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.

Simultaneous GC-ECNICI-MS measurement of nitrite, nitrate and creatinine in human urine and plasma in clinical settings.

Creatinine in urine is a useful biochemical parameter to correct the urinary excretion rate of endogenous and exogenous substances. Nitrite (ONO-) and nitrate (ONO2-) are metabolites of nitric oxide (NO), a signalling molecule with multiple biological functions. Under certain and standardized conditions, the concentration of nitrate in the urine is a suitable measure of whole body NO synthesis. The urinary nitrate-to-nitrite molar ratio (UNOxR) may indicate nitrite-dependent renal carbonic anhydrase (CA) activity. In clinical studies, urine is commonly collected by spontaneous micturition. In those cases the nitrate and nitrite excretion must be corrected for creatinine excretion. Pentafluorobenzyl (PFB) bromide (PFB-Br) is a useful derivatization reagent of numerous inorganic and organic compounds, including urinary nitrite, nitrate and creatinine, for highly sensitive and specific quantitation by GC-MS. Here, we report on the simultaneous PFB-Br derivatization (60min, 50°C) of ONO-, O15NO-, ONO2-, O15NO2-, creatinine (do-Crea) and [methylo-2H3]creatinine (d3-Crea) in acetonic dilutions of native human urine and plasma samples (4:1, v/v) and their simultaneous quantification by GC-MS as PFBNO2, PFB15NO2, PFBONO2, PFBO15NO2, do-Crea-PFB and d3-Crea-PFB, respectively. Electron capture negative-ion chemical ionization (ECNICI) of these derivatives generates anions due to [M-PFB]-, i.e., the starting analytes. Quantification is performed by selected-ion monitoring (SIM) of m/z 46 (ONO-), m/z 47 (O15NO-), m/z 62 (ONO2-), m/z 63 (O15NO2-), m/z 112 (do-Crea), and m/z 115 (d3-Crea). Retention times were 2.97min for PFB-ONO2/PFB-O15NO2, 3.1min for PFB-NO2/PFB-15NO2, and 6.7min for do-Crea-PFB/d3-Crea-PFB. We used this method to investigate the effects of long-term oral NaNO3 or NaCl (serving as placebo) supplementation (each 0.1mmol/kg body weight per day for 3 weeks) on creatinine excretion and UNOxR in 17 healthy young men. Compared to NaCl (n=8), NaNO3 (n=9) supplementation increased UNOxR (1709±355 vs. 369±77, P<0.05). Creatinine excretion did not differ between the groups (6.67±1.34mM vs. 5.72±1.27mM, P=0.57). The method is also applicable to human plasma. In 78 adults patients newly diagnosed for cerebrovascular disease (CVD), there was a close correlation (r=0.9833) between the creatinine concentrations measured in plasma by GC-ECNICI-MS and those measured in serum by an enzymatic assay. Creatinine-corrected plasma nitrate and nitrite concentrations (P=0.035 and P=0.004, respectively) but not their concentrations (P=0.68 and P=0.40, respectively) differ between male (n=54) and female (n=24) CVD patients. No such differences were found between preterm newborn boys (n=25) and girls (n=22). Like in urine, circulating creatinine may be useful to correct for gender-specific differences in plasma nitrite and nitrate in adults. Chronic NaNO3 supplementation to healthy young men does not affect renal CA-dependent nitrite excretion or creatinine synthesis and excretion.

Simultaneous stable-isotope dilution GC-MS measurement of homoarginine, guanidinoacetate and their common precursor arginine in plasma and their interrelationships in healthy and diseased humans.

Low concentrations of L-homoarginine (hArg) in plasma or serum and urine have recently emerged as a novel cardiovascular risk factor. Previously, we reported gas chromatography-mass spectrometry (GC-MS) and GC-tandem MS (GC-MS/MS) methods for the quantitative determination of hArg and Arg in plasma, serum, urine and other biological samples. In these methods, plasma and serum are ultrafiltered by means of commercially available cartridges (10 kDa), and 10-µL ultrafiltrate aliquots are subjected to a two-step derivatization procedure, yielding the methyl ester tri(N-pentafluoropropionyl) derivatives. De novo prepared trideuteromethyl ester hArg (d3Me-hArg) was used as an internal standard. To make the hArg analysis in plasma more convenient, straightforward and cheaper we performed two key modifications: (1) precipitation of plasma proteins by methanol and (2) use of newly prepared and d3Me-hArg as the internal standard. The method was validated and used for the quantitative determination of hArg in human plasma by GC-MS after electron-capture negative-ion chemical ionization (ECNICI) using methane as the reactant gas. Intra-assay accuracy (recovery) and imprecision (relative standard deviation) were within generally accepted ranges (93-109 and 2.3-10 %, respectively). Furthermore, we extended the applicability of this method to guanidinoacetate (GAA). This is of particular importance because hArg and GAA are produced from Arg by the catalytic action of arginine:glycine amidinotransferase (AGAT) also known as glycine:arginine transamidinase (GATM). Using this method, we quantitated simultaneously hArg, Arg and GAA in the selected-ion monitoring mode in 10-µL aliquots of plasma. In plasma samples of 17 non-medicated healthy young men, the concentration of hArg, GAA and Arg was determined to be (mean ± SD) 1.7 ± 0.6, 2.6 ± 0.8, 91 ± 29 µM, respectively. The correlation between hArg and Arg was borderline (r = 0.47, P = 0.06). GAA strongly correlated with Arg (r = 0.82, P < 0.0001) but did not correlate with hArg (r = 0.17, P = 0.52). The plasma concentrations of hArg, GAA and Arg measured in 9 patients suffering from stroke or transitory ischemic attack were 1.8 ± 0.6, 2.7 ± 0.4 and 82 ± 17 µM. The ratio values of the hArg, GAA and Arg concentrations measured after removal of plasma proteins by methanol precipitation or ultrafiltration were 0.94 ± 0.1, 0.94 ± 0.08, and 0.88 ± 0.07, respectively. Simultaneous measurement of hArg and GAA in human plasma may allow assessment of AGAT activity in vivo with respect both to GAA and to hArg and their relationship in health, disease, nutrition and pharmacotherapy.