Structural analysis of C8H6˙+ fragment ion from quinoline using ion-mobility spectrometry/mass spectrometry.

This study investigated the structures of fragment ions derived from the quinoline (C9H7N) radical cation using ion-mobility spectrometry and mass spectrometry. Ion mobility and mass analysis revealed that C8H6˙+ is the primary dissociation product resulting from the loss of HCN during collision-induced dissociation of the quinoline radical cation. The reduced mobility (K0) of the C8H6˙+ fragment product in helium gas was measured over a range of reduced electric fields (E/N = 20.8-27.4 Td) at room temperature. The experimental K0 values indicated that C8H6˙+ is a mixture of phenylacetylene and pentalene radical cations. Furthermore, quantum chemical calculations revealed two potential energy surfaces delineating the loss of HCN from the quinoline radical cation to form phenylacetylene radical cations.

Development of an ion mobility spectrometer using radio-frequency electric field.

This paper describes the development of a new ion mobility spectrometer (IMS) using the radio-frequency (RF) electric field. The proposed IMS has high ion transmission efficiency. Seven connected IMS devices, in which the RF and DC electric fields are created by separate electrodes, are constructed. The ions are confined by the RF electric field and drifted by the DC electric field. The electrodes in each IMS device include short quadrupole electrodes and segmented vane electrodes. The uniform electric field in the IMS is verified by simulated results obtained using SIMION. To measure the exact value of reduced mobility K 0 at low Td (1 Td = 10-17 V cm2), two ion gates are installed in the IMS. By installing the ion gates at suitable positions for eliminating the effect of gas flow, the exact ion velocity through the IMS can be measured. The K 0 values of O2 + and C6H6 + ions are measured as a function of Td. In addition, the K 0 of CH3OCH2 + fragment ions is measured. These K 0 measurement results are consistent with previous results obtained using electrostatic drift tube apparatus. In summary, as our IMS can measure K 0 under low Td conditions, it can be used to better understand the structure of small molecular or fragment ions.

FFA3 Activation Stimulates Duodenal Bicarbonate Secretion and Prevents NSAID-Induced Enteropathy via the GLP-2 Pathway in Rats.

BACKGROUND: Therapy with nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with enteropathy in humans and experimental animals, a cause of considerable morbidity. Unlike foregut NSAID-associated mucosal lesions, most treatments for this condition are of little efficacy. We propose that the endogenously released intestinotrophic hormone glucagon-like peptide-2 (GLP-2) prevents the development of NSAID-induced enteropathy. Since the short-chain fatty acid receptor FFA3 is expressed on enteroendocrine L cells and on enteric nerves in the gastrointestinal tract, we further hypothesized that activation of FFA3 on L cells protects the mucosa from injury via GLP-2 release with enhanced duodenal HCO3- secretion. We thus investigated the effects of synthetic selective FFA3 agonists with consequent GLP-2 release on NSAID-induced enteropathy. METHODS: We measured duodenal HCO3- secretion in isoflurane-anesthetized rats in a duodenal loop perfused with the selective FFA3 agonists MQC or AR420626 (AR) while measuring released GLP-2 in the portal vein (PV). Intestinal injury was produced by indomethacin (IND, 10 mg/kg, sc) with or without MQC (1-10 mg/kg, ig) or AR (0.01-0.1 mg/kg, ig or ip) treatment. RESULTS: Luminal perfusion with MQC or AR (0.1-10 µM) dose-dependently augmented duodenal HCO3- secretion accompanied by increased GLP-2 concentrations in the PV. The effect of FFA3 agonists was inhibited by co-perfusion of the selective FFA3 antagonist CF3-MQC (30 µM). AR-induced augmented HCO3- secretion was reduced by iv injection of the GLP-2 receptor antagonist GLP-2(3-33) (3 nmol/kg), or by pretreatment with the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTRinh-172 (1 mg/kg, ip). IND-induced small intestinal ulcers were dose-dependently inhibited by intragastric administration of MQC or AR. GLP-2(3-33) (1 mg/kg, ip) or CF3-MQC (1 mg/kg, ig) reversed AR-associated reduction in IND-induced enteropathy. In contrast, ip injection of AR had no effect on enteropathy. CONCLUSION: These results suggest that luminal FFA3 activation enhances mucosal defenses and prevents NSAID-induced enteropathy via the GLP-2 pathway. The selective FFA3 agonist may be a potential therapeutic candidate for NSAID-induced enteropathy.

FFA2 activation combined with ulcerogenic COX inhibition induces duodenal mucosal injury via the 5-HT pathway in rats.

Serotonin (5-HT), predominantly synthesized and released by enterochromaffin cells, is implicated in gastrointestinal symptoms such as emesis, abdominal pain, and diarrhea. Because luminal short-chain fatty acids (SCFAs) release 5-HT from enterochromaffin cells, which express the SCFA receptor free fatty acid receptor 2 (FFA2) in rat duodenum, we examined the effects of the selective FFA2 agonist phenylacetamide-1 (PA1) on duodenal 5-HT release with consequent bicarbonate secretion [duodenal bicarbonate secretion (DBS)] and on indomethacin (IND)-induced enteropathy. Intestinal injury was induced by IND (10 mg/kg sc) with or without PA1. We measured DBS in vivo in a duodenal loop perfused with PA1 while measuring 5-HT released in the portal vein. Duodenal blood flow was measured by laser-Doppler flowmetry. IND induced small intestinal ulcers with duodenal sparing. PA1 given with IND (IND + PA1) dose dependently induced duodenal erosions. IND + PA1-induced duodenal lesions were inhibited by the FFA2 antagonist GLPG-0974, ondansetron, or omeprazole but not by RS-23597 or atropine. Luminal perfusion of PA1 augmented DBS accompanied by increased portal blood 5-HT concentrations with approximately eight times more release at 0.1 mM than at 1 µM, with the effects inhibited by coperfusion of GLPG-0974. Luminal PA1 at 1 µM increased, but at 0.1 mM diminished, duodenal blood flow. Cosuperfusion of PA1 (0.1 mM) decreased acid-induced hyperemia, further reduced by IND pretreatment but restored by ondansetron. These results suggest that, although FFA2 activation enhances duodenal mucosal defenses, FFA2 overactivation during ulcerogenic cyclooxygenase inhibition may increase the vulnerability of the duodenal mucosa to gastric acid via excessive 5-HT release and 5-HT3 receptor activation, implicated in foregut-related symptoms such as emesis and epigastralgia.NEW & NOTEWORTHY Luminal free fatty acid receptor 2 agonists stimulate enterochromaffin cells and release serotonin, which enhances mucosal defenses in rat duodenum. However, overdriving serotonin release with high luminal concentrations of free fatty acid 2 ligands such as short-chain fatty acids injures the mucosa by decreasing mucosal blood flow. These results are likely implicated in serotonin-related dyspeptic symptom generation because of small intestinal bacterial overgrowth, which is hypothesized to generate excess SCFAs in the foregut, overdriving serotonin release from enterochromaffin cells.

Neural FFA3 activation inversely regulates anion secretion evoked by nicotinic ACh receptor activation in rat proximal colon.

KEY POINTS: Luminal short-chain fatty acids (SCFAs) influence gut physiological function via SCFA receptors and transporters. The contribution of an SCFA receptor, free fatty acid receptor (FFA)3, to the enteric nervous system is unknown. FFA3 is expressed in enteric cholinergic neurons. Activation of neural FFA3 suppresses Cl(-) secretion induced by nicotinic ACh receptor activation via a Gi/o pathway. Neural FFA3 may have an anti-secretory function by modulating cholinergic neural reflexes in the enteric nervous system. ABSTRACT: The proximal colonic mucosa is constantly exposed to high concentrations of microbially-produced short-chain fatty acids (SCFAs). Although luminal SCFAs evoke electrogenic anion secretion and smooth muscle contractility via neural and non-neural cholinergic pathways in the colon, the involvement of the SCFA receptor free fatty acid receptor (FFA)3, one of the free fatty acid receptor family members, has not been clarified. We investigated the contribution of FFA3 to cholinergic-mediated secretory responses in rat proximal colon. FFA3 was immunolocalized to enteroendocrine cells and to the enteric neural plexuses. Most FFA3-immunoreactive nerve fibres and nerve endings were cholinergic, colocalized with protein gene product (PGP)9.5, the vesicular ACh transporter, and the high-affinity choline transporter CHT1. In Ussing chambered mucosa-submucosa preparations (including the submucosal plexus) of rat proximal colon, carbachol (CCh)-induced Cl(-) secretion was decreased by TTX, hexamethonium, and the serosal FFA3 agonists acetate or propionate, although not by an inactive analogue 3-chloropropionate. Serosal application of a selective FFA3 agonist (N-[2-methylphenyl]-[4-furan-3-yl]-2-methyl-5-oxo-1,4,5,6,7,8-hexahydro-quinoline-3-carboxamide; MQC) dose-dependently suppressed the response to CCh but not to forskolin, with an IC50 of 13 µm. Pretreatment with MQC inhibited nicotine-evoked but not bethanechol-evoked secretion. The inhibitory effect of MQC was reversed by pretreatment with pertussis toxin, indicating that FFA3 acts via the Gi/o pathway. Luminal propionate induced Cl(-) secretion via the cholinergic pathway, which was reduced by MQC, as well as by TTX, hexamethonium or removal of the submucosal plexus. These results suggest that the SCFA-FFA3 pathway has a novel anti-secretory function in that it inhibits cholinergic neural reflexes in the enteric nervous system.

Short-chain fatty acid sensing in rat duodenum.

KEY POINTS: Luminal lipid in the duodenum modulates gastroduodenal functions via the release of gut hormones and mediators such as cholecystokinin and 5-HT. The effects of luminal short-chain fatty acids (SCFAs) in the foregut are unknown. Free fatty acid receptors (FFARs) for long-chain fatty acids (LCFAs) and SCFAs are expressed in enteroendocrine cells. SCFA receptors, termed FFA2 and FFA3, are expressed in duodenal enterochromaffin cells and L cells, respectively. Activation of LCFA receptor (FFA1) and presumed FFA3 stimulates duodenal HCO3(-) secretion via a glucagon-like peptide (GLP)-2 pathway, whereas FFA2 activation induces HCO3(-) secretion via muscarinic and 5-HT4 receptor activation. The presence of SCFA sensing in the duodenum with GLP-2 and 5-HT signals further supports the hypothesis that luminal SCFA in the foregut may contribute towards the generation of functional symptoms. ABSTRACT: Intraduodenal fatty acids (FA) and bacterial overgrowth, which generate short-chain FAs (SCFAs), have been implicated in the generation of functional dyspepsia symptoms. We studied the mechanisms by which luminal SCFA perfusion affects duodenal HCO3(-) secretion (DBS), a measure of mucosal neurohumoral activation. Free fatty acid receptor (FFAR) 1 (FFA1), which binds long-chain FA (LCFA), and SCFA receptors FFA2 and FFA3 were immunolocalised to duodenal enteroendocrine cells. FFA3 colocalised with glucagon-like peptide (GLP)-1, whereas FFA2 colocalised with 5-HT. Luminal perfusion of the SCFA acetate or propionate increased DBS, enhanced by dipeptidyl peptidase-IV (DPPIV) inhibition, at the same time as increasing GLP-2 portal blood concentrations. Acetate-induced DBS was partially inhibited by monocarboxylate/HCO3(-) exchanger inhibition without affecting GLP-2 release, implicating acetate absorption in the partial mediation of DBS. A selective FFA2 agonist dose-dependently increased DBS, unaffected by DPPIV inhibition or by cholecystokinin or 5-HT3 receptor antagonists, but was inhibited by atropine and a 5-HT4 antagonist. By contrast, a selective FFA1 agonist increased DBS accompanied by GLP-2 release, enhanced by DPPIV inhibition and inhibited by a GLP-2 receptor antagonist. Activation of FFA1 by LCFA and presumably FFA3 by SCFA increased DBS via GLP-2 release, whereas FFA2 activation stimulated DBS via muscarinic and 5-HT4 receptor activation. SCFA/HCO3(-) exchange also appears to be present in the duodenum. The presence of duodenal fatty acid sensing receptors that signal hormone release and possibly signal neural activation may be implicated in the pathogenesis of functional dyspepsia.

Change in mutagenic activity of genistein after a nitrite treatment.

This study examined the mutagenic activity of genistein after a nitrite treatment under acidic conditions. Nitrite-treated genistein exhibited mutagenic activity toward Salmonella typhimurium strains TA 100 and TA 98 with or without S9 mix. Nitrite-treated genistein was demonstrated by electron spin resonance to generate radicals. An instrumental analysis showed 3'-nitro-genistein to have been formed in the reaction mixture. However, 3'-nitro-genistein did not exhibit mutagenic activity toward the S. typhimurium strains, suggesting that other mutagens might also have been formed in the reaction mixture. The clastogenic properties of nitrite-treated genistein and 3'-nitro-genistein were examined by a micronucleus test with male ICR mice. Nitrite-treated genistein and 3'-nitro-genistein showed a significantly higher frequency of micronucleated reticulocytes in mice than in the control group. These results suggest that a daily oral intake of genistein and nitrite through foodstuffs might induce the formation of various mutagenic compounds in the body.

High-energy electron transfer dissociation (HE-ETD) using alkali metal targets for sequence analysis of post-translational peptides.

Post-translational modifications (PTMs) of proteins are important in the activation, localization, and regulation of protein function in vivo. The usefulness of electron capture dissociation (ECD) and electron-transfer dissociation (ETD) in tandem mass spectrometry (MS/MS) using low-energy (LE) trap type mass spectrometer is associated with no loss of a labile PTM group regarding peptide and protein sequencing. The experimental results of high-energy (HE) collision induced dissociation (CID) using the Xe and Cs targets and LE-ETD were compared for doubly-phosphorylated peptides TGFLT(p)EY(p)VATR (1). Although HE-CID using the Xe target did not provide information on the amino acid sequence, HE-CID using the Cs target provided all the z-type ions without loss of the phosphate groups as a result of HE-ETD process, while LE-ETD using fluoranthene anion gave only z-type ions from z(5) to z(11). The difference in the results of HE-CID between the Xe and Cs targets demonstrated that HE-ETD process with the Cs target took place much more dominantly than collisional activation. The difference between HE-ETD using Cs targets and LE-ETD using the anion demonstrated that mass discrimination was much weaker in the high-energy process. HE-ETD was also applied to three other phosphopeptides YGGMHRQEX(p)VDC (2: X = S, 3: X = T, 4: X = Y). The HE-CID spectra of the doubly-protonated phosphopeptides (= [M + 2H](2+)) of 2, 3, and 4 using the Cs target showed a very similar feature that the c-type ions from c(7) to c(11) and the z-type ions from z(7) to z(11) were formed via N-C alpha bond cleavage without a loss of the phosphate group.

Unimolecular and collision-induced dissociation of singly-charged mono-bromide silver clusters Ag(x)Br(+) (x = 2, 4, 6, 8, 10).

Relative intensities of singly-charged mono-bromide silver clusters Ag(x)Br(+) formed from sputtering of a pressed pellet of silver bromide were measured by mass spectrometry. The obtained results suggest that the Ag(x)Br(+) clusters have a structural formula of the form Ag(x-1)(+)(AgBr). The relative stability of Ag(x-1)(+)(AgBr) was determined by the intrinsic stability of the remaining metallic portion of the cluster (Ag(x-1)(+)) as predicted by the spherical jellium model (SJM). Unimolecular and high- energy collision-induced dissociation (CID) spectra of Ag(x)Br(+) (x = 2, 4, 6, 8, 10) clusters were also measured. In all of the spectra, the most intense fragment peaks were assigned to the Ag(x-1)(+) ions accompanying the loss of AgBr. The difference in the relative intensities of the Ag(x-1)(+) peaks between unimolecular dissociation and CID spectra led us to conclude that the weakest bond in the excited cluster Ag(x)Br(+*) is the Ag(x-1)(+)-AgBr bond and the structure of Ag(x)Br(+) is a metallic Ag(x-1)(+) ion cluster adduct with AgBr. The primary fragments observed in the CID spectra were also explained by the stabilities of the generated ion products and neutral fragments, both having even delocalized valence electrons. The present results were consistently explained by SJM. The dissociation behavior of Ag(2)Br(+) can be explained on the basis of the calculated thermochemical data.

Development of an ion trap/multi-turn time-of-flight mass spectrometer with potential- lift.

An ion trap/multi-turn time-of-flight (ToF) mass spectrometer with potential-lift has been developed. This system consists of an external ion source, a lens system, an ion trap, a potential-lift, a multi-turn ToF mass spectrometer and a detector. The ion trap consists of hyperbolic electrode cross-sections (Paul trap) and is used as an ion storage device. The potential-lift, which is part of the flight tube, was attached between the ion trap and the multi-turn ToF mass spectrometer. The potential-lift is known to be useful for increasing the kinetic energy of the ions. In order to check the ability of the potential-lift, mass distributions of [(CsI)(n) Cs]+ clusters (n = 1-9) were measured. The relative intensity ratios of the [(CsI)(n)Cs]+ clusters were consistent with the results obtained using other apparatus. To check the properties of the new apparatus, Xe+ isotopes were analyzed using either a linear or multi-turn ToF mass spectrometer. In the linear mode, the mass resolution was 500. In the multi-turn mode, the resolution depended on the number of cycles of the multi-turn ToF mass spectrometer; the mass resolution was 4400 (FWHM) after nine cycles. This new apparatus with a high resolution will be useful for measurements of ion-molecule reactions and photodissociations.

Methylene-bridged bis(benzimidazolium) salt as a highly efficient catalyst for the benzoin reaction in aqueous media.

Benzoin reactions are catalyzed effectively by a methylene-bridged bis(benzimidazolium) salt to yield alpha-hydroxy ketones, and the reactions proceed in water as the aqueous medium under mild conditions.

Efficiencies of singlet oxygen production and rate constants for oxygen quenching in the S1 state of dicyanonaphthalenes and related compounds.

The quantum yield of singlet oxygen ((1)O(2) ((1)Delta(g))) production (Phi(Delta)) in the oxygen quenching of photoexcited states for 1,2-dicyanonaphthalene (1,2-DCNN), 1,4-dicyanonaphthalene (1,4-DCNN) and 2,3-dicyanonaphthalene (2,3-DCNN) in cyclohexane, benzene, and acetonitrile was measured using a time-resolved thermal lens (TRTL) technique, in order to determine the efficiency of singlet oxygen ((1)Delta(g)) production in the first excited singlet state (S(1)), (f(Delta)(S)). The efficiencies of singlet oxygen ((1)Delta(g)) production from the lowest triplet state (T(1)), (f(Delta)(T)), were nearly unity for all DCNNs in all the solvents. The values of f(Delta)(S) were fairly large for 1,2-DCNN (0.33-0.57) and 1,4-DCNN (0.33-0.66), but were close to zero for 2,3-DCNN. Rate constants for oxygen quenching in the S(1) state (k(q)(S)) obtained for these compounds were significantly smaller than diffusion-controlled rate constants. The kinetics for processes leading to production and no production of singlet oxygen is discussed on the basis of the values of f(Delta)(S) and k(q)(S). The results obtained regarding phenanthrene (PH), 9-cyanophenanthrene (9-CNPH), pyrene (PY) and 1-cyanopyrene (1-CNPY) are also discussed.

Development of enzyme immunoassay for detection of DDT.

Dichlorodiphenyltrichloroethane (DDT) is one of the persistent organic pollutants (POPs) widely found in the environment and in the general population. In this study, a direct competitive enzyme immunoassay (EIA) has been developed for the quantitative analysis of DDT. To generate a specific polyclonal antibody for EIA, p, p'-DDT was conjugated to porcine thyroglobulin for rabbit immunization. At optimized EIA conditions, the standard curves ranged from 0.137 to 100 ng/mL with the quantification limit of 0.41 ng/mL. The coefficients of variation (CV%) were 5.42-10.53% for intra-assay and 6.04-7.26% for inter-assay. Cross-reactivities with DDT metabolites (DDTs, including o, p'-DDT, p, p'-DDD, o, p'-DDD, p, p'-DDE, o, p'-DDE, p, p'-dichlorobenzophenone (DCBP), o, p'-DCBP) were investigated. The polyclonal antibody showed relatively low and/or no cross-reactivity with these compounds, and the assay was seen to be highly selective for p, p'-DDT. Moreover, the DDTs could be ranked by their reactivity: DDT > DDD > DDE > DCBP. In addition, the characterization of the polyclonal antibody indicated that the antiserum possesses a high specificity for p, p'-isomers. The results indicated that the developed EIA using this antibody could be a convenient and supplemental analytical tool for monitoring DDT.

Experimental and theoretical study on gas-phase ion/molecule reactions of silver trimer cation, Ag3+, with 12-crown-4.

The reaction mechanisms of silver trimer cation, Ag3+, with 12-crown-4 (12C4) were studied experimentally and theoretically. Using a cylindrical ion trap time-of-flight mass spectrometer, gas-phase ion/molecule reactions of Ag3+ with 12C4 were observed. Metal-ligand complexes of [Ag(12C4)]+, [Ag3(12C4)]+ and [Ag3(12C4)2]+, and of [Ag(12C4)2]+ and [Ag3(12C4)3]+, were observed as the reaction intermediates and terminal products, respectively. The formations of the [Ag12C4]+ and [Ag(12C4)2]+ complexes indicated that the neutral dimer (Ag2) had been eliminated from the trimer cation. From the results of ab initio calculations at the HF/LanL2DZ level of theory and the experiments, it is suggested that three 12C4 molecules can attach to Ag3+ through consecutive reactions and that neutral Ag2 can be easily eliminated from [Ag3(12C4)]+.