Dietary L-arginine Research Articles

Discordant effects of dietary L-arginine on vascular structure and reactivity in hypercholesterolemic rabbits.

We investigated the effect of dietary supplementation of L-arginine (L-Arg), the precursor of endothelial nitric oxide (NO), on endothelium-dependent and endothelium-independent vascular responses, as well as vascular structure, in the abdominal aorta of hypercholesterolemic rabbits. Rabbits were fed (a) normal rabbit chow, (b) 1% cholesterol diet, or (c) 1% cholesterol diet supplemented with 2.25% L-Arg HCl in drinking water. After 10 weeks, the abdominal aorta was harvested for study of vascular reactivity and histomorphometry. L-Arg did not affect serum cholesterol levels. Histomorphometric analysis demonstrated an eightfold reduction in intimal thickening in the abdominal aorta of the arginine-supplemented hypercholesterolemic rabbits. By contrast, the effects on vascular reactivity were subtle. Contraction to norepinephrine (NE) was not altered by hypercholesterolemia or L-Arg. Contraction to acetylcholine (ACh) was increased in hypercholesterolemic animals; this was normalized by dietary arginine supplementation. Relaxation to nitroglycerin (NTG) was not altered by hypercholesterolemia but was attenuated in the arginine-supplemented rabbits. Endothelium-dependent relaxation to ACh was impaired in both hypercholesterolemic groups. Dietary L-Arg has a dramatic antiatherogenic effect in hypercholesterolemic rabbits. This effect is associated with rather slight changes in vascular reactivity that are suggestive of a slight increase in NO elaboration by the endothelium. The discordance between the effects of dietary arginine on vascular structure and reactivity suggests that the antiatherogenic effects of the NO precursor may not be mediated entirely by its effect on the endothelium.

765-1 Platelet Hyperaggregability in Hypercholesterolemic Humans: Reversal by Dietary L-Arginine

We have shown that vascular NO activity is reduced in hypercholesterolemic animals, and that this abnormality can be reversed by dietary L-arginine (Arg) supplementation. A sustained improvement in vascular NO activity is associated with a reduction in platelet reactivity and elevation of platelet cGMP that is reversed by NO synthase inhibition. This study tested the hypothesis that chronic Arg supplementation would inhibit platelet reactivity in hypercholesterolemic humans. Venous blood was collected from normal (NC; n = 11) and hypercholesterolemic (HC; n = 22) volunteers for isolation of plateletrich plasma and aggregometry. Half the HC group received Arg (8.4 g/d) for 2 weeks; aggregometry was performed using collagen (5 μ g/ml) before and after 2 wks of treatment, and after a 2-wk washout. HC platelets were hyperaggregable (71 ± 3 v 56 ± 6 aggregation units [au]; HC v NC; p < 0.03). After 2 wks of Arg, HC platelets were no different in reactivity (64 ± 7 v 61 ± 6 au). This decrease of aggregability was sustained for 2 wks after discontinuing Arg (47 ± 9 v 60 ± 5 au; HC v NC). Figure shows change in au over time in HC-treated v untreated subjects. These studies are consistent with our previous observations in animals that Arg inhibits platelet reactivity. The effect of Arg is likely mediated by its conversion to NO.

765-4 Adhesiveness of Mononuclear Cells is Increased in Hypercholesterolemic Humans, and Reduced by the NO Precursor

Enhanced interaction of mononuclear cells with the endothelium is the first observable event in the development of atherosclerosis. Hypercholesterolemia reduces vascular nitric oxide activity. This dysfunction may promote endothelial monocyte interaction, as NO is a potent inhibitor of adhesion. We have previously shown that dietary L-arginine (Arg) supplementation in hypercholesterolemic rabbits restores NO activity and inhibits monocyte-endothelial cell interaction, in association with a reduction in atherogenesis. Accordingly we developed a functional binding assay to assess the adhesiveness of human mononuclear cells, so as to determine the effects of hypercholesterolemia and L-arginine therapy. We found a 50% increase in adhesion of mononuclear cells (MNC) from hypercholesterolemic (HC) subjects to cultured endothelial cells ex vivo compared to a normocholesterolemic control population (p < 0.0001, n = 20). Increased MNC adhesion was reversed to normal levels by preincubation of the MNC suspension with 10 -5 M sodium nitroprusside (164.4 ± 8.7% vs 97.5 ± 6.9%, P < 0.0005, n = 7), while L-nitroarginine and Arg did not have an effect in vitro . In a double-blinded placebo-controlled study, oral Arg (8.4 g/day) was administered to 7 HC subjects. Over a course of two weeks, this treatment abolished the increased adhesion (158.5 ± 10.9% vs 103.5 ± 4.9% vs 100 ± 5.2%, HCvs Arg vs Control. p < 0.005), while MNC adhesion remained significantly elevated in placebo-treated subjects. The adhesiveness of human mononuclear cells is increased by hypercholesterolemia. The increase in adhesiveness is reversed in vitro by NO donors, and reversed in vivo by treatment with the NO precursor L-arginine.

765-2 Dietary L-Arginine Attenuates Macrophage Infiltration and Intimal Hyperplasia After Balloon Injury

Balloon injury in arteries of hypercholesterolemic animals creates a myointimal lesion that is comprised of macrophages as well as vascular smooth muscle cells. We have shown that chronic administration of L-arginine (Arg) enhances endothelial NO production and inhibits both monocyte-vessel wall interaction and plaque formation. Accordingly, we hypothesized that dietary Arg would reduce lesion formation and macrophage infiltration in hypercholesterolemic animals following balloon injury. NZW rabbits were divided into 3 groups: Normal chow (N), 0.5% cholesterol (Chol), or 0.5% Chol + 2.25% Arg supplementation. Treatment was initiated 6 weeks prior to balloon injury of the left iliac artery. Four weeks after injury, the iliacs were harvested for: assessment of endothelium-dependent relaxation by vasodilation to acetylcholine; histology; and immunohistochemistry to assess macrophage infiltration. Endothelium-dependent relaxation was impaired following injury in the Chol and Arg groups (32 v 4 v 2%, N v Chol v Arg). In the contralateral non-injured arteries, Arg treatment restored endotheliumdependent relaxation (79 v 44 v 64%, N v Chol v Arg). The Chol group showed a 2.5-fold increase in intimal area following injury compared with N, while Arg attenuated lesion formation (0.28 v 0.69 v 0.34 mm 2 , N v Chol v Arg, p < 0.01). Area involved by macrophage increased in the Chol iliacs compared with N, while Arg significantly attenuated macrophage infiltration (2 v 28 v 5%, N v Arg v Chol, p < 0.001). Chronic arginine supplementation reduces intimal proliferation and attenuates macrophage infiltration following balloon injury in hypercholesterolemic animals, and is associated with restored NO-dependent vasodilation.

Dietary L-arginine attenuates blood pressure in mineralocorticoid-salt hypertensive rats.

The present study was designed to investigate the influence of dietary L-arginine supplementation on blood pressure and on ex vivo vascular reactivity in mineralocorticoid-salt (DOCA-salt) hypertensive rats. Systolic blood pressure and heart rate were determined throughout the experimental period in unanaesthetized rats. Plasma and urine electrolyte levels were measured. Vasoconstrictor response to noradrenaline and vasodilator responses to acetylcholine and sodium nitroprusside were evaluated in the isolated perfused mesenteric vascular bed. DOCA-salt hypertensive rats were divided into 2 groups: a control group and a treated group receiving 0.8% L-arginine supplementation in drinking water. Dietary L-arginine supplementation attenuated systolic blood pressure in conscious DOCA-salt hypertensive rats, but did not modify heart rate. Plasma calcium and sodium concentrations and urinary magnesium excretion were decreased by L-arginine supplementation. Noradrenaline-induced vasoconstriction decreased and acetylcholine-induced vasodilatation increased, whereas sodium nitroprusside-induced vasodilatation was not modified, in the L-arginine-supplemented rats. It is concluded that dietary L-arginine supplementation in the diet lowers systolic blood pressure in DOCA-salt hypertensive rats, probably through vascular action.

Arginine-derived nitric oxide reduces fecal oocyst shedding in nude mice infected with Cryptosporidium parvum.

Dietary L-arginine (4%) significantly reduced fecal oocyst shedding in athymic nude mice chronically infected with Cryptosporidium parvum. This effect appeared to be due to an increase in host nitric oxide (NO) production as it was not observed in arginine-supplemented animals administered the NO synthase inhibitor, N-nitro-L-arginine methyl ester. N-Nitro-L-arginine methyl ester alone significantly increased fecal oocyst shedding in chronically infected animals. In in vitro assays, oocyst excystation and sporozoite viability were significantly reduced by the NO donors sodium nitroprusside and S-nitroso-L-acetyl penicillamine in a concentration-dependent manner. These data suggest that arginine-derived NO may reduce the parasite load in experimental cryptosporidiosis.

L-arginine attenuates platelet reactivity in hypercholesterolemic rabbits.

Platelets are capable of producing nitric oxide (NO) through the L-arginine-NO synthase pathway. Acute exposure to supraphysiological concentrations of L-arginine in vitro increases the production of NO by platelets and is associated with an increase in platelet cyclic GMP (cGMP) levels and a reduction in platelet aggregation. The purpose of this study was to determine if chronic oral administration of L-arginine decreases platelet aggregation in hypercholesterolemic animals and to determine if this effect is mediated by the metabolism of L-arginine to NO. Male New Zealand White rabbits were fed normal chow (Con), a 1% cholesterol diet (Chol), or a 1% cholesterol diet supplemented with a sixfold enrichment of dietary L-arginine (Arg) or L-methionine (Met). After 10 weeks, cholesterol levels were equally increased in Chol and Arg animals, whereas plasma arginine levels were doubled in the Arg group. There was no difference in maximum aggregation initiated by ADP (100 mumol/L) between washed platelets from Con, Met, and Chol animals, but aggregation of platelets from Arg animals was significantly decreased (P < .05). In aggregating platelets from Arg animals, cGMP levels were significantly higher than the other groups (P < .05). When platelets were incubated ex vivo with the NO synthase inhibitor NG-monomethyl-L-arginine, the effects of dietary L-arginine were reversed. Chronic dietary supplementation of L-arginine decreases platelet aggregation in hypercholesterolemic rabbits. This effect appears to be due to the metabolism of L-arginine to NO.

Enhanced endothelial adhesiveness in hypercholesterolemia is attenuated by L-arginine.

We have shown that chronic administration of the nitric oxide (NO) precursor L-arginine normalizes NO-dependent vasodilation and markedly inhibits atherogenesis in a hypercholesterolemic rabbit model. We hypothesized that this antiatherogenic effect is due to modulation of endothelial adhesiveness by endothelium-derived NO. New Zealand White rabbits were fed normal chow (Cont), a high-cholesterol diet (Chol), a high-cholesterol diet supplemented with L-arginine (Arg), or a normal diet supplemented with the NO synthase antagonist L-nitroarginine (L-NA) for 2 weeks. In additional studies, some animals receiving L-NA were also treated with hydralazine to normalize blood pressure. After 2 weeks, thoracic aortas were harvested, opened longitudinally, and placed in a culture dish with the endothelial surface exposed to medium containing WEHI 78/24 cells, a monocytoid cell line. After incubation with the monocytoid cells for 30 minutes on a rocking platform, the aortic segments were washed repeatedly to remove nonadherent cells and adherent cells counted by epifluorescent microscopy. Monocytoid cell binding to aortic endothelium was significantly increased in Chol (P < .001 versus Cont); binding was markedly reduced in arginine-fed hypercholesterolemic animals (P < .05, Arg versus Chol). Monocytoid cell binding to aortic endothelium was also significantly increased in L-NA (P < .05); hydralazine normalized blood pressure but did not reduce monocytoid cell binding. To confirm that alterations in NO activity modulate endothelial cell-monocyte interaction, the release of nitrogen oxides (NOx) by thoracic aortas was assessed by a chemiluminescent technique. The concentration of NOx in the conditioned medium from segments of Arg thoracic aortas was significantly greater than that from Cont aortas, whereas that from L-NA aortas was significantly less. Hypercholesterolemia enhances the adhesiveness of aortic endothelium for monocytes; this effect is attenuated by dietary L-arginine. Conversely, inhibition of NO synthesis enhances monocyte binding. The results suggest that endothelium-derived NO plays an important role in regulating the endothelial adhesiveness for monocytes. Alterations in NO activity may play a critical role in atherogenesis.

Dietary arginine prevents atherogenesis in the coronary artery of the hypercholesterolemic rabbit

Objectives. This study was designed to test the hypothesis that long-term oral supplementation of dietary l-arginine (to provide a sustained elevation of nitric oxide activity) would inhibit atherogenesis in hypercholesterolemic rabbits, as assessed by histomorphometric measurements.Background. Endothelium-derived nitric oxide inhibits a number of processes that are critical in atherogenesis. Hypercholesterolemia reduces endothelial nitric oxide activity, and we postulate that this may promote atherogenesis. This reduction in nitric oxide activity can be reversed acutely by intravenous infusion of l-arginine, the precursor of nitric oxide. We show that dietary supplementation of l-arginine abrogates the development of coronary atheroma in hypercholesterolemic rabbits.Methods. Male New Zealand White rabbits were fed normal rabbit chow, 1% cholesterol chow or 1% cholesterol chow with dietary arginine or methionine supplementation to increase their intake of these amino acids sixfold. After 1 or 10 weeks of dietary intervention, the left main and left anterior descending coronary arteries were harvested for histologic study. Plasma cholesterol measurements were elevated to the same degree in all groups of rabbits receiving the 1% cholesterol diet, whereas plasma arginine levels were doubled in the arginine-treated group. High density lipoprotein (HDL) cholesterol values were not affected by arginine treatment.Results. In rabbits receiving the 1% cholesterol diet, with or without methionine supplementation, light and electron microscopy revealed a marked increase from 1 to 10 weeks in the intimal accumulation of macrophages, associated with an increase in the intimal area of the left main coronary artery. By contrast, in arginine-treated hypercholesterolemic rabbits, there was a near absence of adherent monocytes and tissue macrophages and no progression of intimal thickness from 1 to 10 weeks.Conclusions. Dietary supplements of l-arginine prevent intimal thickening in the coronary arteries of hypercholesterolemic rabbits. This antiatherogenic effect is not due to an alteration in plasma total cholesterol, HDL cholesterol or caloric or nitrogen balance. The data are consistent with the hypothesis that nitric oxide has antiatherogenic properties.

Low-dose dietary L-arginine increases plasma interleukin 1α but not interleukin 1β in patients with diabetes mellitus

Oral high-dose arginine supplementation is used for the experimental immunotherapy of tissue trauma and sepsis. Yet the adequate dosage required for immunomodulation has to be established and the toxicity of high-dose arginine has not been fully elucidated. Following a protocol for the treatment of diabetic long-term complications (oral daily doses of 30 mg/kg BW; blind, placebo-controlled prospective study with crossing-over design) we studied plasma levels of interleukins 1α (IL-1α) and 1β reflecting immunostimulation. Arginine supplementation in 29 patients with diabetes mellitus prompted a 2-fold increase of IL-1α from baseline levels ( P < 0.001) while IL-1β was unaffected. Implications for the treated panel of diabetic patients could be a reduction of collagen accumulation by enhanced collagenolysis and clearance of advanced-stage non-enzymatic glycosylation products. Based upon our data, low-dose arginine protocols for further immunotherapeutical studies should be discussed.

Antiatherogenic effects of L-arginine in the hypercholesterolemic rabbit.

The purpose of this study was to determine if chronic administration of L-arginine, the precursor of endothelium-derived relaxing factor (EDRF), normalizes endothelium-dependent relaxation and decreases atherosclerosis in hypercholesterolemic animals. Male rabbits were fed (a) normal rabbit chow; (b) 1% cholesterol diet; or (c) 1% cholesterol diet supplemented by 2.25% L-arginine HCl in drinking water. Arginine supplementation doubled plasma arginine levels without affecting serum cholesterol values. After 10 wk, the thoracic aorta was harvested for studies of vascular reactivity and histomorphometry. Endothelium-dependent relaxations (to acetylcholine and calcium ionophore A23187) were significantly impaired in thoracic aortae from animals fed a 1% cholesterol diet. By contrast, vessels from hypercholesterolemic animals receiving L-arginine supplementation exhibited significantly improved endothelium-dependent relaxations. Responses to norepinephrine or nitroglycerin were not affected by either dietary intervention. Histomorphometric analysis revealed a reduction in lesion surface area and intimal thickness in thoracic aortae from arginine-supplemented animals compared to those from untreated hypercholesterolemic rabbits. This is the first study to demonstrate that supplementation of dietary L-arginine, the EDRF precursor, improves endothelium-dependent vasorelaxation. More importantly, we have shown that this improvement in EDRF activity is associated with a reduction in atherogenesis.

Enhancement of interleukin-2 immunotherapy with L-arginine.

Nutrient substrates have been shown to enhance cell-mediated immunity, but their role as adjuvants to immunotherapy has not been previously determined. This study evaluated L-arginine as an essential substrate for optimal generation of lymphokine-activated killer (LAK) cells. This experiment also assessed supplemental dietary L-arginine as a means to potentiate the host antitumor response to interleukin-2 (IL-2) in a murine neuroblastoma (NRB) model. A/J mice received 1% arginine or isonitrogenous 1.7% glycine in addition to a regular diet 14 days before subcutaneous inoculation with C1300 NRB cells. Twenty-four hours later, animals received low (1 x 10(6) U/kg three times a day) or high (3 x 10(6) U/kg three times a day) doses of IL-2 or saline intraperitoneally for 4 days. On days 4 and 10 post-C1300 NRB inoculation, mice were killed for assessment of natural killer cell and tumor specific cytotoxicity. Remaining animals were followed for tumor incidence, tumor growth, and duration of host survival. Interleukin-2 therapy in mice receiving dietary arginine compared with those receiving glycine resulted in significantly augmented natural killer cell cytotoxicity (day 4) and generation of specific tumoricidal mechanisms (day 10). The addition of dietary arginine to low-dose IL-2 therapy significantly diminished C1300 NRB engraftment (p less than 0.05) and growth (p less than 0.001) and prolonged the duration of host survival (p less than 0.05) compared with the glycine treatment group. In vitro studies demonstrated that L-arginine is an essential substrate for optimal generation of LAK cells. Thus, supplemental dietary L-arginine enhances lymphocyte cytotoxic mechanisms and potentiates IL-2 immunotherapy.

Urinary nitrate excretion in relation to murine macrophage activation. Influence of dietary L-arginine and oral NG-monomethyl-L-arginine.

Murine macrophage oxidation of L-arginine guanidino nitrogen to nitrite/nitrate yields an intermediate effector, possibly nitric oxide, with antimicrobial activity. Total body nitrogen oxidation metabolism (NOM) was measured in vivo by determining the urinary nitrate excretion of mice ingesting a chemically defined nitrite/nitrate-free diet. As reported previously, mycobacterial infection with bacillus Calmétte-Guerin led to a large increase in urinary nitrate excretion. This increase was temporally related to macrophage activation in vivo. The substrate for macrophage nitrogen oxidation metabolism in vitro, L-arginine, was deleted from the diet without ameliorating the urinary nitrate excretion response induced by BCG. This suggested that L-arginine was synthesized endogenously because there are no other known natural substrates for NOM. A competitive inhibitor of NOM, the L-arginine analog, NG-monomethyl-L-arginine was fed to mice in their drinking water. NG-monomethyl-L-arginine ingestion blocked both basal and bacillus Calmétte-Guerin-induced urinary nitrate excretion over a 2-4 week time span. These experimental conditions should prove useful for further investigation on the role of macrophage NOM in host defense against intracellular microorganisms.

Inhibitory Effect of Dietary Arginine on Growth of Ehrlich Ascites Tumor Cells in Mice

The effect of dietary L-arginine on the growth and development of transplantable Ehrlich Ascites tumor cells was examined. Growth of tumor bearing mice was significantly inhibited by feeding a purified casein diet supplemented with 5% arginine. This diet significantly reduced the total number of free tumor cells growing in the peritoneal cavity of mice. Total free tumor cell RNA, DNA, and protein were also significantly reduced. Supplemental arginine approximately doubled the length of time for 50% death of tumor bearing mice. Arginine did not alter respiration as measured by glucose or citrate oxidation. Varying the concentration of supplemental dietary arginine revealed that 3% arginine also significantly retarded the growth of Ehrlich Ascites Tumor Cells. Tumor ornithine decarboxylase activities were significantly reduced by dietary arginine supplementation. Supplemental dietary arginine at 3 or 5% did not significantly affect the growth of non-tumor bearing mice. Dietary arginine may play a critical role in growth of normal as well as neoplastic tissue.

Regulations for Admission to the Army Veterinary Department, 1905.

l-Arginine is a nutritionally essential amino acid for spermatogenesis and plays versatile roles in animal health and can be utilized as a potential agent to improve reproductive performance of boars under high ambient temperature. The present study aimed to determine whether dietary l-arginine could alleviate heat stress-induced infertility in boars. In all, 20 boars (PIC 1040; 248.59±3.84 kg BW and 407.65±6.40 days of age) were selected and randomly assigned to four groups (group 0.0%, basal diet; group 0.6%, 0.8% or 1.0%, basal diet added with 0.6%, 0.8% or 1.0% l-arginine (wt:wt), respectively.) The four diets were made isonitrogenous by addition of appropriate amounts of l-alanine. Boars were pre-fed the corresponding experimental diet for 42 days. Then, the semen characteristics and libido were accessed for 6 weeks during the hot summer period (25.5° to 33.0°C). Results show that dietary l-arginine remarkably improved sperm motility, normality, total sperm number and effective total sperm number. Also, dietary l-arginine improved semen antioxidant capacity, such as decrease of malondialdehyde and 8-Hydroxy-2'-deoxyguanosine content in sperm (P<0.05), increase of the ratio of glutathione and oxidized glutathione, total antioxidant capacity, glutathione peroxidase and catalase activities in seminal plasma (P<0.05). Most of mitochondria contained intact ultrastructure in l-arginine-supplemented group which also accompany with higher ATP content than the 0.0% group. The boars fed 0.8% l-arginine show increased levels of estradiol-17β and testosterone and exhibit improved libido performance than boars in the 0.0% group. Adding dietary l-arginine linearly increased (P=0.002) nitric oxide content (as l-arginine increased). The scrotal surface temperature in the 0.6%, 0.8% and 1.0% group were decreased by 0.9°C, 0.9°C and 0.4°C, respectively, compared with the 0.0% group. l-Arginine levels caused linear effect on semen quality and antioxidant capacity, also caused quadratic effect on libido performance. During the hot summer months, the predicted optimal l-arginine levels for best semen quality and antioxidant capacity was 0.8% to 1.0% and for best libido performance was 0.8%. It can be concluded that l-arginine can be used as an effective agent to alleviate heat stress-induced infertility of boar, and that 0.8% to 1.0% can be considered as the optimum dosage.