Biosynthesis of homoarginine (hArg) and asymmetric dimethylarginine (ADMA) from acutely and chronically administered free <Emphasis Type="SmallCaps">l</Emphasis>-arginine in humans

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthesis, whereas l-arginine (Arg) and l-homoarginine (hArg) serve as substrates for NO synthesis. ADMA and other methylated arginines are generally believed to exclusively derive from guanidine (N ^G)-methylated arginine residues in proteins by protein arginine methyltransferases (PRMTs) that use S-adenosylmethionine (SAM) as the methyl donor. l-Lysine is known for decades as a precursor for hArg, but only recent studies indicate that arginine:glycine amidinotransferase (AGAT) is responsible for the synthesis of hArg. AGAT catalyzes the formation of guanidinoacetate (GAA) that is methylated to creatine by guanidinoacetate methyltransferase (GAMT) which also uses SAM. The aim of the present study was to learn more about the mechanisms of ADMA and hArg formation in humans. Especially, we hypothesized that ADMA is produced by N ^G-methylation of free Arg in addition to the known PRMTs-involving mechanism. In knockout mouse models of AGAT- and GAMT-deficiency, we investigated the contribution of these enzymes to hArg synthesis. Arg infusion (0.5 g/kg, 30 min) in children (n = 11) and ingestion of high-fat protein meals by overweight men (n = 10) were used to study acute effects on ADMA and hArg synthesis. Daily Arg ingestion (10 g) or placebo for 3 or 6 months by patients suffering from peripheral arterial occlusive disease (PAOD, n = 20) or coronary artery disease (CAD, n = 30) was used to study chronic effects of Arg on ADMA synthesis. Mass spectrometric methods were used to measure all biochemical parameters in plasma and urine samples. In mice, AGAT but not GAMT was found to contribute to plasma hArg, while ADMA synthesis was independent of AGAT and GAMT. Arg infusion acutely increased plasma Arg, hArg and ADMA concentrations, but decreased the plasma hArg/ADMA ratio. High-fat protein meals acutely increased plasma Arg, hArg, ADMA concentrations, as well as the plasma hArg/ADMA ratio. In the PAOD and CAD studies, plasma Arg concentration increased in the verum compared to the placebo groups. Plasma ADMA concentration increased only in the PAOD patients who received Arg. Our study suggests that in humans a minor fraction of free Arg is rapidly metabolized to ADMA and hArg. In mice, GAMT and N ^G-methyltransferases contribute to ADMA and hArg synthesis from Arg, whereas AGAT is involved in the synthesis of hArg but not of ADMA. The underlying biochemical mechanisms remain still elusive.     

Effects of sex and estrous cycle on the brain and plasma arginine metabolic profile in rats

l-arginine is a versatile amino acid with a number of bioactive metabolites. Increasing evidence implicates altered arginine metabolism in the aging and neurodegenerative processes. The present study, for the first time, determined the effects of sex and estrous cycle on the brain and blood (plasma) arginine metabolic profile in naïve rats. Female rats displayed significantly lower levels of l-arginine in the frontal cortex and three sub-regions of the hippocampus when compared to male rats. Moreover, female rats had significantly higher levels of l-arginine and γ-aminobutyric acid, but lower levels of l-ornithine, agmatine and putrescine, in plasma relative to male rats. The observed sex difference in brain l-arginine appeared to be independent of the enzymes involved in its metabolism, de novo synthesis and blood-to-brain transport (cationic acid transporter 1 protein expression at least), as well as circulating l-arginine. While the estrous cycle did not affect l-arginine and its metabolites in the brain, there were estrous cycle phase-dependent changes in plasma l-arginine. These findings demonstrate the sex difference in brain l-arginine in the estrous cycle-independent manner. Since peripheral blood has been increasingly used to identify biomarkers of brain pathology, the influences of sex and estrous cycle on blood arginine metabolic profile need attention when experimental research involves female rodents.

     

Semi-rational design of L-amino acid deaminase for production of pyruvate and d-alanine by Escherichia coli whole-cell biocatalyst

In our previous study, one-step pyruvate and d-alanine production from d,l-alanine by a whole-cell biocatalyst Escherichia coli expressing l-amino acid deaminase (Pm1) derived from Proteus mirabilis was investigated. However, due to the low catalytic efficiency of Pm1, the pyruvate titer was relatively low. Here, semi-rational design based on site-directed saturation mutagenesis was carried out to improve the catalytic efficiency of Pm1. A novel high-throughput screening (HTS) method for pyruvate based on 2,4-dinitrophenylhydrazine indicator was then established. The catalytic efficiency (k_cat/K_m) of the mutant V437I screened out by this method was 1.88 times higher than wild type. Next, to improve the growth of the engineered strain BLK07, the genes encoding for Xpk and Fbp were integrated into its genome to construct non-oxidative glycolysis (NOG) pathway. Finally, the CRISPR/Cas9 system was used to integrate the N6-pm1-V437I gene into the genome of BLK07. Pyruvic acid titer of the plasmid-free strain reached 42.20 g/L with an l-alanine conversion rate of 77.62% and a d-alanine resolution of 82.4%. This work would accelerate the industrial production of pyruvate and d-alanine by biocatalysis, and the HTS method established here could be used to screen other Pm1 mutants with high pyruvate titers.

     

Effect of l-Carnosine as adjunctive therapy in the management of children with autism spectrum disorder: a randomized controlled study

l-Carnosine is an amino acid that acts as an anti-oxidant, anti-toxic and neuroprotective agent. There is a paucity of data about the effectiveness of l-Carnosine in the management of autism spectrum disorder (ASD) in children. This study aimed at investigating the effectiveness of l-Carnosine as adjunctive therapy in the management of ASD. This was a randomized controlled trial. Children aged 3–6 years with a diagnosis of mild to moderate ASD were assigned to standard care arm (occupational and speech therapy) and intervention care arm (l-Carnosine, 10–15 mg/kg in 2 divided doses) plus standard care treatment. The children were assessed at the baseline and the end of 2 months for the scores of Childhood Autism Rating Scale, Second Edition—Standard Version (CARS2-ST), Autism Treatment Evaluation Checklist (ATEC), BEARS sleep screening tool and 6-item Gastrointestinal Severity Index (6-GSI). Of the sixty-seven children enrolled, sixty-three children had completed the study. No statistically significant difference (p > 0.05) was observed for any of the outcome measures assessed. Supplementation of l-Carnosine did not improve the total score of CARS2-ST, ATEC, BEARS sleep screening tool and 6-GSI scores of children with ASD. Further investigations are needed with more objective assessments to critically validate the effectiveness of l-Carnosine on ASD children for more decisive results.

     

First enzymological characterization of selenocysteine β-lyase from a lactic acid bacterium,Leuconostoc mesenteroides

We succeeded in expressing selenocysteine β-lyase (SCL) from a lactic acid bacterium, Leuconostoc mesenteroides LK-151 (Lm-SCL), in the soluble fractions of Escherichia coli Rosetta (DE3) using a novel expression vector of pET21malb constructed by ourselves that has both maltose binding protein (MBP)- and 6?×?His-tag. Lm-SCL acted on l-selenocysteine, l-cysteine, and l-cysteine sulfinic acid but showed a high preference for l-selenocysteine. The k_cat and k_cat/K_m values of Lm-SCL were determined to be 108 (min^?1) and 42.0 (min^?1?mM^?1), respectively, and this was enough catalytic efficiency to suggest that Lm-SCL might also be involved in supplying elemental selenium from l-selenocysteine to selenoproteins like other SCLs. The optimum temperature and optimum pH of Lm-SCL were determined to be 37 °C and pH 6.5, respectively. Lm-SCL was stable at 37–45 °C and pH 6.5–7.5. Lm-SCL was completely inhibited by the addition of hydroxylamine, semicarbazide, and iodoacetic acid. The enzyme activity of Lm-SCL was decreased in the presence of various metal ions, especially Cu²⁺. The quaternary structure of Lm-SCL is a homodimer with a subunit molecular mass of 47.5 kDa. The similarity of the primary structure of Lm-SCL to other SCLs from Citrobacter freundii, Escherichia coli, humans, or mouse was calculated to be 47.0, 48.0, 12.5, or 24.0%, respectively. Unlike Ec-SCL, our mutational and molecular docking simulation studies revealed that C362 of Lm-SCL might also catalyze the deselenation of l-selenocysteine in addition to the desulfuration of l-cysteine.

     

l-Arginine increases AMPK phosphorylation and the oxidation of energy substrates in hepatocytes,skeletal muscle cells,and adipocytes

Previous work has shown that dietary l-arginine (Arg) supplementation reduced white fat mass in obese rats. The present study was conducted with cell models to define direct effects of Arg on energy-substrate oxidation in hepatocytes, skeletal muscle cells, and adipocytes. BNL CL.2 mouse hepatocytes, C2C12 mouse myotubes, and 3T3-L1 mouse adipocytes were treated with different extracellular concentrations of Arg (0, 15, 50, 100 and 400 µM) or 400 µM Arg?+?0.5 mM N^G-nitro-l-arginine methyl ester (L-NAME; an NOS inhibitor) for 48 h. Increasing Arg concentrations in culture medium dose-dependently enhanced (P?<?0.05) the oxidation of glucose and oleic acid to CO₂ in all three cell types, lactate release from C2C12 cells, and the incorporation of oleic acid into esterified lipids in BNL CL.2 and 3T3-L1 cells. Arg at 400 µM also stimulated (P?<?0.05) the phosphorylation of AMP-activated protein kinase (AMPK) in all three cell types and increased (P?<?0.05) NO production in C2C12 and BNL CL.2 cells. The inhibition of NOS by L-NAME moderately reduced (P?<?0.05) glucose and oleic acid oxidation, lactate release, and the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) in BNL CL.2 cells, but had no effect (P?>?0.05) on these variables in C2C12 or 3T3-L1 cells. Collectively, these results indicate that Arg increased AMPK activity and energy-substrate oxidation in BNL CL.2, C2C12, and 3T3-L1 cells through both NO-dependent and NO-independent mechanisms.

     

Anti-inflammatory effect of l-cysteine (a semi-essential amino acid) on 5-FU-induced oral mucositis in hamsters

Oral mucositis is an inflammation of the oral mucosa mainly resulting from the cytotoxic effect of 5-fluorouracil (5-FU). The literature shows anti-inflammatory action of l-cysteine (l-cys) involving hydrogen sulfide (H₂S). In view of these properties, we investigate the effect of l-cys in oral mucositis induced by 5-FU in hamsters. The animals were divided into the following groups: saline 0.9%, mechanical trauma, 5-FU 60–40 mg/kg, l-cys 10/40 mg and NaHS 27 µg/kg. 5-FU was administered on days 1st to 2nd; 4th day excoriations were made on the mucosa; 5th–6th received l-cys and NaHS. For data analysis, histological analyses, mast cell count, inflammatory and antioxidants markers, and immunohistochemistry (cyclooxygenase-2(COX-2)/inducible nitric oxide synthase (iNOs)/H₂S) were performed. Results showed that l-cys decreased levels of inflammatory markers, mast cells, levels of COX-2, iNOS and increased levels of antioxidants markers and H₂S when compared to the group 5-FU (p < 0.005). It is suggested that l-cys increases the H₂S production with anti-inflammatory action in the 5-FU lesion.

     

Comparative evaluation of Aspergillus niger strains for endogenous pectin-depolymerization capacity and suitability for d-galacturonic acid production

Pectinaceous agricultural residues rich in d-galacturonic acid (d-GalA), such as sugar beet pulp, are considered as promising feedstocks for waste-to-value conversions. Aspergillus niger is known for its strong pectinolytic activity. However, while specialized strains for production of citric acid or proteins are well characterized, this is not the case for the production of pectinases. We, therefore, systematically compared the pectinolytic capabilities of six A. niger strains (ATCC 1015, ATCC 11414, NRRL 3122, CBS 513.88, NRRL 3, and N402) using controlled batch cultivations in stirred-tank bioreactors. A. niger ATCC 11414 showed the highest polygalacturonase activity, specific protein secretion, and a suitable morphology. Furthermore, d-GalA release from sugar beet pulp was 75% higher compared to the standard lab strain A. niger N402. Our study, therefore, presents a robust initial strain selection to guide future process improvement of d-GalA production from agricultural residues and identifies a high-performance base strain for further genetic optimizations.

     

Design of peptidase‐resistant peptide inhibitors of myosin light chain kinase

Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility including smooth muscle contraction, cell migration, cytokinesis, receptor capping, secretion, etc. Inhibition of MLCK activity in endothelial and epithelial monolayers using cell‐permeant peptide Arg‐Lys‐Lys‐Tyr‐Lys‐Tyr‐Arg‐Arg‐Lys (PIK, P eptide I nhibitor of K inase) allows protecting the barrier capacity, suggesting a potential medical use of PIK. However, low stability of L ‐PIK in a biological milieu prompts for development of more stable L ‐PIK analogues for use as experimental tools in basic and drug‐oriented biomedical research. Previously, we designed PIK1, H‐(N^αMe)Arg‐Lys‐Lys‐Tyr‐Lys‐Tyr‐Arg‐Arg‐Lys‐NH₂, that was 2.5‐fold more resistant to peptidases in human plasma in vitro than L ‐PIK and equal to it as MLCK inhibitor. In order to further enhance proteolytic stability of PIK inhibitor, we designed the set of six site‐protected peptides based on L ‐PIK and PIK1 degradation patterns in human plasma as revealed by ¹H‐NMR analysis. Implemented modifications increased half‐live of the PIK‐related peptides in plasma about 10‐fold, and these compounds retained 25–100% of L ‐PIK inhibitory activity toward MLCK in vitro. Based on stability and functional activity ranking, PIK2, H‐(N^αMe)Arg‐Lys‐Lys‐Tyr‐Lys‐Tyr‐Arg‐D ‐Arg‐Lys‐NH₂, was identified as the most stable and effective L ‐PIK analogue. PIK2 was able to decrease myosin light chain phosphorylation in endothelial cells stimulated with thrombin, and this effect correlated with the inhibition by PIK2 of thrombin‐induced endothelial hyperpermeability in vitro. Therefore, PIK2 could be used as novel alternative to other cell‐permeant inhibitors of MLCK in cell culture‐based and in vivo studies where MLCK catalytic activity inhibition is required. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

A conservative distribution of tridomain NDP-heptose synthetases in actinobacteria

Heptoses are important structural components of Gram-negative bacterium cell wall and participate in bacterial colonization, infection, and immune recognition. Current knowledge of NDP-heptose originating from d-sedoheptulose 7-phosphate in Grampositive bacterium remains limited. Here, in silico analysis suggested that the special tridomain NDP-heptose synthetases with isomerase, kinase, and nucleotidyltransferase activities are conservatively distributed in Actinobacteria class of Gram-positive bacterium. Enzymatical characterization of the tridomain proteins from different strains showed that they are involved in ADP-d-glycero-β-d-manno-heptose biosynthesis despite the unexpected discovery of kinase activities deficient in some proteins. The presence of three types of NDP-heptose synthetases in Gram-positive bacterium suggests that it is also a rich source of heptoses and the heptose moieties may play important roles in vivo. Our work updates the understanding of NDP-heptose biosynthesis in Gram-positive bacterium and lays a solid foundation for further physiological function explorations.

     

Synthesis of novel sugar derived aziridines,as starting materials giving access to sugar amino acid derivatives

d-Erythrosyl aziridines were obtained from d-erythrosyl triazoles either by photolysis or through diazirine intermediates. These were found to undergo rich, high yielding chemistry by reaction with protic acids (HCl, BiI₃/H₂O and trifluoroacetic acid) leading to two types of furanoid sugar α-amino acids, and polyhydroxylprolines. Based on experimental evidence, reaction mechanisms have been proposed for the syntheses.

     

Physicochemical properties of l- and dl-valine: first-principles calculations

At present, physicochemical properties of amino acid molecular crystals are of the utmost interest. The compounds where molecules have different chirality are the focus of particular interest. This paper, presents a study on the structural and electronic properties of crystalline l- and dl-valine within the framework of density functional theory including van der Waals interactions. The results of this study showed that electronic properties of the two forms of valine are similar at zero pressure. Pressure leads to different responses in these crystals which is manifested as various deformations of molecules. The pressure effect on the infrared spectra and distribution of electron density of l- and dl-valine has been studied.

     

The Interactive Approach of Rhizobacteria and l-tryptophan on Growth,Physiology, Tuber Characteristics,and Iron Concentration of Potato (Solanum tuberosum L.)

Iron deficiency is one of the most prevailing micronutrient deficiencies throughout the globe. Iron malnutrition affects billions of people around the world especially children and pregnant women. Its deficiencies can be overcome through microbial biofortification: a process of deliberately increasing desirable nutrients in crop plants. Plant growth-promoting rhizobacteria (PGPR) can improve iron content in edible plant tissues through different direct and indirect mechanisms. Adding plant growth regulators along with rhizobacteria makes it a novel fortification approach. In the current experiment, the interactive effect of two bacterial isolates (O-13 & K-10) alone and in consortium with l-tryptophan in the presence of iron sulfate was evaluated on growth, physiology, tuber characteristics, and iron concentration in potato (Solanum tuberosum L.). Results revealed that inoculation with PGPR and plant growth regulator (PGR) significantly improved the plant height, straw yield, and the number of tubers per plant. Potato (Solanum tuberosum L.) tuber characteristics (starch content, vitamin-C, relative water content) were also improved significantly. O-13, K-10, and l-tryptophan had significantly improved the iron concentration up to 20.59, 33.12, and 28.95%, respectively. However, inoculation with the microbial consortium and l-tryptophan showed a significant increase of up to one-fold in the iron concentration of potato (Solanum tuberosum L.) as compared with uninoculated control. The results suggest that rhizobacteria can help the plant to uptake nutrients from the soil. These findings concluded on the fact that the interactive effect of microbial assisted biofortification and plant growth regulator is a novel, promising, and cost-effective approach to mitigate micronutrient deficiencies especially in resource-limited countries.

     

Simultaneous determination of l-tryptophan impurities in meat products

L-tryptophan has been used as a feed additive for swine and poultry and as a nutrient supplement for humans. However, some impurities in l-tryptophan have been reported as causative components of eosinophilia-myalgia syndrome. Therefore, from a safety perspective, it is important to analyze meat samples for these impurities. This study aims to develop an analytical method for the simultaneous detection of l-tryptophan impurities in meat products using LC–MS/MS. Among the various impurities, detection methods for (S)-2-amino-3-(5-hydroxy-1H-indol-3-yl)propanoic acid (5-hydroxytryptophan) (HTP), 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (MTCA), 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo-[2,3-b]-indole-2-carboxylic acid (PIC), and 1,1′-ethylidenebistryptophan (EBT) and 2-(3-indoylmethyl)-l-tryptophan (IMT) were developed. The developed method allowed simultaneous determination of these four impurities in 5 min. No interferences from the matrix were observed, and the method showed good sensitivity to each analyte. The method detection limit and limit of quantification in meat matrices were below 11.2 and 35.7 μg/kg, respectively.

     

Colorimetric recognition of aromatic amino acid enantiomers by gluconic acid-capped gold nanoparticles

In this work, we prepared gold nanoparticles (AuNPs) by employing gluconic acid (GlcA) as reducing-cum-stabilizing agent. The proposed GlcA-AuNPs successfully worked as a colorimetric sensor for visual chiral recognition of aromatic amino acid enantiomers, namely tyrosine (d/l-Tyr), phenylalanine (d/l-Phe), and tryptophan (d/l-Trp). After adding L-types to GlcA-AuNPs solution, the color of the mixture changed from red to purple (or gray), while no obvious color change occurred on the addition of D-types. The effect can be detected by naked eyes. The particles have been characterized by transmission electron microscopy, Fourier-transform infrared spectroscopy, zeta potential, the dynamic light scattering analysis as well as UV–Vis spectroscopy. This assay can be used to determine the enantiomeric excess of l-Trp in the range from 0 to + 100%. The method has advantages in simplicity, sensitivity, fast response, and low cost.

     

Efficient strategies to enhance plasmid stability for fermentation of recombinant Escherichia coli harboring tyrosine phenol lyase

Objective

To solve the bottleneck of plasmid instability during microbial fermentation of l-DOPA with recombinant Escherichia coli expressing heterologous tyrosine phenol lyase.

Results

The tyrosine phenol lyase from Fusobacterium nucleatum was constitutively expressed in E. coli and a fed-batch fermentation process with temperature down-shift cultivation was performed. Efficient strategies including replacing the original ampicillin resistance gene, as well as inserting cer site that is active for resolving plasmid multimers were applied. As a result, the plasmid stability was increased. The co-use of cer site on plasmid and kanamycin in culture medium resulted in proportion of plasmid containing cells maintained at 100% after fermentation for 35 h. The specific activity of tyrosine phenol lyase reached 1493 U/g dcw, while the volumetric activity increased from 2943 to 14,408 U/L for l-DOPA biosynthesis.

Conclusions

The established strategies for plasmid stability is not only promoted the applicability of the recombinant cells for l-DOPA production, but also provides important guidance for industrial fermentation with improved microbial productivity.

     

Depletion of L-arginine induces autophagy as a cytoprotective response to endoplasmic reticulum stress in human T lymphocytes

L-arginine (L-Arg) deficiency results in decreased T-cell proliferation and impaired T-cell function. Here we have found that L-Arg depletion inhibited expression of different membrane antigens, including CD247 (CD3ζ), and led to an ER stress response, as well as cell cycle arrest at G₀/G₁ in both human Jurkat and peripheral blood mitogen-activated T cells, without undergoing apoptosis. By genetic and biochemical approaches, we found that L-Arg depletion also induced autophagy. Deprivation of L-Arg induced EIF2S1 (eIF2α), MAPK8 (JNK), BCL2 (Bcl-2) phosphorylation, and displacement of BECN1 (Beclin 1) binding to BCL2, leading to autophagosome formation. Silencing of ERN1 (IRE1α) prevented the induction of autophagy as well as MAPK8 activation, BCL2 phosphorylation and XBP1 splicing, whereas led T lymphocytes to apoptosis under L-Arg starvation, suggesting that the ERN1-MAPK8 pathway plays a major role in the activation of autophagy following L-Arg depletion. Autophagy was required for survival of T lymphocytes in the absence of L-Arg, and resulted in a reversible process. Replenishment of L-Arg made T lymphocytes to regain the normal cell cycle profile and proliferate, whereas autophagy was inhibited. Inhibition of autophagy by ERN1, BECN1 and ATG7 silencing, or by pharmacological inhibitors, promoted cell death of T lymphocytes incubated in the absence of L-Arg. Our data indicate for the first time that depletion of L-Arg in T lymphocytes leads to a reversible response that preserves T lymphocytes through ER stress and autophagy, while remaining arrested at G₀/G₁. Our data also show that the L-Arg depletion-induced ER stress response could lead to apoptosis when autophagy is blocked.     

GC–MS and GC–MS/MS measurement of the cardiovascular risk factor homoarginine in biological samples

l-Homoarginine (hArg) has recently emerged as a novel cardiovascular risk factor and to herald a poor prognosis in heart failure patients. Here, we report on the development and thorough validation of gas chromatography–mass spectrometry (GC–MS) and gas chromatography–tandem mass spectrometry (GC–MS/MS) methods for the quantitative determination of hArg in biological samples, including human plasma, urine and sputum. For plasma and serum samples, ultrafiltrate (10 µL; cutoff, 10 kDa) was used. For urine samples, native urine (10 µL) was used. For sputum, protein precipitation by acetone was performed. hArg is derivatized to its methyl ester tri(N-pentafluoropropionyl) derivative; de novo synthesized trideutero-methyl ester hArg is used as the internal standard (IS). Alternatively, [guanidino-¹⁵N₂]-arginine can be used as an IS. Quantitative analyses were performed after electron-capture negative-ion chemical ionization by selected-ion monitoring in GC–MS and selected-reaction monitoring in GC–MS/MS. We obtained very similar hArg concentrations by GC–MS and GC–MS/MS, suggesting that GC–MS suffices for accurate and precise quantification of hArg in biological samples. In plasma and serum samples of the same subjects very close hArg concentrations were measured. The plasma-to-serum hArg concentration ratio was determined to be 1.12 ± 0.21 (RSD, 19 %), suggesting that blood anticoagulation is not a major preanalytical concern in hArg analysis. In healthy subjects, the creatinine-corrected urinary excretion of hArg varies considerably (0.18 ± 0.22 µmol/mmol, mean ± SD, n = 19) unlike asymmetric dimethylarginine (ADMA, 2.89 ± 0.89 µmol/mmol). In urine, hArg correlated with ADMA (r = 0.475, P = 0.040); in average, subjects excreted in the urine about 17.5 times more ADMA than hArg. In plasma of healthy humans, the concentration of hArg is of the order of 2 µM. hArg may be a low-abundance constituent of human plasma proteins. The GC–MS and GC-MS/MS methods we report in this article are useful to study the physiology and pathology of hArg in experimental and clinical settings.     

A higher plant enzyme exhibiting broad acceptance of stereoisomers

An arginase, purified from the leaf of the jack bean, Canavalia ensiformis, can effectively hydrolyze both l- and d-arginine. Arginases, examined from a number of other plant and animal sources, exhibit marked substrate stereospecificity and fail to catabolize d-arginine. In order to provide essential nitrogen, jack bean leaf arginase also catabolizes l-canavanine, an arginine analog that is a predominant nitrogen-storing metabolite of this legume. The ability of arginase to metabolize both stereoisomers of arginine may result from the requirement for this enzyme to exhibit limited substrate specificity in order to hydrolyze both arginine and canavanine.