Characterization of the L-Arginine/Nitric Oxide Pathway and Oxidative Stress in Pediatric Patients with Atopic Diseases.

INTRODUCTION: L-Arginine (Arg) is a semi-essential amino acid. Constitutive and inducible nitric oxide synthase (NOS) isoforms convert Arg to nitric oxide (NO), a potent vaso- and bronchodilator with multiple biological functions. Atopic dermatitis (AD) and bronchial asthma (BA) are atopic diseases affecting many children globally. Several studies analyzed NO in airways, yet the systemic synthesis of NO in AD and BA in children with BA, AD or both is elusive. METHODS: In a multicenter study, blood and urine were obtained from 130 of 302 participating children for the measurement of metabolites of the Arg/NO pathway (BA 31.5%; AD 5.4%; AD + BA 36.1%; attention deficit hyperactivity disorder (ADHD) 12.3%). In plasma and urine amino acids Arg and homoarginine (hArg), both substrates of NOS, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), both inhibitors of NOS, dimethylamine (DMA), and nitrite and nitrate, were measured by gas chromatography-mass spectrometry. Malondialdehyde (MDA) was measured in plasma and urine samples to evaluate possible effects of oxidative stress. RESULTS: There were no differences in the Arg/NO pathway between the groups of children with different atopic diseases. In comparison to children with ADHD, children with AD, BA or AD and BA had higher plasma nitrite (p < 0.001) and nitrate (p < 0.001) concentrations, suggesting higher systemic NO synthesis in AD and BA. Urinary excretion of DMA was also higher (p = 0.028) in AD and BA compared to patients with ADHD, suggesting elevated ADMA metabolization. DISCUSSION/CONCLUSION: The Arg/NO pathway is activated in atopic diseases independent of severity. Systemic NO synthesis is increased in children with an atopic disease. Plasma and urinary MDA levels did not differ between the groups, suggesting no effect of oxidative stress on the Arg/NO pathway in atopic diseases.

An Optimized MRM-Based Workflow of the l-Arginine/Nitric Oxide Pathway Metabolites Revealed Disease- and Sex-Related Differences in the Cardiovascular Field.

Clinical data indicate that low circulating l-homoarginine (HArg) concentrations are associated with cardiovascular (CV) disease, CV mortality, and all-cause mortality. A high number of LC-based analytical methods for the quantification of HArg, in combination with the l-arginine (Arg)-related pathway metabolites, have been reported. However, these methods usually consider a limited panel of analytes. Thus, in order to achieve a comprehensive picture of the Arg metabolism, we described an improved targeted metabolomic approach based on a multiple reaction monitoring (MRM) mass spectrometry method for the simultaneous quantification of the Arg/nitric oxide (NO) pathway metabolites. This methodology was then employed to quantify the plasma concentrations of these analytes in a cohort of individuals with different grades/types of coronary artery disease (CAD) in order to increase knowledge about the role of HArg and its associated metabolites in the CV field. Our results showed that the MRM method here implemented is suitable for the simultaneous assessment of a wide panel of amino acids involved in the Arg/NO metabolic pathway in plasma samples from patients with CV disease. Further, our findings highlighted an impairment of the Arg/NO metabolic pathway, and suggest a sex-dependent regulation of this metabolic route.

Homoarginine and methylarginines independently predict long-term outcome in patients presenting with suspicion of venous thromboembolism.

Endogenous arginine derivatives homoarginine, asymmetric dimethylarginine (ADMA) and symmetric dimethyarginine (SDMA) are independent mortality predictors in atherosclerotic cardiovascular disease (CVD). Our study reports the first analysis, whether homoarginine, ADMA and SDMA predict venous thromboembolism (VTE) recurrence and overall mortality in patients with suspected acute VTE. We assessed serum levels of homoarginine, ADMA and SDMA by LC-MS/MS in 865 individuals from a prospective consecutive cohort of patients with clinical suspicion of VTE. The median follow-up time for mortality was 1196 days. VTE was confirmed by imaging in 418 patients and excluded in 447 patients. Low levels of homoarginine and high levels of ADMA or SDMA independently predicted all-cause mortality after adjustment for sex, age, oral anticoagulants, body mass index, arterial hypertension, diabetes mellitus, smoking, dyslipidemia, chronic heart failure, history of stroke, creatinine and cancer both in patients with VTE and without VTE. Interestingly, none of those parameters was predictive for VTE recurrence. We provide the first report that low circulating levels of homoarginine and high circulating levels of ADMA and SDMA independently predict all-cause mortality in patients with suspected VTE. These parameters might serve as markers of "frailty" and should be considered for future risk stratification approaches in this clinical population. Taking into account that homoarginine supplementation is protective in animal models of CVD and safe in healthy human volunteers, our study provides the basis for future homoarginine supplementation studies in patients with suspected VTE to investigate possible direct protective effects of homoarginine in this population.

L-homoarginine is associated with decreased cardiovascular- and all-cause mortality.

BACKGROUND: Increasing evidence suggests that L-homoarginine, an endogenous analogue of the amino acid L-arginine, may have beneficial effects on vascular homeostasis. We examined whether L-homoarginine is associated with 10-year risk of all-cause and cardiovascular mortality in a black South African population. METHODS: We included 669 black South African participants (mean age 59.5 years), 143 of whom died during the 10-year follow-up period. Mortality data were acquired via verbal autopsy. Plasma L-homoarginine (and other related markers) were analysed with liquid chromatography-tandem mass spectrometry. RESULTS: Survivors had higher L-homoarginine levels compared with nonsurvivors (1.25 µM vs. 0.89 µM; P < .001). Multivariable Cox regression analyses revealed that higher plasma L-homoarginine predicted a reduction in 10-year cardiovascular (hazard ratio [HR] per SD increment, 0.61; 95% CI 0.50 to 0.75) and all-cause (hazard ratio [HR] per SD increment, 0.59; 95% CI 0.41 to 0.84) mortality risk. CONCLUSION: Higher L-homoarginine levels are associated with reduced risk of 10-year cardiovascular and all-cause mortality. Regulation of L-homoarginine levels as a therapeutic target in the management of cardiovascular disease should be investigated.

Comprehensive arginine metabolomics and peripheral vasodilatory capacity in rheumatoid arthritis: A monocentric cross-sectional study.

BACKGROUND: The relationship between plasma arginine metabolites influencing vascular homeostasis and peripheral vasodilatory capacity in rheumatoid arthritis (RA) patients is not known. METHODS: l-arginine (Arg), monomethyl-l-arginine (MMA), l-homoarginine (hArg), asymmetric dimethyl-l-arginine (ADMA), symmetric dimethyl-l-arginine, and l-citrulline (Cit) were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) in 164 RA patients and 100 age- and sex-matched healthy controls without previous cardiovascular events. Log-transformed reactive hyperemia index (Ln-RHI) evaluated by flow-mediated pulse amplitude tonometry (PAT, EndoPAT2000 device) was assessed as surrogate measure of peripheral vasodilatory capacity in RA patients. Ln-RHI values <0.51 indicated peripheral endothelial dysfunction (ED). The relationship between plasma arginine metabolite concentrations, RA descriptors and peripheral vasodilatory capacity was evaluated by bivariate correlation and regression analyses. RESULTS: Plasma ADMA concentrations were significantly higher, and plasma hArg concentrations significantly lower, in RA patients than in controls (0.53 ± 0.09 vs 0.465 ± 0.07 µmol/L and 1.50 ± 0.60 vs 1.924 ± 0.78 µmol/L, respectively; p < 0.001 for both comparisons). Bivariate correlation analysis demonstrated no significant correlation between arginine metabolites and disease descriptors. In regression analysis in RA patients, higher plasma ADMA concentrations were independently associated with presence of ED [OR(95% CI) = 77.3(1.478-4050.005), p = 0.031] and lower Ln-RHI [B coefficient(95% CI) = -0.57(-1.09 to -0.05), p = 0.032]. CONCLUSIONS: ADMA was significantly, albeit weakly, associated with impaired microcirculatory vasodilatory capacity and peripheral endothelial dysfunction in RA. This suggests an important pathophysiological role of this metabolite in the vascular alterations observed in this patient group.

Arginine derivatives in atrial fibrillation progression phenotypes.

Arginine, homoarginine (hArg), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) affect nitric oxide metabolism and altered concentrations are associated with cardiovascular morbidity and mortality. We analyzed these metabolites using liquid chromatography-tandem mass spectrometry in patients with atrial fibrillation (AF) (n = 241) with a focus on heart rhythm at blood withdrawal, AF progression phenotypes, and successful sinus rhythm (SR) restoration (n = 22). AF progression phenotypes were defined as paroxysmal AF with/without low voltage areas (LVA) and persistent AF with/without LVA. While arginine, ADMA, and hArg were within reference limits for healthy controls, SDMA was higher in the AF cohort (0.57 ± 0.12 vs. 0.53 µmol/L (97.5th percentile in reference cohort)). SR restoration in AF patients resulted in normalization of SDMA concentrations (0.465 ± 0.082 vs. 0.570 ± 0.134 µmol/L at baseline, p < 0.001). Patients with AF at the time of blood sampling had significantly lower hArg (1.65 ± 0.51 vs. 1.85 ± 0.60 µmol/L, p = 0.006) and higher ADMA concentrations (0.526 ± 0.08 vs. 0.477 ± 0.08 µmol/L, p < 0.001) compared with AF patients in SR. hArg concentrations were lower in patients with advanced AF progression phenotypes (persistent AF with LVA (p = 0.046)) independent of heart rhythm at blood sampling. Summarizing, arginine metabolism imbalance is associated with AF in general and AF progression and may contribute to associated risk. KEY MESSAGES: • Heart rhythm at blood withdrawal affects ADMA and hArg level in AF patients. • SDMA is higher in AF patients. • SDMA levels normalize after sinus rhythm restoration. • hArg levels decrease in advanced AF progression phenotypes.

GC-MS measurement of spermidine and putrescine in serum of elderly subjects: intriguing association between spermidine and homoarginine.

Gas chromatography-mass spectrometry (GC-MS) methods were developed, validated and used to measure serum spermidine (SPD) and putrescine (PUT) in 9 seropositive Helicobacter pylori (Hp +) and 18 seronegative Helicobacter pylori (Hp -) subjects (31-105 years). Homoarginine (hArg) was also measured by GC-MS. There were no statistical differences (unpaired t test) between the Hp + and Hp - subjects with respect to the serum concentrations of SPD (67.6 ± 40.3 vs. 93.7 ± 37.7 nM, P = 0.109), PUT (220 ± 139 vs. 236 ± 85 nM, P = 0.708) and hArg (1.60 ± 0.64 µM vs. 1.83 ± 0.74 µM, P = 0.554). Serum SPD and hArg concentrations correlated with each other (r = 0.426, P = 0.026, n = 27). The PUT/SPD molar ratio correlated inversely with the hArg concentration (r = - 0.406, P = 0.034, n = 27) and proteinic citrulline (r = - 0.487, P = 0.01, n = 27). These results suggest that SPD and PUT synthesis is associated with hArg formation and protein citrullination in healthy elderly subjects. The mechanisms underlying these associations and their significance remain to be elucidated.

Drugs linked to plasma homoarginine in chronic kidney disease patients-a cross-sectional analysis of the German Chronic Kidney Disease cohort.

BACKGROUND: Elevated plasma concentrations of symmetric and asymmetric dimethylarginine (SDMA and ADMA, respectively) and a lower plasma concentration of the structurally related homoarginine are commonly observed in patients with chronic kidney disease (CKD) and independently predict total mortality as well as progression of renal disease. We aimed to identify drugs that may alter this adverse metabolite pattern in a favourable fashion. METHODS: Plasma ADMA, SDMA, homoarginine and l-arginine were determined by liquid chromatography-tandem mass spectrometry in 4756 CKD patients ages 18-74 years with an estimated glomerular filtration rate (eGFR) of 30-60 mL/min/1.73 m2 or an eGFR >60 mL/min/1.73 m2 and overt proteinuria who were enrolled in the German Chronic Kidney Disease (GCKD) study. Associations between laboratory, clinical and medication data were assessed. RESULTS: Intake of several commonly used drugs was independently associated with plasma concentrations of homoarginine and/or related metabolites. Among these, the peroxisome proliferator-activated receptor alpha (PPAR-α) agonist fenofibrate was associated with the most profound differences in ADMA, SDMA and homoarginine plasma concentrations: 66 patients taking fenofibrate had a multivariable adjusted odds ratio (OR) of 5.83 [95% confidence interval (CI) 2.82-12.03, P < 0.001] to have a plasma homoarginine concentration above the median. The median homoarginine plasma concentration in patients taking fenofibrate was 2.30 µmol/L versus 1.55 in patients not taking the drug (P < 0.001). In addition, fibrates were significantly associated with lower plasma SDMA and higher l-arginine concentrations. In contrast, glucocorticoids were associated with lower plasma homoarginine, with adjusted ORs of 0.52 (95% CI 0.40-0.67, P < 0.001) and 0.53 (95% CI 0.31-0.90, P = 0.018) for prednisolone and methylprednisolone, respectively. CONCLUSIONS: In a large cohort of CKD patients, intake of fenofibrate and glucocorticoids were independently associated with higher and lower plasma homoarginine concentrations, respectively. Effects on plasma homoarginine and methylarginines warrant further investigation as potential mechanisms mediating beneficial or adverse drug effects.

Low homoarginine/SDMA ratio is associated with poor short- and long-term outcome after stroke in two prospective studies.

BACKGROUND: 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. METHODS: 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). RESULTS: 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 < 3; P = 0.007; n = 138), whereas hArg/ADMA did not. CONCLUSION: These results from two prospective stroke studies reveal that hArg/SDMA ratio could prove a valuable blood-based biomarker to discriminate patients with poor short- and long-term outcome, increased neurological impairment, and severe disability after stroke.

[Homoarginine level and methionine-homocysteine balance in patients with ischemic heart disease.]

The level of homoarginine (hArg) in terms of prognostic significance may exceed the natriuretic peptides and other well-known markers according to the latest data about the progression of cardiovascular diseases. The lack of data on the association of hArg levels with levels of other metabolites makes it difficult to understand its role in the pathogenesis of cardiovascular diseases. Relationships of hArg and other amino acids, including methionine (Met) and total homocysteine (tHcy), and their ratio in patients with ischemic heart disease were evaluated. The study included 74 patients with coronary heart disease (57 men and 17 women) aged 62 (57 - 67) years before coronary artery bypass surgery and 27 healthy people of similar age. In patients, the level of hArg was almost 2 times lower (p < 0.05) than in healthy individuals and rates lower than 1.4 µM were in half of them. The statistically significant decrease (p = 0.0025) of the Met/tHcy ratio corresponded to a decrease in the level of hArg. This ratio did not correlate with glucose level or body mass index. Less statistical significance of hArg correlation with levels of Met or tHcy separately was observed. In the subgroup of patients with hAarg level above 2.1 µM, a lower incidence of myocardial infarction was noted. Thus, a low hArg level is associated with impaired metabolism of sulfur-containing amino acids involved in transmethylation reactions, in patients with ischemic heart disease. The Met/tHcy ratio, closely correlating with the level of hArg, apparently reveals a link between the reactions of creatine formation and transmethylation, highlighting a cohort of patients with the most profound and dangerous changes in tissue metabolism.

High plasma guanidinoacetate-to-homoarginine ratio is associated with high all-cause and cardiovascular mortality rate in adult renal transplant recipients.

L-Arginine:glycine amidinotransferase (AGAT) is the main producer of the creatine precursor, guanidinoacetate (GAA), and L-homoarginine (hArg). We and others previously reported lower levels of circulating and urinary hArg in renal transplant recipients (RTR) compared to healthy subjects. In adults, hArg emerged as a novel risk factor for renal and cardiovascular adverse outcome. Urinary GAA was found to be lower in children and adolescents with kidney transplants compared to healthy controls. Whether GAA is also a risk factor in the renal and cardiovascular systems of adults, is not yet known. In the present study, we aimed to investigate the significance of circulating GAA and the GAA-to-hArg molar ratio (GAA/hArg) in adult RTR. We hypothesized that GAA/hArg represents a measure of the balanced state of the AGAT activity in the kidneys, and would prospectively allow assessing a potential association between GAA/hArg and long-term outcome in RTR. The median follow-up period was 5.4 years. Confounders and potential mediators of GAA/hArg associations were evaluated with multivariate linear regression analyses, and the association with all-cause and cardiovascular mortality or death-censored graft loss was studied with Cox regression analyses. The study cohort consisted of 686 stable RTR and 140 healthy kidney donors. Median plasma GAA concentration was significantly lower in the RTR compared to the kidney donors before kidney donation: 2.19 [1.77-2.70] µM vs. 2.78 [2.89-3.35] µM (P < 0.001). In cross-sectional multivariable analyses in RTR, HDL cholesterol showed the strongest association with GAA/hArg. In prospective analyses in RTR, GAA/hArg was associated with a higher risk for all-cause mortality (hazard ratio (HR): 1.35 [95% CI 1.19-1.53]) and cardiovascular mortality (HR: 1.46 [95% CI 1.24-1.73]), independent of potential confounders. GAA but not GAA/hArg was associated with death-censored graft loss in crude survival and Cox regression analyses. The association of GAA and death-censored graft loss was lost after adjustment for eGFR. Our study suggests that in the kidneys of RTR, the AGAT-catalyzed biosynthesis of GAA is decreased. That high GAA/hArg is associated with a higher risk for all-cause and cardiovascular mortality may suggest that low plasma hArg is a stronger contributor to these adverse outcomes in RTR than GAA.

Establishment of reference values for the lysine acetylation marker Nɛ-acetyllysine in small volume human plasma samples by a multi-target LC-MS/MS method.

Cardiovascular disease (CVD) and chronic kidney disease (CKD) constitute substantial burdens for public health. The identification and validation of risk markers for CVD and CKD in epidemiological studies requires frequent adaption of existing analytical methods as well as development of new methods. In this study, an analytical procedure to simultaneously quantify ten endogenous biomarkers for CVD and CKD is described. An easy-to-handle sample preparation requiring only 20 µL of human plasma is followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The method was successfully validated according to established guidelines meeting required criteria for accuracy, precision, recovery, linearity, selectivity, and limits of quantification. The scalability of the method for application in larger cohorts was assessed using a set of plasma samples from healthy volunteers (n = 391) providing first reference values for the recently established biomarker Nɛ-acetyllysine (Nɛ-AcLys). Other biomarkers analyzed were creatinine, ß-aminoisobutyric acid (ß-AIB), carnitine, 1-methylnicotinamide (1-MNA), citrulline, symmetric dimethylarginine (SDMA), asymmetric dimethylarginine (ADMA), homoarginine (hArg), and ornithine. All obtained results are within reference values specified elsewhere. Overall, these results demonstrate the suitability of the method for simultaneous quantification of ten endogenous biomarkers for CVD and CKD in plasma samples from larger cohorts and allow validation of Nɛ-AcLys as a biomarker in large cohorts.

Distinct associations between plasma osteoprotegerin, homoarginine and asymmetric dimethylarginine in chronic kidney disease male patients with coronary artery disease.

High plasma osteoprotegerin (OPG) and asymmetric dimethylarginine (ADMA) and low homoarginine (hArg) predict adverse renal and cardiovascular (CV) outcomes. In patients with chronic kidney disease and stable coronary artery disease, plasma OPG correlated with hArg (r = - 0.37, P = 0.03) and the hArg/ADMA molar ratio (r = - 0.46, P = 0.009), which was maintained upon adjustment for renal function. Elevated OPG levels and decreased hArg/ADMA ratios independently predicted 4-year composite CV and renal endpoints (CV death or progression to dialysis). Thus, high OPG and low hArg/ADMA ratio, albeit interrelated, appear to independently contribute to adverse clinical outcome.

Effect of ranolazine on plasma arginine derivatives and urinary isoprostane 8-iso-PGF2α in patients with myocardial infarction in the randomized RIMINI-Trial.

The purpose of the present study was to assess whether 6-week ranolazine application on top of guideline-based treatment impacts on the arginine/NO pathway and urinary isoprostane 8-iso-PGF2α as marker of oxidative stress in patients directly after a myocardial infarction. 20 patients with unstable angina pectoris and proof of acute cardiac ischemia entered the study. 10 subjects received the study drug ranolazine in addition to standard treatment, the others received only standard treatment. Urine and venous blood were collected before and after treatment. At the end of the study and compared to baseline, homoarginine levels had increased in the control group. This was not the case in ranolazine-patients. Interestingly, in ranolazine-treated-patients arginine plasma levels were significantly higher at the end of the study than at baseline (difference +26 µmol/L, 95% CI 8.6 to 44 µmol/L). ADMA and SDMA levels were not different. Urine levels of the oxidative stress marker 8-iso-PGF2α tended to be lower in ranolazine-treated patients (-144 pmol/mg creatinine). Findings of this hypothesis-driven study give evidence that ranolazine treatment enhances arginine plasma levels and lowers oxidative stress.

Asymmetric dimethylation and citrullination of proteinic arginine and homoarginine synthesis in human Helicobacter pylori infection.

The importance of L-arginine (Arg) and relatives, including L-homoarginine (hArg) and asymmetric dimethylarginine (ADMA), in humans infected with Helicobacter pylori (Hp) is little understood. ADMA is produced by asymmetric dimethylation of the guanidine group of Arg residues in certain proteins and is released by proteolysis. High concentrations of circulating free ADMA are considered a risk factor for morbidity and mortality in adults. This risk is considered to arise from the inhibition of the synthesis of nitric oxide (NO), which is a potent vasodilator and inhibitor of platelet aggregation. In the present study, we quantified by stable isotope dilution gas chromatography-mass spectrometry (GC-MS) the concentration of free (f) and total (t) ADMA, Arg, hArg, lysine (Lys) and the sum of citrulline (Cit) and ornithine (Orn) (6 M HCl, 20 h, 110 °C) in serum samples of apparently healthy elderly subjects (n = 27; age, 31-105 years) who were tested for Hp infection. Nine subjects (5 males, 4 females) were found to be Hp seropositive (Hp+) and 18 subjects (8 males, 9 females) were found to be Hp seronegative (Hp‒). Proteinic (p) concentrations were determined by difference. fADMA (0.493 ± 0.068 vs 0.466 ± 0.081 µM, P = 0.382), pADMA (113 ± 73 vs 76 ± 59 nM, P = 0.169) and tADMA (0.606 ± 0.126 vs 0.543 ± 0.121 µM, P = 0.280) serum concentrations were found not to differ between the Hp+ and Hp- subjects. Serum concentrations of fArg (162 ± 30 vs 177 ± 36 µM, P = 0.471), fhArg (1.600 ± 0.638 vs 1.831 ± 0.742 µM, P = 0.554), and fLys (388 ± 170 vs 395 ± 149 µM, P = 0.700) also did not differ statistically between Hp+ and Hp- subjects. tArg (12.4 ± 1.49 vs 13.0 ± 1.33 mM, P = 0.190), tLys (23.0 ± 2.65 vs. 23.9 ± 2.66 mM, P = 0.456) and tCit + Orn (2.53 ± 0.76 vs 2.63 ± 0.85 mM, P = 0.817) did not differ between Hp+and Hp‒ subjects as well. phArg concentration was close to the limit of quantitation of the method (Hp+: 30 ± 210 nM; Hp-: 42 ± 205 nM), suggesting that hArg is virtually absent in serum proteins of the investigated subjects. pCit + Orn did not differ between infected and non-infected subjects. Our study suggests that Hp infection is not associated with elevated asymmetric dimethylation and citrullination of Arg proteins present in the serum or with the hArg synthesis from free Arg in elderly subjects. However, asymmetric Arg dimethylation was found to correlate inversely with Arg citrullination in Hp- (r2 = 0.408, P = 0.004) but not in Hp+ (r2 = 0.065, P = 0.506), with Arg citrullination decreasing and Arg asymmetric dimethylation increasing with subjects' age.

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.

Is Homoarginine a Protective Cardiovascular Risk Factor?

A series of recent epidemiological studies have implicated the endogenous nonproteinogenic amino acid l-homoarginine as a novel candidate cardiovascular risk factor. The association between homoarginine levels and the risk of adverse cardiovascular outcomes is inverse (ie, high cardiovascular risk is predicted by low rather than high homoarginine levels), which makes it plausible to normalize systemic homoarginine levels via oral supplementation. The emergence of homoarginine as a potentially treatable protective cardiovascular risk factor has generated a wave of hope in the field of cardiovascular prevention. Herein, we review the biochemistry, physiology, and metabolism of homoarginine, summarize the strengths and weaknesses of the epidemiological evidence linking homoarginine to cardiovascular disease and its potential protective cardiovascular effects, and identify priorities for future research needed to define the clinical utility of homoarginine as a prognostic factor and therapeutic target in cardiovascular disease.

Guanidino compound ratios are associated with stroke etiology, internal carotid artery stenosis and CHA2DS2-VASc score in three cross-sectional studies.

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 < .001) in all three cohorts. 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.

Utility of homoarginine in the GC-MS quantification of asymmetric dimethylarginine (ADMA) in human serum proteins.

NG,NG-Dimethyl-L-arginine (asymmetric dimethylarginine, ADMA) is a cardiovascular risk factor. ADMA circulates in blood as a free acid (fADMA) and as constituent of not yet identified proteins (prADMA). We describe here a protocol for the GC-MS quantification of ADMA released from serum proteins using 6 M HCl (110 °C, 20 h). L-Homoarginine (hArg) is useful in measuring digestibility of amino acids in food proteins. We demonstrate that hArg is not present in human serum proteins and is useful in measuring serum prADMA. The concentration of prADMA in elderly subjects is about 90 nM and the average fADMA/prADMA ratio 6:1.

Cross-Sectional Associations between Homoarginine, Intermediate Phenotypes, and Atrial Fibrillation in the Community-The Gutenberg Health Study.

Homoarginine has come into the focus of interest as a biomarker for cardiovascular disease. Atrial fibrillation (AF) causes a substantial increase in morbidity and mortality. Whether circulating homoarginine is associated with occurrence or persistence of AF and may serve as a new predictive biomarker remains unknown. We measured plasma levels of homoarginine in the population-based Gutenberg health study (3761 patients included, of them 51.7% males), mean age 55.6 ± 10.9 years-old. Associations between homoarginine and intermediate electrocardiographic and echocardiographic phenotypes and manifest AF were examined. Patients with AF (124 patients, of them 73.4% males) had a mean age 64.8 ± 8.6 years-old compared to a mean age of 55.3 ± 10.9 in the population without AF (p-value < 0.001) and showed a less beneficial risk factor profile. The median homoarginine levels in individuals with and without AF were 1.9 µmol/L (interquartile range (IQR) 1.5⁻2.5) and 2.0 µmol/L (IQR 1.5⁻2.5), respectively, p = 0.56. In multivariable-adjusted regression analyses homoarginine was not statistically significantly related to electrocardiographic variables. Among echocardiographic variables beta per standard deviation increase was -0.12 (95% confidence interval (CI) -0.23⁻(-0.02); p = 0.024) for left atrial area and -0.01 (95% CI -0.02⁻(-0.003); p = 0.013) for E/A ratio. The odds ratio between homoarginine and AF was 0.91 (95% CI 0.70⁻1.16; p = 0.45). In our large, population-based cross-sectional study, we did not find statistically significant correlations between lower homoarginine levels and occurrence or persistence of AF or most standard electrocardiographic phenotypes, but some moderate inverse associations with echocardiographic left atrial size and E/A. Homoarginine may not represent a strong biomarker to identify individuals at increased risk for AF. Further investigations will be needed to elucidate the role of homoarginine and cardiac function.