(Circulation. 1996;93:2135-2141.)
© 1996 American Heart Association, Inc.
Articles |
Randomized, Double-Blind, Placebo-Controlled Study of Supplemental Oral L-Arginine in Patients With Heart Failure
From the Cardiovascular Division, Department of Medicine, University of Minnesota Medical School, Minneapolis.
Correspondence to Thomas S. Rector, PhD, Box 508 UMHC, University of Minnesota Medical School, 420 Delaware St SE, Minneapolis, MN 55455.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Background Patients with heart failure have reduced peripheral blood flow at rest, during exercise, and in response to endothelium-dependent vasodilators. Nitric oxide formed from L-arginine metabolism in endothelial cells contributes to regulation of blood flow under these conditions. A randomized, double-blind crossover study design was used to determine whether supplemental oral L-arginine can augment peripheral blood flow and improve functional status in patients with moderate to severe heart failure.
Methods and Results Fifteen subjects were given 6 weeks of oral L-arginine hydrochloride (5.6 to 12.6 g/d) and 6 weeks of matched placebo capsules in random sequence. Compared with placebo, supplemental oral L-arginine significantly increased forearm blood flow during forearm exercise, on average from 5.1±2.8 to 6.6±3.4 mL·min-1·dL-1 (P<.05). Furthermore, functional status was significantly better on L-arginine compared with placebo, as indicated by increased distances during a 6-minute walk test (390±91 versus 422±86 m, P<.05) and lower scores on the Living With Heart Failure questionnaire (55±28 versus 42±26, P<.05). Oral L-arginine also improved arterial compliance from 1.99±0.38 to 2.36±0.30 mL/mm Hg (P<.001) and reduced circulating levels of endothelin from 1.9±1.1 to 1.5±1.1 pmol/L (P<.05).
Conclusions Supplemental oral L-arginine had beneficial effects in patients with heart failure. Further studies are needed to confirm the therapeutic potential of supplemental oral L-arginine and to identify mechanisms of action in patients with heart failure.
Key Words: heart failure • endothelium • L-arginine
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Many studies have demonstrated that patients with heart failure have reduced peripheral blood flow at rest and during exercise.1 2 3 Several studies have also demonstrated subnormal peripheral vasodilatation in response to endothelium-dependent vasodilators in heart failure.4 5 6 7 These abnormalities have been related to the severity of the symptoms patients experience and to exercise capacity. Furthermore, improvements in exercise performance with a converting enzyme inhibitor have been associated with increases in peripheral blood flow during exercise.8 Interventions that augment endogenous nitric oxide activity over the long term may also improve peripheral blood flow and have clinical benefits in patients with heart failure.
Endothelial cells produce nitric oxide from
L-arginine via nitric oxide synthase.9 Studies
using tracer amounts of [15N]L-arginine
demonstrated that 
1% of dietary L-arginine appears in
the urine as labeled nitrate, but the effect of changes in dietary
L-arginine on vascular nitric oxide formation is not
known.10 Under normal conditions, an excess of
L-arginine is available for endothelial
cells, on the basis of in vitro determinations of nitric oxide synthase
kinetics. However, in vitro formation of nitric oxide in response to
endothelium-dependent vasodilators can be
improved by L-arginine when L-arginine stores
are depleted or in the presence of
L-glutamine.11 12 Furthermore, studies of
patients with abnormal endothelium-dependent
vasodilatation related to atherosclerosis or heart
transplantation have observed significant improvements during
short-term intravenous infusions of
L-arginine.13 14
Intra-arterial L-arginine also enhanced the
forearm vasodilator response to acetylcholine, an
endothelium-dependent vasodilator, in patients with
heart failure.15 These studies suggest that, under some
conditions, supplemental L-arginine may augment nitric
oxide production.
The objective of this investigation was to determine whether supplemental oral L-arginine improves peripheral blood flow in patients with heart failure at rest or when stimulated by exercise, postischemic hyperemia, or an endothelium-dependent vasodilator. In addition, measures of submaximal exercise tolerance and quality of life were examined to determine whether supplemental oral L-arginine might produce clinical benefits.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Study Design
Seventeen patients who were referred for tertiary treatment of heart failure were enrolled in a two-period randomized crossover study. All patients gave written informed consent. Before data analysis, two subjects were excluded because of protocol noncompliance manifested by excessive dietary salt intake that resulted in an acute weight gain of more than 5 pounds associated with symptomatic deterioration that required adjustments in concurrent therapy. These confounding events occurred during the L-arginine treatment periods and may have biased our estimates of the true L-arginine effects. No similar events were seen during the placebo period. Characteristics of the remaining 15 patients are described in Table 1
. Diabetes mellitus,
hypertension, and overt atherosclerotic peripheral vascular
disease were exclusion criteria, and all participants had total
cholesterol levels 
6.2 mmol/L (240 mg/dL).
|
Seven patients were randomly assigned to placebo followed by L-arginine, and 8 patients were assigned to the opposite sequence of treatments. During the 6-week treatment period, patients were given either 2.8 g L-arginine hydrochloride (Tyson and Associates, Inc) twice daily (n=9) or 4.2 g three times daily (n=6). The initial dose of oral L-arginine was selected to double the estimated average daily dietary intake of L-arginine. However, we did not evaluate the actual dietary intake of L-arginine in these patients. In an attempt to detect a dose effect, we arbitrarily increased the amount of supplemental L-arginine 2.5 times after 10 subjects were enrolled. Significant effects were seen after 10 weeks of oral L-arginine treatment in animals.16 Six-week treatment periods were used in this study to increase the likelihood that these patients with severe heart failure would remain stable for the entire study.
Matching placebo capsules were administered during the 6-week control period to mask the identity of study capsules.
Study Assessments
The following sequence of measurements was made at the end of
each 6-week treatment period. Medications were withheld on the morning
of these evaluations. First, study nurses read standard instructions
and then asked the patients to complete the Living With Heart Failure
questionnaire to ascertain how their heart failure had affected their
quality of life.17 18 The total distance covered during a
6-minute walk test was used as a measure of exercise tolerance during
daily activities.19 20 A standard set of instructions was
read each time the walk test was done. Patients were told to walk as
far as they could in 6 minutes in an isolated 60-foot hallway. During
the walk test, patients were periodically encouraged to go as far as
they could. Rest stops were allowed if symptoms became too
bothersome.
Vascular function was examined at the end of each 6-week period by a
regionally perfused forearm technique. Forearm blood flow was measured
by venous occlusion plethysmography. Patients rested in the supine
position with their nondominant forearm abducted 30° and raised to
midchest position. A mercury-in-Silastic strain gauge was placed
around the upper forearm and connected to a calibrated plethysmograph
to measure forearm blood flow as the rate of change of forearm volume
(mL·min-1·100
mL-1 forearm volume). Venous occlusion
was induced by rapid inflation of a blood pressure cuff placed around
the upper arm to 40 mm Hg (D.E. Hokanson, Inc). Hand blood flow was
excluded by a wrist cuff inflated to suprasystolic pressure
before each measurement, which was the average of three
recordings. Room temperature was constant during each day of
study and did not vary by more than 1°C between study days. Forearm
volume was calculated as the volume of a truncated cone based on wrist
and upper forearm circumference and forearm length. The average forearm
volume was 1097±169 mL.
After measurement of baseline forearm blood flow, forearm exercise was performed with a hand dynamometer set at 15%, 30%, and 45% of each patient's maximal voluntary contraction as has been previously described.21 The exercise protocol consisted of cycles of 5 seconds of contraction followed by 10 seconds of relaxation. Patients exercised for 2 minutes, then forearm blood flow was measured 5 seconds into each relaxation phase during the third minute of exercise. When flow had returned to baseline, a 5-minute period of forearm ischemia was created by inflation of the cuff around the upper arm to suprasystolic pressure. Flow measurements taken immediately after the pressure occlusion was deflated and after 15, 30, 45, and 60 seconds were used to characterize the amount of reactive hyperemia.
A 20-gauge cannula was then inserted under local lidocaine anesthesia into the brachial artery of the same arm to study responses to intra-arterial administration of an endothelium-dependent vasodilator, methacholine, and an endothelium-independent vasodilator, nitroprusside. Patients rested quietly for 30 minutes after catheter insertion before baseline blood samples, blood pressure, and heart rate were obtained. Baseline forearm blood flow was measured, followed by infusion of methacholine at 0.3, 1.5, and 3.0 µg/min, with measurement of flow at the end of each 90-second infusion. When baseline flow was reestablished, the responses to three doses of nitroprusside, 1, 5, and 10 µg/min, were determined in the same manner. Finally, a competitive inhibitor of nitric oxide synthase, L-N-monomethylarginine, was administered to assess basal nitric oxide activity. First, baseline flow was reestablished, then flow was measured at the end of two cumulative 4-minute infusions of L-N-monomethylarginine (4 and 8 µmol/min).
Vascular smooth muscle relaxation can increase arterial compliance as well as lower arteriolar resistance.22 Arterial compliance was estimated by pulse contour analysis.23 24 Pressure waves were obtained by placing a tonometer sensor array (model N-500, Nellcor) over the radial artery of the nondominant arm. Tonometer dispersions were calibrated by use of synchronized automatic blood pressure cuff measurements taken from the contralateral arm. Pressure waveforms from several stable beats captured over 30 seconds were fit by nonlinear least-squares regression to a modified Windkessel model resulting in two separate compliance indexes based on the exponential decay of the diastolic portion of the wave form (C1) and the superimposed decaying sinusoidal waveform (C2).
The effects of L-arginine on several vasoconstrictor pathways that are often activated in patients with heart failure were also examined because of previous reports suggesting that increased nitric oxide activity may interact with the sympathetic nervous system or endothelin.25 26 27 Plasma endothelin was quantified by radioimmunoassay.28 The rabbit antibody used (Peninsula Laboratories, Inc) had 100% cross-reactivity with endothelin-1, 7% with endothelin-2 and -3, and 17% with big endothelin. The normal range for endothelin in our laboratory is 0.2 to 1.9 pmol/L. Plasma norepinephrine was measured by high-performance liquid chromatography with electrochemical detection.29 Our normal plasma norepinephrine ranges from 0.8 to 1.9 nmol/L. Plasma renin activity was determined by a radioimmunoassay with a normal range from 0.12 to 0.85 nmol·h-1·L-1. The concentration of L-arginine in plasma was determined by our hospital laboratory, in which normal values range from 20 to 180 µmol/L.
Safety measures included clinic visits every 3 weeks; blood tests for electrolytes, glucose, insulin, renal function, liver function; and complete blood counts. Intravenous administration of much higher doses of L-arginine hydrochloride have been associated with hyperinsulinemia and changes in potassium concentrations.30 If similar changes occur during oral L-arginine treatment, endothelium-dependent vasodilatation could be affected. Blood urea nitrogen was also increased by oral L-arginine in a previous animal study.16
Statistical Analysis
Data were summarized as mean±SD except in the figures, in which
standard error bars are shown. Measurements obtained after 6 weeks of
oral L-arginine were compared with those obtained after the
6-week placebo control period by ANOVA for repeated measurements that
included estimates of the effects of treatment sequence and dose of
oral L-arginine. Since treatment sequence and
L-arginine dose did not significantly affect the results,
the data were not presented for subgroups defined by these
factors. If changes in neurohormones followed a skewed distribution, a
Wilcoxon signed rank test was used to compare concentrations
during the oral L-arginine and placebo treatment periods. A
value of P.05 was considered statistically
significant.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Clinical Assessments
Supplemental oral L-arginine improved patients' functional status. There was a significant improvement, from 55±28 to 42±26 (P<.05), in the Living With Heart Failure questionnaire score (Fig 1
) and a significant increase,
from 390±91 to 422±86 m (P<.05), in the distance walked
in 6 minutes (Fig 2). Body weight remained stable
(87.7±11.4 versus 87.2±11.4 kg) throughout the 12-week study period.
Mean supine blood pressure decreased slightly, from 82±9 to 80±6 mm
Hg (P<.05), during L-arginine administration.
Supine resting heart rate did not change (81±16 versus 81±13
bpm).
|
|
Peripheral Vascular Function
During forearm exercise, forearm blood flow increased
significantly after the L-arginine treatment period
compared with the placebo period (Fig 3). The average
improvement in forearm blood flow during exercise was 1.5±3.0
mL·min-1·dL-1
(P<.05). Maximum voluntary hand contraction did not change
(35±9 versus 36±10 kg). In contrast to exercise hyperemia,
reactive hyperemia after 5 minutes of arterial
occlusion was not significantly augmented (Fig 4).
|
|
Methacholine was used to examine the effect of oral
L-arginine on the response of the
endothelial nitric oxide pathway when stimulated to
release increased amounts of nitric oxide. Increases in forearm blood
flow in response to methacholine were on average 1.5±4.2
mL·min-1·dL-1
greater with L-arginine than with placebo (Fig 5), but this difference did not attain statistical
significance (P=.33). Responses to exogenous nitric oxide
provided by infusion of nitroprusside were similar during the
L-arginine and placebo periods (Fig 6).
|
|
Vasoconstrictor responses to
L-N-monomethylarginine, an
inhibitor of nitric oxide synthase, were similar during the
L-arginine and placebo treatment periods (Fig 7), suggesting that basal endothelial
nitric oxide activity was not increased by oral L-arginine
supplementation. This finding is consistent with unchanged
forearm blood flow under basal conditions during L-arginine
administration compared with placebo (3.7±1.1 versus 3.8±1.6
mL·min-1 · dL-1,
respectively).
|
Arterial compliance as assessed by pulse contour analysis was improved by administration of oral L-arginine. Compliance estimated from the exponential decay of the diastolic pressure (C1) improved to 2.36±0.30 from 1.99±0.38 mL/mm Hg (P<.001), and the second compliance term in the modified Windkessel model of the circulation (C2) increased from 0.048±0.024 to 0.064±0.026 mL/mm Hg (P<.05).
Biochemical Assessments
There was a significant increase in mean plasma
L-arginine concentration, from 85±21 to 98±28 µmol/L
(P<.05), during administration of supplemental oral
L-arginine. Other data are summarized in Table 2. Small increases in blood urea nitrogen were seen,
which may be related to L-arginine metabolism
in the urea cycle. There were no differences in serum electrolytes.
|
Endothelin levels fell significantly, from 1.9±1.1 to 1.5±1.1 pmol/L (P<.05). Plasma norepinephrine and plasma renin activity were not significantly reduced by oral L-arginine.
Adverse Effects
One patient experienced diarrhea during the placebo period. Two
patients, one in each treatment period, had acute episodes of gout
during the trial. End-point assessments were avoided during these
attacks. No other potential adverse effects were noted.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
This is the first randomized, double-blind study that investigated whether supplemental oral L-arginine could improve peripheral blood flow and functional status in patients with heart failure. The results suggest that oral L-arginine has beneficial effects in patients with moderate to severe heart failure. Patients noted significantly fewer limitations secondary to their heart failure as assessed by the Living With Heart Failure questionnaire and walked farther during a 6-minute walk test. In addition, limb blood flow during exercise and arterial compliance were significantly enhanced by oral L-arginine compared with placebo. Finally, circulating levels of the potent vasoconstrictor endothelin were reduced by L-arginine.
Previous studies in animals and humans with heart failure have indicated that short-term administration of L-arginine can restore vasodilator responses to an endothelial nitric oxide–dependent vasodilator.15 31 This was the first study to examine the effects of oral L-arginine administered over several weeks. Three different assessments were used to determine whether supplemental oral L-arginine could improve peripheral blood flow in patients with heart failure when nitric oxide activity was stimulated. Both forearm exercise and release of a brachial artery occlusion increase shear stress in the forearm vasculature, which can lead to increased formation of nitric oxide from L-arginine.32 33 Muscarinic agonists have served as a pharmacological stimulus to examine the functional status of the endothelial nitric oxide pathway ever since the seminal study of Furchgott and Zawadzki.34 Significant enhancement of exercise hyperemia was observed during L-arginine administration and may have been related to increased nitric oxide activity. However, additional investigations using inhibitors of nitric oxide synthase during exercise are needed to determine whether the observed increase in exercise hyperemia was indeed mediated via the nitric oxide pathway. The effect of supplemental oral L-arginine on methacholine-induced vasodilation was, on average, similar to the increase in exercise hyperemia, but the changes in methacholine responses were more variable than changes in exercise hyperemia. Given greater variation in the effect of L-arginine on methacholine responses, a much larger sample size would be needed to achieve statistical significance and provide a precise estimate of any effect of L-arginine. Postischemic hyperemia was not significantly increased by oral L-arginine. Although peripheral blood flow tended to improve during all three interventions used to stimulate the nitric oxide pathway, the results were somewhat inconsistent, perhaps because of differences in the extent to which nitric oxide contributes to the total vasodilator responses to these three interventions. Overall, these results can neither establish nor preclude the possibility that supplemental L-arginine may be converted to nitric oxide in patients with heart failure when activity of the nitric oxide synthase pathway is increased by different stimuli.
Lack of significant increases in the vasoconstrictor response to L-N-monomethylarginine, an inhibitor of nitric oxide synthase, suggests that oral L-arginine did not substantially enhance basal nitric oxide formation in the forearm vasculature. Resting vasoconstrictor tone as measured by forearm vascular resistance also was not reduced, since basal forearm blood flow did not increase and mean blood pressure was only slightly decreased. Similar responses to nitroprusside during the L-arginine and placebo treatment periods suggest that L-arginine did not alter nitric oxide–induced smooth muscle relaxation.
Indexes of vascular compliance were also significantly improved during the L-arginine treatment period. Reduced vascular compliance has been documented in patients with heart failure by pulse contour analysis as well as other methods.35 36 37 Theoretically, improvements in peripheral vascular compliance could decrease reflection of pressure waves from the periphery, thereby reducing impedance to left ventricular outflow. In addition, better arterial compliance may improve arterial baroreceptor function and decrease sympathetic activity in patients with heart failure. Other studies have suggested that endothelial nitric oxide can attenuate sympathetic nervous system activity.25 38 However, mean plasma norepinephrine concentration was not reduced by L-arginine. This observation, in conjunction with the lack of change in basal forearm vascular resistance, suggests that compliance was not improved by reductions in sympathetic vasoconstrictor tone.
Few reports were found in the literature describing structural alterations in arteries from patients with heart failure. Theoretically, increased circulating levels of growth-promoting factors such as norepinephrine, angiotensin II, and endothelin may alter vascular structure. In infarcted rats, increased medial thickness of intramuscular resistance vessels and increased carotid artery stiffness associated with increased collagen content and adventitial thickness have been demonstrated.39 40 It is possible that L-arginine could have improved vascular compliance via structural changes related to antigrowth activity of nitric oxide and barrier functions of intact endothelium.41 Indexes of arterial compliance based on pulse contour analysis cannot be used to isolate the contributions of hemodynamic versus structural changes. L-Arginine had only minimal effects on blood pressure that would not be expected to greatly improve vascular compliance through a decrease in distending pressure. Nitric oxide has been shown to inhibit the positive inotropic effect of ß-adrenergic stimulation.42 Since our measures of arterial compliance vary in direct proportion to the cardiac output and cardiac output was not directly measured, it is possible that the compliance estimates were confounded by changes in cardiac output. More in-depth investigation of changes in compliance with L-arginine is needed.
Plasma endothelin levels were significantly reduced during the L-arginine treatment period compared with the placebo period. This study did not examine whether decreased formation or increased clearance of endothelin from the circulation could explain this observation. Interestingly, changes in the availability of nitric oxide have been associated with the regulation of endothelin production in vitro.26 27 Since endothelin is a potent vasoconstrictor and mitogenic factor, reduced production of endothelin may be beneficial in patients with heart failure. A recent study of an endothelin receptor antagonist administered to patients with severe heart failure observed significant hemodynamic improvements.43 These investigators also confirmed a strong relationship between plasma endothelin and mean pulmonary vascular resistance. More research is needed to determine whether this potential mechanism accounts for some of the effects of oral L-arginine.
Others have shown that oral L-arginine can improve responses to an endothelium-dependent vasodilator in patients and animals with hypercholesterolemia in the absence of heart failure.16 44 Since the precise degree of elevation of cholesterol or its derivatives, such as oxidized LDL cholesterol, that are needed to alter the activity of the nitric oxide pathway have not been established, it is possible that the effects of L-arginine seen in this study were related to cholesterol.45 However, L-arginine did not change total circulating cholesterol and had similar effects on exercise blood flow, methacholine-induced vasodilation, 6-minute walk test, and quality of life when subgroups defined by the median cholesterol level (4.7 mmol/L, or 180 mg/dL) or a history of ischemic coronary artery disease were examined (data not shown).
Oral L-arginine, 7 g three times a day for 3 days, did not enhance endothelium-dependent forearm vasodilatation in normal subjects who had relatively high cholesterol levels (mean, 5.4 mmol/L).46 Interestingly, platelet aggregation was inhibited by oral L-arginine. The effect of L-arginine on platelet aggregation could be blocked with L-N-monomethylarginine, suggesting that nitric oxide synthase mediated this effect. Platelet aggregation was not evaluated in our study.
Although this investigation was limited to a small sample from a single medical center, the observation of multiple improvements, including increased quality of life, submaximal exercise performance, forearm exercise hyperemia, arterial compliance, and reduced plasma endothelin, warrant further investigations of supplemental oral L-arginine in patients with heart failure. The optimal dosage regimen was not established by this study, and longer studies are needed to determine whether the L-arginine effects persist. A mechanism of action for oral L-arginine could not be established on the basis of these data, which were focused primarily on the forearm vasculature and indirect measures of the nitric oxide pathway. If therapeutic effects can be confirmed by other investigations, studies to identify the mechanism of action may provide valuable insight into the biology of L-arginine.
|
Acknowledgments |
|---|
This work was supported in part by program project grant PO1-HL-32427 from the National Institutes of Health, Bethesda, Md. We gratefully acknowledge our Biochemistry Laboratory under the direction of Dianne Judd, BS, for measurements of endothelin, norepinephrine, and plasma renin activity.
Received October 10, 1995; revision received December 28, 1995; accepted January 2, 1996.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Leithe ME, Margorien RD, Hermiller JB, Unverferth DV, Leier CV. Relationships between central hemodynamics and regional blood flow in normal subjects and in patients with congestive heart failure. Circulation. 1984;69:57-64.
2.
Zelis R, Longhurst J, Capone RJ, Mason DT. A
comparison of regional blood flow and oxygen utilization during dynamic
forearm exercise in normal subjects and patients with congestive heart
failure. Circulation. 1974;50:137-143.
3.
Sullivan MJ, Knight JD, Higginbotham MB, Cobb
FR. Relation between central and peripheral
hemodynamics during exercise in patients with chronic
heart failure: muscle blood flow is reduced with maintenance of
arterial perfusion pressure.
Circulation. 1989;80:769-781.
4.
Kubo SH, Rector TS, Bank AJ, Williams RE, Heifetz
SM. Endothelium-dependent vasodilation is
attenuated in patients with heart failure.
Circulation. 1991;84:1589-1596.
5. Nakamura M, Ishikawa M, Funakoshi T, Hashimoto K, Chiba M, Hiramori K. Attenuated endothelium-dependent peripheral vasodilation and clinical characteristics in patients with chronic heart failure. Am Heart J. 1994;128:1164-1169. [Medline] [Order article via Infotrieve]
6. Katz SD, Biasucci L, Sabba C, Strom JA, Jondeau G, Galvao M, Solomon S, Nikolic SD, Forman R, LeJemtel TH. Impaired endothelium-mediated vasodilatation in the peripheral vasculature of patients with congestive heart failure. J Am Coll Cardiol. 1992;19:918-925. [Abstract]
7. Drexler H, Hayoz D, Munzel T, Hornig B, Just H, Brunner HR, Zelis R. Endothelial function in chronic congestive heart failure. Am J Cardiol. 1992;69:1596-1601. [Medline] [Order article via Infotrieve]
8. Mancini DM, Davis L, Wexler JP, Chadwick B, LeJemtel TH. Dependence of enhanced maximal exercise performance on increased peak skeletal muscle perfusion during long-term captopril therapy in heart failure. J Am Coll Cardiol. 1987;10:845-850. [Abstract]
9. Palmer RMJ, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature. 1988;333:664-666. [Medline] [Order article via Infotrieve]
10.
Castillo L, DeRojas TC, Chapman TE, Vogt J, Burke JF,
Tannenbaum SR, Young VR. Splanchnic metabolism of
dietary arginine in relation to nitric oxide synthesis in normal adult
man. Proc Natl Acad Sci U S A. 1993;90:193-197.
11. Arnal JF, Munzel T, Venema RC, James NL, Bai C, Mitch WE, Harrison DG. Interactions between L-arginine and L-glutamine change endothelial NO production. J Clin Invest. 1995;95:2565-2572.
12. Gold ME, Bush PA, Ignarro LJ. Depletion of arterial L-arginine causes reversible tolerance to endothelium-dependent relaxation. Biochem Biophys Res Commun. 1989;164:714-721. [Medline] [Order article via Infotrieve]
13. Creager MA, Gallagher SJ, Girerd XJ, Coleman SM, Dzau VJ, Cooke JP. L-Arginine improves endothelium-dependent vasodilation in hypercholesterolemic humans. J Clin Invest. 1992;90:1248-1253.
14.
Drexler H, Fischell TA, Pinto FJ, Chenzbraun A, Botas
J, Cooke JP, Alderman EL. Effect of L-arginine on
coronary endothelial function in cardiac
transplant recipients. Circulation. 1994;89:1615-1623.
15.
Hirooka Y, Imaizumi T, Tagawa T, Shiramoto M, Endo T,
Ando S, Takeshita A. Effects of L-arginine on
impaired acetylcholine-induced and ischemic vasodilation of
the forearm in patients with heart failure.
Circulation. 1994;90:658-668.
16. Cooke JP, Singer AH, Tsao P, Zera P, Rowan RA, Billingham ME. Antiatherogenic effects of L-arginine in the hypercholesterolemic rabbit. J Clin Invest. 1992;90:1168-1172.
17. Rector TS, Cohn JN. Assessment of patient outcome with the Minnesota Living With Heart Failure questionnaire: reliability and validity during a randomized, double-blind placebo-controlled trial of pimobendan. Am Heart J. 1992;124:1017-1025. [Medline] [Order article via Infotrieve]
18. Rector TS, Kubo SH, Cohn JN. Validity of the Minnesota Living With Heart Failure questionnaire as a measure of therapeutic response to enalapril or placebo. Am J Cardiol. 1993;71:1106-1107. [Medline] [Order article via Infotrieve]
19. Lipkin DP, Scriven AJ, Crake T, Poole-Wilson PA. Six minute walking test for assessing exercise capacity in chronic heart failure. Br Med J. 1986;292:653-655.
20.
Bittner V, Weiner DH, Yusuf S, Rogers WJ, McIntyre KM,
Bangdiwala SI, Kronenberg MW, Kostis JB, Kohn RM, Guillotte M,
Greenberg B, Woods PA, Bourassa MG. Prediction of mortality and
morbidity with a 6-minute walk test in patients with left
ventricular dysfunction. JAMA. 1993;270:1702-1707.
21.
Arnold JMO, Ribeiro JP, Colucci WS. Muscle blood
flow during forearm exercise in patients with severe heart
failure. Circulation. 1990;82:465-472.
22.
Bank AJ, Wilson RF, Kubo SH, Holte JE, Dresing TJ, Wang
H. Direct effects of smooth muscle relaxation and contraction on
in vivo human brachial artery elastic properties.
Circ Res. 1995;77:1008-1016.
23.
Cohn JN, Finkelstein S, McVeigh G, Morgan D, LeMay L,
Robinson J, Mock J. Noninvasive pulse wave analysis for
the early detection of vascular disease.
Hypertension. 1995;26:503-508.
24.
Watt TB, Burrus C. Arterial pressure
contour analysis for estimating human vascular
properties. J Appl Physiol. 1976;40:171-176.
25.
Zanzinger J, Czachurski J, Seller H. Inhibition
of sympathetic vasoconstriction is a major principle of vasodilation by
nitric oxide in vivo. Circ Res. 1994;75:1073-1077.
26. Saijonmaa O, Ristimaki A, Fyhrquist F. Atrial natriuretic peptide, nitroglycerine and nitroprusside reduce basal and stimulated endothelin production from cultured endothelial cells. Biochem Biophys Res Commun. 1990;173:514-520. [Medline] [Order article via Infotrieve]
27. Boulanger C, Luscher TF. Release of endothelin from the porcine aorta: inhibition by endothelium-derived nitric oxide. J Clin Invest. 1990;85:587-590.
28.
Lerman A, Hildebrand FL, Aarhus LL, Burnett JC.
Endothelin has biological actions at
pathophysiological concentrations.
Circulation. 1991;83:1808-1814.
29. Candito M, Krstulovic C, Sbirazzuoli V, Chambon P. Proposal for the standardization of the calibration method for the assay of plasma catecholamines. J Chromatogr. 1990;526:194-202. [Medline] [Order article via Infotrieve]
30.
Barbul A. Arginine: biochemistry, physiology and
therapeutic implications. J Parent Enteral
Nutr. 1986;10:227-238.
31. Mayhan WG, Rubinstein I. Acetylcholine induces vasoconstriction in the microcirculation of cardiomyopathic hamsters: reversal by L-arginine. Biochem Biophys Res Commun. 1992;184:1372-1377. [Medline] [Order article via Infotrieve]
32.
Gilligan DM, Panza JA, Kilcoyne CM, Waclawiw MA, Casino
PR, Quyyumi AA. Contribution of
endothelium-derived nitric oxide to
exercise-induced vasodilation.
Circulation. 1994;90:2853-2858.
33.
Tagawa T, Imaizumi T, Endo T, Shiramoto M, Harasawa Y,
Takeshita A. Role of nitric oxide in reactive hyperemia
in human forearm vessels. Circulation. 1994;90:2285-2290.
34. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980;288:373-376. [Medline] [Order article via Infotrieve]
35. Finkelstein SM, Cohn JN, Collins VR, Carlyle PF, Shelley WJ. Vascular hemodynamic impedance in congestive heart failure. Am J Cardiol. 1985;55:423-427. [Medline] [Order article via Infotrieve]
36.
Arnold JMO, Marchiori GE, Imrie JR, Burton GL,
Pflugfelder PW, Kostuk WJ. Large artery function in patients
with chronic heart failure. Circulation. 1991;84:2418-2425.
37. Lage SG, Kopel L, Monachini MC, Medeiros CJ, Pileggi F, Polak JF, Creager MA. Carotid arterial compliance in patients with congestive heart failure secondary to idiopathic dilated cardiomyopathy. Am J Cardiol. 1994;74:691-695. [Medline] [Order article via Infotrieve]
38.
Vita JA, Treasure CB, Yeung AC, Vekshtein VI, Fantasia
GM, Fish RD, Ganz P, Selwyn AP. Patients with evidence of
coronary endothelial dysfunction as assessed by
acetylcholine infusion demonstrate marked increase in sensitivity to
constrictor effects of catecholamines.
Circulation. 1992;85:1390-1397.
39. Gaballa MA, Raya TE, Goldman S. Large artery remodeling after myocardial infarction. Am J Physiol. 1995;37:H2092-H3103.
40. Schieffer B, Wollert KC, Berchtold M, Sall K, Schieffer E, Hornig B, Riede UN, Drexler H. Development and prevention of skeletal muscle structural alterations after experimental myocardial infarction. Am J Physiol. 1995;38:H1507-H1513.
41.
Hamon M, Vallet B, Bauters C, Wernert N, McFadden EP,
Lablanche JM, Dupuis B, Bertrand ME. Long-term oral
administration of L-arginine reduces intimal thickening and
enhances neoendothelium-dependent
acetylcholine-induced relaxation after arterial
injury. Circulation. 1994;90:1357-1362.
42.
Hare JM, Loh E, Creager MA, Colucci WS. Nitric
oxide inhibits the positive inotropic response to ß-adrenergic
stimulation in humans with left ventricular
dysfunction. Circulation. 1995;92:2198-2203.
43. Kiowski W, Sutsch G, Hunziker P, Muller P, Kim J, Oechslin E, Schmitt R, Jones R, Bertel O. Evidence for endothelin-1-mediated vasoconstriction in severe chronic heart failure. Lancet. 1995;346:732-736. [Medline] [Order article via Infotrieve]
44. Drexler H, Zeiher AM, Meinzer K, Just H. Correction of endothelial dysfunction in coronary microcirculation of hypercholesterolemic patients by L-arginine. Lancet. 1991;338:1546-1550. [Medline] [Order article via Infotrieve]
45. Stroes ESG, Koomans HA, de Bruin TWA, Rabelink TJ. Vascular function in the forearm of hypercholesterolaemic patients off and on lipid-lowering medication. Lancet. 1995;346:467-471. [Medline] [Order article via Infotrieve]
46. Adams MR, Forsyth CJ, Jessup W, Robinson J, Celermajer DS. Oral L-arginine inhibits platelet aggregation but does not enhance endothelium-dependent dilation in healthy young men. J Am Coll Cardiol. 1995;26:1054-1061.[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  E. N. Dedkova and L. A. Blatter Characteristics and function of cardiac mitochondrial nitric oxide synthase J. Physiol., February 15, 2009; 587(4): 851 - 872. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Bai, L. Sun, T. Yang, K. Sun, J. Chen, and R. Hui Increase in fasting vascular endothelial function after short-term oral L-arginine is effective when baseline flow-mediated dilation is low: a meta-analysis of randomized controlled trials Am. J. Clinical Nutrition, January 1, 2009; 89(1): 77 - 84. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. H. Boger The Pharmacodynamics of L-Arginine J. Nutr., June 1, 2007; 137(6): 1650S - 1655S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. K. Grimble Adverse Gastrointestinal Effects of Arginine and Related Amino Acids J. Nutr., June 1, 2007; 137(6): 1693S - 1701S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. D. Peluffo L-Arginine currents in rat cardiac ventricular myocytes J. Physiol., May 1, 2007; 580(3): 925 - 936. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Thengchaisri, R. Shipley, Y. Ren, J. Parker, and L. Kuo Exercise Training Restores Coronary Arteriolar Dilation to NOS Activation Distal to Coronary Artery Occlusion: Role of Hydrogen Peroxide Arterioscler Thromb Vasc Biol, April 1, 2007; 27(4): 791 - 798. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. H. Zucker Novel Mechanisms of Sympathetic Regulation in Chronic Heart Failure Hypertension, December 1, 2006; 48(6): 1005 - 1011. [Full Text] [PDF]
|
|
|
|
 |
|
 X. Sun and D. D. Ku Allicin in garlic protects against coronary endothelial dysfunction and right heart hypertrophy in pulmonary hypertensive rats Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2431 - H2438. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. P. Schulman, L. C. Becker, D. A. Kass, H. C. Champion, M. L. Terrin, S. Forman, K. V. Ernst, M. D. Kelemen, S. N. Townsend, A. Capriotti, et al. L-Arginine Therapy in Acute Myocardial Infarction: The Vascular Interaction With Age in Myocardial Infarction (VINTAGE MI) Randomized Clinical Trial JAMA, January 4, 2006; 295(1): 58 - 64. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. K. McConell, N. N. Huynh, R. S. Lee-Young, B. J. Canny, and G. D. Wadley L-Arginine infusion increases glucose clearance during prolonged exercise in humans Am J Physiol Endocrinol Metab, January 1, 2006; 290(1): E60 - E66. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. G. Olsson, K. Swedberg, A. L. Clark, K. K. Witte, and J. G.F. Cleland Six minute corridor walk test as an outcome measure for the assessment of treatment in randomized, blinded intervention trials of chronic heart failure: a systematic review Eur. Heart J., April 2, 2005; 26(8): 778 - 793. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Niebauer, A. L. Clark, K. M. Webb-Peploe, R. Boger, and A. J.S. Coats Home-based exercise training modulates pro-oxidant substrates in patients with chronic heart failure Eur J Heart Fail, March 2, 2005; 7(2): 183 - 188. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. G. West, A. Likos-Krick, P. Brown, and F. Mariotti Oral L-Arginine Improves Hemodynamic Responses to Stress and Reduces Plasma Homocysteine in Hypercholesterolemic Men J. Nutr., February 1, 2005; 135(2): 212 - 217. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. L. Gornik and M. A. Creager Arginine and Endothelial and Vascular Health J. Nutr., October 1, 2004; 134(10): 2880S - 2887S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Parnell, J. P. F. Chin-Dusting, J. Starr, and D. M. Kaye In vivo and in vitro evidence for ACh-stimulated L-arginine uptake Am J Physiol Heart Circ Physiol, July 1, 2004; 287(1): H395 - H400. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.E.S. Miner, A. Al-Hesayen, S. Kelly, T. Benson, J.J. Thiessen, V.R. Young, and J.D. Parker L-Arginine Transport in the Human Coronary and Peripheral Circulation Circulation, March 16, 2004; 109(10): 1278 - 1283. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Pernow, F. Bohm, E. Beltran, and A. Gonon L-Arginine protects from ischemia-reperfusion-induced endothelial dysfunction in humans in vivo J Appl Physiol, December 1, 2003; 95(6): 2218 - 2222. [Abstract] [Full Text]
|
|
|
|
 |
|
 R. H Boger The emerging role of asymmetric dimethylarginine as a novel cardiovascular risk factor Cardiovasc Res, October 1, 2003; 59(4): 824 - 833. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. R. Morris, S. M. Morris Jr., W. Hagar, J. van Warmerdam, S. Claster, D. Kepka-Lenhart, L. Machado, F. A. Kuypers, and E. P. Vichinsky Arginine Therapy: A New Treatment for Pulmonary Hypertension in Sickle Cell Disease? Am. J. Respir. Crit. Care Med., July 1, 2003; 168(1): 63 - 69. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Nakamura, S. Al-Ruzzeh, A. H. Chester, A. Dewar, S. Rothery, N. J. Severs, M. H. Yacoub, and M. Amrani Age-related changes in the protective effect of chronic administration of L-arginine on post-ischemic recovery of endothelial function Eur. J. Cardiothorac. Surg., April 1, 2003; 23(4): 626 - 632. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. B. Feldman The Scientific Evidence for a Beneficial Health Relationship Between Walnuts and Coronary Heart Disease J. Nutr., May 1, 2002; 132(5): 1062S - 1101. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Houghton, E. F. Philbin, D. S. Strogatz, M. T. Torosoff, S. A. Fein, P. A. Kuhner, V. E. Smith, and A. A. Carr The presence of African American race predicts improvement in coronary endothelial function after supplementary L-arginine J. Am. Coll. Cardiol., April 17, 2002; 39(8): 1314 - 1322. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Nakamura, I. Schmidt, C. C. Gray, A. Dewar, S. Rothery, N. J. Severs, M. H. Yacoub, and M. Amrani The effect of chronic L-arginine administration on vascular recovery following cold cardioplegic arrest in rats Eur. J. Cardiothorac. Surg., April 1, 2002; 21(4): 753 - 759. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kihara, S. Biro, M. Imamura, S. Yoshifuku, K. Takasaki, Y. Ikeda, Y. Otuji, S. Minagoe, Y. Toyama, and C. Tei Repeated sauna treatment improves vascular endothelial and cardiac function in patients with chronic heart failure J. Am. Coll. Cardiol., March 6, 2002; 39(5): 754 - 759. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Romero, S. M. Suzuka, R. L. Nagel, and M. E. Fabry Arginine supplementation of sickle transgenic mice reduces red cell density and Gardos channel activity Blood, February 15, 2002; 99(4): 1103 - 1108. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Napoli, V. Guardasole, M. Matarazzo, E. A. Palmieri, U. Oliviero, S. Fazio, and L. Sacca Growth hormone corrects vascular dysfunction in patients with chronic heart failure J. Am. Coll. Cardiol., January 2, 2002; 39(1): 90 - 95. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Maxwell and K. A. Bruinsma Uric acid is closely linked to vascular nitric oxide activity: Evidence for mechanism of association with cardiovascular disease J. Am. Coll. Cardiol., December 1, 2001; 38(7): 1850 - 1858. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Schaefer, F. Piquard, S. Doutreleau, B. Mettauer, E. Epailly, B. Eisenmann, J. Lonsdorfer, and B. Geny Reduced exercise capacity is associated with reduced nitric oxide production after heart transplantation J. Thorac. Cardiovasc. Surg., October 1, 2001; 122(4): 821 - 822. [Full Text] [PDF]
|
|
|
|
 |
|
 A. lannuzzi, G. Jannuzzo, C. Sapio, P. Pauciullo, D. Jorio, N. Spampinato, M. Mancini, and P. Rubba L-Arginine Improves Post-Ischemic Vasodilation in Coronary Heart Disease Patients Taking Vasodilating Drugs Journal of Cardiovascular Pharmacology and Therapeutics, June 1, 2001; 6(2): 121 - 127. [Abstract] [PDF]
|
|
|
|
|
|
N. NAGAYA, M. UEMATSU, H. OYA, N. SATO, F. SAKAMAKI, S. KYOTANI, K. UENO, N. NAKANISHI, M. YAMAGISHI, and K. MIYATAKE Short-term Oral Administration of L-Arginine Improves Hemodynamics and Exercise Capacity in Patients with Precapillary Pulmonary Hypertension Am. J. Respir. Crit. Care Med., March 15, 2001; 163(4): 887 - 891. [Abstract] [Full Text]
|
|
|
|
|
|
A. J. Maxwell, H.-K. V. Ho, C. Q. Le, P. S. Lin, D. Bernstein, and J. P. Cooke L-Arginine enhances aerobic exercise capacity in association with augmented nitric oxide production J Appl Physiol, March 1, 2001; 90(3): 933 - 938. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A.C. Mendes Ribeiro, T.M.C. Brunini, J.C. Ellory, and G.E. Mann Abnormalities in L-arginine transport and nitric oxide biosynthesis in chronic renal and heart failure Cardiovasc Res, March 1, 2001; 49(4): 697 - 712. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Kaye, B. A. Ahlers, D. J. Autelitano, and J. P. F. Chin-Dusting In Vivo and In Vitro Evidence for Impaired Arginine Transport in Human Heart Failure Circulation, November 28, 2000; 102(22): 2707 - 2712. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ishibashi, T. Shimada, T. Sakane, N. Takahashi, T. Sugamori, S. Ohhata, S.-i. Inoue, H. Katoh, K. Sano, Y. Murakami, et al. Contribution of endogenous nitric oxide to basal vasomotor tone of peripheral vessels and plasma B-Type natriuretic peptide levels in patients with congestive heart failure J. Am. Coll. Cardiol., November 1, 2000; 36(5): 1605 - 1611. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. H. Chen, J. A. Grantham, J. A. Schirger, M. Jougasaki, M. M. Redfield, and J. C. Burnett Jr. Subcutaneous administration of brain natriuretic peptide in experimental heart failure J. Am. Coll. Cardiol., November 1, 2000; 36(5): 1706 - 1712. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Wu and C. J. Meininger Arginine Nutrition and Cardiovascular Function J. Nutr., November 1, 2000; 130(11): 2626 - 2629. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. D. Katz, K. Balidemaj, S. Homma, H. Wu, J. Wang, and S. Maybaum Acute type 5 phosphodiesterase inhibition with sildenafil enhances flow-mediated vasodilation in patients with chronic heart failure J. Am. Coll. Cardiol., September 1, 2000; 36(3): 845 - 851. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Oomen, M. J. van Erk, E. J. M. Feskens, F. J. Kok, and D. Kromhout Arginine Intake and Risk of Coronary Heart Disease Mortality in Elderly Men Arterioscler Thromb Vasc Biol, September 1, 2000; 20(9): 2134 - 2139. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Hambrecht, L. Hilbrich, S. Erbs, S. Gielen, E. Fiehn, N. Schoene, and G. Schuler Correction of endothelial dysfunction in chronic heart failure: additional effects of exercise training and oral L-arginine supplementation J. Am. Coll. Cardiol., March 1, 2000; 35(3): 706 - 713. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. J. Osterziel, S. M Bode-Boger, O. Strohm, A. E Ellmer, N. Bit-Avragim, D. Hanlein, M. B Ranke, R. Dietz, and R. H Boger Role of nitric oxide in the vasodilator effect of recombinant human growth hormone in patients with dilated cardiomyopathy Cardiovasc Res, January 14, 2000; 45(2): 447 - 453. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y Kanaya, M Nakamura, N Kobayashi, and K Hiramori Effects of L-arginine on lower limb vasodilator reserve and exercise capacity in patients with chronic heart failure Heart, May 1, 1999; 81(5): 512 - 517. [Abstract] [Full Text]
|
|
|
|
|
|
H. Drexler Endothelium as a Therapeutic Target in Heart Failure Circulation, December 15, 1998; 98(24): 2652 - 2655. [Full Text] [PDF]
|
|
|
|
|
|
R. H. Boger, S. M. Bode-Boger, W. Thiele, A. Creutzig, K. Alexander, and J.u. C. Frolich Restoring vascular nitric oxide formation by L-arginine improves the symptoms of intermittent claudication in patients with peripheral arterial occlusive disease J. Am. Coll. Cardiol., November 1, 1998; 32(5): 1336 - 1344. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Lerman, J. C. Burnett Jr, S. T. Higano, L. J. McKinley, and D. R. Holmes Jr Long-term L-Arginine Supplementation Improves Small-Vessel Coronary Endothelial Function in Humans Circulation, June 2, 1998; 97(21): 2123 - 2128. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Feng, X. Lu, A. J Fortin, A. Pettersson, T. Hedner, R. L Kline, and J.M. O Arnold Elevation of an endogenous inhibitor of nitric oxide synthesis in experimental congestive heart failure Cardiovasc Res, March 1, 1998; 37(3): 667 - 675. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||