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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1557.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Plasma Concentration of Asymmetric Dimethylarginine, an Endogenous Inhibitor of Nitric Oxide Synthase, Is Elevated in Monkeys With Hyperhomocyst(e)inemia or Hypercholesterolemia

Rainer H. Böger; Stefanie M. Bode-Böger; Karsten Sydow; Donald D. Heistad; Steven R. Lentz

From the Institute of Clinical Pharmacology (R.H.B., S.M.B.-B., K.S.), Medical School, Hannover, Germany, and the Departments of Internal Medicine (D.D.H., S.R.L.) and Pharmacology (D.D.H.), University of Iowa College of Medicine, and Veterans Affairs Medical Center (D.D.H., S.R.L.), Iowa City, Iowa.

	Abstract

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Abstract—Hyperhomocyst(e)inemia is associated with endothelial dysfunction. Mechanisms responsible for endothelial dysfunction in hyperhomocyst(e)inemia may involve impaired bioavailability of endothelium-dependent nitric oxide. We tested the hypothesis that hyperhomocyst(e)inemia is associated with an elevated plasma concentration of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase. One group of adult cynomolgus monkeys was fed either a control or hyperhomocyst(e)inemic diet for 4 weeks in a randomized crossover design. The second group was fed an atherogenic diet that produces both hyperhomocyst(e)inemia and hypercholesterolemia for 17 months, followed by an atherogenic diet supplemented with B vitamins for 6 months to decrease plasma homocyst(e)ine concentration. Human endothelial cells were used to study the effects of methionine and homocysteine in the presence or absence of B vitamins or the methylation inhibitor S-adenosylhomocysteine on the formation of ADMA and its inactive stereoisomer, symmetric dimethylarginine. The hyperhomocyst(e)inemic diet produced 2- to 3-fold increases in plasma levels of homocyst(e)ine and ADMA (both P<0.05). The atherogenic diet also produced elevated plasma levels of homocyst(e)ine and ADMA (both P<0.05). Supplementation of the atherogenic diet with B vitamins decreased the plasma levels of homocyst(e)ine but did not affect the plasma levels of ADMA or endothelial function. There was a strong correlation between plasma ADMA and homocyst(e)ine and a strong inverse correlation between ADMA and carotid artery relaxation to acetylcholine. ADMA release by cultured endothelial cells was significantly increased in the presence of methionine or homocysteine. This effect was blocked by S-adenosylhomocysteine but not by B vitamins. We conclude that plasma levels of ADMA are elevated in hyperhomocyst(e)inemia. Because ADMA acts as a competitive inhibitor of endothelial nitric oxide synthase, these findings suggest a novel mechanism for impaired endothelial function in hyperhomocyst(e)inemia.

Key Words: nitric oxide • endothelium • asymmetric dimethylarginine • homocysteine

	Introduction

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Hyperhomocyst(e)inemia, which is a risk factor for atherosclerotic vascular disease, is associated with endothelial dysfunction in monkeys¹ and humans.^{2 3} (The term "hyperhomocyst(e)inemia" is used in this article to indicate that plasma homocyst(e)ine assays measure the total concentration of thiol, disulfide, and mixed disulfide adducts of homocysteine.) Mechanisms that produce endothelial dysfunction in hyperhomocyst(e)inemia are not clear but may involve a direct toxic effect of homocyst(e)ine on endothelial cells⁴ and oxidative inactivation of nitric oxide (NO) by homocyst(e)ine.⁵ Another mechanism that may contribute to endothelial dysfunction in atherosclerosis is the generation of asymmetric dimethylarginine (ADMA), an endogenous competitive inhibitor of NO synthase.⁶

Plasma levels of ADMA and its biologically inactive stereoisomer, symmetric dimethylarginine (SDMA), are elevated in hypercholesterolemic rabbits.^{7 8} Elevation of ADMA is associated with reduced activity of NO synthase in this animal model.⁸ Similar observations have been made in patients with peripheral arterial disease and generalized atherosclerosis⁹ and in hypercholesterolemic humans.¹⁰

Dimethylarginines are probably formed from the degradation of methylated proteins.¹¹ A major source of methyl groups used for various methylating reactions is the demethylation of methionine to homocysteine.¹² This methyl group may then be transferred, directly or indirectly, to L-arginine to yield N^G,N^G-dimethyl-L-arginine (ADMA) and/or N^G,N^G'-dimethyl-L-arginine (SDMA).

In the present study, we tested the hypothesis that moderate diet-induced hyperhomocyst(e)inemia is associated with increased plasma concentration of ADMA in cynomolgus monkeys. We measured plasma concentrations of ADMA and determined whether there is an association between elevated ADMA concentration and endothelial dysfunction in monkeys fed a hyperhomocyst(e)inemic diet, an atherogenic diet [which induces hypercholesterolemia and moderate hyperhomocyst(e)inemia], an atherogenic diet supplemented with B vitamins [to decrease homocyst(e)ine levels], or a control diet.^{1 13}

Endothelial cells are capable of synthesizing ADMA and, in minor amounts, SDMA.^{14 15} Therefore, we sought to determine whether the formation of ADMA and SDMA by cultured human endothelial cells is increased in the presence of high concentrations of methionine or homocysteine and whether inhibition of S-adenosylmethionine–dependent methylases reduces this effect. We also investigated the effect of B vitamins on ADMA formation in the presence of elevated homocysteine concentrations in cell cultures.

Our results indicate that plasma concentrations of ADMA are elevated in monkeys with hyperhomocyst(e)inemia and/or hypercholesterolemia and that plasma levels of ADMA correlate strongly with endothelial dysfunction. We have also demonstrated in the present study that human endothelial cells are a source of ADMA and that ADMA formation by endothelial cells is increased in the presence of high methionine or homocysteine levels in a manner reversible by the methylation inhibitor S-adenosylhomocysteine.

	Methods

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Animals and Experimental Protocol
Sixteen adult cynomolgus monkeys (Macaca fascicularis) were studied in 2 separate groups. Details of the study protocols have been published previously.^{1 13} In group 1, 7 monkeys were assigned to receive, in a randomized crossover design, either a control diet (Purina Monkey Chow, Ralston-Purina) or a hyperhomocyst(e)inemic diet enriched in methionine (1.0 g/100 g), relatively depleted of folic acid (0.15 g/100 g), and free of choline, for 4 weeks, followed by the other diet for 4 weeks. In group 2, 9 monkeys were assigned to receive an atherogenic diet for 17 months, followed by an atherogenic diet supplemented with B vitamins (5 mg folic acid, 400 µg vitamin B₁₂, and 20 mg vitamin B₆ daily) for 6 months. The atherogenic diet contained 43% of total calories as fat, 0.7% as cholesterol, and small amounts of B vitamins (1.0 µg vitamin B₁₂, 0.75 mg vitamin B₆, and <25 µg folic acid daily).

After the first experimental period of both studies (4 weeks in group 1 and 17 months in group 2), the animals were sedated with ketamine hydrochloride (25 mg/kg IM) and anesthetized with sodium pentobarbital (30 mg/kg IV). A tracheotomy was performed, and the animals were intubated and ventilated with room air and supplemental oxygen. Venous blood was drawn for the measurement of plasma total cholesterol, homocyst(e)ine, L-arginine, and dimethylarginine concentrations. A nonobstructive multiple–side-hole catheter equipped with a Doppler transducer was inserted into the right femoral artery and positioned in the distal aorta, and the right femoral vein was cannulated for administration of supplemental anesthesia (15 mg/kg IV pentobarbital as needed). Heart rate, respiration, and blood pressure were monitored continuously. Changes in blood flow to the legs were measured in response to intra-arterial infusion of collagen (150 mg/min for 10 minutes) or intra-arterial injection of acetylcholine (3x10^-8, 10^-7, and 3x10^-7 mol/L) by quantitative angiography and Doppler measurement of hindlimb flow velocity. Collagen activates platelet aggregation in vivo,¹⁶ which causes greater reduction of peripheral blood flow when endothelium-mediated inhibition of platelet activation is impaired than when endothelial function is normal.¹⁶ Cineangiograms of the distal descending aorta and the left iliac artery were obtained as described previously.¹ Quantification of arterial lumen diameter was performed by the use of computerized arterial lumen edge detection software as described previously.¹⁶ Velocity of blood flow to the leg was measured with a Doppler transducer at the time of angiography. By measuring the velocity of flow (by Doppler) and aortic mean diameter (by angiography), blood flow to the leg was calculated.

At the end of the procedure, 1 common carotid artery was surgically exposed and ligated proximally and distally, and the isolated segment of artery was removed and placed in oxygenated Krebs’ solution. Removal of 1 carotid artery did not produce stroke or other adverse effects in any monkey and did not alter arterial blood pressure. After the second experimental period (8 weeks in group 1 and 23 months in group 2), animals were anesthetized again, another venous blood sample was drawn, and measurement of the vasomotor response to collagen infusion in vivo was repeated. The remaining carotid artery was removed and placed in oxygenated Krebs’ solution. The animals were killed by administration of sodium pentobarbital (200 mg/kg IV) and exsanguinated. The study protocol was approved by the University of Iowa Animal Care and Use Committee.

Cell Culture
The spontaneously transformed human umbilical vein endothelial cell line ECV304 (American Type Culture Collection)¹⁷ was cultured in medium 199 (GIBCO-BRL) containing 10% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin (GIBCO-BRL). This cell line retains many of the characteristics of primary endothelial cells, including synthesis of angiotensin-converting enzyme, NO, and prostacyclin, and the expression of endothelial adhesion molecules, including intercellular adhesion molecule-1 and lymphocyte function–associated antigen-3. ECV304 cells were maintained in medium 199 until they reached confluence. The cells were washed, and fresh medium was added. Cells were then maintained in culture for an additional 24 hours in the absence or presence of methionine (1x10^-4 to 4x10^-4 mol/L), DL-homocysteine (10^-3 mol/L), DL-homocysteine plus S-adenosylhomocysteine (10^-4 mol/L), or DL-homocysteine plus B vitamins (10^-3 mol/L pyridoxine, 10^-4 mol/L cobalamine, and 10^-4 mol/L folic acid) before the cells and media were harvested for measurement of ADMA and SDMA levels.

Biochemical Analyses
Concentrations of L-arginine and dimethylarginines in plasma and in cell supernatants were determined by high-performance liquid chromatography (HPLC) with precolumn derivatization with o-phthalaldehyde by a modification of a previously described method.⁷ Samples were spiked with 10 µmol/L homoarginine as an internal standard and extracted on CBA solid-phase extraction cartridges (Varian). The eluates were dried under nitrogen, and residues were dissolved in bidistilled water for HPLC analysis. HPLC was carried out on a liquid chromatography system (Gynkotek) consisting of 2 HPLC pumps with a gradient controller (model M 480 HDG), a spectral fluorescence detector RF 1002, and an automatic injector (model GINA 160). Samples and standards were incubated for exactly 30 seconds with the o-phthalaldehyde reagent (5.4 mg/mL o-phthalaldehyde in borate buffer, pH 8.5, containing 0.4% 2-mercaptoethanol) before automatic injection into the HPLC system. Chromatographic separation was performed on a C₆H₅ column (Macherey and Nagel) with the fluorescence monitor set at excitation and emission wavelengths of 340 and 455 nm, respectively. Samples were eluted from the column isocratically with 0.96% citric acid/methanol (2:1 [vol/vol], pH 6.8) at a flow rate of 1 mL/min. The coefficients of variation of this method were 5.2% for within-assay determination and 5.5% for between-assay determination; the detection limit of the assay was 0.1 µmol/L.

Fasting plasma homocyst(e)ine concentrations were measured by HPLC and electrochemical detection, according to the method of Smolin and Schneider,¹⁸ as previously described.^{1 19}

Plasma total cholesterol levels were determined by using methods established by the Lipid Research Centers and standardized by the Centers of Disease Control as described previously.²⁰

Endothelium-Dependent Vascular Function Ex Vivo
After removal of loose connective tissue, the common carotid artery was cut into multiple rings, each 5 mm wide. Carotid artery rings were suspended in organ chambers containing oxygenated Krebs’ buffer maintained at 37°C and connected to force transducers to measure changes in isometric tension. Rings were precontracted to a tension of 1.0 g by stepwise addition of prostaglandin F₂ (1 to 3 µmol/L), and relaxation concentration response curves were generated by cumulative addition of acetylcholine or sodium nitroprusside (each 1 nmol/L to 10 µmol/L).

Calculations and Statistical Analyses
Data are given as mean±SEM. Statistical significance was tested by ANOVA, followed by the Fisher protected least significant difference test. Linear regression curves and correlation coefficients were calculated according to the least squares method. Multiple regression analysis was performed for endothelium-dependent vasodilation ex vivo, with cholesterol, ADMA, and homocyst(e)ine concentrations used as independent variables. A linear ADMA-homocyst(e)ine interaction term was also included as an independent variable to assess the potential interaction between both molecules on endothelial function. Statistical significance was accepted at P<0.05.

	Results

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Effects of Hyperhomocyst(e)inemic Diet on Plasma Homocysteine, Total Cholesterol, L-Arginine, and ADMA Levels
The hyperhomocyst(e)inemic diet increased plasma homocyst(e)ine levels 2.7-fold compared with the control diet (P<0.05, Table). Plasma total cholesterol concentrations were not significantly different between these groups (Table).

View this table: [in this window] [in a new window]   Table 1. Plasma Concentrations of Homocyst(e)ine, Total Cholesterol, and L-Arginine

Plasma ADMA levels were elevated 3-fold in animals fed the hyperhomocyst(e)inemic diet (P<0.05, Figure 1A). Plasma SDMA levels did not differ significantly between the 2 groups of animals (Table). Plasma concentrations of L-arginine were elevated during the hyperhomocyst(e)inemic diet (P<0.05, Table). The L-arginine/ADMA ratio was significantly lower in the group fed the hyperhomocyst(e)inemic diet (P<0.05, Figure 1B).

View larger version (31K): [in this window] [in a new window]   Figure 1. A, Plasma concentrations of the endogenous NO synthase inhibitor ADMA. B, Plasma L-arginine/ADMA ratios. Monkeys were fed hyperhomocyst(e)inemic (HyperHC) or control diets or an atherogenic (AS) diet with or without supplemental B vitamins (Vit). Data are mean±SEM of 7 to 9 animals in each group. *P<0.05 vs control.

Effects of Atherogenic Diet and B Vitamin Supplementation on Plasma Homocyst(e)ine, Total Cholesterol, L-Arginine, and ADMA Levels
The atherogenic diet increased plasma total cholesterol and homocyst(e)ine concentrations (Table).¹³ B vitamin supplementation decreased homocyst(e)ine to control levels but did not significantly affect total cholesterol levels (Table).

ADMA levels were elevated in monkeys fed the atherogenic diet and remained unchanged after B vitamins were added to the diet (Figure 1A). Plasma concentrations of L-arginine and SDMA were not significantly changed by the atherogenic diet with or without supplemental vitamins (Table). The L-arginine/ADMA ratio was significantly decreased in these 2 groups compared with monkeys fed the control diet; vitamin supplementation did not affect this ratio (Figure 1B).

Regression Analysis of Plasma ADMA and Homocyst(e)ine Concentrations
There was a significant correlation between plasma ADMA concentration and plasma homocyst(e)ine concentration in monkeys fed the hyperhomocyst(e)inemic or control diet (R=0.83, P=0.01; n=14; Figure 2A). When all animals were included in the analysis, the correlation was also significant (R=0.40, P=0.02; n=32; Figure 2B).

View larger version (15K): [in this window] [in a new window]   Figure 2. A, Correlation between plasma homocyst(e)ine and ADMA concentrations in monkeys fed control or HyperHC diets. B, Correlation between plasma homocyst(e)ine and ADMA concentrations in monkeys fed 4 different dietary regimens.

Regression Analysis of Plasma ADMA Concentration and Endothelium-Dependent Vasodilator Function Ex Vivo
Isolated carotid arteries from monkeys showed a concentration-dependent relaxation response to acetylcholine, and maximum acetylcholine-induced relaxation was significantly impaired in carotid arteries from hyperhomocyst(e)inemic animals compared with control animals.¹ Maximum relaxation was 80.7±7.7% in monkeys fed the control diet, 48.7±12.2% in monkeys fed the hyperhomocyst(e)inemic diet (P<0.05 versus the control diet), and 53.3±9.4% in monkeys fed the atherogenic diet without supplemental B vitamins.^{1 13} Supplementation of the atherogenic diet with B vitamins did not improve maximum relaxation to acetylcholine (54.6±9.4%), although sensitivity to the lowest concentrations of acetylcholine was modestly increased.¹³

In simple regression analysis, a strong inverse correlation was found between ADMA plasma concentration and maximum acetylcholine-induced vasodilation of carotid artery ex vivo for monkeys fed control or hyperhomocyst(e)inemic diets (R=0.65, P=0.01; n=14). A significant inverse correlation was also observed when data from all animals were included in the analysis (R=0.50, P=0.004; n=32; Figure 3A).

View larger version (18K): [in this window] [in a new window]   Figure 3. A, Correlation between plasma ADMA concentration and endothelium-dependent relaxation of isolated carotid artery segments in response to 10 µmol/L acetylcholine (ACh) ex vivo from monkeys on 4 different dietary regimens. B, Correlation between plasma ADMA levels and blood flow response to intra-arterial infusion of collagen in vivo in monkeys on 4 different dietary regimens.

Acetylcholine-induced relaxation of the carotid artery ex vivo also correlated inversely with the plasma homocyst(e)ine concentration (R=0.42, P=0.02; n=32). Multiple regression analysis indicated that plasma ADMA concentration was the only independent predictor of endothelium-dependent vasodilation ex vivo (R=0.553, P=0.02), whereas plasma homocyst(e)ine or total cholesterol levels were not independent predictors of endothelium-dependent relaxation. Inclusion of an ADMA-homocyst(e)ine interaction term did not increase the correlation coefficient (R=0.554, P=0.03), indicating the absence of an additive effect of ADMA and homocyst(e)ine on endothelial dysfunction of the carotid artery (P=0.82 for interaction).

Regression Analysis of Plasma ADMA and Blood Flow Response to Collagen In Vivo
Intra-arterial infusion of collagen decreased hindlimb blood flow by 42±9% in monkeys fed the hyperhomocyst(e)inemic diet and by 14±11% in monkeys fed the control diet (P<0.01).¹ In monkeys fed the atherogenic diet, reduction in hindlimb blood flow in response to collagen was 30±3% before and 38±5% after supplementation with B vitamins (P=NS).¹³

There was a significant inverse correlation between plasma ADMA concentration and the change in hindlimb blood flow in response to collagen for all animals (R=0.49, P<0.01; Figure 3B) and for monkeys fed the hyperhomocyst(e)inemic or control diet (R=0.56, P<0.05). Plasma ADMA concentration did not correlate with a change in limb blood flow in response to acetylcholine (R=0.14, P=NS).

Effects of Methionine and Homocysteine on ADMA Release by Endothelial Cells
Under control conditions (medium free of methionine and homocysteine), ECV304 human endothelial cells produced 13.7±1.3 pmol of ADMA per microgram of protein over 24 hours and 10.3±3.6 pmol of SDMA per microgram of protein over 24 hours. ADMA formation was concentration-dependently increased by increasing the concentrations of methionine (P<0.05 versus control for 200 and 400 µmol/L, Figure 4). Incubation with 1.0 mmol/L homocysteine also significantly elevated ADMA production (27.8±2.9 pmol/µg protein over 24 hours, P<0.05 versus control). SDMA formation was not significantly affected under the same conditions. The increased production rate of ADMA in the presence of homocysteine was completely reversed in the presence of the methylation inhibitor S-adenosylhomocysteine (13.6±1.8 pmol/µg protein over 24 hours, P<0.05 versus homocysteine; P=NS versus control), but it was not affected by the addition of B vitamins (34.2±4.4 pmol/µg protein over 24 hours, P=NS versus homocysteine).

View larger version (17K): [in this window] [in a new window]   Figure 4. Formation of ADMA by cultured ECV304 human endothelial cells. Confluent endothelial cells were incubated in methionine-free and homocysteine-free medium (control), in the presence of increasing concentrations of exogenous methionine (1x10-4 to 4x10-4 mol/L), and in the presence of homocysteine (10-3 mol/L), homocysteine plus S-adenosylhomocysteine (S-Adeno-hcys, 10-4 mol/L), or homocysteine plus B vitamins (10-3 mol/L pyridoxine, 10-4 mol/L cobalamine, and 10-4 mol/L folic acid). Data are mean±SEM *P<0.05 vs control.

	Discussion

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The major findings of the present study are that (1) plasma levels of ADMA, an endogenous NO synthase inhibitor, are elevated in monkeys with diet-induced hyperhomocyst(e)inemia and/or hypercholesterolemia; (2) there is a significant linear correlation between plasma levels of ADMA and homocyst(e)ine in hyperhomocyst(e)inemic and hypercholesterolemic monkeys; (3) plasma levels of ADMA are inversely correlated with endothelium-dependent relaxation of carotid artery rings ex vivo and with the blood flow response to collagen in vivo; (4) dietary supplementation with B vitamins, which normalizes plasma homocyst(e)ine levels, does not decrease plasma ADMA concentration or restore normal endothelium-dependent vasomotor function in hypercholesterolemic monkeys; and (5) release of ADMA by endothelial cells is increased in the presence of high methionine or homocysteine levels in a manner reversible by the methylation inhibitor S-adenosylhomocysteine.

We observed that plasma concentration of ADMA was elevated in monkeys fed an atherogenic diet that produces hyperhomocyst(e)inemia and hypercholesterolemia. Supplementation of the atherogenic diet with B vitamins decreased plasma homocyst(e)ine to control levels but did not affect the plasma concentration of ADMA or improve endothelium-dependent vasodilatation in these monkeys.¹³ B vitamins are essential cofactors for the transsulfuration of homocyst(e)ine to cystathionine by cystathionine ß synthase (vitamin B₆) and the remethylation of homocyst(e)ine to methionine by methionine synthase (folic acid, vitamin B₁₂), as seen in Figure 5.^{12 21} Thus, dietary supplementation with B vitamins promotes the transsulfuration and remethylation of homocysteine, resulting in decreased plasma levels of homocyst(e)ine.^{21 22} Despite decreasing plasma homocyst(e)ine, however, supplementation with B vitamins may not decrease protein methylation or the generation of ADMA, because intracellular levels of S-adenosylmethionine may actually increase as a consequence of increased turnover of the homocyst(e)ine-methionine pathway (Figure 5).²³

View larger version (15K): [in this window] [in a new window]   Figure 5. Schematic representation of the biochemical pathways potentially linking the demethylation of methionine to form homocyst(e)ine with the generation of ADMA.

In cultured endothelial cells, incubation with methionine concentration-dependently increased ADMA levels. This effect was also induced by homocysteine; it was reversed by the methylation inhibitor S-adenosylhomocysteine, but it was not changed by the addition of B vitamins. Thus, these data in vitro corroborate our findings in monkeys in vivo and further support the proposed model (Figure 5) that N-methylation of L-arginine to ADMA may occur concomitantly with the demethylation of methionine to homocysteine. Wang et al²⁴ recently reported that the addition of homocysteine to endothelial cells in the presence of adenosine and an adenosine deaminase inhibitor can inhibit carboxy methylation of p21^ras by increasing the levels of S-adenosylhomocysteine. These findings are in contrast to our present results, which were obtained in the absence of exogenous adenosine. The differences may be due to different culture conditions and differential regulation of specific methyltransferases. It is very likely that the effects of homocysteine on methylation reactions are dependent on intracellular concentrations of adenosine, methionine, and B vitamins as well as other factors that influence the levels of S-adenosylmethionine and S-adenosylhomocysteine.

Factors unrelated to homocyst(e)ine metabolism may have affected ADMA levels in our in vivo study. We have previously reported that ADMA levels increase in the presence of hypercholesterolemia in animals^{7 8} and humans.¹⁰ Dimethylarginines are excreted by the kidneys and accumulate in chronic renal failure.²⁵ However, accumulation of ADMA has been shown to occur in atherosclerotic humans⁹ and in cholesterol-fed rabbits⁸ in spite of normal renal function. ADMA is metabolized to citrulline by the enzyme dimethylarginine dimethylaminohydrolase (DDAH).²⁶ Inhibition of DDAH produces gradual constriction of vascular segments, which is reversed by L-arginine, further supporting the view that the ratio between endogenous ADMA and L-arginine regulates endothelial NO synthase activity.²⁷ Reduced DDAH activity has been proposed to account for the elevation of ADMA in hypercholesterolemia²⁸ and hyperglycemia.²⁹ We cannot exclude the possibility that modulation of DDAH activity contributed to elevated ADMA concentration in the present study, although preliminary data suggest that homocyst(e)ine does not affect DDAH activity in vitro (R.H.B. et al, unpublished data, 2000).

Our finding that monkeys fed hyperhomocyst(e)inemic diets had significantly elevated plasma ADMA levels is consistent with the hypothesis that methyl groups incorporated into dimethylarginines may be supplied during the demethylation of methionine to homocyst(e)ine. This reaction results in the cleavage from S-adenosylmethionine of 1 methyl group that may be inserted directly or indirectly into L-arginine, thereby forming methylated arginine analogues, such as ADMA and SDMA (Figure 5). Indeed, it has recently been shown that S-adenosylmethionine, an intermediate in the conversion of methionine to homocyst(e)ine, is the source of methyl groups for the methylation of arginine residues within proteins by the enzyme protein-arginine methyltransferase-1 of yeast.³⁰ At least 3 different isoforms of protein-arginine methyltransferases, which have different tissue distribution and different product specificities for ADMA or SDMA, have been characterized.^{31 32} We report in the present study that cultured human endothelial cells produce more ADMA than SDMA under control conditions. This finding, which is in accordance with previous studies,^{14 15} suggests that the methyltransferase present in endothelial cells preferentially methylates arginine in a manner yielding asymmetric dimethylarginine. Selective elevation of ADMA levels, but not of SDMA levels, in hyperhomocyst(e)inemic monkeys may therefore be explained by the altered endothelial metabolism of methylarginines.

Hyperhomocyst(e)inemia is associated with impaired endothelial function in animals¹ and humans.^{2 3 33} However, mechanisms responsible for endothelial dysfunction in hyperhomocyst(e)inemia have remained unclear.³⁴ In high concentrations, homocyst(e)ine is directly toxic to cultured endothelial cells,⁴ and it may decrease endothelial production of NO through oxidative mechanisms.⁵ Our present findings suggest that increased generation of ADMA may be an alternative mechanism of endothelial dysfunction in hyperhomocyst(e)inemia. We observed that the plasma concentration of ADMA was inversely correlated with the acetylcholine-induced relaxation of carotid artery rings ex vivo in hyperhomocyst(e)inemic monkeys. In multiple regression analysis, plasma ADMA was a better predictor of endothelial dysfunction than either plasma homocyst(e)ine or total cholesterol concentrations. No interaction was found between ADMA and homocyst(e)ine in relation to endothelial function, which further supports the hypothesis that homocyst(e)ine may impair endothelial function via ADMA instead of potentiate the detrimental effects of both molecules on endothelial NO formation.

There also was an inverse correlation between plasma ADMA concentration and changes in blood flow in response to collagen, but there was no correlation with changes in hindlimb blood flow in response to acetylcholine in vivo (R=0.014, P=NS). The explanation is not clear for the finding that plasma ADMA correlated well with vascular responses to collagen, but not acetylcholine, but may be related to different mediators for responses to collagen and acetylcholine. It is likely that vasodilatation in response to the activation of platelets by collagen is mediated by NO and thus is inhibited by ADMA. In contrast, the response of resistance vessels to acetylcholine may not be mediated by NO³⁵ and thus may not be inhibited by ADMA.

Despite the 2- to 3-fold elevation of ADMA plasma concentrations in hyperhomocyst(e)inemic monkeys, ADMA levels were still far below those of L-arginine in plasma (2 to 3 µmol/L versus 60 to 100 µmol/L). These levels may seem unlikely to antagonize L-arginine as a substrate for NO synthase.³⁶ Studies in vitro, however, have shown that intracellular concentrations of ADMA are higher in cultured endothelial cells than in the extracellular fluid, which suggests an accumulation of ADMA within cells.¹⁴ We have recently found that ADMA significantly and concentration-dependently inhibits conversion of L-[guanidino-¹⁵N₂]arginine to [¹⁵N]nitrate in primary human endothelial cells within the concentration range between 0.5 µmol/L and 10 µmol/L (R.H.B., S.M.B.-B., unpublished data, 2000). This finding indicates that ADMA concentrations like those reported in the present study are within the steep part of the concentration-effect curve and may well contribute to the modulation of NO synthase activity. Extracellular concentrations of ADMA between 1 and 10 µmol/L inhibit endothelium-dependent vasodilation in isolated blood vessels,^{37 38} and ADMA inhibits the release of NO by cultured endothelial cells¹⁴ and macrophages¹⁵ within the same concentration range. Moreover, in young asymptomatic adults with hypercholesterolemia, elevated plasma concentrations of ADMA are significantly related to the degree of impaired endothelium-dependent forearm vasodilation.¹⁰ Taken together, these studies suggest that ADMA, within the concentration range found in the present study, contributes to the regulation of endothelial NO synthase activity.

From a therapeutic point of view, the observation that lowering homocyst(e)ine plasma concentrations with B vitamins does not improve endothelium-dependent vasodilation may have far-reaching implications. Because a relationship between elevated homocyst(e)ine concentration and cardiovascular disease has been established in epidemiological studies,³⁹ the implicit notion is that lowering homocyst(e)ine levels might reduce cardiovascular risk.^{40 41} The present study implies that this is not necessarily the case. This view is supported by a recent study in which we showed that endothelial dysfunction is not improved by B vitamin treatment in patients with chronic hyperhomocyst(e)inemia and vascular disease.⁴² Preliminary data indicate that these patients also have elevated ADMA plasma concentration (R.H.B., K.S., unpublished data, 2000). Therefore, if methylation of L-arginine is the link between homocyst(e)ine and vascular dysfunction in humans, a beneficial effect would not be expected during supplementation with B vitamins. Prospective interventional clinical trials are necessary to clarify this issue.

	Acknowledgments

 
This work was supported in part by the Office of Research and Development, US Department of Veterans Affairs; by National Institutes of Health grants HL-14388, NS-24621, HL-16066, HL-62984, and OK-25295; and by a grant from the Deutsche Forschungsgemeinschaft (DFG Bo1431/3-1). The authors gratefully acknowledge the excellent technical assistance of B. Schubert, M.T. Suchy, and D.J. Piegors.

	Footnotes

 
Reprint requests to Rainer H. Böger, MD, Institute of Clinical Pharmacology, Hannover Medical School, 30623 Hannover, Germany.

Guest Editor was Peter Libby, Brigham & Women’s Hospital, Boston, Mass.

Received September 2, 1999; accepted February 14, 2000.

	References

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