Since the publication of the original findings of Furchgott and Zawadzki (1) showing that the endothelium is required for the vasodilating effects of acetylcholine (ACh), the physiology of this single cell layer and its role as an active rather than a passive vasomodulator has provoked intense study. Nitric oxide (NO) is responsible for the vasodilating properties of the endothelium (2) and the release of NO from the endothelium may be triggered by such external stimuli as shear stress and chemical modulators, including ACh, bradykinin (BK), and substance P (3).
In healthy blood vessels, NO is produced mainly in endothelial cells from the terminal guanidino nitrogen atom of L-arginine (L-ARG) (4). Because the endogenous concentration of L-ARG is between 100 and 800 μM(5,6), and the Km of the calmodulin-requiring endothelial NO-synthase is <5 μM(7), it is generally believed that under normal conditions there is ample substrate for the enzyme. Much of the data on the effect of in vitro L-ARG addition to animal isolated blood vessels or vascular endothelial cells in culture suggest that further addition of the amino acid does not alter functional endothelium-dependent vasodilatation unless conditions are such that there is a depletion of L-ARG (4,5,8). Whether this is true of human blood vessels is debatable, however; e.g., low concentrations of L-ARG enhance forearm blood flow (FBF) responses to ACh stereospecifically in healthy humans (9,10). When infused intravenously into normal volunteers at high concentrations (500 mg/kg/min), L-ARG reportedly decreases both systolic and diastolic blood pressure (SBP, DBP) (11). This BP-lowering effect may not be NO dependent however, since a vasodilatory effect of high concentrations of D-ARG has also been reported (12).
We initiated the present study of the vascular effects of dietary supplementation with L-ARG in healthy humans after reports of the effective oral use of this semiessential amino acid in infants with carbamyl phosphate synthetase insufficiency, in children with lysinuria or arginosuccinase deficiency, and in adult males for improvement of male spermatogenesis (13). Our data indicate that dietary supplementation of L-ARG 20 g/day every day for 28 days, does not influence vascular function. We therefore extended our studies, investigating whether this null effect could be due to saturation of NO-synthase in the healthy human vessel by adding L-ARG exogenously to subcutaneous arteries isolated from gluteal biopsies performed on healthy humans.
SUBJECTS AND METHODS
Twenty-six (men aged 18-35 years) were recruited by advertisement; 16 participated in the L-ARG dietary supplementation protocol (part 1), and 10 participated in the gluteal biopsy study (part 2). An out-of-pocket expenses fee of AUS$75.00 per session was paid to each subject.
Criteria for study inclusion were nonsmoking status, alcohol intake less than three standard alcohol drinks/day, SBP <140 mm Hg, DBP <90 mm Hg, total plasma cholesterol <5.5 mM, and total plasma triglyceride <2.0 mM. In addition, all volunteers underwent a thorough physical examination and had normal findings on routine hematological and biochemical blood analyses. The study was approved by the Alfred Group of Hospitals Ethics Committee, and written informed consent was obtained from all subjects.
Part 1: Dietary supplementation with L-ARG
Subjects were randomly allocated in a double-blind protocol to receive either L-ARG 20 g/day or placebo for 28 days. Randomization and the preparation of both the active (L-ARG) and placebo oral solutions were performed by the Pharmacy Department, Alfred Hospital, Melbourne, Victoria, Australia. Patients were dispensed their oral solution (14-day supply at a time). The active (L-ARG) solution was arginine HCl (300 g, donated by Ajinomoto, Kawasaki, Japan) added to deionized water (400 ml) and compound hydroxybenzoate solution (5 ml) and made up to 900 ml with either aromatic or orange syrup. The preparation for the placebo oral solution was deionized water (600 ml) and compound hydroxybenzoate solution (5 ml) made up to 900 ml with either aromatic or orange syrup. The purity of L-ARG HCl was 98 +%. Patients were instructed to self-administer 30 ml oral solution (10 g L-ARG) twice daily.
Resting BP and plasma biochemical analysis. Weekly BP measurements were obtained with an automated BP monitor (Critikon Dinamap vital signs monitor). Supine BP measurements were obtained in subjects after a 10-min horizontal rest, and standing BP measurements were obtained after 5 min in the upright position. To the greatest degree possible, BP was measured at the same time on the same day of each week from week 0 (pretreatment) to weeks 1, 2, 3, and 4 (final week of treatment).
Plasma biochemical analysis. Blood samples were obtained just before each plethysmograph session (weeks 0 and 4); 7 ml blood was obtained for analysis of urea, creatinine, liver function tests, and electrolytes (performed Pathology Department, Alfred Hospital). Ten milliliters blood was then obtained and centrifuged, and the plasma was frozen at -20°C until it was dispatched to the Biochemistry Department, Monash University, Clayton, Victoria, Australia for analysis of amino acid by high-performance liquid chromatography.
Forearm venous occlusion plethysmography. To study the effect of dietary supplementation of L-ARG on vascular function, forearm vascular reactivity was measured by forearm venous occlusion plethysmography on day 0, immediately before supplementation with L-ARG (or placebo) and on day 29 after 28 days of oral supplementation. Subjects rested supine throughout the experiment in a comfortable, relaxed environment maintained at a constant temperature of 22°C. The protocol for measurement of FBF was described previously (14,15). FBF was measured by venous occlusion plethysmography with a sealed, alloy-filled (gallium and indium) double-strand strain gauge (Medasonic, Mountain View, CA, U.S.A.). Venous occlusion pressure was 40-50 mm Hg at the proximal end (elbow), and cuff occlusion pressure at the distal (wrist) end was ≈200 mm Hg to arrest all circulation to the hand. The distal cuff was inflated before determination of FBF and left inflated for the duration of all measurements. FBF was calculated for 10 of every 20 s.
The brachial artery was cannulated (3.0 F, 5-cm catheter, Cook, Australia) for intraarterial BP recordings as well as for local sequential infusions of ACh (9.25, 18.5, and 37 μg/min), SNP (0.4, 0.8, and 1.6 μg/min), and the NO-synthase inhibitor NG-monomethyl-L-arginine (L-NMMA 1, 2, and 4 mmol/min). Basal blood flow (the average of three flow measurements before each drug infusion) was obtained after an equilibration period of 40-60 s. Drugs were infused at 2 ml/min for a maximum of 6 min or until the response for three flow measurements reached a plateau (usually by 3 min for ACh and SNP and 5 min for L-NMMA). Rest periods of 5-15 min between concentrations and rest periods of 15 min between drugs were observed. At the concentrations used, neither systemic BP, recorded with a disposable physiological pressure transducer (model CMS-327, Biosensors International Proprietary, Singapore) nor heart rate (HR), recorded with an ECG lead II (Spacelabs, Washington, U.S.A.) was affected. Forearm vascular resistance (FVR) was calculated from the equation: FVR (RU = mean arterial pressure (MAP, mm Hg)/FBF (ml/100ml/min).
Part 2: Isolated gluteal subcutaneous arteries
Buttock skin biopsies were performed on a separate group of volunteers under local anesthesia (lignocaine 1% subcutaneoulsy) and using strict aseptic conditions. Biopsies were immediately placed in ice-cold Krebs solution and transported to the laboratories, where subcutaneous small arteries were isolated with a dissecting microscope (Wild M38, Heerbrugg, Switzerland). When possible, two segments of the same vessel were obtained from any one biopsy. Alternatively, two vessels of similar size, again from the same biopsy, were obtained.
The method used for the preparation of these vessels was described previously (16). Segments (2 mm) of arteries were threaded onto 40-μm diameter stainless-steel wires and mounted as ring preparations in an isometric small vessel myograph (Scientific Concepts, Victoria, Australia). Vessels were bathed in Krebs solution (in mM: NaCl 119, KCl 4.7, KH2PO4 1.18, MgSO4 1.17, NaHCO3 25, CaCl2 2.5, EDTA 0.026 and glucose 5.5) bubbled with carbogen (95% O2/5% CO2) at pH 7.4, and kept at a constant temperature of 37°C.
After an unstretched equilibration period of 30 min, each vessel was stretched to an internal circumference equal to 0.9 × L100 denotes the internal circumference at the level of passive stretch equivalent to a transmural pressure of 100 mm Hg. This was calculated using the length-tension relation obtained in each vessel; 0.9 × L100 was used since it has been shown to be optimal for maximal force production in arteries (17). This procedure ensured that arteries of different internal diameter were stretched to a similar point on their length-tension curve.
Thirty minutes later, the viability of each vessel was confirmed by a 2-min exposure to a high potassium Krebs solution (K+ 124 mM). A cumulative concentration-response curve to NE (10 nM to 10 μM) was then obtained, followed by cumulative concentration-response curves to ACh, (1 nM to 10 μM), and substance P (1 pM to 1 nM), both of which were constructed on arteries contracted to 70-80% maximal constriction by NE (1-3 μM). A 15-min rest period was observed after thorough washout between the construction of each curve. Finally, a single concentration response was obtained to SNP (10 μM), again on arteries precontracted with NE. Vessels were then incubated with either L-(10 μM) or D-ARG (10 μM) for a minimum of 30 min before repetition of the entire protocol in the continued presence of either amino acid.
Part 1 ACh chloride (BDH Chemicals, Poole, Dorset, U.K.) was diluted under sterile conditions with 0.9% saline. SNP (David Bull Laboratories, Victoria, Australia) and L-NMMA (H-arg Me)-OH.AcOH, N methyl-L-arginine.acetate, Clinalfa, Switzerland) were diluted under sterile conditions in 5% dextrose.
Part 2 Drugs used were (-)-NE bitartrate (arterenol), ACh chloride, and L- and D-ARG hydrochloride (all Sigma), and ([Sar9, Met (O2)11]-substance P (AUSPEP). Drugs were made up in distilled water and diluted in Krebs on the day of each experiment.
Part 1. Forearm venous occulsion plethysmography Unless otherwise stated, all results are mean ± SEM. Data were assessed by analysis of variance (ANOVA: one- or two-way and/or repeated-measures when appropriate) followed by a Student's t test (paired when appropriate). Analysis was performed with SigmaStat Statistical Software (Jandel Scientific, San Rafael, CA, U.S.A.) which inherently analyzes data for normality before performing parametric analysis. When data failed to meet normality criteria, nonparametric analysis (Kruskal-Wallis nonparametrics) was applied. In addition, calculation of the area under each individual dose-response curve was also analyzed as a single-value summary for that data set (18).
Part 2: Isolated gluteal subcutaneous arteries Contractile responses were expressed as g force normalized to the internal diameter for subcutaneous arteries. Individual concentration-response curves for each agonist were fitted to a logistic equation of the form E = MAP/(AP × KP), where E is the response and M is the maximum response, A is the concentration eliciting 50% of the maximum response (i.e., EC50), and P is the slope parameter (19). From this equation, the concentrations corresponding to 10-90% (EC10-90) of the maximum response were determined. Data are mean ± SEM and were assessed by repeated-measures ANOVA with SigmaStat Statistical Software (Jandel Scientific), which inherently analyzes data for normality before performing parametric analysis followed by Student's t test, in which p < 0.05 is used as the criterion for statistical significance.
Part 1: Dietary supplementation with L-ARG
The mean age of subjects randomized to receive the active L-ARG supplement was 21.9 ± 0.6 years (n = 8); that of subjects receiving the placebo supplement was 20.9 ± 1.0 years (n = 8). None of the volunteers reported any side effects after ingestion of oral L-ARG/placebo, although several alluded to the unpleasant flavor of the supplement. These complaints were unrelated to whether the treatment was arginine or placebo. Biochemical analyses of plasma obtained before and after treatment showed no effect of treatment on urea, creatinine, electrolyte (sodium, potassium, chloride), or liver function enzyme levels [aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and creatinine (Table 1)].
Plasma levels of all L-α-amino acids, with the exception of tryptophan, asparagine, and glutamine, were analyzed. Although plasma levels of each individual amino acid (including plasma L-ARG levels) (Table 2) were unaffected by either treatment, a significant alteration was observed in the distribution of the total amino acid pool after active, but not placebo, treatment (Mann-Whitney U Test, p < 0.05). Figure 1 shows the frequency distribution of the 17 amino acids analyzed. Amino acids were ordered into frequency bands of concentration (% mol) (Table 2) ranging from 0 to 14% mol. Analysis was performed with the Mann-Whitney U test for nonparametric testing. After L-ARG, but not placebo, supplementation, the frequency distribution of these 17 amino acids was clearly altered. Neither resting supine SBP or DBP or standing SBP or DBP was affected by placebo or L-ARG treatments (Table 3).
FVR Mean basal FBF was not affected by either L-ARG (before vs. after 1.82 ± 0.43 vs. 1.85 ± 0.5 ml/100/ml/min, p > 0.05) or placebo (before vs. after 1.54 ± 0.47 vs. 1.89 ± 0.4 ml/100/ml/min, p > 0.05). Figure 2 shows the mean FBF, MAP, and mean calculated FVR measurements obtained throughout the study, i.e., with and without ACh (9.25, 18.5, and 37 μg/min), SNP (0.4, 0.8 and 1.6 μg/min) and L-NMMA (1, 2, and 4 μmol/min) before and after L-ARG and placebo supplementation. Responses to the drugs used were also expressed as absolute blood flow, absolute difference in blood flow, percent change in blood flow, and corresponding changes in FVR. Neither assessment of these parameters by either repeated-measures ANOVA or area under the dose-response curve (paired t test) showed any effect of either the placebo or the L-ARG treatments (data not shown).
Part 2: Isolated gluteal subcutaneous arteries
Vessel internal diameter at L100 averaged 0.40 ± 0.05 mm (n = 10) in arteries exposed to L-ARG and 0.37 ± 0.03 mm in arteries exposed to D-ARG (n = 9). Both L- and D-ARG (10 μM) significantly shifted the NE concentration-response curve to the right (Fig. 3) without altering maximal contractile force. -log M EC50 values to NE decreased from 7.12 ± 0.15 to 6.66 ± 0.16 (two-way repeated measures ANOVA followed by paired t test, p < 0.05) after L-ARG and from 7.36 ± 0.17 to 6.85 ± 0.18 (p < 0.05) after D-ARG. Neither L- nor D-ARG had any effect on the potency or maximal dilatation generated by either ACh or substance P (Table 4). Similarly, the one-point dilatation to SNP 10 μM was not significantly affected by either L-ARG (from 87.44 ± 3.92 to 94.12 ± 2.38%, Student's paired t test; p < 0.05) or D-ARG (from 89.50 ± 4.70 to 95.76 ± 1.52%, Student's paired t test, p > 0.05).
In light of the augmenting effects of L-ARG on vessels with diminished responses to ACh observed in previous studies (5), we also analyzed the effect of arginine on arteries in which the initial maximal dilatation to ACh (or substance P) was <90% full dilatation (subset A, n = 5) separately from those in which a maximal dilatation >90% was obtained (subset B, n = 5). As shown in Fig. 4, L-ARG significantly increased the potency of ACh in arteries from subset A but not in those from subset B. D-ARG had no such effect. When a similar protocol was followed for substance P, i.e., when arteries in which the initial maximal dilatation to substance P was <90% dilatation were analyzed separately from those in which a maximal dilatation >90% was obtained, neither L- nor D-ARG had any effect on responses to substance P in either subset A (L-ARG, n = 5, p = 0.067) nor B.
Addition of L-ARG to a normal, robust, human vascular system does not improve endothelium-NO-dependent vasodilatation. This was clearly demonstrated in the current study in which both in vivo and in vitro addition of L-ARG had no effect on endothelium-dependent vasodilatation. The other major finding was the confirmation that arginine has vasodilatory properties that are not NO dependent. This was demonstrated by the nonstereospecific ability of both L- and D-ARG to antagonize responses to NE functionally in gluteal subcutaneous arteries.
Our study was initiated on the premise that a dietary supplement of the semiessential amino acid L-ARG improves the symptoms of L-ARG deficiency in infants with carbamyl phosphate synthetase insufficiency and children with lysinuria or arginosuccinase deficiency and improves spermatogenesis in adult males (13). More significantly, L-ARG added to diets of hypercholesterolemic rabbits improved endothelium-mediated responses otherwise impaired (20).
Because the average American diet provides a daily intake of 5.4 g L-ARG (21), administration of 20 g/day used in the current study was clearly supranormal. An a priori expectation was that plasma L-ARG levels would increase with this increased intake, but this did not occur. On the other hand, analysis of the major 17 amino acids in plasma showed a clear alteration after active L-ARG, but not placebo, supplementation. Therefore, we suggest that chronic supplementation of L-ARG leads to a domino effect on many amino acids. Because L-ARG shares competitive transport systems with many of these amino acids (including lysine, ornithine, and cysteine) (13), is a substrate for at least four other enzymes beside the NO-synthase enzyme (22), and is influenced by citrulline and glutamine (23), this effect of increased L-ARG intake may not be surprising. Another possibility is that L-ARG caused a change in appetite (or possibly taste) and thus protein intake, which would probably change the amino acid profile without significantly changing the plasma concentration of any individual amino acid. Our study did not show whether a higher dose of L-ARG would result in an increase in plasma L-ARG levels. Therefore, although the alteration in total amino acid profile after L-ARG intake provides confirmation that the subjects involved were complaint with treatment, it unfortunately does not negate the possibility that an inadequate dose of L-ARG was delivered.
There is one or more possible reasons for the null effect of oral L-ARG on vascular function in the present study. First, chronic oral L-ARG may be channeled into other sites, altering total amino acid profile (as already discussed) without achievement of the concentrations required at the endothelium-smooth muscle compartment. This contrasts with findings of Cooke and colleagues (20) showing that oral administration of L-ARG to hypercholesterolemic rabbits both increases plasma L-ARG levels significantly and improves endothelium-dependent vasodilatation. The difference in the two studies may either be species related (i.e., the metabolic fate of L-ARG may be different in rabbits as compared with humans) or disease related (i.e., an impairment of endothelium function may be required, as in hypercholesterolemic rabbits. Another possible reason for the null effect of oral L-ARG in our study is that the dose used (20 g/day) was insufficient; this dose is four-fold higher than the normal intake in humans as compared with sixfold enrichment of this amino acid in the study of hypercholesterolemic rabbits (20). The final possible explanation is that supplementation of this amino acid to a vascular system that is healthy and robust and already functioning optimally does not augment dilatation because the NO-synthase enzyme is already saturated with the substrate. This possibility is supported by reports of a null effect of intraarterial infusions of L-ARG on either basal FVR (12) or blood flow in response to endothelium-dependent vasodilators such as ACh (24) and methacholine (25) in normal healthy humans. In direct contradiction to this, however, increases in endothelium-dependent vasodilatation have also been reported of intraarterial infusions of L-ARG into forearm resistance arteries of normal healthy humans (9,10). These conflicting findings in the human forearm may be due to factors not related to endothelium function but rather to problems inherent with the in vivo study of vascular function, i.e., of equilibrium, questionable range of the dose-response curve, uncontrolled concentrations, speed of injection, and metabolism (26). To overcome these difficulties, we extended our investigation to studies in the organ bath, in which equilibrium is easily established (26) and in which the effect of L-ARG on the full range of the agonist dose-response curve can be examined. Gluteal subcutaneous arteries were procured from normal, healthy humans and mounted in myographs. This procedure enabled, for the first time, the direct assessment of the effects of L-ARG supplementation on vessels from healthy subjects.
Our first finding was that arginine functionally antagonizes NE nonstereospecifically, consistent with the findings of Calver and co-workers (12), who demonstrated that high concentrations of arginine have direct, nonstereospecific in vivo vasodilator effects on both arteries and veins of the human upper limb. The concentration of arginine we used (10 μM) is comparable to that previously shown to be stereospecific and endothelium-dependent in rabbit isolated aortic rings (27). In these rabbit preparations, stereospecificity was lost only at >10 mM concentrations (28). Therefore, the human subcutaneous artery appears to be more sensitive to the dilator actions of arginine. Because the L-isomer of arginine is the only substrate for the NO-synthase enzyme, we conclude that the observed dilator action of arginine is not dependent on the NO pathway. Although the protocol we used does not exclude a time-dependent, arginine-independent shift on the dose-response curves to NE, this is unlikely since a time-dependent effect was not observed with the other agonists used.
From the myograph experiments, we also demonstrated conclusively that neither L- nor D-ARG supplementation has any influence on vasodilatation mediated by the endothelium-dependent vasodilators ACh and substance P, consistent with the hypothesis that there is ample substrate in an optimally functioning system and that the NO-synthase enzyme is already fully saturated. Also noteworthy is that when the response to the ACh-stimulated release of NO was less than optimal (i.e., when <90% dilatation was observed), L- but not D-ARG, significantly improved the potency of the agonist. Although the same trend was observed for substance P, the improvement caused by L-ARG just failed to reach statistical significance (p = 0.067). In vessels that responded optimally to ACh and substance P (i.e., when >90% dilatation was observed), incubation with L-ARG had no effect. Clearly, although L-ARG has no effect on an optimally functioning system, there was activity when the NO pathway was not functioning at full capacity. This finding parallels reports that L-ARG increases vasodilator responses to ACh in hindlimbs of cholesterol-fed rabbits (in which endothelial dysfunction was observed) but not those of control animals (29) and suggests that L-ARG supplementation plays a role in improving the endothelial dysfunction observed in some disease states (24,25).
Oral supplementation with L-ARG 20 g/day for 28 days does not affect endothelium function in normal healthy adults probably because oral administration of L-ARG at this dose is insufficient, leading only to dissipation of L-ARG to other pathways, as evidenced by the change in total amino acid profile, but having no effect on L-ARG plasma concentrations. It may also be that L-ARG cannot improve a normal endothelium-mediated vasodialation.
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