Skip to main content
  • American Heart Association
  • Science Volunteer
  • Warning Signs
  • Advanced Search
  • Donate

  • Home
  • About this Journal
    • Editorial Board
    • Meet the Editors
    • ATVB Journal History
    • General Statistics
  • All Issues
  • Subjects
    • All Subjects
    • Arrhythmia and Electrophysiology
    • Basic, Translational, and Clinical Research
    • Critical Care and Resuscitation
    • Epidemiology, Lifestyle, and Prevention
    • Genetics
    • Heart Failure and Cardiac Disease
    • Hypertension
    • Imaging and Diagnostic Testing
    • Intervention, Surgery, Transplantation
    • Quality and Outcomes
    • Stroke
    • Vascular Disease
  • Browse Features
    • Cover Art Award
    • ATVB Early Career Award
    • ATVB in Focus
    • Recent Brief Reviews of ATVB
    • Lecture Series
    • Collections
    • Recent Highlights of ATVB
    • Commentaries
    • Browse Abstracts
    • Insight into ATVB Authors
  • Resources
    • Instructions for Authors
    • Online Submission/Peer Review Site
    • Council on ATVB
    • Permissions and Rights Q&A
    • AHA Guidelines and Statements
    • Customer Service and Ordering Information
    • Author Reprints
    • International Users
    • AHA Newsroom
  • AHA Journals
    • AHA Journals Home
    • Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB)
    • Circulation
    • → Circ: Arrhythmia and Electrophysiology
    • → Circ: Genomic and Precision Medicine
    • → Circ: Cardiovascular Imaging
    • → Circ: Cardiovascular Interventions
    • → Circ: Cardiovascular Quality & Outcomes
    • → Circ: Heart Failure
    • Circulation Research
    • Hypertension
    • Stroke
    • Journal of the American Heart Association
  • Facebook
  • LinkedIn
  • Twitter

  • My alerts
  • Sign In
  • Join

  • Advanced search

Header Publisher Menu

  • American Heart Association
  • Science Volunteer
  • Warning Signs
  • Advanced Search
  • Donate

Arteriosclerosis, Thrombosis, and Vascular Biology

  • My alerts
  • Sign In
  • Join

  • Facebook
  • LinkedIn
  • Twitter
  • Home
  • About this Journal
    • Editorial Board
    • Meet the Editors
    • ATVB Journal History
    • General Statistics
  • All Issues
  • Subjects
    • All Subjects
    • Arrhythmia and Electrophysiology
    • Basic, Translational, and Clinical Research
    • Critical Care and Resuscitation
    • Epidemiology, Lifestyle, and Prevention
    • Genetics
    • Heart Failure and Cardiac Disease
    • Hypertension
    • Imaging and Diagnostic Testing
    • Intervention, Surgery, Transplantation
    • Quality and Outcomes
    • Stroke
    • Vascular Disease
  • Browse Features
    • Cover Art Award
    • ATVB Early Career Award
    • ATVB in Focus
    • Recent Brief Reviews of ATVB
    • Lecture Series
    • Collections
    • Recent Highlights of ATVB
    • Commentaries
    • Browse Abstracts
    • Insight into ATVB Authors
  • Resources
    • Instructions for Authors
    • Online Submission/Peer Review Site
    • Council on ATVB
    • Permissions and Rights Q&A
    • AHA Guidelines and Statements
    • Customer Service and Ordering Information
    • Author Reprints
    • International Users
    • AHA Newsroom
  • AHA Journals
    • AHA Journals Home
    • Arteriosclerosis, Thrombosis, and Vascular Biology (ATVB)
    • Circulation
    • → Circ: Arrhythmia and Electrophysiology
    • → Circ: Genomic and Precision Medicine
    • → Circ: Cardiovascular Imaging
    • → Circ: Cardiovascular Interventions
    • → Circ: Cardiovascular Quality & Outcomes
    • → Circ: Heart Failure
    • Circulation Research
    • Hypertension
    • Stroke
    • Journal of the American Heart Association
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
Download PDF
https://doi.org/10.1161/01.ATV.20.6.1557
Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1557-1564
Originally published June 1, 2000
Rainer H. Böger
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Stefanie M. Bode-Böger
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Karsten Sydow
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Donald D. Heistad
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Steven R. Lentz
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & Tables
  • Info & Metrics
  • eLetters

Jump to

  • Article
    • Abstract
    • Methods
    • Results
    • Discussion
    • Acknowledgments
    • References
  • Figures & Tables
  • Info & Metrics
  • eLetters
Loading

Abstract

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.

  • nitric oxide
  • endothelium
  • asymmetric dimethylarginine
  • homocysteine
  • 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.

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 NG,NG-dimethyl-l-arginine (ADMA) and/or NG,NG′-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

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 (3×10−8, 10−7, and 3×10−7 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 (1×10−4 to 4×10−4 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 F2α (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

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:
  • View inline
  • View popup
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⇓).

Figure 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
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⇓).

Figure 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
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⇓).

Figure 3.
  • Download figure
  • Open in new tab
  • Download powerpoint
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).

Figure 4.
  • Download figure
  • Open in new tab
  • Download powerpoint
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 (1×10−4 to 4×10−4 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

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 β 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

Figure 5.
  • Download figure
  • Open in new tab
  • Download powerpoint
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 p21ras 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 animals7 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.

References

  1. ↵
    Lentz SR, Sobey CG, Piegors DJ, Bhopatkar MY, Faraci FM, Malinow MR, Heistad DD. Vascular dysfunction in monkeys with diet-induced hyperhomocyst(e)inemia. J Clin Invest. 1996;98:24–29.
    OpenUrlCrossRefPubMed
  2. ↵
    Celermajer DS, Sorensen K, Ryallis M, Robinson J, Thomas O, Leonard JV, Deanfield JE. Impaired endothelial function occurs in the systemic arteries of children with homozygous homocystinuria but not in their heterozygous parents. J Am Coll Cardiol. 1993;22:854–858.
    OpenUrlPubMed
  3. ↵
    Tawakol A, Omland T, Gerhard M, Wu JT, Creager MA. Hyperhomocyst(e)inemia is associated with impaired endothelium-dependent vasodilation in humans. Circulation. 1997;95:1119–1121.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    de Groot PG, Willems C, Boers GHJ, Gonsalves MD, van Aken WG, van Mourik JA. Endothelial cell dysfunction in homocystinuria. Eur J Clin Invest. 1983;13:405–410.
    OpenUrlCrossRefPubMed
  5. ↵
    Stamler JS, Osborn JA, Jaraji O, Ralbani LE, Mullins M, Singel D, Loscalzo J. Adverse effects of homocysteine are modulated by endothelium-derived relaxing factor and related oxides of nitrogen. J Clin Invest. 1993;91:308–318.
  6. ↵
    Vallance P, Leone A, Calver A, Collier J, Moncada S. Endogenous dimethylarginine as an inhibitor of nitric oxide synthesis. J Cardiovasc Pharmacol. 1992;20(suppl 12):S60–S62.
  7. ↵
    Bode-Böger SM, Böger RH, Kienke S, Junker W, Frölich JC. Elevated L-arginine/dimethylarginine ratio contributes to enhanced systemic NO production by dietary L-arginine in hypercholesterolemic rabbits. Biochem Biophys Res Commun. 1996;219:598–603.
    OpenUrlCrossRefPubMed
  8. ↵
    Böger RH, Bode-Böger SM, Phivthong-ngam L, Böhme M, Brandes RP, Mügge A, Frölich JC. Dietary L-arginine slows the progression of atherosclerosis in cholesterol-fed rabbits: comparison with lovastatin. Circulation. 1997;96:1282–1290.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Böger RH, Bode-Böger SM, Thiele W, Junker W, Alexander K, Frölich JC. Biochemical evidence for impaired nitric oxide synthesis in patients with peripheral arterial occlusive disease. Circulation. 1997;95:2068–2074.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Böger RH, Bode-Böger SM, Szuba A, Tsao PS, Chan JR, Tangphao O, Blaschke TF, Cooke JP. Asymmetric dimethylarginine: a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Circulation. 1998;98:1842–1847.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    McDermott JR. Studies on the catabolism of NG-methylarginine, NG, N′G-dimethylarginine, and NG, NG-dimethylarginine in the rabbit. Biochem J. 1976;154:179–184.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Malinow MR. Homocyst(e)ine and arterial occlusive diseases. J Intern Med. 1994;2336:603–617.
    OpenUrl
  13. ↵
    Lentz SR, Malinow MR, Piegors DJ, Bhopatkar-Teredesai M, Faraci FM, Heistad DD. Consequences of hyperhomocyst(e)inemia on vascular function in atherosclerotic monkeys. Arterioscler Thromb Vasc Biol. 1997;17:2930–2934.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    Böger RH, Bode-Böger SM, Tsao PS, Lin PS, Chan JR, Cooke JP. The endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) exerts pro-atherosclerotic effects in cultured human endothelial cells. Circulation. 1997;96(suppl I):I-1588. Abstract.
  15. ↵
    Fickling SA, Leone AM, Nussey SS, Vallance P, Whitley GSJ. Synthesis of NG, NG dimethylarginine by human endothelial cells. Endothelium. 1993;1:137–140.
    OpenUrlCrossRef
  16. ↵
    Kaul S, Heistad DD, Mügge A, Armstrong ML, Piegors DJ, Lopez AG. Vascular responses to platelet activation in normal and atherosclerotic primates in vivo. Arterioscler Thromb. 1991;11:1745–1751.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Takahashi K, Sawasaki Y. Rare spontaneously transformed human endothelial cell line provides useful new research tool. In Vitro Cell Dev Biol. 1992;28A:380–382.
  18. ↵
    Smolin LA, Schneider JA. Measurement of total plasma cysteamine using high-performance liquid chromatography with electrochemical detection. Anal Biochem. 1988;168:374–379.
    OpenUrlCrossRefPubMed
  19. ↵
    Malinow MR, Kang SS, Taylor LM, Wong PKW, Coull B, Inhara T, Mukerjee D, Sexton G, Upson B. Prevalence of hyperhomocyst(e)inemia in patients with peripheral arterial occlusive disease. Circulation. 1989;79:1180–1188.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Lauer RM, Lee J, Clarke WR. Factors affecting the relationship between childhood and adult cholesterol levels: the Muscatine study. Pediatrics. 1988;82:309–318.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Ubbink JB, Vermaak WJH, van der Merwe A, Becker PJ, Delport R, Potgieter HC. Vitamin requirements for the treatment of hyperhomocysteinemia in humans. J Nutr. 1994;124:1927–1933.
  22. ↵
    Malinow MR. Hyperhomocysteinemia: a common and easily reversible risk factor for occlusive atherosclerosis. Circulation. 1990;81:2004–2006.
    OpenUrlFREE Full Text
  23. ↵
    Miller JW, Nadeau MR, Smith J, Smith D, Selhub J. Folate-deficiency-induced homocysteinemia in rats: disruption of S-adenosylmethionine’s co-ordinate regulation of homocysteine metabolism. Biochem J. 1994;298:415–419.
  24. ↵
    Wang H, Yoshizumi M, Lai K, Tsai JC, Perrella MA, Haber E, Lee ME. Inhibition of growth and p21ras methylation in vascular endothelial cells by homocysteine but not cysteine. J Biol Chem. 1997;272:25380–25385.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Vallance P, Leone A, Calver A, Collier J, Moncada S. Accumulation of an endogenous inhibitor of NO synthesis in chronic renal failure. Lancet. 1992;339:572–575.
    OpenUrlCrossRefPubMed
  26. ↵
    MacAllister RJ, Fickling SA, Whitley GSJ, Vallance P. Metabolism of methylarginines by human vasculature: implications for the regulation of nitric oxide synthesis. Br J Pharmacol. 1994;112:43–48.
    OpenUrlPubMed
  27. ↵
    MacAllister RJ, Parry H, Kimoto M, Ogawa T, Russell RJ, Hodson H, Whitley GSJ, Vallance P. Regulation of nitric oxide synthesis by dimethylarginine dimethylaminohydrolase. Br J Pharmacol. 1996;119:1533–1540.
    OpenUrlCrossRefPubMed
  28. ↵
    Ito A, Tsao PS, Adimoolam S, Kimoto M, Ogawa T, Cooke JP. Novel mechanism for endothelial dysfunction: dysregulation of dimethylarginine dimethylaminohydrolase. Circulation. 1999;99:3092–3095.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Ito A, Asagami T, Tsao PS, Adimoolam S, Kimoto M, Tsuji H, Reaven GM, Cooke JP. Dysregulation of dimethylarginine dimethylaminohydrolase: a mechanism of endothelial dysfunction in diabetes mellitus. Circulation. 1999;100(suppl I):I-473. Abstract.
  30. ↵
    Gary JD, Lin WJ, Yang MC, Herschman HR, Clarke S. The predominant protein-arginine methyltransferase from Saccharomyces cerevisiae. J Biol Chem. 1996;271:12585–12594.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Scott HS, Antonarakis SE, Lalioti MD, Rossier C, Silver PA, Henry MF. Identification and characterization of two putative human arginine methyltransferases (HRMT1L1 and HRMT1L2). Genomics. 1998;48:330–340.
    OpenUrlCrossRefPubMed
  32. ↵
    Tang J, Gary JD, Clarke S, Herschman HR. PRMT3, a type I protein arginine N-methyltransferase that differs from PRMT1 in its oligomerization, subcellular localization, substrate specificity, and regulation. J Biol Chem. 1998;273:16935–16945.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Woo KS, Chook P, Lolin YI, Cheung ASP, Chan LT, Sun YY, Sanderson JE, Metrewelic C, Celermajer DS. Hyperhomocysteinemia is a risk factor for arterial endothelial dysfunction in humans. Circulation. 1997;96:2542–2544.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Lentz SR. Homocysteine and vascular dysfunction. Life Sci. 1997;61:1205–1215.
    OpenUrlCrossRefPubMed
  35. ↵
    Mügge A, Lopez JA, Piegors DJ, Breese KR, Heistad DD. Acetylcholine-induced vasodilatation in rabbit hindlimb in vivo is not inhibited by analogues of L-arginine. Am J Physiol. 1991;260:H242–H247.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Harrison DG. Cellular and molecular mechanisms of endothelial cell dysfunction. J Clin Invest. 1997;100:2153–2157.
    OpenUrlCrossRefPubMed
  37. ↵
    Faraci FM, Brian JE, Heistad DD. Response of cerebral blood vessels to an endogenous inhibitor of nitric oxide synthase. Am J Physiol. 1995;269:H1522–H1527.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Kurose I, Wolf R, Grisham MB, Granger DN. Effects of an endogenous inhibitor of nitric oxide synthesis on postcapillary venules. Am J Physiol. 1995;268:H2224–H2231.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Stampfer MJ, Malinow R, Willett WC, Newcomer LM, Upson B, Ullmann D, Tishler PV, Hennekens CH. A prospective study of plasma homocyst(e)ine and risk of myocardial infarction in US physicians. JAMA. 1992;268:877–881.
    OpenUrlCrossRefPubMed
  40. ↵
    Malinow MR, Duell PB, Hess DL, Anderson PH, Krueger WD, Phillipson BE, Gluckman RA, Block PC, Upson BM. Reduction of plasma homocyst(e)ine levels by breakfast cereal fortified with folic acid in patients with coronary heart disease. N Engl J Med. 1998;338:1009–1015.
    OpenUrlCrossRefPubMed
  41. ↵
    Robinson K, Arheart K, Refsum H, Brattström L, Boers G, Ueland P, Palma-Reis R, Meleady R, Daly L, Witteman J, Graham I. Low circulating folate and vitamin B6 concentrations: risk factors for stroke, peripheral vascular disease, and coronary artery disease: European Comac Group. Circulation. 1998;97:437–443.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Hornig B, Bode-Böger SM, Arakawa N, Sydow K, Drexler H, Frölich JC, Böger RH. Lack of improvement of homocyst(e)ine-induced endothelial dysfunction during supplementation with B vitamins in patients with arterial occlusive disease. Circulation. 1999;100(suppl I):I-757. Abstract.
View Abstract
Back to top
Previous ArticleNext Article

This Issue

Arteriosclerosis, Thrombosis, and Vascular Biology
June 2000, Volume 20, Issue 6
  • Table of Contents
Previous ArticleNext Article

Jump to

  • Article
    • Abstract
    • Methods
    • Results
    • Discussion
    • Acknowledgments
    • References
  • Figures & Tables
  • Info & Metrics
  • eLetters

Article Tools

  • Print
  • Citation Tools
    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 and Steven R. Lentz
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1557-1564, originally published June 1, 2000
    https://doi.org/10.1161/01.ATV.20.6.1557

    Citation Manager Formats

    • BibTeX
    • Bookends
    • EasyBib
    • EndNote (tagged)
    • EndNote 8 (xml)
    • Medlars
    • Mendeley
    • Papers
    • RefWorks Tagged
    • Ref Manager
    • RIS
    • Zotero
  •  Download Powerpoint
  • Article Alerts
    Log in to Email Alerts with your email address.
  • Save to my folders

Share this Article

  • Email

    Thank you for your interest in spreading the word on Arteriosclerosis, Thrombosis, and Vascular Biology.

    NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

    Enter multiple addresses on separate lines or separate them with commas.
    Plasma Concentration of Asymmetric Dimethylarginine, an Endogenous Inhibitor of Nitric Oxide Synthase, Is Elevated in Monkeys With Hyperhomocyst(e)inemia or Hypercholesterolemia
    (Your Name) has sent you a message from Arteriosclerosis, Thrombosis, and Vascular Biology
    (Your Name) thought you would like to see the Arteriosclerosis, Thrombosis, and Vascular Biology web site.
  • Share on Social Media
    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 and Steven R. Lentz
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1557-1564, originally published June 1, 2000
    https://doi.org/10.1161/01.ATV.20.6.1557
    del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Related Articles

Cited By...

Subjects

  • Basic, Translational, and Clinical Research
    • Mechanisms

Arteriosclerosis, Thrombosis, and Vascular Biology

  • About ATVB
  • Instructions for Authors
  • AHA CME
  • Meeting Abstracts
  • Permissions
  • Email Alerts
  • Open Access Information
  • AHA Journals RSS
  • AHA Newsroom

Contact the Editorial Office:
email: atvb@atvb.org

Information for:
  • Advertisers
  • Subscribers
  • Subscriber Help
  • Institutions / Librarians
  • Institutional Subscriptions FAQ
  • International Users
American Heart Association Learn and Live
National Center
7272 Greenville Ave.
Dallas, TX 75231

Customer Service

  • 1-800-AHA-USA-1
  • 1-800-242-8721
  • Local Info
  • Contact Us

About Us

Our mission is to build healthier lives, free of cardiovascular diseases and stroke. That single purpose drives all we do. The need for our work is beyond question. Find Out More about the American Heart Association

  • Careers
  • SHOP
  • Latest Heart and Stroke News
  • AHA/ASA Media Newsroom

Our Sites

  • American Heart Association
  • American Stroke Association
  • For Professionals
  • More Sites

Take Action

  • Advocate
  • Donate
  • Planned Giving
  • Volunteer

Online Communities

  • AFib Support
  • Garden Community
  • Patient Support Network
  • Professional Online Network

Follow Us:

  • Follow Circulation on Twitter
  • Visit Circulation on Facebook
  • Follow Circulation on Google Plus
  • Follow Circulation on Instagram
  • Follow Circulation on Pinterest
  • Follow Circulation on YouTube
  • Rss Feeds
  • Privacy Policy
  • Copyright
  • Ethics Policy
  • Conflict of Interest Policy
  • Linking Policy
  • Diversity
  • Careers

©2018 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited. The American Heart Association is a qualified 501(c)(3) tax-exempt organization.
*Red Dress™ DHHS, Go Red™ AHA; National Wear Red Day ® is a registered trademark.

  • PUTTING PATIENTS FIRST National Health Council Standards of Excellence Certification Program
  • BBB Accredited Charity
  • Comodo Secured