Yoshihito Ohba

Evaluation of chemiluminescence reagents for selective detection of reactive oxygen species

In order to evaluate the chemiluminescence (CL) reagents for selective detection of reactive oxygen species (ROS), we comprehensively measured the CL responses of 20 CL reagents (three luminol derivatives, two imidazopyrazinone derivatives, eight lophine derivatives, six acridinium ester derivatives and lucigenin) against six types of ROS (superoxide anion: O(2)(*-), hydroxyl radical: *OH, hydrogen peroxide: H(2)O(2), hypochlorite anion: ClO(-), singlet oxygen: (1)O(2), and nitric oxide: NO). As a result of the screening, it was found that nine CL reagents selectively detected O(2)(*-) while one CL reagent selectively detected *OH. However, no CL reagent had selectivity on the detection of H(2)O(2), ClO(-), (1)O(2) and NO. Our screening results could help to select the most suitable CL reagent for selective determination of different ROS. As an application study, 4-methoxyphenyl-10-methylacridinium-9-carboxylate (MMAC), one of the acridinium ester derivatives, showed high selectivity on the detection of O(2)(*-), and thus was applied to the assay of superoxide dismutase (SOD) activity. The dynamic range and detection limit of the developed CL assay were 0.1-10 and 0.06 U mL(-1), respectively. Significant correlation (r=0.997) was observed between the results by the CL assay using MMAC and the spectrophotometric assay using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt.

Phenylglyoxal and glyoxal as fluorogenic reagents selective for N-terminal tryptophan-containing peptides

Abstract Methods are described for the determination of N -terminal tryptophan-containing peptides in which phenylglyoxal and glyoxal are used as fluorogenic reagents. Phenylglyoxal and glyoxal react selectively with the peptides when heated in a strongly acidic medium in the presence of potassium hexacyanoferrate(III) and in a moderately acidic medium, respectively, at 100°C for min to produce fluorescence. The detection limit of N -terminal tryptophan-containing small peptides tested are 118–165 pmol ml −1 in the reaction mixtures. The reaction mixture of tryptophylalanine, a model peptide, with glyoxal afforded a single peak when separated by reversed-phase liquid chromatography whereas that with phenylglyoxal gave several peaks.

Liquid chromatography of fatty acids with chemiluminescence detection

Abstract This paper reviews high-performance liquid chromatographic (HPLC) methods with chemiluminescence (CL) detection for the analysis of biologically important fatty acids. A CL detection method dose not need an exciting light, which can eliminate any scattering of light source. Therefore, the method is highly sensitive owing to a large signal-to-noise ratio (S/N). Further, the method has advantages over other detection method for HPLC—high selectivity, wide dynamic range, and relatively simple instrumentation. Through these advantages, the HPLC with CL detection has increasing importance in the areas of clinical and biomedical analyses. In this review, CL dereivatization reagents for fatty acids and their utilization to the CL detection methods for HPLC were discussed in detail.

A validated HPLC-fluorescence method with a semi-micro column for routine determination of homocysteine, cysteine and cysteamine, and the relation between the thiol derivatives in normal human plasma.

A semi-micro column HPLC-fluorescence method for routine determination of thiol derivatives such as homocysteine (Hcy), cysteine (Cys) and cysteamine (CA) is described. The thiol derivatives labeled with ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F) were isocratically separated within 12 min on a semi-micro ODS column (Daisopak-SP-120-5-ODS-BP) with a mixture of 25 mm acetate buffer (pH 2.00) and CH(3)CN as a mobile phase. The purity and similarity of SBD-thiols by a multi-wavelength fluorescence detector were more than 92.3 and 96.7%. The detection limits of Hcy, Cys and CA at a signal-to-noise ratio of 3 were 0.16, 0.47 and 0.03 microm, respectively. Furthermore validation parameters such as accuracy, precision and robustness of the proposed method showed satisfactory results. Almost 850 plasma sample injections (range 572-1076, n = 3) for a column could be performed without differences in retention time and peak heights of labels. As an application of the proposed method, the determination of thiol derivatives in normal human plasma (n = 103) was demonstrated. The correlation coefficients between Hcy vs Cys and Hcy vs CA were 0.38 and -0.35, respectively.

Preparation and characterization of poly(l-phenylalanine) chiral stationary phases with varying peptide length.

Three new chiral stationary phases with different lengths of l-phenylalanine peptide were prepared by solid-phase synthesis with tert-butoxycarbonyl (Boc)-l-phenylalanine on silica. The effect of phenylalanine peptide length on enantioselectivity was studied. The best separation of R/S-warfarin was achieved by the chiral stationary phase with intermediate peptide length. These stationary phases were found to exist mainly in alpha-helical conformation by using FT-IR spectra. The end-capping reagents for the N-terminus of the peptide were also evaluated.

Evaluation of lophine derivatives as L-012 (luminol analog)-dependent chemiluminescence enhancers for measuring horseradish peroxidase and H2O2.

8-Amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione (L-012) was recently synthesized as a new chemiluminescence (CL) probe; the light intensity and the sensitivity of L-012 are higher than those of other CL probes such as luminol. Previously, our group developed four lophine-based CL enhancers of the horseradish peroxidase (HRP)-catalyzed CL oxidation of luminol, namely 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI), 2-(4-hydroxyphenyl)-4,5-di(2-pyridyl)imidazole (HPI), 4-(4,5-diphenyl-1H-imidazol-2-yl)phenylboronic acid (DPA), and 4-[4,5-di(2-pyridyl)-1H-imidazol-2-yl]phenylboronic acid (DPPA), and showed that DPPA was suitable for the photographic detection of HRP. In this study, we replaced luminol with L-012 and evaluated these as L-012-dependent CL enhancers. In addition, to detect HRP and/or H2O2 with higher sensitivity, each detection condition for the L-012-HRP-H2O2 enhanced CL was optimized. All the derivatives enhanced the L-012-dependent CL as well as luminol CL; HPI generated the highest enhanced luminescence. Under optimized conditions for HRP detection, the detection limit of HRP was 0.08 fmol. By contrast, the detection limit of HRP with the enhanced L-012-dependent CL using 4-iodophenol, which is a common enhancer of luminol CL, was 1.1 fmol. With regard to H2O2 detection, the detection limits for enhanced CL with HPI and 4-iodophenol were 0.29 and 1.5 pmol, respectively. Therefore, it is demonstrated that HPI is the most superior L-012-dependent CL enhancer.

Alkoxyphenylglyoxals as fluorogenic reagents selective for guanine and its nucleosides and nucleotides in liquid chromatography

Abstract Glyoxal analogues which have a phenyl moiety substituted with electron-donating methoxyl group(s) or a methylenedioxyl group, react selectively with guanine and its nucleosides and nucleotides in phosphate buffer (pH 7.0) at 37°C for 5–7 min to give the corresponding fluorescent derivatives. The derivatives can be separated by reversed-phase liquid chromatography. 3,4-Dimethoxyphenylglyoxal is most sensitive; the detection limits (S/N = 3) for guanine and its nucleosides and nucleotides vary in the range 40–400 fmol per injection volume depending on the guanine-related compounds.

Assay of human platelet guanylate cyclase activity by high-performance liquid chromatography with fluorescence derivatization

A selective and sensitive high-performance liquid chromatography (HPLC) method with fluorescence derivatization for the assay of guanylate cyclase (GC) activity is described. GTP and cGMP, which are the substrate and the product of GC, respectively, and other guanine-containing compounds are selectively converted by the reaction with (3,4-dimethoxyphenyl)glyoxal to the fluorescent derivatives. The derivatives were separated by reversed-phase HPLC. The limit of detection at a signal-to-noise ratio of 3 for cGMP was 10 fmol on the column. The sensitivity of this method was less than that of the conventional radioisotopic method, but this method is simple and convenient. Human platelet GC activity was measured, and the effects of some compounds were investigated.

Aminopyrazine analogues as chemiluminescence derivatization reagents for pyruvic acid

Aminopyrazine analogues were studied as sensitive and selective chemiluminescence derivatization reagents for pyruvic acid. These analogues reacted with pyruvic acid under acidic conditions at 100 degrees C to produce Cypridina luciferin derivatives, which exhibit chemiluminescence by reaction with hydrogen peroxide in the presence of potassium t-butoxide in dimethylformamide. Of the four aminopyrazine analogues (2-amino-5-phenylpyrazine, 2-amino-5-(4-hydroxyphenyl)pyrazine, 2-amino-5-(3,4, 5-trimethoxyphenyl)pyrazine, and 2-aminoquinoxaline), in the present test 2-amino-5-(3,4,5-trimethoxyphenyl) pyrazine was the most sensitive for pyruvic acid, and the chemiluminescence intensity was about four times higher than that obtained with aminopyrazine.