Pharmacology Journal

The three isoforms of nitric oxide synthase (NOS), neuronal NOS (nNOS, NOS 1), inducible NOS (iNOS, NOS 2) and endothelial (eNOS, NOS 3), convert l-arginine to nitric oxide (NO) and l-citrulline. NO is a vasoactive compound that induces vasodilation of arterial and venous blood vessels. In endothelial cells, l-arginine is transported via the cell membrane by cationic amino acid transporters that are colocalized with eNOS. The Michaelis–Menten constant (K_m) for eNOS is 3 µM l-arginine. This is at least one order of magnitude lower than the normal plasma concentrations of l-arginine, which are usually in the range 60–140 µM. Nevertheless, oral supplementation with l-arginine has been shown to enhance NO-mediated vasodilation in several clinical studies, but not in all. One possible explanation for this ‘arginine paradox’ is the presence of an endogenous inhibitor of NOS, which may shift the steep part of the substrate–activity curve of NOS towards higher l-arginine levels. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of all three isoforms of NOS and it is circulating at low µM concentrations in humans. The ratio of l-arginine over ADMA (arginine/ADMA ratio) is one determinant of NO production by NOS. Once produced, NO activates soluble guanylyl cyclase (sGC) in smooth muscle cells, which leads to elevated intracellular cyclic guanosine monophosphate (cGMP). In human blood vessels this mechanism results in vasodilation. This process is essential for endothelial function, and disturbed NO production in the human endothelium contributes to endothelial dysfunction.

The semiessential amino acid l-arginine is part of the human diet and only 5–15% of plasma arginine originate from de novo synthesis. After oral administration, l-arginine is subject to extensive presystemic and systemic elimination, i.e. by bacteria in the gut and arginases in the gut and liver, respectively. The non-essential amino acid l-citrulline is not subject to presystemic elimination but to systemic metabolism. l-Citrulline is converted to l-argininosuccinate by argininosuccinate synthase and subsequently to l-arginine by argininosuccinate lyase. It may therefore serve as an l-arginine precursor.

The aim of this study was to investigate the pharmacokinetic (PK) and pharmacodynamic (PD) effects of different oral doses of l-arginine and l-citrulline in humans subjects with impaired NO elaboration secondary to elevated ADMA concentrations.

Twenty healthy, non-obese volunteers (13 male, 7 female) were included in this study. They were recruited from a group of 168 clinically healthy humans screened for fasting plasma ADMA concentration. Subjects were eligible if they had ADMA concentrations within the highest quartile of the distribution of the screened population. All participants had normal clinical history and physical examination, 12-lead electrocardiogram, haematological and biochemical screen. Diabetes, obesity, hypertension, cardiovascular disease, liver or kidney disease, current infections or smoking were exclusion criteria. None of the volunteers received any drugs that might alter amino acid or vitamin status, and dietary habits were kept constant during the study. A history of hormone replacement therapy (HRT) was known in two female participants. HRT was stopped 21 days prior to receiving the first dose of study drug. Written informed consent was obtained from all participants. The study protocol was approved by the Ethics Committee of the Hamburg Board of Physicians, and the investigation was conducted in accordance with the Declaration of Helsinki.

Study design

In a randomized, double-blind, placebo-controlled cross-over design participants received either l-citrulline 0.75 g twice daily, l-citrulline 1.5 g twice daily, l-citrulline 3 g twice daily, l-arginine immediate-release (IR) 1.0 g tid, l-arginine sustained-release (SR) 1.6 g twice daily, or placebo for 7 days each. The study periods were separated by wash-out phases of 1 week, and the sequence of the medications was randomly chosen in each participant. On day 7 of each medication phase, venous blood samples were drawn from an antecubital vein for PK analyses at 0, 0.5, 1, 2, 4, 6, 8, 12, 16 and 24 h. On day 7 only a single dose, equivalent to half of the total daily dose, was administered. The twice daily or three times daily dosing was administered on days 1 through 6. At baseline and on day 7 (at 4 h after dosing) additional blood and urine samples were collected for ADMA plasma concentrations and urinary nitrate and cGMP excretion rates, respectively. Finally, at baseline and at 4 h after dosing on day 7, endothelial function was assessed by flow-mediated vasodilation (FMD) testing of the brachial artery as detailed below.

Biochemical analyses

Plasma l-arginine and l-citrulline concentrations were determined by liquid chromatography (LC)-tandem mass spectrometry (MS) analysis as described previously. Briefly, a 50-µl aliquot of plasma was spiked with stable isotope-labelled l-citrulline and l-arginine, which served as internal standards. Protein was precipitated with 100 µl of methanol, filtrated through a 0.22-µm hydrophilic membrane (Multiscreen HTS^TM; Millipore, Molsheim, France), derivatized with butanolic HCl (1 N, 65°C, 17 min) and analysed by LC-tandem MS. Quantification was performed by selected reaction monitoring of the respective daughter ions of analytes and internal standards (Waters, Eschborn, Germany). Plasma ADMA was analysed by enzyme-linked immunosorbent assay (ELISA), as previously described. Urinary nitrate levels were determined by GC-MS as described elsewere. Urinary cGMP was analysed by ELISA. Urinary excretion rates of nitrate and cGMP were corrected for creatinine excretion.

Pharmacokinetic analyses

PK parameters (C_max, T_max, C_min, AUC) were calculated for each dose of l-arginine and l-citrulline after 1 week of oral supplementation. After l-citrulline supplementation, PK parameters were calculated for l-arginine and l-citrulline plasma concentrations, whereas PK parameters were calculated only for l-arginine concentrations after l-arginine supplementation. Areas under the plasma concentration–time curve (AUC) were calculated for up to 24 h. To account for the circadian rhythms of endogenous l-arginine and l-citrulline concentrations, plasma concentrations following l-arginine and l-citrulline administration at each time point were corrected for individual baseline and placebo data prior to calculation of C_max, T_max, C_min and AUC values. Even for corrected data, calculation of half-life was still not possible. All PK calculations were performed using WinNonlin (v. 5.0; Pharsight Corp., Mountain View, CA, USA).

Vascular function testing

Methods of assessing endothelium-dependent vasodilation followed the principles set by the International Brachial Artery Reactivity Task Force. Endothelial function was assessed in the volunteers’ right arm in a quiet, temperature-controlled room (22°C) by high-resolution ultrasound (12 MHz linear array transducer; Siena, Siemens, Germany). Longitudinal scans of the brachial artery were obtained approximately 5 cm proximal of the antecubital fossa. The transmit focus zone was set at the depth of the anterior wall. Anatomical landmarks and snapshot images were used to assess FMD in the same vessel section on each study day and at each time point. A view of a 5-cm longitudinal section of the brachial artery was recorded for time periods of 30 s at baseline and during peak reactive hyperaemia (60 s after deflation of a blood pressure cuff previously inflated to 50 mmHg above the volunteer’s systolic blood pressure for 5 min). Each 30-s recording was digitalized (Vascular Imager 4.1.3; Medical Imaging Applications LLC, IA, USA) at a rate of 10 high-resolution frames per second (= 300 frames per recording), by using specialized software (Brachial Analyser 4.1.3; Medical Imaging Applications LLC). FMD was calculated as the percent change in diameter 1 min after cuff release relative to the baseline diameter before cuff release. Ultrasound studies and image analysis were performed separately by independent investigators in an observer-blinded fashion. The mean intraindividual coefficient of variation of the arterial diameter at the baseline measurements obtained on the six separate study days was 4.65%.

Statistical analyses

All data are given as mean ± SEM, together with 95% confidence intervals for the mean differences (CI). Statistical comparisons were made by Student’s t-test (two-tailed) for paired data. Statistical analysis was performed with SPSS (release 10 for Windows; Chicago, IL, USA).

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