Asymmetrical dimethyl arginine (ADMA) is an endogenous non-selective inhibitor of nitric oxide synthase that may influence the severity of organ failure and the occurrence of shock secondary to an infectious insult. Levels may be genetically determined by a promoter polymorphism in a regulatory gene encoding dimethylarginine dimethylaminohydrolase II (DDAH II), which functions by metabolising ADMA to citrulline. The aim of this study was to examine the association between ADMA levels and the severity of organ failure and shock in severe sepsis and also to assess the influence of a promoter polymorphism in DDAH II on ADMA levels.
A prospective observational study was designed, and 47 intensive care unit (ICU) patients with severe sepsis and 10 healthy controls were enrolled. Serum ADMA and IL-6 were assayed on admission to the ICU and seven days later. Allelic variation for a polymorphism at position -449 in the DDAH II gene was assessed in each patient. Clinical and demographic details were also collected.
On day 1 more ADMA was detectable in the ICU group than in the control group (p = 0.005). Levels subsequently increased during the first week in ICU (p = 0.001). ADMA levels were associated with vasopressor requirements on day one (p = 0.001). ADMA levels and Sequential Organ Failure Assessment scores were directly associated on day one (p = 0.0001) and day seven (p = 0.002). The degree of acidaemia and lactaemia was directly correlated with ADMA levels at both time points (p < 0.01). On day seven, IL-6 was directly correlated with ADMA levels (p = 0.006). The variant allele with G at position -449 in the DDAH II gene was associated with increased ADMA concentrations at both time points (p < 0.05).
Severity of organ failure, inflammation and presence of early shock in severe sepsis are associated with increased ADMA levels. ADMA concentrations may be influenced by a polymorphism in the DDAH II gene.
Asymmetric dimethylarginine (ADMA), present in human serum, is an endogenous inhibitor of nitric oxide synthase and contributes to vascular disease. Dimethylarginine dimethylaminohydrolase (DDAH) is an ADMA degrading enzyme that has two isoforms: DDAHI and DDAHII. We sought to determine whether serum ADMA levels in type 2 diabetes are influenced by common polymorphisms in the DDAH1 and DDAH2 genes.
Relevant clinical parameters were measured and peripheral whole blood obtained for serum and genetic analysis on 343 participants with type 2 diabetes. Serum ADMA concentrations were determined by mass spectroscopy. Twenty six tag SNPs in the DDAH1 and 10 in the DDAH2 gene were genotyped in all subjects and tested for association with serum ADMA levels. Several SNPs and haplotypes in the DDAH genes were strongly associated with ADMA levels. Most significantly in the DDAH1 gene, rs669173 (p = 2.96×10−7), rs7521189 (p = 6.40×10−7), rs2474123 (p = 0.00082) and rs13373844 (p = 0.00027), and in the DDAH2 gene, rs3131383 (p = 0.0029) and the TGCCCAGGAG haplotype (p = 0.0012) were significantly associated with ADMA levels. Sub-analysis by diabetic retinopathy (DR) status revealed these variants were associated with ADMA levels predominantly in participants without DR. Combined analysis of the most strongly associated SNPs in DDAH1 (rs669173) and DDAH2 (rs3131383) revealed an additive effect (p = 1.37×10−8) on ADMA levels.
Genetic variation in the DDAH1 and 2 genes is significantly associated with serum ADMA levels. Further studies are required to determine the pathophysiological significance of elevated serum ADMA in type 2 diabetes and to better understand how DDAH gene variation influences ADMA levels.
Increased plasma concentrations of the endogenous nitric oxide (NO) synthase inhibitor, asymmetric dimethylarginine (ADMA), decreased arginine bioavailability, and mitochondrial dysfunction have been reported in adult sepsis. We studied whether ADMA, arginine, and carnitine metabolism (a measure of mitochondrial dysfunction) are altered in pediatric sepsis and whether these are clinically useful biomarkers.
Prospective, observational study
Pediatric intensive care unit at an academic medical center
Ninety patients ≤ 18 years-old—30 with severe sepsis or septic shock compared with thirty age-matched febrile and thirty age-matched healthy controls.
Measurements and Main Results
Plasma ADMA and whole blood arginine, citrulline, ornithine, and acylcarnitine:free carnitine (AC:FC) ratio were measured daily for septic patients and once for controls using tandem mass spectrometry. Plasma ADMA concentration (median, IQR µmol/L) on day 1 was lower in severe sepsis and septic shock (0.38, 0.30–0.56) compared with febrile (0.45, 0.40–0.59) and healthy (0.60, 0.54–0.67) controls (p<0.001), though decreased ADMA was predominantly found in neutropenic patients. Day 1 arginine was lower in septic (10, IQR 7–20 µmol/L) compared with healthy patients (32, IQR 23–40; p<0.001), and the arginine:ornithine ratio was decreased in sepsis, indicating increased arginase activity (an alternative pathway for arginine metabolism). The arginine:ADMA and AC:FC ratios did not differ between septic and control patients. ADMA was inversely correlated with organ dysfunction by PELOD score (r=−0.50, p=0.009), interleukin-6 (r=−0.55, p=0.01), and interleukin-8 (r=−0.52, p=0.03) on admission. Arginine, arginine:ADMA, and AC:FC were not associated with organ dysfunction or outcomes.
ADMA was decreased in pediatric sepsis and was inversely associated with inflammation and organ dysfunction. This suggests that inhibition of NO synthase by ADMA accumulation is unlikely to impact sepsis pathophysiology in septic children despite decreased arginine bioavailability. We did not find an association of ADMA with altered carnitine metabolism, nor were ADMA, arginine, and AC:FC useful as clinical biomarkers.
Nitric oxide; nitric oxide synthase; arginine; carnitine; sepsis; intensive care units, pediatric
The endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is an independent predictor of cardiovascular and overall mortality. Moreover, elevated ADMA plasma concentrations are associated with the extent of hypertension. However, data from small-sized clinical trials and experimental approaches using murine transgenic models have revealed conflicting results regarding the impact of ADMA and its metabolizing enzyme dimethylarginine dimethylaminohydrolase (DDAH) in the pathogenesis of hypertension.
Therefore, we investigated the role of ADMA and DDAH1 in hypertension-induced end organ damage using the uninephrectomized, deoxycorticosterone actetate salt, and angiotensin II-induced hypertension model in human DDAH1 (hDDAH1) overexpressing and wild-type (WT) mice. ADMA plasma concentrations differed significantly between hDDAH1 and WT mice at baseline, but did not significantly change during the induction of hypertension. hDDAH1 overexpression did not protect against hypertension-induced cardiac fibrosis and hypertrophy. In addition, the hypertension-induced impairment of the endothelium-dependent vasorelaxation of aortic segments ex vivo was not significantly attenuated by hDDAH1 overexpression. However, hDDAH1 mice displayed an attenuated hypertensive inflammatory response in renal tissue, resulting in less hypertensive renal injury.
Our data reveal that hDDAH1 organ-specifically modulates the inflammatory response in this murine model of hypertension. The lack of protection in cardiac and aortic tissues may be due to DDAH1 tissue selectivity and/or the extent of hypertension by the used combined model. However, our study underlines the potency of hDDAH1 overexpression in modulating inflammatory processes as a crucial step in the pathogenesis of hypertension, which needs further experimental and clinical investigation.
Hyperhomocysteinemia is a cardiovascular risk factor that is associated with the nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA). Using mice transgenic for overexpression of the ADMA-hydrolyzing enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH1), we tested the hypothesis that overexpression of DDAH1 protects from adverse structural and functional changes in cerebral arterioles in hyperhomocysteinemia.
Methods and Results
Hyperhomocysteinemia was induced in DDAH1 transgenic (DDAH1 Tg) mice and wild-type littermates using a high methionine/low folate (HM/LF) diet. Plasma total homocysteine was elevated approximately 3-fold in both wild-type and DDAH1 Tg mice fed the HM/LF diet compared with the control diet (P<0.001). Plasma ADMA was approximately 40% lower in DDAH1 Tg mice compared with wild-type mice (P<0.001) irrespective of diet. Compared with the control diet, the HM/LF diet diminished endothelium-dependent dilation to 10 µmol/L acetylcholine in cerebral arterioles of both wild-type (12±2 vs. 29±3%; P<0.001) and DDAH1 Tg (14±3 vs. 28±2%; P<0.001) mice. Responses to 10 µmol/L papaverine, a direct smooth muscle dilator, were impaired with the HM/LF diet in wild-type mice (30±3 vs. 45±5%; P<0.05) but not DDAH1 Tg mice (45±7 vs. 48±6%). DDAH1 Tg mice also were protected from hypertrophy of cerebral arterioles (P<0.05) but not from accelerated carotid artery thrombosis induced by the HM/LF diet.
Overexpression of DDAH1 protects from hyperhomocysteinemia-induced alterations in cerebral arteriolar structure and vascular muscle function.
amino acids; nitric oxide synthase; endothelium; vasodilation; thrombosis
Methylated arginines are endogenous analogues of L-arginine, the substrate for nitric oxide (NO) synthase. Asymmetric dimethylarginine (ADMA) interferes with NO formation, causing endothelial dysfunction. ADMA is a predictor of cardiovascular events and mortality in humans. It is eliminated primarily by enzymatic activity of dimethylarginine dimethylaminohydrolase (DDAH).
We investigated whether human DDAH-1 (hDDAH-1) transgenicity protects from ischemic tissue damage in temporary middle cerebral artery occlusion (tMCAO) in mice. Infarct sizes did not significantly differ between hDDAH-1 transgenic (TG) mice and wild-type littermates (WT). As expected, ADMA plasma concentrations were significantly decreased, cerebral hDDAH expression and protein significantly increased in transgenic animals. Interestingly, neither brain tissue DDAH activity nor ADMA concentrations were different between TG and WT mice. In contrast, muscular DDAH activity was generally lower than in brain but significantly increased in TG mice.
Our study demonstrates that hDDAH-1 transgenic mice are not protected from ischemic cerebral tissue damage in tMCAO. This lack of protection is due to high basal cerebral DDAH activity, which is not further increasable by transgenic overexpression of DDAH.
Previous studies suggest that nitric oxide (NO) may modulate insulin-induced uptake of glucose in insulin-sensitive tissues. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase (NOS). We hypothesized that a reduction in endogenous ADMA would increase NO synthesis and thereby enhance insulin sensitivity.
Methods and Results
To test this hypothesis we employed a transgenic mouse in which we overexpressed human dimethylarginine dimethylaminohydrolase (DDAH-I). The DDAH-I mice had lower plasma ADMA at all ages (22–70 weeks) by comparison to wild-type (WT) littermates. With a glucose challenge, WT mice showed a prompt increase in ADMA, whereas DDAH-I mice had a blunted response. Furthermore, DDAH-I mice had a blunted increase in plasma insulin and glucose levels after glucose challenge, with a 50% reduction in the insulin resistence index, consistent with enhanced sensitivity to insulin. In liver, we observed an increased Akt phosphorylation in the DDAH-I mice after i.p. glucose challenge. Incubation of skeletal muscle from WT mice ex vivo with ADMA (2μM) markedly suppressed insulin-induced glycogen synthesis in fast-twitch but not slow-twitch muscle.
These findings suggest that the endogenous NOS inhibitor ADMA reduces insulin sensitivity, consistent with previous observations that NO plays a role in insulin sensitivity.
Arteriosclerosis; nitric oxide; asymmetric dimethylarginine; dimethylarginine dimethylaminohydrolase; glucose
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of endothelial nitric oxide synthase, which was associated with insulin resistance. Dimethylarginine dimethylaminohydrolase (DDAH) is the major determinant of plasma ADMA. Examining data from the DIAGRAM+ (Diabetes Genetics Replication And Meta-analysis), we identified a variant (rs9267551) in the DDAH2 gene nominally associated with type 2 diabetes (P = 3×10−5).
initially, we assessed the functional impact of rs9267551 in human endothelial cells (HUVECs), observing that the G allele had a lower transcriptional activity resulting in reduced expression of DDAH2 and decreased NO production in primary HUVECs naturally carrying it. We then proceeded to investigate whether this variant is associated with insulin sensitivity in vivo. To this end, two cohorts of nondiabetic subjects of European ancestry were studied. In sample 1 (n = 958) insulin sensitivity was determined by the insulin sensitivity index (ISI), while in sample 2 (n = 527) it was measured with a euglycemic-hyperinsulinemic clamp. In sample 1, carriers of the GG genotype had lower ISI than carriers of the C allele (67±33 vs.79±44; P = 0.003 after adjusting for age, gender, and BMI). ADMA levels were higher in subjects carrying the GG genotype than in carriers of the C allele (0.68±0.14 vs. 0.57±0.14 µmol/l; P = 0.04). In sample 2, glucose disposal was lower in GG carriers as compared with C carriers (9.3±4.1 vs. 11.0±4.2 mg×Kg−1 free fat mass×min−1; P = 0.009).
A functional polymorphism of the DDAH2 gene may confer increased risk for type 2 diabetes by affecting insulin sensitivity throughout increased ADMA levels.
Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase inhibitor that blocks nitric oxide production, while congestive heart failure is associated with increased plasma and tissue ADMA content. Increased plasma ADMA is a strong and independent predictor of all-cause mortality in the community and the strongest predictor of mortality in patients after myocardial infarction. Recent studies demonstrated that dimethylarginine dimethylaminohydrolase-1 (DDAH1) is the critical enzyme for ADMA degradation and thereby plays an important role in maintaining cardiovascular nitric oxide bioavailability. Interestingly, activation of the farnesoid X receptor (FXR) through the bile acid ursodeoxycholic acid (UDCA) or synthetic FXR agonists, such as GW4064, can increase DDAH1 expression. Thus, modulating DDAH1 activity through FXR receptor agonists such as UDCA could be a therapeutic target for treating reduced nitric oxide bioavailability in congestive heart failure and other cardiovascular diseases.
Nitric oxide (NO) production is reduced in renal disease, partially due to decreased endothelial NO production. Evidence indicates that NO deficiency contributes to cardiovascular events and progression of kidney damage. Two possible causes of NO deficiency are substrate (l-arginine) limitation and increased levels of circulating endogenous inhibitors of NO synthase (particularly asymmetric dimethylarginine [ADMA]). Decreased l-arginine availability in chronic kidney disease (CKD) is due to perturbed renal biosynthesis of this amino acid. In addition, inhibition of transport of l-arginine into endothelial cells and shunting of l-arginine into other metabolic pathways (e.g. those involving arginase) might also decrease availability. Elevated plasma and tissue levels of ADMA in CKD are functions of both reduced renal excretion and reduced catabolism by dimethylarginine dimethylaminohydrolase (DDAH). The latter might be associated with loss-of-function polymorphisms of a DDAH gene, functional inhibition of the enzyme by oxidative stress in CKD and end-stage renal disease, or both. These findings provide the rationale for novel therapies, including supplementation of dietary l-arginine or its precursor l-citrulline, inhibition of non-NO-producing pathways of l-arginine utilization, or both. Because an increase in ADMA has emerged as a major independent risk factor in end-stage renal disease (and probably also in CKD), lowering ADMA concentration is a major therapeutic goal; interventions that enhance the activity of the ADMA-hydrolyzing enzyme DDAH are under investigation.
arginase; asymmetric dimethylarginine; cardiovascular events; dimethylarginine dimethylaminohydrolase; l-arginine transporters
Endothelial dysfunction participates in the development and progression of salt-sensitive hypertension. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS). The objectives of this study were to investigate the impact of a high salt diet on the PRMT/ADMA/DDAH (protein arginine methyltransferases; dimethylarginine dimethylaminohydrolase) pathway in Dahl salt-sensitive (DS) rats and SS-13BN consomic (DR) rats, and to explore the mechanisms that regulate ADMA metabolism independent of blood pressure reduction. Plasma levels of nitric oxide (NO) in DS rats given a high salt diet and subjected to intragastric administration of hydralazine (SH + HYD group) were lower than those given a normal salt diet (SN group). There were significant decreases in expression and activity of dimethylarginine dimethylaminohydrolase (DDAH) and endothelial NO synthase (eNOS) in DS rats given a high diet (SH group) in comparison to the SN group. The activity of DDAH and expression of eNOS in the SH + HYD group decreased more significantly than SN group. The mRNA expression of DDAH-1 and DDAH-2 were lowest in the SH group. The results suggest that salt, independent of blood pressure, can affect the PRMT-1/ADMA/DDAH system to a certain degree and lead to endothelial dysfunction in Dahl salt-sensitive rats.
endothelial dysfunction; asymmetric dimethylarginine; dimethylarginine; dimethylaminohydrolase; endothelial nitrite oxide synthase; oxidative stress
Asymmetric NG,NG-dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, is regulated by the enzymatic participants of synthetic and metabolic processes, i.e., type I protein N-arginine methyltransferase (PRMT) and dimethylarginine dimethylaminohydrolase (DDAH). Previous reports have demonstrated that circulating ADMA levels can vary in patients with type 1 and type 2 diabetes mellitus (T2DM). White adipose tissue expresses the full enzymatic machinery necessary for ADMA production and metabolism; however, modulation of the activities of adipose ADMA-related enzymes in T2DM remains to be determined.
A rodent model of T2DM using 11- and 20-week old Goto-Kakizaki (GK) rats was used. The expression and catalytic activity of PRMT1 and DDAH1 and 2 in the white adipose tissues (periepididymal, visceral and subcutaneous fats) and femur skeletal muscle tissue were determined by immunoblotting, in vitro methyltransferase and in vitro citrulline assays.
Non-obese diabetic GK rats showed low expression and activity of adipose PRMT1 compared to age-matched Wistar controls. Adipose tissues from the periepididymal, visceral and subcutaneous fats of GK rats had high DDAH1 expression and total DDAH activity, whereas the DDAH2 expression was lowered below the control value. This dynamic of ADMA-related enzymes in white adipose tissues was distinct from that of skeletal muscle tissue. GK rats had lower levels of serum non-esterified fatty acids (NEFA) and triglycerides (TG) than the control rats. In all subjects the adipose PRMT1 and DDAH activities were statistically correlated with the levels of serum NEFA and TG.
Activities of PRMT1 and DDAH in white adipose tissues were altered in diabetic GK rats in an organ-specific manner, which was reflected in the serum levels of NEFA and TG. Changes in adipose ADMA-related enzymes might play a part in the function of white adipose tissue.
Protein N-arginine methyltransferase 1; Dimethylarginine dimethylaminohydrolase 1 and 2; Non-esterified fatty acids; Triglycerides; Type 2 diabetes mellitus
Purpose of Review
Nitric oxide (NO) deficiency occurs by multiple mechanisms and contributes to the pathogenesis of progression of chronic kidney disease (CKD) and its cardiovascular complications. This article concentrates on recent developments on the regulation of the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) in chronic kidney disease and on the importance of the NO synthases in kidney disease progression, particularly in diabetic nephropathy.
The increased plasma ADMA seen in renal disease is generally predictive of severity of CKD progression and cardiovascular risk. However, some assumptions about the control of ADMA have been challenged; the primacy of the kidney as a metabolic organ for plasma ADMA regulation coming under scrutiny and the relative importance of the 2 isoforms of the ADMA-metabolizing enzymes dimethylarginine dimethylaminohydrolases (DDAHs) is being reevaluated. Alterations in nitric oxide synthases also contribute to CKD progression with the endothelial isoform playing a major role in diabetic nephropathy.
Improving our understanding of ADMA regulation is important since pharmacologic targeting of DDAH is underway. The major role of eNOS-derived NO in diabetic nephropathy should lead to novel therapies. The beneficial actions of dietary nitrate supplementation on blood pressure and kidney disease are of considerable clinical relevance.
asymmetric dimethylarginine; dimethylarginine dimethylaminohydrolase; nitric oxide synthase
Nitric oxide (NO) is a potent signaling molecule that needs to be tightly regulated to maintain metabolic and cardiovascular homeostasis. The nitric oxide synthase (NOS)/Dimethylarginine dimethylaminohydrolase (DDAH)/Asymmetric Dimethylarginine (ADMA) pathway is central to this regulation. Specifically, the small molecule ADMA competitively inhibits NOS, thus lowering NO levels. The majority of ADMA is physiologically metabolized by DDAH, thus maintaining NO levels at physiological concentration. However, under pathophysiological conditions, DDAH activity is impaired, in part as a result of its sensitivity to oxidative stress. Therefore, the application of high throughput chemical screening for the discovery of small molecules that could restore or enhance DDAH activity might have significant potential in treating metabolic and vascular diseases characterized by reduced NO levels, including atherosclerosis, hypertension, and insulin resistance. By contrast, excessive generation of NO (primarily driven by iNOS) could play a role in idiopathic pulmonary fibrosis (IPF), sepsis, migraine headaches, and some types of cancer. In these conditions, small molecules that inhibit DDAH activity might be therapeutically useful. Here, we describe optimization and validation of a highly reproducible and robust assay successfully used in a high throughput screen for DDAH modulators.
nitric oxide; asymmetric dimethylarginine; diabetes; hypertension; idiopathic pulmonary fibrosis
Nitric oxide (NO) is an important mediator of host defence and of vascular tone. In septic shock, upregulation of inducible NO synthase leads to the production of vast amounts of NO, which contribute to pathogen elimination but also to inappropriate vasodilation and to loss of vascular resistance. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthases shown to contribute to the regulation of vascular tone. ADMA was recently identified as a marker of organ dysfunction and mortality in intensive care patients and as a novel cardiovascular risk factor. In the present issue of Critical Care, a study by O'Dwyer and colleagues identifies ADMA as a potential regulator of NO production in septic shock. Being an inhibitor of NO production, ADMA may at least partly counteract pathological hypotension, but at the same time may impair the NO-dependent host defence. A mechanism is proposed by which the interplay between ADMA and inducible NO synthase activity is mediated. ADMA levels should be determined in future studies evaluating the regulation of NO in the intensive care setting.
Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, is known as mediator of endothelial cell dysfunction and atherosclerosis. Circulating ADMA levels are correlated with cardiovascular risk factors such as hypercholesterolemia, arterial hypertension, diabetes mellitus, hyperhomocysteinemia, age and smoking. Accordingly, clinical studies found evidence that increased ADMA levels are associated with a higher risk of cerebrovascular events. After the acute event of ischemic stroke, levels of ADMA and its analog symmetric dimethylarginine (SDMA) are elevated through augmentation of protein methylation and oxidative stress. Furthermore, cleavage of ADMA through dimethylarginine dimethylaminohydrolases (DDAHs) is reduced. This increase of dimethylarginines might be predictive for adverse clinical outcome. However, the definite role of ADMA after acute ischemic stroke still needs to be clarified. On the one hand, ADMA might contribute to brain injury by reduction of cerebral blood flow. On the other hand, ADMA might be involved in NOS-induced oxidative stress and excitotoxic neuronal death. In the present review, we highlight the current knowledge from clinical and experimental studies on ADMA and its role for stroke risk and ischemic brain injury in the hyperacute stage after stroke. Finally, further studies are warranted to unravel the relevance of the close association of dimethylarginines with stroke.
asymmetric dimethylarginine (ADMA); symmetric dimethylarginine (SDMA); nitric oxide (NO); nitric oxide synthase (NOS); ischemic stroke
Elevated plasma levels of asymmetric dimethylarginine (ADMA) has been reported to be associated with insulin resistance and micro/macrovascular diabetic complications, and may predict cardiovascular events in type 2 diabetic patients. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is the major enzyme eliminating ADMA in humans, but the effect of genetic variations in DDAH1 on type 2 diabetes and its long-term outcome are unknown.
From July 2006 to June 2009, we assessed the association between polymorphisms in DDAH1 and type 2 diabetes in 814 consecutive unrelated subjects, including 309 type 2 diabetic patients and 505 non-diabetic individuals. Six single nucleotide polymorphisms (SNPs) in DDAH1, rs233112, rs1498373, rs1498374, rs587843, rs1403956, and rs1241321 were analyzed. Plasma ADMA levels were determined by high performance liquid chromatography. Insulin sensitivity was assessed by the homeostasis model assessment of insulin resistance (HOMA-IR).
Among the 6 SNPs, only rs1241321 was significantly associated with a decreased risk of type 2 diabetes (AA vs GG+AG, OR = 0.64, 95% CI 0.47-0.86, p = 0.004). The association remained unchanged after adjustment for plasma ADMA level. The fasting plasma glucose and log HOMA-IR tended to be lower in subjects carrying the homozygous AA genotype of rs1241321 compared with the GG+AG genotypes. Over a median follow-up period of 28.2 months, there were 44 all-cause mortality and 50 major adverse cardiovascular events (MACE, including cardiovascular death, non-fatal myocardial infarction and stroke). Compared with the GG and AG genotypes, the AA genotype of rs1241321 was associated with reduced risk of MACE (HR = 0.31, 95% CI: 0.11-0.90, p = 0.03) and all-cause mortality (HR = 0.18, 95% CI: 0.04-0.80, p = 0.02) only in subgroup with type 2 diabetes. One common haplotype (GGCAGC) was found to be significantly associated with a decreased risk of type 2 diabetes (OR = 0.67, 95% CI = 0.46-0.98, p = 0.04).
Our results provide the first evidence that SNP rs1241321 in DDAH1 is associated with type 2 diabetes and its long-term outcome.
Genetic and pharmacological studies have shown that impairment of the nitric oxide (NO) synthase (NOS) pathway is associated with hypertension and insulin-resistance (IR). In addition, inhibition of NOS by the endogenous inhibitor, asymmetric dimethylarginine (ADMA), may also result in hypertension and IR. On the other hand, overexpression of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes ADMA, in mice is associated with lower ADMA, increased NO and enhanced insulin sensitivity. Since DDAH carries a farnesoid X receptor (FXR)-responsive element, we aimed to upregulate its expression by an FXR-agonist, INT-747, and evaluate its effect on blood pressure and insulin sensitivity.
Methods and Results
In this study, we evaluated the in vivo effect of INT-747 on tissue DDAH expression and insulin sensitivity in the Dahl rat model of salt-sensitive hypertension and IR (Dahl-SS). Our data indicates that high salt (HS) diet significantly increased systemic blood pressure. In addition, HS diet downregulated tissue DDAH expression while INT-747 protected the loss in DDAH expression and enhanced insulin sensitivity compared to vehicle controls.
Our study may provide the basis for a new therapeutic approach for IR by modulating DDAH expression and/or activity using small molecules.
L-Arginine (ARG) is converted to nitric oxide (NO) and L-citrulline (CIT) by endothelial nitric oxide synthase which is competitively inhibited by asymmetric dimethylarginine (ADMA). We have developed a liquid chromatography-mass spectrometric method for the simultaneous determination of endogenous ARG, labeled ARG (15N4-ARG), CIT, ADMA, and its inactive isomer, symmetric dimethylarginine (SDMA) in biological samples.
Concentrations of unlabeled ARG, 15N4-ARG, CIT, ADMA, and SDMA in EA.hy926 human endothelial cell lysate, cell incubation media, rat plasma or rat urine were measured by hydrophilic-interaction liquid chromatography electrospray tandem mass spectrometry. 13C6-ARG, D4-CIT and D7-ADMA were used as internal standards for ARG, CIT and dimethylarginines, respectively.
The calibration curves of ARG, 15N4-ARG, CIT, ADMA, and SDMA were linear and independent of several sample matrices. Intra- and inter-day variabilities for the quantification of all the compounds were below 15 % in quality control samples. Application of this method to determine the uptake as well as efflux of these compounds was illustrated through in vitro cell study by exposing human endothelial cells to 15N4-ARG, which allowed the observation of generation of 15N3-CIT and 15N3-ARG in the cell lyate. Use of these isotopes adds insights into the cellular handling of endogenous vs. exogenous ARG. Application of this method for rat plasma and rat urine assays was demonstrated after ARG oral supplementation in rats.
An LC-MS/MS method was developed to quantify 6 ARG-related compounds simultaneously, utilizing 3 separate internal standards. This assay allows concurrent monitoring of uptake, efflux and metabolic processes when isotope-labeled ARG and CIT are measured, and can be applied for determination of these compounds in rat plasma and rat urine.
L-arginine; L-citrulline; asymmetric dimethylarginine; symmetric dimethylarginine; LC-MS/MS
Acute lung injury (ALI) is associated with severe alterations in lung structure and function and is characterized by hypoxemia, pulmonary edema, low lung compliance and widespread capillary leakage. Asymmetric dimethylarginine (ADMA), a known cardiovascular risk factor, has been linked to endothelial dysfunction and the pathogenesis of a number of cardiovascular diseases. However, the role of ADMA in the pathogenesis of ALI is less clear. ADMA is metabolized via hydrolytic degradation to L-citrulline and dimethylamine by the enzyme, dimethylarginine dimethylaminohydrolase (DDAH). Recent studies suggest that lipopolysaccharide (LPS) markedly increases the level of ADMA and decreases DDAH activity in endothelial cells. Thus, the purpose of this study was to determine if alterations in the ADMA/DDAH pathway contribute to the development of ALI initiated by LPS-exposure in mice. Our data demonstrate that LPS exposure significantly increases ADMA levels and this correlates with a decrease in DDAH activity but not protein levels of either DDAH I or DDAH II isoforms. Further, we found that the increase in ADMA levels cause an early decrease in nitric oxide (NOx) and a significant increase in both NO synthase (NOS)-derived superoxide and total nitrated lung proteins. Finally, we found that decreasing peroxynitrite levels with either uric acid or Manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTymPyp) significantly attenuated the lung leak associated with LPS-exposure in mice suggesting a key role for protein nitration in the progression of ALI. In conclusion, this is the first study that suggests a role of the ADMA/DDAH pathway during the development of ALI in mice and that ADMA may be a novel therapeutic biomarker to ascertain the risk for development of ALI.
Nitration; Superoxide; Arginine metabolism
Dysfunctions of the L-arginine (L-arg)/nitric-oxide (NO) pathway are suspected to be important for the pathogenesis of multiple organ dysfunction syndrome (MODS) in septic shock. Therefore plasma concentrations of L-arg and asymmetric dimethylarginine (ADMA) were measured in 60 patients with septic shock, 30 surgical patients and 30 healthy volunteers using enzyme linked immunosorbent assay (ELISA) kits. Plasma samples from patients with septic shock were collected at sepsis onset, and 24 h, 4 d, 7 d, 14 d and 28 d later. Samples from surgical patients were collected prior to surgery, immediately after the end of the surgical procedure as well as 24 h later and from healthy volunteers once. In comparison to healthy volunteers and surgical patients, individuals with septic shock showed significantly increased levels of ADMA, as well as a decrease in the ratio of L-arg and ADMA at all timepoints. In septic patients with an acute liver failure (ALF), plasma levels of ADMA and L-arg were significantly increased in comparison to septic patients with an intact hepatic function. In summary it can be stated, that bioavailability of NO is reduced in septic shock. Moreover, measurements of ADMA and L-arg appear to be early predictors for survival in patients with sepsis-associated ALF.
Nitric oxide (NO) is generated from L-arginine by NO synthases, of which three forms have been identified: endothelial, inducible and neuronal (eNOS, iNOS and nNOS, respectively). The arginine metabolite asymmetric dimethylarginine (ADMA) is a potent, noncompetitive inhibitor of nNOS, while its congener NG-monomethyl-L-arginine (L-NMMA) is a less potent, competitive inhibitor. In rat neurons large amounts of ADMA are found, suggesting its importance in modulatin neuronal activity.
Humans generate approximately 300 μmol (∼60 mg) ADMA per day. It is released from myelin basic proteins that are highly expressed in neuronal tissue. ADMA is mainly degraded by the action of the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which exists in two isoforms. DDAH1 is highly expressed in brain, suggesting specific function in this area. The presence of nNOS and DDAH1 in brain suggests that ADMA may have specific CNS activity and be more than an unregulated metabolite.
Increased NO production - either prior to or concurrently with opioid administration - results in an enhanced rate and extent of development of tolerance to morphine in mice. NO produces an alteration in the μ-opioid receptor that increases constitutive receptor activity. It thereby reduces the ability of a selective μ-opioid agonist to activate the μ-opioid receptor; these in vitro molecular effects occur in a time course consistent with the in vivo development of antinociceptive tolerance in mice. Amongst many other synthetic NOS inhibitors of varying specificity, 7-nitroindazole (7-NI) has been shown to have a high affinity (IC50 0.71 μM) to nNOS. Selective blockade of nNOS by 7-NI attenuated morphine withdrawal in opiate dependent rats, suggesting nNOS as a viable target for development of pharmacotherapies.
We hypothesize that, by inhibiting nNOS and reducing NO levels, ADMA may decrease μ-opiate receptor constitutive activity, resulting in alteration of the analgesic dose-response curve of morphine.
addiction; opiates; heroine; nociception; NOS; Asymmetrical dimethylarginine; NG-monomethyl-L-arginine
l-Arginine (Arg) is oxidized to l-citrulline and nitric oxide (NO) by the action of endothelial nitric oxide synthase (NOS). In contrast, protein-incorporated Arg residues can be methylated with subsequent proteolysis giving rise to methylarginine compounds, such as asymmetric dimethylarginine (ADMA) that competes with Arg for binding to NOS. Most ADMA is degraded by dimethylarginine dimethyaminohydrolase (DDAH), distributed widely throughout the body and regulates ADMA levels and, therefore, NO synthesis. In recent years, several studies have suggested that increased ADMA levels are a marker of atherosclerotic change, and can be used to assess cardiovascular risk, consistent with ADMA being predominantly absorbed by endothelial cells. NO is an important messenger molecule involved in numerous biological processes, and its activity is essential to understand both pathogenic and therapeutic mechanisms in kidney disease and renal transplantation. NO production is reduced in renal patients because of their elevated ADMA levels with associated reduced DDAH activity. These factors contribute to endothelial dysfunction, oxidative stress and the progression of renal damage, but there are treatments that may effectively reduce ADMA levels in patients with kidney disease. Available data on ADMA levels in controls and renal patients, both in adults and children, also are summarized in this review.
asymmetric dimethylarginine (ADMA); arginine (Arg); children; dimethylarginine dimethylaminohydrolase (DDAH); endothelial dysfunction; kidney; methylarginines; nitric oxide; oxidative stress; renal failure
To examine the hemodynamic determinants of dysregulated arginine metabolism in patients with acute decompensated heart failure and explore possible mechanism of arginine dysregulation in human heart failure.
Accumulating methylated arginine metabolites and impaired arginine bioavailability have been associated with heart failure, but the underlying pathophysiology remains unclear.
We prospectively determined plasma levels of asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, and global arginine bioavailability ratio [GABR=arginine/(ornithine+citrulline)] by tandem mass spectrometry in subjects with advanced decompensated heart failure in the intensive care unit (“ADHF”, n=68) and with stable chronic heart failure (“CHF”, n=57).
Compared to CHF subjects, plasma ADMA was significantly higher (median[interquartile range]: 1.29 [1.04–1.77] versus 0.87 [0.72–1.05] μM, p<0.0001), and GABR significantly lower (0.90 [0.69–1.22] versus 1.13 [0.92–1.37], p=0.002) in ADHF subjects. Elevated ADMA and diminished GABR were associated with higher systolic pulmonary artery pressure (sPAP) and higher central venous pressure, but not with other clinical or hemodynamic indices. We further observed myocardial levels of dimethylarginine dimethylaminohydrolase-1 (DDAH-1) were increased in CHF without elevated sPAP (<50mmHg), but diminished with elevated sPAP (≥50mmHg, difference with sPAP<50 mmHg, p=0.02).
Dysregulated arginine metabolism was observed in advanced decompensated heart failure, particularly with pulmonary hypertension and elevated intracardiac filling pressures. Compared to control hearts, we observed higher amounts of ADMA-degradation enzyme DDAH-1 (but similar amounts of ADMA-producing enzyme, PRMT-1) in the human failing myocardium.
Nitric oxide synthase; asymmetric dimethylarginine; heart failure; pulmonary hypertension
Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is mainly metabolized by NG,NG-dimethylarginine dimethylaminohydrolase (DDAH). We investigated whether altered cavernosal ADMA–DDAH metabolism might cause impairment of erection in rat model of atherosclerosis (AS). Male Sprague–Dawley rats (3 months old) were divided into an AS group and a normal control (Con) group (n=20 in each group). The AS rats received AS-prone treatment (6 weeks of 1% cholesterol diet plus early 2 weeks of NG-nitro-L-arginine methyl ester (3 mg ml−1 per day) treatment). After 6 weeks, rats underwent cavernosometry measuring the maximal intracavernosal pressure/mean arterial pressure (ICP/MAP) ratios as a surrogate marker of erectile function. The amount of cavernosal ADMA was assessed by immunoblot analysis and correlated with the ICP/MAP. Isoform-specific DDAH expression was compared by immunohistochemistry. Cavernosal DDAH and NOS activity were measured. Cavernosal malondialdehyde levels were assayed to determine the degree of lipid peroxidation. Compared to the controls, the AS rats had signs of impaired erectile function. Higher cavernosal ADMA was observed in the AS rats. The cavernosal ADMA had a moderately negative correlation with the ICP/MAP. Immunohistochemistry revealed the expression of both isoforms was not affected by the presence of AS. However, significantly diminished DDAH as well as NOS activity was observed in the AS group. In addition, elevated cavernosal malondialdehyde levels were noted in the AS rats. Our study showed that decreased cavernosal DDAH activity is the cause of cavernosal ADMA accumulation leading to reduced cavernosal NOS activity and impairment of erectile function.
erectile dysfunction; atherosclerosis; rats; asymmetric dimethylarginine; dimethylarginine dimethylaminohydrolase