Systemic Effects of Homoarginine Supplementation on Arginine Metabolizing Enzymes in Rats with Heart Failure with Preserved Ejection Fraction.

A restoration of low homoarginine (hArg) levels in obese ZSF1 rats (O-ZSF1) before (S1-ZSF1) and after (S2-ZSF1) the manifestation of heart failure with preserved ejection fraction (HFpEF) did not affect the worsening of cardiac HFpEF characteristics. Here, potential regulation of key enzymes of arginine metabolism in other organs was analyzed. Arginase 2 (ARG2) was reduced >35% in the kidney and small intestine of hArg-supplemented rats compared to O-ZSF1. Glycine amidinotransferase (GATM) was 29% upregulated in the kidneys of S1-ZSF1. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) levels were reduced >50% in the livers of O-ZSF1 but restored in S2-ZSF1 compared to healthy rats (L-ZSF1). In the skeletal muscle, iNOS was lower in O-ZSF1 and further decreased in S1-ZSF1 and S2-ZSF1 compared to L-ZSF1. iNOS levels were lower in the liver of the S2-ZSF1 group but higher in the kidneys of S1-ZSF1 compared to L-ZSF1. Supplementation with hArg in an in vivo HFpEF model resulted in the inhibition of renal ARG2 and an increase in GATM expression. This supplementation might contribute to the stabilization of intestinal iNOS and ARG2 imbalances, thereby enhancing barrier function. Additionally, it may offer protective effects in skeletal muscle by downregulating iNOS. In the conceptualization of hArg supplementation studies, the current disease progression stage as well as organ-specific enzyme regulation should be considered.

Homoarginine and creatine deficiency do not exacerbate murine ischaemic heart failure.

AIMS: Low levels of homoarginine and creatine are associated with heart failure severity in humans, but it is unclear to what extent they contribute to pathophysiology. Both are synthesized via L-arginine:glycine amidinotransferase (AGAT), such that AGAT-/- mice have a combined creatine and homoarginine deficiency. We hypothesized that this would be detrimental in the setting of chronic heart failure. METHODS AND RESULTS: Study 1: homoarginine deficiency-female AGAT-/- and wild-type mice were given creatine-supplemented diet so that both had normal myocardial creatine levels, but only AGAT-/- had low plasma homoarginine. Myocardial infarction (MI) was surgically induced and left ventricular (LV) structure and function assessed at 6-7 weeks by in vivo imaging and haemodynamics. Study 2: homoarginine and creatine-deficiency-as before, but AGAT-/- mice were given creatine-supplemented diet until 1 week post-MI, when 50% were changed to a creatine-free diet. Both groups therefore had low homoarginine levels, but one group also developed lower myocardial creatine levels. In both studies, all groups had LV remodelling and dysfunction commensurate with the development of chronic heart failure, for example, LV dilatation and mean ejection fraction <20%. However, neither homoarginine deficiency alone or in combination with creatine deficiency had a significant effect on mortality, LV remodelling, or on any indices of contractile and lusitropic function. CONCLUSIONS: Low levels of homoarginine and creatine do not worsen chronic heart failure arguing against a major causative role in disease progression. This suggests that it is unnecessary to correct hArg deficiency in patients with heart failure, although supra-physiological levels may still be beneficial.

The Amino Acid Homoarginine Inhibits Atherogenesis by Modulating T-Cell Function.

BACKGROUND: Amino acid metabolism is crucial for inflammatory processes during atherogenesis. The endogenous amino acid homoarginine is a robust biomarker for cardiovascular outcome and mortality with high levels being protective. However, the underlying mechanisms remain elusive. We investigated the effect of homoarginine supplementation on atherosclerotic plaque development with a particular focus on inflammation. METHODS: Female ApoE-deficient mice were supplemented with homoarginine (14 mg/L) in drinking water starting 2 weeks before and continuing throughout a 6-week period of Western-type diet feeding. Control mice received normal drinking water. Immunohistochemistry and flow cytometry were used for plaque- and immunological phenotyping. T cells were characterized using mass spectrometry-based proteomics, by functional in vitro approaches, for example, proliferation and migration/chemotaxis assays as well as by super-resolution microscopy. RESULTS: Homoarginine supplementation led to a 2-fold increase in circulating homoarginine concentrations. Homoarginine-treated mice exhibited reduced atherosclerosis in the aortic root and brachiocephalic trunk. A substantial decrease in CD3+ T cells in the atherosclerotic lesions suggested a T-cell-related effect of homoarginine supplementation, which was mainly attributed to CD4+ T cells. Macrophages, dendritic cells, and B cells were not affected. CD4+ T-cell proteomics and subsequent pathway analysis together with in vitro studies demonstrated that homoarginine profoundly modulated the spatial organization of the T-cell actin cytoskeleton and increased filopodia formation via inhibition of Myh9 (myosin heavy chain 9). Further mechanistic studies revealed an inhibition of T-cell proliferation as well as a striking impairment of the migratory capacities of T cells in response to relevant chemokines by homoarginine, all of which likely contribute to its atheroprotective effects. CONCLUSIONS: Our study unravels a novel mechanism by which the amino acid homoarginine reduces atherosclerosis, establishing that homoarginine modulates the T-cell cytoskeleton and thereby mitigates T-cell functions important during atherogenesis. These findings provide a molecular explanation for the beneficial effects of homoarginine in atherosclerotic cardiovascular disease.

Reference Interval for Serum L-Homoarginine Determined with Enzyme-Linked Immunosorbent Assay in the Population-Based Study of Health in Pomerania.

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.

Effects of homoarginine supplementation on heart and skeletal muscle of rats with heart failure with preserved ejection fraction.

AIM: Heart failure with preserved ejection fraction (HFpEF) is associated with left ventricular stiffness, impaired diastolic relaxation, and severe exercise intolerance. Decreased homoarginine (hArg) levels are an independent predictor of mortality in cardiovascular disease and correlate with impaired exercise performance. We recently reported alterations in arginine, hArg, and related amino acids in obese ZSF1 rats (O-ZSF1), with a HFpEF phenotype. Although low hArg is associated with diastolic dysfunction in humans, potential effects of hArg supplementation were not tested yet. METHODS AND RESULTS: At an age of 6 weeks, 12 O-ZSF1 were randomized into two groups: (1) O-ZSF1 rats supplemented with hArg in their drinking water (sO-ZSF1) or (2) O-ZSF1 rats receiving no hArg supplementation (O-ZSF1). At an age of 32 weeks, effects of primary prevention by hArg supplementation on echocardiographic, histological, and functional parameters of heart and skeletal muscle were determined. Lean ZSF1 rats (L-ZSF1) served as controls. hArg supplementation did not prevent impairment of diastolic relaxation (E/e': O-ZSF1 21 ± 3 vs. sO-ZSF1 22 ± 3, P = 0.954, L-ZSF1 18 ± 5) but resulted in more cardiac fibrosis (histological collagen staining: +57% in sO-ZSF1 vs. O-ZSF1, P = 0.027) and increased collagen gene expression (Col1a1: +48% in sO-ZSF1 vs. O-ZSF1, P = 0.026). In contrary, right ventricular function was preserved by hArg supplementation (TAPSE (mm): O-ZSF1 1.2 ± 0.3 vs. sO-ZSF1 1.7 ± 0.3, P = 0.020, L-ZSF1 1.8 ± 0.4). Musculus soleus maximal specific muscle force (N/cm2 ) in O-ZSF1 (30.4 ± 0.8) and sO-ZSF1 (31.9 ± 0.9) was comparable but significantly reduced compared with L-ZSF1 (36.4 ± 0.7; both P < 0.05). Maximal absolute muscle force (g) (O-ZSF1: 177.6 ± 7.8, sO-ZSF1: 187.8 ± 5.0, L-ZSF1: 181.5 ± 7.9, all P > 0.05) and cross-sectional fibre area (arbitrary units) (O-ZSF1: 1697 ± 57, sO-ZSF1: 1965 ± 121, L-ZSF1: 1691 ± 104, all P > 0.05) were not altered. CONCLUSIONS: Preservation of physiological hArg level in HFpEF may not be suited to prevent alterations in left ventricular and skeletal muscle function and structure. However, hArg supplementation may be beneficial for right ventricular function especially in pulmonary hypertension in HFpEF. We may speculate that clinically observed decreased hArg level are not the cause but the consequence of a yet unrecognized pathomechanism that underpins HFpEF.

Population kinetics of homoarginine and optimized supplementation for cardiovascular risk reduction.

Homoarginine is an endogenous amino acid whose levels are reduced in patients with renal, cardio- and cerebrovascular disease. Moreover, low homoarginine concentrations independently predict morbidity and mortality in these patients. Besides endogenous synthesis, homoarginine is also a constituent of the human diet. The objective of the present study was to analyze the kinetics of orally supplemented homoarginine in human plasma by means of a pharmacometric approach. We developed a pharmacometric model to evaluate different dosing regimens, especially the regimen of 125 mg once weekly, based on a previous clinical study (n = 20). The model was adapted to account for differences in baseline homoarginine plasma concentrations between healthy and diseased individuals. A novel dosing regimen of 25 mg once daily led to higher attainment of homoarginine reference concentrations using clinical trial simulations. With 25 mg/day, the trough concentration of only 6% of the older and 3.8% of the younger population was predicted to be below the target concentration of 2.0-4.1 µmol/L. In synopsis, the new dosing regimen recapitulates the kinetics of homoarginine in healthy individuals optimally.

Expression of cardiovascular-related microRNAs is altered in L-arginine:glycine amidinotransferase deficient mice.

In humans and mice, L-arginine:glycine amidinotransferase (AGAT) and its metabolites homoarginine (hArg) and creatine have been linked to cardiovascular disease (CVD), specifically myocardial infarction (MI) and heart failure (HF). The underlying molecular and regulatory mechanisms, however, remain unclear. To identify potential pathways of cardiac AGAT metabolism, we sequenced microRNA (miRNA) in left ventricles of wild-type (wt) compared to AGAT-deficient (AGAT-/-) mice. Using literature search and validation by qPCR, we identified eight significantly regulated miRNAs in AGAT-/- mice linked to atherosclerosis, MI and HF: miR-30b, miR-31, miR-130a, miR-135a, miR-148a, miR-204, miR-298, and let-7i. Analysis of Gene Expression Omnibus (GEO) data confirmed deregulation of these miRNAs in mouse models of MI and HF. Quantification of miRNA expression by qPCR in AGAT-/- mice supplemented with creatine or hArg revealed that miR-30b, miR-31, miR-130a, miR-148a, and miR-204 were regulated by creatine, while miR-135a and miR-298 showed a trend of regulation by hArg. Finally, bioinformatics-based target prediction showed that numerous AGAT-dependent genes previously linked to CVD are likely to be regulated by the identified miRNAs. Taken together, AGAT deficiency and hArg/creatine supplementation are associated with cardiac miRNA expression which may influence cardiac (dys)function and CVD.

Arginine:Glycine Amidinotransferase Is Essential for Creatine Supply in Mice During Chronic Hypoxia.

Objective: Chronic hypoxia induces pulmonary and cardiovascular pathologies, including pulmonary hypertension (PH). L-arginine:glycine amidinotransferase (AGAT) is essential for homoarginine (hArg) and guanidinoacetate synthesis, the latter being converted to creatine by guanidinoacetate methyltransferase. Low hArg concentrations are associated with cardiovascular morbidity and predict mortality in patients with PH. We therefore aimed to investigate the survival and cardiac outcome of AGAT knockout (Agat -/-) mice under hypoxia and a possible rescue of the phenotype. Methods: Agat -/- mice and wild-type (WT) littermates were subjected to normoxia or normobaric hypoxia (10% oxygen) for 4 weeks. A subgroup of Agat -/- mice was supplemented with 1% creatine from weaning. Survival, hematocrit, blood lactate and glucose, heart weight-to-tibia length (HW/TL) ratio, hArg plasma concentration, and Agat and Gamt expression in lung, liver, and kidneys were evaluated. Results: After 6 h of hypoxia, blood lactate was lower in Agat -/--mice as compared to normoxia (p < 0.001). Agat -/- mice died within 2 days of hypoxia, whereas Agat -/- mice supplemented with creatine and WT mice survived until the end of the study. In WT mice, hematocrit (74 ± 4 vs. 55 ± 2%, mean ± SD, p < 0.001) and HW/TL (9.9 ± 1.3 vs. 7.3 ± 0.7 mg/mm, p < 0.01) were higher in hypoxia, while hArg plasma concentration (0.25 ± 0.06 vs. 0.38 ± 0.12 µmol/L, p < 0.01) was lower. Agat and Gamt expressions were differentially downregulated by hypoxia in lung, liver, and kidneys. Conclusion: Agat and Gamt are downregulated in hypoxia. Agat-/- mice are nonviable in hypoxia. Creatine rescues the lethal phenotype, but it does not reduce right ventricular hypertrophy of Agat-/- mice in hypoxia.

Homoarginine- and Creatine-Dependent Gene Regulation in Murine Brains with l-Arginine:Glycine Amidinotransferase Deficiency.

l-arginine:glycine amidinotransferase (AGAT) and its metabolites homoarginine (hArg) and creatine have been linked to stroke pathology in both human and mouse studies. However, a comprehensive understanding of the underlying molecular mechanism is lacking. To investigate transcriptional changes in cerebral AGAT metabolism, we applied a transcriptome analysis in brains of wild-type (WT) mice compared to untreated AGAT-deficient (AGAT-/-) mice and AGAT-/- mice with creatine or hArg supplementation. We identified significantly regulated genes between AGAT-/- and WT mice in two independent cohorts of mice which can be linked to amino acid metabolism (Ivd, Lcmt2), creatine metabolism (Slc6a8), cerebral myelination (Bcas1) and neuronal excitability (Kcnip3). While Ivd and Kcnip3 showed regulation by hArg supplementation, Bcas1 and Slc6a8 were creatine dependent. Additional regulated genes such as Pla2g4e and Exd1 need further evaluation of their influence on cerebral function. Experimental stroke models showed a significant regulation of Bcas1 and Slc6a8. Together, these results reveal that AGAT deficiency, hArg and creatine regulate gene expression in the brain, which may be critical in stroke pathology.

Analysis of L-arginine:glycine amidinotransferase-, creatine- and homoarginine-dependent gene regulation in the murine heart.

L-arginine:glycine amidinotransferase (AGAT) and its metabolites creatine and homoarginine (HA) have been linked to cardiovascular pathologies in both human and murine studies, but the underlying molecular mechanisms are poorly understood. Here, we report the first analysis of heart transcriptome variation using microarrays in an AGAT-deficient (AGAT-/-) mouse model to evaluate AGAT-, creatine- and HA-dependent gene regulation. Our data revealed significant differences of gene expression between AGAT-/- and wild-type (WT) mice, affecting cardiac energy metabolism (Fbp2, Ucp2), cardiac hypertrophy and fibrosis (Nppa, Ctgf), immune response (Fgl2), and the conduction system of the heart (Dsc2, Ehd4, Hcn2, Hcn4, Scn4a, Scn4b). All of these genes being expressed on WT level in creatine-supplemented mice. Using in silico analysis based on the GEO database we found that most of these candidate genes (Ctgf, Dsc2, Fbp2, Fgl2, Hcn2, Nppa)  revealed significant alterations in a WT mouse model of myocardial infarction underlining a pathophysiological relationship between AGAT metabolism and cardiovascular disease.

Muscle phenotype of AGAT- and GAMT-deficient mice after simvastatin exposure.

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 < 0.01 and P < 0.001, respectively). Simvastatin-induced motor impairment was exacerbated in AGAT-deficient mice compared with AGAT-overexpressing GAMT-/- mice and therefore revealed an effect independent of Cr. But Cr supplementation itself improved muscle strength independent of AGAT expression (normalized grip strength: 55.8 ± 2.9% vs 72.5% ± 3.0%, P < 0.01). Homoarginine supplementation did not affect statin-induced myopathy in AGAT-deficient mice. Our results from clinical and animal studies suggest that AGAT expression/activity and its product Cr influence statin-induced myopathy independent of each other. The interplay between simvastatin treatment, AGAT expression and activity, and Cr seems to be complex. Further clinical pharmacological studies are needed to elucidate the underlying mechanism(s) and to evaluate whether supplementation with Cr, or possibly GAA, in patients under statin medication may reduce the risk of muscular side effects.

Myeloid-Derived Suppressor Cells Mediate Immunosuppression After Cardiopulmonary Bypass.

OBJECTIVES: Cardiopulmonary bypass is associated with severe immune dysfunctions. Particularly, a cardiopulmonary bypass-related long-lasting immunosuppressive state predisposes patients to a higher risk of postoperative complications, such as persistent bacterial infections. This study was conducted to elucidate mechanisms of post-cardiopulmonary bypass immunosuppression. DESIGN: In vitro studies with human peripheral blood mononuclear cells. SETTING: Cardiosurgical ICU, University Research Laboratory. PATIENTS: Seventy-one patients undergoing cardiac surgery with cardiopulmonary bypass (enrolled May 2017 to August 2018). INTERVENTIONS: Peripheral blood mononuclear cells before and after cardiopulmonary bypass were analyzed for the expression of immunomodulatory cell markers by real-time quantitative reverse transcription polymerase chain reaction. T cell effector functions were determined by enzyme-linked immunosorbent assay, carboxyfluorescein succinimidyl ester staining, and cytotoxicity assays. Expression of cell surface markers was assessed by flow cytometry. CD15 cells were depleted by microbead separation. Serum arginine was measured by mass spectrometry. Patient peripheral blood mononuclear cells were incubated in different arginine concentrations, and T cell functions were tested. MEASUREMENTS AND MAIN RESULTS: After cardiopulmonary bypass, peripheral blood mononuclear cells exhibited significantly reduced levels of costimulatory receptors (inducible T-cell costimulator, interleukin 7 receptor), whereas inhibitory receptors (programmed cell death protein 1 and programmed cell death 1 ligand 1) were induced. T cell effector functions (interferon γ secretion, proliferation, and CD8-specific cell lysis) were markedly repressed. In 66 of 71 patients, a not yet described cell population was found, which could be characterized as myeloid-derived suppressor cells. Myeloid-derived suppressor cells are known to impair immune cell functions by expression of the arginine-degrading enzyme arginase-1. Accordingly, we found dramatically increased arginase-1 levels in post-cardiopulmonary bypass peripheral blood mononuclear cells, whereas serum arginine levels were significantly reduced. Depletion of myeloid-derived suppressor cells from post-cardiopulmonary bypass peripheral blood mononuclear cells remarkably improved T cell effector function in vitro. Additionally, in vitro supplementation of arginine enhanced T cell immunocompetence. CONCLUSIONS: Cardiopulmonary bypass strongly impairs the adaptive immune system by triggering the accumulation of myeloid-derived suppressor cells. These myeloid-derived suppressor cells induce an immunosuppressive T cell phenotype by increasing serum arginine breakdown. Supplementation with L-arginine may be an effective measure to counteract the onset of immunoparalysis in the setting of cardiopulmonary bypass.

Evidence by GC-MS that lysine is an arginase-catalyzed metabolite of homoarginine in vitro and in vivo in humans.

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.

Cognitive performance of 20 healthy humans supplemented with L-homoarginine for 4 weeks.

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.

A Label-Free Continuous Fluorescence-Based Assay for Monitoring Ornithine Decarboxylase Activity with a Synthetic Putrescine Receptor.

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 (>107 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 ( kcat = 0.12 s-1 and KM = 24 µM) and inhibition constants of the two ODC inhibitors α-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.

Oral supplementation with L-homoarginine in young volunteers.

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.

Homoarginine levels are regulated by L-arginine:glycine amidinotransferase and affect stroke outcome: results from human and murine studies.

BACKGROUND: Endogenous arginine homologues, including homoarginine, have been identified as novel biomarkers for cardiovascular disease and outcomes. Our studies of human cohorts and a confirmatory murine model associated the arginine homologue homoarginine and its metabolism with stroke pathology and outcome. METHODS AND RESULTS: Increasing homoarginine levels were independently associated with a reduction in all-cause mortality in patients with ischemic stroke (7.4 years of follow-up; hazard ratio for 1-SD homoarginine, 0.79 [95% confidence interval, 0.64-0.96]; P=0.019; n=389). Homoarginine was also independently associated with the National Institutes of Health Stroke Scale+age score and 30-day mortality after ischemic stroke (P<0.05; n=137). A genome-wide association study revealed that plasma homoarginine was strongly associated with single nucleotide polymorphisms in the L-arginine:glycine amidinotransferase (AGAT) gene (P<2.1 × 10(-8); n=2806), and increased AGAT expression in a cell model was associated with increased homoarginine. Next, we used 2 genetic murine models to investigate the link between plasma homoarginine and outcome after experimental ischemic stroke: (1) an AGAT deletion (AGAT(-/-)) and (2) a guanidinoacetate N-methyltransferase deletion (GAMT(-/-)) causing AGAT upregulation. As suggested by the genome-wide association study, homoarginine was absent in AGAT(-/-) mice and increased in GAMT(-/-) mice. Cerebral damage and neurological deficits in experimental stroke were increased in AGAT(-/-) mice and attenuated by homoarginine supplementation, whereas infarct size in GAMT(-/-) mice was decreased compared with controls. CONCLUSIONS: Low homoarginine appears to be related to poor outcome after ischemic stroke. Further validation in future trials may lead to therapeutic adjustments of homoarginine metabolism that alleviate stroke and other vascular disorders.

Isoprostanes and neuroprostanes: total synthesis, biological activity and biomarkers of oxidative stress in humans.

Isoprostanes (IsoPs) and neuroprostanes (NeuroPs) are formed in vivo by a free radical non-enzymatic mechanism involving peroxidation of arachidonic acid (AA, C20:4 n-6) and docosahexaenoic acid (DHA, C22:6 n-3) respectively. This review summarises our research in the total synthesis of these lipid metabolites, as well as their biological activities and their utility as biomarkers of oxidative stress in humans.

Reference intervals for plasma L-arginine and the L-arginine:asymmetric dimethylarginine ratio in the Framingham Offspring Cohort.

L-arginine, as a precursor of NO synthesis, has attracted much scientific attention in recent years. Experimental mouse models suggest that L-arginine supplementation can retard, halt, or even reverse atherogenesis. In human studies, supplementation with L-arginine improved endothelium-dependent vasodilation. However, L-arginine levels are best interpreted in the context of levels of asymmetric dimethylarginine (ADMA), a competitive inhibitor of NO synthase. Thus, reference limits for circulating L-arginine and the L-arginine:ADMA ratio may help to determine the nutritional state of individuals at high cardiovascular risk in light of increased ADMA levels. We defined reference limits for plasma L-arginine in 1141 people and for the L-arginine:ADMA ratio in 1138 relatively healthy individuals from the Framingham Offspring Cohort. Plasma L-arginine and ADMA concentrations were determined by using a stable isotope-based LC-MS/MS method. The reference limits (2.5th and 97.5th percentiles) for plasma L-arginine were 41.0 µmol/L (95% CI = 39.5-42.5 µmol/L) and 114 µmol/L (95% CI = 112-115 µmol/L), whereas corresponding reference limits (2.5th and 97.5th percentiles) for the L-arginine:ADMA ratio were 74.3 µmol/L (95% CI = 71.1-77.3 µmol/L) and 225 µmol/L (95% CI = 222-228 µmol/L). Plasma L-arginine was positively associated with the estimated glomerular filtration rate (eGFR) and blood glucose levels, whereas the L-arginine:ADMA ratio was positively associated with eGFR and diastolic blood pressure but inversely associated with homocysteine and (log)C-reactive protein. We report reference levels for plasma L-arginine and for the L-arginine:ADMA ratio that may be helpful for evaluation of the effects of L-arginine supplementation in participants with an impaired L-arginine/NO pathway.