Identification and characterization of dolichyl dolichoate, a novel isoprenoic derivative in bovine thyroid.

Inspection of the TLC pattern of the neutral lipid fraction of bovine thyroid reveals, in addition to cholesteryl esters and dolichyl fatty acid esters, the presence of a not yet identified compound in the most apolar lipid region. This unknown compound was purified on a preparative scale by silicic acid column chromatography, Lipidex-5000 gel-filtration chromatography and preparative thin-layer chromatography. By chemical (hydrolysis and reduction) and spectroscopic (ultraviolet, infrared, NMR, mass spectroscopy) methods this lipid was identified as dolichyl dolichoate. The homologue pattern was analyzed, by HPLC and FDMS, respectively.

Interaction of myeloperoxidase with diclofenac. Inhibition of the chlorinating activity of myeloperoxidase by diclofenac and oxidation of diclofenac to dihydroxyazobenzene by myeloperoxidase.

The chlorinating activity of myeloperoxidase, isolated from human polymorphonuclear neutrophils, was inhibited by the non-steroidal anti-inflammatory drug diclofenac (Voltaren). The concentration of diclofenac needed for 50% inhibition was 20 microM, a value comparable with IC50 values found for other drugs. Diclofenac did not react with HOCl nor with H2O2 but was oxidized in the presence of myeloperoxidase and H2O2 to an orange-coloured unstable product. The rate of oxidation was proportional to the enzyme concentration and to the concentration of diclofenac. but independent of the H2O2 concentration. Presumably both Compound I and Compound II, two intermediates formed during the reaction cycle of myeloperoxidase with H2O2 are able to oxidize diclofenac. In these redox reactions, the active short-living Compound I is reduced to Compound II, thereby inhibiting the chlorinating activity of the enzyme. Analysis by Fast Atom Bombardment mass spectrometry showed that in the presence of H2O2 myeloperoxidase oxidizes diclofenac to dihydroxyazobenzene.

The role of distonic ions in the formation of CH3NH 3 (+) and (CH 3) 2NH 2 (+) from the molecular ions of octopamine and synephrine.

It is shown by mass-analyzed ion kinetic energy spectrometry that the metastably decomposing molecular ions of octopamine (p-HOC6H4CH(OH)CH2NH2) and synephrine (p-HOC6H4CH(OH)CH2NHCH3) yield only protonated methylamine and dimethylamine, respectively, as product ions. From deuterium labeling and variation of the internal energy of the molecular ions, experimental support has been obtained that these product ions are generated via the occurrence of a distonic ion-neutral complex. In the case of octopamine, this complex would consist of a nitrogen-protonated aminomethyl radical and p-hydroxylbenzaldehyde in which the former species abstracts the aldehydic or phenolic hydrogen atom from the latter to give protonated dimethylamine.

In-source decay of hyperbranched polyesteramides in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Hyperbranched polyesteramides (DA2), prepared from hexahydrophthalic anhydride (D) and diisopropanolamine (A) have been characterized, by use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), field desorption (FD)-MS, and electrospray ionization (ESI)-MS. MALDI of polyesteramides produces protonated molecules. The spectra show a complex chemical composition distribution and end-group distribution which are mainly composed of two series of homologous oligomers DnA(n)+1 - mH2O and DnA(n) - mH2O, where m = 1-2. Signals from protonated molecules DnAn+1 and DnAn are almost absent in the MALDI spectrum, whereas these ions are responsible for the base peak of DnA(n)+1 - mH2O and DnA(n) - mH2O (m = 1-2) clusters in the ESI spectrum. The absence of -OH end-groups signals in the MALDI spectrum is due to a metastable decay of protonated DnA(n)+1 and DnAn ions in the ion source of the MALDI mass spectrometer prior to ion extraction. In-source decay results in the formation of protonated lower DnA(n)+1 - mH2O and DnA(n) - mH2O oligomers and their corresponding neutrals, leading to wrong conclusions concerning the relative end-group distribution as a function of the degree of polymerization and the chemical composition.

Chemical characterization and comparative cellular effects of meta-iodobenzyl guanidine and benzyl guanidine.

meta-Iodobenzyl guanidine (MIBG) combines the structural properties of the neuron-blocking agents bretylium and guanethidine and is being used increasingly for various clinical applications. Different samples of MIBG were assayed for possible contamination with benzyl guanidine (BG). Fast-atom-bombardment mass spectrometry (FAB-MS) analysis showed a prominent but variable m/z 150 signal, corresponding to a protonated BG. The MS/MS fragmentation pattern of these [M + H]+ ions was similar to that obtained from FAB-MS-generated, protonated BG, confirming the proposed molecule and associated structures. RP-HPLC analysis of both guanidines, however, excluded the possibility of contamination of MIBG with BG. It was therefore concluded that the BG signal was an artifact of the FAB-MS procedure. In addition, the importance of the meta-substituted iodine for the biological activity of MIBG was investigated. Three different biochemical and cell-biological properties of MIBG were compared with those of its precursor MIBA and BG. The assays used were: inhibition of the catecholamine "Uptake I" system in SK-N-SH neuroblastoma and PC-12 pheochromocytoma cells, inhibition of mitochondrial respiration, and general cytotoxicity in L1210 leukemia cells. Of the drugs tested, MIBG was the most efficient in Uptake I inhibition and was more toxic in survival assays, but as compared with BG it was almost equipotent in inhibiting mitochondrial respiration. These findings contribute to a further elucidation of the mechanism by which MIBG exerts its various actions.

2-Mercaptoethanesulfonate-cysteine disulfide excretion following the administration of 2-mercaptoethanesulfonate--a pitfall in the diagnosis of sulfite oxidase deficiency.

In the urine of a neonate with respiratory insufficiency and convulsions a positive sulfite reaction was found, which is suggestive of sulfite oxidase deficiency. The nitroprusside reaction also was positive. More detailed investigations showed that both tests were positive due to the administration of 2-mercaptoethanesulfonate, a mucolytic drug. The patient's urine contained an acidic amino acid with a column chromatographic behaviour like S-sulfocysteine. The high-voltage electrophoretic mobility was slightly different. This compound was isolated from the urine and identified as the mixed disulfide of 2-mercaptoethanesulfonate and cysteine. Its identity was proven with field desorption mass spectrometry, a technique which is suitable for the analysis of sulfonic acid derivatives.

Artifact formation due to ethyl thio-incorporation into silylated steroid structures as determined in doping analysis.

Trimethylsilylation of target substances in a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide and ethanethiol is frequently applied for the application of gas chromatography-mass spectrometry (GC-MS) in steroid analysis. However, artifacts were formed when using this mixture to silylate the steroids androsterone and etiocholanolone obtained from a urine matrix. The artifacts were identified as ethyl thio-containing products of the respective trimethylsilyl derivatives. The conversion of the studied products increased slowly as a function of time, was dependent on the presence of the urine matrix and was significantly accelerated by adding diethyl disulfide to the reagent before incubation. Also ethyl thio-incorporation into testosterone and epitestosterone was established. A mechanism for ethyl thio-incorporation is proposed. The conversion achieved after 120-h sample storage at room temperature was insufficient to significantly influence the analysis of androsterone and etiocholanolone under the studied conditions. However, the results provide fundamental insight into the mechanism of silylation and the occurring side-reactions. Moreover, when investigating the formation of new metabolites, the ethyl thio-incorporation can lead to misinterpretation.

Gas-phase ion chemistry of Glu/Met systems.

A combined chemical ionisation and tandem mass spectrometry (MS/MS) approach has been used for investigation of the gas-phase ion chemistry of systems containing the amino acids Glu and Met, and the dipeptides gamma-Glu-Met and Met-Glu. The metastable fragmentation of the protonated dimer, (Glu)2H(+), reveals an intracluster reaction leading to the elimination of the Glu residue. The main features of the ion-molecule reactions observed in the systems containing Glu and Glu + Met can be described in terms of sequential adduct formation. The results obtained for the thermal dehydration of Glu were used to rationalise the formation of the proton-bound structures (Glu-H2O...H(+)...(Glu-H2O) and (Glu-H2O)3-H(+). The adduct ions, [(Glu-H2O) + H + Glu](+) and [(Glu-H2O) + H + Met](+), and further association products were also observed. The results lead to a reconsideration of the structural aspects proposed earlier for these species in the sense that they suggest that the systems correspond to a mixture of isomeric covalent and proton-bound structures. The thermal effects on the decomposition of the neutral (gamma-Glu-Met) and its protonated form, (gamma-Glu-Met)H(+), at m/z 279 were investigated, and dramatic changes in the MI spectra of the m/z 279 ion with temperature were found. A mechanistic explanation for the observed evolution of higher mass ion peaks in the mass spectra is developed.

Re-examination of cellular cyclic beta-1,2-glucans of Rhizobiaceae: distribution of ring sizes and degrees of glycerol-1-phosphate substitution.

Gel-filtration and thin layer chromatography of low molecular weight carbohydrates from culture filtrates of Agrobacterium radiobacter, Isolate II, have shown, that next to the neutral beta-1,2-glucan fraction a major acidic fraction was present which was found to be glycerophosphorylated cyclic beta-1,2-glucans. Re-examination of cyclic beta-1,2-glucan preparations which had been obtained by extraction of Rhizobium cells with hot phenol-water also showed these acidic modified beta-1,2-glucans to be present. Cyclic beta-1,2-glucans from R. leguminosarum (9 strains) and of R. phaseoli (1 strain) had ring size distribution with degrees of polymerisation (DPs) of 19 and 20 as major ring sizes of which a minor part was glycerophosphorylated; beta-1,2-glucans of R. trifolii (3 strains) had ring sizes with DPs measuring 19-22 as prominent components which were largely unsubstituted, and R. meliloti (7 strains) had beta-1,2-glucans with ring size distributions extending to still higher DPs of 19-25 of which the major part appeared to be glycerophosphorylated.

Peptide bond formation in gas-phase ion/molecule reactions of amino acids: a novel proposal for the synthesis of prebiotic oligopeptides.

There is a general fascination with regard to the origin of life on Earth. There is an intriguing possibility that prebiotic precursors of life occurred in the interstellar space and were then transported to the early Earth by comets, asteroids and meteorites. It is probable that some part of the prebiotic molecules may have been generated by gas-phase ion/molecule reactions. Here we show experimentally that gaseous ion/molecule reactions of the amino acids, Glu and Met, may promote the synthesis of protonated dipeptides such as (Glu-Glu)H(+) and (Glu-Met)H(+) and their chemical growth to larger protonated peptides.

FAB and tandem mass spectrometry for endorphin- and ACTH peptides of molecular weight to 2000.

Four beta-endorphins (beta-endorphin 6-17, 2-17, 1-16, and 1-17) and two adrenocorticotropic hormone (ACTH) peptides (ACTH 1-10 and (1-16)-NH2) were studied by using fast atom bombardment coupled with tandem mass spectrometry. The capability to reproduce metastable ion and collisionally activated decomposition spectra on two different commercial sector mass spectrometers in two different laboratories was found to be acceptable (deviations in relative abundance are less than +/- 50%). The endorphin peptides fragment metastably or upon collisional activation to give abundant B-series ions as well as Y-series ions, whereas Y-series ions are the principal ionic species produced upon the desorption by fast atom bombardment. The ACTH peptides also fragment to give Y-series ions, but of relatively low abundance compared to those from the endorphins. For both sets of peptides, high-energy collisionally activated decomposition and metastable ion decomposition daughter ion spectra are precise, structurally informative--even for peptides up to m/z 2000--and complementary to spectra of daughter ions produced by desorption ionization alone.

Antimicrobially active alkaloids from Tabernaemontana chippii.

From Tabernaemontana chippii root bark, forty-five alkaloids were isolated; thirty-four were fully characterized by means of their spectral data and/or co-tlc; eight alkaloids were new, four of them being 3-hydroxy derivatives of known dimeric voacamine type alkaloids. Most of the twenty-six known alkaloids belonged to the corynanthean, ibogan, or bisindole classes. The structures of eleven other alkaloids--all minor--were only partially elucidated, most of them being new alkaloids. All the dimeric alkaloids were shown to possess strong antimicrobial activity against gram-positive bacteria and moderate to weak activity against gram-negative bacteria.

Methodology for the development of a drug library based upon collision-induced fragmentation for the identification of toxicologically relevant drugs in plasma samples.

The possibility of creating a robust mass spectral library with use of high-performance liquid chromatography-atmospheric pressure-electrospray ionization (HPLC-AP-ESI) for the identification of drugs misused in cases of clinical toxicology has been examined. Factors reported as influencing the fragmentation induced by "source transport region collision induced dissociation" (CID) have been tested in this study (i.e. solvent, pH, different acids or buffer salts and their concentration, different organic modifiers and the modifier concentration). The tests performed on a few "model drugs" were analysed with use of two different single quadrupole instruments. The large number of mass spectra obtained appears to be affected by the mobile phase conditions to only a minor extent. This also holds for the mass spectra obtained at two different instruments (laboratories). Subsequently breakdown curves have been measured for about 20 randomly chosen drugs by variation of the kinetic energy of their ions in the CID zone through changing the fragmenter voltage. These breakdown curves were used to optimize the fragmenter voltage for each drug. The optimized fragmenter voltages were then applied by use of a variably ramped fragmenter voltage to acquire mass spectra for the library. The chromatographic separations were run on a Zorbax Stable bond column using a 10-mM ammonium formate-acetonitrile gradient method. Spiked blank serum and patient samples with a total of 40 different drugs were extracted with use of a standard basic liquid-liquid extraction (LLE) method. A search of significant peaks in the chromatogram by application of the developed mass spectral library is shown to result in a more than 95% positive identification. reserved.

Characterization of two pterin derivatives isolated from Methanoculleus thermophilicum.

Methanoculleus thermophilicum was shown to contain two pterin derivatives. The structures of these pterin derivatives were established from amino acid analysis, 1H-NMR and fast-atom bombardment mass spectrometry data. One of the pterins was identified as tatiopterin-O, an aspartyl derivative of methanopterin with a proton at position 7 of the pterin moiety. The other pterin, which we named thermopterin, differed in the structure of the aniline group, containing two additional hydroxyl residues. The IUPAC name of thermopterin is N-[-1'-(2"-amino-4"-hydroxy-6"-pteridinyl)ethyl]-4- [2',3',4',5'-tetrahydroxypent-1'-yl(5'----1") O-alpha-ribofuranosyl-5"-phosphoric acid]-2,5-dihydroxyaniline, in which the phosphate group is esterified with alpha-hydroxyglutarylaspartic acid.

Prenatal diagnosis of a thyroglobulin synthesis defect in goats.

A strain of goats with congenital goitre due to a thyroglobulin (Tg) synthesis defect was studied. All goats excreted low molecular weight iodinated material (LOMWIOM) in their urine, but non-goitrous goats excreted in the LOMWIOM fraction less than 5 micrograms iodine per 24 h and the goitrous ones more than 15 micrograms iodine per 24 h. Prenatal diagnosis of the Tg synthesis defect in goats is possible since non-goitrous goats pregnant with goitrous kids excrete in the LOMWIOM fraction more than 10 micrograms iodine per 24 h while non-goitrous goats pregnant with non-goitrous kids excrete 10 micrograms or less iodine per 24 h as LOMWIOM. In 24 out of 25 cases a correct diagnosis could be made in the last 47 days of pregnancy. We argue that prenatal diagnosis of analogous defects in man may also be possible, using the excretion of LOMWIOM in maternal urine as yard-stick. By means of field desorption mass spectrometry (FOMS) and high performance liquid chromatography (HPLC) monoiodohistidine was identified as the major component of the LOMWIOM fraction in the urine of goitrous goats.

Nanostructures via noncovalent synthesis: 144 hydrogen bonds bring together 27 components.

This paper describes the spontaneous and reversible assembly of approximately 20 kDa synthetic hydrogen-bonded assemblies via the formation of 144 cooperative hydrogen bonds. These nanostructures ( approximately 3.0 x 5.5 nm), consisting of 27 different components, have been carefully characterized using a combination of (1)H NMR spectroscopy, MALDI-TOF MS using Ag(+)-labeling, gel permeation chromatography, and CD spectroscopy.

Convergent synthesis of noncovalent metallodendrimers containing hydrophobic dendrons at the periphery.

The noncovalent synthesis of "layer-block" metallodendrimers containing hydrophobic shells of covalent dendritic wedges at the periphery is described. Starting from first- and second-generation Fréchet wedges having phosphines at their focal point, convergent dendritic growth yields third- and fourth-generation metallodendrimers in which the coordination of nitriles, pyridines, and phosphines to SCS Pd(II) pincers is used as the assembly motif. In this convergent growth, the number of terminal hydrophobic phosphine wedges increases with generation. The solubility of the dendritic structures in apolar organic solvents such as chloroform and dichloromethane increases accordingly, in contrast to previously reported metallodendrimers. All dendritic structures were characterized by (1)H and (31)P NMR spectroscopy, elemental analysis, and MALDI-TOF mass spectrometry.

Studies on the mechanism of the vitamin K-dependent carboxylation reaction. Carboxylation without the concurrent formation of vitamin K 2,3-epoxide.

In addition to the three known forms of vitamin K (vitamin K quinone, vitamin K hydroquinone, and vitamin K epoxide), a fourth metabolite, hydroxyvitamin K, was found in reaction mixtures containing a vitamin K-dependent carboxylating enzyme system. When sulfite was added to such reaction mixtures, the formation of hydroxyvitamin K was substantially enhanced, whereas no epoxide was formed anymore. The vitamin K-dependent carboxylation was stimulated at these sulfite concentrations. Vitamin K hydroquinone could be replaced by t-butylhydroperoxide and also under these conditions the carboxylation was enhanced by sulfite. In the presence of peroxidase, the carboxylation reaction was blocked, whereas hydroxyvitamin K could still be detected in the reaction mixtures, even in the absence of sulfite. These observations lead us to the hypothesis that the carboxylation of glutamic acid residues is coupled to the heterolytic cleavage of a peroxide bond with the concurrent formation of vitamin epoxide.

Verification of the position of the phosphate group in some synthetic phosphopeptides by fast-atom bombardment and tandem mass spectrometry.

Some synthetically obtained linear and cyclic phosphopeptides of low molecular weight have been studied by fast-atom bombardment and tandem mass spectrometry to verify the position of the phosphate group in these compounds. Based upon the occurrence/non-occurrence of loss of phosphoric acid from low abundance fragment ions induced by low- and high-energy collisions with target gases, it is shown that the position of the phosphate group in the phosphopeptides studied can be determined unequivocally.