Tomoyuki Oe

A novel controlled local drug delivery system for inner ear disease.

Purpose: Our goal is to develop a novel drug delivery system that can potentially improve clinical outcomes compared to current methods of dosing drugs such as dexamethasone or gentamicin. This system focuses on a single local application to the inner ear via the round window membrane.

Determination of cellular redox status by stable isotope dilution liquid chromatography/mass spectrometry analysis of glutathione and glutathione disulfide

Oxidation of glutathione (GSH) to glutathione disulfide (GSSG) occurs during cellular oxidative stress. The redox potential of the 2GSH/GSSG couple, which is determined by the Nernst equation, provides a means to assess cellular redox status. It is difficult to accurately quantify GSH and GSSG due to the ease with which GSH is oxidized to GSSG during sample preparation. To overcome this problem, a stable isotope dilution liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) method has been developed using 4-fluoro-7-sulfamoylbenzofurazan (ABD-F) derivatization. ABD-F derivatization of the GSH thiol group was rapid, quantitative, and occurred at room temperature. The LC/MRM-MS method, which requires no sample clean-up, was validated within the calibration ranges of 5 to 400 nmol/mL in cell lysates for GSH and 0.5 to 40 nmol/mL in cell lysates for GSSG. Calibration curves prepared by adding known concentrations of GSH and GSSG to cell lysates were parallel to the standard curve prepared in buffers. GSH and GSSG concentrations were determined in two monocyte/macrophage RAW 267.4 cell lines with or without 15-LOX-1 expression (R15LO and RMock cells, respectively) after treatment with the bifunctional electrophile 4-oxo-2(E)-nonenal (ONE). R15LO cells synthesized much higher concentrations of the lipid hydroperoxide, 15(S)-hydroperoxyeicosatetraenoic acid (15-HPETE), which undergoes homolytic decomposition to ONE. GSH was depleted by ONE treatment in both RMock and R15LO cells, leading to significant increases in their redox potentials. However, R15LO cells had higher GSH concentrations (most likely through increased GSH biosynthesis) and had increased resistance to ONE-mediated GSH depletion than RMock cells. Consequently, R15LO cells had lower reduction potentials at all concentrations of ONE. GSSG concentrations were higher in R15LO cells after ONE treatment when compared with the ONE-treated RMock cells. This suggests that increased expression of 15(S)-HPETE modulates the activity of cellular GSH reductases or the transporters involved in removal of GSSG.

Determination of γ-glutamylglutathione and other low-molecular-mass biological thiol compounds by isocratic high-performance liquid chromatography with fluorimetric detection

Abstract A method was developed for the simultaneous determination of γ-glutamylglutathione (γ-GluGSH) and other low-molecular-mass thiol compounds (cysteine, cysteamine, homocysteine, cysteinylglycine, γ-glutamylcysteine, glutathione and N-acetylcysteine) using high-performance liquid chromatography combined with precolumn fluorescence labeling with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F). These SBD-labeled thiol compounds were separated within 35 min on a Cosmosil 5C-18AR column with isocratic elution using 75 m M sodium citrate buffer (pH 2.90)–methanol (98:2) and detected fluorimetrically (ex. 386 nm, em. 516 nm). The calibration graphs using 2-mercaptoethanol as an internal standard showed good linearity in the range from 20 pmol to 10 nmol for all thiol compounds examined. The application of this method for the quantitative determination of thiol compounds in the urine from γ-glutamyl transpeptidase-deficient mice was also demonstrated. This method is sufficiently simple, rapid and sensitive for the determination of γ-GluGSH and other low-molecular-mass thiol compounds in biological samples.

Sensitive ferrocene reagents for derivatization of thiol compounds in high-performance liquid chromatography with dual-electrode coulometric dectection

Three N-substituted maleimides possessing ferrocene as an electrophore were prepared and evaluated for pre-column derivatization of thiol compounds in high-performance liquid chromatography with dual-electrode coulometric detection. The utility of these reagents was investigated by employing N-acetyl-L-cysteine as a model compound. Among the three, N-(ferrocenyl) maleimide was the most favourable reagent with respect to reactivity, stability and electrochemical properties. The dual-electrode coulometric detection of the adduct showed high selectivity and sensitivity with a detection limit of 0.06 pmol. The proposed method is applicable to the determination of glutathione in biological specimens.

Mass Spectrometric Characterization of Modifications to Angiotensin II by Lipid Peroxidation Products, 4-Oxo-2(E)-nonenal and 4-Hydroxy-2(E)-nonenal

The octapeptide angiotensin II (Ang II; Asp(1)-Arg(2)-Val(3)-Tyr(4)-Ile(5)-His(6)-Pro(7)-Phe(8)) is the primary active hormone of the renin/angiotensin system (RAS) and has been implicated in various cardiovascular diseases. Numerous structure-activity relationship studies have identified Asp(1), Arg(2), and His(6) of Ang II to be critical for its biological activity and receptor binding. From the reactions of Ang II with lipid peroxidation-derived aldehydes, 4-oxo-2(E)-nonenal (ONE) or 4-hydroxy-2(E)-nonenal (HNE), we have identified the major modifications to the N-terminus, Asp(1), Arg(2), and His(6) of Ang II by liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption ionization-time-of-flight/MS (MALDI-TOF/MS). The identities of ONE- and HNE-modified Ang II were confirmed by tandem mass spectrometry (MS/MS) and postsource decay (PSD)-TOF/MS before and after the reaction with sodium borohydride. In the reaction with ONE, a pyruvamide-Ang II that formed via oxidative decarboxylation of N-terminal Asp was detected as the most abundant product after 48 h of incubation. It was followed by Arg-modified [Arg(2)(ONE-H(2)O)]-Ang II and the N-terminal-modified 4-ketoamide form of [N-ONE]-Ang II. The Michael addition products of [His(6)(HNE)]-Ang II were the most abundant products in the beginning of the reaction with HNE, followed by the dehydrated Michael addition products of [His(6)(HNE-H(2)O)]-Ang II. [His(6)(HNE)]-Ang II was dehydrated to [His(6)(HNE-H(2)O)]-Ang II during the prolonged incubation, and [His(6)(HNE-H(2)O)]-Ang II became the major products after 7 days. The model reactions of N(α)-tert-butoxycarbonyl (tBoc)-Arg with ONE and tBoc-His with HNE were performed and compared with the Ang II reaction. tBoc-Arg readily reacted with ONE to produce a compound analogous to [Arg(2)(ONE-H(2)O)]-Ang II, which confirmed Arg as one of the important target nucleophiles of ONE. However, tBoc-His exclusively formed a Michael addition product upon the reaction with HNE. The unexpected formation of [His(6)(HNE-H(2)O)]-Ang II can be explained by the proximity of His(6) to C-terminal carboxylate in the specific conformation of Ang II, which facilitates the dehydration of Michael addition products. Therefore, our results suggest a possible discrepancy in the adduction chemistry of ONE and HNE for model amino acids and endogenous bioactive peptides, which is governed by the microenvironment of peptides, such as the specific amino acid sequence and conformation. Such stable ONE- and HNE-derived modifications to Ang II could potentially modulate its functions in vivo by disrupting the interaction with Ang II type 1 (AT(1)) receptor and/or inhibiting the enzyme activity of aminopeptidase A (APA), which cleaves the N-terminal Asp residue of Ang II to generate Ang III.

Non-invasive proteomic analysis of human skin keratins: screening of methionine oxidation in keratins by mass spectrometry.

Keratins are the main constituent of human skin and have been identified as major oxidative target proteins. However, there has been a lack of studies aimed at identifying the oxidation sites of keratins because of the difficulties associated with their insolubility and handling. Here, we introduce a mass spectrometry (MS)-based proteomic methodology to screen oxidative modifications in human skin keratins. Human skin proteins were obtained non-invasively by tape stripping and solubilized in SDS buffer, followed by purification and digestion using the modified filter-aided sample preparation method. The tryptic peptides were then analyzed by MALDI-TOF/MS, LC-ESI/MS, and MS/MS. PMF analyses have identified keratins K1 and K10 as the major proteins of human skin. Met(259), Met(262), Met(296), and Met(469), located in the α-helical rod domain of K1, were the most susceptible sites to oxidation induced by hydrogen peroxide in vitro and in vivo. Our results indicate a potential use of the identified methionine residues as biomarkers of oxidative skin damage. The present methodology is the first MS-based approach to detecting oxidative modifications in keratins obtained directly from human skin and can be easily applied to the monitoring of other keratin modifications in various skin conditions.

Sensitive ferrocene reagents for derivatization of amines for high-performance liquid chromatography with electrochemical detection

Six reagents possessing ferrocene as an electrophore were prepared and evaluated for pre-column derivatization of amino compounds for their determination by high-performance liquid chromatography with electrochemical detection. The utility of these reagents was investigated employing phenethylamine as a model compound. Among these six, N-succinimidyl 3-ferrocenylpropionate was the best with respect to reactivity, stability and electrochemical properties. The developed method was applied to the determination of putrescine formed from ornithine by ornithine decarboxylase.

Ferrocene Derivatization Reagents for Optical Resolution of Carboxylic Acids by High-performance Liquid Chromatography with Electrochemical Detection

Abstract New derivatization methods using chiral ferrocene reagents have been developed for the optical resolution of carboxylic acids by high-performance liquid chromatography with electrochemical detection. Two chiral derivatization reagents, 1-ferrocenylethylamine and 1-ferrocenylpropylamine, were readily prepared from acetylferrocene and propionylferrocene in two steps, respectively. Condensation of carboxylic acids with the chiral reagent was effected in the presence of water-soluble carbodiimide and 1-hydroxybenzotriazole. The diastereomeric amides formed from N-acetylamino acid and α-arylpropionic acid enantiomers were efficiently resolved by reversed-phase chromatography and showed the satisfactory sensitivity at +0.45 V vs. an Ag/AgCl reference electrode with a detection limit of 0.5 pmole (S/N=5).

Determination of the platinum drug cis-amminedichloro(2-methylpyridine)platinum(II) in human urine by liquid chromatography–tandem mass spectrometry

A validated stable isotope dilution liquid chromatography-tandem mass spectrometry assay for the novel platinum drug cis-amminedichloro(2-methylpyridine)platinum(II) (ZD0473) in human urine has been developed. This method uses selected reaction monitoring on the transition of m/z 393 [M+NH(4)](+) to m/z 304 [M+NH(4)-NH(3)-2 x H(35)Cl](+) for ZD0473, and m/z 400 [M+NH(4)](+) to m/z 310 [M+NH(4)-NH(3)-H(35)Cl-(2)H(35)Cl](+) for the internal standard [(2)H(7)]ZD0473. Standard curves were prepared over the range from 0.15 to 50 microg/ml. The lower limit of quantitation was 0.2 microg/ml for 100 microl of urine. This simple, rapid, reliable, and sensitive method of quantitation displayed acceptable accuracy and precision over the 3 days of assay validation. A novel platinum adduct was formed during the storage of ZD0473 in human urine. The adduct did not correspond to any of the typical sulfhydryl adducts that have been identified previously for platinum drugs. Formation of the adduct was prevented by the addition of 50% (w/v) sodium chloride to the urine. The assay can be used to quantify intact ZD0473 in the urine of subjects dosed with this new platinum drug.

Lgr4 Controls Specialization of Female Gonads in Mice

ABSTRACT Leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is a type of membrane receptor with a seven-transmembrane structure. LGR4 is homologous to gonadotropin receptors, such as follicle-stimulating hormone receptor (Fshr) and luteinizing hormone/choriogonadotropin receptor (Lhcgr). Recently, it has been reported that Lgr4 is a membrane receptor for R-spondin ligands, which mediate Wnt/beta-catenin signaling. Defects of R-spondin homolog (Rspo1) and wingless-type MMTV integration site family, member 4 (Wnt4) cause masculinization of female gonads. We observed that Lgr4−/− female mice show abnormal development of the Wolffian ducts and somatic cells similar to that in the male gonads. Lgr4−/− female mice exhibited masculinization similar to that observed in Rspo1-deficient mice. In Lgr4−/− ovarian somatic cells, the expression levels of lymphoid enhancer-binding factor 1 (Lefl) and Axin2 (Axin2), which are target genes of Wnt/beta-catenin signaling, were lower than they were in wild-type mice. This study suggests that Lgr4 is critical for ovarian somatic cell specialization via the cooperative signaling of Rspo1 and Wnt/beta-catenin.