Characterization of supported liquid extraction as a sample pretreatment method for eicosanoids and related metabolites in biological fluids.

Sample pretreatment is an important process in liquid chromatography-mass spectrometry-based quantitative lipidomics. Reversed-phase solid phase extraction (RP-SPE) has been widely used for analyzing various types of samples, including aqueous samples such as cell culture media, plasma, serum, urine, and other biological fluids. Because lipid mediators are often protein-bound, prior deproteinization is necessary for their effective recovery. Deproteinization is typically performed by the addition of organic solvents, which requires time-consuming evaporation-reconstitution, or dilution with aqueous solvents before RP-SPE; however, both of these approaches compromise the analytical performance. As a potential alternative, we attempted to utilize supported liquid extraction (SLE), an automation-compatible variant of liquid-liquid extraction, for the determination of eicosanoids and related metabolites in aqueous samples. We screened 81 different sample diluent-eluent conditions and found that the use of 0.1% formic acid-water as the diluent and 0.1% formic acid-methyl acetate as the eluent enabled the optimum recovery of a variety of eicosanoids, except for peptide leukotrienes. The optimized SLE method efficiently removed protein from human plasma, while phospholipids and neutral lipids were modestly recovered. Moreover, the proposed method exhibited a quantitative performance comparable to that of typical ordinary RP-SPE method in the analysis of human platelets stimulated with thrombin receptor-activating peptide 6. Thus, we propose SLE as an attractive option for rapid lipid mediator extraction from aqueous samples.

A comprehensive quantification method for eicosanoids and related compounds by using liquid chromatography/mass spectrometry with high speed continuous ionization polarity switching.

Fatty acids and related metabolites, comprising several hundreds of molecular species, are an important target in disease metabolomics, as they are involved in various mammalian pathologies and physiologies. Selected reaction monitoring (SRM) analysis, which is capable of monitoring hundreds of compounds in a single run, has been widely used for comprehensive quantification. However, it is difficult to monitor a large number of compounds with different ionization polarity, as polarity switching requires a sub-second period per cycle in classical mass spectrometers. In the present study, we developed and evaluated a comprehensive quantification method for eicosanoids and related compounds by using LC/MS with high-speed continuous ionization polarity switching. The new method employs a fast (30ms/cycle) continuous ionization polarity switching, and differentiates 137 targets either by chromatography or by SRM transition. Polarity switching did not affect the lower limits of quantification, which ranged similarly from 0.5 to 200pg on column. Lipid extracts from mouse tissues were analyzed by this method, and 65 targets were quantitatively detected in the brain, including 6 compounds analyzed in the positive ion mode. We demonstrated that a fast continuous ionization polarity switching enables the quantification of a wide variety of lipid mediator species without compromising the sensitivity and reliability.

Novel swine model of transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a life-threatening complication of blood transfusion. Antibodies against human leukocyte antigens in donors' plasma are the major causes of TRALI. Several animal models of TRALI have been developed, and the mechanism underlying TRALI development has been extensively investigated using rodent models. Although sheep models of nonimmune TRALI have been developed, large-animal models of antibody-mediated TRALI are not yet available. STUDY DESIGN AND METHODS: To develop a swine model of TRALI, male Clawn strain miniature pigs were used. A monoclonal antibody (MoAb) against swine leukocyte antigens (SLAs) Class I (4G8, 0.3 or 1.0 mg/kg body weight [BW]) and a control antibody (1.0 mg/kg BW) were injected into the peripheral vein after priming with or without 1 µg/kg BW lipopolysaccharide (LPS; n = 3 each). Lung injury was assessed using PaO2 /FiO2 (P/F) ratio and by chest X-ray imaging. Histopathologic analysis was also conducted. RESULTS: Lung injury could be induced by injecting 4G8 at an amount of 1.0 mg/kg BW, after LPS. The P/F ratio 90 minutes after the administration of 4G8 significantly decreased (p < 0.05). Bilateral infiltration was shown in chest X-ray imaging. Lung injury was confirmed by histopathologic analysis. CONCLUSION: Lung injury in pigs was successfully induced by anti-SLA MoAb. Priming with LPS is a prerequisite for inducing lung injury and the amount of the antibody is a critical condition.

Organelle-localizable fluorescent chemosensors for site-specific multicolor imaging of nucleoside polyphosphate dynamics in living cells.

ATP and its derivatives (nucleoside polyphosphates (NPPs)) are implicated in many biological events, so their rapid and convenient detection is important. In particular, live cell detection of NPPs at specific local regions of cells could greatly contribute understanding of the complicated roles of NPPs. We report herein the design of two new fluorescent chemosensors that detect the dynamics of NPPs in specific regions of living cells. To achieve imaging of NPPs on plasma membrane surfaces (2-2Zn(II)), a lipid anchor was introduced into xanthene-based Zn(II) complex 1-2Zn(II), which was previously developed as a turn-on type fluorescent chemosensor for NPPs. Meanwhile, for subcellular imaging of ATP in mitochondria, we designed rhodamine-type Zn(II) complex 3-2Zn(II), which possesses a cationic pyronin ring instead of xanthene. Detailed spectroscopic studies revealed that 2-2Zn(II) and 3-2Zn(II) can sense NPPs with a several-fold increase of their fluorescence intensities through a sensing mechanism similar to 1-2Zn(II), involving binding-induced recovery of the conjugated form of the xanthene or pyronin ring. In live cell imaging, 2-2Zn(II) containing a lipid anchor selectively localized on the plasma membrane surface and detected the extracellular release of NPPs during cell necrosis induced by streptolysin O. On the other hand, rhodamine-type complex 3-2Zn(II) spontaneously localized at mitochondria inside cells, and sensed the local increase of ATP concentration during apoptosis. Multicolor images were obtained through simultaneous use of 2-2Zn(II) and 3-2Zn(II), allowing detection of the dynamics of ATP in different cellular compartments at the same time.

Rational design of FRET-based ratiometric chemosensors for in vitro and in cell fluorescence analyses of nucleoside polyphosphates.

Ratiometric fluorescence sensing is a useful technique for the precise and quantitative analysis of biological events occurring under complex conditions, such as those inside cells. We report herein the design of new ratiometric chemosensors for nucleoside polyphosphates such as ATP that are based on binding-induced modulation of fluorescence resonance energy transfer (FRET) coupled with a turn-on fluorescence-sensing mechanism. We designed these new FRET-based ratiometric chemosensors by utilizing spectral overlap changes to modulate the FRET efficiency. Introduction of coumarin fluorophores as the FRET donors into a binuclear zinc complex as the FRET acceptor provided the ratiometric chemosensors. These chemosensors exhibited a clear dual-mission signal change upon binding with strong affinity (K(app) ≈ 10(6)-10(7) M(-1)) to nucleoside polyphosphates in aqueous solution, whereas no detectable emission change was observed with monophosphates and phosphodiester species or various other anions. These chemosensors were used for real-time fluorescence monitoring of enzyme reactions such as saccharide synthesis by glycosyltransferase and phosphorylation by protein kinase, both of which involve nucleoside polyphosphates as substrates. The utility of ratiometric sensing by chemosensors was further demonstrated in a fluorescence-imaging study of the nucleoside polyphosphates inside living cells, wherein we ratiometrically visualized the stimulus-responsive concentration change of ATP, an indicator of the cellular energy level.

Turn-on fluorescence sensing of nucleoside polyphosphates using a xanthene-based Zn(II) complex chemosensor.

Fluorescence sensing with small molecular chemosensors is a versatile technique for elucidation of function of various biological substances. We now report a new fluorescent chemosensor for nucleoside polyphosphates such as ATP using metal-anion coordination chemistry. The chemosensor 1-2Zn(II) is comprised of the two sites of 2,2'-dipicolylamine (Dpa)-Zn(II) as the binding motifs and xanthene as a fluorescent sensing unit for nucleoside polyphosphates. The chemosensor 1-2Zn(II) selectively senses nucleoside polyphosphates with a large fluorescence enhancement (F/F(o) > 15) and strong binding affinity (K(app) approximately = 1 x 10(6) M(-1)), whereas no detectable fluorescence change was induced by monophosphate species and various other anions. The 'turn-on,' fluorescence of 1-2Zn(II) is based on a new mechanism, which involves the binding-induced recovery of the conjugated form of the xanthene ring from its nonfluorescent deconjugated state which was formed by an unprecedented nucleophilic attack of zinc-bound water. The selective and highly sensitive ability of 1-2Zn(II) to detect nucleoside polyphosphates enables its bioanalytical applications in fluorescence visualization of ATP particulate stores in living cells, demonstrating the potential utility of 1-2Zn(II).

Cooperation between artificial receptors and supramolecular hydrogels for sensing and discriminating phosphate derivatives.

This study has successfully demonstrated that the cooperative action of artificial receptors with semi-wet supramolecular hydrogels may produce a unique and efficient molecular recognition device not only for the simple sensing of phosphate derivatives, but also for discriminating among phosphate derivatives. We directly observed by confocal laser scanning microscopy that fluorescent artificial receptors can dynamically change the location between the aqueous cavity and the hydrophobic fibers upon guest-binding under semi-wet conditions provided by the supramolecular hydrogel. On the basis of such a guest-dependent dynamic redistribution of the receptor molecules, a sophisticated means for molecular recognition of phosphate derivatives can be rationally designed in the hydrogel matrix. That is, the elaborate utilization of the hydrophobic fibrous domains, as well as the water-rich hydrophilic cavities, enables us to establish three distinct signal transduction modes for phosphate sensing: the use of (i) a photoinduced electron transfer type of chemosensor, (ii) an environmentally sensitive probe, and (iii) an artificial receptor displaying a fluorescence resonance energy transfer type of fluorescent signal change. Thus, one can selectively sense and discriminate the various phosphate derivatives, such as phosphate, phospho-tyrosine, phenyl phosphate, and adenosine triphosphate, using a fluorescence wavelength shift and a seesaw type of ratiometric fluorescence change, as well as a simple fluorescence intensity change. It is also shown that an array of the miniaturized hydrogel is promising for the rapid and high-throughput sensing of these phosphate derivatives.

Recognition and fluorescence sensing of specific amino acid residue on protein surface using designed molecules.

Many biological processes are mediated by surface recognition between proteins. Small molecules that recognize and bind a specific region of a protein surface may be promising agents for disrupting certain protein-protein surface interactions, which consequently leads to regulation of cellar functions. This article describes our recent efforts toward the development of the designed small molecules, which can recognize histidine or phosphorylated amino acid residues on peptide surfaces in a sequence-selective manner. These results demonstrate that cooperative metal-ligand interaction is powerful for tight and selective binding to the specific amino acid residues of proteins in aqueous medium.