L-arginine-induced vasodilation in healthy humans: pharmacokinetic–pharmacodynamic relationship

Abstract

Aims

Administration of l-arginine by intravenous infusion or via oral absorption has been shown to induce peripheral vasodilation in humans, and to improve endothelium-dependent vasodilation. We investigated the pharmacokinetics and pharmacokinetic-pharmacodynamic relationship of l-arginine after a single intravenous infusion of 30 g or 6 g, or after a single oral application of 6 g, as compared with the respective placebo, in eight healthy male human subjects.

Methods

l-arginine levels were determined by h.p.l.c. The vasodilator effects of l-arginine were assessed non-invasively by blood pressure monitoring and impedance cardiography. Urinary nitrate and cyclic GMP excretion rates were measured as non-invasive indicators of endogenous NO production.

Results

Plasma l-arginine levels increased to (mean±s.e.mean) 6223±407 (range, 5100–7680) and 822±59 (527–955) μmol l⁻¹ after intravenous infusion of 30 g and 6 g l-arginine, respectively, and to 310±152 (118–1219) μmol l⁻¹ after oral ingestion of 6 g l-arginine. Oral bioavailability of l-arginine was 68±9 (51–87)%. Clearance was 544±24 (440–620), 894±164 (470–1190), and 1018±230 (710–2130) ml min⁻¹, and elimination half-life was calculated as 41.6±2.3 (34–55), 59.6±9.1 (24–98), and 79.5±9.3 (50–121) min, respectively, for 30 g i.v., 6 g i.v., and 6 g p.o. of l-arginine. Blood pressure and total peripheral resistance were significantly decreased after intravenous infusion of 30 g l-arginine by 4.4±1.4% and 10.4±3.6%, respectively, but were not significantly changed after oral or intravenous administration of 6 g l-arginine. l-arginine (30 g) also significantly increased urinary nitrate and cyclic GMP excretion rates by 97±28 and 66±20%, respectively. After infusion of 6 g l-arginine, urinary nitrate excretion also significantly increased, (nitrate by 47±12% [P < 0.05], cyclic GMP by 67±47% [P = ns]), although to a lesser and more variable extent than after 30 g of l-arginine. The onset and the duration of the vasodilator effect of l-arginine and its effects on endogenous NO production closely corresponded to the plasma concentration half-life of l-arginine, as indicated by an equilibration half-life of 6±2 (3.7–8.4) min between plasma concentration and effect in pharmacokinetic-pharmacodynamic analysis, and the lack of hysteresis in the plasma concentration-versus-effect plot.

Conclusions

The vascular effects of l-arginine are closely correlated with its plasma concentrations. These data may provide a basis for the utilization of l-arginine in cardiovascular diseases.

Introduction

l-arginine is the physiological precursor of nitric oxide (NO), an important mediator of vasodilation and inhibition of platelet aggregation via increased formation of cyclic GMP [1]. NO is synthesized in endothelial cells from the terminal guanidino nitrogen of l-arginine by the activity of the endothelial, calcium-dependent isoform of NO synthase [2]. NO is rapidly oxidized to NO−2 and NO−3 in vivo [3], and NO−3 is subsequently eliminated via the kidneys [4], as is cyclic GMP [5]. Quantification of the urinary excretion rates of NO−3 and cyclic GMP has therefore been shown to be a suitable non-invasive method to assess NO formation rates in human subjects in vivo [6–8].

It has previously been shown that an intravenous infusion of 14–30 g of l-arginine induces vasodilation and inhibition of platelet aggregation [6, 8], and improves acetylcholine-induced, endothelium-dependent vasodilation [9, 10]. In atherosclerotic patients, intravenous infusion of 30 g l-arginine induces peripheral vasodilation [7], resulting in improved nutritive muscle blood flow of the calves [11]. Chronic oral administration of 5–8 g of l-arginine 2–3 times per day during 2–6 weeks has also resulted in improved endothelium-dependent vasodilation [12], inhibition of platelet aggregation [13], and in reduced monocyte adhesiveness for the endothelium [14] in humans. l-arginine has also been used for other purposes like stimulation of growth hormone release from the pituitary gland [15]. Physiological uptake of l-arginine with usual diets is about 4–6 g day⁻¹ [16]. The daily doses of l-arginine utilized in these studies significantly exceed physiological uptake of this semi-essential amino acid by 3–8-fold.

However, few data are known on the pharmacokinetic parameters and the pharmacokinetic-pharmacodynamic relationship of l-arginine in humans. One study in healthy human subjects investigated the pharmacokinetics of a multi-amino acid formulation used for the supplementation of polytraumatic patients [17]; however, the dose of l-arginine which was administered in this study was much lower than the doses used to induce NO-dependent vasodilation or growth hormone secretion. To characterize better the pharmacokinetic behaviour of l-arginine in healthy humans after acute intravenous and oral administration, we performed a double-blind, placebo-controlled dose-comparison study in healthy human subjects. Measurements were made during the time interval during which vasodilator effects can be expected after l-arginine infusion, in order to analyze the pharmacokinetic-pharmacodynamic relationship of l-arginine.

Methods

Subjects and study design

Eight healthy male human subjects (mean age, 25.2±0.2 years, height, 184.4±3.7 cm, weight, 77.9±2.6 kg) participated in this double-blind, placebo controlled study. All subjects underwent a physical examination and a routine laboratory screening (peripheral blood cell count, serum creatinine, sodium, potassium, chloride, ALT and AST activities) and gave their written informed consent before inclusion into the study. The protocol had previously been approved by the Institutional Review Board for Studies in Humans. The study consisted of 5 study days for each subject in randomized sequence with at least 7 days of washout between them. Before each study day, the participants were subjected to a standardized diet with a reduced nitrate content (∼32 mg nitrate day⁻¹) for 24h. On each day, they received either an intravenous infusion of l-arginine (30 g or 6 g l-arginine-hydrochloride (Fresenius, Bad Homburg, Germany) dissolved in 150 ml of physiological saline) or placebo (150 ml of physiological saline) during 30 min, or 6 g l-arginine or placebo (12 capsules of 0.5 g of l-arginine-hydrochloride or lactose).

At the beginning of each study day the subjects emptied their bladder. Mild oral volume loading with herb tea (using demineralized water) was started with 3 ml kg⁻¹ of body weight initially, and continued during the entire study period with 1–2 ml kg⁻¹ of body weight h⁻¹. Self-adhesive ring electrodes were fixed around the thorax and left there during the entire study period for continuous registration of the impedance cardiogram (see below). A blood pressure cuff was fixed to the right arm for blood pressure registration. Haemodynamic measurements were started after at least 60 min of adaptation, during which the subjects remained in the supine position. Baseline blood pressure was calculated as the mean of five measurements during the last 20 min before start of the l-arginine infusion.

Plasma samples were drawn into vacutainers containing sodium EDTA at baseline and 5, 10, 15, 20, 25, 30, 35, 40, 45, 60, 90, 150, and 210 min after the start of the infusion. On the days with capsule intake, time points for plasma generation were 0, 30, 60, 90, 150, and 210 min. Subjects came back to the laboratory on the next morning when additional plasma samples were drawn at 16, 20, and 24 h after drug intake. On the infusion days, urine samples were collected during 1 h immediately before the start of the infusion (baseline), at the end of the infusion (30 min), as well as 60, 120, and 180 min after the start of the infusion. On the days on which the drugs were applied orally, urine samples were collected during 1 h immediately before drug intake (baseline), as well as 60, 120, 180, and 240 min after drug intake.

Quantification of urinary NO−3 and cGMP

Urinary NO−3 was determined as its pentafluorobenzyl (PFB) derivative by gas chromatography-mass spectrometry (GC-MS) as described previously [18, 19]. Aliquots (1 ml) of urine were spiked with [¹⁵N]-NO−3 (MSD Isotopes Merck Frosst, Montreal, Canada) as internal standard. Reduction of nitrate to nitrite was performed prior to derivatization under alkaline conditions (5 weight% ammonium chloride buffer adjusted to pH 8.8 by sodium borate) by incubating sample or standard with 5 mg of cadmium (10 min, 20° C). These samples (100 μl) were treated with 400 μl of acetone and 5 μl of PFB bromide, and the mixture was allowed to react for 60 min at 50° C. Acetone was then removed under nitrogen, and reactants were extracted by vortexing with 1000 μl of toluene. Aliquots (1 μl) thereof were injected into the GC-MS instrument. GC-MS was carried out on a Hewlett Packard MS Engine 5989 series II (Waldbronn, Germany). A fused-silica capillary column DB-5 MS (30 m×0.25 mm i.d., 0.25 μm film thickness) from J&W Scientific (Rancho Cordova, CA) was used with helium as the carrier gas (70 kPa). Negative ions were produced by chemical ionization using methane as the reactant gas (200 Pa) at an electron energy of 230 eV and an electron current of 300 μA. Quantitation was performed by selected ion monitoring at m/z 46 for endogenous NO−2/NO−3 and m/z 47 for the internal standard. The detection limit of the method was 20 fmol nitrate. Intra- and interassay variabilities were below 3.8%.

For the determination of cGMP, urine samples were diluted 1:500 in phosphate buffered saline and acetylated by a mixture of acetic acid anhydride/triethylamine. Cyclic GMP content was measured by radioimmunoassay using [¹²⁵I]-cGMP as a tracer and globulin precipitation. The detection limit of the assay was 160 fmol ml⁻¹. Intra- and interassay variabilities were 3.5% and 8.1%, respectively.

Urinary and plasma creatinine was determined spectrophotometrically with the alkaline picric acid method in an automatic analyzer (Beckman, Galway, Ireland). The urinary excretion rates of NO−3 and cGMP were corrected by urinary creatinine concentration in order to limit the variability due to differences in renal excretory function as described previously [4].

Determination of plasma l-arginine concentrations

Plasma l-arginine concentrations were determined by high-performance liquid chromatography (h.p.l.c.) using pre-column derivatization with o-phthalaldehyde (OPA) as described previously [20]. Plasma samples and standards were extracted on CBA solid phase extraction cartridges (Varian, Harbor City, CA, USA). The eluates were dried under nitrogen and residues dissolved in double distilled water for h.p.l.c. analysis (Gynkotek, Munich, Germany). Samples and standards were incubated for exactly 30 s with the OPA reagent (5.4 mg ml⁻¹ OPA in borate buffer, pH 8.5, containing 0.4% 2-mercaptoethanol) before automatic injection into the h.p.l.c. system. Chromatographic separation was performed on a C₆H₅ column (Macherey and Nagel, Düren, Germany) with the fluorescence monitor set at λ^ex = 340 nm and λ^em = 455 nm. Samples were isocratically eluted from the column with 0.96% citric acid/methanol (2:1 v/v, pH 6.8) at a flow rate of 1 ml min⁻¹. The coefficients of variation of the method had previously been determined as 5.2% within-assay and 5.5% between-assay; the detection limit of the assay was 0.1 μmol l⁻¹.

Haemodynamic measurements

Systemic arterial blood pressure was measured by the standard sphygmomanometric method using an automatic device (Boso digital II, Bosch und Sohn, Jungingen, Germany). Non-invasive determination of cardiac output (CO) was performed by impedance cardiography [21]. The patients were attached to an impedance cardiograph as described by Belardinelli et al. [22]. Briefly, a constant sinusoidal alternating current (100 MHz, 4 mA) was applied between two thoracic electrodes placed on the level of the jugular fossa and lower chest. The associated voltage is detected by two inner electrodes positioned 5 cm apart from the outer electrode pair and parallel to the current path. This voltage is transmitted to an amplifier and a thoracic impedance signal is produced. Thoracic impedance was registered from beat to beat with an impedance cardiograph (BMT, Stuttgart, Germany) and transferred online to an IBM-compatible computer. Data analysis was performed offline using 5 min mean values. Stroke volume was automatically calculated for each heartbeat according to the equation described by Kubicek et al. [21]. There is good correlation of intraindividual changes in cardiac output obtained by impedance cardiography and echo-Doppler cardiography [23]. Total peripheral resistance was calculated as TPR = 80×(mean blood pressure)/CO [dyn s cm⁻⁵]. Baseline values were calculated as the mean of all measurements during the last 20 min prior to the start of l-arginine infusion or oral l-arginine intake.

Pharmacokinetic and pharmacokinetic/-dynamic (PK/PD) analysis

l-arginine plasma concentration-time curves were constructed by subtracting baseline fasting l-arginine levels as assessed at the corresponding time points during and after placebo administration from the concentrations obtained after l-arginine administration. Pharmacokinetic analysis was performed by use of a one or two compartment model, or a non-compartmental body model using nonlinear regression assuming linear kinetics. The model that showed the best fit to the data was chosen for further analysis. Terminal elimination half-life was calculated by extrapolating l-arginine plasma concentrations to infinity using the measured concentration at 24 h after drug intake. Areas under the concentration-time curves (AUC values) were calculated using the linear trapezoidal rule of the TOPFIT program [24] for data extrapolated to infinity. Oral bioavailability was calculated from comparison of the AUC values for intravenous and oral administration of 6 g of l-arginine.

PK/PD analysis was performed on individual plasma l-arginine/TPR data for the intravenous 30 g dose using a partially nonparametric approach [25, 26]. Changes in TPR were too small for both administration routes of the 6 g dose to allow PK/PD analysis. As the maximum plasma concentration and the maximum decrease in TPR occurred almost simultaneously, a model directly linking the effect to the plasma compartment was chosen. The accuracy of model selection was then verified by testing for the presence of hysteresis in the sequential concentration-versus-effect plot.

Calculations and statistics

Data are given as mean±s.e.mean Statistical significance was tested using analysis of variance followed by Fisher’s protected least significant difference test for comparisons between the treatment groups. Linear regression curves and correlation coefficients were obtained by the least squares method. Statistical significance was accepted at the 0.05 level of probability.

Results

Urinary nitrate and cyclic GMP excretion rates

Baseline hourly urinary nitrate and cyclic GMP excretion rates were 68.7±4.7 μmol mmol⁻¹ creatinine and 30.1±1.6 nmol mmol⁻¹ creatinine, respectively. There were no significant differences at baseline between the 5 study days. Infusion of 30 g l-arginine induced a maximum increase in urinary nitrate excretion by 96.5±28.4% and in cyclic GMP excretion by 65.8±20.0% (each P < 0.05 vs baseline; Figure 1). Infusion of 6 g l-arginine resulted in maximum increases 46.8±11.7% in nitrate (P < 0.05), but a highly variable and not statistically significant response in cyclic GMP excretion (66.9±46.6%, P = NS). These maximum increases in urinary nitrate and cyclic GMP excretion rates were noted in urines collected from 0–30 min after intravenous infusion of l-arginine. The respective maximum increases in nitrate and cyclic GMP after oral administration of 6 g l-arginine were also highly variable and not statistically significant (32.1±18.0% and 31.8±36.3% respectively each, P = NS vs baseline). After oral l-arginine, peak urinary nitrate excretion was observed in urines collected from 60–120 min, and peak urinary cyclic GMP excretion was observed in urines collected from 30–60 min. Urinary nitrate and cyclic GMP excretion rates before and after intravenous l-arginine infusion showed a close linear correlation (r = 0.59, P < 0.01). Neither intravenous nor oral placebo had any significant effects on nitrate or cyclic GMP excretion rates.

Figure 1.

Urinary nitrate excretion rates of nitrate (a) and cyclic GMP (b) in healthy human subjects before and after the infusion of 30 g (•) or 6 g (▪) l-arginine or placebo (□), or oral ingestion of 6 g l-arginine (▴) or placebo (▵). Values represent mean±s.e.mean of n = 8 subjects. *P < 0.05 vs baseline.

After intravenous infusion, urinary nitrate and cyclic GMP excretion rates peaked in the first urine sample after the end of the infusion (30 min), whereas after oral l-arginine administration, the peak in urinary nitrate excretion was delayed until 120 min after intake (Figure 1).

Haemodynamic effects of l-arginine

Infusion of 30 g l-arginine significantly reduced diastolic (maximum, −9.0±2.3 mm Hg; P < 0.05) and systolic blood pressure (maximum, −9.2±1.8 mm Hg; P < 0.05; Figure 2), but induced only minor changes in heart rate. 6 g l-arginine had no significant effect on diastolic or systolic blood pressure, neither after intravenous nor after oral administration. Neither intravenous nor oral placebo had any significant effects on blood pressure and heart rate.

Figure 2.

Effects of intravenous l-arginine (30 mg (•), 6 mg (▪)) and placebo (□) on systolic and diastolic blood pressure in healthy human subjects. Values represent mean±s.e.mean of n = 8 subjects. The shaded area represents the duration of the infusion. *P < 0.05 vs baseline.

Impedance cardiographic measurements showed a significant increase in cardiac output after 30 g l-arginine, but no significant changes after 6 g l-arginine or placebo (Figure 3a). Total peripheral resistance significantly decreased after intravenous infusion of 30 g l-arginine, but showed no significant changes after intravenous or oral administration of 6 g l-arginine, nor after placebo (Figure 3b).

Figure 3.

Effects of intravenous infusion of 30 g (•) or 6 g (▪) of l-arginine, or placebo (□) on cardiac output (a) and total peripheral resistance (b) in healthy human subjects. Values represent mean±s.e.mean of n = 8 subjects. *P < 0.05 vs baseline.

Pharmacokinetic parameters of intravenous and oral l-arginine

During the intravenous infusion of 30 g l-arginine, endogenous plasma concentrations increased from 71±4 μmol l⁻¹ to a maximum plasma concentration of 6223±407 μmol l⁻¹ at t_max = 30 min. Peak plasma l-arginine concentrations were 822±59 μmol l⁻¹ at t_max = 22 min after infusion of 6 g l-arginine, and 310±152 μmol l⁻¹ at t_max = 90 min after oral administration of 6 g l-arginine. Figure 4 shows the time course of mean l-arginine plasma concentrations after the three different dosages of l-arginine. Pharmacokinetic data analysis was performed after allowing for baseline l-arginine concentrations determined at the same time points during and after placebo administration.

Figure 4.

Time course of plasma l-arginine concentrations after intravenous or oral administration of l-arginine in healthy human subjects.

Pharmacokinetic data for l-arginine were best described by a non-compartmental model (r = 0.993); they are given in Table 1. There was evidence for nonlinear pharmacokinetics, as evidenced by a dose-dependent elimination half life (42±2 min, 60±9 min, and 76±9 min for 30 g i.v., 6 g i.v., and 6 g p.o., respectively). Moreover, AUC (0, ∞) values showed a proportional increase with increasing doses of l-arginine (r = 0.975, P < 0.05). Comparison of AUC for intravenous and oral administration of 6 g l-arginine revealed that l-arginine was incompletely bioavailable after oral administration; absolute bioavailability of oral l-arginine was 68±9%.

Table 1.

Pharmacokinetic parameters of intravenous and oral l-arginine.

Pharmacokinetic-pharmacodynamic relationship

The temporal pattern of the changes in l-arginine plasma concentration was closely mirrored in the time course of the changes in urinary nitrate excretion and total peripheral resistance after the infusion of 30 g l-arginine (Figure 5a). The relationship between pharmacokinetic and -dynamic data were best described using a linear effect model in which the effect was linked directly to l-arginine plasma concentration (r = 0.993). There was also a significant linear correlation between urinary nitrate excretion rates and plasma l-arginine concentrations at midpoint of the urine sampling interval (r = 0.604, P < 0.05). The mean plasma concentration-effect relationship for intravenous infusion of 30 g l-arginine is shown in Figure 5b. No hysteresis was present in the plasma concentration-effect plot. The equilibration half-life between the l-arginine plasma concentration and effect was calculated to be 5.8±2.3 min.

Figure 5.

(a) Comparison of the time course of plasma l-arginine concentrations, total peripheral resistance, and urinary nitrate excretion rates in healthy human subjects after intravenous infusion of 30 g of l-arginine. (b) Plot of plasma l-arginine concentrations against effect (change in total peripheral resistance).

Discussion

The salient findings of our study are that l-arginine concentration-dependently induces vasodilation in healthy human subjects. This haemodynamic effect is paralleled by increased urinary excretion rates of nitrate, the final oxidative metabolite of NO, and its second messenger cyclic GMP. Pharmacokinetic analysis showed evidence for dose-related kinetics of l-arginine. Oral bioavailability of l-arginine is about 70%, and the maximum plasma concentration reached after oral ingestion of l-arginine is considerably lower than after intravenous infusion, due to the delay in absorption from the intestinal tract. In PK/PD analysis there is evidence for a direct link between the vasodilator effect of l-arginine and its plasma concentration.

We and others have previously demonstrated that l-arginine induces peripheral vasodilation during intravenous infusion in healthy human subjects [6, 8, 27]. Similar vasoactive effects of a single intravenous infusion of l-arginine have also been observed in patients with hypercholesterolaemia [9] and coronary [10] or generalized atherosclerosis [7]. In several studies, l-arginine has also been used in an oral dosage form in patients with cardiovascular diseases. Clarkson et al. [12] found improved endothelium-dependent brachial artery vasodilation after 4 weeks of 21 g day⁻¹ of oral l-arginine in hypercholesterolaemic subjects. Adams et al. [14] administered 21 g l-arginine day⁻¹ for 3 days to young patients with coronary artery disease; they found improved endothelium-dependent vasodilation and reduced monocyte adhesion after l-arginine as compared with placebo. Rector et al. [28] gave 5.6 to 12.6 g day⁻¹ of l-arginine during 6 weeks to patients with heart failure and found significantly improved limb blood flow and functional performance of the patients during exercise testing. Although l-arginine has been applied intravenously or orally in these and other studies in patients, the pharmacokinetic data of l-arginine are largely unknown. Investigation of the oral bioavailability of l-arginine as well as its pharmacokinetics are an important basis for further clinical studies.

In the present study, the temporal pattern of l-arginine plasma concentration closely corresponded to the temporal pattern of its vasodilator effect, i.e., the reduction in total peripheral resistance and blood pressure. Pharmacokinetic/pharmacodynamic modelling indicated that the effect (reduction in TPR) was directly linked to l-arginine plasma concentration, Alternative models using an indirect link of the effect, e.g. to a tissue compartment, or using a separate effect compartment, less closely represented the data. The presence of a direct link between l-arginine plasma levels and its haemodynamic effect was further confirmed by the lack of hysteresis in the concentration-effect plot. A counterclockwise hysteresis loop would have been expected if a delay in equilibration between plasma l-arginine concentration and its concentration at the effect site would have occurred, if a metabolite of l-arginine would be responsible for the effect, or it the effect would be mediated by an indirect mechanism like protein synthesis [25]. This, taken together with the short equilibration half-life between l-arginine plasma concentration and the effect, suggests a direct vasodilator action of l-arginine within the vasculature.

The mechanism by which exogenous l-arginine induces vasodilation may involve stimulation of endogenous, endothelial NO formation, as suggested by studies in which the urinary excretion rate of nitrate, the final oxidative metabolite of NO, was increased [6, 8]. The present study further supports this hypothesis, as we found a close linear relationship between l-arginine plasma levels and urinary nitrate excretion rates. In other studies, increased exhalation of NO was reported as an indicator for enhanced endogenous NO formation after l-arginine administration [29]. We here report that the urinary excretion rates of nitrate and cGMP, two index molecules for endogenous NO formation in vivo [4], are increased in a dose-related manner after intravenous infusion of l-arginine. The maximum elevation of urinary nitrate and cGMP excretion rates occurred within 30–60 min after the end of l-arginine infusion [6, 7]. 90 min after the end of the infusion, these index metabolites had returned to the basal range again.

The hypothesis that l-arginine induces NO elaboration by the endothelium and thereby causes vasodilation is in agreement with the present observation of a direct link between l-arginine plasma levels and vasodilation. Endothelial NO synthase is continuously stimulated by shear stress induced by the blood streaming along the endothelial surface [30]. NO is released from the endothelium almost instantaneously after stimulation, and NO itself has a very short biological half-life in the range of a few seconds [31]. It is rapidly inactivated by oxidation to nitrite and nitrate [3, 4]. These biochemical observations correspond well to the results of the biochemical measurements performed in the present study. We found that the elevation of urinary nitrate excretion corresponded closely to the time pattern of l-arginine plasma levels, as did the change in urinary excretion of the second messenger, cyclic GMP, and the haemodynamic response.

The fact that l-arginine increases NO release in vivo has been called the ‘l-arginine paradox’, because compared with the K_m value of the endothelial NO synthase (∼2.9 μm [39]) physiological fasting l-arginine plasma levels should be high enough to saturate the enzyme with substrate [16]. This paradox may be resolved by several observations: Firstly, the K_m value was determined in vitro in a crude enzyme preparation [32]; in vivo, however, factors like intracellular compartmentalization of l-arginine and NO synthase may differ, potentially reducing the availability of substrate for the enzyme. Moreover, the presence of endogenous compounds competing with l-arginine for the enzyme binding site may also cause relative substrate depletion. One such endogenous competitive inhibitor is asymmetric dimethylarginine (ADMA); it has been reported to be present in human plasma [33] and in cultured human endothelial cells [34, 35]. Whether ADMA downregulates NO elaboration in healthy humans, in whom ADMA plasma levels are low [33, 36], remains undetermined. Finally, l-arginine uptake may be a crucial step limiting intracellular l-arginine availability in vivo [37].

Besides stimulating NO production, l-arginine is known to exert other effects which may contribute to its vasodilator properties, like stimulation of growth hormone secretion [15] and insulin release [27]. However, the peak in growth hormone secretion after l-arginine infusion occurs later than the peak in vasodilation [own, unpublished observation]. Interestingly, these endocrine effects of l-arginine may may also cause secondary increases in NO release: Many of the physiological effects of growth hormone have been shown to occur via local production of IGF-1 [38], which in turn induces endothelium-dependent vasodilation that can be blocked by NO synthase inhibitors [39]. We have recently shown that NO may be responsible for the haemodynamic effects of recombinant growth hormone in patients with acquired growth hormone deficiency [40]. Likewise, the haemodynamic effects of insulin are mediated at least in part via NO [41]. Furthermore, unspecific vasodilation has been shown to occur at l-or d-arginine concentrations considerably higher than those achieved in the present study [42]. Enhanced peripheral blood flow itself, as induced by (unspecific) vasodilation may increase endothelial NO release via elevating shear stress at the endothelial surface [43].

Comparison of the AUC values for the oral or intravenous administration of 6 g l-arginine showed that the bioavailability of l-arginine is ∼70% after oral ingestion. Using stable-isotope-labeled l-arginine, Castillo et al. [44] also found incomplete bioavailability of orally ingested l-arginine (∼38%). In contrast, Matera et al. [17] reported complete bioavailability of oral l-arginine in healthy humans. They administered in a very low daily dose (100 mg day⁻¹), contained in a complex polyaminoacid formulation intended for use as an oral supplement in intensive care medicine, for 7 days. In the same study, these authors reported a terminal elimination half-life of ∼1.2 h, which is in agreement with our results obtained with considerably higher doses of l-arginine. Half-life calculation in our study was somewhat hampered by the long time interval between the sampling times at 210 and 960 min. This was caused by logistic reasons which did not allow us to keep the subjects in the lab overnight. The significance of half life calculations was potentially reduced by this time pattern, but there is still a relatively close correspondence between our findings and those reported by others. Noeh et al. [45] studied tissue distribution of l-arginine in rats after intraperitoneal application; they found close correlation between the rise in plasma arginine levels and arginine concentrations in NO-generating organs like aorta, heart, and vena cava. In their study, l-arginine half-life was ∼1 h in plasma, and 1–2 h in the various tissues.

The rate of utilization of l-arginine after absorption in the splanchnic region and the factors affecting it are of considerable interest, since l-arginine uptake and metabolism in the liver may affect systemic bioavailability of this amino acid. There is evidence that the activity of the l-arginine uptake mechanism (y⁺-transporter for basic amino acids) is low in hepatocytes as compared with other cell types [46]. Accordingly, Felig & Wahren [47] found no concentration gradient between the portal and hepatic vein in the postabsorptive state. Taken together, these studies suggest that l-arginine metabolism in the liver is functionally separated from whole-body l-arginine metabolism. Blanchier et al. [48] showed that a minor part of an oral l-arginine dose administered to healthy rats is metabolized by enterocytes. These findings may explain why the majority of an orally administered l-arginine dose was systemically available in the present study. Even outside the liver, l-arginine can be a substrate for several metabolic pathways: Ingested arginine can be a source of ornithine in the intestine [49]; it may serve to replenish arginine which is lost during hepatic urea synthesis [50]; it may undergo decarboxylation to agmatine in the kidney and the brain [51], or be used by the NO synthase to generate NO and citrulline [52]. Castillo et al. [53] previously demonstrated that part of orally administered [¹⁵N]-l-arginine is converted to urinary [¹⁵N]-NO₃⁻ in healthy humans, indicating that l-arginine taken up from the splanchnic region is being used as a substrate for the NO synthase. Rhodes et al. [54] confirmed this finding by demonstrating [¹⁵N]-nitrite enrichment in plasma after intravenous infusion of [¹⁵N]-l-arginine in healthy human subjects. Although from their tracer studies Leaf et al. [55] calculated that below 0.1% of the administered l-arginine dose were converted via NO to nitrate, Rhodes et al. [54] estimated that about 90% of circulating nitrite is derived from the l-arginine:NO pathway in fasted humans. Taken together, these studies confirm that exogenous l-arginine acts as a substrate for NO synthesis in normal humans.

The pharmacokinetic data generated in the present study provide evidence for dose-dependent pharmacokinetics of l-arginine: With increasing dose, both the elimination half-life and the apparent volume of distribution decreased. One explanation for this observation may be a saturation of l-arginine uptake into cells by the highest dose (30 g). Extremely high plasma levels of l-arginine after this dose may have exceeded the renal threshold for l-arginine reabsorption, resulting in l-arginine overflow into the urine. In contrast to the 30 g dose of l-arginine, steady state was reached during the infusion of 6 g of l-arginine, as indicated by the plateau of l-arginine plasma concentration during the final 10 min of the infusion period. Therefore, the half-life calculated after the infusion of 6 g l-arginine may represent most closely the ‘true’ physiological half-life of this amino acid. Single doses of about 6 g l-arginine administered 2–3 times a day may be a reasonable approach to long-term studies with this amino acid for cardiovascular disease.

In conclusion, our present study provides pharmacokinetic data for l-arginine after intravenous and oral administration. We have shown that the vascular effects of l-arginine in healthy human subjects are closely correlated with its plasma concentrations. These data may provide a basis for the utilization of l-arginine in cardiovascular diseases.