Efficient nitroso group transfer from N-nitrosoindoles to nucleotides and 2'-deoxyguanosine at physiological pH. A new pathway for N-nitrosocompounds to exert genotoxicity.
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The endogenous formation of N-nitrosoindoles is of concern since humans are exposed to a variety of naturally occurring and synthetic indolic compounds. As part of a study to evaluate the genotoxicity of N-nitrosoindoles, the reactions of three model compounds with purine nucleotides and 2'-deoxyguanosine at physiological pH were investigated. The profiles of reaction products were identical for each of the N-nitrosoindoles and three distinct pathways of reaction could be discerned. These pathways were: (i) depurination to the corresponding purine bases, (ii) deamination, coupled with depurination, to give hypoxanthine and xanthine, and (iii) formation of the novel nucleotide 2'-deoxyoxanosine monophosphate and its corresponding depurination product oxanine in reactions with 2'-deoxyguanosine monophosphate. 2'-Deoxyoxanosine and oxanine were observed in reactions with 2'-deoxyguanosine. Further studies showed that formation of all of these products could be rationalized by an initial transnitrosation step. These results suggest that, in contrast to many other genotoxic N-nitrosocompounds which are known to alkylate DNA, the genotoxicity of N-nitrosoindoles is likely to arise through transfer of the nitroso group to nucleophilic sites on the purine bases. All of the products resulting from transnitrosation by N-nitrosoindoles are potentially mutagenic. These findings reveal a new pathway for N-nitrosocompounds to exert genotoxicity. (+info)
Studies of structure and mechanism in acetonitrile chemical ionization tandem mass spectrometry of polyunsaturated fatty acid methyl esters.
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Recently it has been shown that acetonitrile chemical ionization tandem mass spectrometry (CI-MS/MS) is a rapid, on-line means to determine double bond position in fatty acid methyl esters (FAME). The mechanism of this gas phase condensation reaction has been studied. Evidence of the (1-methyleneimino)-1-ethenylium ion (m/z 54), formed upon the reaction of acetonitrile with itself, adding across the double bond in a [2 + 2] cycloaddition reaction is observed. When this nascent complex undergoes collision-induced dissociation, two diagnostic ions emerge. One of these ions results from loss of the hydrocarbon end of the FAME, whereas the other ion results from loss of the methyl ester end, and when considered together, the diagnostic ions localize the positions of the double bonds in the FAME. Several labeling and MS/MS/MS experiments on the two diagnostic ions were performed to determine a plausible fragmentation mechanism of the stable (1-methyleneimino)-1-ethenylium-FAME complex. The first generation product ions, or diagnostic ions, appear to be formed though a charge-driven mechanism, whereas the second generation product ions are formed via charge-remote fragmentations. Plausible mechanisms for the formation and subsequent dissociation of the diagnostic ions are presented for the monounsaturated, diunsaturated, and polyunsaturated (3 or more double bonds) FAME. (+info)
Stimulation of tolbutamide hydroxylation by acetone and acetonitrile in human liver microsomes and in a cytochrome P-450 2C9-reconstituted system.
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Organic solvents are often used to solubilize lipophilic new chemical entities before their addition to in vitro test systems such as microsomal stability or cytochrome P-450 (CYP) inhibition. However, the effect of these organic solvents on the test systems is not usually characterized. This study was initiated to evaluate the effect of acetonitrile and acetone, in addition to other organic solvents, on the tolbutamide hydroxylation activity of CYP2C9 in both human liver microsomes and a CYP2C9-reconstituted system. Both acetonitrile and acetone significantly stimulated the NADPH-dependent tolbutamide hydroxylation by nearly 2- to 3-fold in human liver microsomes and CYP2C9-reconstituted system when incubated at 2 and 4% final solvent concentrations. When cumene hydroperoxide was used instead of NADPH, both acetone and acetonitrile significantly inhibited tolbutamide hydroxylation. This NADPH-dependent stimulatory effect was further evaluated by examining the effect of a series of other organic solvents with different carbon chain lengths and various functional groups, including hydroxyl, ketone, and aldehyde. Unlike acetone, two other ketone-containing solvents, methyl ethyl ketone (2-butanone) and diethyl ketone (3-pentanone) failed to significantly enhance tolbutamide hydroxylation. Other solvents tested, including methanol, ethanol, propanol, 1-butanol, 2-butanol, 1-pentanol, 2-pentanol, acetaldehyde, and dimethyl sulfoxide significantly inhibited NADPH-dependent tolbutamide hydroxylation. Overall, the stimulatory effect of both acetonitrile and acetone on tolbutamide hydroxylation was found to be primarily due to a consistent increase in V(max), whereas K(m) was unchanged in both human liver microsomes and the reconstituted CYP2C9 system. These data suggest that acetone and acetonitrile stimulate NADPH-mediated tolbutamide hydroxylation via the CYP reductase and not by modifying the affinity of tolbutamide for the CYP2C9 enzyme. (+info)
Substrate-dependent effect of acetonitrile on human liver microsomal cytochrome P450 2C9 (CYP2C9) activity.
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Acetonitrile is an organic solvent commonly used to increase the solubility of lipophilic substrates for in vitro studies. In this study, we examined its effect on four reactions (diclofenac hydroxylation, tolbutamide methyl hydroxylation, phenytoin hydroxylation, and celecoxib methyl hydroxylation) catalyzed by human liver microsomes and by the recombinant CYP2C9. In both cases, the effect of acetonitrile on activity was found to be substrate-dependent. Namely, it increased diclofenac 4'-hydroxylase and tolbutamide methyl hydroxylase activities, but decreased celecoxib methyl hydroxylase activity in a concentration-dependent manner. By comparison, hydroxylation of phenytoin was resistant to its effect. The presence of acetonitrile (3%, v/v) gave rise to a lower K(m) and a higher V(max) for diclofenac hydroxylase in both liver microsomes and recombinant CYP2C9 preparations (87 and 52% increase in V(max)/K(m) ratio, respectively). On the other hand, the inhibitory effect of the solvent (1%, v/v) toward celecoxib hydroxylase was characterized by a decrease in V(max) (human liver microsomes) or a change in both K(m) and V(max) (rCYP2C9), leading to 25 and 46% decrease in V(max)/K(m) for both systems. The results of this study underscore the need for careful evaluation of solvent effects before initiation of inhibition or cytochrome P450 reaction phenotyping studies. (+info)
High-throughput method development approaches for bioanalytical mass spectrometry.
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A rational approach to the development and optimization of solid-phase extraction (SPE) methods is described. The semiautomated scheme allows for the simultaneous testing of multiple chemistries using a custom multiple-sorbent 96-well method development plate. Optimized extraction conditions for up to five analytes are determined in a single 2.5-h experiment. The experiment can be tailored to determine SPE conditions (including wash protocols) for related analytes. Data obtained by liquid chromatography-atmospheric pressure ionization-mass spectrometry allows the quantitation of absolute recovery and selection of the best extraction conditions for approximately 100 analytes of diverse structure. Optimized extraction protocols yielding at least 80% recovery are determined for 81% of the analytes. For 96% of the analytes screened, extraction conditions resulting in recoveries of > or = 60% are determined. The most generic set of SPE conditions consist of either C8 or C18 sorbent with an eluent composition of acetonitrile with 5mM nitric acid added. (+info)
High-performance liquid chromatographic determination of amphotericin B in a liposomal pharmaceutical product and validation of the assay.
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A validated high-performance liquid chromatographic method is presented to quantitate amphotericin B (AB) in a liposomal pharmaceutical formulation. The analysis is based on the chromatographic separation of AB and 1-amino-4-nitronaphthalene (the internal standard) on a C18 muBondapac reversed-phase column with a mobile phase consisting of a mixture of acetonitrile and 0.02 M ethylenediamine tetra-acetic acid disodium salt at pH 5.0 (45:55, v/v). The chromatographic analysis time is less than 10 min, and the validation of the assay shows that it is selective, accurate, and linear for the concentration range of 2.50 to 7.50 microg/mL with a detection limit of 0.00500 microg/mL. The within-day and between-day relative standard deviation values are 1.26% (n = 18) and 1.25% (n = 8), respectively. The method described conforms to the validation of compendial methods used for finished pharmaceutical products in general and offers a reliable, quick, and cost-effective procedure for examining the consistency or quality-control analysis of AB in liposomal products. It can also be applied for the determination of AB in other nonliposomal lipid-based drug delivery systems that are on the market. (+info)
Simultaneous determination of sweeteners and preservatives in preserved fruits by micellar electrokinetic capillary chromatography.
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A micellar electrokinetic capillary method for the simultaneous determination of the sweeteners dulcin, aspartame, saccharin, and acesulfame-K and the preservatives sorbic acid; benzoic acid; sodium dehydroacetate; and methyl-, ethyl-, propyl-, isopropyl-, butyl-, and isobutyl-p-hydroxybenzoate in preserved fruits is developed. These additives are ion-paired and extracted using sonication followed by solid-phase extraction from the sample. Separation is achieved using a 57-cm fused-silica capillary with a buffer comprised of 0.05 M sodium deoxycholate, 0.02 M borate-phosphate buffer (pH 8.6), and 5% acetonitrile, and the wavelength for detection is 214 nm. The average recovery rate for all sweeteners and preservatives is approximately 90% with good reproducibility, and the detection limits range from 10 to 25 microg/g. Fifty preserved fruit samples are analyzed for the content of sweeteners and preservatives. The sweeteners found in 28 samples was aspartame (0.17-11.59 g/kg) or saccharin (0.09-5.64 g/kg). Benzoic acid (0.02-1.72 g/kg) and sorbic acid (0.27-1.15 g/kg) were found as preservatives in 29 samples. (+info)
Lactobacillus arizonensis sp. nov., isolated from jojoba meal.
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Five strains of simmondsin-degrading, lactic-acid-producing bacteria were isolated from fermented jojoba meal. These isolates were facultatively anaerobic, gram-positive, non-motile, non-spore-forming, homofermentative, rod-shaped organisms. They grew singly and in short chains, produced lactic acid but no gas from glucose, and did not exhibit catalase activity. Growth occurred at 15 and 45 degrees C. All strains fermented cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannitol, D-mannose, melibiose, D-ribose, salicin, D-sorbitol, sucrose and trehalose. Some strains fermented L-(-)-arabinose and L-rhamnose. D-Xylose was not fermented and starch was not hydrolysed. The mean G+C content of the DNA was 48 mol%. Phylogenetic analyses of 16S rDNA established that the isolates were members of the genus Lactobacillus. DNA reassociation of 45% or less was obtained between the new isolates and the reference strains of species with G+C contents of about 48 mol%. The isolates were differentiated from other homofermentative Lactobacillus spp. on the basis of 16S rDNA sequence divergence, DNA relatedness, stereoisomerism of the lactic acid produced, growth temperature and carbohydrate fermentation. The data support the conclusion that these organisms represent strains of a new species, for which the name Lactobacillus arizonensis is proposed. The type strain of L. arizonensis is NRRL B-14768T (= DSM 13273T). (+info)