was due to increased blood flow velocity because the femoral artery diameter remained unchanged [9]. It then became interesting to know whether the action of L-arginine increased muscle capillary blood flow. This was addressed with a further clinical study in which we performed serial measurements of muscle capillary blood flow by using positron emission tomography. A single systemic infusion with 30 g of L-arginine significantly increased nutritive muscle blood flow, whereas 8 g of L-arginine had no significant effect [63].

Clinical pharmacokinetics and pharmacodynamics

We determined the pharmacokinetics of single intravenous doses of L-arginine (30 and 6 g) with those of oral L-arginine (6 g). After an intravenous infusion, peak plasma L-arginine levels are achieved within 20-30 min, and after oral administration within 60 min. Orally administered L-arginine is rapidly and almost completely absorbed via active uptake by the intestinal y+-transporter system for cationic amino acids. Data for oral bioavailability vary between 21±4% and 68±9%. The half-life of L-arginine was 1.5-2 h after an oral dose of 6 g [10]. These data and the increased nutritive tissue blood flow as well as our observation that blood flow remained elevated for 1–2 h after the end of L-arginine infusion convinced us to give patients with peripheral arterial disease a therapy with intravenous L-arginine (three doses for 8 g/day) for 3 weeks. Claudication distance was significantly increased and absolute and pain-free walking distances were improved, whereas the control group showed no significant changes [16]. Other investigators have also shown that oral L-arginine supplementation improves clinical symptoms of vascular disease: Ceremuzynski et al. [22] showed beneficial effects of oral administration of 6 g L-arginine for 3 days in patients with stable angina. Lerman et al. [48] showed that 6 months of oral L-arginine (3 g TID) resulted in a significantly improved angina symptom score and improved coronary blood flow response to acetylcholine in 26 patients with small-vessel coronary artery disease. In contrast, Blum et al. [6] administered oral L-arginine chronically to individuals with established coronary artery disease. In this randomized, double-blind crossover study, the investigators failed to find an effect of 9 g of daily L-arginine for 1 month on flow-mediated brachial artery dilation or cell adhesion molecule expression. However, patients were on an optimized medical treatment including cholesterol-lowering, and flow-mediated vasodilation was normal at baseline.

Side effects

L-arginine has generally been well tolerated by healthy volunteers and patients when administered via the intravenous or oral route in doses <30 g. When high doses of L-arginine are given intravenously, local irritation and phlebitis may occur because of the high osmolality of the solution [44]. The vasodilator action of L-arginine may lead to hypotension [55], but usually the blood pressure-lowering effect of L-arginine is relatively low. Because the L-arginine hydrochloride solution is acidic, a sudden drop in blood pH may cause metabolic acidosis, which has been associated with arrhythmias [57]. Flushing and other dermal side effects have been reported [54]. After oral L-arginine, which has a bitter taste in higher doses, nausea and vomiting were reported in about 3% of the patients [18].

Mechanisms of action of L-arginine effects on NO production in humans

Both acute and chronic administration of L-arginine evoke benefits in improving endothelial function, but what may be the underlying mechanism? The precise molecular mechanisms by which L-arginine improves endothelial function remain puzzling. Providing the enzyme NOS with substrate because of lowered availability of L-arginine does not appear to be rate limiting: The intracellular levels of the amino acid are in the millimolar range [37], whereas the enzyme's K M for substrate is in the micromolar range (2.9 μmol/l [19]. This biochemical discrepancy was termed the "arginine paradox." For this reason, other possible explanations have been proposed to explain the effects of L-arginine.

Arginase (the enzyme that converts arginine to ornithine and urea) activity may alter the cellular levels of L-arginine, leading to decreased NO production. Wei et al. [73] have shown in animal models that the upregulation of arginase may contribute to smooth muscle proliferation, resulting in endothelial dysfunction. L-arginine is first converted to N G-hydroxy-L-arginine by eNOS, which inhibits arginase and may thereby increase intracellular steady-state levels of L-arginine [20]. Another interesting possible mechanism is that oxidized LDL and lysophosphatidycholine decrease L-arginine transport into endothelial cells [43, 45]. This may be the reason for the beneficial effects of L-arginine in patients with hypercholesterolemia. L-arginine competes with other cationic amino acids for transport into cells, especially L-glutamine, and increased L-arginine may increase intracellular substrate concentration by competitively enhancing cellular uptake in this setting.

Another possibility could be the compartmentalization of L-arginine in the cytoplasm so that local concentrations in the vicinity of endothelial NOS may be lower than expected from L-arginine levels in whole cell homogenates [51]. Extracellular L-arginine may be preferentially utilized by NOS within this microenvironment.

Another explanation for the L-arginine paradox may be the presence of endogenously occurring analogs of L-arginine, asymmetric dimethylarginine (ADMA) and N G-monomethyl-L-arginine (L-NMMA), which exert biological activity by competitively inhibiting NO synthase activity. Today we know that an elevation in plasma ADMA occurs in hypercholesterolemia and that the elevation in ADMA correlates with endothelial dysfunction [15]. Elevations in plasma ADMA may play a role in hyperhomocyst(e)-inemia, hypertension, diabetes mellitus, insulin resistance, and chronic heart failure, resulting in diminished NOS activity. Inhibition of NOS activity may be overcome by excess substrate and could explain how L-arginine improves endothelial function in patients with vascular disease [17]. Several studies have provided convincing data to support the hypothesis that ADMA could be a potential novel cardiovascular risk factor [53, 71, 76]. This implicates further therapeutic options for L-arginine supplementation.

Very recently, Suschek et al. [66] discussed the critical role of L-arginine in endothelial cell survival during oxidative stress and showed that arginine concentrations corresponding to physiological serum levels do not allow for optimal endothelial iNOS activity and would therefore impair the endothelial iNOS-mediated stress response, increasing the risk of endothelial dysfunction. Even small concentration changes within the physiological range will lead to increased iNOS activity and thus improve the antioxidative effects of L-arginine.

Other experiments show that L-arginine has direct antioxidant effects and that this is due to the alpha-amino group, a chemical moiety different from that necessary for NO biosynthesis [46, 72]. By simply acting as an antioxidant, L-arginine may scavenge superoxide or restore the crucial cofactor tetrahydrobiopterin (BH4) and thereby prevent eNOS-mediated superoxide production in an “uncoupled” status. We could show in patients with peripheral arterial occlusive disease (PAOD) and hyperhomocyst(e)inemia that L-arginine significantly improved flow-dependent vasodilation and reduced oxidative stress as reflected by decreased urinary levels of the established biomarker of oxidative stress 8-iso-prostaglandin \({\text{F}}_{{2\alpha }} \) [67].

Proposed indirect mechanisms by which L-arginine increases bioactive NO in the vasculature are equally diverse. L-arginine increases insulin and growth hormone (GH) secretion. We were able to show that 30 g of L-arginine induced a rapid release of insulin and a delayed release of GH [11]. During coinfusion of somatostatin, release of both hormones was blocked; however, somatostatin inhibited only the late response but not the early increase in NO production. We conclude that GH contributes to the prolonged NO-dependent vasodilation with high doses of L-arginine via IGF-1. This was not found during oral L-arginine supplementation in human subjects.

In addition, L-arginine stimulates histamine release from mast cells [35], which also evokes a vasodilator response. Lastly, L-arginine can undergo decarboxylation to L-agmatine, which induces clonidine-like effects and which has also been shown to inhibit NO synthesis at millimolar concentrations [34].

Evidence for therapeutic use of L-arginine?

NO plays an integral role in endothelial function, and the biochemical pathways associated with its production are well understood. Most of the evidence to enhance the production of NO in endothelial tissue by dietary supplementation came from studies with oral L-arginine. Oral L-arginine feeding in animal studies has especially shown beneficial effects. In animals with hypercholesterolemia, L-arginine appears to inhibit the progression of atherosclerotic plaques and preserve endothelial function. In addition, L-arginine affects other mediators of atherosclerosis, including inflammatory cells and platelets. Although animal model data are positive, results from human studies have been inconclusive. Table 1 summarizes 20 studies of intravenous L-arginine use in humans. Four of the 20 human studies showed no vascular health benefit from intravenous application. The selection of the studies was not complete because studies with intravenous L-arginine represent a wide spectrum. Without exception, studies were positive in hypercholesterolemia and coronary artery disease. Hypercholesterolemia is known to increase ADMA, a condition that may be overcome with supplemental L-arginine by increasing the L-arginine/ADMA molar ratio. Dosing also represents a heterogenous spectrum.

Table 1 Effects of intravenous L-arginine in humans (not complete; HC hypercholesterolemia, Con control, IV intravenous, FMD flow-mediated dilation, CAD coronary artery disease, TPR total peripheral resistance, CO cardiac output, MBP mean blood pressure, PET positron emission tomography, RF renal failure, RPF renal plasma flow, PPH primary pulmonary hypertension, PAOD peripheral arterial disease, PVR peripheral vascular resistance, PAP pulmonary arterial pressure, ESRF end-stage renal failure, HD hemodialysis)
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Table 2 summarizes published studies on oral L-arginine. These are of higher impact with respect to L-arginine as a therapeutic agent because the oral application enables us to get the drug into the patient in a more comfortable way. In hypercholesterolemia we had five positive studies and one negative with the HeartBar, an arginine-enriched compound (Unither Pharma, Silver Spring, MD, USA). For coronary artery disease we had one negative: Blum et al. [6] conducted a randomized, double-blind crossover study with 9 g daily for 1 month and assessed flow-mediated brachial artery dilation and cell adhesion molecule expression. In this study it was unclear whether NO production was increased. The study subjects were on full cardiovascular medication with statins and ACE inhibitors, both of which have been shown to improve endothelial function. In addition, β-blockers and aspirin as an antioxidant were used. This may have confounded possible effects of L-arginine itself. In chronic heart failure, Chin-Dusting et al. [25] described a negative result with 20 g/day L-arginine for 28 days. In contrast to intravenous application in hypertension, oral L-arginine was given by Lekakis et al. [47] with a positive result, but only 6 g were given acutely. Studies in patients with diabetes, hyperhomocyst(e)inemia, and peripheral arterial disease and in healthy elderly people and smokers were without exception positive. Oomen et al. [56] conducted an observational study based on dietary history calculated as L-arginine intake of 4 g/day, which is within the normal average intake of 5 g/day of L-arginine. In healthy controls, Adams et al. [2] showed a positive effect on platelet aggregation and brachial artery dilation. Blum et al. [5] observed a lack of effect on major endocrine hormones and lipid profile; this finding supports the safety of oral L-arginine administration, but the study failed to show a beneficial effect on brachial artery dilation, cell adhesion molecules, or NO synthesis in 10 healthy postmenopausal women [7]. The dosing of oral L-arginine represents a wide spectrum, from two HeartBars per day for 1 week [2] to 24 g/day of L-arginine for 8 weeks [11]. The longest application was given by Chan et al. [23], who gave 14–21 g/day of L-arginine for 3 months.

Table 2 Effects of oral L-arginine in humans (HC hypercholesterolemia, Con control, FMD flow-mediated dilation, CAD coronary artery disease, TPR total peripheral resistance, CO cardiac output, MBP mean blood pressure, PET positron emission tomography, RF renal failure, RPF renal plasma flow, PPH primary pulmonary hypertension, PAOD peripheral arterial disease, PVR peripheral vascular resistance, PAP pulmonary arterial pressure, ESRF end-stage renal failure, HD hemodialysis, MI myocardial infarction, CHF chronic heart failure, BiD twice a day, QD once a day, TiD three times daily, PAOD peripheral arterial occlusive disease, HRQL health-related quality life)
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So where do we stand with clinical testing regarding L-arginine as a therapeutic agent?

To look at the four processes of drug therapy (Fig. 1), we have the first problem: Is the drug L-arginine getting into the patient? L-arginine in oral form could be given in capsules, which, for example, means 42 huge capsules of 500 mg three times a day for a total amount of 21 g. Another possibility is L-arginine intake as a powder in water or in the form of HeartBar.

Fig. 1
figure 1

The fate of L-arginine in humans

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As to the pharmacokinetic process that involves absorption, distribution, and elimination, we have data from about 18 healthy subjects. This could be considered as phase 1, in which usually 20–80 healthy volunteers participate. Data on the bioavailability vary between 20% and 60%. So the second problem is, how much of the L-arginine dose, which varies between 5 g (this is within the range of average arginine intake with food) and 24 g, reaches the systemic circulation and is available for distribution to the site of action?

To address the question of whether the drug L-arginine produces the required pharmacological effect and to shed light on the underlying mechanisms, we have to admit that we have no clear explanation for the benefit in prior studies and that we need future studies to address underlying mechanisms. Today, we have reached phase 2 of human testing: About 548 subjects and patients received oral L-arginine, and we have results about the effectiveness and safety aspects. The problem is the heterogeneity of the published studies concerning dose and outcome parameters. What should we consider to be a pharmacodynamic effect, and what do we mean by the therapeutic effect? Is an increase in NO synthesis measured by a variety of methods (for example, GC-MS as the gold standard, Griess assay, cGMP as the second messenger, and so on) the appropriate surrogate parameter? Is the improvement of flow-mediated dilation a real clinical benefit? How long should the effect of L-arginine last? We are standing only at the beginning of clinical trial design, and we need larger, randomized clinical trials with an appropriate L-arginine dose of sufficiently long duration to fully elucidate the effects of dietary L-arginine supplementation on vascular health.