Asymmetric Dimethylarginine Infusion Research Articles

Increased Circulating ADMA in Young Male Rats Caused Cognitive Deficits and Increased Intestinal and Hippocampal NLRP3 Inflammasome Expression and Microbiota Composition Alterations: Effects of Resveratrol.

Endothelial dysfunction is characterized by disturbances in nitric oxide (NO) bioavailability and increased circulating asymmetric dimethylarginine (ADMA) due to the enormous release of free radicals. Increased circulating ADMA may cause endothelial dysfunction and a variety of clinical disorders, such as liver and kidney disease. Young male Sprague-Dawley rats at postnatal day 17 ± 1 received continuous ADMA infusion via an intraperitoneal pump to induce endothelial dysfunction. Four groups of rats (n = 10 per group) were allocated: control, control and resveratrol, ADMA infusion, and ADMA infusion and resveratrol groups. Spatial memory, NLR family pyrin-domain-containing 3 (NLRP3) inflammasome, cytokine expression, tight junction proteins in the ileum and dorsal hippocampus, and microbiota composition were examined. We found cognitive deficits; increased NLRP3 inflammasome in the plasma, ileum, and dorsal hippocampus; decreased ileum and dorsal hippocampal cytokine activation and tight junction proteins; and microbiota composition alterations in the ADMA-infusion young male rats. Resveratrol had beneficial effects in this context. In conclusion, we observed NLRP3 inflammasome activation in peripheral and central dysbiosis in young male rats with increased circulating ADMA, and found that resveratrol had beneficial effects. Our work adds to the mounting evidence that inhibiting systemic inflammation is a promising therapeutic avenue for cognition impairment, probably via the gut-brain axis.

Enhanced dimethylarginine degradation improves coronary flow reserve and exercise tolerance in Duchenne muscular dystrophy carrier mice.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by null mutations in dystrophin and characterized by muscle degeneration. Cardiomyopathy is common and often prevalent at similar frequency in female DMD carriers irrespective of whether they manifest skeletal muscle disease. Impaired muscle nitric oxide (NO) production in DMD disrupts muscle blood flow regulation and exaggerates postexercise fatigue. We show that circulating levels of endogenous methylated arginines including asymmetric dimethylarginine (ADMA), which act as NO synthase inhibitors, are elevated by acute necrotic muscle damage and in chronically necrotic dystrophin-deficient mice. We therefore hypothesized that excessive ADMA impairs muscle NO production and diminishes exercise tolerance in DMD. We used transgenic expression of dimethylarginine dimethylaminohydrolase 1 (DDAH), which degrades methylated arginines, to investigate their contribution to exercise-induced fatigue in DMD. Although infusion of exogenous ADMA was sufficient to impair exercise performance in wild-type mice, transgenic DDAH expression did not rescue exercise-induced fatigue in dystrophin-deficient male mdx mice. Surprisingly, DDAH transgene expression did attenuate exercise-induced fatigue in dystrophin-heterozygous female mdx carrier mice. Improved exercise tolerance was associated with reduced heart weight and improved cardiac β-adrenergic responsiveness in DDAH-transgenic mdx carriers. We conclude that DDAH overexpression increases exercise tolerance in female DMD carriers, possibly by limiting cardiac pathology and preserving the heart's responses to changes in physiological demand. Methylated arginine metabolism may be a new target to improve exercise tolerance and cardiac function in DMD carriers or act as an adjuvant to promote NO signaling alongside therapies that partially restore dystrophin expression in patients with DMD.NEW & NOTEWORTHY Duchenne muscular dystrophy (DMD) carriers are at risk for cardiomyopathy. The nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is released from damaged muscle in DMD and impairs exercise performance. Transgenic expression of dimethylarginine dimethylaminohydrolase to degrade ADMA prevents cardiac hypertrophy, improves cardiac function, and improves exercise tolerance in DMD carrier mice. These findings highlight the relevance of ADMA to muscular dystrophy and have important implications for therapies targeting nitric oxide in patients with DMD and DMD carriers.

Young rats with increased circulatory asymmetric dimethylarginine exhibited spatial deficit and alterations in dorsal hippocampus brain-derived neurotrophic factor and asymmetric dimethylarginine: Effects of melatonin

Increased plasma concentration of asymmetric dimethylarginine (ADMA) can be encountered in chronic inflammatory disease, liver damage, renal failure, and multiple organ failure. In addition, an association between circulating ADMA and all-cause mortality has been reported. Male Sprague-Dawley rats, postnatal day (PND) 17 ± 1, received continuous ADMA infusion via an intraperitoneal pump. Spatial performance, as well as plasma and dorsal hippocampus ADMA and brain-derived neurotrophic factor (BDNF) concentration, were examined and the effect of melatonin was tested. We found that a 4-week continuous ADMA infusion in young rats caused spatial deficit. Furthermore, increased ADMA concentration and decreased BDNF expression were found in the plasma and dorsal hippocampus. Melatonin protected against these effects, alleviating spatial deficit and reducing the changes in plasma and dorsal hippocampus ADMA and BDNF concentration.

Abstract 415: Transgenic Expression of Dimethylarginine Dimethylaminohydrolase Attenuates Exercise-induced Fatigue in Duchenne Muscular Dystrophy Carrier Mice

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in dystrophin and characterized by muscle degeneration, cardiomyopathy, and impaired muscle nitric oxide (NO) production that disrupts muscle blood flow regulation and leads to excessive post-exercise fatigue. Interestingly, circulating levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) are elevated in dystrophin-deficient subjects. Therefore, we hypothesized that excessive circulating ADMA impairs muscle NO production and thus negatively impacts exercise tolerance in DMD. The objectives of this study were to determine whether increased circulating ADMA is itself sufficient to affect exercise performance, and whether transgenic modulation of ADMA metabolism could improve exercise-induced fatigue in the mdx mouse model of DMD. Although infusion of exogenous ADMA did impair exercise performance in healthy, wild-type mice, transgenic expression of dimethylarginine dimethylaminohydrolase 1 (DDAH), the enzyme that degrades ADMA, did not affect exercise-induced fatigue in dystrophin-deficient male mdx mice. Surprisingly, DDAH transgene expression did attenuate exercise-induced fatigue in dystrophin-heterozygous female mdx carrier mice. This improvement in exercise tolerance was associated with reduced heart weight, improved cardiac contractile function, and improved chronotropic responsiveness to beta-adrenergic stimulation in DDAH-transgenic female mdx carriers. In contrast, DDAH transgene expression did not significantly affect skeletal muscle pathology in female mdx carriers. We conclude that DDAH transgene expression improves exercise tolerance in dystrophin-heterozygous females, possibly by limiting pathological cardiac remodeling and helping to maintain heart performance. These findings emphasize the importance of methylated arginines to the development of cardiomyopathy in female DMD carriers, and have interesting implications for current genetic therapies that may only partially restore dystrophin expression in the hearts of male DMD patients.

Acetylation of asymmetric and symmetric dimethylarginine: an undercharacterized pathway of metabolism of endogenous methylarginines.

Increased levels of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are associated with cardiovascular and renal diseases. We and others have shown that both ADMA and SDMA can be Nα-acetylated to form asymmetric and symmetric Nα-acetyldimethylarginine (Ac-ADMA and Ac-SDMA). The current study further investigated this undercharacterized metabolic pathway. ADMA and SDMA were infused in C57/BL6 mice for 3 days using osmotic minipumps. Half of the mice underwent bilateral nephrectomy 24 h before completion of the infusion. Plasma and tissue levels of Ac-ADMA and Ac-SDMA were detected by liquid chromatography-tandem mass spectrometry. ADMA and SDMA infusion resulted in a 3.6-fold increase in plasma Ac-ADMA and a 21-fold increase in plasma Ac-SDMA levels, respectively. Plasma Ac-ADMA and Ac-SDMA levels were dramatically increased after bilateral nephrectomy. The highest baseline tissue concentrations of Ac-ADMA and Ac-SDMA in wild-type mice were detected in the liver, kidney, small intestine, pancreas and spleen. Incubation of the tissue lysates with ADMA and SDMA resulted in increased levels of the corresponding Nα-acetylated products only in the liver, kidney and small intestine. Our results show that overload of ADMA or SDMA leads to an increase in plasma Ac-ADMA and Ac-SDMA levels. This observation is consistent with the hypothesis that Ac-ADMA and Ac-SDMA are formed directly from ADMA and SDMA in vivo. The increase in plasma Ac-ADMA and Ac-SDMA concentrations after bilateral nephrectomy suggests that both compounds are predominantly eliminated via the kidneys. We demonstrated that acetylation of ADMA and SDMA occurs primarily in the liver, kidney and small intestine.

Role of the endogenous nitric oxide inhibitor asymmetric dimethylarginine (ADMA) and brain-derived neurotrophic factor (BDNF) in depression and behavioural changes: clinical and preclinical data in chronic kidney disease.

Patients with chronic kidney disease (CKD) exhibit a high prevalence of neuropsychiatric alterations, including depression and behavioural changes. CKD is also associated with decreased physical activity not fully explained by co-morbidities. In patients without CKD, the brain-derived neurotropic factor (BDNF) as well as the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) had been suspected to be involved in major depression. The aim of our study was to examine the role of ADMA and BDNF in the behaviour of haemodialysis patients (CKD5D) as well as in a rat model of 5/6 nephrectomy and chronic ADMA infusion alone. Eleven (5F/6M) CKD5D patients underwent Beck Depression Inventory (BDI) testing along with analysis of ADMA and BDNF. Male Sprague-Dawley rats were randomly assigned to four groups: (i) saline infusion; (ii) ADMA (250 µg/kg/day) infusion via osmotic mini pumps; (iii) 5/6 nephrectomy; (iv) untreated controls. After 28 days, the animals underwent behavioural tests measuring anxiety, locomotion and investigative behaviour. Animals were sacrificed, blood samples were drawn and analysed and hippocampal immunohistology for BDNF was performed. In CKD5D patients, decreased BDNF levels correlated with higher scores of depression (Pearson r = -0.8156, P = 0.002). ADMA infusion led to a significant decrease of BDNF while the decrease of BDNF in 5/6 nephrectomy was not significant. However, an attenuated hippocampal BDNF expression could be detected in 5/6 nephrecomized animals. Decreased spontaneous locomotor activity was shown in ADMA-infused rats [15.9 (13.5-26.1) lines crossed/min] and 5/6 nephrectomy [14.6 (6.1-20.2) lines crossed/min] when compared with controls [32.5 (15.3-42.4) lines crossed/min]. Anxiety-like behaviour tested by hole investigation time was significantly more pronounced in 5/6 nephrectomy [24 (6-44) s] when compared with ADMA infusion [64 (28-93) s] and controls [33 (26-65) s]. Progressive renal failure in rats is accompanied by a marked increase of ADMA and a decrease in BDNF. 5/6 nephrectomy leads to significantly decreased exploratory behaviour and locomotion. Both behaviours could be reproduced by ADMA infusion alone. Indicators of anxiety were more pronounced in ADMA-infused animals when compared with 5/6 nephrectomized rats. Furthermore, an inverse relationship of BDNF and BDI in 11 CKD5D patients was shown.

Abstract 162: Detection of N-alpha-acetyltransferase Activity Towards Endogenous Inhibitor of Nitric Oxide Synthase Asymmetric Dimethylarginine in the Liver and Kidneys

Background: Endogenous inhibitor of nitric oxide synthase asymmetric dimethylarginine (ADMA) has been proposed as a risk factor and mediator of cardiovascular diseases. ADMA can undergo hydrolysis by dimethylarginine dimethylaminohydrolase or be processed through an alternative pathway by alanine:glyoxylate aminotransferase 2. We and other have also shown that ADMA can be N-acetylated to form asymmetrical Nα-acetyldimethylarginine (Ac-ADMA). The goal of the study was to characterize this novel pathway in vivo and identify the organs, which are responsible for the ADMA-acetylating activity. Results: We infused ADMA in mice for three days. Control mice received saline infusion. Half of the mice underwent bilateral nephrectomy 24 hours before completion of the infusion, the rest had a sham surgery. ADMA infusion resulted in a 3.5 fold rise in plasma Ac-ADMA levels in the sham-operated mice (1.35 ± 0.45 nmol/L vs. 4.77 ± 0.88 nmol/L). In nephrectomized mice plasma Ac-ADMA levels were markedly increased even after saline infusion (45.08 ± 9.25 nmol/L) and further raised 5-fold after ADMA infusion (229.65 ± 91.30 nmol/L). We assessed tissue distribution of Ac-ADMA in mice and found the highest levels in pancreas, small intestine, liver and kidney. We incubated the lysates of those organs with ADMA for 24 hours and estimated production of Ac-ADMA. Only liver and kidney lysates showed significant increase in Ac-ADMA levels upon addition of ADMA (from 0.03 ± 0.01 nmol/μg protein to 0.18 ± 0.06 nmol/μg protein and from 0.06 ± 0.01 nmol/μg protein to 0.11 ± 0.02 nmol/μg protein). Conclusions: We showed that overload of ADMA leads to increase in plasma Ac-ADMA levels. This observation is consistent with the hypothesis that Ac-ADMA is formed from ADMA in vivo. The striking rise in plasma Ac-ADMA concentrations after bilateral nephrectomy suggests that Ac-ADMA is predominantly eliminated via the kidneys. We demonstrated that liver and kidney are the organs, in which acetylation of ADMA takes place. The high levels of Ac-ADMA in pancreas and intestine may be due to accumulation of Ac-ADMA in those organs. In the next step we want to identify the enzyme responsible for acetylation of ADMA and explore the clinical implications of this novel pathway of ADMA metabolism.

Abstract 19058: Role of Agxt2 in Metabolism of Adma in the Settings of Acute Elevation of Systemic Adma Levels

Background: Endogenous inhibitor of nitric oxide synthases asymmetric dimethylarginine (ADMA) has been proposed to serve as an independent cardiovascular risk factor. The major pathway of ADMA degradation is hydrolysis catalyzed by dimethylarginine dimethylaminohydrolase (DDAH). ADMA can also be metabolized through a different pathway, by alanine:glyoxylate aminotransferase 2 (AGXT2), with the formation of α-keto-δ-(N,N-dimethylguanidino)valeric acid (DMGV). The goal of this study was to better characterize the role of the AGXT2 pathway in metabolism of ADMA in vivo using a recently developed assay for detection of DMGV in biological samples. Methods: ADMA (250 μmol x kg-1 x d-1) was infused in C57/BL6 mice for 3 days using osmotic minipumps implanted intraperitoneally. We performed bilateral nephrectomy in some of the mice 24 hours before the end of ADMA infusion and sample collection. Measurements of ADMA and DMGV were performed LC-MS/MS. Results: Infusion of ADMA for 3 days resulted in a 3.4 fold increase in plasma and in a 5.2 fold increase in urine ADMA levels. Elevation of ADMA concentration coincided with elevation of plasma and urine DMGV levels (2.9 and 3.9 folds respectively). Bilateral nephrectomy in the mice, which did not undergo ADMA infusion, did not increase plasma ADMA levels, while bilateral nephrectomy in the mice, which underwent ADMA infusion, led to a 1.7 fold increase in plasma ADMA levels and a 17.6 fold increase in DMGV levels. Neither bilateral nephrectomy nor infusion of ADMA for 3 days changed the AGXT2 mRNA levels in the liver. Conclusions: Infusion of ADMA leads to increased flux through the AGXT2 pathway of ADMA metabolism in vivo. The observation that each 1 μmol / mmol creatinine increase in ADMA level in urine leads to about 1 μmol / mmol creatinine increase in urine DMGV level suggests that AGXT2 is a major enzyme regulating ADMA levels in urine. The marked increase in DMGV levels in the mice lacking both kidneys suggests significant extrarenal production of DMGV in addition to absent renal excretion. Lack of increase in plasma ADMA levels after nephrectomy in mice infused with saline suggests that ADMA-metabolyzing ezymes are able to compensate for lack of renal excretion of ADMA in our model.

Abstract 219: Agxt2 Plays Important Role in Regulation of Urine Adma Levels After Acute Adma Loading in Mice

Background Multiple epidemiological studies demonstrated increased levels of an endogenous inhibitor of nitric oxide synthases asymmetric dimethylarginine (ADMA) in cardiovascular diseases. In addition to the well characterized pathway of ADMA hydrolysis by dimethylarginine dimethylaminohydrolase (DDAH) ADMA can also be metabolized through an alternative pathway by alanine:glyoxylate aminotransferase 2 (AGXT2), which converts ADMA to α-keto-δ-(N,N-dimethylguanidino)valeric acid (DMGV). The goal of this study was to better characterize the role of AGXT2 pathway in metabolism of ADMA in vivo using a recently developed assay for detection of DMGV in biological samples. Methods ADMA (250 μmol x kg-1 x d-1) was infused in C57/BL6 mice for 3 days using osmotic minipumps implanted intraperitoneally. We performed bilateral nephrectomy in some of the mice 24 hours before the end of ADMA infusion and sample collection. Urine was collected for 24 hours in metabolic cages. Blood was collected by cardiac puncture. Measurements of ADMA and DMGV were performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results Intraperitoneal infusion of ADMA for 3 days resulted in an increase in plasma ADMA levels from 0.46±0.045 to 1.57±0.40 μM (p<0.05) and in urine ADMA levels from 36.4±3.4 to 188.6±31.5 μmol / mmol creatinine (p<0.05). Elevation of ADMA concentration coincided with elevation of plasma DMGV levels from 0.21±0.025 to 0.61±0.14 μM (p<0.05) and urine DMGV levels from 41.4±3.2 to 162.5±32.9 μmol / mmol creatinine (p<0.05). Bilateral nephrectomy in the mice, which underwent ADMA infusion, lead to a 1.7 fold increase in plasma ADMA levels and a 17.6 fold increase in DMGV levels compared with the mice from the sham group. Conclusions Infusion of ADMA leads to increased flux through the AGXT2 pathway of ADMA metabolism in vivo. The observation that each 1 μmol / mmol creatinine increase in ADMA level in urine leads to a ~1 μmol / mmol creatinine increase in urine DMGV level suggests that AGXT2 is a major enzyme regulating ADMA levels in urine. The marked increase in DMGV levels in the mice lacking both kidneys suggests significant extrarenal production of DMGV in addition to absent renal excretion.

Effect of chronic elevated asymmetric dimethylarginine (ADMA) levels on granulopoiesis

The endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is elevated in both animal models of chronic inflammatory disorders as well as in patients with chronic inflammatory disease. In vivo data suggest that ADMA can increase the number of circulating monocytes and possibly affect their adhesion potential in vitro. The aim of our study was to evaluate possible effects of chronically elevated levels of ADMA on white blood cell count (WBC), leukocyte subsets, and WBC distribution pattern using a model of chronic exogenous ADMA infusion. Male Sprague-Dawley rats (n = 20, 10 weeks of age) were randomized to receive either (1) isotonic saline or (2) ADMA applied by osmotic mini pumps. After 28 days of infusion, all animals were sacrificed for blood and tissue sampling. WBC count, flow cytometry for subtype assessment, and histological assessment were performed. Over a time period of 28 days, continuous ADMA infusion significantly increased mean plasma levels (1.26 ± 0.07 μmol/l) as compared to saline infusion (0.57 ± 0.02 μmol/l). Clinical side effects were not observed. Despite a physiologically relevant rise in ADMA plasma levels, measured by decrease of the L-arginine/AMDA ratio-a surrogate parameter of NO production capacity-there was no effect on WBC count or pattern of leukocyte subsets. Numbers and morphology of peripheral blood cells as well as number of NK-cells leveling liver and spleen were not affected by chronic ADMA infusion. Chronically elevated ADMA levels in otherwise healthy rats did not affect WBC counts or leukocyte subsets. Furthermore, anemia frequently found in patients with progressive renal failure and elevated ADMA levels, was not observed. In a chronic inflammatory state, elevated ADMA levels themselves are rather the result than the cause of the underlying inflammatory process.

Asymmetric dimethylarginine may mediate increased heat pain threshold in experimental chronic kidney disease

Thermal sensitivity in uraemia is decreased. Non-selective synthetic nitric oxide synthase (NOS) inhibitors significantly attenuate thermal hyperalgesia in preclinical models. The aim of our study was to evaluate the effect of experimental uraemia, which is associated with an increase of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA), on thermal sensitivity in rats. Furthermore, we intended to study the effect of chronic ADMA infusion alone on thermal sensitivity. Male Sprague-Dawley rats (n = 54), 10 weeks old, weight 370-430 g, were randomly assigned to three groups receiving either (i) isotonic saline or (ii) ADMA via osmotic mini pumps or (iii) underwent 5/6 nephrectomy (Nx). After 14 days, 50% of all animals from all groups underwent thermal sensitivity testing and terminal blood draw. After 28 days, the remaining animals underwent the same procedures. Thermal sensitivity examination was performed by the hot-plate test, measuring time from heat exposition to first paw licking or jumping of the animal. While the median [interquartile range] latency time between heat exposition to first paw licking or jumping of the animal in the NaCl infusion group remained unchanged between Day 14 (8.4 [6.75-11.50] s) and Day 28 (7.35 [6.10-7.90] s) both, ADMA infusion and 5/6 nephrectomy tended to increase the thermal pain threshold at Day 14 (9.25 [6.55-12.18] s) and (9.50 [5.8 ± 11.0] s), respectively, compared to NaCl on Day 14 (8.4 [6.75-11.50] s). This difference became statistical significant at Day 28 where the median latency time in the ADMA group (13.10 [11.85-15.95] s) and in the 5/6 Nx group (13.50 [10.85-17.55] s) were significantly higher than in the NaCl group (7.35 [6.10-7.90] s). Induction of progressive renal failure in rats by 5/6 nephrectomy, which is accompanied by a marked increase of the serum levels of the endogenous NOS inhibitor ADMA, leads to a significantly increased heat pain threshold at 28 days. The sole infusion of ADMA into healthy rats leads to the same increase in heat pain threshold.

Altered Asymmetric Dimethyl Arginine Metabolism in Allergically Inflamed Mouse Lungs

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), causes uncoupling of NOS leading to generation of reactive nitrogen species, such as peroxynitrite. The lung generates a significant amount of ADMA, potentially contributing to plasma ADMA levels that have been related to endothelial dysfunction. ADMA infusion causes increased collagen deposition in lungs, suggesting that it could influence the development of chronic lung diseases such as fibrosis, chronic obstructive pulmonary disease, and asthma. To explore the link between endogenous ADMA and asthma, we determined the levels of ADMA, enzymes implicated in its metabolism, and peroxynitrite in murine models of allergic airway inflammation (AAI) resembling asthma. ADMA levels and nitrosative stress were found to be positively correlated in cytosol and mitochondria during AAI. This was associated with increased expression of protein-arginine methyltransferase-2, an ADMA-synthesizing enzyme, and reduced expression of dimethylarginine dimethylaminohydrolase-2, an ADMA-degrading enzyme, in bronchial epithelia. Increased nitrotyrosine similarly localized to the bronchial epithelium, as well as in infiltrated inflammatory cells. Administration of L-arginine, which was expected to compete with ADMA and reverse the uncoupling/inhibition of NOS, restored normal ADMA metabolism, along with the expected reduction of nitrosative stress in lung. Because dimethylarginine dimethylaminohydrolase-2 function is known to be negatively related to oxidative stress, this may represent a feed-forward loop effect. We conclude that a delicate balance between ADMA-metabolizing enzymes is disturbed in bronchial epithelium during AAI, potentially causing increased nitrosative stress in a self-propagating cycle. This represents a potential therapeutic target in asthma.

Low arginine/asymmetric dimethylarginine ratio deteriorates systemic hemodynamics and organ blood flow in a rat model

Both arginine and asymmetric dimethylarginine (ADMA) play a crucial role in the arginine-nitric oxide pathway. Low arginine and high ADMA levels can be found in critically ill patients after major surgery. The aim of this study was to evaluate the effects of low arginine plasma concentrations in combination with high ADMA plasma concentrations on hemodynamics and organ blood flow. Randomized, placebo-controlled animal laboratory investigation. Male Wistar rats (n = 21), anesthetized. Rats were randomly assigned to three groups: a control group, an ADMA group, or an arginase (ASE)/ADMA group. In the control group, rats received (at t = 0) an intravenous (IV) infusion of 1.5 mL 0.9% NaCl during a 20-minute period. After 60 minutes (t = 60), rats received an IV bolus of 1.0 mL 0.9% NaCl. In the ADMA group, rats received an IV infusion of 1.5 mL 0.9% NaCl during a 20-minute period and at t = 60 an IV bolus of 1.0 mL ADMA (20 mg/kg). In the ASE/ADMA group, rats received an IV infusion of 1.5 mL ASE (3200 IU) solution during a 20-minute period and at t = 60 an IV bolus of 1.0 mL ADMA (20 mg/kg). Infusion of ADMA (20 mg/kg) and ASE (3200 IU) resulted in increased plasma ADMA levels and decreased arginine levels. During the whole experiment, systemic hemodynamics (heart rate, mean arterial pressure [MAP], and cardiac output) were measured. In addition, organ blood flow was measured at t = 90 and t = 180 minutes, using fluorescent microspheres. Compared with the control group, MAP and systemic vascular resistance were increased after infusion of ADMA. Infusion of ASE in combination with ADMA significantly deteriorated systemic hemodynamics (MAP, cardiac output, stroke volume, and systemic vascular resistance) and organ blood flow through the kidney and spleen. In addition, an initial decrease in arterial flow, followed by a later major increase, and panlobular apoptosis and necrosis of the liver was observed. The current study shows that low arginine plasma levels in combination with high ADMA plasma levels deteriorates systemic hemodynamics and reduces blood flow through the kidney and spleen and liver. These data suggest that a diminished nitric oxide production may be involved in the onset of organ failure.

Elevated Asymmetric Dimethylarginine Alters Lung Function and Induces Collagen Deposition in Mice

Increasing evidence suggests that lung mechanics and structure are maintained in part by an intimate balance between the L-arginine-metabolizing enzymes nitric oxide synthase (NOS) and arginase. Asymmetric dimethylarginine (ADMA) is a competitive endogenous inhibitor of NOS. The role of ADMA in the regulation of NOS and arginase in the airways has not yet been explored. Our objective was to investigate the role of ADMA in lung physiology. A murine model of continuous subcutaneous ADMA infusion via osmotic minipump was used for assessment of elevated ADMA in vivo, and primary lung fibroblasts were used for in vitro assessments. Two weeks after minipump placement, animals were anesthetized and mechanically ventilated, and lung mechanical responses were evaluated. Lungs were assessed histologically and biochemically for collagen content, arginase activity, and arginase protein levels. Lung lavage fluid was assessed for cellularity, nitrite, urea, and cytokine concentrations. ADMA infusion resulted in significantly enhanced lung resistance and decreased dynamic compliance in response to methacholine. These physiologic changes were associated with significantly increased lung collagen content in the absence of inflammation. Significant decreases in lung fluid nitrite were accompanied by elevated lung fluid urea and arginase activity in lung homogenates. These changes were reversed in mice 4 weeks after completion of ADMA administration. In addition, treatment of primary mouse lung fibroblasts with ADMA stimulated arginase activity and collagen formation in vitro. These data support the idea that ADMA may play a role in airway diseases, including asthma and pulmonary fibrosis, through NOS inhibition and enhancement of arginase activity.

Asymmetric Dimethylarginine and Cerebrovascular Disorders in Humans

To the Editor: We read with great interest the recent article by Dr Kielstein and colleagues1 dealing with the cerebrovascular effects of the endogenous nitric oxide (NO) synthase inhibitor, asymmetric dimethylarginine (ADMA), in humans. The results of their study demonstrated that systemic infusion of ADMA increased arterial stiffness and decreased total cerebral perfusion independent of blood pressure changes. The authors proposed that ADMA modulated vascular compliance and decreased cerebral blood flow and might be involved in the pathogenesis of cerebrovascular diseases in humans. There is evidence that ADMA may actively participate in the …

ADMA Increases Arterial Stiffness and Decreases Cerebral Blood Flow in Humans

Preclinical studies have revealed that the endogenous nitric oxide synthase inhibitor, asymmetric dimethylarginine (ADMA), increases vascular tone in cerebral blood vessels. Marked elevations of ADMA blood levels were found in patients with diseases characterized by decreased cerebral perfusion, such as ischemic stroke. Arterial stiffness is an independent predictor of stroke and other adverse cardiovascular events. The aim of this study was to investigate the influence of a systemic subpressor dose of ADMA on arterial stiffness and cerebral perfusion in humans. Using a double-blind, vehicle-controlled study design, we allocated 20 healthy men in random order to infusion of either ADMA (0.10 mg ADMA/kg per min) or vehicle over a period of 40 minutes. Arterial stiffness was assessed noninvasively by pulse wave analysis. All volunteers underwent measurement of cerebral perfusion by dynamic contrast-enhanced perfusion magnetic resonance imaging of the brain. Infusion of ADMA significantly decreased total cerebral perfusion by 15.1+/-4.5% (P=0.007), whereas blood flow in the vehicle group increased by 7.7+/-2.8% (P=0.02). ADMA also increased arterial stiffness as assessed by measurement of the augmentation index (-12.6+/-1.9 to -9.6+/-1.5, P=0.007). Our results document for the first time that subpressor doses of ADMA increase vascular stiffness and decrease cerebral perfusion in healthy subjects. Thus, ADMA is an important endogenous modulator of cerebral vascular tone and may be involved in the pathogenesis of cerebrovascular disease.

Effects of asymmetric dimethylarginine (ADMA) infusion in humans

Increased blood concentrations of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) can be found in patients with cardiovascular risk factors such as age, hypertension, diabetes, insulin resistance, hypercholesterolemia, hypertriglyceridemia, chronic kidney disease, and hyperhomocystinemia. ADMA has been shown to be a strong and independent predictor of cardiovascular and overall mortality in selected patient populations. Furthermore, in patients with chronic kidney disease, it is a strong and independent risk marker for decrease of renal function, progression to end-stage renal disease, and mortality. Infusion of exogenous ADMA in humans helped to elucidate its role in the pathogenesis of endothelial dysfunction. Pathophysiologically relevant concentrations of ADMA have been shown to decrease heart rate and cardiac output and to increase systemic vascular resistance and pulmonary vascular resistance. ADMA decreases effective renal plasma flow and increases renovascular resistance in a dose-related manner. Moreover, administration of ADMA causes significant sodium retention and blood pressure increase. These studies also revealed that ADMA is less potent than the synthetic NOS inhibitor N G-nitro-L-arginine methyl ester (L-NAME) and behaves differently in respect to onset of action, which can be explained by the different routes of elimination as well as different cellular transport mechanisms. Collectively these results document that ADMA has well-defined effects on cardiovascular and renal function in healthy subjects. It is therefore conceivable that ADMA causes sustained changes in vascular function through an intracellular action in endothelial cells at blood concentrations found in patients with cardiovascular pathology.

Liquid Chromatography–Tandem Mass Spectrometry Method for the Analysis of Asymmetric Dimethylarginine in Human Plasma

Nitric oxide (NO) is essential in numerous physiologic processes and may be involved in related pathologic processes. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of isoforms of NO synthase in humans (1). ADMA originates from protein arginine methylation by protein arginine methyltransferases after protein hydrolysis (2). Enzymatic hydrolysis by dimethylarginine dimethylaminohydrolases to dimethylamine and citrulline is the major pathway for elimination of ADMA (3). Circulating ADMA is altered in patients with cardiovascular and neurologic diseases, erectile dysfunction, and many other disorders (4)(5)(6), and increased circulating ADMA independently predicts future cardiovascular events and mortality (7)(8). Short-time infusion of ADMA affects hemodynamics and cardiac function in humans (3)(9). Analytical methods for the measurement of ADMA include HPLC, capillary electrophoresis, ELISA, and mass spectrometry (MS). The commonly used HPLC methods with fluorescence detection (10) measure o -phthaldialdehyde derivatives of ADMA. These derivatives are not stable and must be analyzed on-line. Moreover, ADMA must be separated by chromatographic means from its biologically inactive isomer, symmetric dimethylamine (SDMA). Thus, these HPLC methods have long analysis times of up to 45 min. Alternatively, ADMA and SDMA can be analyzed by gas chromatography–mass spectrometry (11)(12), which shows different fragmentation patterns for the respective ions. These methods include several extraction and derivatization steps and require considerable analysis times. Recently, Kirchherr and Kuhn-Velten (13) developed a mass spectrometric method that omits derivatization procedures; however, chromatographic separation of ADMA from SDMA is still required for this method. Here we report a simple and rapid liquid chromatography–tandem MS (LC-MS/MS)-based method with 1 derivatization step and sample pretreatment consisting of protein precipitation. This is the first LC-MS–based method that separates methylated arginine derivatives by specific fragmentation patterns instead of by chromatographic separation. Analyses were performed on a Varian 1200L Triple Quadrupole …

Cardiovascular Effects of Asymmetric Dimethylarginine

To the Editor: Kielstein et al1 describe the effects of the endogenous nitric oxide synthase inhibitor, asymmetric dimethylarginine (ADMA), in humans in vivo but do not make reference to two previous human studies.2,3 In the original paper describing the presence of ADMA in human plasma and its accumulation in chronic renal failure, Vallance et al2 also described the effects of ADMA in isolated blood vessels, on blood pressure in the guinea pig, and on forearm blood flow in healthy human subjects. They described an 8.3% fall in forearm blood flow after an 8 μmol/min ADMA infusion.2 It was this finding that led …

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 µmol/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.