Elevated levels of asymmetric dimethylarginine in chronic heart failure: a pathophysiologic link between oxygen radical load and impaired vasodilator capacity and the therapeutic effect of allopurinol.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide-dependent vasodilation. In 113 patients with chronic heart failure (CHF) and 26 controls, ADMA level was studied in relation to peripheral blood flow and vasodilator capacity. Further, the effects of allopurinol on concentrations of reactive oxygen species (ROS) and ADMA and peripheral vasodilator capacity were tested in a double-blind design. ADMA level was found to be elevated in CHF patients as compared with controls and increased in parallel with New York Heart Association (NYHA) class and exercise capacity (all P < 0.0001). The level of ADMA predicted resting blood flow (P < 0.05) and postischemic vasodilator capacity (P < 0.001). Sixty eight patients died during the follow-up period. The level of ADMA predicted survival after multivariable adjustment (P = 0.04). Allopurinol reduced uric acid (UA) concentration (P < 0.001) and decreased ROS concentration (allantoin, P < 0.01). Allopurinol lowered ADMA concentration (P = 0.02); postischemic vasodilation as well as endothelium-dependent vasodilation (both P < 0.05) improved. ADMA may be a pathophysiologic factor that is modulated by ROS accumulation and contributes to impaired vascular regulation in CHF.

Negative association of L-arginine methylation products with oocyte numbers.

BACKGROUND: The aim of this study was to determine the concentrations of L-arginine and methylarginines in follicular fluid obtained from women participating in our IVF program and to find clinical correlates of these biochemical parameters. METHODS: Follicular fluid was obtained from 108 women by ultrasonography guided transvaginal puncture following controlled ovarian hyperstimulation. Follicular fluid L-arginine, asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and monomethylarginine (MMA) concentrations were determined with liquid chromatography-tandem mass spectrometry. The integrated index of arginine methylation (arg-MI) was calculated according to the formula: arg-MI = (ADMA + SDMA)/MMA. RESULTS: There were significant inverse relationships between IVF embryo number and follicular fluid L-arginine (r = -0.507, P < 0.001), ADMA (r = -0.356, P < 0.024), SDMA (r = -0.347, P < 0.028), MMA (r = -0.449, P < 0.004) and to L-arginine/ADMA ratio (r = -0.328, P < 0.031). By contrast, arg-MI was directly related to IVF embryo number (r = 0.426, P < 0.006). Moreover, the number of IVF oocytes was also inversely related to ADMA (r = -0.202, P < 0.037) and MMA (r = -0.384, P < 0.012) and positively to arg-MI (r = 0.450, P < 0.03). CONCLUSIONS: The elevated levels of follicular fluid l-arginine and methylarginines appear to have an adverse influence on the reproductive processes as reflected by a reduction in the number of oocytes and embryos conceived. In contrast, the integrated methylation index proved to be positively correlated to the above parameters of fertilization.

Organic nitrates in cardiovascular disease.

Therapeutic activation of the vascular NO/cGMP pathway is induced by a variety of stimuli/mediators including physical activity, supplementation with the precursor L-arginine and organic nitrates which generate NO in the vasculature. The necessity of an enzymatic reduction for NO generation from these drugs as well as differences in the activity of the NO/cGMP pathway within the vascular tree determine the unique hemodynamic changes elicited by organic nitrates. These changes include preferential venodilation, vessel-size specific arterial dilation and improvement of the aortic distensibility and Windkessel-function. Some animal experiments and clinical investigations suggest that nitrates may also be endowed with cardioprotective and/or vasoprotective effects. "Early entry" therapy with nitrates do not significantly improve survival in myocardial infarction but increases the beneficial effects of the ACE-inhibitor enalapril by 50%. Furthermore, nitrates have been shown to improve survival in heart failure, but prognostic effects in stable angina pectoris are unknown. Short-term experimental and clinical investigations suggest that nitrate tolerance induced by nitroglycerin is associated with toxic effects in the vasculature, but this is not true for pentaerythrityl tetranitrate and isosorbide mononitrate. The observed endothelial dysfunction induced by a continuous treatment with nitroglycerin may be an additional risk for patients who receive continuous nitroglycerin to treat conditions such as unstable angina and acute heart failure. In general, nitrates are remarkably safe drugs and are well tolerated. Appropriate clinical trials are needed to answer the question whether nitrates can do more than symptomatic relief in cardiovascular disease.

[Influence of L-arginine on development of atherosclerosis: what is the therapeutically assured?]. / Einfluss von L-Arginin auf die Arterioskleroseentwicklung: Was ist therapeutisch gesichert?

L-arginine is the substrate for the enzyme nitric oxide synthase (NOS), which is responsible for the production of nitric oxide (NO), an endogenous messenger molecule involved in many of the processes associated with the development of atherosclerosis. Acute and chronic administration of L-arginine has been shown to improve endothelial function in animal models of hypercholesterolemia and atherosclerosis. Therefore, a lot of studies were conducted to elucidate whether dietary L-arginine supplementation can augment NO production in man and thereby improve vascular health. In this review the results of studies of intravenous and oral L-arginine supplementation with a colorful spectrum of doses, study duration and surrogate parameters of endothelial function are summarized. The pharmacokinetics of L-arginine have been investigated, side effects are rare and mostly mild and dose-dependent. Several possible mechanisms of action of L-arginine are discussed. An assessment of L-arginine as a therapeutic agent from the point of view of a clinical pharmacologist is given.

Low dialysance of asymmetric dimethylarginine (ADMA)--in vivo and in vitro evidence of significant protein binding.

BACKGROUND: Increased blood levels of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) predict cardiovascular mortality in patients with end-stage renal disease. Despite its low molecular weight, available information on the impact of hemodialysis (HD) on ADMA plasma levels is controversial. METHODS: We assessed plasma concentrations, dialyzer clearance and total amount of ADMA removed in 30 patients with end-stage renal disease during regular HD. In addition, plasma ADMA levels were assessed in 10 patients with acute renal failure treated with extended HD. RESULTS: Regular HD decreased plasma creatinine (from 774 +/- 42 to 312 +/- 17 micromol/l) and urea (from 24.5 +/- 1.5 to 8.4 +/- 0.5 mmol/l) concentrations significantly (both p < 0.001), whereas plasma ADMA remained unchanged (4.35 +/- 0.19 vs. 4.76 +/- 0.24 micromol/l). ADMA clearance was 92 +/- 6 ml/min, and the total amount removed in the spent dialysate was 37 +/- 4 micromol. The clearances of creatinine (161 +/- 3 ml/min) and of urea (173 +/- 3 ml/min) were significantly higher. Furthermore, even during extended HD, plasma ADMA concentrations did not decrease significantly (1.73 +/- 0.22 vs. 1.63 +/- 0.18 micromol/l). CONCLUSION: In conclusion, dialysance of ADMA is markedly lower than expected from its molecular weight because of significant protein binding of the substance. Since markedly increased ADMA blood concentrations have been linked to cardiovascular complications due to atherosclerosis in patients with ESRD, new strategies should be evaluated to remove this putative uremic toxin.

Subpressor dose asymmetric dimethylarginine modulates renal function in humans through nitric oxide synthase inhibition.

Increased blood concentrations of the endogenous nitric oxide (NO) synthase inhibitor asymmetric dimethylarginine (ADMA) have been linked to high blood pressure and to cardiovascular mortality. We evaluated the effects of a subpressor ADMA dose on NO production, renal hemodynamics, sodium handling and active renin and noradrenalin plasma concentrations in 12 healthy subjects (age 26 +/- 1 year) using a double-blind placebo-controlled study design. Infusion of ADMA caused a significant decrease in plasma cyclic guanosine monophosphate (cGMP) levels, i.e. the second messenger of NO (from 6.1 +/- 0.4 to 4.3 +/- 0.3 pmol/l; p < 0.05). In parallel, effective renal plasma flow (ERPF) decreased while renovascular resistance (RVR) increased significantly (ERPF from 667 +/- 9 to 603 +/- 10 ml/min/1.73 m2; RVR from 79 +/- 2 to 91 +/- 2 ml/min/mm Hg; both p < 0.05 vs. baseline). Infusion of placebo did not cause significant changes in plasma cGMP levels, ERPF and RVR (cGMP from 5.7 +/- 0.5 to 5.9 +/- 0.6 pmol/l; ERPF from 665 +/- 12 to 662 +/- 11 ml/min/1.73 m2; RVR from 79 +/- 2 to 78 +/- 2 ml/min/mm Hg; all non-significant). Moreover, urinary sodium excretion was significantly lower with infusion of ADMA as compared with placebo infusion (128 +/- 8 vs. 152 +/- 7 micromol/min; p < 0.05). In contrast, blood pressure, active renin and noradrenalin plasma concentrations did not change significantly with either infusion protocol. Acute infusion of a subpressor ADMA dose modulates several aspects of renal function in humans without affecting the activity of the renin-angiotensin and sympathetic system. Whether chronic (intrarenal) NO synthase inhibition in individuals with increased ADMA blood levels may cause persistent renal vasoconstriction and sodium retention must be evaluated.

Endogenous nitric oxide synthase inhibitors and renal perfusion in patients with heart failure.

BACKGROUND: Patients with heart failure are characterized by impaired nitric oxide-dependent endothelial vasodilation and, in addition, by reduced renal perfusion. DESIGN: We assessed blood concentrations of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) as well as renal haemodynamics to compare normotensive patients with mild heart failure (n = 12, seven males, 70 +/- 1 years, 72.0 +/- 2.7 kg, 92 +/- 2 mmHg, NYHA I/II) and healthy subjects matched with respect to gender, age and body weight (n = 12, seven males, 69 +/- 2 years, 72.7 +/- 2.5 kg, 88 +/- 2 mmHg). RESULTS: Plasma ADMA concentration and renovascular resistance (RVR) were significantly higher (P < 0.01) and effective renal plasma flow (ERPF) significantly lower (P < 0.01) in the patients with heart failure (ADMA 4.18 +/- 0.42 micro mol L-1, RVR 159 +/- 12 mmHg mL-1 min-1, ERPF 381 +/- 26 mL min-1 1.73 m-2) as compared with the healthy controls (ADMA 2.38 +/- 0.11 micro mol L-1, RVR 117 +/- 8 mmHg mL-1 min-1, ERPF 496 +/- 19 mL min-1 1.73 m-2). In contrast, plasma concentrations of l-arginine, homocysteine, symmetric dimethylarginine (i.e. the biologically inactive stereoisomer of ADMA) and plasma renin activity were not significantly different in both groups studied. In the multiple regression analysis, only plasma ADMA concentrations independently predicted reduced ERPF (r = -0.57; P < 0.003). CONCLUSIONS: In normotensive patients with heart failure plasma ADMA concentrations are markedly increased and related to reduced renal perfusion. Thus accumulation of this endogenous nitric oxide inhibitor may play a role in renal pathology in these patients.

Endothelium-dependent vasodilation is independent of the plasma L-arginine/ADMA ratio in men with stable angina: lack of effect of oral L-arginine on endothelial function, oxidative stress and exercise performance.

OBJECTIVES: This study was designed to determine the effect of two weeks' treatment with L-arginine on the ratio of plasma L-arginine to asymmetric dimethylarginine (ADMA), oxidative stress, endothelium-dependent vasodilatation to acetylcholine, exercise performance and heart rate variability in men with stable angina. BACKGROUND: The ratio of plasma L-arginine:ADMA has been proposed as a determinant of endothelium-dependent dilation; dietary supplementation with L-arginine has been shown to improve endothelium-dependent vasodilation and symptoms in some conditions. METHODS: Men (n = 40) with stable angina, at least one epicardial coronary artery with a stenosis >50% and a positive exercise test were randomized to receive L-arginine (15 g daily) or placebo for two weeks according to a double-blind parallel-group design. Plasma L-arginine, ADMA, 8-epi-prostaglandin F2alpha (a marker of oxidative stress) and forearm vasodilator responses to brachial artery infusion of nitroprusside and acetylcholine (+/-L-arginine) were measured. A standard Bruce protocol exercise test was performed before and at the end of the treatment period. RESULTS: Plasma L-arginine increased after oral L-arginine, whereas ADMA remained unchanged, leading to an increase in the L-arginine/ADMA ratio of 62 +/- 11% (mean +/- SE, p < 0.01). Despite a significant enhancement in acetylcholine response by intra-arterial L-arginine at baseline, this response remained unchanged after oral L-arginine. Measures of oxidative stress and exercise performance after L-arginine/placebo were similar in placebo and active groups. CONCLUSIONS: In men with stable angina, an increase in plasma L-arginine/ADMA ratio after two weeks' oral supplementation with L-arginine is not associated with an improvement in endothelium-dependent vasodilatation, oxidative stress or exercise performance.

The clinical pharmacology of L-arginine.

L-Arginine (2-amino-5-guanidinovaleric acid) is the precursor of nitric oxide, an endogenous messenger molecule involved in a variety of endothelium-mediated physiological effects in the vascular system. Acute and chronic administration of L-arginine has been shown to improve endothelial function in animal models of hypercholesterolemia and atherosclerosis. L-Arginine also improves endothelium-dependent vasodilation in humans with hypercholesterolemia and atherosclerosis. The responsiveness to L-arginine depends on the specific cardiovascular disease studied, the vessel segment, and morphology of the artery. The pharmacokinetics of L-arginine have recently been investigated. Side effects are rare and mostly mild and dose dependent. The mechanism of action of L-arginine may involve nitric oxide synthase substrate provision, especially in patients with elevated levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine. Endocrine effects and unspecific reactions may contribute to L-arginine-induced vasodilation after higher doses. Several long-term studies have been performed that show that chronic oral administration of L-arginine or intermittent infusion therapy with L-arginine can improve clinical symptoms of cardiovascular disease in man.

Elevation of asymmetrical dimethylarginine may mediate endothelial dysfunction during experimental hyperhomocyst(e)inaemia in humans.

Hyperhomocyst(e)inaemia is associated with endothelial dysfunction in animals and humans. Mechanisms responsible for endothelial dysfunction in hyperhomocyst(e)inaemia are poorly understood, but may involve impaired bioavailability of endothelium-derived nitric oxide (NO). We hypothesized that acute elevation of homocyst(e)ine by oral methionine loading may stimulate the formation of asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, due to a transmethylation reaction during the formation of homocyst(e)ine from methionine. We studied nine healthy human subjects (five males, four females) aged 29+/-2 years. Flow-mediated vasodilation (FMD) in the brachial artery (endothelium-dependent) and vasodilation induced by nitroglycerine (endothelium-independent) were measured with high-resolution ultrasound before and 8 h after oral methionine (100 mg/kg in cranberry juice) or placebo (cranberry juice), on separate days and in random order. Plasma homocyst(e)ine and ADMA concentrations were measured by specific HPLC methods. After a methionine bolus, elevation of homocyst(e)ine (28.4+/-3.5 micromol/l) was associated with an increased plasma concentration of ADMA (2.03+/-0.18 micromol/l) and reduced FMD (1.54+/-0.92%). Placebo had no effect on these parameters. There was a significant inverse linear relationship between ADMA concentration and FMD (r=-0.49; P<0.05), which was stronger than the relationship between the homocyst(e)ine concentration and FMD (r=-0.36; not significant). We conclude that acute elevation of the homocyst(e)ine concentration impairs vascular endothelial function by a mechanism in which an elevated concentration of ADMA may be involved. This finding may have importance for understanding the mechanism(s) leading to homocyst(e)ine-associated vascular disease, and its potential treatment.

Relationship of asymmetric dimethylarginine to dialysis treatment and atherosclerotic disease.

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of endothelial nitric oxide (NO) synthase. Its concentration is elevated in patients with end-stage renal disease (ESRD), in part because it is excreted via the kidneys. In addition, ADMA is degraded by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which hydrolyzes ADMA to L-citrulline and dimethylamine. Activity of DDAH is decreased by oxidized low density lipoprotein (LDL) or tumor necrosis factor-alpha (TNF-alpha) in vitro yielding increased levels of ADMA. Furthermore, plasma levels of ADMA are elevated in hyperhomocyst(e)inemia and in hypertensive patients on a high salt diet. Data from several experimental studies suggest that ADMA concentrations in a pathophysiologically high range (3 to 10 micromol/L) significantly inhibit vascular NO formation by NO synthase in the presence of L-arginine in isolated human blood vessels, cultured macrophages, and in cultured endothelial cells. It has been well demonstrated that ADMA accumulates in chronic renal failure. Although there is controversy concerning the absolute concentration of ADMA, all authors found a two- to sixfold increase in ADMA levels in patients in chronic renal failure as compared to controls. Different dialysis treatment strategies differentially affect ADMA levels. The presence of atherosclerosis is associated with higher ADMA levels in patients with normal renal function as well as in dialysis patients, but this phenomenon may be unrelated to renal handling of ADMA. Reduced NO elaboration secondary to accumulation of ADMA may be an important pathogenic factor for atherosclerosis in chronic renal failure and ADMA may be a new uremic toxin. Clinical studies on the effect of ADMA are needed to further elucidate its pathophysiological role in atherosclerosis and uremia.

Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: a prospective study.

BACKGROUND: The plasma concentration of asymmetrical dimethylarginine (ADMA), an inhibitor of nitric-oxide synthase, which has been linked to endothelial dysfunction and atherosclerosis in the general population, is raised in patients with end-stage renal disease and could contribute to the high cardiovascular risk in patients with chronic renal failure. We investigated the relation between cardiovascular risk factors and plasma ADMA concentration in a cohort of haemodialysis patients (n=225), and tested the predictive power of ADMA for mortality and cardiovascular outcomes. METHODS: Patients had standard dialysis three times a week. We accurately recorded cardiovascular events over a mean follow-up of 33.4 months (SD 14.6); these events were reviewed by a panel of physicians. We identified correlates of plasma ADMA by univariate and multivariate analyses. FINDINGS: On univariate analysis, ADMA concentration in plasma was directly related to concentrations of fibrinogen and L-arginine in plasma, duration of dialysis treatment, and serum cholesterol concentration, and was inversely related to serum albumin concentration. On multivariate analysis, only plasma fibrinogen (p=0.0001) and serum albumin (p=0.04) concentrations were independently related to plasma ADMA concentration (multiple r=0.44, p=0.0001). 83 patients died, 53 (64%) by cardiovascular causes. In a Cox's proportional-hazards model, plasma ADMA ranked as the second factor predicting overall mortality (hazard ratio 1.26, 95% Cl 1.11-1.41, p=0.0001) and cardiovascular events (1.17, 1.04-1.33, p=0.008). INTERPRETATION: In haemodialysis patients, plasma ADMA is a strong and independent predictor of overall mortality and cardiovascular outcome. These findings lend support to the hypothesis that accumulation of ADMA is an important risk factor for cardiovascular disease in chronic renal failure.

An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes.

OBJECTIVES: We sought to determine whether asymmetric dimethylarginine (ADMA) inhibits nitric oxide (NO) elaboration in cultured human endothelial cells and whether this is associated with the activation of oxidant-sensitive signaling mediating endothelial adhesiveness for monocytes. BACKGROUND: Endothelial NO elaboration is impaired in hypercholesterolemia and atherosclerosis, which may be due to elevated concentrations of ADMA, an endogenous inhibitor of NO synthase. METHODS: Human umbilical vein endothelial cells (ECV 304) and human monocytoid cells (THP-1) were studied in a functional binding assay. Nitric oxide and superoxide anion (O2-) were measured by chemiluminescence; ADMA by high pressure liquid chromatography; monocyte chemotactic protein-1 (MCP-1) by ELISA and NF-KB by electromobility gel shift assay. RESULTS: Incubation of endothelial cells with ADMA (0.1 microM to 100 microM) inhibited NO formation, which was reversed by coincubation with L-arginine (1 mM). The biologically inactive stereoisomer symmetric dimethylarginine did not inhibit NO release. Asymmetric dimethylarginine (10 microM) or native low-density lipoprotein cholesterol (100 mg/dL) increased endothelial O2- to the same degree. Asymmetric dimethylarginine also stimulated MCP-1 formation by endothelial cells. This effect was paralleled by activation of the redox-sensitive transcription factor NF-KB. Preincubation of endothelial cells with ADMA increased the adhesiveness of endothelial cells for THP-1 cells in a concentration-dependent manner. Asymmetric dimethylarginine-induced monocyte binding was diminished by L-arginine or by a neutralizing anti-MCP-1 antibody. CONCLUSIONS: We concluded that the endogenous NO synthase inhibitor ADMA is synthesized in human endothelial cells. Asymmetric dimethylarginine increases endothelial oxidative stress and potentiates monocyte binding. Asymmetric dimethylarginine may be an endogenous proatherogenic molecule.

Asymmetric dimethylarginine, derangements of the endothelial nitric oxide synthase pathway, and cardiovascular diseases.

Analogues of L-arginine that are chemically modified at the terminal guanidino nitrogen group, such as Nomega-monomethy-L-arginine (L-NMMA), have been used for nitric oxide synthase inhibition. However, L-NMMA and other methylated L-arginine analogues are also endogenously formed. Among these, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) have been shown to be the most abundant. Like L-NMMA, ADMA is an inhibitor of NO synthase, whereas SDMA is inactive. ADMA is synthesized by N-methyltransferases, a family of enzymes that methylate L-arginine residues within specific proteins. Free ADMA is released during proteolytic cleavage of methylated proteins; it can be detected in plasma and urine, but its intracellular concentrations appear to be much higher. ADMA is metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), and inhibition of DDAH activity has been shown to lead to increased ADMA levels and endothelial dysfunction. Plasma levels of ADMA are elevated in endstage renal failure, in atherosclerosis and hypercholesterolemia, in hypertension, and in heart failure. Although the molecular cause for elevation of ADMA concentration in these diseases has not been fully elucidated, evidence is accumulating that ADMA is one cause of endothelial dysfunction in these diseases. Moreover, it may be a marker or even a risk factor for cardiovascular disease. Therefore, pharmacological modulation of ADMA concentration may be a novel therapeutic target in cardiovascular diseases.

Drug-drug interactions in medical patients: effects of in-hospital treatment and relation to multiple drug use.

OBJECTIVES: Adverse drug reactions (ADRs) are a major cause of hospital admissions, thereby leading to significant medical and economical problems. Drug interactions contribute to a major part of ADRs, especially in elderly patients and in patients under polymedication. As a peculiarity of the German health care system the general practitioner is less involved in patient care during hospital stay than in other countries. Consequently, changes in medication at the transition point from out-patient to in-patient care and back may contribute to drug-related problems. PATIENTS: In the present study we investigated potential interactions in 169 consecutive patients with the diagnosis CHD (coronary heart disease) or COLD (chronic obstructive lung disease) who were admitted to the University Hospitals of Hannover and Magdeburg. METHODS: For each patient, potential interactions between prescribed drugs at admission, at discharge, and 3 months after discharge were assessed by using drug interaction data bases. RESULTS: We found that the number of drugs taken per patient as well as the number of interactions per patient are higher during hospitalization than before admission (pre-admission), and fall back after the hospital stay (post-discharge), but not to the pre-admission level. The number of potential interactions was significantly correlated in a polynomial manner to the number of drugs taken by each patient. The number of patients without a potential interaction was 44.4% pre-admission, 39.6% at discharge, and 39.1% post-discharge. Patients with potential interactions had a mean of 2.8, 2.7, and 2.4 interactions at each of the time points. Drug classes mainly involved with potential interactions were kaliuretic diuretics (recorded by 43.3% at discharge), ACE inhibitors (30.3%), anticoagulants and aggregation inhibitors (20.4%) and digitalis glycosides (14.7%). Regarding the frequency of the interaction categories, 68-70% of the potential interactions demand clinical attention, while 1-2% are life-threatening. 17-19% may result in therapeutic benefit; 10-12% are without clinical relevance. CONCLUSIONS: The risk of drug interactions increases exponentially with the number of drugs given to a patient. Although we have no data on the fraction of interactions that became clinically manifest, our study indicates that prescribing fewer drugs can reduce the risk of suffering from sickness secondary to drug interactions. Taking this risk into account may help to improve the quality of drug treatment and to save costs.

Endogenous nitric oxide synthase inhibitors are responsible for the L-arginine paradox.

L-Arginine, the substrate of nitric oxide (NO) synthases (NOSs), is found in the mammalian organism at concentrations by far exceeding K(M) values of these enzymes. Therefore, additional L-arginine should not enhance NO formation. In vivo, however, increasing L-arginine concentration in plasma has been shown repeatedly to increase NO production. This phenomenon has been named the L-arginine paradox; it has found no satisfactory explanation so far. In the present work, evidence for the hypothesis that the endogenous NOS inhibitors methylarginines, asymmetric dimethylarginine being the most powerful (IC(50) 1.5 microM), are responsible for the L-arginine paradox is presented.

LDL cholesterol upregulates synthesis of asymmetrical dimethylarginine in human endothelial cells: involvement of S-adenosylmethionine-dependent methyltransferases.

Asymmetrical dimethylarginine (ADMA) is an endogenous nitric oxide synthase inhibitor. It is formed by protein arginine N-methyltransferases (PRMTs), which utilize S-adenosylmethionine as methyl group donor. ADMA plasma concentration is elevated in hypercholesterolemia, leading to endothelial dysfunction and producing proatherogenic changes of endothelial cell function. Four different isoforms of human PRMTs have been identified. Because the release of ADMA from human endothelial cells is increased in the presence of native or oxidized LDL cholesterol, we investigated the potential involvement of PRMT activity and gene expression in this effect. We found that the production of ADMA by human endothelial cells is upregulated in the presence of methionine or homocysteine and inhibited by either of the methyltransferase inhibitors S-adenosylhomocysteine, adenosine dialdehyde, or cycloleucine. This effect is specific for ADMA but not symmetrical dimethylarginine. The upregulation of ADMA release by native and oxidized LDL is abolished by S-adenosylhomocysteine and by the antioxidant pyrrollidine dithiocarbamate. Furthermore, a methyl-(14)C label is transferred from S-adenosylmethionine to ADMA but not symmetrical dimethylarginine, in human endothelial cells. The expression of PRMTs is upregulated in the presence of native or oxidized LDL. Our data suggest that the production of ADMA by human endothelial cells is regulated by S-adenosylmethionine-dependent methyltransferases. This activity is upregulated by LDL cholesterol, which may be due in part to the enhanced gene expression of PRMTs. In concentrations reached by stimulation of methyltransferases (5 to 50 micromol/L), ADMA significantly inhibited the formation of (15)N-nitrite from L-[guanidino-(15)N(2)]arginine. These findings suggest a novel mechanism by which ADMA concentration is elevated in hypercholesterolemia, leading to endothelial dysfunction and atherosclerosis.

Plasma concentration of asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, is elevated in monkeys with hyperhomocyst(e)inemia or hypercholesterolemia.

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.

L-arginine supplementation in hypercholesterolemic rabbits normalizes leukocyte adhesion to non-endothelial matrix.

UNLABELLED: L-arginine slows the development of atheromatous lesions, improves endothelium-dependent relaxation, and reduces the vascular superoxide anion production in hypercholesterolemic rabbits. These beneficial effects have been attributed to L-arginine-dependent formation of nitric oxide within the endothelial layer; a direct effect of L-arginine on other cells, however, has not been investigated. We hypothesised that in hypercholesterolemia L-arginine also specifically acts via a direct inhibitory effect on leukocytes, without affecting endothelial cells. The action of L-arginine was compared to vitamin E and the HMG CoA reductase inhibitor lovastatin which are known to attenuate progression of atherosclerosis. Rabbits were fed cholesterol enriched diet and from week five on lovastatin (10 mg/day), vitamin E (300 mg/d) or L-arginine (2% in drinking water) were given. After 16 weeks, blood cholesterol concentration was determined and leukocyte adhesion to cotton wool was measured. In order to exclude any endothelium-mediated effects an adhesion assay to endothelial cells was avoided. Cholesterol-enriched diet increased plasma cholesterol concentration (19+/-3 vs. 1427+/-117 mg/dl). Cholesterol levels were not affected by L-arginine (1344+/-163 mg/dl) or vitamine E (1312+/-243 mg/dl). Lovastatin treatment reduced cholesterol concentration by 35% as compared to the cholesterol group (899+/-51, p<0.05 vs. cholesterol). Cholesterol diet significantly increased leukocyte adhesion to cotton wool (16+/-3% vs 27+/-4%, p<0.05). Lovastatin or vitamine E had no effect on leukocyte adhesion (31+/-4%, 39+/-5), whereas L-arginine completely normalized adhesion (8.8+/-3%). CONCLUSION: Rabbits fed high cholesterol diet have increased leukocyte adhesion, which is not affected by lovastatin or vitamine E treatment, but prevented by L-arginine supplementation. A direct inhibitory effect of L-arginine on leukocyte adhesion may contribute to the beneficial effects observed with this substance.

Asymmetric dimethylarginine increases mononuclear cell adhesiveness in hypercholesterolemic humans.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is elevated in hypercholesterolemia. This study was designed to determine the role of ADMA in the increased mononuclear cell adhesiveness observed in human hypercholesterolemia. In patient studies, plasma ADMA levels were determined by high-performance liquid chromatography. Functional mononuclear leukocyte adhesion assays were performed in parallel, and flow cytometry was used to characterize bound monocytes and T lymphocytes. Hypercholesterolemic patients were then placed on an oral L-arginine regimen of 14 or 21 g/d and studied over 12 weeks. In cell culture studies, bovine aortic endothelial cells were incubated with varied concentrations of ADMA. Monocytoid cells were cocultured with these bovine aortic endothelial cells, and their adhesiveness was assessed by use of a binding assay. Flow cytometry was used to quantify adhesion molecule expression. Plasma ADMA levels and adhesiveness of mononuclear cells (specifically, monocytes and T lymphocytes) were elevated in hypercholesterolemic patients. Adhesiveness was inversely correlated with the plasma L-arginine/ADMA ratio. Oral administration of L-arginine normalized plasma L-arginine/ADMA ratios and attenuated monocyte and T-lymphocyte adhesiveness. ADMA had no direct effect on the adhesiveness of mononuclear cells. However, monocytes became hyperadhesive when cocultured with ADMA-exposed endothelial cells. In human hypercholesterolemia, the plasma L-arginine/ADMA ratio is inversely correlated with mononuclear cell adhesiveness. Restoration of the L-arginine/ADMA ratio to control levels normalizes mononuclear cell adhesiveness. Our studies suggest that the elaboration of endothelium-derived nitric oxide affects the behavior of circulating T lymphocytes and monocytes.