(Circulation. 1996;94:498-506.)
© 1996 American Heart Association, Inc.
Articles |
Effects of Dietary L-Arginine on Atherosclerosis and Endothelium-Dependent Vasodilatationin the Hypercholesterolemic Rabbit
Response According to Treatment Duration, Anatomic Site, and Sex
the Department of Medicine (R.W.J., H.M.), University of Sydney, and the Department of Biochemistry (D.S.), Royal Prince Alfred Hospital, Sydney, Australia.
Correspondence to Dr Richmond W. Jeremy, Department of Medicine, Rm 495, Blackburn Building D06, University of Sydney, Sydney, NSW 2006, Australia.
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Abstract |
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Background Nitric oxide (NO) may protect arteries against atherosclerosis. In the present study, we examined whether di-etary L-arginine, the precursor of NO, could chronically preserve endothelium-dependent vasodilatation in vivo and/or limit atherogenesis.
Methods and Results Rabbits were randomized according to sex to receive 2% dietary cholesterol, with or without L-arginine (2.25% solution), for 7 or 14 weeks. Hindlimb vasodilator responses to acetylcholine and nitroprusside were measured with an electromagnetic flow probe. Atherosclerosis was measured with planimetry of aortic lesions stained with Oil-Red-O. In rabbits administered L-arginine, plasma arginine levels increased to 483±30 µmol/L at 3 weeks (mean±SEM, P<.0001 versus control animals) but declined to 224±25 µmol/L at 7 weeks (P=.02) and to 100±23 µmol/L at 14 weeks (NS versus control animals). At 7 weeks, peak hindlimb conductance in response to acetylcholine in cholesterol-fed males was 249±49% of baseline compared with 332±9% in control animals (P=.04), but peak response in arginine-fed rabbits (314±24%) did not differ from that of control animals. At 14 weeks, peak responses to acetylcholine were equally reduced in males fed cholesterol with (266±21%, P=.02 versus control) or without (263±13%, P=.01 versus control) L-arginine. Similar impairment of endothelium-dependent vasodilatation was seen in females at 14 weeks. Vasodilator responses to nitroprusside did not differ from those of control animals in any treatment group. After 14 weeks, atherosclerosis was less in the descending aorta of arginine-fed males (16±4% surface area) than that of males fed cholesterol only (42±8%, P=.04), but no treatment benefit was seen in the ascending aorta or in females.
Conclusions Dietary L-arginine supplementation causes an early rise in plasma arginine levels, with limitation of atherosclerosis in the descending aorta and preservation of endothelium-dependent vasodilatation in resistance arteries, but this treatment effect is not sustained. Dietary L-arginine may not be of long-term benefit in the prevention of atherosclerosis in humans.
Key Words: atherosclerosis • arginine • cholesterol • endothelium • sex
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Introduction |
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Endothelium-derived relaxing factor (EDRF), which is thought to be nitric oxide (NO), appears to play an important role in protecting the arterial wall against injury, including during ischemia and reperfusion.1 2 There is evidence that NO can inhibit platelet aggregation and adhesion3 4 5 and reduce vascular smooth muscle cell proliferation.6 It is possible, therefore, that NO may provide an endogenous defense against atherosclerosis.
Experimental7 8 9 and human10 11 12 13 14 studies show that endothelium-dependent arterial vasodilator responses are abnormal in the presence of atherosclerosis or hypercholesterolemia. Vasodilator responses are abnormal in both the conduit and resistance arteries. Acute administration of the substrate for NO synthase, L-arginine,15 16 has been reported to restore endothelium-dependent vasodilatation in hypercholesterolemic individuals.17 18 19 20 21 22 These observations raise the possibility that chronic administration of L-arginine may preserve endothelium-dependent vasodilatation and even limit development of atherosclerosis in hypercholesterolemic patients. There is evidence that dietary supplementation with L-arginine can partially restore in vitro vasodilator responses to acetylcholine in aortic rings from hypercholesterolemic rabbits.23 Some reduction in the extent of atherosclerosis in the descending aorta23 and in the coronary24 and carotid25 arteries has also been described in rabbits treated with L-arginine. These observations support the hypothesis that NO is antiatherogenic and suggest that dietary supplementation with L-arginine may even be a useful treatment for humans.
Before this potentially beneficial treatment can be applied to humans, a number of additional questions must be answered, such as whether the treatment effect with L-arginine is sustained; whether endothelium-dependent vascular responses are restored in vivo, particularly in the resistance arteries; whether L-arginine uniformly limits atherogenesis throughout the arterial system or there is regional variation in response; and whether treatment effects are similar for males and females. In the present study, we addressed these questions to further evaluate the potential therapeutic use of L-arginine.
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Methods |
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Ethics
The study design was approved by the Animal Research Ethics Committee of the University of Sydney, and the housing and care of the rabbits were supervised by the professional staff of the university animal research facility.
Experimental Groups
Adult New Zealand White rabbits (2.5 to 3.5 kg) were initially fed with standard chow and water ad libitum for 2 weeks during adaptation in the animal research facility. During the study period, rabbits in each treatment group gained weight, with an average weight gain of 4% of baseline weight after 7 weeks. The effects on blood chemistry of L-arginine supplementation were studied in pilot groups of male rabbits fed standard chow plus L-arginine (n=7) or standard chow plus 2% cholesterol plus L-arginine (n=8) for 7 weeks. Blood samples were obtained from an ear vein before the diet and after 1, 3, and 7 weeks of the diet for measurement of cholesterol and arginine levels. All blood samples were obtained at 10:00 to 11:00 AM, and blood lipids were measured with a Hitachi 747 autoanalyzer with the use of enzymatic reagents for cholesterol (1 127 578, Boehringer Mannheim) and triglyceride (1 128 027). Plasma arginine levels were measured after protein precipitation with the use of reverse-phase HPLC with cation exchange and postcolumn derivatization with o-phthaldehyde, followed by fluorescence detection. Subsequently, rabbits were randomly allocated within same-sex groups to receive one of three diets: standard chow ("normal chow" group), standard chow plus 2% cholesterol, or standard chow plus 2% cholesterol plus 2.25% L-arginine in drinking water. The duration of treatment was either 7 weeks (males only) or 14 weeks (males and females). In each group, blood cholesterol and arginine levels were measured in venous blood samples obtained at the time of hemodynamic study.
Measurement of Vascular Reactivity
Hindlimb vascular reactivity was studied in males after 7 and 14 weeks and in females after 14 weeks of dietary treatment and compared with sex-matched control animals. On the day of the study, the rabbits were anesthetized with sodium pentobarbital (45 to 60 mg/kg IV) before being placed on a heating pad warmed to 38°C. The right femoral artery was exposed, and a double-lumen catheter was inserted and advanced to the distal abdominal aorta, just above the bifurcation. The right femoral artery was ligated just distal to the catheter entry site. Systemic arterial pressure was monitored (Statham P23Db) via one lumen, and the other lumen was used for injection of drugs. The left femoral artery was exposed, and an electromagnetic flow probe (Carolina Instruments) was placed around the vessel. Absolute zero reference was established by transient occlusion of the artery, and the presence of brisk hyperemia confirmed that there was no kinking or obstruction of the artery. Arterial pressure and femoral blood flow were recorded continuously on chart paper. The rabbits were anticoagulated for the experiment with 2000 IU heparin IV.
Hindlimb vasodilator responses to acetylcholine and sodium nitroprusside were examined. Acetylcholine (62.5 ng to 25 000 ng) was injected into the distal aorta, and arterial pressure and left femoral artery flow were monitored continuously. The peak flow response to each dose of acetylcholine was recorded. A rest period of at least 2 minutes was allowed between each dose for arterial flow to return to control levels, and blank injections of physiological saline were performed to exclude flow artifacts. On completion of the acetylcholine dose-response study, baseline femoral artery flow and arterial pressure were again recorded before graded doses of sodium nitroprusside (0.25 to 250 µg) were injected into the descending aorta. Peak left femoral artery flow responses to each dose were again recorded, with at least 2 minutes allowed between doses for rest. The effective arterial perfusion pressure to the hindlimb was calculated as the difference between the mean aortic pressure and a zero-flow pressure of 15 mm Hg. Hindlimb conductance was calculated as the quotient of mean femoral artery flow and perfusion pressure. Changes in hindlimb vascular conductance were calculated as the percentage increase in conductance relative to baseline levels in each rabbit.
Measurement of Atherosclerosis
On completion of the vascular reactivity experiments, the rabbits were administered a lethal dose of sodium pentobarbital. The thoracic cavity was opened, and the aorta was removed en bloc from the level of the aortic valve to the diaphragm. After removal of perivascular tissues, the aorta was bisected longitudinally and washed in physiological saline before being stained in 2% Oil-Red-O solution for 5 minutes. The aorta was then washed in 60% isopropyl alcohol and mounted at its in vivo length on a plastic sheet for color photography. Total intimal surface area and the extent of atherosclerosis (area of the red-stained intimal lesions) were measured with computer planimetry. The left subclavian artery was used as the border between the arch and descending aorta. All measurements were made by one investigator, who was blinded to the treatment groups.
Drugs
L-Arginine hydrochloride and acetylcholine chloride were obtained from Sigma Chemical Co, and sodium nitroprusside was obtained from David Bull Labs. Cholesterol was supplied by ICN Biomedicals. Drug solutions were freshly prepared on the morning of each experiment and kept at 4°C until required.
Statistical Analysis
Hemodynamic variables during baseline conditions and during the drug studies were compared with ANOVA with Newman-Keuls comparison of group mean values.26 Dose-response relations for acetylcholine and nitroprusside were compared between treatment groups with two-way ANOVA for repeated measures.27 Only data from rabbits that had complete dose-response relations documented for each drug were included. The extent of atherosclerosis was compared between groups with the use of Student's t test. Group results are reported as mean and SEM. A value of P<.05 was considered statistically significant.
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Biochemistry
Serial measurements of plasma arginine levels in the pilot groups receiving L-arginine in drinking water are shown in Fig 1A
. Baseline plasma arginine levels in normal control animals were 116±14 µmol/L. Dietary L-arginine supplementation resulted in an increase in plasma arginine levels during the first 3 weeks. The initial increase in plasma arginine was similar in rabbits receiving normal chow (424±62 µmol/L, P=.02 versus control animals) and those receiving the cholesterol diet (519±46 µmol/L, P<.001 versus control animals). After 7 weeks, plasma arginine levels had decreased in both groups, although plasma levels were still elevated relative to control animals (normal chow, 211±15 µmol/L; cholesterol-fed, 188±9 µmol/L; both P=.02 versus control animals). The plasma arginine levels in the experimental groups at the time of hemodynamic study are compared in Fig 1B. At 7 weeks, the L-arginine–fed males still had elevated plasma arginine levels (224±25 µmol/L), but after 14 weeks, there was no difference in plasma arginine levels between rabbits fed cholesterol alone and those fed cholesterol plus L-arginine. There were no sex differences in plasma arginine levels.
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The serum cholesterol, triglyceride, and arginine levels in each of the dietary treatment groups at the time of hemodynamic and anatomic study are summarized in Table 1. There were no differences in these parameters between male and female control animals, and the pooled results are given in the Table. After 14 weeks, male rabbits had higher serum cholesterol levels than did those fed for 7 weeks. Females had slightly lower total cholesterol levels than males. Serum cholesterol levels did not differ between groups with and those without L-arginine supplementation. In rabbits receiving L-arginine for 7 weeks, plasma arginine levels were approximately twice control levels (P=.02) at the time of hemodynamic study. In contrast, rabbits receiving L-arginine for 14 weeks had plasma arginine levels that were similar to control values at the time of hemodynamic study.
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Hindlimb Vascular Conductance
Baseline hemodynamic data for treatment groups after 14 weeks are shown in Table 2. Hemodynamic variables after 7 weeks were similar to those after 14 weeks. Baseline systemic arterial pressures and heart rates were comparable in the control group and the cholesterol-fed groups, but baseline heart rate and mean arterial pressure were slightly lower in the arginine-fed males (Table 2). There was a small decrease in both mean arterial pressure and heart rate after acetylcholine studies in each group.
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Changes in hindlimb conductance in response to acetylcholine are shown for males (Fig 2). Among control animals, the peak conductance response was 332±9% of baseline conductance. After 7 weeks (Fig 2A), hypercholesterolemic rabbits without L-arginine had a peak conductance response of 249±49% (P=.04 versus control animals). In contrast, those receiving L-arginine had a peak response (314±24%) that did not differ significantly (NS) from that of control animals. After 14 weeks (Fig 2B), the peak conductance response to acetylcholine was equally impaired in hypercholesterolemic rabbits with L-arginine (266±21%, P<.05 versus control animals) and those without L-arginine supplement (263±13%, P<.01 versus control animals).
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The effects of nitroprusside on hindlimb vascular conductance are shown for males (Fig 3). In the control animals, the peak vasodilator response to nitroprusside was an increase in conductance to 372±22% of baseline conductance. In the hypercholesterolemic rabbits, the dose-response curves to nitroprusside did not differ significantly from those of control animals. After 7 weeks (Fig 3A), peak conductance in those without L-arginine was 313±22% of baseline (NS versus control animals), and in those with L-arginine, the peak response was 351±195 (NS versus control). After 14 weeks (Fig 3B), hypercholesterolemic rabbits without L-arginine had a peak conductance of 362±20%, and those with L-arginine had a peak conductance of 317±40% (both NS versus control animals).
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The responses of the hindlimb vasculature to acetylcholine and nitroprusside are shown for females (Fig 4). In female control animals, the peak response to acetylcholine was 335±18% of baseline, and the dose-response curve did not differ significantly from that of male control animals (Fig 4A). In cholesterol-fed females, the peak response to acetylcholine was reduced to 251±11% (P<.01 versus control animals). As with the males, dietary L-arginine did not appear to preserve vasodilator responses to acetylcholine in hypercholesterolemic females after 14 weeks. The peak vasodilator response to acetylcholine in females fed L-arginine was 241±23% (P<.05 versus control animals). The dose-response curves to nitroprusside were similar in male and female control animals (Fig 4B). The peak vasodilator response to nitroprusside was 313±22% in those not fed L-arginine and 351±19% in those fed L-arginine (both NS versus control animals).
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Anatomic Extent of Atherosclerosis
A comparison of the proportions of intimal surface area covered by atherosclerotic lesions for the different treatment groups is given in Fig 5. Among males, the extent of atherosclerosis in the ascending aorta after 7 weeks was similar in those with and those without L-arginine treatment. The extent of atherosclerosis in the descending aorta of those receiving L-arginine (12±4% of surface area) was less than that in those receiving cholesterol alone (28±8%), but the difference was not significant (P=.15). Atherosclerosis was more extensive after 14 weeks, but treatment with L-arginine did not reduce atherosclerosis in the ascending aorta. In the descending aorta, the extent of atherosclerosis was less in the group fed L-arginine (16±4%) than in the group fed cholesterol only (42±8%, P=.037). Among females, the extent of atherosclerosis in the ascending aorta was similar to that observed in males. No reduction in atherosclerosis was seen in females receiving L-arginine. In the descending aorta, the extent of atherosclerosis after 14 weeks (16±4%) was less than that observed in males, but no reduction in atherosclerosis was evident in the females fed L-arginine (16±2%).
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Discussion |
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In the present study, we evaluated the potential therapeutic use of dietary L-arginine supplementation to prevent atherosclerosis and maintain normal endothelium-dependent vascular reactivity in the presence of hypercholesterolemia. Although L-arginine treatment increases plasma arginine levels and appears to preserve endothelium-dependent vasodilatation, this beneficial treatment response is not maintained. After 14 weeks, plasma arginine levels had returned to control values despite continued oral supplementation, and endothelium-dependent vasodilatation was equally impaired in hypercholesterolemic rabbits with and without L-arginine supplementation. Atherosclerosis was reduced in males, but this was limited to the descending thoracic aorta, and no benefit was seen in females.
Arginine Metabolism
The first major finding of this study was that chronic dietary L-arginine supplementation does not result in a sustained increase in plasma arginine levels. We employed the same L-arginine dose that was used in previous studies,23 25 and plasma arginine levels increased markedly during the first 3 weeks of treatment, to levels similar to those reported previously. After 7 weeks, plasma arginine levels had declined but were still approximately twice control levels, but after 14 weeks, there was no difference between control animals and arginine-fed animals. It is noteworthy that Cooke et al23 also found a nonsignificant difference in plasma arginine levels between control and arginine-fed groups after 10 weeks.
Our findings suggest that a homeostatic mechanism restores plasma arginine levels to control values, despite continued oral intake of excess L-arginine. These findings are supported by those of Boger et al,28 who fed L-arginine to rats for 5 months. After an initial increase, urinary excretion of NO3- (as an index of NO metabolism) decreased to control levels despite continued treatment with L-arginine. The two principal mechanisms of arginine homeostasis, other than metabolism by nitric oxide synthase, appear to be metabolism by arginase29 30 and altered urinary excretion. Studies in adult humans showed that total body arginine homeostasis is related to the rate of degradation by arginase,31 and experimental studies indicated that the activity of hepatic arginase is directly related to the concentration of arginine substrate.32 33 Excess arginine intake is also associated with increased urinary excretion of the amino acid, as the renal tubular reabsorption of arginine exhibits a transport maximum.34 When pigs were fed excess arginine, a marked increase in urinary arginine excretion was observed.35 In humans, acute intravenous loading with L-arginine resulted in a marked increase in plasma arginine levels, which rapidly returned to normal after 3 hours, but oral loading resulted in only a small increase in plasma arginine levels.36
Endothelium-Dependent Vasodilatation
The second major finding of this study was that dietary L-arginine supplementation results in only temporary preservation of endothelium-dependent vasodilatation in vivo. Numerous studies have demonstrated abnormal endothelium-dependent vasodilator responses in both conduit and resistance arteries of hypercholesterolemic animals7 8 9 and humans.10 11 12 13 14 As have most other groups, we have used acetylcholine as a pharmacological stimulus to endothelial NO production. Some clinical data suggest that abnormal vasodilator responses to acetylcholine may result from an abnormality of the muscarinic receptor rather than NO metabolism.10 However, subsequent studies that also showed abnormal vasodilator responses to substance P indicate a more generalized abnormality of endothelial NO metabolism in hypercholesterolemic individuals.37
Acute administration of L-arginine can restore endothelium-dependent vasodilatation both in vitro and in vivo.17 18 19 20 21 22 Acute infusion studies are, however, associated with very high and nonphysiological blood arginine levels, and some of the acute vasodilator response to L-arginine observed in these studies may be due to a direct, endothelium-independent mechanism.38 39 More recently, two groups examined the effects of long-term dietary L-arginine supplementation on endothelium-dependent vasodilatation in vitro. In the first study, Cooke et al23 showed that dietary L-arginine supplementation was associated with partial restoration of endothelium-dependent vasodilatation of aortic ring segments. Similar findings were reported by Boger et al,25 although these investigators found more severe impairment of vascular reactivity in hypercholesterolemic animals than did Cooke et al. These findings are provocative, but their interpretation is tempered by the observation that L-arginine treatment was also associated with less intimal thickening in the aorta. The better vasodilator responses in the L-arginine–treated group may therefore reflect less structural abnormality of the aortic wall.
The present study differs from the two previous di-etary studies in that vascular reactivity was examined in vivo and that vascular responses were studied in the hindlimb resistance vessels to avoid any confounding influence of atherosclerosis. Our findings support the hypothesis that an increase in plasma arginine levels is associated with preservation of endothelium-dependent vasodilatation. Unfortunately, although a beneficial treatment effect was observed at 7 weeks, the subsequent decline in plasma arginine levels was paralleled by impairment of endothelium-dependent vasodilatation at 14 weeks. A recent study by Singer et al40 showed only a slight effect of 10 weeks of L-arginine treatment on endothelium-dependent vasodilatation in ring segments from the abdominal aorta. Although the authors did not report plasma arginine levels, their findings are consistent with the decline in plasma arginine levels documented in the present study.
The mechanisms by which endothelium-dependent vasodilatation is impaired in the presence of hypercholesterolemia and how it may be restored by L-arginine remain uncertain. The two major possibilities are substrate limitation of NO synthesis and accelerated degradation of NO by oxygen radicals. There are reasons to doubt a direct influence of plasma arginine levels on the rate of endothelial NO synthesis in vivo. First, in cultured bovine endothelial cells, intracellular arginine concentrations of 200 to 800 µmol/L41 42 are greatly in excess of the Km for NO synthase,43 and intracellular arginine concentrations in vivo may be even higher.42 Second, kinetic studies with [14C]L-arginine suggest that the intracellular concentration of arginine is not rate limiting for NO synthase.44 Third, recycling of the 
-amino group from citrulline to arginine may be sufficient to maintain adequate intracellular concentrations of arginine for NO synthesis,45 and endothelial cells incubated in arginine-free medium remain capable of NO synthesis for prolonged periods of time.15 41 Nevertheless, there is evidence that L-arginine supplementation does result in increased endothelial NO synthesis,25 but this may not reflect a substrate effect of L-arginine for NO synthase. It appears that L-arginine can independently reverse an inhibitory effect of L-glutamine on NO synthase,46 and this could explain the observations of increased NO synthesis.
The hypothesis that NO production is substrate limited in hypercholesterolemic individuals is also questionable based on bioassay experiments, which show that NO production is actually increased in the arteries of hypercholesterolemic rabbits.47 48 Impaired endothelium-dependent vasodilator responses might be due to accelerated degradation of NO, possibly through a reaction with oxygen free radicals49 50 or oxidized LDL particles.51 52 There is evidence that superoxide production is increased in the aortas of hypercholesterolemic rabbits53 54 and that treatment with L-arginine is associated with a reduction in vascular superoxide release.25 In addition, L-arginine or NO may reduce oxidation of LDL55 56 and thereby reduce degradation of endothelial NO. These potential antioxidant effects may represent a mechanism of benefit from L-arginine treatment that is distinct from its function as a substrate for endothelial NO synthesis.
Limitation of Atherosclerosis
The third major finding of this study is that dietary supplementation with L-arginine was associated with less atherosclerosis in the descending aorta of male rabbits. After 7 weeks, some reduction in atherosclerosis was seen in the L-arginine group, but the treatment effect was not statistically significant, most likely reflecting the limited degree of atherosclerosis in both groups at this stage. After 14 weeks, atherosclerosis had progressed in both the cholesterol and cholesterol plus L-arginine groups, but a significant treatment effect of L-arginine was observed. This latter finding is concordant with that of previous studies,23 25 and the degree of protection appears to be similar to the previous findings.
The mechanism of the apparent benefit of L-arginine in limiting atherosclerosis also is not yet defined, but it may involve modulation of macrophage NO synthesis or reduction in oxidation of LDL. The monocyte/macrophage system plays a central role in the pathogenesis of atherosclerosis. Although dietary L-arginine has been shown to reduce monocyte adhesion to the endothelium of hypercholesterolemic rabbits after 2 weeks,57 it is unknown whether this effect persists in the long term. The protective effect of L-arginine may not be mediated by increased endothelial NO synthesis but rather by increased NO synthesis in the macrophages. Unlike the endothelium, the rate of NO synthesis in macrophages is dependent on extracellular concentrations of L-arginine,58 59 60 and macrophages cannot recycle citrulline to arginine.61 There is evidence that NO synthesized by the macrophage may inhibit oxidative modification of LDL and thereby reduce their atherogenic potential.55 56 The role of macrophage NO synthesis should be viewed with caution, as, under some circumstances, macrophage NO can promote oxidative modification of LDL62 and synthesis of large amounts of NO by the macrophage is associated with tissue injury.63 Unrestrained synthesis of NO by macrophages therefore may not be entirely beneficial.
Although L-arginine treatment appears to be associated with limitation of atherosclerosis, it is quite possible that this is only a temporary phenomenon. If increased plasma arginine levels do influence macrophage NO metabolism or platelet adhesion to the endothelium, then this beneficial effect will be lost after plasma arginine levels return to control values. It is quite possible that with longer periods of hypercholesterolemia, the extent of atherosclerosis in the arginine-fed group would catch up with that of the cholesterol-only group.
Despite the apparent benefit on atherosclerosis in the descending aorta, there was no evidence of limitation of atherosclerosis in the ascending aorta, even at 7 weeks. It is noteworthy that a previous study also reported greater treatment benefit in the more distal regions of the descending aorta.23 This may be due to regional variation in activity of endothelial NO synthase in the aorta, and there is some evidence from studies in the rat aorta that such variation does exist.64 Alternatively, hemodynamic stress in the ascending aorta and arch, with consequent microinjury of the endothelium, may be so great that any benefit from L-arginine on regional NO metabolism is insufficient to overcome this injury, and development of atherosclerosis proceeds rapidly.
Sex and L-Arginine Response
The degree of impairment of endothelium-dependent vasodilatation was similar in male and female rabbits after 14 weeks, and no preservation of vascular reactivity was seen in the arginine-treated females. The extent of atherosclerosis in the ascending aorta was similar in the two sexes, but atherosclerosis in the descending aorta was much less in females. Unlike the males, there was no evidence of a treatment benefit of L-arginine in females. This may simply reflect a smaller expected treatment effect in animals with less extensive disease, but there is also evidence for sex differences in NO metabolism in the aorta. In both rats65 66 and rabbits,67 aortic endothelial NO synthesis in the basal state and in response to pharmacological stimuli is greater in females than in males. This difference is particularly marked in the descending aorta and appears to be related to the plasma estradiol concentration.68 The effects of estrogens on NO metabolism in the endothelium, and possibly at other sites, may outweigh any effect of excess L-arginine.
Conclusions
The results of the present study support a limited benefit of dietary L-arginine supplementation in preserving endothelium-dependent vasodilatation in vivo and in limiting atherosclerosis in hypercholesterolemic males, but the treatment effect is not sustained. Clinical trials of L-arginine or pharmacological NO donors do not appear to be warranted until sustained preservation of endothelium-dependent vasodilatation and limitation of atherosclerosis can be demonstrated. Future studies should also address the issue of whether L-arginine or NO donors can attenuate progression of preexisting early atherosclerotic lesions, as exist in humans.
In recent years, impaired endothelium-dependent vasodilatation has been interpreted as a marker of early ath-erosclerosis, and changes in endothelium-dependent vascular reactivity might serve as markers of changes in the severity of atherosclerosis. The disparities between abnormal endothelium-dependent vasodilatation in the microvasculature and the extent of atherosclerosis in the aorta seen in the present study suggest that different mechanisms may be responsible for the two phenomena. This issue has also been raised by the findings of Singer et al.40 These observations suggest that changes in endothelium-dependent vasodilatation may not be a useful clinical marker of progression or otherwise of atherosclerotic disease in humans.
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This work was supported by grant G91S3298 from the National Heart Foundation of Australia.
Received November 28, 1995; revision received January 16, 1996; accepted January 22, 1996.
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