Nitric Oxide Synthase Inhibitor Asymmetric Dimethylarginine Research Articles

Differences in l-arginine metabolism and asthma morbidity among asthma patients with and without obstructive sleep apnea

BackgroundImbalance in l-arginine and nitric oxide (NO) metabolism has been implicated in the pathophysiology of asthma and obstructive sleep apnea (OSA), and both diseases impact the other’s morbidity. We sought to determine whether l-arginine/NO metabolism differs between adults with asthma with or without comorbid OSA, and its association with asthma morbidity.MethodsThis is a cross-sectional study of 322 adults with asthma recruited in Denver, CO and New York City, NY. Data were collected on OSA status, spirometry, and metrics of asthma control and morbidity. l-Arginine metabolites were quantified in patient serum. Bivariate analyses and multiple regression were performed to determine differences between l-arginine metabolism, OSA and association with asthma morbidity.ResultsAmong the 322 participants, 92 (28.5%) had OSA. The cohort was 81.6% female, 23.4% identified as Black and 30.6% as Latino. Patients with asthma and OSA had significantly higher serum concentrations of NO synthase inhibitor asymmetric dimethylarginine (ADMA) (p-value = 0.019), lower L-arginine to ornithine ratios (p-value = 0.003), and increased ornithine (p-value = 0.001) and proline levels (p-value < 0.001) compared to those without OSA. In adjusted models, OSA was associated with worse asthma control, adjusted mean difference in asthma control questionnaire of 0.36 (95% confidence interval [CI]: 0.06 to 0.65), and asthma quality of life questionnaire, adjusted mean difference: − 0.53 (95% CI: − 0.85 to − 0.21), after adjusting for relevant covariates including body mass index and L-arginine metabolites.ConclusionsAdults with asthma and OSA had increased ADMA, an inhibitor of nitric oxide synthase, and greater metabolism of l-arginine via the arginase pathway compared to those with asthma alone, indicating a possible shared pathophysiological mechanism of these diseases.

Metabolic Pattern of Systemic Sclerosis: Association of Changes in Plasma Concentrations of Amino Acid-Related Compounds With Disease Presentation.

ObjectiveAmino acids (AA) and their derivatives play an integral role in the synthesis of structural and regulatory elements in human organisms and therefore pathologies such as systemic sclerosis that may alter the blood pattern of these compounds. This study aimed to evaluate changes in plasma concentrations of amino acid-related metabolites in systemic sclerosis in a search for potential biomarkers and mechanisms of the disease.MethodsPlasma samples from 42 patients diagnosed with systemic sclerosis (SSc) according to the 2013 American College of Rheumatology and European League Against Rheumatism ACR/EULAR classification criteria were compared to 27 matched healthy controls. Liquid chromatography/mass spectrometry was applied for the analysis of 36 amino acid-related metabolites.ResultsThe analysis of plasma AA metabolite patterns revealed the number of changes including an increase (20%) in concentrations of NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) in SSc vs. healthy subjects. Furthermore, SSc patients had higher glutamine, proline, betaine, 1-methylhistidine, and methylnicotinamide levels, while the concentration of tryptophan was lower. The specific metabolic pattern was identified for several aspects of disease presentation. Most interestingly NOS inhibitor L-NAME was elevated in patients with diffuse systemic sclerosis or telangiectasia.ConclusionsThese results provide further evidence for the involvement of endothelium-dependent pathways in the mechanisms and presentation of SSc. Endothelial dysfunction biomarkers may be useful in the assessment of presentation and prognosis in SSc.

Fourteen-Day Study of Praliciguat, a Soluble Guanylate Cyclase Stimulator, in Patients with Diabetes and Hypertension

Background: Praliciguat (IW-1973), a soluble guanylate cyclase stimulator, has been shown to augment nitric oxide (NO) signaling and reduce fasting plasma glucose and proteinuria in an animal model of diabetic nephropathy. In healthy adults, repeated oral doses of 15-40 mg were tolerated and lowered blood pressure (BP). Methods: This Phase 2a, double-blind, placebo (PBO)-controlled study assessed the effects of oral praliciguat in 26 patients with type 2 diabetes mellitus (T2DM) and hypertension on stable glycemic and BP-lowering therapy. Most (69%) were also on statins. Two praliciguat regimens were tested: (1) 40 mg QD for days 1-14 (N=10), and (2) 20 mg BID for days 1-7 then 40 mg QD for days 8-14 (N=10). Assessments included serum chemistry, fasting plasma glucose, BP and heart rate, platelet function, reactive hyperemia index (RHI), plasma biomarkers, and adverse events (AEs). Results: Results were similar for both praliciguat regimens and are here combined. After 14 days of treatment, praliciguat-treated patients had notable differences in least squares mean change from baseline (95% CI) relative to PBO in 24-hour mean arterial pressure [-4.7 mmHg (-10, 0.8)], heart rate [2.9 bpm (-0.7, 6.5)], fasting glucose [-13 mg/dL (-32, 7)], HOMA-IR [-26 (-58, 7)], total serum cholesterol [-26 mg/dL (-44, -7)], and LDL cholesterol [-20 mg/dL (-37, -3)]. Praliciguat had no effect on platelet function or RHI. Among the exploratory biomarkers tested, levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine were lower in praliciguat vs. PBO recipients. Both praliciguat dose regimens were generally well tolerated. Except for one serious AE of upper gastrointestinal hemorrhage, AEs in praliciguat-treated patients were mild. Conclusion: In this initial trial, praliciguat had beneficial effects on BP and metabolic parameters in T2DM patients with hypertension on standard therapies. Further clinical investigation of praliciguat in T2DM patients is warranted. Disclosure J.P. Hanrahan: Employee; Self; Ironwood. J.D. Wakefield: Employee; Self; Ironwood Pharmaceuticals, Inc.. Stock/Shareholder; Self; Ironwood Pharmaceuticals, Inc.. P.J. Wilson: None. P. Miller: None. J. Chickering: None. L. Morrow: Stock/Shareholder; Self; ProSciento. Employee; Spouse/Partner; Eli Lilly and Company. M.L. Hall: Employee; Self; Ironwood Pharmaceuticals, Inc.. Stock/Shareholder; Self; Ironwood Pharmaceuticals, Inc. M. Currie: Employee; Self; Ironwood Pharmaceuticals, Inc. G. Milne: Employee; Self; Ironwood Pharmaceuticals, Inc.. Stock/Shareholder; Self; Ironwood Pharmaceuticals, Inc.. Employee; Spouse/Partner; Catabasis Pharmaceuticals. Stock/Shareholder; Spouse/Partner; Catabasis Pharmaceuticals. A.T. Profy: Other Relationship; Self; Ironwood Pharmaceuticals.

DYNAMICS OF ASYMMETRIC DIMETHYLARGININE AFTER RENAL DENERVATION IN PATIENTS WITH RESISTANT HYPERTENSION

Objective: The recent experimental and clinical studies evidence a potential bidirectional relationship between sympathetic activity and the endogenous circulating NO synthase inhibitor asymmetric dimethylarginine (ADMA). However, the data are controversial. The purpose of our study was to assess the change in ADMA level after renal sympathetic denervation (RSDN) in patients with resistant hypertension (RAH). Design and method: We examined 19 patients (7 males) with verified RAH [mean age – 59 (28–70) years], who underwent the RSDN by standard protocol (Simplicity, Medtronic). ADMA level was measured by immune-enzyme assay (ADMA direct ELISA Kit, Immunodiagnostic AG, Bensheim, Germany, reference values 0.26–0.64 mcmol/l) at baseline before the RSDN (the first patient was enrolled in 2013), 6 and 12 months after the operation (the last patient was examined in 2015). Statistical analysis was carried out using the program SPSS 17.0, the data are presented as median (25%;75%). Results: At baseline ADMA was 0.81 (0.3; 1.98) mcmol/l. The increased values were found in 1 male and 9 females (chi-square = 1.9; p = 0.2) without any gender differences compared to those with normal ADMA level. ADMA level remained unchanged at 6-month follow-up: 0.83 (0.35; 0.94) mcmol/l (p = 0.91). Although it decreased by 12-month follow-up visit – 0.66 (0.23; 0.99) mcmol/l, the changes were non-significant (p = 0.6, repeated measures, one-way ANOVA). Conclusions: Half of RAH patients (53%) demonstrate increased ADMA indicating worse prognosis. Renal denervation failed to decrease ADMA level. However, small sample size and a trend toward improvement of endothelial function deserves further investigation in order to understand who may benefit from the RSDN procedure in the very high risk population with resistant hypertension.

Role of nitric oxide synthase inhibitor in the skeletal muscle contractile dysfunction of type 2 diabetic rats

Objective To investigate the role of endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) in the contractile dysfunction of skeletal muscle in diabetic rats and on which the therapeutic effects of L-Arginine. Methods Type 2 diabetic rats were induced by high fat diet and a single intraperitoneal injection of streptozotocin (STZ, 30 mg/kg), followed by high fat diet for 8 weeks. Specific twicth tension and specific tetanic tension of soleus (SOL) and extensor digitorum longus (EDL) isolated from control and diabetic rats were detected by electric stimulation to reflect contractile function of skeletal muscle. ADMA content of skeletal muscle was analyzed by enzyme linked immunosorbent assay (ELISA), and activities of dimethylarginie dimethylaminohydrolase (DDAH) and NOS, NO content were measured by colorimetry. The protein expression of ADMA synthetase protein arginine methyl transferase 1 (PRMT1) and ADMA hydrolase DDAH and NOS were determined detected by Western blott-ing. Oral glucose tolerance test and protein expressions of phosphorylated insulin receptor substrate 1(p-IRS-1) and protein kinase B (p-Akt) as well as the membrane transportation of glucose transporter 4 (Glut4) were measured to reflect insulin resistance. Results In comparison with control rats, specific twicth tension and tetanic tension of SOL and EDL in diabetic rats were significantly decreased (P<0.01), indicating the contractile dysfunction. Increased ADMA content (P<0.05), decreased DDAH and NOS activities as well as NO content (P<0.01) in comparison with up-regulated protein expression of PRMT1 and down-regulated protein expression of DDAH, endothelial NOS (eNOS) and neuronal NOS (nNOS)(P<0.05) were observed in the skeletal muscle of diabetic rats compared to control rats, indicating that the pathway of PRMT1/ADMA/DDAH/ NOS/NO was disordered in the skeletal muscle of diabetic rats. Furthermore, the glucose tolerance, both IRS-1 and Akt protein phosphorylation as well as the membrane translocation of Glut4 were decreased (P<0.05), indicating the insulin resistance in diabetic rats. Treatment with L-Arginine for 8 weeks not only significantly improved the contractile dysfunction but also reversed the disorder of ADMA signaling pathway and insulin resistance in skeletal muscle of diabetic rats compared to untreated diabetic rats. Conclusions The accumulation of endogenous NOS inhibitor ADMA contributes to the contractile dysfunction of skeletal muscle in diabetic rats, the underlying mechanism may be related to insulin resistance. Key words: Asymmetric dimethylarginine; Type 2 diabetes mellitus; Contractile dysfunction; Insulin resistance; L-arginine

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.

Pharmacological assessment of ibuprofen arginate on platelet aggregation and colon cancer cell killing

Nonsteroidal anti-inflammatory drugs (NSAIDs), including ibuprofen, are amongst the most commonly used medications and produce their anti-inflammatory and analgesic benefits by blocking cyclooxygenase (COX)-2. These drugs also have the potential to prevent and treat cancer and some members of the class including ibuprofen can produce anti-platelet effects. Despite their utility, all NSAIDs are associated with increased risk of cardiovascular side effects which our recent work suggests could be mediated by increased levels of the endogenous NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) leading to reduced endothelial NOS activity and associated endothelial cell dysfunction. ADMA is a cardiotoxic hormone and biomarker of cardiovascular risk whose effects can be prevented by l-arginine. The ibuprofen salt, ibuprofen arginate (Spididol®) was created to increase drug solubility but we have previously established that it not only effectively blocks COX-2 but also provides an arginine source able to reverse the effects of ADMA in vitro and in vivo. Here we have gone on to explore whether the formulation of ibuprofen with arginine influences the potency and efficacy of the parent molecule using a range of simple in vitro assays designed to test the effects of NSAIDs on (i) platelet aggregation and (iii) colon cancer cell killing. Our findings demonstrate that ibuprofen arginate retains these key functional effects of NSAIDs with similar or increased potency compared to ibuprofen sodium, further illustrating the potential of ibuprofen arginate as an efficacious drug with the possibility of improved cardiovascular safety.

Serum L-arginine and dimethylarginine levels in migraine patients with brain white matter lesions

Background/Aim Migraine is a risk factor for the formation of silent brain white matter lesions (WMLs) that are possibly ischemic in nature. Although dysfunction of the L-arginine/nitric oxide (NO) pathway has been associated with oxidative stress and endothelial dysfunction in migraine, its role in WML development has not been specifically investigated. Thus, this prospective study aimed to measure the serum concentrations of the NO substrate L-arginine, the NO synthase inhibitor asymmetric dimethylarginine (ADMA), and the L-arginine transport regulator symmetric dimethylarginine (SDMA) in migraine patients in a headache-free period. Methods All participants underwent MR imaging to assess for the presence of WMLs on fluid-attenuated inversion recovery imaging. Altogether 109 migraine patients (43 with lesions, 66 without lesions) and 46 control individuals were studied. High-performance liquid chromatography was used to quantify L-arginine, ADMA and SDMA serum concentrations. Migraine characteristics were investigated, and participants were screened for risk factors that can lead to elevated serum ADMA levels independent of migraine. Results Migraine patients and controls did not differ in regard to vascular risk factors. Migraineurs with WMLs had a longer disease duration ( p < 0.001) and a higher number of lifetime headache attacks ( p = 0.005) than lesion-free patients. Higher L-arginine serum levels were found in both migraine subgroups compared to controls ( p < 0.001). Migraine patients with WMLs showed higher ADMA concentrations than lesion-free patients and controls ( p < 0.001, for both). In migraineurs, the presence of WMLs, aura and increasing age proved to be significant predictors of increased ADMA levels ( p = 0.008, 0.047 and 0.012, respectively). SDMA serum levels of lesional migraineurs were higher than in nonlesional patients ( p < 0.001). The presence of lesions and increasing age indicated an increased SDMA level ( p = 0.017 and 0.001, respectively). Binary logistic regression analysis showed that ADMA level ( p = 0.006), increasing age ( p = 0.017) and the total number of lifetime migraine attacks ( p = 0.026) were associated with an increased likelihood of exhibiting WMLs. There was no significant effect of age on ADMA and SDMA concentrations in controls. Conclusions Elevated ADMA levels may impact the pathogenesis of migraine-related WMLs by influencing cerebrovascular autoregulation and vasomotor reactivity. Higher SDMA concentrations may indirectly influence NO synthesis by reducing substrate availability. Elevated L-arginine serum levels might reflect an increased demand for NO synthesis.

Evidence that links loss of cyclooxygenase-2 with increased asymmetric dimethylarginine: novel explanation of cardiovascular side effects associated with anti-inflammatory drugs.

Cardiovascular side effects associated with cyclooxygenase-2 inhibitor drugs dominate clinical concern. Cyclooxygenase-2 is expressed in the renal medulla where inhibition causes fluid retention and increased blood pressure. However, the mechanisms linking cyclooxygenase-2 inhibition and cardiovascular events are unknown and no biomarkers have been identified. Transcriptome analysis of wild-type and cyclooxygenase-2(-/-) mouse tissues revealed 1 gene altered in the heart and aorta, but >1000 genes altered in the renal medulla, including those regulating the endogenous nitric oxide synthase inhibitors asymmetrical dimethylarginine (ADMA) and monomethyl-l-arginine. Cyclo-oxygenase-2(-/-) mice had increased plasma levels of ADMA and monomethyl-l-arginine and reduced endothelial nitric oxide responses. These genes and methylarginines were not similarly altered in mice lacking prostacyclin receptors. Wild-type mice or human volunteers taking cyclooxygenase-2 inhibitors also showed increased plasma ADMA. Endothelial nitric oxide is cardio-protective, reducing thrombosis and atherosclerosis. Consequently, increased ADMA is associated with cardiovascular disease. Thus, our study identifies ADMA as a biomarker and mechanistic bridge between renal cyclooxygenase-2 inhibition and systemic vascular dysfunction with nonsteroidal anti-inflammatory drug usage. We identify the endogenous endothelial nitric oxide synthase inhibitor ADMA as a biomarker and mechanistic bridge between renal cyclooxygenase-2 inhibition and systemic vascular dysfunction.

Endogenous Nitric-Oxide Synthase Inhibitor ADMA after Acute Brain Injury

Previous results on nitric oxide (NO) metabolism after traumatic brain injury (TBI) show variations in NO availability and controversial effects of exogenous nitric oxide synthase (NOS)-inhibitors. Furthermore, elevated levels of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) were reported in cerebro-spinal fluid (CSF) after traumatic subarachnoid hemorrhage (SAH). Therefore, we examined whether ADMA and the enzymes involved in NO- and ADMA-metabolism are expressed in brain tissue after TBI and if time-dependent changes occur. TBI was induced by controlled cortical impact injury (CCII) and neurological performance was monitored. Expression of NOS, ADMA, dimethylarginine dimethylaminohydrolases (DDAH) and protein-arginine methyltransferase 1 (PRMT1) was determined by immunostaining in different brain regions and at various time-points after CCII. ADMA and PRMT1 expression decreased in all animals after TBI compared to the control group, while DDAH1 and DDAH2 expression increased in comparison to controls. Furthermore, perilesionally ADMA is positively correlated with neuroscore performance, while DDAH1 and DDAH2 are negatively correlated. ADMA and its metabolizing enzymes show significant temporal changes after TBI and may be new targets in TBI treatment.

Biological Functional Relevance of Asymmetric Dimethylarginine (ADMA) in Cardiovascular Disease

There is growing evidence that increased levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) may contribute to endothelial dysfunction. Studies in animal models as well as in humans have suggested that the increase in ADMA occurs at a time when vascular disease has not yet become clinically evident. ADMA competitively inhibits NO elaboration by displacing l-arginine from NO synthase. In a concentration-dependent manner, it thereby interferes not only with endothelium-dependent, NO-mediated vasodilation, but also with other biological functions exerted by NO. The upshot may be a pro-atherogenic state. Recently, several studies have investigated the effect of various therapeutical interventions on ADMA plasma concentrations.

Treatment with the Nitric Oxide Donor SNP Increases Triiodothyronine Levels in Hyper- and Hypothyroid Sprague-Dawley Rats

Nitric oxide pathway might play a crucial role in the pathophysiology of thyroid dysfunctions. This study aimed to investigate the impact of nitric oxide (NO) on hypothyroid and hyperthyroid Sprague-Dawley rats under controlled diet. Furthermore, the effects of the nitric oxide donor sodium nitroprusside (SNP) on thyroid dysfunctions were also assessed. Sprague-Dawley rats (n=107) were subdivided into normal diet and high-fat diet (HFD) groups and grouped into controls, hypothyroid, hyperthyroid, and SNP treated groups. Hypothyroidism was induced through propylthiouracil, whereas hyperthyroidism by triiodothyronine (T3). After 12 weeks of T3 treatment, serum nitric oxides (NOX), endogenous asymmetric dimethylarginine (ADMA), body weight and food intake were analyzed. Hypothyroid rats showed decreased serum T3 levels, hyperthyroid rats increased T3 compared to controls. Diet had no impact on T3. Thyroid dysfunctions were accompanied by changes in calorie intake and body weight. Serum NOX was significantly reduced in normal diet hypothyroid rats. SNP administration compensated the decrease and markedly increased T3. NO synthase inhibitor ADMA levels were significantly higher in the HFD control group than in the normal diet controls. ADMA was declined in both hypothyroid groups and increased in normal diet hyperthyroid rats. An association of thyroid dysfunctions with reduced bioavailability of NO and alterations of ADMA levels could be established. Treatment with the NO donor SNP resulted in an increase of serum T3 levels. These results demonstrate that the NO pathway is implicated in thyroid dysfunctions, which may be of clinical relevance.

Asymmetric Dimethylarginine in Response to Recombinant Tissue-Type Plasminogen Activator and Erythropoietin in Acute Stroke

In the German Multicenter Erythropoietin (EPO) Stroke Trial, patients not receiving thrombolysis most likely benefited from EPO on clinical recovery, whereas a combination of rtPA and EPO was associated with increased mortality. We investigated whether the combination of rtPA and EPO increased release of the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA), and thereby potentially deteriorated ischemic stroke outcome, as suggested from experimental data. ADMA was determined in serum samples from 90 patients of the German Multicenter EPO Stroke Trial taken at days 1 (within 6 hours after symptom onset), 2, 3, 4, and 7 after stroke using high-performance liquid chromatography-tandem mass spectrometry. ADMA was analyzed for the different treatment groups (EPO, n=25; placebo, n=30; rtPA+placebo, n=18; EPO+rtPA, n=17). Clinical outcome was expressed as difference between National Institutes of Health Stroke Scale at baseline and 90 days. ADMA levels significantly increased during the observation time in EPO, EPO+rtPA, and placebo groups (P<0.05). A treatment effect on ADMA levels was revealed by repeated measures ANOVA only in the rtPA+placebo group (P=0.027). Here, ADMA levels were decreased compared with the placebo group (P<0.05). Both the EPO and the rtPA+placebo groups in the Hannover subgroup of the EPO trial had better outcome than the placebo group (P<0.05). Our data underscore the potential benefit of EPO in ischemic stroke. The hypothesis from experimental data, that EPO treatment increases ADMA in stroke patients, was disproved. Further studies are needed to clarify whether decreased ADMA might contribute to therapeutic rtPA effects.

Does Oxidative Stress Play a Critical Role in Cardiovascular Complications of Kawasaki Disease?

The aim of the present work was to evaluate the contribution of the different reactive oxidizing species to systemic oxidative stress in the whole blood of patients with Kawasaki disease (KD). This is a rare generalized systemic vasculitis typical of the early childhood characterized by inflammation and endothelial dysfunction with a high risk for cardiovascular fatal events. We found that, compared to age-matched healthy donors, blood from KD patients showed increased production of oxygen- and nitrogen-derived species as detected by electron paramagnetic resonance (EPR) spin probing with the cyclic hydroxylamine 1-hydroxy-3-carboxy-pyrrolidine. The (•)NO pathway involvement was also confirmed by the decreased concentrations of the endogenous (•)NO synthase inhibitor asymmetric dimethyl-arginine and the increased amounts of 3-nitrotyrosine in plasma. Further, increased plasma yields of the proinflammatory enzyme myeloperoxidase were also observed. The appearance of circulating red blood cell alterations typically associated with oxidative imbalance and premature aging (e.g., decrease of total thiol content, glycophorin A, and CD47 expression, as well as increase of phosphatidylserine externalization) has also been detected. Collectively, our observations lead to hypothesize that the simultaneous oxidative and nitrative stress occurrence in the blood of KD patients may play a pathogenetic role in the cardiovascular complications often associated with this rare disease.

Nitric oxide and l-arginine metabolism in a devascularized porcine model of acute liver failure

In acute liver failure (ALF), the hyperdynamic circulation is believed to be the result of overproduction of nitric oxide (NO) in the splanchnic circulation. However, it has been suggested that arginine concentrations (the substrate for NO) are believed to be decreased, limiting substrate availability for NO production. To characterize the metabolic fate of arginine in early-phase ALF, we systematically assessed its interorgan transport and metabolism and measured the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) in a porcine model of ALF. Female adult pigs (23-30 kg) were randomized to sham (N = 8) or hepatic devascularization ALF (N = 8) procedure for 6 h. We measured plasma arginine, citrulline, ornithine levels; arginase activity, NO, and ADMA. Whole body metabolic rates and interorgan flux measurements were calculated using stable isotope-labeled amino acids. Plasma arginine decreased >85% of the basal level at t = 6 h (P < 0.001), whereas citrulline and ornithine progressively increased in ALF (P < 0.001 and P < 0.001, vs. sham respectively). No difference was found between the groups in the whole body rate of appearance of arginine or NO. However, ALF showed a significant increase in de novo arginine synthesis (P < 0.05). Interorgan data showed citrulline net intestinal production and renal consumption that was related to net renal production of arginine and ornithine. Both plasma arginase activity and plasma ADMA levels significantly increased in ALF (P < 0.001). In this model of early-phase ALF, arginine deficiency or higher ADMA levels do not limit whole body NO production. Arginine deficiency is caused by arginase-related arginine clearance in which arginine production is stimulated de novo.

Pilot Study of the Association of the DDAH2 −449G Polymorphism with Asymmetric Dimethylarginine and Hemodynamic Shock in Pediatric Sepsis

BackgroundGenetic variability in the regulation of the nitric oxide (NO) pathway may influence hemodynamic changes in pediatric sepsis. We sought to determine whether functional polymorphisms in DDAH2, which metabolizes the NO synthase inhibitor asymmetric dimethylarginine (ADMA), are associated with susceptibility to sepsis, plasma ADMA, distinct hemodynamic states, and vasopressor requirements in pediatric septic shock.Methodology/Principal FindingsIn a prospective study, blood and buccal swabs were obtained from 82 patients ≤18 years (29 with severe sepsis/septic shock plus 27 febrile and 26 healthy controls). Plasma ADMA was measured using tandem mass spectrometry. DDAH2 gene was partially sequenced to determine the −871 6g/7g insertion/deletion and −449G/C single nucleotide polymorphisms. Shock type (“warm” versus “cold”) was characterized by clinical assessment. The −871 7g allele was more common in septic (17%) then febrile (4%) and healthy (8%) patients, though this was not significant after controlling for sex and race (p = 0.96). ADMA did not differ between −871 6g/7g genotypes. While genotype frequencies also did not vary between groups for the −449G/C SNP (p = 0.75), septic patients with at least one −449G allele had lower ADMA (median, IQR 0.36, 0.30–0.41 µmol/L) than patients with the −449CC genotype (0.55, 0.49–0.64 µmol/L, p = 0.008) and exhibited a higher incidence of “cold” shock (45% versus 0%, p = 0.01). However, after controlling for race, the association with shock type became non-significant (p = 0.32). Neither polymorphism was associated with inotrope score or vasoactive infusion duration.Conclusions/SignificanceThe −449G polymorphism in the DDAH2 gene was associated with both low plasma ADMA and an increased likelihood of presenting with “cold” shock in pediatric sepsis, but not with vasopressor requirement. Race, however, was an important confounder. These results support and justify the need for larger studies in racially homogenous populations to further examine whether genotypic differences in NO metabolism contribute to phenotypic variability in sepsis pathophysiology.

Dimethylarginine dimethylaminohydrolase 1 regulates nerve growth factor‐promoted differentiation of PC12 cells in a nitric oxide‐dependent but asymmetric dimethylargenine‐independent manner

There are significant morphological and biochemical alterations during nerve growth factor (NGF)-promoted neuronal differentiation, and the process is regulated by molecules, including nitric oxide (NO). Dimethylarginine dimethylaminohydrolase (DDAH) is thought to play a critical role in regulating NO production via hydrolyzing the endogenous NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA). Thus, we tested the role of DDAH in NGF-promoted differentiation of PC12 (pheochromocytoma) cells. The present results show that both mRNA and protein levels of DDAH1 were increased, whereas those of DDAH2 were decreased, during NGF-promoted cell differentiation. Both the DDAH activity and the ADMA level in cultured medium were unchanged in this process. NGF promoted neurite formation and induced the expression of microtubule-associated protein 2 (MAP2), a neuronal marker, which were both significantly repressed by DDAH1 silence with small interfering RNA but not by DDAH2 silence. The expressions of three isoforms of NOS were markedly upregulated after NGF stimulation with a time course similar to that of DDAH1, which were attenuated by DDAH1 silence. Conversely, overexpression of DDAH1 accelerated neurite formation in PC12 cells, concomitantly with upregulating the expression of three NOS isoforms. In summary, our data reveal the critical regulatory effect of DDAH1 on NGF-promoted differentiation of PC12 cells in an NOS/NO-dependent but ADMA-independent manner.

The transhepatic endotoxin gradient is present despite liver cirrhosis and is attenuated after transjugular portosystemic shunt (TIPS).

BackgroundTranslocation of gut-derived bacterial products such as endotoxin is a major problem in liver cirrhosis.MethodsTo assess the hepatic clearance of bacterial products in individuals with cirrhosis, we tested concentrations of Gram-negative bacterial lipopolysaccharide (LPS), LPS-binding protein (LBP), and the precursor of nitric oxide (NO), L-arginine, in a cohort of 8 stable patients with liver cirrhosis before and after elective transjugular portosystemic shunt (TIPS) implantation, including central venous, hepatic venous, and portal venous measurements.ResultsUsing an adapted LPS assay, we detected high portal venous LPS concentrations (mean 1743 ± 819 pg/mL). High concentrations of LPS were detectable in the central venous blood (931 ± 551 pg/mL), as expected in persons with cirrhosis. The transhepatic LPS gradient was found to be 438 ± 287 pg/mL, and 25 ± 12% of portal LPS was cleared by the cirrhotic liver. After TIPS, central venous LPS concentrations increased in the hepatic and central veins, indicating shunting of LPS with the portal blood through the stent. This paralleled a systemic increase of L-arginine, whereas the NO synthase inhibitor asymmetric dimethylarginine (ADMA) remained unchanged, suggesting that bacterial translocation may contribute to the pathogenesis of circulatory dysfunction post-TIPS.ConclusionsThis study provides quantitative estimates of the role of the liver in the pathophysiology of bacterial translocation. The data indicate that the cirrhotic liver retains the capacity for clearance of bacterial endotoxin from the portal venous blood and that TIPS implantation attenuates this clearance. Thus, increased endotoxin concentrations in the systemic circulation provide a possible link to the increased encephalopathy in TIPS patients.

Elevated concentrations of Nɛ-homocysteinyl-lysine isopeptide in acute myocardial infarction: links with ADMA formation

A homocysteine (Hcy) metabolite, thioester Hcy-thiolactone, whose reaction with protein lysine residues affords N-homocysteinylated proteins, has been implicated in cardiovascular disease. Proteolytic turnover of N-homocysteinylated proteins generates the isopeptide Nɛ-homocysteinyl-lysine (N-Hcy-Lys). We determined N-Hcy-Lys in serum and a NO syntase inhibitor asymmetric dimethylarginine (ADMA), as well as symmetric dimethylarginine (SDMA) and glutathione in plasma by high performance liquid chromatography in 52 consecutive patients with acute myocardial infarction (AMI) recruited within the first 12 h following onset of chest pain. Associations of N-Hcy-Lys with markers of thrombin generation, oxidative stress, enhanced inflammation, fibrinolysis, and autoantibodies against homocysteinylated proteins were also analyzed. N-Hcy-Lys concentrations, detectable in 45 (86.5%) patients (>0.1 μmol/L), were 127.3% higher compared with healthy controls. N-Hcy-Lys correlated with ADMA (r=0.50; p<0.001), SDMA (r=0.43; p<0.01), glutathione (r=0.37; p<0.05), fibrinogen (r=0.39; p<0.01) and plasminogen activator inhibitor-1 (r=0.32; p<0.05), but not with plasma total Hcy, anti-N-Hcy-protein autoantibodies, vitamin B(12), folate, interleukin-6, plasma thrombin-antithrombin (TAT) complexes, prothrombin fragments F1+2 or 8-isoprostaglandin F(2α) a marker of oxidative stress. N-Hcy-Lys is increased in AMI patients and its formation is linked with the nitric oxide synthase inhibitor ADMA.

Immunoregulatory effects of the flavonol quercetin in vitro and in vivo

Atherosclerosis is known to be an inflammatory disease. Dendritic cells (DCs) are essential for the regulation of the immune system. Up to 10% of the cells in atherosclerotic plaques are DCs. The cardiovascular protective effects of flavonoids (tea, wine) may be mediated by anti-inflammatory mechanisms that affect DC regulation. We aimed to characterize the impact of the flavonol quercetin on DC activity and differentiation in vitro and in vivo. For the in vitro experiments, we used murine DCs and endothelial cells to study adhesion properties. For all other experiments (DC phagocytosis capacity, DC maturation, DC differentiation (BDCA-1/-2) and NF-kB-activation), human monocyte-derived DCs were used. The cells were incubated with quercetin (10μmol/L) ± oxLDL (10μg/mL) between 24 and 48h. For in vivo experiments, eight healthy male volunteers took 500mg of quercetin twice daily over 4weeks, five healthy male volunteers served as control. Before and after intake, blood samples were collected. Peripheral blood leukocytes were isolated (analyses of DC differentiation), and plasma was immediately frozen. Quercetin reduced DC adhesion (-42%; p<0.05) and expression of CD11a (-21%; p<0.05). OxLDL-induced DC differentiation was partially inhibited by quercetin (BDCA-1-29%; BDCA-2-33%; p<0.05). These effects were achieved by compensation of oxLDL-induced up-regulation of NF-kB by quercetin. The 4-week treatment with quercetin resulted in relevant plasma levels (2.47μmol/L) and reduced BDCA-2+DCs in the peripheral blood by 42% (p<0.05) as well as systemic levels of the NO-synthase inhibitor asymmetric dimethylarginine (-31%, p<0.05). In vitro, quercetin reduced DC adhesion and oxLDL-induced DC differentiation. In vivo, quercetin reduced circulating plasmacytoid DCs and systemic ADMA-levels. The immunoregulatory effects of quercetin may contribute to the anti-atherosclerotic potential of flavonols.