Determination of nicotinamide N-oxide—a human excretory product

A method for the determination of nicotinamide N-oxide has been developed. It is based on the ability of the N-oxide to function as an electron acceptor in the xanthine oxidase catalyzed oxidation of xanthine. In simple mixtures the N-oxide can be converted quantitatively to nicotinamide and the latter determined by the cyanogen bromide method. The conversion is not always quantitative in complex mixtures, such as urine; an isotope dilution variation on the basic method permits the determination of the N-oxide in such situations. The basic method is applicable over the range 0.02–0.3 μmole of nicotinamide N-oxide.The new method has been used to verify the prominent excretory role of nicotinamide N-oxide in rodents. Application of the method to a study of human urines has permitted the detection of the N-oxide as an excretory metabolite in man. Only vanishingly small quantities of the N-oxide are excreted under normal conditions. However after the ingestion of 200 mg of nicotinamide, significant quantities of the N-oxide are detectable in human urine. Urine samples obtained from a number of other mammalian species contained little or no detectable nicotinamide N-oxide.     

Protostrychnine,a new alkaloid from Strychnos nux-vomica

The tertiary bases from a sample of Strychnos nux-vomica contain, as well as the expected strychnine and brucine, an unusually high proportion of 4-hydroxy and 4-hydroxy-3-methoxy compounds. The biosynthetic implications of the isolation of a new alkaloid, 12β,13α-dihydro-12α-hydroxyisostrychnine, named protostrychnine, are discussed.     

Liver metabolic reactions: tertiary amine N-dealkylation, tertiary amine N-oxidation, N-oxide reduction, and N-oxide N-dealkylation. II. N,N-dimethylaniline

Rat liver microsomal preparations enzymatically catalyze the N-demethylation and N-oxidation of dimethylaniline as well as the N-demethylation of dimethylaniline-N-oxide. Both compounds were used as substrates and the formation of formaldehyde and N-oxide were determined.Both demethylation and N-oxidation of dimethylaniline are dependent on NADPH. This cofactor also increases the demethylation of dimethylaniline-N-oxide, although it is not an absolute requirement. Nicotinamide increases the rate of formation of formaldehyde and N-oxide from dimethylaniline by a factor of about 4 and decreases the N-oxide demethylation by the same factor. The cofactor optimum consists of NADPH, nicotinamide, and magnesium ions for the demethylation and N-oxidation of dimethylaniline, and of NADPH alone for the demethylation of its N-oxide. The kinetic constants of the three test reactions have been determined under these optimal cofactor requirements.Various agents strongly influence the rates of product formation of the three test reactions studied. SH-blocking agents, the chelating agent EGTA, as well as nicotinamide influence the rates of formaldehyde formation from dimethylaniline and N-oxide demethylation in an opposite way. This demonstrates that, in the tertiary amine demethylation of dimethylaniline, a C-oxidation pathway is operative in addition to an N-oxidation pathway with subsequent N-oxide demethylation. The following influences on the actual metabolic reactions could be deduced from the effects of agents on the test reactions: SKF 525-A inhibits and phenobarbital pretreatment stimulates N-oxide demethylation; EDTA inhibits both the latter reaction and N-oxidation; EGTA and nicotinamide stimulate C-oxidation and inhibit N-oxide demethylation; SH-blocking agents inhibit C-oxidation and stimulate both N-oxidation and N-oxide demethylation.Quantitative and qualitative species differences with respect to cofactor requirement and effect of SKF 525-A have been observed between rat and pig liver microsomes. In addition, profound differences in subcellular localization and metabolic rates between dimethylaniline and other substrates are known. Thus it is unlikely that the three metabolic reactions dealt with in this report are characteristic of tertiarr amine N-dealkylation in general.     

Alkaloids from Melodinus yunnanensis

Ten monoterpenoid indole alkaloids, namely meloyine, 19S-methoxytubotaiwine N₄-oxide, 16,19-epoxy-Δ¹⁴-vincanol, 14β-hydroxymeloyunine, meloyunine, Δ¹⁴-vincamenine N₄-oxide, 16β,21β-epoxy-vincadine, 14β,15β-20S-quebrachamine, 3-oxo-voaphylline, 2α,7α-dihydroxy-dihydrovoaphylline, and 32 known alkaloids were isolated from leaves and twigs of Melodinus yunnanensis. Their structures were elucidated based on 1- and 2-D NMR, FTIR, UV, and MS spectroscopic data. Meloyine I showed weak cytotoxic activity against four human cancer cell lines: MCF-7 breast cancer, SMMC-7721 hepatocellular carcinoma, HL-60 myeloid leukemia, and A-549 lung cancer.     

Quenching of singlet oxygen by alkaloids and related nitrogen heterocycles

Fifteen plant alkaloids and related heterocyclic compounds were tested for their ability to quench singlet oxygen. Most of the compounds showed high activity; brucine and strychnine were especially efficient quenchers. Brucine, at a concentration of ca 2.6 x 10^?5 M, is capable of inactivating half the singlet oxygen molecules it encounters. This quenching may serve in nature to protect plants from the deleterious effects of singlet oxygen or other reactive oxidants.     

Nicotine N-oxides

Nicotine-1-N-oxide, trans and cis isomers of nicotine-1′-N-oxide and of nicotine-1,1′-di-N-oxide have been prepared and characterised by NMR, MS and reduction to nicotine. The trans and cis isomers of nicotine-1′-N-oxide have been identified in leaves, stems and roots of Nicotiana tabacum, N. affinis and N. sylvestris.     

Interactions between Allosteric Modulators and 4-DAMP and Other Antagonists at Muscarinic Receptors: Potential Significance of the Distance between the N and Carboxyl C Atoms in the Molecules of Antagonists

Allosteric enhancement of the affinity of muscarinic receptors for their ligands offers a new way to influence cholinergic neurotransmission. The structure of the allosteric binding domain(s) and the features of agonists, antagonists and modulators which determine the occurrence of either positive or negative cooperativity require clarification. We tested interactions between allosteric modulators alcuronium, strychnine and brucine and eight antagonists at muscarinic receptors expressed in CHO cells. In experiments with unlabeled antagonists, all three modulators enhanced the affinity for 4-diphenylacetoxy-N-dimethylpiperidinium (4-DAMP) at the M₂ receptors, and strychnine did so also at the M₄ receptors. Positive interactions were also observed between alcuronium and L-hyoscyamine (M₂) and scopolamine (M₂), between strychnine and butylscopolamine (M₄), L-hyoscyamine (M₂ and M₄) and scopolamine (M₄), and between brucine and scopolamine (M₂). Positive effects of alcuronium, strychnine and brucine on the affinity of the M₂ receptors for 4-DAMP have been confirmed by direct measurements of the binding of [³H]-4-DAMP. A comparison of molecular models of several antagonists which are esters revealed that antagonists in which the distance between the N and the carboxyl C atoms corresponds to five chemical bonds are more likely to display positive cooperativity with alcuronium at the M₂ receptors than the antagonists in which the N-carboxyl C distance corresponds to four chemical bonds.     

(−)-Epilamprolobine and its N-oxide,lupin alkaloids from Sophora tomentosa

From the fresh leaves of Sophora tomentosa, three new lupin alkaloids, (?)-epilamprolobine, (+)-epilamprolobine N-oxide and 5-(3′-methoxycarbonylbutyroyl)aminomethyl-trans-quinolizidine N-oxide, have further been isolated along with (+)-matrine, (+)-matrine N-oxide, (+)-sophocarpine N-oxide, (?)-anagyrine, (?)- baptifoline, (?)-cytisine, (?)-N-methylcytisine, (?)-N-formylcytisine, (?)-N-acetylcytisine and (±)-ammodendrine. The absolute configurations of (+)-epilamprolobine N-oxide (1R:5R:6S) and (?)-epilamprolobine (5R:6S) have also been established by spectroscopic data and by comparison with synthetic (+)-epilamprolobine (5S:6R)derived from (?)-lupinine (5R:6R). (?)-Epilamprolobine is a diastereomer of (+)-lamprolobine (5R:6R) in Lamprolobium fruticosum and 5-(3′-methoxycarbonylbutyroyl) aminomethyl-trans-quinolizidine N-oxide is presumed to be an artefact. A biosynthetic pathway for the formation of (?)-epilamprolobine is also proposed.     

Hyoscyamine N-oxide in Atropa belladonna

Separated organs of Atropa belladonna have been examined for their total alkaloid, hyoscyamine and hyoscyamine N-oxide contents during ontogenesis. Marked fluctuations in N-oxide content were observed, the highest being found in the ripe fruit. [G-³H]-atropine was fed to A. belladonna fruits and radioactively labelled hyoscyamine N-oxide isolated.     

Ultra-performance liquid chromatography-tandem mass spectrometric method for the determination of strychnine and brucine in mice plasma

A selective, simple and efficient method-ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for determination of two toxic alkaloids, namely strychnine and brucine in mice plasma. The UPLC separation was carried out using a 1.7 μm BEH C(18) column (50 mm × 2.1 mm) with a mobile phase consisting of methanol:0.1% formic acid (25:75, v/v), hence providing high efficiency, high resolution and excellent peak shape for the analytes and internal standard. The method was validated over the range of 2.48-496.4 ng/ml for strychnine and 2.64-528 ng/ml for brucine, respectively. Intra- and inter-day accuracy ranged from 95.0% to 107.9% for strychnine, 93.4% to 103.3% for brucine, and the precisions were within 13.8%. The extraction recoveries of both the two alkaloids exceed 81.9%. With a simple and minor sample preparation procedure and short run-time (<3 min), the proposed method was applicable for the pharmacokinetic and toxicological analysis of strychnine and brucine in vivo.     

Synthesis and characterization of 5-hydroxymethyl-5-methyl-pyrroline N-oxide and its derivatives

Synthesis and spin trapping behavior of three novel DMPO derivatives, namely 5-hydroxymethyl-5-methyl-pyrroline N-oxide (HMMPO), 5-(2-furanyl)-oxymethyl-5-methyl-pyrroline N-oxide (FMMPO), and 5-(2-pyranyl)-oxymethyl-5-methyl-pyrroline N-oxide (PMMPO) towards different oxygen- and carbon-centered radicals are described. The stabilizing effect of a series of cyclodextrins on the superoxide adducts was tested.     

Two simple tetrahydroisoquinoline alkaloid N-oxides from cacti

Tehuanine N-oxide was isolated from Pachycereus pringlei, and deglucopterocereine N-oxide was isolated from Pterocereus gaumeri. These     

(+)-5,17-dehydromatrine N-oxide,a new alkaloid in Euchresta japonica

A new lupin alkaloid, (+)-5,17-dehydromatrine N-oxide, was isolated from the fresh aerial parts of Euchresta japonica. Its structure was confirmed by spectrometric data and by direct comparison with a synthetic sample, prepared from (+)-sophoranol ((+)-5-hydroxymatrine). It was also concluded that (+)-5,17-dehydromatrine N-oxide and (+)-matrine N-oxide possess the same configuration with respect to the asymmetric nitrogen by NMR spectra.     

(+)-5α,9α-dihydroxymatrine,a new lupin alkaloid from Euchresta horsfeldii

A new lupin alkaloid (+)-5α,9α-dihydroxymatrine has been isolated from the leaves of Euchresta horsfeldii (E. formosana cv riukiuensis) together with matrine, matrine N-oxide, sophoranol, sophoranol N-oxide, anagyrine, N-methylcytisine, N-formylcytisine and cytisine.     

Increased sensitivity of xeroderma pigmentosum cells to some chemical carcinogens and mutagens

The clone-forming capacity and level of DNA repair was examined on normal human cells and repair-deficient Xeroderma pigmentosum (XP) fibroblasts exposed to various chemical carcinogens and mutagens.The cultured fibroblasts were treated for 90 min with the carcinogenic and mutagenic 4-nitroquinoline 1-oxide (4NQO), 4-hydroxyaminoquinoline 1-oxide (4HAQO), 2-methyl-4-nitroquinoline 1-oxide (2-Me-4NQO), 3-methyl-4-nitropyridine 1-oxide 3-Me-4NPO) and the non-carcinogenic 6-nitroquinoline 1-oxide (6NQO). The response of the cells to the N-oxides was compared to that induced by the mutagen and carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and UV-irradiation.The XP cells showed (1) a reduced level of DNA repair synthesis when exposed to various carcinogenic N-oxides, (2) no unscheduled DNA synthesis following 6NQO and (3) a normal degree of DNA repair synthesis after treatment with MNNG.When the clone-forming capacity was examined the XP cells exhibited (1) a higher increased sensitivity to the various carcinogenic N-oxides, (2) no reduction in the clone formation following 6NQO and (3) a sensitivity virtually comparable to that of normal cells after treatment with MNNG.The results suggest a link between extent of DNA damage, level of DNA repair and degree of sensitivity in human cells exposed to various chemical carcinogens and which induce DNA alterations that cannot be repaired by DNA repair synthesis.     

Flavin-containing monooxygenase mediated metabolism of benzydamine in perfused brain and liver

Benzydamine (BZY) N-oxidation mediated by flavin-containing monooxygenase (FMO) was evaluated in perfused brain and liver. Following 20 min of perfusion with modified Ringer solution, the infusion of BZY into brain or liver led to production of BZY N-oxide. BZY N-oxide, a metabolite of BZY oxidized exclusively by FMO, was mostly recovered in the effluent without undergoing further metabolism or reduction back to the parent substrate. The BZY N-oxide formation rate increased as the infusion concentration of BZY increased both in perfused brain and perfused liver. BZY N-oxidation activities in perfused rat brain and liver were 4.2 nmol/g brain/min and 50 nmol/g liver/min, respectively, although the BZY N-oxidation activity in brain homogenates was one 4000th that in liver homogenates. This is the first study of FMO activity in brain in situ.     

Novel N-oxide derivatives of benzyltetrahydroisoquinoline alkaloid from the tubers of Stephania viridiflavens H.S. Lo et M. Yang

An investigation on the phytochemistry of the medicinal plant Stephania viridiflavens H.S. Lo et M. Yang led to isolate two new naturally occurring benzyltetrahydroisoquinoline alkaloids, (+)-1S, 2R-laudanidine-N_β-oxide 2 and (+)-1S, 2S-laudanidine-N_α-oxide 3, along with four known benzyltetrahydroisoquinoline alkaloids: (+)-laudanidine 1, (+)-reticuline 4, (+)-1S, 2R-reticuline-N_β-oxide 5 and (+)-1S, 2S-reticuline-N_α-oxide 6. The structure and the stereochemistry of these compounds were determined on the basis of spectroscopic methods and also confirmed by partial synthesis. To examine putative acetycholinesterase (AChE) inhibitory or cytotoxic activities, various bioassays were performed, the N-oxide derivatives (5 and 6) demonstrated more potent cytotoxicity than the corresponding free base.     

N-Demethylation and N-oxidation of imipramine in rat thoracic aortic endothelial cells

The aim of this study was to examine whether cultured rat thoracic aortic endothelial cells (TAECs) have the ability to metabolize the tertiary amine, imipramine. In rat TAECs, imipramine was biotransformed into N-demethylate and N-oxide by cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO), respectively. The intrinsic clearance (V _max/K _m) for the N-oxide formation was approximately five times as high as that for the N-demethylate formation, indicating that oxidation by CYP was much higher than that by FMO. Moreover, we suggest that CYP2C11 and CYP3A2 are key players in the metabolism to N-demethylate in rat TAECs using the respective anti-rat CYP antibodies (anti-CYP2C11 and anti-CYP3A2). The presence of CYP2C11 and CYP3A2 proteins was also confirmed in cultured rat TAECs using a polyclonal anti-CYP antibody and immunofluorescence microscopy. In contrast, the formation rate of N-oxide at pH 8.4 was higher than that at pH 7.4. Inhibition of N-oxide formation by methimazole was found to be the best model of competitive inhibition yielding an apparent K _i value of 0.80 μmol/L, demonstrating that N-oxidation was catalyzed by FMO in rat TAECs. These results suggest that rat TAEC enzymes can convert substrates of exogenous origin such as imipramine, indicating that TAECs have an important function for metabolic products, besides hepatic cells.     

Cytochromes of the trimethylamine N-oxide anaerobic respiratory pathway of Escherichia coli

Escherichia coli grown anaerobically with trimethylamine N-oxide (TMAO) as a terminal electron acceptor develops a new cytochrome pathway in addition to the aerobic respiratory pathways which are still formed. Formate, NADH, and possibly other substrates derived from glucose, supply electrons to this pathway. Cytochromes with α-absorption peaks at about 548, 552, 554 and 557 nm are rapidly reoxidized by TMAO in a reaction which is not inhibited by 2-n-heptyl-4-hydroxyquinoneN-oxide. CuSO₄ inhibits the reoxidation by TMAO of the first two of these cytochromes. This suggests that the pathway of electron transfer leading to the reduction of TMAO is: substrates → cytochromes 548,552 → cytochromes 554,557 → trimethylamine-N-oxide reductase → TMAO. These cytochromes, but not those of the aerobic respiratory pathways, are reoxidized by the membrane-impermeant oxidant ammonium persulfate in intact cells. This suggests that the cytochromes of the TMAO reduction pathway and / or trimethylamine-N-oxide reductase are situated at the periplasmic surface of the cytoplasmic membrane of E. coli.     

Lupin alkaloids from sophora chrysophylla

A new lupin alkaloid, (?)-mamanine N-oxide, was isolated from Sophora chrysophylla together with 18 known alkaloids including some unusual lupin alkaloids such as kuraramine, lamprolobine, epilamprolobine, epilamprolobine N-oxide, (+)-mamanine and (?)-pohakuline. It was also shown that the alkaloid constituents of S. chrysophylla differed considerably in the leaves, stems and seeds.