Cytotoxic aporphine alkaloids from Cassytha filiformis.

Purification of a cytotoxic crude alkaloid extract of Cassytha filiformis led to the isolation of four known aporphine alkaloids: neolitsine, dicentrine, cassythine (= cassyfiline) and actinodaphnine. Their structures were determined by analysis of spectroscopic data. All isolated alkaloids were tested for their cytotoxic activities on cancer and non-cancer cell lines in vitro. Neolitsine was the most active against HeLa and 3T3 cells (IC 50 :21.6 microM, and 21.4 microM, respectively). Cassythine and actinodaphnine showed the highest activity against Mel-5 (IC 50 : 24.3 microM and 25.7 microM, respectively) and HL-60 (IC 50 : 19.9 microM and 15.4 microM, respectively). This is the first report on the cytotoxic activity of C. filiformis extract and of neolitsine and cassythine. Furthermore, the complete NMR data of cassythine and actinodaphnine are given here for the first time.

ent-trachyloban-3beta-ol, a new cytotoxic diterpene from Croton zambesicus.

The dichloromethane extract of leaves of Croton zambesicus (Euphorbiaceae) showing in vitro cytotoxicity against human cervix carcinoma cells was investigated in order to identify its active compounds. A bio-guided fractionation by HSCCC followed by MPLC led us to isolate a trachylobane diterpene, ent-trachyloban-3beta-ol, with cytotoxic properties (IC50 on HeLa cells = 7.3 microg/ml). This is the first report on the cytotoxicity of a trachylobane diterpene.

Structure characterization of flavonoid O-diglycosides by positive and negative nano-electrospray ionization ion trap mass spectrometry.

Isomeric flavonoid O-diglycosides were analyzed by positive and negative nano-electrospray ionization (ESI) ion trap mass spectrometry (ITMS) in order to evaluate whether the two most common interglycosidic linkage types, i.e. 1 --> 2 and 1 --> 6, found for glycosides containing a rhamnosylglucose glycan part can be differentiated. In the positive ion mode the degree of internal glucose residue loss was found to be strongly dependent on the aglycone type and was very pronounced for aglycones of the flavanone type. The relative abundance of the Y-type ions formed by fragmentation at glycosidic bonds only allows one to infer the interglycosidic linkage types in the case of flavone O-diglycosides. In contrast, the negative ion mode makes a clear differentiation between a rutinoside (1 --> 6) and a neohesperidoside (1 --> 2) glycan residue possible for all aglycone types. The neohesperidose-containing compounds could be characterized by additional product ions. When the compounds were dissolved in pure methanol a molecular radical ion was found to be the base peak in nano-ESI.

Radiosterilization of cefotaxime: investigation of potential degradation compounds by liquid chromatography-electrospray mass spectrometry.

The nonvolatile radiolytic compounds produced by irradiation of cefotaxime were studied by a liquid chromatography-electrospray mass spectrometry method. Full scan LC-MS was first performed in order to obtain the m/z value of the protonated molecules of all detected peaks. LC-MS-MS was then carried out on the compounds of interest. A comparison between the MS-MS spectrum of cefotaxime and those of the radiolytic compounds showed that their fragmentation patterns were very similar suggesting that they were structural analogues of the main drug. The examination of the two main fragmentation pathways also permitted the location of the modified substructures. Moreover, it was shown that some stereoisomers appeared with the irradiation process. The complete fragmentation pattern of cefotaxime was studied by MSn and used to obtain information about the structure of the radiolytic compounds. A complete structure was proposed for four of these.

Oxidative catabolism of alpha-tocopherol in rat liver microsomes.

The goal of this study was to clarify the mechanism responsible for the catabolism of alpha-tocopherol. The vitamin, bound to albumin, was incubated with rat liver microsomes and appeared to be broken down. Optimal production of the metabolite was obtained when 1 mg of microsomal protein was incubated with 36 microM of alpha-tocopherol in the presence of 1.5 mM of NADPH. Chromatographic and mass spectrometric analyses of the metabolite led to the conclusion that it consists of an omega-acid with an opened chroman ring, although we could not perform nuclear magnetic resonance analysis to confirm this. Our data show that alpha-tocopherol is omega-oxidized to a carboxylic acid and that this process can occur in rat liver microsomes in the presence of NADPH and O2. The oxidation to the quinone structure appears to be a subsequent event that may be artifactual and/or catalyzed by a microsomal enzyme(s).

Determination of flavone, flavonol, and flavanone aglycones by negative ion liquid chromatography electrospray ion trap mass spectrometry.

Eleven naturally occurring flavonoid aglycones, belonging to the representative flavone, flavonol, and flavanone types were separated by high performance liquid chromatography and analyzed on-line with negative ion electrospray ionization tandem mass spectrometry (ESI-MS/MS). In order to resolve the MS/MS spectra obtained, each compound was reinvestigated by direct loop injections using an ion trap mass spectrometer. The MSn spectra obtained allowed us to propose plausible schemes for their fragmentation supported by the analysis of five complementary synthetic flavonoid aglycones. The negative ion ESI-MS/MS behavior of the different aglycones investigated in this study revealed interesting differences when compared with the previously described patterns obtained using various ionization techniques in positive ion. Thus, concerning the retro Diels-Alder (RDA) fragmentation pathways, several structurally informative anions appeared highly specific of the negative ion mode. In addition, a new lactone-type structure, instead of a ketene, was proposed for a classic RDA diagnostic ion. We also observed unusual CO, CO2, and C3O2 losses which appear to be characteristic of the negative ion mode. All these results and these unusual neutral losses show that the negative ion mode was a powerful complementary tool of the positive ion mode for the structural characterization of flavonoid aglycones by ESI-MS/MS.

Assessment of [18F]fluoroethylflumazenil metabolites using high-performance liquid chromatography and tandem mass spectrometry.

A simple procedure using HPLC and tandem mass spectrometry has been developed for the determination of fluoroethylflumazenil metabolites. Samples were precipitated with acetonitrile, evaporated to dryness followed by reconstitution with methanol. As mobile phase, 50 mM ammonium formate-methanol (58:42, v/v) was used. The method is valid both for cold and radiolabelled metabolites. Various cold metabolites (hydroxylated and/or dealkylated) were identified in rat and human microsome preparations. Radiolabelled metabolites arise from two or more transformations including hydroxylation. The methodology developed can be applied for further characterisation of metabolites, and for the determination of non metabolised [18F]fluoroethylflumazenil in routine clinical analysis.

Characterization of fluorescent and nonfluorescent peptide siderophores produced by Pseudomonas syringae strains and their potential use in strain identification.

Nonfluorescent highly virulent strains of Pseudomonas syringae pv. aptata isolated in different European countries and in Uruguay produce a nonfluorescent peptide siderophore, the production of which is iron repressed and specific to these strains. The amino acid composition of this siderophore is identical to that of the dominant fluorescent peptide siderophore produced by fluorescent P. syringae strains, and the molecular masses of the respective Fe(III) chelates are 1,177 and 1,175 atomic mass units. The unchelated nonfluorescent siderophore is converted into the fluorescent siderophore at pH 10, and colors and spectral characteristics of the unchelated siderophores and of the Fe(III)-chelates in acidic conditions are similar to those of dihydropyoverdins and pyoverdins, respectively. The nonfluorescent siderophore is used by fluorescent and nonfluorescent P. syringae strains. These results and additional mass spectrometry data strongly suggest the presence of a pyoverdin chromophore in the fluorescent siderophore and a dihydropyoverdin chromophore in the nonfluorescent siderophore, which are both ligated to a succinamide residue. When chelated, the siderophores behave differently from typical pyoverdins and dihydropyoverdins in neutral and alkaline conditions, apparently because of the ionization occurring around pH 4.5 of carboxylic acids present in beta-hydroxyaspartic acid residues of the peptide chains. These differences can be detected visually by pH-dependent changes of the chelate colors and spectrophotochemically. These characteristics and the electrophoretic behavior of the unchelated and chelated siderophores offer new tools to discriminate between saprophytic fluorescent Pseudomonas species and fluorescent P. syringae and P. viridiflava strains and to distinguish between the two siderovars in P. syringae pv. aptata.

Effects of 1,4-benzoxazin-3-one derivatives from maize on survival and fecundity of Metopolophium dirhodum (Walker) on artificial diet.

Effects of 2-beta-D-glucopyranosyloxy-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-Glc) and DIMBOA-Glc N-O-methylated (HDMBOA-Glc), two compounds present in high concentration in maize, were tested on the aphid Metopolophium dirhodum reared on artificial diet. HDMBOA-Glc and DIMBOA-Glc decrease survival of adults with an LD50 of I mM and 5.6 mM, respectively, after 72 hr of feeding. These compounds also decrease the fecundity of the aphids at concentrations of 2 mM and 1 mM, respectively. At concentrations of 2 mM HDMBOA-Glc and 8 mM DIMBOA-Glc in the diet, the average lifetime fecundity of 10 females is near zero. Offspring mortality on diet with 2 mM DIMBOA-Glc is significantly higher than with the control diet. In contrast, HDMBOA-Glc has no effect on the survival of offspring. The possibility that these compounds protect Poaceae against aphids is discussed.

Identification of undescribed medium-chain acylcarnitines present in urine of patients with propionic and methylmalonic acidemias.

In urine of patients with propionyl-CoA carboxylase deficiency or with methylmalonic acidemia, carnitine esters of 2-methyl-branched fatty acids of all chain lengths between 4 and 9 atoms of carbon were identified during the acute phase of the diseases. The chemical structure of these compounds was obtained by gas chromatography-mass spectrometry analysis of their fatty acid moieties in their free and picolinyl ester forms. We suggest mechanisms for the biosynthesis of these branched fatty acids, and their accumulation in urine during episodes of caloric imbalance.

Variation of DIMBOA and related compounds content in relation to the age and plant organ in maize.

We report the variation of all 1,4-benzoxazin-3-one derivatives content detectable in maize with plant age in roots and aerial parts. Our results show that the concentration of hydroxamic acids, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc) and its 8-methoxylated analogue (DIM2BOA-Glc) is high after seed germination and then decreases with plant age. Nevertheless, these compounds continue to be biosynthesised during 6-10 days after germination. Variation in concentration of N-O-methylated DIMBOA-Glc (HDMBOA-Glc) is similar to the one of hydroxamic acids in aerial parts. On the contrary, in roots, its concentration remains relatively stable with plant age. After 10 days, HDMBOA-Glc becomes the main compound in roots. This compound is also present in higher concentration than hydroxamic acids in the oldest leaf of 20-day-old maize. The presence of four other DIMBOA related compounds in maize plants depends on variety, age and tissue. The role of these compounds in plant resistance to aphids is discussed.

Identification of new medium-chain acylcarnitines present in urine of a patient with medium-chain acyl-CoA dehydrogenase deficiency.

Previously undescribed medium-chain acylcarnitines were identified in a urine sample from a patient with medium-chain acyl-CoA dehydrogenase deficiency. These are the 4-methylvaleryl, 4- and 5-methylhexanoyl, 6-methylheptanoyl-, 6-methyloctanoyl-, 4,5-dimethylhexanoyl- and 4,7-decadienoylcarnitines. Their chemical structures were obtained by gas chromatographymass spectrometry analysis of their fatty acid moieties as picolinyl esters.

Structural assignment of permethylated oligosaccharide subunits using sequential tandem mass spectrometry.

The sequential tandem mass spectrometry (MSn) capabilities offered by quadrupole ion trap instruments have been explored in a systematic study of permethylated oligosaccharides. Under collision-induced dissociation, protonated molecular species generated in the electrospray ionization mode yield simple and predictable mass spectra. Information on sequence, branching, and, to some extent, interglycosidic linkages can be deduced from fragments resulting from the cleavage of glycosidic bonds. Simple rules for the structural assignment of carbohydrates have been established for the fragmentation of protonated species and subunits thereof and corroborated by 18O-labeling experiments. Moreover, sequential tandem mass spectrometry was demonstrated to allow the straightforward structural characterization of unknown carbohydrate moieties by comparing their CID spectra with those of a set of references. As the collision-induced dissociation patterns are not dependent on the number of prior tandem mass spectrometric steps, structures can be unambiguously assigned by match of the spectra. These findings establish the basis of MSn performed on a quadrupole ion trap instrument for elucidating structures of large carbohydrates, which can be virtually degraded in the mass spectrometer into smaller entities in one or several steps. This powerful technique has been applied, used in conjunction with specific CD3 labeling, to the characterization of series of subunits generated from fucosylated and sialylated oligosaccharides, which are among the most important structures as far as biological activities are concerned.

Isolation and characterization of the major form of polyprenyl-phospho-mannose from Mycobacterium smegmatis.

We isolated from the endogenous polyprenyl-phospho-sugar pool of Mycobacterium smegmatis two mannose-containing compounds, i.e., a partially saturated C35-octahydroheptaprenyl-P-mannose and a fully unsaturated C50-decaprenyl-P-mannose. The relative amount of C35-polyprenyl-P-mannose in mycobacterial cells was comparable to that of decaprenyl- P-pentoses and, at least, an order of magnitude higher than that of C50-decaprenyl-P-mannose. The major form of mycobacterial polyprenyl-P-mannose was structurally characterized by combined gas chromatography-mass spectrometry, fast-atom bombardment tandem mass spectrometry and proton-nuclear magnetic resonance spectroscopy as beta-d-mannopyranosyl-monophospho-(C35)octahydroheptapren ol of which all three isoprene units have Z ( cis ) configuration. The differences in the structure and cellular concentrations of the mycobacterial mannosyl-P-polyprenols reflect distinct biochemical pathways of the two compounds and suggest the existence of specific GDP-Man:polyprenyl-P mannosyltransferases (synthetases) able to distinguish between C35-octahydroheptaprenyl- and C50-decaprenyl- phosphates of mycobacteria. Since the 6'-O-mycoloylated form of C35-octahydroheptaprenyl-P-mannose isolated from M. smegmatis is apparently involved in mycolate rather than mannosyl transfer reactions, we speculate that a catabolic pathway responsible for degradation of C35-P-mannose and recycling C35-octahydroheptaprenyl phosphate might exist in mycobacteria.

An electrospray-ionization tandem mass spectrometry method for determination of the anomeric configuration of glycosyl 1-phosphate derivatives.

A rapid, simple, and sensitive method is described for the determination of the anomeric configuration of sugar 1-phosphates, sugar nucleotides, and polyisoprenyl-phospho-sugars. Negative-ion electrospray ionization of picomole amounts of glycosyl 1-phosphate derivatives produces an intense signal of the [M-H]-deprotonated molecule which, by collision-induced dissociation, decomposes in a characteristic manner depending on cis/trans configuration of the 2-hydroxyl and phosphate groups of the glycosyl residue. A distinct feature of the product ion spectra of glycosyl 1-P and polyisoprenyl-P-sugars with cis configuration is the presence of abundant ions that correspond to the [M-H2O-H]- dehydration product and the [R-PO4-(C2H3O]- fragment arising from a cleavage across the sugar ring, where R is -H or -polyprenyl/dolichyl for glycosyl 1-P and polyisoprenyl-P-sugar, respectively. These two fragments, [M-H2O-H]- and [R-PO4-(C2H3O)]- are absent from the product ion spectra of sugar 1-P and polyisoprenyl-P-sugars with trans configuration. For sugar nucleotides, compounds with cis configuration produce, in tandem mass spectrometry, only one abundant fragment of nucleoside monophosphate, whereas those with trans configuration give nucleoside diphosphate as a major fragment ion. Accordingly, the anomeric configuration of a glycosyl 1-phosphate derivative can be easily determined by using electrospray-ionization tandem mass spectrometry provided that the glycosyl residue of known absolute configuration has a free 2-hydroxyl group and no other charge location.

Identification of new medium-chain acylcarnitines present in normal human urine.

Analysis of urinary medium-chain acylcarnitines extracted on C18 cartridges and gas chromatography mass spectrometry of their fatty acid moieties as picolinyl esters allowed the determination of the chemical structure of previously unidentified acylcarnitines in normal human urine. These are the 2,6-dimethylheptanoyl-, the 2,6-dimethyl-5-heptenoyl-, and the trans- and cis-3,4-methylene heptanoylcarnitines, also named 3-cyclopropane octanoylcarnitines. Assessment of the structure of these cyclopropane derivatives was obtained by 1H and 13C nuclear magnetic resonance spectroscopy. In addition, other acylcarnitines were tentatively identified.

Structural analysis of permethylated oligosaccharides by electrospray tandem mass spectrometry.

Electrospray tandem mass spectrometry used in conjunction with reversed-phase liquid chromatography was applied to characterize permethylated oligosaccharides. N-Acetylhexosamine-containing carbohydrates yielded under these conditions promoted molecular ions which underwent extensive fragmentation, even under low-energy collision-induced dissociation. MS/MS spectra of [M + H]+ ions are characterized by simple fragmentation patterns which result from cleavage of the glycosidic bonds and thus allow a straightforward interpretation. A systematic study of various oligosaccharides showed that information on sugar sequence and branching could be obtained. The nature of the substituent linked in position 3 of HexNAc-containing fragments could easily be assigned as the result of a specific secondary fragmentation process. The nature of some internal fragments was established on the basis of MS/MS spectra of derivatives 18O-labeled at the reducing end. Furthermore, MS/MS experiments carried out on fragment ions were proven to be useful for the structural characterization of oligosaccharide subunits. Thus, this approach constitutes a powerful tool for the structural assignment of moieties derived from larger glycans.

alpha-Oxidation of 3-methyl-substituted fatty acids in rat liver. Production of formic acid instead of CO2, cofactor requirements, subcellular localization and formation of a 2-hydroxy-3-methylacyl-CoA intermediate.

alpha-Oxidation of 3-methyl-substituted fatty acids in rat liver was studied in intact and permeabilized rat hepatocytes, and in homogenates and subcellular fractions. The experiments revealed that the primary end product of alpha-oxidation is formic acid, which is then converted to CO2. Rates of alpha-oxidation identical to those observed in intact hepatocytes were obtained in the permeabilized hepatocytes and liver homogenates when ATP, Mg2+ and CoA, and Fe2+, 2-oxoglutarate and ascorbate were added, suggesting that alpha-oxidation involves a fatty acid activation reaction and a dioxygenase reaction. Subcellular fractionation by differential and density gradient centrifugation demonstrated that alpha-oxidation is confined to peroxisomes, which produce formic acid that is converted to CO2, mainly in the cytosol. alpha-Oxidation in broken cell systems went hand in hand with the formation of a 2-hydroxy-3-methylacyl-CoA ester. Formation of the metabolite was strictly dependent on the presence of the above-mentioned cofactors, was confined to peroxisomes and was inhibited by fenoprofen and propyl gallate, inhibitors of alpha-oxidation in intact cells, indicating that the 2-hydroxyacyl-CoA ester is a bona fide intermediate of alpha-oxidation. Selective omission of cofactors from the reaction mixture and analysis of the incubation mixtures for 3-methyl fatty acids, 3-methyl fatty acyl-CoAs and their respective 2-hydroxy derivatives revealed that the activation reaction precedes the dioxygenase (hydroxylase) reaction. Our experiments demonstrate that alpha-oxidation is a peroxisomal process that consists of at least three reactions: fatty acid activation, hydroxylation and the reaction(s) involved in the release of formic acid.

Analysis by fast-atom bombardment tandem mass spectrometry of phosphatidylcholine isolated from heart mitochondrial fractions: Evidence of incorporation of monohydroxylated fatty acyl moieties.

Phosphatidylcholine (PC) is one of the main phospholipids present in mitochondrial membranes. According to current knowledge, the predominant fatty acyl moieties in this phospholipid are 16, 18, 20, or 22 carbon atoms long with chains that contain only carbon and hydrogen atoms. We have conducted a detailed analysis of the fatty acid substituents of the phospholipids present in mitochondrial fractions by using fast-atom bombardment tandem PC extracted from mitochondrial fractions of rat heart. The structure of one of these monohydroxylated fatty acids has been elucidated and corresponded to 12-hydroxy 9-octadecenoic acid. Indications that concern the structure of the five other monohydroxylated fatty acids are presented. These monohydroxylated fatty acyl groups are preferentially associated in the PC molecule with C-18 and C-20 fatty acyl moieties. We present arguments to suggest that the formation of these compounds is probably not due to a free-radical initiated mechanism. The potential implication of these monohydroxylated fatty acids in several physiological functions is suggested by the fact that free hydroxylated fatty acids that are identical or closely related to those found in the mitochondrial fractions possess various biological activities.

Determination of the anomeric configuration of glycosyl esters of nucleoside pyrophosphates and polyisoprenyl phosphates by fast-atom bombardment tandem mass spectrometry.

Collision-induced dissociation of the deprotonated molecules of glycosyl esters of nucleoside pyrophosphates and polyisoprenyl (dolichyl and polyprenyl) phosphates results in distinct fragmentation patterns that depend on cis-trans configuration of the phosphodiester and 2″ (or 2', respectively)-hydroxyl groups of the glycosyl residue. At the collision-offset voltage of 0. 5 V, sugar nucleotides with cis configuration produce only one very abundant fragment of nucleoside monophosphate, whereas compounds with trans configuration give weak signals for nucleoside di- and mono-phosphates and their dehydration products. These fragmentation patterns are largely preserved at higher collision energy, with the exception that, for sugar nucleotides with trans configuration, the characteristic signals are much more abundant and a novel diagnostic fragment of [ribosyl(deoxyribosyl)-5'-P2O5 - H](-) is generated. In the case of polyisoprenyl-P-sugars, polyisoprenyl phosphate ion is the only fragment observed for compounds with trans configuration, whereas in compounds with cis configuration, this ion is accompanied by another abundant fragment, which is derived from the cleavage across the sugar ring and corresponds to [polyisoprenyl-PO4-(C2H3O)](-). The relative intensity ratio of the latter ion to the [polyisoprenyl-HPO4](-) ion is close to 1 for compounds with cis configuration, but it is only about 0. 01 for compounds with trans configuration. This ratio may serve, therefore, as a diagnostic value for determination of the anomeric configuration of glycosyl esters of polyisoprenyl phosphates. It is proposed that the observed differences in fragmentation patterns of cis-trans sugar nucleotides and polyisoprenyl-P-sugars could be explained in terms of kinetic stereoelectronic effect, and a speculative mechanism of fragmentation of compounds with trans configuration is presented. For compounds with cis configuration, formation of a hydrogen bond between the C-2″(2') hydroxyl and the phosphate group could play a crucial role in directing the specific fragmentation reactions. Consequently, the described empirical rules would hold only for compounds that have a free 2″(2')-hydroxyl group and no alternative charge location. Owing to its simplicity, sensitivity, and tolerance of impurities, fast-atom bombardment-tandem mass spectrometry represents a suitable method for determination of the anomeric linkage of glycosyl esters of nucleoside pyrophosphates and polyisoprenyl phosphates if the absolute configuration of glycosyl residue is known and the compound fulfills the above-mentioned requirements.