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Background Low levels of the endogenous amino acid L-homoarginine are a risk factor for cardiovascular morbidity and mortality. For individual risk prediction, commercially available test systems are mandatory. This study aims at formulating sex- and age-specific reference intervals of serum L-homoarginine determined with an ELISA. Methods We determined reference intervals for serum L-homoarginine stratified by age and sex in a sample of 1285 healthy participants of the Study of Health in Pomerania (SHIP)-TREND cohort after exclusion of participants with cardiovascular diseases, diabetes mellitus, hypertension, metabolic syndrome, elevated liver enzymes, chronic kidney disease stages III or IV, or body mass index >25 kg/m2. Serum L-homoarginine was determined applying a commercially available ELISA. Results The reference cohort included 836 women (median age 41, 25th and 75th percentiles are 32 and 50 years) and 449 men (median age 38, 25th, and 75th percentiles are 30 and 49 years). The median serum concentration of L-homoarginine was 1.93 (25th 1.49; 75th 2.60) µmol/L in women and 2.02 (25th 1.63; 75th 2.61) µmol/L in men (P = 0.04). The reference intervals (2.5th to 97.5th percentile) were 0.89–5.29 µmol/L for women and 1.09–3.76 µmol/L for men. The L-homoarginine serum concentration declined over age decades in both sexes and a notable interaction with sex hormone intake in women was observed. Conclusions The novelty of our study is that we determined reference intervals specific for the L-isomer being lower than those previously reported for homoarginine in SHIP and thus might be helpful in identifying individuals suitable for oral L-homoarginine supplementation.

Guanidino compounds, including asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and L-homoarginine (hArg), have been associated with cardio- and cerebrovascular events and risk. We aimed to study if low hArg/ADMA and hArg/SDMA ratios are associated with mortality and outcome after stroke. In two prospective cohorts of acute stroke patients from Germany and the UK, we analyzed hArg, ADMA, and SDMA to determine hArg/ADMA and hArg/SDMA ratios. The guanidino compound levels were associated with mortality, adverse events, and neurological impairment, i.e., National Institutes of Health Stroke Scale (NIHSS) and modified Rankin Scale (mRS). During 7.4 years, high hArg/ADMA and hArg/SDMA ratios were both associated with a reduction in all-cause mortality in patients with ischemic stroke in a UK stroke cohort (hArg/ADMA: hazard ratio (HR) 0.75 [95% confidence interval (CI) 0.62–0.92]; n = 394; P = 0.006; hArg/SDMA: HR 0.68 [0.54–0.85]; n = 394; P = 0.001). In a German stroke cohort, patients with high hArg/SDMA ratio experienced fewer adverse events compared with those with low hArg/SDMA ratios within 30 days after stroke (HR 0.73 [0.57–0.92]; n = 135; P = 0.009), whereas hArg/ADMA was not predictive. Furthermore, hArg/SDMA ratios inversely correlated with the degree of neurological impairment (NIHSS) (r = − 0.27; P = 0.001; n = 138). Lower hArg/SDMA ratios were also found in dependent (mRS 3-6) compared with independent patients (mRS

Introduction Guanidino compounds, including l -homoarginine ( l -hArg), symmetric dimethylarginine (SDMA), asymmetric dimethylarginine (ADMA) and l -arginine ( l -Arg) are associated with mortality, fatal strokes, stroke incidence, and atherosclerosis. Objectives We aimed to study the association of guanidino compounds ( l -hArg/ADMA and l -hArg/SDMA) with stroke etiology, internal carotid artery (ICA) stenosis and CHA2DS2-VASc score in patients with cerebrovascular disease. Methods We analyzed l -hArg, SDMA, ADMA, l -Arg, and compound molar ratios, i.e. l -hArg/ADMA and l -hArg/SDMA, in 272 patients with cerebrovascular disease in a cross-sectional discovery cohort and two cross-sectional validation cohorts of acute stroke patients from Germany (n = 137) and UK (n = 394). The guanidino compound levels were compared with clinical, imaging, and ultrasound parameters. Results Low l -hArg/ADMA and l -hArg/SDMA molar ratios predicted territorial infarcts (OR 1.74; 95% CI 1.34–2.26 and OR 1.64; 95% CI 1.26–2.15, respectively) and were associated with stroke subtypes due to large vessel disease or cardio-embolism (OR 1.52; 95% CI 1.12–2.06 and OR 2.01; 95% CI 1.35–3.00, respectively) in meta-analysis of the discovery and validation cohort data. In line with these results, a low l -hArg/ADMA and l -hArg/SDMA molar ratio was found in patients with ICA stenosis (OR 0.73; 95% CI 0.55–0.97 and OR 0.69; 95% CI 0.50–0.94, respectively) in the discovery and validation cohort. Furthermore, guanidino compound ratios (i.e. l -hArg/ADMA and l -hArg/SDMA) were strongly correlated with CHA2DS2-VASC score (p Discussion The results from these three cross-sectional studies reveal that guanidino compound ratios (i.e. l -hArg/ADMA and l -hArg/SDMA) can discriminate stroke etiologies, predict ICA stenosis and estimate risk prediction in patients with cerebrovascular disease.

Statin-induced myopathy affects more than 10 million people worldwide. But discontinuation of statin treatment increases mortality and cardiovascular events. Recently, l-arginine:glycine amidinotransferase (AGAT) gene was associated with statin-induced myopathy in two populations, but the causal link is still unclear. AGAT is responsible for the synthesis of l-homoarginine (hArg) and guanidinoacetate (GAA). GAA is further methylated to creatine (Cr) by guanidinoacetate methyltransferase (GAMT). In cerebrovascular patients treated with statin, lower hArg and GAA plasma concentrations were found than in non-statin patients, indicating suppressed AGAT expression and/or activity (n = 272, P = 0.033 and P = 0.039, respectively). This observation suggests that statin-induced myopathy may be associated with AGAT expression and/or activity in muscle cells. To address this, we studied simvastatin-induced myopathy in AGAT- and GAMT-deficient mice. We found that simvastatin induced muscle damage and reduced AGAT expression in wildtype mice (myocyte diameter: 34.1 ± 1.3 µm vs 21.5 ± 1.3 µm, P = 0.026; AGAT expression: 1.0 ± 0.3 vs 0.48 ± 0.05, P = 0.017). Increasing AGAT expression levels of transgenic mouse models resulted in rising plasma levels of hArg and GAA (P

Elevated levels of arginine derivatives in the NO pathway, such as asymmetric dimethylarginine (ADMA), are related to disease severity and reduced exercise capacity in heart failure (HF). We investigated the influence of exercise intervention on these parameters and on L-arginine (L-Arg) and L-homoarginine (L-hArg) in HF with preserved ejection fraction (HFpEF) patients.Sixty-two patients (65 ± 6 years) were included in this analysis and randomized to supervised endurance/resistance training (ET) or to usual care (UC). EDTA-plasma was analysed for NO metabolites.There were baseline associations for adjusted values of maximum workload with ADMA (r= -0.322, p = 0.028) and L-Arg/ADMA ratio (r = 0.331, p = 0.015), and for the 6-min walk test (6MWT) with ADMA (r= -0.314, p = 0.024) and L-Arg/ADMA ratio (r = 0.346, p = 0.015). No significant differences between UC and ET changes of NO parameters were observed at 3-month follow-up. Higher L-hArg levels were associated with a greater improvement in peak oxygen uptake (peak [Formula: see text]OExercise intervention did not influence NO parameters in HFpEF patients, but L-hArg was related to change in peak [Formula: see text]O

l-Homoarginine (hArg) is biosynthesized from l-arginine (Arg) and l-lysine (Lys) by arginine:glycine amidinotransferase (AGAT). AGAT also catalyzes the formation of guanidinoacetate (GAA) from Arg and glycine (Gly). GAA is converted to creatine (N-methyl guanidinoacetate) by guanidinoacetate N-methyl-transferase (GAMT). Low circulating and excretory concentrations of hArg are associated with worse cardiovascular outcome and mortality. hArg is a poor substrate of nitric oxide synthase (NOS) and a weak inhibitor of arginase. The metabolism of hArg in humans is little investigated. Previously, we found that orally administered hArg (125 mg/day) increased the plasma concentration of hArg, but not of Arg, the substrate of NOS, in healthy subjects. We newly analyzed the plasma samples collected in that study for Lys and other amino acids. Repeated measures ANOVA revealed statistically significant differences between the groups (P = 0.008) with respect to plasma Lys concentration which increased by about 8% after a 4-week hArg supplementation. In vitro, recombinant human arginase and bovine liver arginase I were demonstrated by a specific and sensitive stable-isotope GC-MS assay to hydrolyze hArg to Lys. Our results suggest that Lys is a metabolite of hArg produced by the hydrolytic activity of arginase. Arginase may play a key role in hArg homeostasis in humans.

Aims Low blood concentrations of the naturally occurring amino acid L-homoarginine (L-hArg) are related to impaired cardiovascular outcome and mortality in humans and animals. L-hArg is a weak substrate of nitric oxide synthase and an inhibitor of arginases in vitro. The aim of our study was to obtain kinetic and dynamic data after oral L-hArg supplementation. Methods In a double-blind, randomized, placebo-controlled crossover study, 20 young volunteers received 125 mg L-hArg once daily for 4 weeks. Kinetic parameters (Cmax, Tmax and AUC0-24h) were calculated after ingestion of single and multiple doses of oral supplementation as primary endpoint. Secondary endpoints that were evaluated were routine laboratory, L-arginine, asymmetric dimethylarginine (ADMA), pulse wave velocity (PWV), augmentation index (AIx), flow-mediated vasodilatation (FMD), corticospinal excitability, i.e. motor threshold (MT), and cortical excitability, i.e. intracortical inhibition (ICI) and facilitation (ICF). Results One hour after ingestion (Tmax), L-hArg increased the baseline L-hArg plasma concentration (2.87 ± 0.91 μmol l−1, mean ± SD) by 8.74 ± 4.46 [95% confidence intervals 6.65; 10.9] and 17.3 ± 4.97 [14.9; 19.6] μmol l−1 (Cmax), after single and multiple doses, respectively. Once-only and 4 weeks of supplementation resulted in AUCs0-24h of 63.5 ± 28.8 [50.0; 76.9] and 225 ± 78.5 [188; 2624] μmol l−1*h, for single and multiple doses, respectively. Routine laboratory parameters, L-arginine, ADMA, PWV, AIx, FMD, MT, ICI and ICF did not change by L-hArg supplementation compared to baseline. Conclusion Once daily orally applied 125 mg L-hArg raises plasma L-hArg four- and sevenfold after single dose and 4 weeks of supplementation, respectively, and is safe and well tolerated in young volunteers.

Background Pulmonary arterial hypertension (PAH) is a rare progressive disease with a 3-year mortality rate of approx. 45% in incident patients. The prostacyclin, endothelin-1 (ET-1), and nitric oxide (NO) pathways are validated therapeutic targets, however the underlying pathomechanisms are not yet fully understood. In the present study, we investigated metabolites of the NO pathway (e.g. l-arginine, asymmetric dimethylarginine (ADMA), and homoarginine), which are potentially involved in the pathophysiology of PAH. Methods Newly diagnosed, treatment-naive incident PAH patients were recruited from six centers and followed for three years. In longitudinal analyses we investigated the prognostic potential of these markers. Cross-sectional analysis was later used to study associations between prognostic relevant markers and clinical phenotypes of PAH. Results Among 108 enrolled patients (53±17years, mean±SD), 76 had idiopathic (i)PAH. Kaplan–Meier survival analysis and adjusted Cox proportional hazard models identified homoarginine as an independent predictor of mortality (HR:0.51, CI:0.28–0.94 for PAH and HR:0.41 CI:0.19–0.88 for iPAH), but not l-arginine or ADMA. Homoarginine was lower in 27 patients who died during the follow-up, i.e. 1.26±0.48 vs. 1.64±0.69μmol/L, P P Conclusion Homoarginine was found an independent predictor for mortality in newly diagnosed PAH and iPAH patients. Further experimental studies are necessary to elucidate the involvement of homoarginine in the pathophysiology of PAH and its potential role as a therapeutic option for these patients.

Low plasma homoarginine (HA) is an emerging biomarker for cardiovascular disease and an independent predictor of mortality in patients with heart failure.1 Plasma levels appear to reflect cardiac dysfunction, positively correlating with ejection fraction and inversely with circulating brain natriuretic peptide.2 However, whether this outcome is a bystander or cause-and-effect has yet to be established. Within the context of stroke, a direct causal relationship has been inferred because normal mice pretreated with 14 mg/L HA had a smaller stroke size.3 In the present study, we show for the first time that dietary supplementation with HA improves cardiac function in the setting of chronic heart failure, suggesting a novel preventive strategy and inferring that low HA levels may be inherently detrimental because of a loss of this effect. We first confirmed that oral supplementation of C57BL/6J mice (Harwell, UK) with 14 mg/L L-HA hydrochloride (Sigma-Aldrich) in the drinking water for 4 weeks increased HA concentrations in both plasma (0.29±0.03 vs 0.89±0.07 µmol/L) and myocardial tissue (17.6±2.7 vs 48.8±6.8 nmol/g protein; n=5–10; P

Guanidino compounds, including l-homoarginine (l-hArg), symmetric dimethylarginine (SDMA), asymmetric dimethylarginine (ADMA) and l-arginine (l-Arg) are associated with mortality, fatal strokes, stroke incidence, and atherosclerosis.We aimed to study the association of guanidino compounds (l-hArg/ADMA and l-hArg/SDMA) with stroke etiology, internal carotid artery (ICA) stenosis and CHAWe analyzed l-hArg, SDMA, ADMA, l-Arg, and compound molar ratios, i.e. l-hArg/ADMA and l-hArg/SDMA, in 272 patients with cerebrovascular disease in a cross-sectional discovery cohort and two cross-sectional validation cohorts of acute stroke patients from Germany (n = 137) and UK (n = 394). The guanidino compound levels were compared with clinical, imaging, and ultrasound parameters.Low l-hArg/ADMA and l-hArg/SDMA molar ratios predicted territorial infarcts (OR 1.74; 95% CI 1.34-2.26 and OR 1.64; 95% CI 1.26-2.15, respectively) and were associated with stroke subtypes due to large vessel disease or cardio-embolism (OR 1.52; 95% CI 1.12-2.06 and OR 2.01; 95% CI 1.35-3.00, respectively) in meta-analysis of the discovery and validation cohort data. In line with these results, a low l-hArg/ADMA and l-hArg/SDMA molar ratio was found in patients with ICA stenosis (OR 0.73; 95% CI 0.55-0.97 and OR 0.69; 95% CI 0.50-0.94, respectively) in the discovery and validation cohort. Furthermore, guanidino compound ratios (i.e. l-hArg/ADMA and l-hArg/SDMA) were strongly correlated with CHAThe results from these three cross-sectional studies reveal that guanidino compound ratios (i.e. l-hArg/ADMA and l-hArg/SDMA) can discriminate stroke etiologies, predict ICA stenosis and estimate risk prediction in patients with cerebrovascular disease.

Homoarginine (hArg) is a non-essential amino acid that was identified as a risk marker for cardiovascular disease. Several analytical methods have been described for the quantification of hArg in biological samples. The aim of this study was to compare a liquid chromatography-tandem mass spectrometric (LC-MS/MS) approach with a commercially available enzyme-linked immunosorbent assay (ELISA). Determination of hArg concentrations in ELISA calibration standards measured by both methods revealed a correlation coefficient r (2) of 0.99, for LC-MS/MS calibrators r (2) was 0.997. However, linear regression analysis between the two assays for hArg concentrations in human plasma samples revealed a correlation coefficient r (2) of 0.78. Plasma concentrations obtained from LC-MS/MS are on average 29 % higher than those by ELISA. We investigated the hArg-isobaric N (ε)-trimethyllysine as potential source for the higher observed values, but evaluation of mass spectra indicated that N (ε)-trimethyllysine did not interfere with hArg quantification in our LC-MS/MS method. Both quantification methods were applied to measure hArg in (1) a case-control study of acute coronary syndrome and (2) L-arginine:glycine amidinotransferase-deficient mice. Our LC-MS/MS and the commercially available ELISA assay are suitable for hArg measurement in human and mouse plasma, but different reference values for each method need to be considered.

l -homoarginine ( l -hArg) is an endogenous non-proteinogenic amino acid. Low l -hArg concentrations are associated with increased all-cause mortality, fatal strokes, and worse outcome after stroke. On the other hand, oral supplementation with l -hArg in mice improved neurological deficits and preserved cardiac function in experimental models of stroke and heart failure, respectively. Recently, oral supplementation with 125 mg daily l -hArg capsules in healthy volunteers demonstrated increased l -hArg plasma levels. Therefore, oral l -hArg supplementation could represent a potential treatment for patients with cerebrovascular disease. In addition to vascular physiology, animal studies have suggested that l -hArg might play a role in synapse function, neurotransmitter metabolism and cognitive training. However the direct influence of l -hArg on cognitive function has not been studied so far. In this study, cognitive performance in healthy humans was analyzed concerning memory, learning, and attention following supplementation with placebo or l -hArg for 4 weeks. Our results did not reveal any effects on cognition, neither impairment nor improvement, upon l -hArg supplementation. Therefore, potential l -hArg treatment is not expected to cause any acute neurocognitive or behavioral side effects.

Polyamines play an important role in cell growth, differentiation, and cancer development, and the biosynthetic pathway of polyamines is established as a drug target for the treatment of parasitic diseases, neoplasia, and cancer chemoprevention. The key enzyme in polyamine biosynthesis is ornithine decarboxylase (ODC). We report herein an analytical method for the continuous fluorescence monitoring of ODC activity based on the supramolecular receptor cucurbit[6] uril (CB6) and the fluorescent dye trans-4-[4-(dimethylamino) styryl]-1-methylpyridinium iodide (DSMI). CB6 has a significantly higher binding constant to the ODC product putrescine (>10(7)M(-1)) than to the substrate L-ornithine (340 M-1). This enables real-time monitoring of the enzymatic reaction through a continuous fluorescence change caused by dye displacement from the macrocycle by the formed product, which allowed a straightforward determination of enzyme kinetic parameters (k(cat) = 0.12 s(-1) and K-M = 24 mu M) and inhibition constants of the two ODC inhibitors alpha-difluoromethylornithine (DFMO) and epigallocatechin gallate (EGCG). The potential for high-throughput screening (HTS) was demonstrated by excellent Z' factors (>0.9) in a microplate reader format, and the sensitivity of the assay is comparable to or better than most established complementary methods, which invariably have the disadvantage of not being compatible with direct implementation and upscaling to HTS format in the drug discovery process.

Background The endogenous amino acid homoarginine predicts mortality in cerebro‐ and cardiovascular disease. The objective was to explore whether homoarginine is associated with atrial fibrillation ( AF ) and outcome in patients with acute chest pain. Methods and Results One thousand six hundred forty‐nine patients with acute chest pain were consecutively enrolled in this study, of whom 589 were diagnosed acute coronary syndrome ( ACS ). On admission, plasma concentrations of homoarginine as well as brain natriuretic peptide ( BNP ), and high‐sensitivity assayed troponin I (hsTnI) were determined along with electrocardiography ( ECG ) variables. During a median follow‐up of 183 days, 60 major adverse cardiovascular events ( MACEs ; 3.8%), including all‐cause death, myocardial infarction, or stroke, were registered in the overall study population and 43 MACEs (7.5%) in the ACS subgroup. Adjusted multivariable Cox regression analyses revealed that an increase of 1 SD of plasma log‐transformed homoarginine (0.37) was associated with a hazard reduction of 26% (hazard ratio [ HR ], 0.74; 95% CI , 0.57–0.96) for incident MACE and likewise of 35% ( HR , 0.65; 95% CI , 0.49–0.88) in ACS patients. In Kaplan–Meier survival curves, homoarginine was predictive for patients with high‐sensitivity assayed troponin I (hsTnI) above 27 ng/L ( P QT c duration ( P AF ( OR , 0.83; 95% CI , 0.71–0.95). Conclusion Low plasma homoarginine was identified as a risk marker for incident MACEs in patients with acute chest pain, in particular, in those with elevated hsTnI. Impaired homoarginine was associated with prevalent AF . Further studies are needed to investigate the link to AF and evaluate homoarginine as a therapeutic option for these patients.

l-Arginine is the substrate of endothelial nitric oxide (NO) synthase forming NO which inherits various biological cardio-protective functions. The dimethylarginines asymmetric (ADMA) and symmetric dimethylarginine (SDMA) can impair the synthesis of NO and are elevated in patients with cardiovascular disease, including heart failure (HF). We investigated the association between dimethylarginines and HF risk in a case-cohort study of the European Prospective Investigation into Cancer and Nutrition (n = 27,548), comprising a random subcohort (n = 2224 including 19 HF cases), and all remaining HF cases (n = 176) that occurred within 8.3 years of follow-up. Serum concentrations of dimethylarginines were measured using liquid chromatography-tandem mass spectrometry. Hazards ratios (HRs) and 95% confidence intervals (CI) were estimated across quartiles and per doubling of ADMA and SDMA concentrations using Cox’s proportional hazards regression. After multivariable adjustment, each doubling of ADMA was associated with a 60% higher HF risk (HR [95% CI] 1.60 [1.10–2.31]). Between SDMA and HF risk a U-shaped association was observed (HR [95% CI] for the second, third and fourth quartile compared to the first: 0.52 [0.33–0.82], 0.63 [0.40–0.99], and 0.71 [0.46–1.10], p for nonlinearity

Background: Low circulating homoarginine has been associated with adverse cardiovascular (CV) outcome and mortality in patients at risk and in the general population. The present study aimed to define plasma homoarginine reference intervals from a representative population sample to improve risk stratification between healthy individuals and individuals at risk. Methods: We determined age- and sex-specific reference intervals for circulating plasma homoarginine in a subgroup of 786 healthy participants (no CV disease or risk factors) of the Gutenberg Health Study. Homoarginine concentrations were measured using a validated liquid chromatography-tandem mass spectrometry method. Results: Median EDTA plasma homoarginine concentration was 1.88 [25th; 75th percentile, 1.47; 2.41] μmol/L, with lower concentrations in women (1.77 [1.38; 2.26] μmol/L) than in men (2.01 [1.61; 2.56] μmol/L; p Conclusions: We provide plasma homoarginine reference intervals in men and women of the general population. The determination of homoarginine levels might be favorable for individual risk stratification.

L-Homoarginine (hArg) is an endogenous amino acid which has emerged as a novel biomarker for stroke and cardiovascular disease. Low circulating hArg levels are associated with increased mortality and vascular events, whereas recent data have revealed positive correlations between circulating hArg and metabolic vascular risk factors like obesity or blood glucose levels. However, it is unclear whether hArg levels are causally linked to metabolic parameters. Therefore, the aim of our study was to investigate whether hArg directly influences body weight, blood glucose, glucose tolerance or insulin sensitivity. Here, we show that hArg supplementation (14 and 28 mg/mL orally per drinking water) ameliorates blood glucose levels in mice on high-fat diet (HFD) by a reduction of 7.3 ± 3.7 or 13.4 ± 3.8 %, respectively. Fasting insulin concentrations were slightly, yet significantly affected (63.8 ± 11.3 or 162.1 ± 39.5 % of control animals, respectively), whereas body weight and glucose tolerance were unaltered. The substantial augmentation of hArg plasma concentrations in supplemented animals (327.5 ± 40.4 or 627.5 ± 60.3 % of control animals, respectively) diminished profoundly after the animals became obese (129.9 ± 16.6 % in control animals after HFD vs. 140.1 ± 8.5 or 206.3 ± 13.6 %, respectively). This hArg-lowering effect may contribute to the discrepancy between the inverse correlation of plasma hArg levels with stroke and cardiovascular outcome, on the one hand, and the direct correlation with cardiovascular risk factors like obesity and blood glucose, on the other hand, that has been observed in human studies. Our results suggest that the glucose-lowering effects of hArg may reflect a compensatory mechanism of blood glucose reduction by hArg upregulation in obese individuals, without directly influencing body weight or glucose tolerance.