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.     

Killing of Bacillus spores is mediated by nitric oxide and nitric oxide synthase during glycoconjugate–enhanced phagocytosis

Nitric oxide (NO) is a signaling and defense molecule of major importance. NO endows macrophages with bactericidal, cytostatic as well as cytotoxic activity against various pathogens. Bacillus spores can produce serious diseases, which might be attenuated if macrophages were able to kill the spores on contact. Present research was carried out to study whether glycoconjugates stimulated NO and nitric oxide synthase (NOS2) production during phagocytosis killing of Bacillus spores. Murine macrophages exposed to glycoconjugate-treated spores induced NOS2 and NO production that was correlated with high viability of macrophages and killing rate of bacterial spores. Increased levels of inducible NOS2 and NO production by macrophages in presence of glycoconjugates suggested that the latter provide an activation signal directed to macrophages. Glycoconjugates were shown to exert a protective influence, sparing macrophages from spore-induced cell death. In presence of glycoconjugates, macrophages efficiently kill the organisms. Without glycoconjugate activation, murine macrophages were ineffective at killing Bacillus spores. These results suggest that glycoconjugates promote killing of Bacillus spores by blocking spore-induced macrophage cell death, while increasing their activation level and NO and NOS2 production. Glycoconjugates suggest novel antimicrobial approaches to prevention and treatment of infection caused by bacterial spores.     

Chronic administration of a nitric oxide synthase inhibitor,Nω-nitro-l-arginine,and drug-induced increase in cerebellar cyclic GMP in vivo

N-nitro-l-arginine (N^G-nitro-l-arginine) is a potent nitric oxide synthase inhibitor which crosses the blood brain barrier and does not undergo extensive metabolism in vivo. In this study, effect of chronic pretreatment of N-nitro-l-arginine (75 mg/kg, i.p., twice daily for 7 days) on the harmaline- (100 mg/kg, s.c.), picrotoxin- (4 mg/kg, s.c.), pentylenetetrazole- (50 mg/kg, i.p.), andl-glutamic acid- (400 g/10 l/mouse, i.c.v.) induced increase in cerebellar cGMP was assessed. All the four drugs produced significant increase in cerebellar cGMP in vehicle pretreated control animals. Cerebellar cGMP increase induced by harmaline, picrotoxin, andl-glutamic acid was attentuated in N-nitro-l-arginine pretreated animals. These results indicate that in vivo cerebellar cGMP levels are increased by the prototype excitatory amino acid receptor agonist,l-glutamic acid and also by the drugs which augment the excitatory amino acid transmission. Furthermore, parenteral chronic administration of N-nitro-l-arginine blocks NO synthase in the brain and hence cerebellar cGMP response in chronic N-nitro-l-arginine treated animals could be used as a tool to assess the physiological functions of nitric oxide in vivo.Part of this work was presented at the Experimental Biology 93 FASEB Meeting at New Orleans, March 1993.     

Inducible nitric oxide synthase inhibitor aminoguanidine, differently affects Morris water maze tasks of ovariectomized and naïve female rats

The role of ovarian hormones, nitric oxide, and their interaction on learning and memory has been widely investigated. The objective of present study was to investigate different effects of chronic administration of inducible nitric oxide synthase inhibitor, aminoguanidine (AM) on learning and memory of ovariectomized (OVX) and na?ve (Sham) female rats. Thirty-two rats were divided into four groups: 1) Sham, 2) OVX, 3) Sham-AM and 4) OVX-AM. The animals of Sham-AM and OVX-AM chronically received 100 mg/kg/day of aminoguanidine during 8 weeks before 5 test days. The animals in Sham and OVX groups received 1 ml/kg saline instead of aminoguanidine. The animals were tested in Morris water maze and the escape latency and traveled path to reach the platform were compared between groups. On the fifth day, the platform was removed, and the animals were allowed to swim for 60 s ( prob trial). The time spent in the target quadrant (Q1) was compared between groups.Results showed that the escape latency and traveled path in OVX group were significantly higher than in the Sham group (p<0.01). Both escape latency and traveled path in the Sham-AM group was significantly higher than in the Sham group (p<0.01) however, there was no significant difference between OVX-AM and OVX groups.The time spent by the animals of OVX group in the target quadrant (Q1) during the probe trial was significantly lower than that in the Sham group (p<0.01). The animals of the Sham-AM group spent shorter times in the target quadrant in comparison with the Sham group (p<0.01). There was no significant difference between the OVX and OVX-AM groups in the time spent in tarthe get quadrant. It is concluded that the effect of aminoguanidine on learning and memory is different in the presence or absence of ovarian hormones but it needs further investigation.     

Synthesis,nitric oxide release,and α-glucosidase inhibition of nitric oxide donating apigenin and chrysin derivatives

α-Glucosidase (AG) play crucial roles in the digestion of carbohydrates. Inhibitors of α-glucosidase (AGIs) are promising candidates for the development of anti-diabetic drugs. Here, five series of apigenin and chrysin nitric oxide (NO)-donating derivatives were synthesised and evaluated for their AG inhibitory activity and NO releasing capacity in vitro. Except for 9a–c, twelve compounds showed remarkable inhibitory activity against α-glucosidase, with potency being better than that of acarbose and 1-deoxynojirimycin. All organic nitrate derivatives released low concentrations of NO in the presence of l-cysteine. Structure activity relationship studies indicated that 5-OH, hydrophobic coupling chain, and carbonyl groups of the coupling chain could enhance the inhibitory activity. Apigenin and chrysin derivatives therefore represents a new class of promising compounds that can inhibit α-glucosidase activity and supply moderate NO for preventing the development of diabetic complications.     

Nitric oxide and cytochrome oxidase: substrate,inhibitor or effector?

Endogenously produced nitric oxide (NO) controls oxygen consumption by inhibiting cytochrome c oxidase, the terminal electron acceptor of the mitochondrial electron transport chain. The oxygen-binding site of the enzyme is an iron/copper (haem a3/CuB) binuclear centre. At high substrate (ferrocytochrome c) concentrations, NO binds reversibly to the reduced iron in competition with oxygen. At low substrate concentrations, NO binds to the oxidized copper. Inhibition at the haem iron site is relieved by dissociation of the NO from the reduced iron. Inhibition at the copper site is relieved by oxidation of the bound NO and subsequent dissociation of nitrite from the enzyme. Therefore, NO can be a substrate, inhibitor or effector of cytochrome oxidase, depending on cellular conditions.     

Nitric oxide control of cardiac function: is neuronal nitric oxide synthase a key component?

Nitric oxide (NO) has been shown to regulate cardiac function, both in physiological conditions and in disease states. However, several aspects of NO signalling in the myocardium remain poorly understood. It is becoming increasingly apparent that the disparate functions ascribed to NO result from its generation by different isoforms of the NO synthase (NOS) enzyme, the varying subcellular localization and regulation of NOS isoforms and their effector proteins. Some apparently contrasting findings may have arisen from the use of non-isoform-specific inhibitors of NOS, and from the assumption that NO donors may be able to mimic the actions of endogenously produced NO. In recent years an at least partial explanation for some of the disagreements, although by no means all, may be found from studies that have focused on the role of the neuronal NOS (nNOS) isoform. These data have shown a key role for nNOS in the control of basal and adrenergically stimulated cardiac contractility and in the autonomic control of heart rate. Whether or not the role of nNOS carries implications for cardiovascular disease remains an intriguing possibility requiring future study.     

Activation of macrophage nuclear factor-κB and induction of inducible nitric oxide synthase by LPS

Background  

Chronic lung disease (CLD) of prematurity is a major problem of neonatal care. Bacterial infection and inflammatory response have been thought to play an important role in the development of CLD and steroids have been given, with some benefit, to neonates with this disease. In the present study, we assessed the ability of lipopolysaccharide (LPS) to stimulate rat alveolar macrophages to produce nitric oxide (NO), express inducible nitric oxide synthase (iNOS) and activate nuclear factor-κB (NF-κB) in vitro. In addition, we investigated the impact of dexamethasone and budesonide on these processes.     

Preliminary screening of Houttuynia cordata for biological potential and chemical components using TLC and GC–MS analysis

Journal of Plant Biochemistry and Biotechnology - The objective of the study is to ascertain the biological potential and to identify the bioactive compounds of the four crude extract hexane,...     

Neuronal nitric oxide synthase immunopositive neurons in cat claustrum—a light and electron microscopic study

Nitric oxide is a unique neurotransmitter, which participates in many physiological and pathological processes in the organism. Nevertheless there are little data about the neuronal Nitric Oxide Synthase immunoreactive (nNOS-ir) neurons and fibers in the dorsal claustrum (DC) of a cat. In this respect the aims of this study were: (1) to demonstrate nNOS-ir in the neurons and fibers of the DC; (2) to describe their light microscopic morphology and distribution; (3) to investigate and analyze the ultrastructure of the nNOS-ir neurons, fibers and synaptic terminals; (4) to verify whether the nNOS-ir neurons consist a specific subpopulation of claustral neurons; (5) to verify whether the nNOS-ir neurons have a specific pattern of organization throughout the DC. For demonstration of the nNOS-ir the Avidin-Biotin-Peroxidase Complex method was applied. Immunopositive for nNOS neurons and fibers were present in all parts of DC. On the light microscope level nNOS-ir neurons were different in shape and size. According to the latter they were divided into three groups-small (with diameter under 15 mum), medium-sized (with diameter from 16 to 20 mum) and large (with diameter over 21 mum). Some of nNOS-ir neurons were lightly-stained while others were darkly-stained. On the electron microscope level the immunoproduct was observed in neurons, dendrites and terminal boutons. Different types of nNOS-ir neurons differ according to their ultrastructural features. Three types of nNOS-ir synaptic boutons were found. As a conclusion we hope that the present study will contribute to a better understanding of the functioning of the DC in cat and that some of the data presented could be extrapolated to other mammals, including human.     

Metabolomic and elemental analysis of camel and bovine urine by GC–MS and ICP–MS

Recent studies from the author’s laboratory indicated that camel urine possesses antiplatelet activity and anti-cancer activity which is not present in bovine urine. The objective of this study is to compare the volatile and elemental components of bovine and camel urine using GC–MS and ICP–MS analysis. We are interested to know the component that performs these biological activities. The freeze dried urine was dissolved in dichloromethane and then derivatization process followed by using BSTFA for GC–MS analysis. Thirty different compounds were analyzed by the derivatization process in full scan mode. For ICP–MS analysis twenty eight important elements were analyzed in both bovine and camel urine. The results of GC–MS and ICP–MS analysis showed marked difference in the urinary metabolites. GC–MS evaluation of camel urine finds a lot of products of metabolism like benzene propanoic acid derivatives, fatty acid derivatives, amino acid derivatives, sugars, prostaglandins and canavanine. Several research reports reveal the metabolomics studies on camel urine but none of them completely reported the pharmacology related metabolomics. The present data of GC–MS suggest and support the previous studies and activities related to camel urine.     

Synthesis of biflavones having a 6-O-7’’ linkage and effects on cyclooxygenase-2 and inducible nitric oxide synthase

In order to establish anti-inflammatory potential of biflavonoids, 17 biflavone derivatives having a 6-O-7″ linkage were synthesized and their effects on cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were evaluated. The basic molecule (6-O-7″ biflavone) potently inhibited COX-2-mediated PGE₂ production (IC₅₀: < 2 μM), being less active on iNOS-mediated NO production (IC₅₀: > 50 μM) from lipopolysaccharide-treated RAW 264.7 cells, a mouse macrophage cell line. Generally, the hydroxyl/methoxyl substitution(s) on the basic biflavone (6-O-7″) reduced the inhibitory activity of PGE₂ production, while the effects on NO production were varied. It is suggested that the basic biflavone (6-O-7″) may have a potential for new anti-inflammatory agent.     

NG-methylarginine, an inhibitor of endothelium-derived nitric oxide synthesis, is a potent pressor agent in the guinea pig: does nitric oxide regulate blood pressure in vivo?

Nitric oxide is a major endothelium-derived vascular smooth muscle relaxing factor; its synthesis from L-arginine is selectively inhibited by L-NG-methylarginine. To assess whether basal nitric oxide release contributes to blood pressure regulation in vivo, we have investigated the cardiovascular effects of L-NG-methylarginine in the anesthetized guinea pig. L-NG-methylarginine (0.1-10 mg/kg, i.v. bolus) elicited a sustained, dose-dependent, increase in arterial pressure and a moderate bradycardia. L-arginine (30 mg/kg i.v.) prevented or reversed the pressor effect of L-NG-methylarginine, while atropine (2 mg/kg) abolished the associated bradycardia. In contrast, L-arginine did not attenuate the pressor effect of norepinephrine or angiotensin. Our findings suggest that basal nitric oxide production is sufficient to modulate peripheral vascular resistance; hence nitric oxide may play a role in arterial pressure homeostasis.     

Comparative metabolite profiling and fingerprinting of medicinal licorice roots using a multiplex approach of GC–MS,LC–MS and 1D NMR techniques

Glycyrrhiza glabra, commonly known as licorice, is a popular herbal supplement used for the treatment of chronic inflammatory conditions and possesses anticancer and antiviral activities. This species contains a plethora of phytochemicals including terpenoids, saponins, flavonoids, polyamines and polysaccharides. The full complement of bioactive compounds has yet to be elucidated, a step necessary in order to explain its medicinal use. There are over 30 species in the Glycyrrhiza genus world-wide, most of which have been little characterized in terms of phytochemical or pharmacological properties. Here, large scale multi-targeted metabolic profiling and fingerprinting techniques were utilized to help gain a broader insight into Glycyrrhiza species chemical composition. UV, MS and NMR spectra of extracted components were connected with NMR, MS, and multivariate analyses data from Glycyrrhiza glabra, Glycyrrhiza uralensis, Glycyrrhiza inflata and Glycyrrhiza echinata. Major peaks in ¹H NMR and MS spectra contributing to the discrimination among species were assigned as those of glycyrrhizin, 4-hydroxyphenyl acetic acid, and glycosidic conjugates of liquiritigenin/isoliquiritigenin. Primary metabolites profiling using GC–MS revealed the presence of cadaverine, an amino acid, exclusively found in G. inflata roots. Both LC–MS and NMR were found effective techniques in sample classification based on genetic and or geographical origin as revealed from derived PCA analysis.     

Reversal of cyanide inhibition of cytochrome c oxidase by the auxiliary substrate nitric oxide: an endogenous antidote to cyanide poisoning?

Nitric oxide (NO) is shown to overcome the cyanide inhibition of cytochrome c oxidase in the presence of excess ferrocytochrome c and oxygen. Addition of NO to the partially reduced cyanide-inhibited form of the bovine enzyme is shown by electron paramagnetic resonance spectroscopy to result in substitution of cyanide at ferriheme a3 by NO with reduction of the heme. The resulting nitrosylferroheme a3 is a 5-coordinate structure, the proximal bond to histidine having been broken. NO does not simply act as a reversibly bound competitive inhibitor but is an auxiliary substrate consumed in a catalytic cycle along with ferrocytochrome c and oxygen. The implications of this observation with regard to estimates of steady-state NO levels in vivo is discussed. Given the multiple sources of NO available to mitochondria, the present results appear to explain in part some of the curious biomedical observations reported by other laboratories; for example, the kidneys of cyanide poisoning victims surprisingly exhibit no significant irreversible damage, and lethal doses of potassium cyanide are able to inhibit cytochrome c oxidase activity by only approximately 50% in brain mitochondria.     

De novo design of transmembrane helix–helix interactions and measurement of stability in a biological membrane

Membrane proteins regulate a large number of cellular functions, and have great potential as tools for manipulation of biological systems. Developing these tools requires a robust and quantitative understanding of membrane protein folding and interactions within the bilayer. With this in mind, we have designed a series of proteins to probe the net thermodynamic contribution of well-known sequence motifs to transmembrane helix-helix association in a biological membrane. The proteins were designed from first principles (de novo) using current knowledge about membrane insertion and stabilizing interaction motifs. A simple poly-Leu “scaffold” was decorated with individual helix interaction motifs (G-XXX-G, polar residues, heptad repeat) to create transmembrane helix–helix interactions of increasing strength. The GALLEX assay, an in vivo assay for measurement of transmembrane helix self-association, was combined with computational methods to characterize the relative strength and mode of interaction for each sequence. In addition, the apparent free energy contribution (ΔΔG^app) of each motif to transmembrane helix self-association was measured in a biological membrane, results that are the first of their kind for these de novo designed sequences, and suggest that the free energy barrier to overcoming weak association is quite small (< 1.4 kcal mol^− 1) in a natural membrane. By quantifying and rationalizing the contribution of key motifs to transmembrane helix association, our work offers a route to direct the design of novel sequences for use in biotechnology or synthetic biology (e.g. molecular switches) and to predict the effects of sequence modification in known transmembrane domains (for control of cellular processes).     

Enzyme–substrate complexes of allosteric citrate synthase: Evidence for a novel intermediate in substrate binding

The citrate synthase (CS) of Escherichia coli is an allosteric hexameric enzyme specifically inhibited by NADH. The crystal structure of wild type (WT) E. coli CS, determined by us previously, has no substrates bound, and part of the active site is in a highly mobile region that is shifted from the position needed for catalysis. The CS of Acetobacter aceti has a similar structure, but has been successfully crystallized with bound substrates: both oxaloacetic acid (OAA) and an analog of acetyl coenzyme A (AcCoA). We engineered a variant of E. coli CS wherein five amino acids in the mobile region have been replaced by those in the A. aceti sequence. The purified enzyme shows unusual kinetics with a low affinity for both substrates. Although the crystal structure without ligands is very similar to that of the WT enzyme (except in the mutated region), complexes are formed with both substrates and the allosteric inhibitor NADH. The complex with OAA in the active site identifies a novel OAA-binding residue, Arg306, which has no functional counterpart in other known CS-OAA complexes. This structure may represent an intermediate in a multi-step substrate binding process where Arg306 changes roles from OAA binding to AcCoA binding. The second complex has the substrate analog, S-carboxymethyl-coenzyme A, in the allosteric NADH-binding site and the AcCoA site is not formed. Additional CS variants unable to bind adenylates at the allosteric site show that this second complex is not a factor in positive allosteric activation of AcCoA binding.