BUNSEKI KAGAKU Abstracts

We have been developing fluorescent indicators for a variety of molecular processes in living cells, including small molecules, known as second messengers, and protein phosphorylation reactions. This is because fluorescence imaging could be the most powerful technique available for observing the spatial and temporal dynamics of molecular processes in living cells, if fluorescent indicators for the relevant molecular processes become available. We have visualized the spatial and temporal dynamics of these molecular events in single living cells using our fluorescent indicators. The present fluorescent indicators are becoming an indispensable tool for understanding the complex mechanism of cellular signaling and for screening pharmaceuticals that inhibit or promote molecular processes in a cell.

For monitoring of hazardous organic compounds, we have been developing an on-line analytical instrument based on a supersonic jet resonance enhanced multi-photon ionization (REMPI) mass spectrometry. We have attempted to apply this technique to monitor transient emission phenomena of hazardous organic compounds in exhaust gas. It has been proven to be possible to conduct real-time monitoring of gaseous compounds in the atmosphere by our instrument with continuous sample introduction. We demonstrated that the signals of monochlorobenzene in a chimney could be detected. Also the delay time between gas emission and detection through a 23 m long stainless-steel pipe kept at 200 degrees Celsius was 150 seconds. It was proven that there were two different kinds of emission mechanisms of monochlorobenzene in the chimney.

There has been great interest in micro-chemical chips for miniaturizing chemical systems and integrating various chemical processes. These devices have many advantages, including a short analysis time ; a reduction of the sample, reagents, and waste volumes ; a more effective reaction due to the large specific interfacial area ; and a smaller space requirement. In order to realize general chemical processes in a microchip, including solvent extraction and reaction, etc., fluid controls of various solutions are important. However, multiphase parallel microflows become unstable due to small pressure fluctuations. In this paper, control methods of multiphase parallel microflows and micro solvent extraction using multiphase parallel microflows are introduced. Firstly, the fundamental physical properties in micro space are explained. Secondly, fluid control methods utilizing the fundamental physical properties are introduced. Thirdly, actual multiphase parallel microflows are introduced. Finally, micro solvent extraction using multiphase parallel microflows is introduced.

Solvent-extraction spectrometory is widely used to determine polyoxyethylene non-ionic surfactants (NS). The method is based on the formation of a complex between a NS and cobalt(III) or iron(III) thiocyanate. We reported on a continuous-flow analysis (CFA) with iron(III) thiocyanate to simplify the procedure and to reduce the amount of reagents in manual solvent-extraction spectrometory. Toluene and a water sample containing NS were flowed to an extraction coil (i.d. 1 mm×3.12 m) at 2.02, 2.90 mL min⁻¹, respectively, to extract NS into toluene. Then, toluene containing NS and a potassium thiocyanate (6 M)-iron(III) chloride (24 mM) mixed solution were flowed to the reaction coil (i.d. 1 mm×1.04 m) at 2.02, 0.42 mL min⁻¹, respectively. Those flows were segmented with air bubbles. The absorbance of NS-iron(III) thiocyanate complex formed in the toluene layer was measured at 505 nm. The detection limit of this CFA was 2 μmol L⁻¹ as heptaoxyethylene dodecyl ether. Good results were obtained in recovery tests using river water.

Tetrahydrocurcumin has been of interest, the antioxidative activity of which has been reported to be higher than that of curcumin. In an attempt to investigate the function, we studied the stability of tetrahydrocurcumin in aqueous solutions and the acid dissociation properties, and compared them with those of curcumin. In aqueous solutions of pH 3.0〜10.0 under air - saturated conditions, curcumin showed complete decomposition within l h. At pH 8.0, curcumin decomposed rapidly with a half-life time of 1.0 min. On the other hand, tetrahydrocurcumin did not show any detectable decomposition within 2 h, showing higher stability in aqueous solutions under air. The acid-dissociation constants of tetrahydrocurcumin were estimated to be pK_a1＝8.59±0.06, pK_a2＝9.53±0.01, pK_a3＝10.74±0.02, which are slightly larger than those of curcumin (pK_a1＝8.47±0.032, pK_a2＝9.43±0.025, pK_a3＝10.59±0.03) by 0.1 pK_a unit.

Several kinds of commercially available insoluble organic pigments were analyzed by laser desorption/ionization (LDI), LDI on silicon (DIOS), matrix-assisted LDI (MALDI) time-of-flight mass spectrometry, and EI and CI ion-trap MS with a direct sample probe. The results showed that the pigment molecules were detected as cation radicals and a mixture of cation radicals and protonated molecules by LDI, DIOS, and MALDI in a positive mode. The molecular ions obtained from EI and CI were cation radicals and protonated molecules in the positive ion mode, respectively. In a negative mode, anion radicals and deprotonated molecules were generated by LDI, DIOS, and MALDI, while anion radicals were mainly detected by EI and CI. In particular, negative LDI and CI showed simpler mass spectra with fewer fragment ions. EI- and CI- MSⁿ were shown to be convenient and effective methods to determine the structures of organic pigments with low molecular weight.

A highly selective, sensitive, and practically useful spot test of aluminum(III) has been developed. In this method, a 5 μL of ethanol–aqueous (1＋1) sample solution containing metal chelates with 2,2'-dihydroxyazobenzene (DHAB) is loaded on a hydrophobic surface of an octadecylsilanized (ODS) silica plate, and thin-layer chromatography (TLC) is directly performed without drying the sample drop. The fluorescent aluminum(III) chelate remains selectively at the loading position, whereas the excess reagent, DHAB, and the chelates of the other metal ions are eluted. The fluorescent spot, which is observed only at the loading position, retains the circular shape of the initial interface between the sample drop and the plate. Immediately performing chromatographic separation after sample loading is effective for avoiding aluminum(III) contamination, which is caused by elution from the ODS-silica, because of the slow rate of formation of the aluminum(III)-DHAB complex. Furthermore, the matrix tolerance of the proposed method is superior to that of the previous method, in which chromatographic separation is performed after drying the sample drop. The visual detection limit is 1.0 μg/L. The calibration curve assessed with the densitometric responses with an excitation beam (505 nm) is linear over the concentration range up to 27 μg/L. The detection limit, which is calculated with 3.3σ at a concentration of 1.4 μg/L, is 0.2 μg/L (n＝5). The proposed method has been applied to the determination of parts per billion levels of aluminum(III) ion in river-water samples.

The present study was examined the effect of tyramine-induced hydroxyl radical (･OH) generation. A flexibly mounted microdialysis technique was used to detect the generation of ･OH in in vivo rat hearts. To measure the level of ･OH, sodium salicylate in Ringer's solution (0.5 nmol/μL/min) was infused directly through a microdialysis probe to detect the generation of ･OH as reflected by the nonenzymatic formation of 2,3-dihydroxybenzoic acid (DHBA). To confirm the generation of ･OH by the Fenton-type reaction, iron(II) was infused through a microdialysis probe. A positive linear correlation between iron(II) and the formation of 2,3-DHBA (R²＝0.983) was observed.