Synthesis of homoagmatine and GC-MS analysis of tissue homoagmatine and agmatine: evidence that homoagmatine but not agmatine is a metabolite of pharmacological L-homoarginine in the anesthetized rat.

Low L-homoarginine (hArg) concentrations in human blood and urine are associated with renal and cardiovascular morbidity and mortality, yet the underlying mechanisms and the biological activities of hArg are elusive. In humans and rats, hArg is metabolized to L-lysine. The aim of the present work was to study hArg metabolism to agmatine (Agm) and homoagmatine (hAgm) in the anesthetized rat. Using a newly developed and validated GC-MS method and a newly synthesized and structurally characterized hAgm we investigated the metabolism of i.p. administered hArg (0, 20, 220, 440 mg/kg) to hAgm and Agm in lung, kidney, liver and heart in anesthetized rats. Our study provides unequivocal evidence that hArg is metabolized to hAgm but not to Agm. Whether hAgm derived from hArg's metabolism may contribute to the pathophysiological significance of endogenous hArg and for the favoured effects of pharmacological hArg remains to be demonstrated. The biology of hArg warrants further investigations.

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.

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.

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.

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.

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.

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.

Development and validation of GC-MS methods for the comprehensive analysis of amino acids in plasma and urine and applications to the HELLP syndrome and pediatric kidney transplantation: evidence of altered methylation, transamidination, and arginase activity.

We developed and validated gas chromatography-mass spectrometry (GC-MS) methods for the simultaneous measurement of amino acids and their metabolites in 10-µL aliquots of human plasma and urine. De novo synthesized trideutero-methyl esters were used as internal standards. Plasma proteins were precipitated by acidified methanol and removed by centrifugation. Supernatants and native urine were evaporated to dryness. Amino acids were first esterified using 2 M HCl in methanol and then amidated using pentafluoropropionic anhydride for electron-capture negative-ion chemical ionization. Time programmes were used for the gas chromatograph oven and the selected-ion monitoring of specific anions. The GC-MS methods were applied in clinical studies on the HELLP syndrome and pediatric kidney transplantation (KTx) focusing on L-arginine-related pathways. We found lower sarcosine (N-methylglycine) and higher asymmetric dimethylarginine (ADMA) plasma concentrations in HELLP syndrome women (n = 7) compared to healthy pregnant women (n = 5) indicating altered methylation. In plasma of pediatric KTx patients, lower guanidinoacetate and homoarginine concentrations were found in plasma but not in urine samples of patients treated with standard mycophenolate mofetil-based immunosuppression (MMF; n = 22) in comparison to matched patients treated with MMF-free immunosuppression (n = 22). On average, the global arginine bioavailability ratio was by about 40% lower in the MMF group compared to the EVR group (P = 0.004). Mycophenolate, the major pharmacologically active metabolite of MMF, is likely to inhibit the arginine:glycine amidinotransferase (AGAT), and to enhance arginase activity in leukocytes and other types of cell of MMF-treated children.

Asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and homoarginine (hArg): the ADMA, SDMA and hArg paradoxes.

NG-Methylation of L-arginine (Arg) residues in certain proteins by protein arginine methyltransferases and subsequent proteolysis yields NG-monomethyl-L-arginine (MMA), NG,NG-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA) and NG,N'G-dimethyl-L-arginine (symmetric dimethylarginine, SDMA). Biological MMA, ADMA and SDMA occur as free acids in the nM-range and as residues of proteins of largely unknown quantity. Arginine:glycine amidinotransferase (AGAT) catalyzes the synthesis of L-homoarginine (hArg) from free Arg and L-lysine. Biological hArg is considered to occur exclusively as free acid in the lower µM-range. Nitric oxide synthase (NOS) catalyzes the conversion of Arg (high affinity) and hArg (low affinity) to nitric oxide (NO) which is a pleiotropic signaling molecule. MMA, ADMA and SDMA are inhibitors (MMA > ADMA â‰« SDMA) of NOS activity. Slightly elevated ADMA and SDMA concentrations and slightly reduced hArg concentrations in the circulation are associated with many diseases including diabetes mellitus. Yet, this is paradox: (1) free ADMA and SDMA are weak inhibitors of endothelial NOS (eNOS) which is primarily responsible for NO-related effects in the cardiovascular system, with free hArg being a poor substrate for eNOS; (2) free ADMA, SDMA and hArg are not associated with oxidative stress which is considered to induce NO-related endothelial dysfunction. This ADMA/SDMA/hArg paradox may be solved by the assumption that not the free acids but their precursor proteins exert biological effects in the vasculature, with hArg antagonizing the effects of NG-methylated proteins.

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.

GC-MS quantification of urinary symmetric dimethylarginine (SDMA), a whole-body symmetric l-arginine methylation index.

Circulating and excretory NG,N´G-dimethyl-l-arginine (symmetric dimethylarginine, SDMA) and NG,NG-dimethyl-l-arginine (asymmetric dimethylarginine, ADMA) are cardiovascular risk factors. Despite close chemical structures, the gas chromatography-mass spectrometry (GC-MS) measurement of SDMA is remarkably more difficult than that of ADMA for as yet unknown reasons. Here, we describe an improved GC-MS method for the quantitative determination of SDMA in human urine using commercially available NG,N´G-di-[2H3]methyl-l-arginine (d6-SDMA) as internal standard. The method is based on a single derivatization step with pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C) to N,N,N,O-tetrakis-pentafluoropropionyl derivatives, electron-capture negative-ion chemical ionization and selected-ion monitoring of the mass-to-charge (m/z) ions of m/z 456 for SDMA and m/z 462 for d6-SDMA.

GC-MS determination of nitrous anhydrase activity of bovine and human carbonic anhydrase II and IV.

The most widely recognized activity of the large family of the metalloenzyme carbonic anhydrases (CAs) is the diffusion-controlled hydration of CO2 to HCO3- and one proton, and the less rapid dehydration of HCO3- to CO2: CO2 + H2O ⇆ HCO3- + H+. CAs also catalyze the reaction of water with other electrophiles such as aromatic esters, sulfates and phosphates, thus contributing to lending to CAs esterase, sulfatase and phosphatase activity, respectively. Renal CAII and CAIV are involved in the reabsorption of nitrite, the autoxidation product of the signalling molecule nitric oxide (NO): 4 NO + O2 + 2 H2O → 4 ONO- + 4 H+. Bovine and human CAII and CAIV have been reported to exert nitrite reductase and nitrous anhydride activity: 2 NO2- + 2 H+ ⇆ [2 HONO] ⇆ N2O3 + H2O. In the presence of L-cysteine, NO may be formed. In the literature, these issues are controversial, mainly due to analytical shortcomings, i.e., the inability to detect authentic HONO and N2O3. Here, we present a gas chromatography-mass spectrometry (GC-MS) assay to unambiguously detect and quantify the nitrous anhydrase activity of CAs. The assay is based on the hydrolysis of N2O3 in H218O to form ON18O- and 18ON18O-. After pentafluorobenzyl bromide derivatization and electron capture negative-ion chemical ionization of the pentafluorobenzyl nitro derivatives, quantification is performed by selected-ion monitoring of the anions with mass-to-charge (m/z) ratios of 46 (ONO-), m/z 48 (ON18O- and 18ONO-), m/z 50 (18ON18O-) and m/z 47 (O15NO-, internal standard).

Effects of single and combined metformin and L-citrulline supplementation on L-arginine-related pathways in Becker muscular dystrophy patients: possible biochemical and clinical implications.

The L-arginine/nitric oxide synthase (NOS) pathway is considered to be altered in muscular dystrophy such as Becker muscular dystrophy (BMD). We investigated two pharmacological options aimed to increase nitric oxide (NO) synthesis in 20 male BMD patients (age range 21-44 years): (1) supplementation with L-citrulline (3 × 5 g/d), the precursor of L-arginine which is the substrate of neuronal NO synthase (nNOS); and (2) treatment with the antidiabetic drug metformin (3 × 500 mg/d) which activates nNOS in human skeletal muscle. We also investigated the combined use of L-citrulline (3 × 5 g/d) and metformin (3 × 500 mg/d). Before and after treatment, we measured in serum and urine samples the concentration of amino acids and metabolites of L-arginine-related pathways and the oxidative stress biomarker malondialdehyde (MDA). Compared to healthy subjects, BMD patients have altered NOS, arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) pathways. Metformin treatment resulted in concentration decrease of arginine and MDA in serum, and of homoarginine (hArg) and guanidinoacetate (GAA) in serum and urine. L-Citrulline supplementation resulted in considerable increase of the concentrations of amino acids and creatinine in the serum, and in their urinary excretion rates. Combined use of metformin and L-citrulline attenuated the effects obtained from their single administrations. Metformin, L-citrulline or their combination did not alter serum nitrite and nitrate concentrations and their urinary excretion rates. In conclusion, metformin or L-citrulline supplementation to BMD patients results in remarkable antidromic changes of the AGAT and GAMT pathways. In combination, metformin and L-citrulline at the doses used in the present study seem to abolish the biochemical effects of the single drugs in slight favor of L-citrulline.

Results, meta-analysis and a first evaluation of UNOxR, the urinary nitrate-to-nitrite molar ratio, as a measure of nitrite reabsorption in experimental and clinical settings.

We recently found that renal carbonic anhydrase (CA) is involved in the reabsorption of inorganic nitrite (NO2-), an abundant reservoir of nitric oxide (NO) in tissues and cells. Impaired NO synthesis in the endothelium and decreased NO bioavailability in the circulation are considered major contributors to the development and progression of renal and cardiovascular diseases in different conditions including diabetes. Isolated human and bovine erythrocytic CAII and CAIV can convert nitrite to nitrous acid (HONO) and its anhydride N2O3 which, in the presence of thiols (RSH), are further converted to S-nitrosothiols (RSNO) and NO. Thus, CA may be responsible both for the homeostasis of nitrite and for its bioactivation to RSNO/NO. We hypothesized that enhanced excretion of nitrite in the urine may contribute to NO-related dysfunctions in the renal and cardiovascular systems, and proposed the urinary nitrate-to-nitrite molar ratio, i.e., UNOxR, as a measure of renal CA-dependent excretion of nitrite. Based on results from clinical and experimental animal studies, here, we report on a first evaluation of UNOxR. We determined UNOxR values in preterm neonates, healthy children, and adults, in children suffering from type 1 diabetes mellitus (T1DM) or Duchenne muscular dystrophy (DMD), in elderly subjects suffering from chronic rheumatic diseases, type 2 diabetes mellitus (T2DM), coronary artery disease (CAD), or peripheral arterial occlusive disease (PAOD). We also determined UNOxR values in healthy young men who ingested isosorbide dinitrate (ISDN), pentaerythrityl tetranitrate (PETN), or inorganic nitrate. In addition, we tested the utility of UNOxR in two animal models, i.e., the LEW.1AR1-iddm rat, an animal model of human T1DM, and the APOE*3-Leiden.CETP mice, a model of human dyslipidemia. Mean UNOxR values were lower in adult patients with rheumatic diseases (187) and in T2DM patients of the DALI study (74) as compared to healthy elderly adults (660) and healthy young men (1500). The intra- and inter-variabilities of UNOxR were of the order of 50% in young and elderly healthy subjects. UNOxR values were lower in black compared to white boys (314 vs. 483, P = 0.007), which is in line with reported lower NO bioavailability in black ethnicity. Mean UNOxR values were lower in DMD (424) compared to healthy (730) children, but they were higher in T1DM children (1192). ISDN (3 × 30 mg) decreased stronger UNOxR compared to PETN (3 × 80 mg) after 1 day (P = 0.046) and after 5 days (P = 0.0016) of oral administration of therapeutically equivalent doses. In healthy young men who ingested NaNO3 (0.1 mmol/kg/d), UNOxR was higher than in those who ingested the same dose of NaCl (1709 vs. 369). In LEW.1AR1-iddm rats, mean UNOxR values were lower than in healthy rats (198 vs. 308) and comparable to those in APOE*3-Leiden.CETP mice (151).

Metabolism and distribution of pharmacological homoarginine in plasma and main organs of the anesthetized rat.

L-Homoarginine (hArg) and guanidinoacetate (GAA) are produced from L-arginine (Arg) by the catalytic action of arginine:glycine amidinotransferase. Guanidinoacetate methyltransferase methylates GAA on its non-guanidine N atom to produce creatine. Arg and hArg are converted by nitric oxide synthase (NOS) to nitric oxide (NO). NO is oxidized to nitrite and nitrate which circulate in the blood and are excreted in the urine. Asymmetric dimethylarginine (ADMA), an NOS inhibitor, is widely accepted to be exclusively produced after asymmetric N G-methylation of Arg residues in proteins and their regular proteolysis. Low circulating and urinary hArg concentrations and high circulating concentrations of ADMA emerged as risk markers in the human renal and cardiovascular systems. While ADMA's distribution and metabolism are thoroughly investigated, such studies on hArg are sparse. The aim of the present pilot study was to investigate the distribution of exogenous hArg in plasma, liver, kidney, lung, and heart in a rat model of takotsubo cardiomyopathy (TTC). hArg hydrochloride solutions in physiological saline were injected intra-peritoneally at potentially pharmacological, non-toxic doses of 20, 220, or 440 mg/kg body weight. Vehicle (saline) served as control. As hArg has been reported to be a pro-oxidant, plasma and tissue malondialdehyde (MDA) was measured as a biomarker of lipid peroxidation. hArg administration resulted in dose-dependent maximum plasma hArg concentrations and distribution in all investigated organs. hArg disappeared from plasma with an elimination half-life ranging between 20 and 40 min. hArg administration resulted in relatively small changes in the plasma and tissue content of Arg, GAA, ADMA, creatinine, and of the NO metabolites nitrite and nitrate. Remarkable changes were observed for tissue GAA, notably in the kidney. Plasma and tissue MDA concentration did not change upon hArg administration, suggesting that even high-dosed hArg is not an oxidant. The lowest hArg dose of 20 mg/kg bodyweight increased 25-fold the mean hArg maximum plasma concentration. This hArg dose seems to be useful as the upper limit in forthcoming studies on the putative cardioprotective effects of hArg in our rat model of TTC.

Comprehensive analysis of the L-arginine/L-homoarginine/nitric oxide pathway in preterm neonates: potential roles for homoarginine and asymmetric dimethylarginine in foetal growth.

L-Arginine (Arg) and L-homoarginine (hArg) are precursors of nitric oxide (NO), a signalling molecule with multiple important roles in human organism. In the circulation of adults, high concentrations of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) and low concentrations of hArg emerged as cardiovascular risk factors. Yet, the importance of the Arg/hArg/NO pathway, especially of hArg and ADMA, in preterm neonates is little understood. We comprehensively investigated the Arg/hArg/NO pathway in 106 healthy preterm infants (51 boys, 55 girls) aged between 23 + 6 and 36 + 1 gestational weeks. Babies were divided into two groups: group I consisted of 31 babies with a gestational age of 23 + 6 - 29 + 6 weeks; group II comprised 75 children with a gestational age of 30 + 0 - 36 + 1 weeks. Plasma and urine concentrations of ADMA, SDMA, hArg, Arg, dimethylamine (DMA) which is the major urinary ADMA metabolite, as well as of nitrite and nitrate, the major NO metabolites, were determined by GC-MS and GC-MS/MS methods. ADMA and hArg plasma levels, but not the hArg/ADMA molar ratio, were significantly higher in group II than in group I: 895 ± 166 nM vs. 774 ± 164 nM (P = 0.001) for ADMA and 0.56 ± 0.04 µM vs. 0.48 ± 0.08 µM (P = 0.010) for hArg. There was no statistical difference between the groups with regard to urinary ADMA (12.2 ± 4.6 vs 12.8 ± 3.6 µmol/mmol creatinine; P = 0.61) and urinary SDMA. Urinary hArg, ADMA, SDMA correlated tightly with each other. Urinary excretion of DMA was slightly higher in group I compared to group II: 282 ± 44 vs. 247 ± 35 µmol/mmol creatinine (P = 0.004). The DMA/ADMA molar ratio in urine was tendentiously higher in neonates of group I compared to group II: 27 ± 13 vs. 20 ± 5 (P = 0.065). There were no differences between the groups with respect to Arg in plasma and to nitrite and nitrate in plasma and urine. In preterm neonates, ADMA and hArg biosynthesis increases with gestational age without remarkable changes in the hArg/ADMA ratio or NO biosynthesis. Our study suggests that ADMA and hArg are involved in foetal growth.

The role of L-arginine/L-homoarginine/nitric oxide pathway for aortic distensibility and intima-media thickness in stroke patients.

Asymmetric dimethylarginine (ADMA) and L-homoarginine (hArg) are L-arginine (Arg) metabolites derived from different pathways. Protein arginine N-methyltransferase (PRMT) and subsequent proteolysis of proteins containing methylarginine residues release ADMA. Arginine:glycine amidinotransferase (AGAT) converts Arg to hArg and guanidinoacetate (GAA). While high concentrations of ADMA and low concentrations of hArg in the blood have been established as cardiovascular risk markers, the cardiovascular relevance of GAA is still unexplored. Arg and hArg are substrates and ADMA is an inhibitor of nitric oxide (NO) synthase (NOS). The cardiovascular effects of ADMA and hArg have been related to NO, a potent endogenous vasodilator. ADMA and hArg are considered to exert additional, not yet explored, presumably NO-unrelated effects and to act antagonistically in the renal and cardiovascular systems. Although the physiological role of Arg, ADMA, hArg and NO for endothelial function in small- and medium-sized arteries has been intensively studied in the past, the clinical relevance of aortic wall remodeling still remains unclear. Here, we evaluated potential relation between aortic distensibility (AD) or aortic intima-media thickness (aIMT) and circulating ADMA, hArg, GAA, and the NO metabolites nitrite and nitrate in the plasma of 78 patients (24 females, 54 males; aged 59 ± 14 years) with recent ischemic stroke or transient ischemic attack (TIA). All biochemical parameters were determined by stable-isotope dilution gas chromatography-mass spectrometry. AD and aIMT were measured by transesophageal echocardiography. Arg, hArg, ADMA and GAA median plasma concentrations (µM) were determined to be 61, 1.43, 0.50 and 2.16, respectively. hArg, ADMA and GAA correlated closely with Arg. Nitrite, nitrate and creatinine median plasma concentrations (µM) were 2.49, 48.7, and 84.1, respectively. Neither AD (2.61 vs. 1.85 10-6 × cm2 × dyn-1, P = 0.064) nor aIMT (1.25 vs. 1.13 mm, P = 0.596) differed between females and males. The hArg/ADMA molar ratio (r = -0.351, P = 0.009), nitrate (r = 0.364, P = 0.007) and nitrite (r = 0.329, P = 0.015) correlated with aIMT but not with AD. Arg, hArg, ADMA and GAA correlated with aIMT but not with AD. The results demonstrate a strong relation between the Arg/NO pathway and aortic atherosclerosis but not with AD suggesting different mechanisms underlying the two aspects of aortic wall remodeling.

Low plasma homoarginine concentration is associated with high rates of all-cause mortality in renal transplant recipients.

In renal transplant recipients (RTR), we recently found that low urinary excretion of homoarginine (hArg) is associated with mortality and graft failure. However, it is not known whether such prospective associations also hold true for plasma concentrations of hArg. In the present study, we therefore determined plasma concentrations of hArg in the same cohort, i.e. in 687 RTR (functioning graft ≥1 year), and in 140 healthy donors, before and after kidney donation. Plasma hArg concentrations were significantly lower in RTR compared to healthy controls [1.24 (0.95-1.63) µM vs. 1.58 (1.31-2.03) µM, P < 0.001], and kidney donation resulted in a decrease in plasma hArg concentration to 1.41 (1.10-1.81) µM (P < 0.001). In RTR, multivariable linear regression analysis revealed BMI (ß = 0.124), heart rate (ß = -0.091), pre-emptive transplantation (ß = 0.078), antidiabetic medication (ß = -0.091), eGFR (ß = 0.272), plasma PTH (ß = -0.098), uric acid (ß = 0.137), alkaline phosphatase (ß = -0.100), HDL (ß = -0.111), NT-pro-BNP (ß = -0.166), and urinary urea excretion (ß = 0.139) as main determinants of plasma hArg (all P < 0.05). In RTR, plasma hArg concentration was inversely associated with all-cause [hazard ratio (HR) 0.59 (95% CI 0.50-0.70), P < 0.001] and cardiovascular mortality [HR 0.50 (0.39-0.66), P < 0.001], both expressed per standard deviation change in log-transformed hArg, independent of potential confounders. To conclude, our results suggest that the kidney is a major hArg production site and an important modulator of hArg homeostasis in the renal and cardiovascular systems. Moreover, low plasma hArg is independently associated with increased risk of cardiovascular mortality in RTR, which corroborates the cardiovascular importance of preserving kidney function after transplantation.

Gas chromatographic-mass spectrometric analysis of the tripeptide glutathione in the electron-capture negative-ion chemical ionization mode.

The dicarboxylic tripeptide glutathione (GSH) is the most abundant intracellular thiol. GSH analysis by liquid chromatography is routine. Yet, GSH analysis by gas chromatography is challenged due to thermal instability and lacking volatility. We report a high-yield laboratory method for the preparation of (2)H-labeled GSH dimethyl ester ((d3Me)2-GSH) for use as internal standard (IS) which was characterized by LC-MS/MS. For GC-MS analysis, the dimethyl esters of GSH and the IS were derivatized with pentafluoropropionic (PFP) anhydride. Electron-capture negative-ion chemical ionization of the (Me)2-(PFP)3-GSH provided high sensitivity. We encourage increasing use of GC-MS in the analysis of amino acids as their Me-PFP derivatives in the ECNICI mode.

Discovery and microassay of a nitrite-dependent carbonic anhydrase activity by stable-isotope dilution gas chromatography-mass spectrometry.

The intrinsic activity of carbonic anhydrase (CA) is the hydration of CO2 to carbonic acid and its dehydration to CO2. CA may also function as esterase and phosphatase. Recently, we demonstrated that renal CA is mainly responsible for the reabsorption of nitrite (NO2(-)) which is the most abundant reservoir of the biologically highly potent nitric oxide (NO). By means of a stable-isotope dilution GC-MS method, we discovered a novel CA activity which strictly depends upon nitrite. We found that bovine erythrocytic CAII (beCAII) catalyses the incorporation of (18)O from H2 (18)O into nitrite at pH 7.4. After derivatization with pentafluorobenzyl bromide, gas chromatographic separation and mass spectrometric analysis, we detected ions at m/z 48 for singly (18)O-labelled nitrite ((16)O=N-(18)O(-)/(18)O=N-(16)O(-)) and at m/z 50 for doubly (18)O-labelled nitrite ((18)O=N-(18)O(-)) in addition to m/z 46 for unlabelled nitrite. Using (15)N-labelled nitrite ((15)NO2 (-), m/z 47) as an internal standard and selected-ion monitoring of m/z 46, m/z 48, m/z 50 and m/z 47, we developed a GC-MS microassay for the quantitative determination of the nitrite-dependent beCAII activity. The CA inhibitors acetazolamide and FC5 207A did not alter beCAII-catalysed formation of singly and doubly (18)O-labelled nitrite. Cysteine and the experimental CA inhibitor DIDS (a diisothiocyanate) increased several fold the beCAII-catalysed formation of the (18)O-labelled nitrite species. Cysteine, acetazolamide, FC5 207A, and DIDS by themselves had no effect on the incorporation of (18)O from H2 (18)O into nitrite. We conclude that erythrocytic CA possesses a nitrite-dependent activity which can only be detected when nitrite is used as the substrate and the reaction is performed in buffers of neutral pH values prepared in H2 (18)O. This novel CA activity, i.e., the nitrous acid anhydrase activity, represents a bioactivation of nitrite and may have both beneficial (via S-nitrosylation and subsequent NO release) and possibly adverse (via C- and N-nitrosylation) effects in living organisms.